Biotechnology
Working with Micro-organisms
Intermediate 2
5493
June 1999
HIGHER STILL
Biotechnology
Working with Micro-organisms
Intermediate 2
Support Materials
INTERMEDIATE 2 BIOTECHNOLOGY
UNIT 2
WORKING WITH MICRO-ORGANISMS
STUDENT LABORATORY MANUAL
UNIT 2: WORKING WITH MICRO-ORGANISMS (INT 2)
STUDENT LABORATORY MANUAL
Contents
Guide to Candidate
Microbiological procedures
1: Preparation of work space
2: Pouring plates
3: Subculturing micro-organisms
3.1: Loop transfer of micro-organisms
3.1.1 solid to solid
3.1.2 liquid to solid
3.1.3 solid to liquid
3.1.4 liquid to liquid
3.2: Streak plate inoculations
3.2.1. solid to solid
3.2.2 liquid to solid
3.3: Plate to plate subculture of fungal mycelium
3.4: Use of sterile swabs to sample the environment
4: Separating micro-organisms
5: Microscopic examination of micro-organisms
5.1: Magnification
5.2: Setting up a microscope
5.3: Microscopic examination of pond water/ hay infusion/yeast
5.3.1 Cavity slide preparation
5.3.2 Bright field microscopy
5.3.3 Dark field microscopy
5.3.4 Phase contrast microscopy
5.4: Calculation of specimen size using a microscope
6: Staining
6.1: Preparation of a smear of bacteria or yeast from a solid culture
6.2: Observation of bacteria using the simple stain methylene blue
6.3: Observation of filamentous fungi using lactophenol blue
6.4: Negative staining of yoghurt with nigrosin
6.5: Staining of root nodules to observe Rhizobium
6.6: Vital staining of yeast with neutral red
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Biotechnology: Working with Micro-Organisms (Int 2) Student Laboratory Manual
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GUIDE TO CANDIDATE
This unit of Intermediate 2 Biotechnology, ‘Working with Micro-organisms’ is a
practical unit in which you will learn some techniques of handling and studying
micro-organisms in the laboratory. These include plate pouring, subculturing microorganisms, separating a mixed culture, staining and microscopy.
To achieve this unit, you must:
• know and understand how microbiological, microscopy and staining techniques
are carried out in a given way; and
• become competent in carrying out a range of microbiological and microscopy
techniques.
To achieve Outcome 1, you must pass a written end of unit assessment which will test
your knowledge and understanding of the theory behind the practical techniques. The
information you need for this is given in the introduction to each activity.
To achieve Outcome 2, you must become proficient in practical microbiological
techniques. You will have to practise them a number of times until you are familiar
with the procedures. Your teacher/lecturer will show you the techniques described in
this laboratory manual.
Each time you perform a technique, you must
• prepare for the work as you have been shown by your teacher or lecturer
• carry out the techniques safely and according to instructions
• keep a record of the work you do
• record your results and observations clearly.
You should keep the record of your work and your observations/results in a laboratory
diary/notebook and make an entry each time you carry out a piece of work or observe
results. The diary can be kept in a simple notebook or jotter and should be submitted
as evidence for Outcome 2.
Each entry should have:
• the date when the work was carried out
• a heading
• a brief description of the technique you carried out including incubation times and
temperatures if appropriate
• observations or results recorded in an appropriate manner (words, diagram, table
etc)
• discussion of results if appropriate.
An example of a diary entry is given below.
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Example of laboratory diary entry.
1.6.98
Subculturing, liquid to solid.
Micrococcus luteus labelled ‘AA, M luteus, 27.5.98’, was aseptically
transferred from a broth culture to an agar plate and plated out to obtain
single colonies using the streak plate method. The plate was incubated
upside down at 30°C for 72 hours and then placed in a refrigerator till it
could be examined.
The technique was also carried out with Saccharomyces cerevisiae but it
was incubated for five days at 30°C.
8.6.98
Results
Organism
Single colonies
Contamination
M luteus
Yes
No
S cerevisiae
Yes
Yes
Discussion
A green fungus contaminated the S cerevisiae plate. A
spore from the air possibly entered the plate during the
process of plating out and developed into the fungal
colony when the plate was incubated. The contaminated
plate cannot be used for further subculture as spores
from the fungal colony may have contaminated the S
cerevisiae colonies.
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MICROBIOLOGICAL PROCEDURES
Introduction
The aim of this unit is to acquire some of the basic technical skills used in the study of
microbes.
In the practicals carried out in this unit, you will be working with living microorganisms, mainly bacteria and fungi. Although the organisms you will be using are
considered safe, there is always a small risk of contamination and so all organisms
must always be treated as if they could cause disease and you must follow the
recommended safety procedures.
The aims of these safety procedures are:
• to avoid bringing contaminating organisms to the laboratory bench
• to avoid contaminating laboratory cultures
• to avoid contaminating yourself, your colleagues and your surroundings
• to avoid taking contaminating organisms out of the laboratory.
Good Working Practice
To achieve the above aims, you must develop good working practice by following the
procedures below and all further safety instructions throughout the unit.
General safety procedures in the laboratory
•
Do not eat, drink or smoke in the laboratory.
•
Do not lick fingers or labels, use only self adhesive labels.
•
Avoid touching the face.
•
Tie back long hair.
•
Cover any cuts and grazes with a waterproof plaster.
•
Wear a lab coat and keep it fastened.
•
Speak quietly and avoid unnecessary movement around the laboratory.
•
Report all accidents, no matter how trivial, to the teacher/lecturer.
•
Do not pipette by mouth.
•
Keep doors and windows closed.
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On entering the microbiology laboratory
1. Hang up outdoor coats.
2. Leave bags at the side of the room taking only essentials to the workbench.
3. Wash hands thoroughly.
4. Dry hands using disposable paper towels.
5. Cover any wounds with a waterproof plaster.
6. Put on a lab coat.
7. Collect disinfectant and paper towel.
8. Swab bench with disinfectant.
You are now ready to start work. Do not touch your bag, outdoor wear etc.
without first washing your hands. Do not leave the laboratory wearing a lab
coat.
Bench Management
1. Swab bench with disinfectant at start and end of work using a paper towel.
2. Sit on the lab stool with legs under the bench.
3. Organise the equipment you are using so that it is within easy reach.
4. Keep bench as uncluttered as possible.
5. Do not lay contaminated materials on bench.
6. Report all spillages and breakage to the teacher or lecturer in charge.
Before leaving the microbiology laboratory
1. Swab bench with disinfectant.
2. Place stool under bench.
3. Remove and put away lab coat.
4. Wash hands thoroughly.
5. Collect outdoor coat and leave laboratory.
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1: PREPARATION OF WORK SPACE
Preparing yourself
When working with micro-organisms, it is important to develop what is known as
aseptic technique so that you do not become contaminated with the micro-organisms
you are working with nor do you contaminate your cultures. Before starting work you
must wash your hands thoroughly with soap and water. This mechanically removes
many of the organisms on your skin. Drying with a fresh paper towel prevents
transfer of organisms from a towel used by other people.
You should then put on a lab coat to protect your own clothes. In the event of a spill
and contamination occurs, a lab coat can be carefully removed.
Preparing the work space
The surface on which you work must not absorb any fluid or have cracks which could
harbour micro-organisms. Some micro-organisms can survive for a considerable
length of time in the environment and if spilled on to a rough or absorbent surface
could present a potential source of contamination both to others using the laboratory
and to cultures. If the bench surface is unsuitable, it should be covered with
Benchkote or other non absorbent material.
Whatever the surface, before starting work you must swab your work space with
disinfectant to reduce the risk of contamination of your cultures. You must keep the
working area tidy and uncluttered in order to minimise the risk of accident. Have
only the essentials on the bench.
While carrying out the experiments in this unit, you will use equipment with microorganisms which will have to be disposed of safely. You should always have a
disposal container of disinfectant within easy reach. If you have to stretch, there is
a risk of knocking over apparatus or dropping organisms from a pipette for example.
A Bunsen burner is essential to the technique of aseptic transfer. You should place it
on the bench so that you can reach it easily (the layout of equipment will be discussed
later). The Bunsen flame provides an updraught which carries air upwards away from
the operator reducing the risk of contamination.
Air movement should be kept to a minimum in microbiology laboratories to prevent
contamination. While practical work is being performed, doors and windows should
be kept shut.
In professional microbiology laboratories, specialised cabinets are used for the
transfer of micro-organisms for safety reasons. Transfer chambers are such
cabinets. The operator works with their hands and forearms in the chamber,
transferring microorganisms, preparing sterile media etc. A lit Bunsen within the
cabinet maintains an airflow into the cabinet so helps prevent contamination.
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Lamina flow chambers or cabinets are more sophisticated types of transfer
chamber. They incorporate fans which control airflow and sometimes filters which
prevent any micro-organisms leaving the chamber. In cases where scientists are
working with extremely dangerous organisms, they work in specialised laboratories
whose atmosphere is maintained at a pressure lower than that of its surroundings,
preventing escape of laboratory air to the outside.
Fig. 1: Example of a transfer chamber
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Questions: Preparation of workspace
1. State two things you must do before starting to prepare your work space.
2. Why must the surface of a work area be non-absorbent?
3. Give one reason for swabbing the work surface with disinfectant before and after
use.
4. When setting out your work space, what precaution should you take to avoid the
risk of knocking over equipment?
5. Explain why doors and windows in a microbiology lab should be kept shut.
6. Why should a microbiologist always carry out transfer work in the region of a
lighted Bunsen burner?
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Activity 1: Preparation of workspace
Materials required
Materials required by each student:
Benchkote and non-absorbent tape if required.
Disinfectant and paper towel
Discard jar with disinfectant
Bunsen burner
Wire loop
Cultures and media
Instructions
The following applies to right-handed operators. Left-handed people should reverse
the arrangement on the bench.
1. Tie back long hair.
2. Wash and dry hands thoroughly.
3. Cover any cuts/grazes with waterproof plaster.
4. Put on lab coat.
5. Collect the materials indicated above.
6. Attach benchkote to the bench if necessary using sticky tape.
7. Swab the surface of your work space with disinfectant using the paper towel.
8. Place your notes, pen etc. away from the immediate area in which you are
carrying out the work.
9. Place the wire loop to the right of the bench so that you can reach it with ease.
10. Place the Bunsen burner on a heat resistant mat centrally so that you can reach it
with ease but not so close that you are likely to burn yourself.
11. Place cultures and media to the left but still within easy reach. Place discard jar
with disinfectant to the right within easy reach.
12. In your laboratory diary/notebook, draw a labelled diagram of the bench set up.
13. Once you have completed an operation place the completed materials to your
right.
You must set up your bench in this way each time you carry out a microbiology
practical.
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2: POURING PLATES
Micro-organisms are grown in or on a culture medium containing all the required
nutrients for growth. Culture media can be liquid or solid. Liquid or broth media
although convenient have some disadvantages. Growths usually do not exhibit
characteristic appearances in them and, except when they are designed for a specific
biochemical test, they are of limited use in identifying species. Also, contaminating
organisms cannot be seen readily in liquid media and liquid media are more difficult
to handle in ways which avoid formation of aerosols.
On solid media, micro-organisms grow to form discrete colonies (See Fig.2). Each
colony is a clone of cells originating from a single organism and represents the growth
of a single species. Micro-organisms can be identified by the appearance exhibited
by the different colonies.
Fig. 2: Bacterial colonies on a nutrient agar plate
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Agar
Liquid media can be made solid by the addition of agar. Agar has no nutritional
value nor does it inhibit growth, it is simply a solidifying agent.
The melting and solidifying points of agar are not the same . At the concentrations
normally used, most agars melt at about 95°C when heated but solidify only when
cooled to about 42°C. This low solidifying point allows heat-sensitive nutrients to be
mixed with molten agar at temperatures as low as 45°C before pouring. The high
melting point ensures that the medium remains solid at all laboratory incubation
temperatures.
Pouring Agar Plates
Agar plates are prepared by pouring liquid agar at 55°C into sterile Petri dishes and
allowing it to solidify.
Before pouring, unopened plates are labelled on the underside using an indelible
pen or wax pencil with initials, date and type of agar. This prevents confusion should
lids inadvertently be swapped. The plates are then placed the right way up ready to
receive molten agar.
Sterile nutrient agar in universal bottles or other containers is first melted by heating
to 100°C then cooled in a water bath to 55°C. Using aseptic technique, a bottle neck
is flamed and the agar poured gently into a Petri dish on a flat surface, raising the lid
of the dish only far enough for the mouth of the bottle to enter. The lid is then
replaced and the plate left undisturbed until the agar has cooled and set.
It is essential that the surface of the medium should be dry in order to maintain
single colonies. Condensation produced from the cooling of the agar can make the
agar surface wet but is normally reduced by pouring the agar at 55°C and if necessary
the plates can be dried open and upside down (see Fig.3) in an undisturbed area.
Any plates showing lumps, bubbles or growth should be discarded. Acceptable
plates should be stored upside down until required (see Fig.4).
Plates are always incubated and stored in the inverted position to prevent
condensation dropping on to the agar surface.
Fig.3: Drying of plate
Fig.4: Storing of plates
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Questions: Pouring plates
1. On what part of the plate do you write the label?
2. Name the three details you should record in the label.
3. What precaution do you take before pouring the agar?
4. How do you avoid bubbles on the surface of the agar?
5. How do you prevent excess condensation gathering on the lid of the plate after the
agar is poured?
6. Where and how should the plates be stored?
7. Name the four characteristics well poured plates should possess.
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Activity 2: Pouring plates
Materials required
Materials required by each student:
Lab coat
Eye protection
Disinfectant and paper towels
Discard jar with disinfectant
3 bottles of sterile nutrient agar
3 sterile plastic Petri dishes
Bunsen burner and mat
Materials to be shared:
Benchkote if necessary
Water bath with water at 55°C
Fine tipped indelible marker pens
Autoclavable bags for disposal of contaminated plates
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat and use eye protection.
2. Collect the Petri dishes, Bunsen burner and mat.
3. Label the empty sterile Petri dishes on the base with name, date and type of agar.
(N.B. if the lid comes off, the plate is no longer sterile and you must discard it).
4. Light the Bunsen burner.
5. Collect one bottle of sterile molten agar from the water bath. Check it is not too
hot and that it has not started to solidify.
6. Place a Petri dish right way up on the bench.
7. Check that the top of the bottle of agar is loose.
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8. Hold the bottle of Agar in the left hand.
9. Unscrew and remove the cap of the bottle with the little finger of the right hand.
10. Pass the neck of the bottle backwards and forwards through a blue Bunsen flame.
11. With the right hand, lift the lid of the Petri dish a little and gently pour in the
molten agar.
For copyright reasons, the image that appeared on this page in print is
unavailable.
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12. Replace the lid of the Petri dish.
13. Replace the cap of the bottle and put it down.
14. Swirl the plate very gently to distribute the agar evenly. (N.B. The agar must not
touch the lid of the plate and must have a smooth surface with no bubbles).
15. Repeat for the other 2 bottles and plates.
16. Leave the agar to solidify.
17. Once cool, turn the plates upside down.
18. Record briefly the procedure you have carried out in your lab diary/notebook.
19. After a few days, examine your plates. Record your observations in a table. For
each plate, make a note of the following characteristics in your lab
diary/notebook:
•
smooth surface to agar?
•
even layer of agar?
•
base of Petri dish covered?
•
contamination?
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Unit 2: Working with Micro-organisms (Int 2)
Title: Pouring plates
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Plates labeled on underside
•
Agar cooled to pouring temperature
•
Aseptic technique satisfactory
•
Plates dried
•
Plates incubated.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on characteristics of plates:
•
Smooth surface to agar
•
Even layer of agar
•
Base of Petri dish covered
•
Contamination.
Teacher/lecturer’s signature:
Date:
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Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
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3: SUBCULTURING MICRO-ORGANISMS
In the laboratory, micro-organisms are usually grown or cultured in liquid medium
(broth) or on solid medium (agar plates or slopes). Growth of bacteria and yeasts
shows as cloudiness or turbidity in the broth although sometimes bacteria grow as a
layer on the surface of the broth or at the bottom of the culture tube.
The growth on plates depends upon how the plate has been inoculated. (Fig. 5).
simple streak plate
(smear plate)
streak plate
Fig. 5: Types of inoculations on agar plates
Subculturing is the aseptic transfer of micro-organisms from a culture to fresh
medium. The freshly inoculated medium is then incubated at the temperature
appropriate for growing the organism.
There are four subculturing procedures with which you should become familiar. They
are:
• solid to solid: the transfer of bacteria or fungi from an agar slope or plate culture
to an agar plate
• solid to liquid: the transfer of bacteria or fungi from an agar slope or plate culture
to a broth
• liquid to solid: the transfer of bacteria or fungi from a broth culture to an agar
slope or plate
• liquid to liquid: the transfer of bacteria or fungi from a broth culture to a broth.
Containers of culture media to be inoculated must be labelled with initials, date and
name of organism. To prevent possible confusion, plates are marked on the underside
while tubes and bottles must be labelled on the side. Lids are not labelled.
The use of aseptic technique minimises the risk of contamination of cultures and also
reduces the risk of micro-organisms from the laboratory cultures escaping to the
environment.
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You will use a wire loop, a straight wire, forceps or a scalpel to subculture bacteria or
fungi or to transfer specimens to a slide. The wire loop is the basic tool of the
microbiologist and you should learn to handle it correctly.
Flame sterilisation of instruments
Metal instruments used to transfer micro-organisms are sterilised using red heat in a
Bunsen burner before and after use. They must be heated till red hot to make sure
that any contaminating bacterial spores are destroyed.
Good flaming technique is very important to avoid contamination of the
surrounding air with aerosols.
A Bunsen burner is lit and the air hole opened fully to provide a blue flame. The
operator holds the loop between thumb and fingers as if holding a pencil very loosely,
at an angle that is almost vertical. (Fig. 6)
Fig. 6: Holding a wire loop
For copyright reasons, this image is unavailable
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The loop is placed in the light blue cone of the flame (Fig. 7). Positioning the loop in
this cool area of the flame allows any liquid to dry out and prevents formation of
aerosols. Aerosols are fine liquid or solid particles that are dispersed into the air and
might contain micro-organisms.
Fig. 7: Placing a loop in blue cone of flame
For copyright reasons, this image is unavailable
After any liquid material has evaporated, the loop is drawn slowly up into the hottest
region of the flame (immediately above the light blue cone) and held there until it is
red hot. (Fig. 8)
Fig. 8: Drawing loop into hottest region of flame
For copyright reasons, this image is unavailable
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The loop is then withdrawn from the flame and allowed to cool before touching
microorganisms. (Fig. 9)
Fig. 9: Cooling the loop
For copyright reasons, this image is unavailable
Straight wires may be sterilised in the same way.
Aseptic transfer operations
Once the loop has been sterilised and cooled, it is used to remove organisms (the
inoculum) from a culture and inoculate sterile growth medium. When working with
micro-organisms, aseptic techniques are used to avoid escape of organisms to the
surroundings and contamination of the culture by micro-organisms from the
environment.
To minimise the chances of contamination, cultures and media are exposed to the air
for the minimum time it takes to perform a manipulation or to make observations. All
subculturing procedures are carried out close to a Bunsen flame.
After flaming, the loop is not put down until the procedure has been completed.
Lids of Petri dishes are never completely removed. They are opened just enough to
allow entry of the loop to perform the manipulation and minimise exposure to the air.
Liquid cultures are disturbed as little as possible to reduce the risk of aerosol
formation. Lids from cultures are never placed on the bench surface where
contamination might occur. They are removed from the bottle or tube using the little
finger, held there while manipulation of the culture takes place and then replaced.
The rest of the hand is free to carry out the manipulation. Lids are tightened to prevent
spillage before incubation.
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To remove any contaminating organisms on the neck of a bottle, it is passed through
a hot Bunsen flame on removal and before replacement of the lid.
The loop must be flamed to red heat when the subculturing procedure is
finished.
The table below summarises potential points of contamination and the techniques
employed to minimise the risk.
Contamination risk
Precaution
Inoculating loop
Flame and cool.
Do not lay down loop until procedure is
complete.
Work close to Bunsen flame.
Opening of Petri dish (solid medium)
Open lid for as short a time as possible.
Open lid just enough to insert wire loop.
Opening of liquid cultures (bottles, tubes) Hold the lid in crook of little finger –
never place on work bench.
Pass neck through a hot Bunsen flame
before insertion and after withdrawal of
loop to kill any contaminating organisms.
Incubation of cultures
After inoculation, cultures are incubated at a given temperature. Petri dishes should
be placed upside down to prevent condensation dropping on to cultures. The lids of
Petri dishes should be secured to the base with diagonal strips of sellotape. Bottles
should be stored upright in a container which will prevent them being knocked over.
Subculture results
Subculturing has been successful when the transferred organisms have grown in the
new medium without contamination.
Contaminating organisms can usually be observed on plate cultures as single colonies
with a different appearance. In a broth culture, it is impossible to tell by simply
observing the broth whether the culture is pure. The liquid culture must be streaked
out on an agar plate to determine if there are any contaminants.
In the practical work associated with this course, you will generally be working with
known organisms and with pure cultures. A pure culture is one in which there is
only one type of organism.
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3.1: Loop transfer of micro-organisms
When working with micro-organisms, you must ‘think safety’ all the time and
constantly be aware of points at which contamination might occur and take
measures to minimise the risks.
Subculturing procedures
In order to achieve aseptic transfer of micro-organisms, there are a number of
procedures that must be followed. These techniques will be demonstrated.
•
Inoculating loops must be sterilised by flaming before and after use see ‘Flame
sterilisation of instruments).
•
Once flamed, the loop must never be put down but held in the hand and allowed to
cool. Inoculating instruments should not be waved around.
•
The necks of tubes and bottles must be flamed after removing and before
replacing a lid.
•
Caps of bottles must never be put down.
•
Cultures should be exposed to the air for as short a time as possible.
•
Lids of Petri dishes should be raised only enough to admit the wire loop and
permit the manipulation.
Disposal of Contaminated Materials
It is essential that contaminated materials be disposed of safely and without risk to
anyone. Cultures and contaminated equipment such must be sterilised before
disposal. Technical staff will carry out these procedures.
You will be directed as to where to place materials for disposal. Make sure that you
follow instructions for disposal of contaminated equipment and materials.
In the event of a spill, you must always inform the teacher/lecturer or
technician.
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Questions: Subculturing micro-organisms
1. Describe the correct way to hold a wire loop.
2. When flaming a wire loop, the loop should first be placed in the pale blue cone of
the Bunsen flame. Why?
3. How do you judge when the loop is sterilised?
4. Why must the loop be cooled before subculturing?
5. State two precautions that should be taken to prevent contamination of liquid
media and cultures.
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6. What process must be carried out at the end of every subculturing procedure using
a wire loop?
7. Explain why the lids of Petri dishes and bottles should not be used to label
cultures.
8. What three pieces of information should always be used on culture labels?
9. Why is it important to incubate or refrigerate plate cultures upside down?
10. Why should cultures be sterilised before final disposal?
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Activity 3.1.1: Loop transfer, solid to solid
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 plate culture Micrococcus luteus
1 plate culture Saccharomyces cerevisiae (yeast)
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
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Activity 3.1.1: Loop transfer, solid to solid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the bases of the Petri dishes containing sterile nutrient agar with initials,
date, name of microorganism and s/s (solid to solid).
4. Turn plate upright.
5. Hold the loop in the right hand.
6. Flame the loop and allow to cool. Do not put down loop or wave it around.
7. Lift the lid a little of the Petri dish containing the inoculum with the left hand.
8. Touch a single colony with the wire loop.
9. Withdraw loop. Do not put down loop or wave it around.
10. Replace lid of Petri dish.
11. Partially lift the lid of the Petri dish containing the solid medium.
For copyright reasons, the image that appeared on this page in print is
unavailable
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12. Holding the loop parallel with the surface
of the agar, smear the inoculum across the
medium as shown.
13. Replace the lid of the Petri dish.
14. Flame loop, and place on heat resistant mat.
15. Secure the lid of the Petri dish to the base with sellotape.
16. Incubate plate upside down at 30°C for 72 hours.
17. Repeat for the other inocula.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
•
•
•
whether growth is present
a description of the growth
whether the plate is contaminated.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
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Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
30
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
31
Activity 3.1.2: Loop transfer, liquid to solid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 broth culture Micrococcus luteus
1 broth culture Saccharomyces cerevisiae (yeast)
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
32
Activity 3.1.2: Loop transfer, liquid to solid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat and use eye protection.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the underside of plates with initials, date, name of microorganism and l/s
(liquid to solid).
4. Turn the plate upright.
5. Loosen the tops of the universals containing the broth cultures so that they can be
removed easily.
6. Hold the loop in the right hand.
7. Flame the loop and allow to cool. Do not put down loop or wave it around.
8. Lift the universal containing the inoculum with the left hand.
9. Remove the lid of the universal with the little finger of the right hand. Do not put
down the lid.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
33
10. Flame neck of universal.
11. Insert the loop into the culture broth (bring bottle to loop, not loop to the bottle)
and withdraw. Take care not to touch the sides of the bottle or its mouth.
12. Flame the neck of the universal.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
34
13. Replace the lid on the universal using the little finger. Turn the bottle, not the lid.
14. Place the universal on the bench.
15. With the left hand, partially lift the lid of the Petri dish containing the solid
medium.
16. Holding the loop parallel with the surface
of the agar, smear the inoculum across the
medium as shown.
17. Replace lid of Petri dish.
18. Flame loop and place on heat resistant mat.
19. Secure the lid to the base with diagonal strips of sellotape.
20. Incubate plates upside down at 30°C for 72 hours.
21. Repeat for the other inocula.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
•
•
•
•
whether growth is present
a description of the growth
whether there are isolated single colonies
whether the plate is contaminated.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
35
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
36
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
e.
f.
g.
h.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
37
Activity 3.1.3: Loop transfer, solid to liquid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 plate culture Micrococcus luteus
1 plate culture Saccharomyces cerevisiae (yeast)
Wire loop
4 universals of sterile nutrient broth
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
38
Activity 3.1.3: Loop transfer, solid to liquid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat and use eye protection.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the universals containing sterile nutrient broth with initials, date, name of
microorganism and s/l (solid to liquid).
4. Loosen the tops of all the universals so that they can be removed easily.
5. Hold the loop in the right hand.
6. Flame the loop and allow to cool. Do not put down loop or wave it around.
7. Lift the lid slightly of the Petri dish containing the inoculum with the left hand.
8. Touch a single colony with the wire loop.
9. Withdraw loop. Do not put down loop or wave it around!
10. Replace lid of Petri dish.
11. Lift a universal of sterile nutrient broth in the left hand.
12. Remove the lid of the universal with the little finger of the right hand which still
holds the charged wire loop. Do not put down the lid.
For copyright reasons, the imagse that appeared on this page in print are
unavailable
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
39
13. Flame the neck of the uinversal.
14. Insert the loop charged with inoculuminto the sterile broth (bring the bottle to the
loop), agitate gently, touch on the inside of the universal and withdraw.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
40
15. Flame the neck of the universal.
16. Replace the lid on the universal using the little finger of the right hand. (Turn the
bottle, not the lid.)
17. Place universal on bench.
18. Flame the loop and place on a heat resistant mat.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
41
19. Tighten lid of universal to make secure but do not overtighten.
20. Incubate inoculated universal at 30°C for 72 hours.
21. Repeat once with the same inoculum and twice with a second culture as the
inoculum.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
•
•
whether growth is present
a description of the growth (heavy or light, spread evenly throughout broth or
concentrated at top or foot etc.).
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
42
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
i.
j.
k.
l.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
43
Activity 3.1.4: Loop transfer, liquid to liquid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 broth culture Micrococcus luteus
1 broth culture Saccharomyces cerevisiae (yeast)
Wire loop
4 universals of sterile nutrient broth
2 bottles sterile water
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
44
Activity 3.1.4: Loop transfer, liquid to liquid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat and use eye protection
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the universals containing sterile nutrient broth with initials, date, name of
microorganism and l/l (liquid to liquid).
4. Loosen the tops of all the universals so that they can be removed easily.
5. Hold the loop in the right hand.
6. Flame the loop and allow to cool. Do not put down loop or wave it aroound.
For copyright reasons, the image that appeared on this page in print is
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
45
7. Lift the universal containing the inoculum with one hand.
8. Remove the lid of the universal with the right hand. Do not put down the lid.
9. Flame neck of universal.
10. Insert the loop into the culture broth and withdraw (bring bottle to loop; not loop
to bottle). Take care not to touch the sides of the bottle or its mouth.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
46
11. Flame the neck of the universal.
12. Replace the lid on the universal using the little finger. Turn the bottle, not the lid.
13. Place the universal on the bench.
14. Lift a universal of sterile nutrient broth in the left hand.
15. Remove the lid of the universal with the little finger of the right hand which still
holds the charged wire loop. Do not put down the lid. Turn the bottle, not the lid.
16. Flame the neck of the universal.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
47
17. Insert the loop charged with inoculum into the sterile broth (bring bottle to loop.)
Agitate gently, touch on the inside of the universal and withdraw.
18. Flame the neck of the universal.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
48
19. Replace the lid on the universal using the little finger of the right hand. Turn the
bottle, not the lid.
20. Place universal on bench.
21. Flame the loop and place on a heat resistant mat.
22. Tighten lid of universal to make secure but do not overtighten.
23. Repeat once with the other inoculum and twice more using sterile water as the
inoculum to check aseptic technique.
24. Incubate the inoculated universals at 30 °C for 72 hours.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Record briefly the procedure you have carried out in your lab diary/notebook. . After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
•
•
whether growth is present
a description of the growth (heavy or light, spread evenly throughout broth or
concentrated at top or foot etc.).
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
49
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
50
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
m. Preparation for work is in
accordance with given
specifications.
n.
o.
p.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
51
3.2: Streak plate inoculations
The term ‘streaking out’ or ‘plating out’ is applied to the inoculation of solid media
in Petri dishes using a technique involving successive strokes with a wire loop. The
aim of the procedure is to obtain isolated single colonies. The loop is charged with a
small amount of culture and several strokes in series are then made on the surface of
the medium with the aim of diluting the bacteria so that single cells are distributed on
the surface. The loop is flamed between strokes to reduce the number of organisms
and increase the chance of isolating single cells. On incubation, each single cell
divides successively to produce a single colony. In this way, pure cultures can be
obtained. (Fig.10)
Fig.10: Streak plate showing single colonies
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
52
Questions: Streak plate inoculations
1. Why do microbiologists use the technique of ‘streaking out’?
2. When performing ‘streaking out’, the loop is flamed between strokes. Why?
3. What is meant by the term ‘pure culture’?
4. How is a bacterial colony formed?
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
53
Activity 3.2.1: Streak plate inoculation, solid to solid
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 plate culture Micrococcus luteus
1 plate culture Saccharomyces cerevisiae (yeast)
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
54
Activity 3.2.1: Streak plate inoculation, solid to solid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the bases of the Petri dishes containing sterile nutrient agar with initials,
date, name of microorganism and s/s.
4. Hold the loop in the right hand.
5. Flame the loop and allow to cool. Do not put down loop or wave it around.
6. Lift the lid a little of the Petri dish containing the inoculum with the left hand.
7. Touch a single colony with the wire loop.
8. Withdraw loop. Do not put down loop or wave it around!
9. Replace lid of Petri dish.
10. Partially lift the lid of the Petri dish containing the solid medium.
For copyright reasons, the imagethat appeared on this page in print is
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
55
11. Holding the loop parallel with the surface
of the agar, smear the inoculum backwards
and forwards across a small area of the
medium
12. Flame loop and allow to cool.
13. Turn plate. Streak loop from A across the
surface of the agar in three parallel lines.
A
14. Flame the loop and allow to cool.
A
15. Turn plate. Streak loop from B across the
surface of the agar in three parallel lines.
B
16. Flame loop and allow to cool.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
56
A
17. Turn plate. Streak loop from C to D across
the surface of the agar as shown.
D
B
C
18. Replace lid of Petri dish.
19. Flame loop and place on heat resistant mat.
20. Repeat for second inoculum.
21. Incubate plate upside down at 30°C for 72 hours.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
•
•
•
•
whether growth is present
a description of the growth
whether there are isolated single colonies
whether the plate is contaminated.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
57
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
58
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
q.
r.
s.
t.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
59
Activity 3.2.2: Streak plate inoculation, liquid to solid
Materials required
Materials required by each student:
Lab coat
Eye protection
Disinfectant and paper towels
Discard jar with disinfectant
Bunsen burner and mat
1 broth culture Micrococcus luteus
1 broth culture Saccharomyces cerevisiae (yeast)
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
60
Activity 3.2.2: Streak plate inoculation, liquid to solid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear lab coat and eye protection.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the underside of plates with initials, date name of microorganism and l/s.
4. Turn the plate upright.
5. Loosen the tops of the universals containing the broth cultures so that they can be
removed easily.
6. Hold the loop in the right hand.
7. Flame the loop and allow to cool. Do not put down loop or wave it around.
8. Lift the universal containing the inoculum with the left hand.
9. Remove the lid of the universal with the right hand. Do not put down the lid.
10. Flame neck of universal.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
61
11. Insert the loop into the culture (bring bottle to\loop, not loop to bottle) and
withdraw. Take care not to touch the side of the bottle or its mouth.
12. Flame the neck of the universal.
13. Replace the lid on the universal using the little finger. Turn bottle, not lid.
14. Place the universal on the bench.
15. With the left hand, partially lift the lid of the Petri dish containing the solid
medium.
For copyright reasons, the images that appeared on this page in print are
unavailable.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
62
16. Holding the loop parallel with the surface
of the agar, smear the inoculum backwards
and forwards across a small area of the
medium
17. Flame loop and allow to cool.
18. Turn plate. Streak loop from A across the
surface of the agar in three parallel lines.
A
19. Flame the loop and allow to cool.
A
20. Turn plate. Streak loop from B across the
surface of the agar in three parallel lines.
B
21. Flame loop and allow to cool.
A
22. Turn plate. Streak loop from C to D across
the surface of the agar as shown.
D
B
C
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
63
23. Replace lid of Petri dish.
24. Flame loop and place on heat resistant mat.
25. Repeat for second inoculum.
26. Incubate plate upside down at 30°C for 72 hours.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
•
•
•
•
whether growth is present
a description of the growth
whether there are isolated single colonies
whether the plate is contaminated.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
64
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
u.
v.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
w. Record of work is clear and
accurate.
x.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
65
3.3: Plate to plate subculture of fungal mycelium
It is sometimes difficult to pick up fungal mycelium with a wire loop or straight wire.
Plate to plate inoculation is therefore often carried out using a sterile scalpel.
The scalpel is first sterilised by covering the blade in ethanol, lighting it in a Bunsen
flame and allowing it to burn off. It is then used to cut through the fungal mycelium
and agar below to extract a small block. This is then transferred aseptically to sterile
agar and incubated. The wide end of a sterile Pasteur pipette or a cork borer flamed in
ethanol may also be used for the transfer operation.
Questions: Plate to plate subculture of fungal mycelium
1. What is meant by ‘fungal mycelium’?
2. Why is a scalpel used to subculture fungal mycelium?
3. Describe one difference apart from the use of a scalpel between subculturing
fungal mycelium and subculturing bacteria from solid to solid?
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
66
Activity 3.3: Plate to plate subculture of fungal mycelium
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 plate culture Penicillium roquefortii or Mucor heimalis
Small volume of ethanol in a covered beaker
Scalpel
2 plates of sterile malt agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
67
Activity 3.3: Plate to plate subculture of fungal mycelium
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear lab coat and use eye protection.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the bases of the Petri dishes containing sterile malt agar with initials, date,
name of micro-organism.
4. Hold the scalpel in the right hand.
5. Keep the ethanol at a safe distance from the Bunsen flame.
6. Dip the scalpel blade in ethanol, place in Bunsen flame briefly and allow ethanol
to burn off. Do not put down scalpel.
7. Lift the lid a little of the Petri dish containing the inoculum with the left hand.
8. Using the tip of the scalpel blade cut a square shape in the fungal mycelium (Fig.
11). This may be at the edge of the colony if there is heavy spore production.
9. Insert the blade under the square you have cut and lift a cube of agar with fungal
mycelium on top on to the blade of the scalpel.
10. Withdraw scalpel from Petri dish. Do not put down scalpel or wave it around!
11. Replace lid of Petri dish.
12. Partially lift the lid of the Petri dish containing the sterile malt agar medium.
13. Place the cube of agar on your scalpel blade on to the centre of the sterile agar
(Fig.11). It does not matter whether it is upright or upside down.
14. Withdraw scalpel.
15. Dip the scalpel blade in ethanol, flame as above and place on heat resistant mat.
16. Incubate plate upside down at 30°C for 72 hours or at room temperature for 5
days..
17. Repeat for second inoculum.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
• whether growth is present
• a description of the growth
• whether the plate is contaminated.
Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual
68
Source of inoculum
Inoculum placed on sterile agar
Fig.11: Transferring a block of agar with mycelium.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
y.
z.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
aa. Record of work is clear and
accurate.
bb. Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
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3.4: Use of sterile swabs to sample the environment
The numbers and types of bacteria and fungi present on a surface such as the
laboratory bench can be estimated by swabbing the area with a sterile swab or cotton
wool bud which has been dampened with sterile water. The swab is then rubbed
across a sterile agar plate which is then incubated.
Plates must be sealed with tape and not opened after incubation since the organisms
that grow are unknown.
Swabs are not sterilised in the Bunsen flame. You may receive them pre-sterilised,
wrapped in aluminium foil or you may use commercial ones directly.
Questions: Use of sterile swabs to sample the environment
1. Why are swabs not sterilised in a Bunsen flame?
2. What precaution must you take when removing a swab from its sterile pack?
3. Why must you seal plates inoculated with swabs of the environment before
incubation and dispose of them unopened?
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Activity 3.4: Use of sterile swabs to sample the environment
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Sterile swabs
Disinfectant and paper towels
Discard jar with disinfectant
1 plates of sterile malt agar
I plate sterile nutrient agar
Bijoux bottle of sterile water
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Sellotape
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Activity 3.4: Use of sterile swabs to sample the environment
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the bases of the Petri dishes containing sterile agar with initials, date and
area to be sampled.
4. Taking care not to touch the cotton wool end, remove a sterile swab from the pack
and hold it in your right hand.
5. Lift a bottle of sterile water in the left hand. Do not put down swab.
6. Remove the lid of the bottle with the little finger of the right hand which is still
holding the swab.
7. Flame the neck of the bottle.
8. Insert and withdraw the cotton wool bud.
9. Flame the neck of the bottle and replace the lid. Do not put down swab or wave it
around!
10. Rub the swab on the area to be tested as directed by your teacher/lecturer for 10 –
15 seconds.
11. Partially lift the lid of the agar plate and gently rub the swab across the surface of
the agar as shown. Take care not to break the agar
surface.
12. Replace lid of Petri dish.
13. Sellotape lid to base of plate with diagonal strips.
14. Incubate plate upside down at room temperature for 72
hours.
15. Repeat for second inoculum.
Record briefly the procedure you have carried out in your
lab diary/notebook. After incubation, take a note of your
observations. These could include a diagram and/or table
and should include the following:
•
•
whether growth is present
a description of the growth.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
cc. Preparation for work is in
accordance with given
specifications.
dd. Techniques are carried out in
accordance with safe practice
and given specifications.
ee. Record of work is clear and
accurate.
ff. Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Organism:
Type of inoculation:
Teacher/lecturer’s signature:
Date:
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4: SEPARATING MICRO-ORGANISMS
When professional microbiologists isolate micro-organisms from the environment or
an infected person, it is extremely rare to obtain a pure culture. It is therefore
necessary to separate micro-organisms. Plating or streaking (see Fig. 10) can be used
to achieve this. Using isolated single colonies as inocula for further streak plates,
pure cultures can be obtained.
Streaking out a mixed broth culture on an agar plate and incubating it to obtain single
colonies of different types of bacteria or yeasts can simulate this.
Fig. 10: Streak plate showing individual colonies
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Questions: Separating micro-organisms
1. What is meant by a ‘mixed culture’?
2. Explain why the procedure of streaking out can be used to separate microorganisms in a mixed culture.
3. Describe how you could obtain pure cultures on agar plates from a mixed broth
culture.
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Activity 4: Separating micro-organisms
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with disinfectant
1 mixed broth culture of Phaffia (a red yeast) and Saccharomyces cerevisiae (a
creamy white yeast)
Wire loop
2 plates of sterile malt agar
Materials to be shared:
Benchkote if necessary
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Instructions
These instructions are for right-handed people. If you are left handed, please reverse
the instructions accordingly.
1. Wear a lab coat.
2. Prepare your work space on the bench, collect the materials and set them out
correctly on the bench.
3. Label the bases of the Petri dishes containing sterile malt agar with initials, date,
name of culture.
4. Using the mixed broth culture as the inoculum, streak out applying the method
you have used previously.
5. Incubate the plates at room temperature for 48 hours.
6. Repeat for second plate.
Record briefly the procedure you have carried out in your lab diary/notebook. After
incubation, take a note of your observations. These could include a diagram and/or
table and should include the following:
• whether growth is present
• a description of the growths
• whether there are isolated single colonies of each type of organism.
If you have obtained well isolated single colonies, use these as inocula for further
streak plates using the method you have used previously to obtain pure cultures.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Separating a mixed culture
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Loop/scalpel flamed correctly
•
Plates labeled on underside
•
Aseptic technique satisfactory
•
Plates incubated appropriately
•
Single colonies of both types present
•
Contaminated materials disposed of
appropriately
•
Surface swabbed with disinfectant.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growths
•
Whether there are isolated single colonies of
each type of organism.
Organisms:
Teacher/lecturer’s signature:
Date:
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5: Microscopic examination of micro-organisms
Micro-organisms are so small that they you cannot see them without the aid of a
microscope. Microscopes are used to produce an enlarged image of objects too small
to be seen with the naked eye.
Although microscopes produced by different manufacturers may look quite unlike
each other, they all work on the same principle and consist essentially of similar
working parts.
To obtain the clearest image of micro-organisms using a microscope, you must learn
how to set it up properly. You must first know the principles on which the
microscope works.
Fig. 12: Structure of the microscope
For copyright reasons, this image on is unavailable.
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Part
Eyepiece lens
Description
It fits into the body tube. It contains lenses which magnify the
image produced by the objective lens and produces the image
the operator sees. Eyepiece lenses usually have a magnification
of x10. You may use x15 to observe bacteria.
Objectives
Contain lenses which magnify the specimen. Objective lenses
bear a coloured ring which indicates their order of
magnification. Microscopes usually have objective lenses of
power x10 and x40. They may also have an oil immersion
objective (x90 or x100).
Stage
A flat platform on which the specimen is placed. It has a hole
in the centre which allows light to pass up through the
specimen. There are usually two side clips which hold the slide
in position.
Condenser
Contains lenses which focus the light into a cone. A condenser
focus control allows the condenser to be moved up and down so
that light is focused at the specimen. The condenser may not be
present in some microscopes.
Iris diaphragm
The iris diaphragm can be opened or closed to control the angle
of the cone of light passing through the condenser. It makes the
specimen brighter and the image clearer.
Mirror
Can be adjusted to reflect light up on to the specimen. One side
of the mirror is flat, the other is concave. The flat side is used
if a condenser is present. The mirror may be replaced with an
electrical light source.
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Principles of the light microscope
• Visible light passes through a substage condenser which focuses the light into a
sharp cone.
• The light passes through the opening in the stage into the slide illuminating the
specimen.
• The light passes through the objective lens and forms a magnified image of the
specimen which is usually darker than the background.
• The eyepiece lens magnifies this image further and creates the image that the user
sees.
Oil immersion
To observe bacteria clearly with a microscope, it is usually necessary to use an
objective lens of x100. The lens must be immersed in a drop of oil which is placed on
the slide. This helps direct more light into the objective lens.
Questions: Structure and function of the microscope
1. What is the purpose of the microscope?
2. What part of the microscope controls the angle of light that passes up through the
condenser?
3. State the function of the stage on a microscope.
4. Name three parts of the microscope that contain lenses.
5. Why would you adjust the focus controls?
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5.1: Magnification
The magnifying power is the degree of enlargement; that is, the number of times the
image appears greater than the original specimen.
To calculate this, multiply together the separate magnifying powers of the objective
and eyepiece lenses. For example, the total magnification of a microscope fitted with
a x10 eyepiece lens and a x10 objective lens is 10 x 10 = x100. Similarly, if a x40
objective lens was being used on the same microscope, the total magnification would
be 10 x 40 = x400.
Questions: Magnification
Complete the following table.
Eyepiece lens
magnification
Objective lens
magnification
Total
magnification
x15
x10
x150
x10
x10
x40
x10
x15
x400
x1000
x40
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Activity 5.2: Setting up a microscope
Microscopes are expensive. Please treat them with care and make sure you
clean them before putting them away.
Materials
Materials required by each student
Microscope
Bench lamp
Prepared slides of micro-organisms
Lens tissue
Instructions
1. Clean the lenses and all other glass surfaces on the microscope with lens tissue to
remove dirt and greasy marks as you have been shown.
2. Feel beneath the stage for the iris diaphragm control and set it to about half open.
3. Turn the condenser focus control and note the movement of the condenser. Raise
the condenser fully, then lower 1 – 2mm. Focus the condenser as shown by your
teacher/lecturer.
4. Place the slide on the stage, specimen uppermost and manoeuvre the slide until the
specimen is above the light source.
5. Turn the x 10 objective lens till it clicks into place above the slide.
6. Switch on the bench lamp and place it about 20cm from the microscope. Shine
the light directly on to the plane surface of the mirror, not the concave.
7. Turn the rough focus knob till the objective lens is as close as possible to the
stage.
8. Look down the eyepiece lens and slowly turn the rough focus knob till the
material on the slide comes in to focus.
9. Turn the fine focus knob till the image becomes sharp. The specimen should now
be sharply focused but may be unevenly illuminated.
10. Look down the eyepiece and move the mirror till the field of view becomes
brightly and evenly filled with light. The specimen should now be in sharp focus
and evenly illuminated but may be too bright.
11. Remove the eyepiece, look down the body tube and adjust the iris till the disc of
light takes up ¾ of the circle.
12. Replace the eyepiece. The microscope should now be set correctly under x10
magnification.
13. Manoeuvre the slide until the desired part of the specimen is in view.
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14. Adjust the fine focus if necessary.
15. If appropriate, make a labelled diagram in your notebook/diary. Include a heading
and magnification.
16. Rotate the turret to bring the x40 objective lens above the slide.
DO NOT ADJUST THE ROUGH FOCUS KNOB
17. Remove one eyepiece, look down the body tube and adjust the iris till the disc of
light takes up ¾ of the circle. Replace the eyepiece.
18. Using only the fine focus knob make the image sharp.
19. Record briefly in your lab diary/notebook the procedure you have carried out and
make drawings of the specimen. Make sure your diagrams have headings and that
the magnification is included.
The following applies only if you are using x100 objective.
20. Turn the turret till the x 40 and x 100 lenses are at equal distances from the slide.
21. Carefully place a single drop of immersion oil on the slide. If immersion oil
drips on to any other part of the microscope, wipe it off immediately and
inform your teacher/lecturer or the technician.
22. Rotate the turret to place the x100 objective lens in the oil on the slide.
23. DO NOT ADJUST THE ROUGH FOCUS KNOB
24. Remove one eyepiece, look down the eyepiece tube and adjust the iris till the disc
of light takes up ¾ of the circle. Replace the eyepiece.
25. Using the fine focus control make the image sharp. Make drawings of the
specimen.
26. When you have finished working with the microscope, clean the lenses thoroughly
as you have been shown before putting it away.
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5.3: Microscopic examination of yeast, pond water and hay infusion
Water in a pond or stream contains many different types of micro-organisms. In this
practical, you will use a cavity slide which has a little pit in its centre to examine a
suspension of yeast or pond water. The drop is suspended from a coverslip into the
cavity to make a ‘hanging drop preparation’. This prevents the micro-organisms from
being flattened and allows you to observe them moving around.
You may also examine a ‘hay infusion’. This is prepared by placing three or four
pieces of clean dry hay or lawn cuttings in a Petri dish and covering them with pond
or stream water. The lid is then replaced and the dish is kept at room temperature for
one to two weeks away from direct sunlight.
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Activity 5.3.1: Cavity slide preparation
Materials
Materials required by each student
Lab coat
Disinfectant and paper towels
Discard jar with disinfectant
Lens tissue
Cavity slide and coverslip
Vaseline
Cocktail sticks
Pasteur pipette (plugged) and bulb
Yeast suspension, pond water or hay infusion
Materials to be shared
Benchkote if necessary
Instructions
Instructions are for right handed people. If you are left handed, please reverse
instructions accordingly.
1. Wear a lab coat.
2. Clean slides and coverslips using lens tissue.
3. Using a cocktail stick place a rim of vaseline round the edge of a coverslip.
4. Place a bulb on a Pasteur pipette.
5. Holding the pipette in the right hand, remove the lid from the stock culture with
the left hand and draw up some liquid into the pipette.
6. Place one drop on to the centre of the coverslip (Fig 13) and discard the pipette
into a jar of disinfectant.
7. Lower the cavity slide on to the coverslip and quickly invert the slide and
coverslip (Fig 13).
vaseline
pond
water
coverslip
slide
invert
slide
coverslip
Fig 13: To make a cavity slide preparation
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5.3.2: Bright field microscopy
In bright field microscopy, most of the contrast (differences between the brightness of
the field and its background) is produced by the specimen absorbing light. Much of
the direct light is either not absorbed or misses the specimen altogether. The resulting
field of view therefore appears fairly brightly lit.
Activity 5.3.2: Examination of a cavity slide preparation using bright field
microscopy
Materials (5.3 .2, 5.3.3, 5.3.4)
Materials required by each student
Lab coat
Disinfectant and paper towels
Discard jar with disinfectant
Microscope
Cavity slide (hanging drop) preparation
Materials to be shared
Benchkote if necessary
Instructions
1. Using the instructions for setting up the microscope given earlier, examine the
prepared hanging drop under x10 objective (it helps to focus on the edge of the
drop first) and then under x40 objective.
2. In your lab diary/notebook record briefly the procedure and note whether the
sample contains one or many types of micro-organisms. If there is a variety,
record in a table the numbers of algae, protozoa and bacteria that you observe and
which type of micro-organism is most common in your sample.
3. Keep your slide preparation to view under dark field and phase contrast
illumination.
4. When finished with slide and coverslip, dispose into discard jar.
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5.3.3: Dark field microscopy
In the previous practical, you will have noticed that many micro-organisms are almost
colourless and it is very difficult to observe them using bright field microscopy. Dark
field illumination is a technique by which the organisms appear brightly illuminated
on a dark background. A special condenser is used which prevents light rays from
entering the objective lens unless they have bounced off organisms on the slide.
You can get some idea of what dark field microscopy is like by closing the iris
diaphragm and illuminating the specimen from above.
Activity 5.3.3: Examination of a cavity slide preparation using dark field
microscopy
Instructions
1. View the slides you have prepared for the previous practical under dark ground
illumination.
2. In your lab diary/notebook, record the differences you observe in the field of view
(the circular area you observe when looking down the eyepiece) and which type of
organisms you can see more clearly.
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5.3.4: Phase Contrast Microscopy
It is difficult for a human eye to distinguish details inside cells because light intensity
changes only slightly when light passes through living cells. The technique of phase
contrast microscopy makes a living cell appear to have a different intensity to its
background. The observer can then distinguish cell contents more clearly.
Activity 5.3.4: Examination of a cavity slide preparation using phase contrast
microscopy
Instructions (if a phase contrast microscope is available).
1. View the slides you have prepared for the previous practical under phase contrast
microscope.
2. In your lab diary/notebook, record the differences you observe in the field of view
and the organisms.
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5.4: Calculation of specimen size using a microscope
Specimens examined under the microscope are generally too small to be seen with the
naked eye. They are measured in micrometres. Viruses may be measured in
nanometres (nm). In general, viruses are too small to be seen with the light
microscope.
1µm = 10-3 millimetres (one thousandth of a millimetre)
= 10-6metres (one millionth of a metre)
1nm = 10-6 millimetres (one millionth of a millimeter)
= 10-9 metres (one thousand millionth of a metre).
The following table indicates the sizes of different types of cells.
Table: Sizes of micro-organisms
Size
Units
1mm
10–3 m
100µm
10–4 m
10µm
10–5 m
1µm
10–6 m
Organism
Algae
Protozoa
Fungi
Bacteria
100nm
10-7m
Viruses
10nm
10-8m
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Measuring objects using a microscope
Before you can measure the dimensions of a micro-organism, you must determine the
size of the microscope field of view (the circular area observed when you look down
the eyepiece). To accomplish this, you must use a special slide on which is printed a
millimetre scale graduated in hundredths of a millimetre. This is called a stage
micrometer. A stage micrometer (Fig.14) is a very small ruler with each division
equal to 0.01mm (10µm).
Fig. 14: Stage micrometer (micrometer slide)
It is not possible, however, to line up specimens on a slide next to this.
To measure the sizes of specimens accurately, you must use an eyepiece micrometer
(a small disc of glass marked with a scale which is placed in an eyepiece). When this
is in place, you can see its graduations and the slide on the stage in the same field of
view (Fig.15). You can line up the object being observed alongside the graduations
on the eyepiece micrometer and measure the length. (Fig.16).
Fig. 15: Eyepiece micrometer
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Fig. 16: Measuring cells against eyepiece micrometer
Graduations on the eyepiece are arbitrary (i.e. they have no units) and the actual
length represented by each one will vary with the objective being used. For each
objective, the eyepiece micrometer must be calibrated against the distance measured
on a stage micrometer (Fig.17).
The eyepiece micrometer is calibrated for each objective by superimposing its scale
on that of the stage micrometer. The number of stage micrometer divisions that are
equivalent to a number of eyepiece divisions is noted and the following calculation
carried out.
One eyepiece unit
=
number of stage micrometer divisions
Number of eyepiece divisions
x
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95
Fig. 17: Eyepiece micrometer over stage micrometer under different objectives
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Questions: Calculation of specimen size using a microscope
1. Give the symbols for the following units: metre, millimetre, micrometre.
2. How many millimetres are in 1 metre?
3. How many micrometres are in 1 metre?
4. How many micrometres are in 1 millimetre?
5. Copy and complete the following table.
Metres
millimetres
micrometres
3
3000 (3x103 )
3000000 (3x106 )
0.5
70
425
0.8
39
0.59
0.02
6
26
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Activity 5.4.1: Calculation of specimen size using a microscope
Materials required
Materials required by each student
Microscope set up with eyepiece micrometer
Stage micrometer slide
Prepared slides of micro-organisms
Lens tissue
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Activity 5.4.1: Calculation of specimen size using a microscope
Instructions
Calibration of eyepiece micrometer
1. Set up microscope on low power with the stage micrometer in focus.
2. Looking down the eyepiece, superimpose the eyepiece micrometer graduations on
the stage micrometer scale (Fig. 17).
3. Adjust until the ‘zero’ marks on each scale are superimposed.
4. Count the number of eyepiece divisions (x) which are equal to a number of
graduations (y) on the stage micrometer. If you have any doubt which scale is
which, rotate the eyepiece and you will see that scale turning. Each stage
micrometer graduation equals 10µm. In your lab notebook/diary, briefly record
what you have done. Carry out the following calculation using your own values
for x and y.
Calculation
x eyepiece units or divisions (epu)
5
6
1 epu
=
=
=
y micrometer units
y
x
10µm
y
x
/x
10µm
Example x4 objective(see Fig. 17a)
32 eyepiece units (epu)
1 epu
1 epu
=
=
=
100 micrometer units
100
10µm
/32 x
31.3µm
Repeat the above for the high power objective.
Carry out the calculation to calibrate the eyepiece micrometer at high power in
your notebook/diary.
Measurement of specimens
7 Remove the stage micrometer.
8 Using low power, focus a slide carrying the organism to be measured.
9 Measure the length of the organism against the eyepiece micrometer graduations.
10 Carry out the following calculation.
Calculation
Length of cell
1epu (for x4)
Length of cell
=
a epu
=
31.3µm
=
a x 31.3 µm
=
31.3a µm
11. Repeat for two other cell types at low power and also at high power.
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Activity 5.4.2: Exercise in calculation of specimen size
Use the examples given in Activity 5.4.1 as a guide for these calculations.
a) Calibration of eyepiece micrometer.
Calibrate the eyepiece micrometer for x10 and x40 objectives in Fig.17b and 17c.
b) Measurement of specimens
Fig. 18 shows Amoeba and Paramoecium cells as seen under the x10 objective and
yeast cells under the x40 objective, with an eyepiece micrometer in place. Measure
the lengths of the cells against the eyepiece micrometer.
Using your answers and the values you have calculated in part a), work out the
lengths of each type of cell.
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Fig.18 (a): Amoeba cells x10 objective
Fig.18 (b): Paramecium cells x10 objective
Fig.18 (c): Yeast cells x40 objective
Fig.18: Measurement of microscopic specimens
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6: STAINING
In general, the cytoplasm of protozoa, fungi and bacteria is almost colourless so it is
difficult to see them using an ordinary microscope. It is important to be able to see
bacteria because the size, shape and grouping of the cells help to identify them.
To observe bacteria and yeasts better, the microbiologist first fixes them to a glass
slide then stains them with a dark stain to provide a contrast to the bright background.
Protozoa and fungal hyphae can be examined by staining the living organism.
Fixation
Bacteria must be fixed to a slide to prevent them being washed off when stain is
applied. A smear of bacteria is made on a clean slide and dried thoroughly in air. It is
then heated gently in a flame. This kills vegetative cells, sticks them to the slide,
makes them more permeable to stain and prevents them from breaking down. The
microbiologist is therefore examining dead cells.
Staining
A solution of dye is applied to the fixed smear.
Simple stains confer the same colour on all the cells which then appear a different
colour to the background. Methylene blue stains cells blue. Carbol fuchsin and
safranin stain cells red.
Negative stains colour the background. The micro-organisms remain unstained and
stand out brightly against a dark background. Common negative stains are India ink
and nigrosin.
Vital stains are used to show up live cells. When irrigated with a vital stain like eosin
or neutral red, cells such as Paramoecium and yeast will take up the stain by
phagocytosis. When observed under the microscope for a period of time, the
background at first appears red and the cells colourless. Gradually, the living cells
take up the dye with the result that live cells become red and the background becomes
paler. The contrast of the cells is thus improved.
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Activity 6.1: Preparation of a smear of bacteria or yeast from a solid culture
Materials
Materials required by each student
Lab coat
Eye protection
Disinfectant and paper towels
Discard jar with disinfectant
Lens tissue
3 glass slides
Labels
Plate cultures of yeast and named bacteria
Loop
Forceps
Bottle of sterile water (bijoux or universal)
Bunsen burner and mat
Materials to be shared
Benchkote if necessary
Instructions (Fig.19)
1. Wear a lab coat and use eye protection.
2. Collect materials.
3. Clean slide thoroughly using lens tissue. If necessary pass it through a hot Bunsen
flame to remove grease.
4. Label one end of the slide with initials, date and organism (use a self adhesive
label, wax pencil or glass marker).
5. Flame loop.
6. Using aseptic technique, transfer a loopful of sterile water on to the centre of the
slide.
7. Flame loop.
8. Using aseptic technique, transfer a very small part of a single colony from the
plate culture into the water and mix well, making sure that the smear is not too
thick.
9. Flame loop and place it on a heat resistant mat.
10. Using forceps to hold the slide and with the film downwards, pass the smear
through a yellow/blue flame several times to ‘fix’ it.
11. Place on a heat resistant mat to cool.
The smear is now ready to be stained.
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Fig. 19: Preparation of a smear of bacteria or yeast
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105
6.2: Observation of bacteria using the simple stain methylene blue
The purpose of simple staining is to show up the shape and arrangement of bacterial
cells. The bacteria take up the stain and they appear coloured. You will use the
simple stain, methylene blue. Staining should be carried out on a rack placed over a
sink or draining dish.
Activity 6.2: Staining a smear preparation with a simple stain
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with disinfectant
Fixed smears of Spirillum serpens, Bacillus subtilis and Micrococcus luteus and yeast
Methylene blue stain
Blotting paper (fibre free)
Distilled water bottle
Staining rack and draining dish
Forceps
Microscope
Immersion oil
Materials to be shared
Benchkote if necessary
Instructions (Fig. 20)
1. Wear a lab coat and eye protection.
2. Collect materials.
3. Place previously fixed slides on staining rack over sink or draining dish.
4. Flood with methylene blue for 3 minutes.
5. Holding the slide at an angle with forceps over a sink or draining dish, wash well
with distilled water.
6. Blot dry between two layers of fibre free blotting paper, taking care not to rub off
the cells.
7. Allow the slide to dry in air.
8. Examine under the microscope using the oil immersion lens if possible.
Otherwise, use the highest magnification available.
9. In your lab diary/notebook, briefly record the procedure and draw diagrams of the
organisms you examined. Make sure that you record the name of the organism
and the magnification used.
10. When finished, dispose of slides, gloves etc. as instructed.
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Flood slide with stain
Wash slide with distilled water
Fig. 20: Staining a smear preparation with a simple stain
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108
Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleaned on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Organism:
Stain:
Teacher/lecturer’s signature:
Date:
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6.3: Observation of the filamentous fungi, Mucor and Penicillium using
Lactophenol Blue
Lactophenol blue stains the fungal cytoplasm. The fungal walls appear colourless
against a light blue background. The fungal hyphae should be well teased out.
Activity 6.3: Staining a filamentous fungus using Lactophenol blue
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with disinfectant
Covered beaker containing ethanol
Plate cultures of Mucor and Penicillium
2 slides and coverslips
Lens tissue
Lactophenol blue (note: keep lactophenol off the skin)
Forceps & mounted needle
Microscope
Bunsen Burner
Mat
Materials to be shared
Benchkote if necessary
Instructions (Fig. 21)
1. Wear a lab coat and eye protection.
2. Collect materials.
3. Clean slide.
4. Place a drop of lactophenol blue in middle of slide.
5. Dip forceps in ethanol and replace lid.
6. Place forceps briefly in the Bunsen flame and allow ethanol to burn off
completely.
7. Allow to cool.
8. Partially lift the lid of the Petri dish and use forceps to remove a small piece of the
fungal colony
9. Replace lid.
10. Place fungus in lactophenol blue and tease out well using forceps and needle.
11. Flame forceps and needle with ethanol.
12. Using the mounted needle to support the coverslip, carefully lower it over the
preparation. Take care to avoid producing bubbles.
13. Examine under the microscope using the x40 objective lens.
14. In your lab diary/notebook, briefly record the procedure and make drawings of the
organisms you observe. Remember to note the name of the organism and the
magnification used.
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Fig. 21: Staining a filamentous fungus
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112
Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
e.
f.
g.
h.
Preparation for work is in
accordance with given
specifications
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleaned on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Organism:
Stain:
Teacher/lecturer’s signature:
Date:
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6.4: Negative staining of yoghurt with nigrosin
Nigrosin is a negative stain which colours the background. Yoghurt is used as a
source of bacteria. The bacteria appear colourless on a blue-black background.
Activity 6.4: Observation of bacteria in yoghurt using a negative stain
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with disinfectant
Live yoghurt
Small beaker
Nigrosin stain
Lens tissues
3 glass slides
Wire loop
Sterile water
Materials to be shared
Benchkote if necessary
Instructions (Fig. 22)
1. Wear a lab coat.
2. Collect materials.
3. Using aseptic technique, mix a little yoghurt with an equal quantity of distilled
water in a beaker.
4. Flame loop.
5. Place a loopful of the mixture at one end of a glass slide.
6. Flame loop.
7. Add one loopful of nigrosin stain and mix thoroughly using the wire loop.
8. Flame loop.
9. Prepare the film as described in Fig. 22. Do not fix with heat.
10. When absolutely dry, examine under the highest magnification available. If
possible, use oil immersion.
11. Note briefly in your lab diary/notebook the procedure you have carried out and
make drawings of the organisms you observe. Remember to title the diagrams
and record the magnification.
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1. Mix nigrosin with yoghurt
2. Place the end of slide B on the surface of slide A and pull it slowly towards the
drop of stain and yoghurt
3. The drop will then run along the edge of slide B
4. Push slide B quickly along slide A, dragging the drop behind – do this once only
Fig. 22: Negative staining of yoghurt
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116
Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
i.
j.
k.
l.
Preparation for work is in
accordance with given
specifications
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleaned on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Organism:
Stain:
Teacher/lecturer’s signature:
Date:
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6.5: Staining of root nodules to observe Rhizobium
Leguminous plants such as peas and clover form a symbiotic relationship with the
bacteria, Rhizobium. Root nodules form when the bacteria are present (Fig. 23). You
will examine a stained smear of the root nodules for Rhizobium. The bacteria appear
as irregular X, Y, club, pear or star shaped bacteroids.
Fig.23: Rhizobium root system
Activity 6.5: Staining and observation of Rhizobium in root nodules
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with disinfectant
3 glass slides
Lens tissues
Wire loop
Forceps
Sterile distilled water
Root nodules on legume roots
Crystal violet stain
Staining rack and dish
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118
Materials to be shared
Benchkote if necessary
Instructions
1. Wear a lab coat and eye protection.
2. Collect materials.
3. Clean slides
4. Using forceps, remove a medium sized clean nodule from the root and place in a
drop of water on the slide.
5. Crush the nodule with a second slide and mash it in a drop of water.
6. Using aseptic technique, make a smear of suspension on a fresh clean slide.
7. When dry, flood with stain and leave for ten seconds.
8. Wash with water.
9. Allow to dry.
10. Examine under the highest magnification available, if possible oil immersion.
11. Note the procedure briefly in your lab diary/notebook and make drawings of the
organisms you observe. Remember to title the diagrams and note the
magnification used.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
m. Preparation for work is in
accordance with given
specifications
n.
o.
p.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleaned on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Organism:
Stain:
Teacher/lecturer’s signature:
Date:
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6.6: Vital staining
Vital stains are used to show up live cells. When irrigated with a vital stain such as
neutral red, living yeast cells become red as they take up the stain. The background
becomes paler as the stain enters the cells.
Activity 6.6: Staining of yeast with the vital stain, neutral red
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with disinfectant
Bunsen burner and mat
Lens tissues
Glass slide and coverslip
Wire loop
Sterile water
Plate culture of yeast
Neutral red
Blotting paper
Microscope
Materials to be shared
Benchkote if necessary
Instructions
1. Wear a lab coat and eye protection
2. Collect materials.
3. Clean slide and coverslip.
4. Using aseptic technique, transfer two loopfuls of sterile water to the centre of the
slide.
5. Using aseptic technique, transfer a small amount of yeast from a single colony
into the water on the slide and mix.
6. Carefully lower the coverslip.
7. Using the Pasteur pipette, draw up a little neutral red.
8. Slowly release the stain along one edge of the coverslip as shown by your
teacher/lecturer.
9. Place the edge of the blotting paper against the edge of the coverslip opposite to
draw through the stain.
10. Observe under high power.
11. Record the colour of the background and the colour of the cells at five minute
intervals for a period of twenty minutes.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
q.
r.
s.
t.
Preparation for work is in
accordance with given
specifications
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleared on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Organism:
Stain:
Teacher/lecturer’s signature:
Date:
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123
Questions: Staining
1. Simple staining improves contrast. What does this mean?
2. Give two reasons for fixing a bacterial smear preparation.
3. Name two simple stains.
4. Explain what the term ‘negative stain’ means.
5. Name the type of stain which is taken up only by living organisms.
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INTERMEDIATE 2 BIOTECHNOLOGY
UNIT 2
WORKING WITH MICRO-ORGANISMS
TEACHER/LECTURER GUIDE
UNIT 2: WORKING WITH MICRO-ORGANISMS (INT 2)
TEACHER/LECTURER GUIDE
Contents
Background to unit
Microbiological procedures
1: Preparation of work space
2: Pouring plates
3: Subculturing micro-organisms
3.1: Loop transfer of micro-organisms
3.2: Streak plate inoculations
3.3: Plate to plate subculture of fungal mycelium
3.4: Use of sterile swabs to sample the environment
4: Separating micro-organisms
5: Microscopic examination of micro-organisms
5.1: Magnification
5.2: Setting up a microscope
5.3: Microscopic examination of pond water/ hay infusion/yeast
5.3.1 Cavity slide preparation
5.3.2 Bright field microscopy
5.3.3 Dark field microscopy
5.3.4 Phase contrast microscopy
5.4: Calculation of specimen size using a microscope
6: Staining
6.1: Preparation of a smear of bacteria or yeast from a solid culture
6.2: Observation of bacteria using the simple stain methylene blue
6.3: Observation of filamentous fungi using lactophenol blue
6.4: Negative staining of yoghurt with nigrosin
6.5: Staining of root nodules to observe Rhizobium
6.6: Vital staining of yeast with neutral red
Answers to questions
Record of Attainment
Checklists
Resources
BACKGROUND TO UNIT
This unit of Intermediate 2 Biotechnology, ‘Working with Micro-organisms’ is a
practical unit in which the student will learn some techniques of handling and
studying micro-organisms in the laboratory. These include plate pouring,
subculturing micro-organisms, separating a mixed culture, staining and microscopy.
Since working with micro-organisms is covered by the COSHH regulations, it is
necessary to have suitable and sufficient control measures in place which have been
developed as a result of assessing the risks involved. An appropriate code of practice
on Safety in Microbiology can be adopted to meet these requirements.
To achieve this unit, the student must:
• know and understand why microbiological, microscopy and staining techniques
are always carried out in a given way; and
• become competent in performing a range of microbiological techniques and in
microscopy.
To achieve Outcome 1, the student must pass a written end of unit assessment which
will test knowledge and understanding of the theory behind the practical techniques.
The information needed for this is given in the Student Notes.
To achieve Outcome 2, the student must become competent in practical
microbiological techniques.
The time allowed for this unit is generous in order to allow the student to have ample
opportunity to practise the techniques a number of times until he/she becomes very
familiar with the procedures. The teacher/lecturer should teach students the
techniques included in the laboratory manual.
Each time the student performs a technique, he/she must
• prepare for the work
• carry out the techniques safely and according to instructions
• keep a record of the work done
• record results and observations clearly.
The student should keep a record of work and observations/results in a laboratory
diary/notebook and make an entry each time he/she carries out a piece of work or
observes results. The diary can be kept in a simple notebook or jotter.
Each entry should have:
• the date when the work was carried out
• a heading
• a brief description of the technique the student carried out including incubation
times and temperatures
• observations or results recorded in an appropriate manner (words, diagram, table
etc)
• discussion of results if appropriate.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
1
An example of a diary entry is given below.
Outcome 2 should only be awarded when the teacher/lecturer is satisfied that the
student is competent in the techniques. It is not enough that he/she performs them
satisfactorily on only one occasion.
Checklists for Outcome 2 are provided in the Student Lab Manual and in this Guide.
Range of Outcome 2
Each student must be competent in the following techniques:
• Plate pouring
• Subculturing micro-organisms. Students should be able to correctly remove
inoculum from solid medium (agar) and liquid medium (broth), and correctly
inoculate solid medium (agar) and liquid medium (broth). The Student Lab
Manual includes instructions for a range of subculturing techniques (Activities
3.1.1 – 3.1.4, 3.2.1 – 3.2.2, 3.3 and 3.4) which provide a range of alternatives
through which this part of the Outcome can be achieved.
• Separating a mixed culture
• Staining and microscopy. Activities 6.2, 6.3, 6.4, 6.5 and 6.6 provide alternatives
through which this part of the Outcome can be achieved.
Advice on Classroom Management for Assessment
Since students will be carrying out techniques several times, the class could be
divided so that different students are assessed for different alternatives on different
occasions. It is not necessary to supervise a single student through a whole procedure
unless the student is experiencing difficulty with the techniques. Rather, the
teacher/lecturer could observe small groups of students unobtrusively.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
2
Example of laboratory diary entry.
1.6.98
Subculturing, liquid to solid.
Micrococcus luteus labeled ‘AA, M luteus, 27.5.98’, was aseptically
transferred from a broth culture to an agar plate and plated out to obtain
single colonies using the streak plate method. The plate was incubated
upside down at 30°C for 72 hours and then placed in a refrigerator till it
could be examined.
The technique was also carried out with Saccharomyces cerevisiae but it
was incubated for five days at 30°C.
8.6.98
Results
Organism
Single colonies
Contamination
M luteus
Yes
No
S cerevisiae
Yes
Yes
Discussion
A green fungus contaminated the S cerevisiae plate. A
spore from the air possibly entered the plate during the
process of plating out and developed into the fungal
colony when the plate was incubated. The contaminated
plate cannot be used for further subculture as spores from
the fungal colony may have contaminated the S cerevisiae
colonies.
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
4
MICROBIOLOGICAL PROCEDURES
Theory and practical work for students is contained the Student Lab Manual. The
Student Summary Notes contain the information necessary for the Outcome 1
Assessment. The following is additional information for teachers and lecturers.
Good working practice
The aim of this unit is for the student to acquire some of the basic technical skills used
in the study of microbes.
All organisms must always be treated as potential pathogens. Teachers and lecturers
should emphasise continually that students must follow safety procedures. If they do
not, they cannot achieve the unit.
Students must be made aware of the principles of Safety in Microbiology through the
Student Lab Manual on Good working practice and the principles in a code of
practice.
Safety is best guaranteed if routines are followed and become ‘second nature’ to the
students. Teachers/lecturers must observe students continually with regard to
ensuring safety and in assessing each student's competency in the practical techniques.
Classroom Management
This applies to all practicals in this unit.
Each should have his/her own set of equipment if possible and be able to sit
comfortably at the work bench with legs below the bench to protect them if there is a
spillage.
Students should move around as little as possible and should work quietly.
The teacher/lecturer should demonstrate each technique. It may be advantageous to
teach the method to small groups to ensure that each student can observe the process
clearly.
Before starting each practical, students should be familiar with the underlying
theory and understand the reasons for the safety control measures.
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6
1: PREPARATION OF WORK SPACE
Type and purpose of activity
This activity can be used to:
• develop knowledge and understanding of how to prepare the work space for
Outcome 1, PC a
• illustrate good working practice
• provide evidence for the assessment of Outcome 2:
PC a preparation of work is in accordance with given specifications
PC c the record of work is clear and accurate.
Background information
The surface on which microbiological work is carried out must not be able to absorb
any fluid or have cracks which could harbour micro-organisms. Some microorganisms can survive for a considerable length of time in the environment and if
spilled on to a rough or absorbent surface could present a potential source of
contamination both to others using the laboratory and to cultures. A sealed wooden
surface is suitable. If the bench surface is unsuitable, it must be covered with
Benchkote or some other non-absorbent material.
Whatever the surface, before starting work it must be swabbed with disinfectant to
reduce the risk of contamination of cultures. The working area must be kept tidy and
uncluttered in order to minimise the risk of accident. Allow only the essentials on the
bench.
While carrying out the experiments in this unit, equipment will become contaminated.
Contaminated items must be disposed of safely. There must always be a disposal
container within easy reach. If the student has to stretch, there is a risk of dropping
the apparatus or leaking organisms from a pipette for example.
A Bunsen burner is essential to the technique of aseptic transfer. It should be placed
on the bench within easy reach of the operator. The Bunsen flame provides an
updraught which carries air away from the operator reducing the risk of breathing in
contaminated air.
Classroom Management
The teacher/lecturer should demonstrate a well set up bench and emphasise the
importance of good bench management from both the safety angle and efficiency of
working.
Students must prepare the workspace each time they carry out microbiology
practical. The teacher/lecturer should make students fully aware that they must do
this for each practical to achieve PC a (preparation for work is in accordance with
given specifications).
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
7
ASSESSMENT/MARKING GUIDELINES
PC a:
PC c:
Teachers/lecturers should observe students following instructions
successfully and check that bench is set up as specified in the
instructions on each occasion they carry out practical work.
The note in the lab book is in accordance with student advice and is
clear and accurate
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
8
2: POURING PLATES
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of pouring agar plates for Outcome 1, PCa.
Background information
Refer to Student Notes.
Classroom Management
The teacher/lecturer should demonstrate the technique of pouring plates, if possible to
small groups of students at a time.
It should be emphasised to students that if spilled, hot agar adheres to skin and
can cause a serious burn.
Students should be reminded that a checklist is included in the Student Lab Manual
which can serve as a guide for achievement of this part of the range of Outcome 2.
ASSESSMENT/MARKING GUIDELINES
To achieve Outcome 2, students must complete this practical satisfactorily.
All students must therefore be made fully aware that they must record this activity and
its results in a laboratory diary or notebook.
A checklist is included.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Pouring plates
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Plates labeled on underside
•
Agar cooled to pouring temperature
•
Aseptic technique satisfactory
•
Plates dried
•
Plates incubated.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on characteristics of plates:
•
Smooth surface to agar
•
Even layer of agar
•
Base of Petri dish covered
•
Contamination.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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3: SUBCULTURING MICRO-ORGANISMS
Background information
Subculturing is the transfer of microorganisms from one medium to another. It
involves the aseptic removal of organisms from one culture medium followed by
aseptic inoculation of another culture medium and is a technique essential to many
biotechnological procedures.
Students should be able to correctly remove inoculum from solid medium (agar) and
liquid medium (broth), and correctly inoculate solid medium (agar) and liquid
medium (broth). The Student Lab Manual include instructions for a range of
subculturing techniques (Activities 3.1.1 – 3.1.4, 3.2.1 – 3.2.2, 3.3 and 3.4) which
provide a range of alternatives through which this part of the Outcome can be
achieved.
The time allowed for this unit is generous because it is intended that students carry out
these procedures repeatedly and be given the opportunity to become competent in
them. They should be aware of the rationale behind each procedure and be conscious
of safety considerations at all times.
Classroom Management for Activities 3.1.1 – 4.2 and 6.1 – 6.6
The teacher/lecturer must stress the importance of safety procedures.
Students should develop a constant awareness of safety considerations and
vehicles or entry points for contamination when working with micro-organisms.
They must also understand and be encouraged to practise the importance of good
flaming technique.
The following should therefore be emphasised continually.
• Inoculating loops or needles must be sterilised by flaming before and after use.
•
Flaming must be carried out correctly.
•
Once flamed, the loop must never be put down (or it could become contaminated)
but held in the hand and allowed to cool. Loops should not be waved around.
•
The necks of tubes and bottles must be flamed after removing and before
replacing a lid.
•
Caps of bottles must never be put down (to prevent contamination of or by them).
•
Cultures should be exposed to the air for as short a time as possible.
•
Lids of Petri dishes should be raised only enough to admit the wire loop and
permit the manipulation.
•
Plates inoculated with swabs of the environment must be sealed.
•
Certain sites must not be sampled.
It should be emphasised to students that any spillage must be reported to the
teacher or lecturer in charge.
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Incubation, refrigeration and disposal
Students must be given clear instructions regarding incubation, refrigeration and
disposal of materials. They must distinguish clearly between contaminated and noncontaminated waste and never dispose of contaminated material in a noncontaminated area. Consult an appropriate code of practice for further advice.
Activities 3.1.1 – 3.1.4: Loop transfer of micro-organisms.
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of subculturing for Outcome 1, PC a
• develop competence in the techniques of handling micro-organisms.
Background information
Students will carry out solid to solid, solid to liquid, liquid to solid and liquid to liquid
inoculations using a wire loop.
It is essential that they learn to hold and flame the wire loop correctly to
minimise the risk of aerosol formation.
Background information regarding the rationale of the procedures is described fully in
the student support notes/background information.
Activities 3.2.1 and 3.2.2: Streak plate inoculations
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of subculturing for Outcome 1, PC a
• develop competence in the techniques of handling micro-organisms.
The term ‘streaking out’ or ‘plating out’ is applied to the inoculation of solid media
in Petri dishes using a technique involving successive strokes with a wire loop. The
aim of this process is to obtain single colonies that can be further subcultured if
required. The loop is charged with a small amount of culture and several strokes in
series are then made on the surface of the medium with the aim of diluting the
bacteria so that single cells are distributed on the surface. The loop is flamed between
each series of strokes to reduce the number of organisms and increase the chance of
isolating bacterial cells. On incubation, each single cell divides successively to
produce a single colony. In this way, pure cultures can be obtained.
The Petri dish should be turned through 90° between each series of strokes to allow
the loop to be held in a safe and comfortable position which remains the same for
each series of streaks. This is not shown well in the diagrams in the Student Lab
Manual. It may help some students to draw the streaking pattern on the underside of
the Petri dish with a fine tipped marker pen.
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Activity 3.3: Plate to plate subculture of fungal mycelium
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of subculturing for Outcome 1, PC a
• develop competence in the techniques of handling micro-organisms.
Background information
It is sometimes difficult to pick up fungal mycelium with a wire loop or straight wire.
Plate to plate inoculation is therefore often carried out using a sterile scalpel.
The scalpel may be sterilised by heating the tip in a Bunsen flame until red hot, as
with a wire loop. The blade may have to be held in the flame longer than a loop so
there is a risk that the handle may become hot.
Preferably, a scalpel wrapped in foil and sterilised by autoclaving may be provided.
The student must dip the scalpel blade in alcohol, place it in the Bunsen flame,
remove it from the flame and allow the alcohol to burn off and let the blade cool
before use. This does not sterilise the blade since bacterial spores will resist these
temperatures but avoids the problem of the scalpel handle becoming too hot to handle.
The scalpel is then used to cut through the fungal mycelium and agar below to extract
a small block. This is then transferred aseptically to sterile agar and incubated.
Alternatively, transfer may be carried out using a cork borer flamed in ethanol or the
wide end of a sterile Pasteur pipette. Here, a mounted needle flamed with ethanol
may be required to assist the transfer.
Young cultures (5 days) which are not sporulating heavily should be provided as
inocula. Each inoculum should be taken from a non- sporing region possibly towards
the edge of a colony to reduce the risk of contamination.
Activity 3.4: Use of sterile swabs to sample the environment
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of subculturing for Outcome 1, PC a
• develop competence in the techniques of handling micro-organisms.
Background information
The numbers and types of bacteria and fungi present on a surface such as the
laboratory bench or floor can be estimated by swabbing the area with a sterile swab or
cotton wool bud which has been dampened with sterile water. The swab is then
rubbed across a sterile agar plate which is then incubated.
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Plates must be sealed with tape and not opened after incubation since the organisms
that grow are unknown. Each Petri dish must be sealed diametrically using
transparent adhesive tape and incubated base uppermost. Lids should not be sealed to
bases around the circumference as this may create anaerobic conditions.
The relevant code of practice should be consulted regarding sites from which samples
should not be taken.
ASSESSMENT/MARKING GUIDELINES
From the range of activities, each student should demonstrate competence in
• removal of inocula from solid media
• removal of inocula from liquid media
• inoculation of solid media
• inoculation of liquid media.
All students must be made fully aware that they must record these activities and their
results in a laboratory diary or notebook to achieve PCs c and d.
A checklist is included for guidance.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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4: SEPARATING MICRO-ORGANISMS
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of subculturing for Outcome 1
• develop competence in the techniques of handling micro-organisms.
Background information
In this experiment, a mixed broth culture is streaked out on an agar plate then
incubated to obtain single colonies of different types of bacteria.
See Technical Guide for advice on organisms.
Classroom Management
See 3: ‘Subculturing micro-organisms’.
ASSESSMENT/MARKING GUIDELINES
All students must be made fully aware that they must record these activities and their
results in a laboratory diary or notebook to achieve PCs c and d.
A checklist is included for guidance.
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Unit 2: Working with Micro-organisms (Int 2)
Title: Separating a mixed culture
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Loop/scalpel flamed correctly
•
Plates labeled on underside
•
Aseptic technique satisfactory
•
Plates incubated appropriately
•
Single colonies of both types present
•
Contaminated materials disposed of
appropriately
•
Surface swabbed with disinfectant.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growths
•
Whether there are isolated single colonies of
each type of organism.
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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5: MICROSCOPIC EXAMINATION OF MICRO-ORGANISMS
Type and purpose of activity
This activity can be used to:
• develop knowledge and understanding of the principles of the light microscope for
Outcome 1, PC b
• develop competence in using a microscope.
Background information
The microscope
For students to observe micro-organisms they must be able to obtain the best possible
performance from the microscope.
The structure and function of the parts of the microscope are given in the Student Lab
Manual.
In summary, the microscope consists of the following.
A stand which bears:
• a body tube which is raised and lowered with coarse/fine adjustment
mechanisms; sometimes the stage moves and the body tube is fixed; the nosepiece
carries objectives.
• a stage on which slides to be examined are placed.
• An optical system consisting of three parts.
• A substage condenser fitted with an iris diaphragm. The condenser collects a
wide source of light from the mirror or electric bulb and condenses it to provide a
cone of rays of sufficiently wide angle to fill the objective being used. The iris
diaphragm controls the angle of the cone of light passing through the condenser.
• Objective lenses mounted on a rotating turret: low power lenses (x4, x10 or both),
a high power lens (x40) and possibly an oil immersion lens (x100).
• The eyepiece or ocular lens at the upper end of the body tube. Magnification is
usually x10 (x15 may be used in conjunction with x40 to view bacteria without oil
immersion).
Three focussing mechanisms :
• a rack and pinion for adjusting the condenser assembly
• a coarse adjustment for raising/lowering the body tube
• a fine adjustment for raising/lowering the body tube.
A light source. In school microscopes this is usually provided by a mirror tilted to
reflect light through the objective lenses. Some microscopes may have a built-in
lamp. In bench lamps, a 60 watt opal bulb should be used.
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Principles of the light microscope
• Visible light passes through a substage condenser which focuses the light into a
sharp cone.
• The light passes through the opening in the stage into the slide holding the
specimen and may be absorbed, reflected or refracted by the specimen.
• The light passes through the objective lens and forms a magnified image of the
specimen which is darker than the background (called the real image because it
can be projected on to a screen but is not seen by the microscopist).
• The image becomes an object for the eyepiece lens which magnifies the image a
second time to create a virtual image in space seen only by the observer.
5.1: Magnification
Type and purpose of activity
This activity can be used to:
• develop knowledge and understanding of how magnification is calculated in a
light microscope for Outcome 1.
Background information
The magnifying power is the degree of enlargement; that is, the number of times the
image appears greater than the original specimen.
To calculate this, multiply together the separate magnifying powers of the objective
and eyepiece lenses. For example, the total magnification of a microscope fitted with
a x10 eyepiece lens and a x10 objective lens is 10 x 10 = x100. Similarly, if a x40
objective lens was being used on the same microscope, the total magnification would
be 10 x 40 = x400.
Resolution
Resolving power or resolution is the ability to reveal closely adjacent structural details
as separate and distinct. The resolving power of an optical microscope determines the
amount of structural detail that can be observed and is limited by the wavelength of
visible light.
The resolving power sets the limit of useful magnification which for a light
microscope is about x1500. Although lenses can be produced to produce much
greater magnification than this, they will reveal no more detail and is described as
‘empty magnification’. A good quality light microscope will resolve down to 2 µm.
Definition
Definition is the capacity of an objective to render the outline of the image of the
object clear and distinct. It depends on the elimination of optical aberrations inherent
in the glass of the lenses.
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Activity 5.2: Setting up a microscope
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of principles of the light microscope for
Outcome 1, PC b
• develop competence in using a microscope.
Background information
Students must become familiar with the structure of the microscope and become
competent at using it to view micro-organisms. The instructions provided in the
Student Lab Manual should allow them to do this. However, the following
adjustments will provide even better results and could be used if demonstration
microscopes are being set up for a class.
Precise adjustment of the condenser
The condenser is adjusted correctly when it is focusing an image of the lamp on the
specimen. To do this:
1. Set up the microscope as far as instruction 12 in the Student Activity Guide.
2. Place a pencil against the surface of the bulb (stand alone or integrated).
3. Look down the microscope and move the pencil slowly over the lamp surface till
you can see it (possibly as a blurred image).
4. Adjust the condenser till the pencil point comes into sharp focus. The condenser
is now focused correctly.
The condenser should be adjusted each time the microscope is set up and each time
the objective is changed.
Precise adjustment of iris
The Student Manual instructions include correct adjustment of the iris. It should be
emphasised to the student that the iris must be adjusted each time the objective is
changed.
Condenser centring
Some student microscopes have centring condensers i.e. the condenser has to be
adjusted so that the cone of light is in line with the objectives.
When the iris is closed down, the circle of light that can be seen should be central
within the body tube. If it is not, use the centring screws to centre the circle of light.
When it is symmetric with the body tube, it is correctly positioned. There should be
no need to readjust it although if handled roughly the condenser may be knocked off
centre.
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Care of the microscope
Dust from the air, grease from fingers, liquids and chemicals from slides all damage
microscopes. Students should develop a routine of cleaning all the glass surfaces of
the microscope with lens tissue before, during and after use. It should be stressed to
them that if immersion oil is left on lenses, it hardens and blurs the image. Ordinary
tissue should not be used as it may scratch the glass surfaces of the lenses and leave
fibres behind which obscure the image.
Only trained staff should carry out maintenance of the focusing mechanisms, oiling of
moving parts and cleaning of the internal components of the lenses. SSERC can
provide technician training in maintaining and servicing microscopes.
Cleaning the objective
The front surface of an objective is likely to become marked by dust and fingerprints
and require regular cleaning. It can be cleaned as follows.
1. Carefully unscrew the objective from the nosepiece and examine the front lens
(the one which is nearest the stage).
2. Fold a clean sheet of lens tissue into a small pad and moisten it with xylol
substitute.
3. Gently wipe the surface of the lens to remove dirt and grease. Take care that the
tissue is not too wet.
4. Polish the surface of the lens with a clean sheet of lens tissue.
Note: if immersion oil has dried on the lens, it may require several attempts to remove
it.
Cleaning the eyepiece
The top lens of the eyepiece often becomes marked with dust and grease. It should be
cleaned in the same way as above. The other lens surfaces of an eyepiece should not
need cleaning.
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Oil immersion
Oil immersion objectives are preferable to view bacteria satisfactorily because of their
greater magnification and resolution properties. If they are not available, use a x15
eyepiece with a x40 objective to give x600 magnification to observe bacterial shapes.
The x100 objective works very close to the object being viewed. Light rays passing
through the glass slide to the air above are refracted so that many of them miss the
front lens of the objective, diminishing both the clarity and brightness of the image.
Immersion oil is used to reduce this refraction. Immersion oil has the same refractive
index as optical glass. When oil fills the space between the slide and the objective,
more light rays pass into the objective and the microscopist observes a much
improved image.
Instructions for viewing a slide using oil immersion are provided in the Student
Activity Notes, ‘Setting up a microscope’.
Immersion oil is messy and it is important to stress to students that they must handle it
carefully and sparingly. They must not put it on lenses other than the oil immersion
lens and if it is spilled on any other part of the microscope it should be wiped off
immediately with lens tissue. Oil immersion lenses should be cleaned with lens tissue
immediately after use.
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Common problems and their solutions
Problem
Image is not sharp
Dirty marks
marks move when slide is
moved
marks move when eyepiece is rotated in drawtube
marks move when
objective is rotated in
nosepiece
Marks appear when lamp
is focused
Too bright
Bright specimen on dark
background
‘Worms’
Dark edged circles
Uneven illumination
Too dark
Image goes slowly out of
focus
Jagged lines
Cause
Dirty objective
Slide upside down
Solution
Clean with xylol substitute
Invert slide
Dirt or grease on slide
Clean both sides with lens
tissue
Clean top lens with xylol
substitute and lens tissue
Dirt or grease on eyepiece
Dirt on objective
Clean front lens with lens
tissue and xylol substitute
Dirt on lamp surface
Clean lamp or defocus
condenser slightly
Iris too far open
Lamp too bright or too
close
Light shining on top of
specimen
Fibres on slide, eyepiece or
objective
Air bubbles
Close down iris slightly
Change bulb or move lamp
further away
Adjust position of lamp
Mirror and/or lamp askew
Objective not properly
clicked into position
Filter tray blocking light
into condenser
Iris closed down too far
Lamp too weak or too
distant
Filter tray obstructing light
into condenser
Condenser not focused
properly
Insufficient friction in
focus control mechanism
Coverslip edge or broken
part of coverslip
Clean with fibre free lens
tissue
Move slide and examine
area without air bubbles or
make fresh slide
Adjust mirror and /or lamp
Rotate nosepiece
Swing in or out
Open iris
Change bulb or move lamp
closer
Swing in or out
Focus condenser
Adjust friction n focus
control (likely to require
trained operator)
Move slide to examine
different region. If c/slip
broken, make new slide
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Classroom Management for Activities 5.2 – 6.6
The teacher/lecturer should demonstrate how to set up a microscope and prepare each
type of slide.
Students should be reminded to clean their microscopes after use.
Activities 5.3.1 – 5.3.4: microscopic examination of yeast, pond water and hay
infusion by bright field, dark field and phase contrast microscopy.
These would be useful as extension activities.
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of principles of the light microscope and
magnification for Outcome 1, PC b
• develop competence in using a microscope.
Activity 5.3.1: Cavity slide preparation
Background information
Use pond water from a non-contaminated pond or stream to make a hanging drop
preparation. It should be rich in microscopic life and students should be able to
observe a variety of micro-organisms. Alternatively, a solution of yeast may be used.
Activity 5.3.2: Bright field microscopy
Background information
In bright field microscopy, the specimen is illuminated directly from the light source
presenting a brightly lit field of view.
Activity 5.3.3: Dark field microscopy
Background information
Dark ground illumination renders visible organisms which do not show up in
unstained preparations under bright field microscopy.
Using a special condenser, the specimen is illuminated by oblique light only. Rays do
not enter the objective unless scattered by objects e.g. cells which have a different
refractive index from the medium in which they are suspended.
Alternatively, you can get some idea of what dark field microscopy is like by closing
the iris diaphragm and illuminating the specimen from above.
Organisms appear brightly illuminated on a dark background
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Activity 5.3.4: Phase contrast microscopy
Background information
The human eye can detect differences in amplitude of light waves but not differences
in phase.
Amplitude controls light intensity.
Using bright field microscopy it is difficult to observe unstained cells because light
intensity is similar before and after light passes through a living cell since the
amplitude is not changed.
Phase oscillation of light waves, however, is changed by passage through a cell. The
human eye cannot detect differences in phase.
Essentially, the phase contrast microscope converts differences in phase to differences
in amplitude so that a living cell appears to have a different intensity to its background
and internal details of cells can be distinguished.
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5.4: Calculation of specimen size using a microscope
Background information
Specimen size
The specimens examined under the microscope are generally too small to be seen with
the naked eye. They are measured in micrometres (µm). Viruses may be measured in
nanometres (nm). In general, viruses are too small to be seen with the light
microscope.
1mm = 10-3metres (one thousandth of a metre)
1µm = 10-3 millimetres (one thousandth of a millimetre)
= 10-6metres (one millionth of a metre)
1nm = 10-3micrometres (one thousandth of a micrometre)
= 10-6 millimetres (one millionth of a millimeter)
= 10-9 metres (one thousand millionth of a metre).
The following table indicates the sizes of different types of cells.
Table: Sizes of micro-organisms
Size
Units
1mm
10–3 m
100µm
10–4 m
10µm
10–5 m
1µm
10–6 m
Organism
Algae
Protozoa
Fungi
Bacteria
100nm
10-7m
Viruses
10nm
10-8m
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Activity 5.4.1: Calculation of specimen size using a microscope
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2.
• develop knowledge and understanding of principles of the light microscope and
magnification and specimen size for Outcome 1, PC b
• develop competence in using a microscope.
Background information
See ‘Student Lab Manual’ for information on ‘measuring objects using a microscope’.
Activity 5.4.2 is a written exercise based on actual recorded measurements and can be
used in addition to or as a substitute for Activity 5.4.1.
Activities 5.4.1 and 5.4.2 are fairly advanced activities and may be suitable as
extension exercises for more able students. Description of an alternative practical
activity is given below.
Alternative practical activity
Simple micrometers printed on acetate may be purchase from standard suppliers and
mounted on a glass slide.
Using this, a student can measure and record the diameter of the field of view under
different objectives.
By replacing the micrometer slide with a specimen, the student can estimate the
length of the specimen as a proportion of the diameter of the field of view. He/she
can then calculate an estimated length using the diameter previously recorded for that
objective.
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Activity 5.4.2: Exercise on calculation of specimen size
Type and purpose of activity
This activity can be used to:
• develop knowledge and understanding of principles of the light microscope and
magnification and specimen size for Outcome 1, PC b
Background information
This is a written exercise in which the student uses diagrams to calibrate an eyepiece
micrometer for different objectives and to measure different specimens.
This may be best suited to more able students.
Answers
Calibration of eyepiece micrometer
x 10 objective (Fig. 17b)
80 eyepiece units (epu)
=
100 micrometer units
1 epu
=
100
=
12.5µm
100 eyepiece units (epu)
=
30 micrometer units
1 epu
=
30
1 epu
=
3µm
/80
x
10µm
X40 objective (Fig. 17c)
/100
x
10µm
Measurement of specimens
Amoeba (Fig 18a) x10 objective
Length of Amoeba cell
=
40 eyepiece units (epu)
1epu for 10 objective
=
12.5µm
Length of cell
=
40 x 12.5 µm
=
500 µm
=
500 µm
Length of Amoeba cell
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Measurement of specimens
Paramoecium (Fig 18b) x10 objective
Length of Paramoecium cell
=
15 eyepiece units (epu)
1epu for 10 objective
=
12.5µm
Length of cell
=
15 x 12.5 µm
=
187.5 µm
=
187.5 µm
Length of yeast cell
=
5 eyepiece units (epu)
1epu for 40 objective
=
3 µm
Length of cell
=
5 x 3 µm
=
15 µm
=
15 µm
Length of Paramoecium cell
Measurement of specimens
Yeast (Fig 18c) x40 objective
Length of yeast cell
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6: STAINING
Background information
Most micro-organisms have a refractive index which differs only slightly from glass
so it is difficult to observe them by bright field microscopy. The purpose of staining
is to improve contrast between the organisms and the bright background. Simple
staining confers the same colour on all cells. Differential staining (such as Gram’s
stain) colours bacteria differentially. Students are required to carry out simple
staining only in this unit.
Simple stains are simple basic dyes e.g. methylene blue, methyl violet or basic fuchsin
that are applied in a watery solution to the cells. The coloured, positively charged
cation of the basic dye combines firmly with negatively charged groups in bacterial
protoplasm, particularly with phosphate groups. After application, the preparation is
washed to remove excess dye from the slide. The cells retain the dye and when
examined under the microscope appear coloured in contrast to a bright background.
Acidic dyes have coloured anions and do not stain bacteria strongly (except at very
acid pH values). The technique of negative staining uses acidic dyes. Bacteria appear
light against a dark background.
Vital stains e.g. neutral red and eosin, are taken up by living cells by the process of
phagocytosis.
Classroom management
Students should prepare for working with micro-organisms. The teacher/lecturer
should emphasise that aseptic technique must always be used when manipulating
micro-organisms.
The teacher/lecturer should demonstrate each technique.
Smears of a variety of organisms will be used for staining. Students may be provided
with prepared smears or make their own preparations. Students should receive clear
instructions regarding disposal of materials.
When working with stains, care must be taken to prevent hands from becoming
stained. Well fitting gloves disposable gloves should be worn if available. A staining
rack (made from glass rods and rubber tubing (Fig.1) and dish per pair of students
avoids crowding around sinks.
Fig. 1: A staining rack
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Activity 6.1: Preparation of a fixed smear of bacteria or yeast from a solid
culture
Background information
Before making a smear, slides should be thoroughly cleaned using lens tissue.
Smears or films are made by aseptically mixing a little growth from a single colony
with a loopful of sterile water on a slide. It is important that only a minute quantity of
organism is placed on the slide and that the water/organism mixture is thoroughly
emulsified. The mixture is then spread evenly and thinly across the centre of the
slide. Beginners tend to pick up too much material and thus make too thick a
smear. Individual cells can only be distinguished in a thin film.
Fixation
Before staining, bacteria must be fixed to the slide.
Fixing kills the vegetative cells, fixes them to the slide, makes them more permeable
to the stain and prevents autolysis.
The film can be fixed by passing the dried slide, smear downwards, three times
through a blue/yellow flame or by heating the underside of the glass slide. Here, the
smear is uppermost and the slide held for a few seconds in the top of the Bunsen
flame till the slide becomes hot. Fixation has taken place when the slide is just too
hot to be borne on the back of the hand. Slides should be held with forceps and care
must be taken not to hold the slide too long in the flame. Eye protection should be
worn as a precaution.
After cooling, the smears are ready to be stained.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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6.2: Observation of bacteria using the simple stain methylene blue
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2, PC a and b
• develop knowledge and understanding of simple staining techniques for
Outcome 1, PC b.
Background information
When stained with methylene blue, bacteria appear blue against a bright background.
The nuclei of yeast cells can often be observed as dark object in the cytoplasm. Cell
walls of both bacteria and yeast do not take up the stain and can often be noted as
colourless areas between cells.
The stain is poured directly on to the slide. After the due time, the dye is washed off
with water using a water bottle. The slide is held at an angle with forceps over the
draining dish and water directed on to it from the bottle.
The slide can then be dried between two sheets of fibre free blotting paper or left to
dry in air. Drying may be completed by heating gently over a Bunsen flame.
Bacteria should be viewed under oil immersion. Otherwise, use of a x15 eyepiece
lens with a x40 objective lens should allow observation.
6.3: Observation of the filamentous fungi, Mucor and Penicillium using
Lactophenol Blue
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2, PC
• develop knowledge and understanding of simple staining techniques for
Outcome 1, PC b.
Safety note
Lactophenol blue is poisonous by absorption and should be kept off skin. The
following control risk.
• Good technique
• Use of small volumes
• Use of a dropping bottle
• Use of a staining rack
• Well fitting disposable gloves should be worn if available.
Background information
The fungal cytoplasm takes up the blue stain. Colourless hyphal walls can be
observed against a light blue background. Fungal cultures should be 5 – 7 days old to
allow observation of hyphae. Old cultures possess too many spores which mask the
filaments.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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The fungal hyphae must be well teased out. When applying the coverslip, support it
with a mounted needle and lower it gently to avoid bubbles.
Remove excess stain with the edge of a piece of blotting paper placed against one
edge of the coverslip. Take care not to remove too much.
Activity 6.4: Negative staining of yoghurt with nigrosin
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of simple staining techniques for
Outcome 1, PC b.
Background information
‘Live’ yoghurt must be used. Bacteria can be seen as bright objects against a dark
background.
Correct preparation of nigrosin is critical to success.
Activity 6.5: Staining of root nodules to observe Rhizobium
Type and purpose of activity
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of simple staining techniques for
Outcome 1, PC b.
Background information
Rhizobium can be observed from root nodule preparations as described in the student
guide.
Activity 6.6: Staining of yeast with the vital stain, neutral red.
This activity can be used to:
• provide evidence for Outcome 2
• develop knowledge and understanding of simple staining techniques for
Outcome 1, PC b.
Background information
Use a growing culture of yeast (or live protozoa). Only live cells take up the vital
stain. The cells at first appear colourless against a red background but as they take up
the stain by phagocytosis, living cells become red in contrast to the background which
pales in colour. This can be observed over a period of about twenty minutes. Neutral
red turns yellow in acid condition. Food vacuoles can be observed as yellow.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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ASSESSMENT/MARKING GUIDELINES
To achieve Outcome 2, students must successfully stain and observe micro-organisms
by one of the methods in Activities 6.2, 6.3, 6.4, 6.5 or 6.6.
All students must be made fully aware that they must record these activities and their
results in a laboratory diary or notebook to achieve PCs c and d.
A checklist is included for guidance.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
40
Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleaned on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Answers to Questions
1.: Preparation of work space
1. Wash hands and put on a lab coat.
2. A non-absorbent surface is required to ensure that micro-organisms cannot
penetrate the surface but can be removed easily.
3. The surface is disinfected before and after use to reduce the number of potential
contaminants.
4. All equipment should be within easy reach of the operator.
5. Windows and doors should be kept closed to minimise draughts and reduce the risk
of contamination.
6. The Bunsen burner provides an updraught which carries air upwards and away
from the operator reducing the risk of contamination.
2: Pouring plates
1. Labels should be written on the base of plates.
2. Three details that should be included in labels are: name, date and type of agar.
3. Necks of bottles should be flamed before pouring agar.
4. Bubbles on the surface of the agar can be avoided by ensuring it is cooled to the
appropriate temperature , not agitating it and pouring and swirling it gently.
5. Excess condensation on lids can be prevented by ensuring that the temperature of
the agar is low when poured and by drying the plates.
6. Plates should be stored upside down.
7. Plates should have a smooth surface, even layer of agar, the base of the dish
should be covered and there should be no contamination.
3.1: Loop transfer of micro-organisms
1. A wire loop should be held loosely in the hand in a similar way to holding a pencil.
It should be held almost vertically to prevent backflow of liquid and should never
be waved around.
2. The pale blue cone of the Bunsen flame is at a lower temperature than the rest of
the flame. This allows any liquid on the loop to evaporate without spurting off the
loop and prevents formation of aerosols containing live organisms.
3. The loop is sterilised when it has become red hot.
4. The loop must be cooled before subculturing to avoid killing the organisms.
5. a) necks of bottles should always be flamed and b) lids should never be put down
on bench.
6. The loop must be flamed at the end of every subculturing procedure.
7. Lids could be transferred inadvertently.
8. Cultures should always be labelled with name or initials, date and organism.
9. Condensation is thus prevented from dropping on to the surface of the agar plates.
10.Cultures must be sterilised before final disposal to ensure that they do not pose a
risk of contamination.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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3.2: Streak plate inoculation
1. The streaking out technique is used to obtain single colonies.
2. The loop is flamed between strokes to reduce the number of micro-organisms on
the loop.
3. A pure culture contains only one type of organism.
4. A colony is produced from a single cell which divides repeatedly.
3.3: Plate to plate transfer of fungal mycelium
1. Fungal mycelium is the hyphal growth a fungus produces giving it a hairy
appearance.
2. It is more difficult to pick up the fungal mycelium with loops and needles.
3. Fungal mycelium is not streaked across the plate but inoculated in a central
position to allow the colony to grow outwards.
3.4: Use of sterile swabs to sample the environment
1. Swabs are made of cotton wool and would burn in a Bunsen flame.
2. Make sure you do not touch the swab end. Only touch the handle.
3. The organisms which grow are unknown and may be dangerous.
4: Separating micro-organisms
1. A mixed culture contains more than one type of organism.
2. Streaking or plating out will dilute and separate micro-organisms until single cells
are deposited on the plate.
3. Streak out to obtain isolated single colonies; use an isolated single colony as the
inoculum for further streaking out.
5: Structure and function of the light microscope
1. The purpose of the light microscope is to view organisms or structures too small
to be seen with the naked eye.
2. The iris controls the angle of the cone of light which passes through the
condenser.
3. The stage supports the slide.
4. Three parts of the microscope which contain lenses are the eyepiece, the
objectives and the condenser.
5. The focus controls are adjusted to make the image sharp.
5.1: Magnification
Eyepiece lens
magnification
x15
x10
X10
x10
x15
Objective lens
magnification
x10
x10
x40
X100
x40
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
Total
magnification
x150
X100
x400
x1000
X600
43
5.4: Measuring objects using a microscope
1. Metre = m; millimetre = mm; micrometre = µm.
2. There are one thousand millimetres in a metre.
3. There are one million micrometres in a metre.
4. There are one thousand micrometres in a millimetre.
5.
Metres
millimetres
micrometres
3
3
3000 (3x10 )
3000000 (3x106 )
0.5
500 (5x102 )
500,000 (5x105 )
-2
0.07 (7x10 )
70
70,000 (7x104 )
0.000425 (4.25x10-4)
0.425 (4.25x10-1)
425
-4
0.0008 (8x10 )
0.8
800 (8x102 )
-5
-2
0.000039 (3.9x10 )
0.039 (3.9x10 )
39
-4
0.00059 (5.9x10 )
0.59
590 (5.9x102 )
0.02
20
20,000 (2x104 )
0.000006 (6x10-6)
0.006 (6x10-3)
6
-2
0.026 (2.6x10 )
26
26,000 (2.6x104 )
6: Staining
1. Improving the contrast means that there is a greater differentiation between cells
and background.
2. Fixing a smear attaches the cells to the slide, kills the cells and makes them more
permeable to stain.
3. Methylene blue, crystal violet.
4. A negative stain does not stain the cells. They appear bright on a dark
background.
5. A vital stain.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Unit 2:Working with Micro-organisms
Record of Attainment
Class
Year
Outcome 2
Candidate
Outcome 1
score
From
solid
subculturing
From
To solid To
liquid
liquid
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
Separating
a mixed
culture
Staining
and
microscopy
O2
achieved
Unit
achieved
45
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
46
Unit 2: Working with Micro-organisms (Int 2)
Title: Pouring plates
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Plates labeled on underside
•
Agar cooled to pouring temperature
•
Aseptic technique satisfactory
•
Plates dried
•
Plates incubated.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on characteristics of plates:
•
Smooth surface to agar
•
Even layer of agar
•
Base of Petri dish covered
•
Contamination.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
48
Unit 2: Working with Micro-organisms (Int 2)
Title: Subculturing micro-organisms
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out in
accordance with safe practice
and given specifications.
Record of work is clear and
accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Instructions followed accurately
•
Aseptic technique satisfactory
•
Technique carried out in accordance
with safe practice
•
Inoculating instrument sterilised
correctly
•
Removal of inoculum satisfactory
•
Inoculation method satisfactory
•
Plates/ broths incubated appropriately
•
Satisfactory growth
•
No growth if inoculated with sterile
water.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growth
•
Contamination.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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50
Unit 2: Working with Micro-organisms (Int 2)
Title: Separating a mixed culture
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
a.
b.
c.
d.
Preparation for work is in
accordance with given
specifications.
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Loop/scalpel flamed correctly
•
Plates labeled on underside
•
Aseptic technique satisfactory
•
Plates incubated appropriately
•
Single colonies of both types present
•
Contaminated materials disposed of
appropriately
•
Surface swabbed with disinfectant.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of technique including
incubation times and temperatures.
Comments on:
•
Whether growth is present
•
Description of growths
•
Whether there are isolated single colonies of
each type of organism.
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Unit 2: Working with Micro-organisms (Int 2)
Title: Staining and Microscopy
Name of candidate
_____________________________________________________
PERFORMANCE CRITERIA
e.
f.
g.
h.
Preparation for work is in
accordance with given
specifications
Techniques are carried out
in accordance with safe
practice and given
specifications.
Record of work is clear
and accurate.
Results and relevant
observations are reported
clearly.
GUIDANCE
•
Hands washed
•
Protective clothing worn
•
Workspace prepared appropriately.
•
Aseptic technique is used in preparation of
slide
•
Slide stained according to instructions
•
Microscope cleaned before use
•
Microscope is set up correctly
•
Slides are disposed of according to instructions
•
Microscope is cleaned on completion of work.
ACHIEVED
Entry in lab diary/notebook:
•
Date work carried out
•
Heading
•
Brief description of procedure including name
of micro-organism/sample and the
magnification used.
•
Description/labelled diagram of microorganisms observed.
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
53
RESOURCES
Videos
‘Aseptic Techniques’: illustrates principles and techniques necessary for aseptic
microbiology work. There are demonstrations in a university laboratory setting and
three industrial settings, the Courage Brewery, Roche Products Ltd and the Public
Health Laboratories.
‘An Introduction to Microscopy’: covers the basic optical properties of the
microscope and demonstrates the correct procedure to set up bright field illumination.
It shows the technique of oil immersion and describes the calibration of the eyepiece
graticule.
Available from Shotlist, The EBS Trust, 36-38 Mortimer Street, London W1N 7RB.
Tel: 0171 765 4635; Fax: 0171 580 6246; e-mail: mail@ebstrust.u-net.com
Web sites
National Centre for Biotechnology Education: http://www.ncbe.reading.ac.uk
‘The microbial world’, a resource compiled by Jim Deacon at Edinburgh University
which covers bacteria, fungi and viruses:
http://helios.bto.ed.ac.uk/bto/microbes/microbes.htm.
Useful addresses
SSERC (Scottish Schools Equipment Research Centre)
St Mary’s Land, 23 Holyrood Road, Edinburgh EH8 8AE
Tel: 0131 558 8180; Fax 0131 558 8191; e-mail: sserc@mhie.ac.uk.
Science and Plants for Schools (SAPS) Biotechnology Scotland, Room 7.05,
Darwin Building, King’s Buildings, University of Edinburgh, EH9 3JR
Tel: 0131 650 7124; Fax: 0131 650 8650
CCAP (Culture Collection of Algae and Protozoa)
Institute of Freshwater Ecology, Windermere Laboratory, Far Sawrey, Ambleside,
Cumbria LA22 0LP
Tel: 01539 442468; Fax: 01539 446914; e-mail: cccap@ife.com.uk
Club Bio, Portland Press Ltd, Commerce Way, Colchester CO2 8HP
Tel: 01206 796351
BBSRC Science Club, Polaris House, North Star Avenue, Swindon SN2 1UH
Tel: 01793 413200 or 413302
Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide
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INTERMEDIATE 2 BIOTECHNOLOGY
UNIT 2
WORKING WITH MICRO-ORGANISMS
STUDENT SUMMARY NOTES
UNIT 2: WORKING WITH MICRO-ORGANISMS (INT 2)
STUDENT SUMMARY NOTES
Contents
Microbiological procedures
1: Preparation of work space
2: Pouring plates
3: Subculturing micro-organisms
4: Separating micro-organisms
5: Microscopic examination of micro-organisms
Magnification
Specimen size
6: Staining
MICROBIOLOGICAL PROCEDURES
All organisms must always be treated as if they could cause disease so recommended
safety procedures must be followed.
The aims of these safety procedures are:
• to avoid bringing contaminating organisms to the laboratory bench
• to avoid contaminating laboratory cultures
• to avoid contaminating yourself, your colleagues and your surroundings
• to avoid taking contaminating organisms out of the laboratory.
Good Working Practice
To achieve the above aims, the following good working practice must be observed.
General safety procedures in the laboratory
•
Do not eat, drink or smoke.
•
Do not lick fingers or labels.
•
Avoid touching the face.
•
Tie back long hair.
•
Cover any cuts and grazes with a waterproof plaster.
•
Wear a lab coat and keep it fastened.
•
Speak quietly and avoid unnecessary movement around the laboratory.
•
Report all accidents, no matter how trivial, to the teacher/lecturer.
•
Do not pipette by mouth.
•
Keep doors and windows closed.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
1
On entering the microbiology laboratory
9. Hang up outdoor coats.
10. Leave bags at the side of the room taking only essentials to the workbench.
11. Wash hands thoroughly.
12. Dry hands using disposable paper towels.
13. Cover any wounds with a waterproof plaster.
14. Put on a lab coat.
15. Collect disinfectant and paper towel.
16. Swab bench with disinfectant.
17. Do not touch your bag, outdoor wear etc. without first washing your hands.
18. Do not leave the laboratory wearing a lab coat.
Bench Management
7. Swab bench with disinfectant at start and end of work using a paper towel.
8. Sit on the lab stool with legs under the bench.
9. Organise the equipment you are using so that it is within easy reach.
10. Keep bench as uncluttered as possible.
11. Do not lay contaminated materials on bench.
12. Report all spillages and breakage to the teacher or lecturer in charge.
Before leaving the microbiology laboratory
1. Swab bench with disinfectant.
2. Place stool under bench.
3. Remove and put away lab coat.
4. Wash hands thoroughly.
5. Collect outdoor coat and leave laboratory.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
2
1: PREPARATION OF WORK SPACE
Preparing yourself
Before starting work you must wash your hands thoroughly with soap and water. You
should then put on a lab coat to protect your own clothes.
Preparing the work space
The surface on which you work must be smooth and non absorbent to ensure it cannot
harbour micro-organisms. If the bench surface is unsuitable, it should be covered
with Benchkote or other non absorbent material.
Before starting work you must swab your work space with disinfectant to reduce the
risk of contamination of your cultures.
You should always have a disposal container of disinfectant within easy reach.
A Bunsen burner is essential to the technique of aseptic transfer. The Bunsen flame
provides an updraught which carries air upwards away from the operator reducing the
risk of contamination.
Air movement should be kept to a minimum in microbiology laboratories to prevent
contamination. While practical work is being performed, doors and windows should
be kept shut.
In professional microbiology laboratories, specialised cabinets are used for the
transfer of micro-organisms for safety reasons. Transfer chambers are such cabinets.
The operator works with their hands and forearms in the chamber, transferring
microorganisms, preparing sterile media etc. Lamina flow chambers or cabinets are
more sophisticated types of transfer chamber.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
3
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
4
2: POURING PLATES
Agar
Liquid media can be made solid by the addition of agar.
The melting and solidifying points of agar are not the same. At the concentrations
normally used, most agars melt at about 95°C when heated but solidify only when
cooled to about 42°C.
Pouring Agar Plates
Agar plates are prepared by pouring liquid agar at 55°C into sterile Petri dishes and
allowing it to solidify.
Before pouring, unopened plates are labelled on the underside using an indelible pen
or wax pencil with initials, date and type of agar. This prevents confusion should lids
inadvertently be swapped. The plates are then placed the right way up ready to
receive molten agar.
Sterile nutrient agar in universal bottles or other containers is first melted by heating
to 100°C then cooled in a water bath to 55°C. Using aseptic technique, a bottle neck
is flamed and the agar poured gently into a Petri dish on a flat surface, raising the lid
of the dish only far enough for the mouth of the bottle to enter. The lid is then
replaced and the plate left undisturbed until the agar has cooled and set.
It is essential that the surface of the medium should be dry in order to maintain single
colonies. Condensation produced from the cooling of the agar can make the agar
surface wet but is normally reduced by pouring the agar at 55°C and if necessary the
plates can be dried open and upside down in an undisturbed area.
Plates are always incubated and stored in the inverted position to prevent
condensation dropping on to the agar surface.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
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Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
6
3: SUBCULTURING MICRO-ORGANISMS
Subculturing is the aseptic transfer of micro-organisms from a culture to fresh
medium.
The use of aseptic technique minimises the risk of contamination of cultures and also
reduces the risk of micro-organisms from the laboratory cultures escaping to the
environment.
Flame sterilisation of instruments
Metal instruments used to transfer micro-organisms are sterilised using red heat in a
Bunsen burner before and after use. They must be heated till red hot to make sure that
any contaminating bacterial spores are destroyed.
Good flaming technique is very important to avoid contamination of the surrounding
air with aerosols.
A Bunsen burner is lit and the air hole opened fully to provide a blue flame. The
operator holds the loop between thumb and fingers as if holding a pencil very loosely,
at an angle that is almost vertical.
Fig. 1: Holding a wire loop
For copyright reasons, the image is unavailable.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
7
The loop is placed in the light blue cone of the flame. Positioning the loop in this cool
area of the flame allows any liquid to dry out and prevents formation of aerosols.
Aerosols are fine liquid or solid particles that are dispersed into the air and might
contain micro-organisms.
Fig. 2: Placing a loop in blue cone of flame
For copyright reasons, the image is unavailable.
After any liquid material has evaporated, the loop is drawn slowly up into the hottest
region of the flame (immediately above the light blue cone and held there until it is
red hot.
Fig. 3: Drawing loop into hottest region of flame
For copyright reasons, the image is unavailable.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
8
The loop is then withdrawn from the flame and allowed to cool before touching
microorganisms.
Fig. 4: Cooling the loop
For copyright reasons, the image is unavailable.
Straight wires may be sterilised in the same way.
Aseptic transfer operations
Once the loop has been sterilised and cooled, it is used to remove organisms (the
inoculum) from a culture and inoculate sterile growth medium. When working with
micro-organisms, aseptic techniques are used to avoid escape of organisms to the
surroundings and contamination of the culture by micro-organisms from the
environment.
To minimise the chances of contamination, cultures and media are exposed to the air
for the minimum time it takes to perform a manipulation or to make observations. All
subculturing procedures are carried out close to a Bunsen flame.
After flaming, the loop is not put down until the procedure has been completed.
Lids of Petri dishes are never completely removed. They are opened just enough to
allow entry of the loop to perform the manipulation and minimise exposure to the air.
Liquid cultures are disturbed as little as possible to reduce the risk of aerosol
formation. Lids from cultures are never placed on the bench surface where
contamination might occur. They are removed from the bottle or tube using the little
finger, held there while manipulation of the culture takes place and then replaced.
The rest of the hand is free to carry out the manipulation. Lids are tightened before
incubation to prevent spillage.
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To remove any contaminating organisms on the neck of a bottle, it is passed through a
hot Bunsen flame on removal and before replacement of the lid.
The loop must be flamed to red heat when the subculturing procedure is finished.
The table below summarises potential points of contamination and the techniques
employed to minimise the risk.
Contamination risk
Precaution
Inoculating loop
Flame and cool.
Do not lay down loop until procedure is
complete.
Work close to Bunsen flame.
Opening of Petri dish (solid medium)
Open lid for as short a time as possible.
Open lid just enough to insert wire loop.
Opening of liquid cultures (bottles, tubes) Hold the lid in crook of little finger –
never place on work bench.
Pass neck passed through a hot Bunsen
flame before insertion and after
withdrawal of loop to kill any
contaminating organisms.
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4: SEPARATING MICRO-ORGANISMS
When professional microbiologists isolate micro-organisms from the environment or
an infected person, it is extremely rare to obtain a pure culture. It is therefore
necessary to separate micro-organisms. Plating or streaking can be used to achieve
this. Using isolated single colonies as inocula for further streak plates, pure cultures
can be obtained.
The term ‘streaking out’ or ‘plating out’ is applied to the inoculation of solid media in
Petri dishes using a technique involving successive strokes with a wire loop. The aim
of the procedure is to obtain isolated single colonies. The loop is charged with a small
amount of culture and several strokes in series are then made on the surface of the
medium with the aim of diluting the bacteria so that single cells are distributed on the
surface. The loop is flamed between strokes to reduce the number of organisms and
increase the chance of isolating single cells. On incubation, each single cell divides
successively to produce a single colony (Fig. 5). In this way, pure cultures can be
obtained.
Fig. 5: Streak plate showing single colonies
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Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
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5: MICROSCOPIC EXAMINATION OF MICRO-ORGANISMS
Micro-organisms are so small that they you cannot see them without the aid of a
microscope. Microscopes are used to produce an enlarged image of objects too small
to be seen with the naked eye.
Although microscopes produced by different manufacturers may look quite unlike
each other, they all work on the same principle and consist essentially of similar
working parts.
Principles of the light microscope
• Visible light passes through a substage condenser which focuses the light into a
sharp cone.
• The light passes through the opening in the stage into the slide illuminating the
specimen.
• The light passes through the objective lens and forms a magnified image of the
specimen which is darker than the background.
• The eyepiece lens magnifies this image further and creates the image that the user
sees.
Magnification
The magnifying power is the degree of enlargement; that is, the number of times the
image appears greater than the original specimen.
To calculate this, multiply together the separate magnifying powers of the objective
and eyepiece lenses. For example, the total magnification of a microscope fitted with
a x10 eyepiece lens and a x10 objective lens is 10 x 10 = x100. Similarly, if a x40
objective lens was being used on the same microscope, the total magnification would
be 10 x 40 = x400.
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Specimen size
Specimens examined under the microscope are generally too small to be seen with the
naked eye. They are measured in micrometres. Viruses may be measured in
nanometres (nm). In general, viruses are too small to be seen with the light
microscope.
1µm = 10-3 millimetres (one thousandth of a millimetre)
= 10-6metres (one millionth of a metre)
1nm = 10-6 millimetres (one millionth of a millimeter)
= 10-9 metres (one thousand millionth of a metre).
The following table indicates the sizes of different types of cells.
Table: Sizes of micro-organisms
Size
Units
1mm
10–3 m
100µm
10–4 m
10µm
10–5 m
1µm
10–6 m
Organism
Algae
Protozoa
Fungi
Bacteria
100nm
10-7m
Viruses
10nm
10-8m
When observing an object under the microscope, it appears larger the greater the
magnification.
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6: STAINING
In general, the cytoplasm of protozoa, fungi and bacteria is almost colourless so it is
difficult to observe such cells using an ordinary microscope. It is important to be able
to see bacteria because the size, shape and grouping of the cells help to identify them.
To observe bacteria and yeasts better, the microbiologist first fixes them to a glass
slide then stains them with a dark stain to provide a contrast to the bright background.
Protozoa and fungal hyphae can be examined by staining the living organism.
Fixation
Bacteria must be fixed to a slide to prevent them being washed off when stain is
applied. A smear of bacteria is made on a clean slide and dried thoroughly in air. It is
then heated gently in a flame. This kills vegetative cells, sticks them to the slide,
makes them more permeable to stain and prevents them from breaking down. The
microbiologist is therefore examining dead cells.
Staining
A solution of dye is applied to the fixed smear.
Simple stains confer the same colour on all the cells which then appear a different
colour to the background. Methylene blue stains cells blue. Carbol fuchsin and
safranin stain cells red.
Negative stains colour the background. The micro-organisms remain unstained and
stand out brightly against a dark background. Common negative stains are India ink
and nigrosin.
Vital stains are used to show up live cells. Paramecium and yeast, for example, will
take up eosin by phagocytosis. When observed under the microscope for a period of
time, the background at first appears red and the cells colourless. Gradually, the
living cells take up the dye with the result that live cells become red and the
background becomes paler. The contrast of the cells is thus improved.
Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
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Biotechnology: Working with Micro-organisms (Int 2) Student summary notes
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INTERMEDIATE 2 BIOTECHNOLOGY
UNIT 2
WORKING WITH MICRO-ORGANISMS
TECHNICAL GUIDE
UNIT 2: WORKING WITH MICRO-ORGANISMS (INT 2)
TECHNICAL GUIDE FOR LABORATORY MANUAL
Contents
Background to unit
1: Preparation of work space
2: Pouring plates
3: Subculturing micro-organisms
3.1: Loop transfer of micro-organismssolid to solid
3.1.3 liquid to solid
3.1.3 solid to liquid
3.1.4 liquid to liquid
3.2: Streak plate inoculations
3.2.1. solid to solid
3.2.2 liquid to solid
3.3: Plate to plate subculture of fungal mycelium
3.4: Use of sterile swabs to sample the environment
4: Separating micro-organisms
5: Microscopic examination of micro-organisms
5.2: Setting up a microscope
5.3: Microscopic examination of pond water/ hay infusion/yeast
5.3.1 Cavity slide preparation
5.3.2 Bright field microscopy
5.3.3 Dark field microscopy
5.3.4 Phase contrast microscopy
5.4: Calculation of specimen size using a microscope
6: Staining
6.1: Preparation of a smear of bacteria or yeast from a solid culture
6.2: Observation of bacteria using the simple stain methylene blue
6.3: Observation of filamentous fungi using lactophenol blue
6.4: Negative staining of yoghurt with nigrosin
6.5: Staining of root nodules to observe Rhizobium
6.6: Vital staining of yeast with neutral red
BACKGROUND TO UNIT
This unit of Intermediate 2 Biotechnology, ‘Working with Micro-organisms’ is a
practical unit in which the student will learn some techniques of handling and
studying micro-organisms in the laboratory. These include plate pouring,
subculturing micro-organisms, separating a mixed culture, staining and microscopy.
Since working with micro-organisms is covered by the COSHH regulations, it is
necessary to have suitable and sufficient control measures in place which have been
developed as a result of assessing the risks involved. An appropriate code of practice
on Safety in Microbiology can be adopted to meet these requirements.
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1: PREPARATION OF WORK SPACE
Materials required
Materials required by each student:
Benchkote and non-absorbent tape if required.
Disinfectant and paper towel
Discard jar with freshly diluted clear phenolic disinfectant
Bunsen burner
Wire loop
Cultures and media
Benchkote need only be used if laboratory work surfaces are absorbent and therefore
unsuitable for microbiological practical work. Depending on the numbers of students
involved, the benchkote can either be cut to size of an individual work space or be cut
to cover several work spaces. Alternatively, individual boards of kitchen work
surface type material could be used.
Advice on disinfectants
For swabbing benches:
• freshly diluted ampholytic surfactants (e.g. Gerrard ‘ASAB’/Harris ‘BAS’ or
similar) or 1% hypochlorite solutions (a good quality commercial bleach e.g.
Domestos or laboratory sodium hypochlorite) available in bottles, sprays and/or
beakers. Use with paper towels for swabbing.
For discard jars:
• Clear phenolic disinfectant such as Stericol or Hycolin. Stericol is available from
AJ Beveridge, 5 Bonnington Road Lane, Edinburgh EH6 5BP, tel. 0131 553 5555.
For dealing with spillages:
• Clear phenolic disinfectant such as Stericol or Hycolin should be available for
dealing with spillages. A spills kit should be on hand whenever microbiological
work is carried out. Advice on dealing with spillages can be found in a suitable
Code of Practice.
Advice on lab coats/eye protection
Lab coats should be worn for all microbiology practicals.
Plastic aprons do not afford adequate protection as they do not absorb the spilled
material but simply redirect it.
Eye protection should be worn in accordance with local guidance from employers.
Gloves of appropriate type for working with stains.
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2: POURING PLATES
Materials required
Materials required by each student:
Lab coat
Eye protection
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
3 bottles of sterile nutrient agar
3 sterile plastic Petri dishes
Bunsen burner and mat
Materials to be shared:
Benchkote if necessary
Water baths with water at 55°C (1 or 2, depending on class size).
Fine tipped indelible marker pens.
Autoclavable bags (or bucket of clear phenolic) for disposal of contaminated plates.
Preparation of materials
Agar – from Oxoid or other standard scientific supplier.
Instructions for the preparation of 100cm3 agar are given below. These can be scaled
up to the appropriate volumes.
Preparing agar
Materials
Nutrient agar powder
Spatula
Weighing boat
Weighing scales
Distilled water
Measuring cylinder
Conical flask
Heat resistant glove
Cotton wool
Aluminium foil
Bunsen burner, tripod and mat
5 universal bottles and lids
Autoclave tape
Water bath
Marker pen
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Instructions
1. Wear eye protection and use heat resistant gloves.
2. Measure out the required weight of nutrient agar powder to make up 100cm3
‘
solution’ (see container for correct weight).
3. Add 100cm3 distilled water to a conical flask.
4. Add agar powder to the cold distilled water and swirl around.
5. Make cotton wool stopper.
6. Place stopper in neck of conical flask and cover with aluminium foil (this prevents
the cotton wool catching fire when over a Bunsen flame).
7. Place the conical flask containing the agar in a water bath.
8. Heat over a Bunsen flame, gently swirling occasionally (Use a heat resistant
glove) until it dissolves fully. (N.B. The solution must be heated until the
liquid is transparent; otherwise the agar will not set evenly).
9. Pour the molten agar into universal or McCartney bottles leaving a space of 1–
2cm at the top.
10. Put caps on loosely.
11. Place in basket for sterilisation.
12. Sterilise in a pressure cooker or autoclave under pressure at 15psi (pounds per
square inch).
13. Tighten lids after autoclaving when cool.
The agar can be stored in this form for several months.
When required, lids should be loosened slightly and bottles of agar should be heated
to 100°C until molten then placed in a water bath at 55°C. It is important that the
water level in the water bath lies above that of the agar to prevent solidification.
To pour the agar, each student should collect a bottle from the water bath.
Note: To prepare large quantities of agar plates for classes, agar may be prepared as
follows.
Add the appropriate quantity of agar powder to up to 500cm3 water in a bottle and
autoclave. Pour plates directly from the bottle when the agar has cooled to the
appropriate temperature. Volumes greater than 500cm3 should not be made as the
efficacy of sterilisation procedures cannot be guaranteed for such volumes.
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3: SUBCULTURING MICRO-ORGANISMS
A number of different subculturing techniques are described in the laboratory manual.
Materials required for each practical are listed below. Since many of them are
common to several practicals, the ‘preparation of materials’ section covers all the
subculturing techniques.
Preparation of materials
Nutrient agar plates
Nutrient agar powder is available from Oxoid or other standard scientific suppliers.
Plates can be poured from flasks of sterilised agar cooled to 55°C using aseptic
procedures (see Activity 2, ‘Pouring plates’).
Malt agar plates
Malt agar is available in powder form from Oxoid and other standard biological
suppliers.
Nutrient broths
Nutrient or peptone broth powder is available from Oxoid and other standard
scientific suppliers. Instructions as to quantities to be added to deionised or distilled
water will be found on the containers. The broth should be made up and dispensed in
10 cm3 volumes to universal bottles, then sterilised by autoclaving.
Bijoux bottles of sterile water
Place 2 – 3 cm3 of distilled water in Bijoux bottles and sterilise by autoclaving.
Tighten lids.
Sterile swabs
Wrap 2 or 3 swabs facing the same direction loosely in foil and seal with autoclave
tape. Sterilise by autoclaving.
Plate cultures of bacteria and yeast to be used as inocula
Plate cultures to be used as inocula should have well isolated single colonies. This
can be achieved by following the instructions in the student lab manual for Activity
3.2 ‘Subculturing – streak plate inoculation’. Plate cultures can be stored for up to
four weeks in a refrigerator or two weeks at room temperature providing they do not
show any signs of contamination.
Plate cultures of Penicillium and Mucor to be used as inocula
Plate cultures of the fungi to be used should be prepared by inoculating the centre of
a sterile malt agar plate with a fragment of fungus from the stock culture. This can be
achieved using a sterile wire loop or needle or by using a sterile scalpel as described
in the student lab manual for Activity 3.3. ‘Plate to plate culture of fungal mycelium’.
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Fungal mycelium will grow out from the centre of the plate followed by the
production of spores. Plate cultures of fungi can be stored for up to four weeks in a
refrigerator or one week at room temperature providing they do not show any signs of
contamination. Young cultures should be provided for the students so that they can
identify the mycelium rather than spores to use for their inoculations.
Only pure cultures should be provided to the students as inocula. Contaminated
plates should be discarded.
Broth cultures to be used as inocula
Pure broth cultures must be provided as inocula. These can be prepared by following
the instructions in the student lab manual for Activity 3.1.3, ‘Loop transfer – solid to
liquid’. It is very important that a well isolated single colony is used as the inoculum
to minimise the risk of contamination. Broth cultures should be stored for no more
than a week and be used only once since it is not possible to identify from their
appearance whether or not they are contaminated.
Disposal of cultures and contaminated materials
Cultures should be sterilised by autoclaving and disposed of as soon as possible after
use in accordance with a suitable Code of Practice.
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Activity 3.1.1: Loop transfer, solid to solid
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 plate culture Micrococcus luteus with isolated single colonies
1 plate culture Saccharomyces cerevisiae (yeast) with isolated single colonies
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Activity 3.1.2: Loop transfer, liquid to solid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 broth culture Micrococcus luteus
1 broth culture Saccharomyces cerevisiae (yeast)
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
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Activity 3.1.3: Loop transfer, solid to liquid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 plate culture Micrococcus luteus with isolated single colonies
1 plate culture Saccharomyces cerevisiae (yeast) with isolated single colonies
Wire loop
4 universals of sterile nutrient broth
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Activity 3.1.4: Loop transfer, liquid to liquid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 broth culture Micrococcus luteus
1 broth culture Saccharomyces cerevisiae (yeast)
Wire loop
4 universals of sterile nutrient broth
2 bottles sterile water
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
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Activity 3.2.1: Streak plate inoculation, solid to solid
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 plate culture Micrococcus luteus with isolated single colonies
1 plate culture Saccharomyces cerevisiae (yeast) with isolated single colonies
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Activity 3.2.2: Streak plate inoculation, liquid to solid
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 broth culture Micrococcus luteus
1 broth culture Saccharomyces cerevisiae (yeast)
Wire loop
4 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
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Activity 3.3: Plate to plate subculture of fungal mycelium
Materials required
Materials required by each student:
Lab coat
Eye protection
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 plate culture Penicillium roquefortii or Mucor heimalis
Small volume of ethanol in a covered beaker
Scalpel
2 plates of sterile malt agar
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Activity 3.4: Use of sterile swabs to sample the environment
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Sterile swabs
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 plates of sterile malt agar
I plate sterile nutrient agar
Bijoux bottle of sterile water
Materials to be shared:
Benchkote if necessary
Incubator at 30°C
Autoclavable bags for disposal of contaminated plates
Fine tipped marker pens
Sellotape
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Activity 4: Separating micro-organisms
Materials required
Materials required by each student:
Lab coat
Bunsen burner and mat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
1 mixed broth culture of Phaffia (a red yeast) and Saccharomyces (a creamy white
yeast)
Wire loop
2 plates of sterile nutrient agar
Materials to be shared:
Benchkote if necessary
Autoclavable bags for disposal of contaminated plates.
Fine tipped marker pens
Preparation of materials
Sterile nutrient agar plates
See ‘Technical Guide’ for ‘Subculturing micro-organisms’.
Mixed broth culture of Phaffia and Saccharomyces
It is best to grow pure broth cultures of each organism and mix them aseptically
immediately prior to student use rather than inoculate one broth with both organisms.
Note that Phaffia grows at room temperature, not 30°C. Saccharomyces will grow at
room temperature also.
It is sometimes difficult to achieve separation from a broth culture using these
organisms. As a back-up, mixed plate cultures with isolated colonies can be prepared
for students to further subculture. These plates are best prepared by streaking out a
pure culture of Phaffia and then streaking a pure culture of Saccharomyces along the
same inoculation lines. After incubation at room temperature, these should show
isolated colonies of each type which can be used to streak new plates.
If Phaffia is not available, use Micrococcus luteus and E coli as organisms in the
mixed culture.
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5. MICROSCOPIC EXAMINATION OF MICRO-ORGANISMS
Activity 5.2: Setting up a microscope
Materials required
Materials required by each student
Microscope
Bench lamp
Prepared slides of micro-organisms such as Amoeba and Chlorella
Lens tissue
Preparation of materials
Basic care of the microscope is described in the teacher/lecturer guide.
Activity 5.3: Microscopic examination of yeast, pond water and hay infusion
(hanging drop)
Materials
Materials required by each student
Lab coat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Lens tissue
Cavity slide and coverslip
Vaseline
Cocktail sticks
Pasteur pipette (plugged) and bulb
Pond water and hay infusion
Materials to be shared
Benchkote if necessary
Preparation of materials
A fresh solution of yeast should be made up in nutrient broth.
Pond water can be collected from a non-contaminated pond or stream. Some plant
material should be collected if possible as this is a rich source of micro-organisms.
A hay infusion is prepared by placing three or four pieces of clean dry hay or lawn
cuttings in a Petri dish and covering them with pond or stream water from a noncontaminated source. The lid is then replaced and the dish is kept at room temperature
for one to two weeks away from direct sunlight.
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Activities 5.3.1, 5.3.2 and 5.3.3: Bright field, dark field and phase contrast
microcopy
If available, microscopes should be set up for dark field and phase contrast
microscopy according to manufacturers’ instructions.
Materials
Materials required by each student
Lab coat
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Microscope
Cavity slide (hanging drop) preparation
Materials to be shared
Benchkote if necessary
Activity 5.4: Calculation of specimen size using a microscope
Materials required
Materials required by each student
Microscope set up with eyepiece micrometer
Stage micrometer slide
Prepared slides of micro-organisms to be measured e.g. Paramecium, Euglena, yeast.
Lens tissue
Preparation of materials
To insert the eyepiece micrometer, remove the eyepiece from the body tube, unscrew
the top lens, drop in the micrometer and screw back the top lens. Replace the
eyepiece in the body tube.
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6: STAINING
Further information regarding the safety of stains can be obtained from SSERC’s
HAZARDOUS CHEMICALS MANUAL and CD.
Activity 6.1: Preparation of a smear of bacteria or yeast from a solid culture.
Materials
Materials required by each student
Lab coat
Eye protection
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Lens tissue
3 glass slides
Labels
Plate cultures of yeast and named bacteria with single colonies
Loop
Forceps
Bijoux bottle of sterile water
Bunsen burner and mat
Materials to be shared
Benchkote if necessary
Activity 6.2: Observation of bacteria using the simple stain methylene blue
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Fixed smears of Spirillum, Bacillus subtilis and Micrococcus luteus and yeast
Methylene blue stain
Blotting paper (fibre free)
Distilled water bottle
Staining rack and dish
Forceps
Microscope
Immersion oil
Materials to be shared
Benchkote if necessary
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Preparation of materials
The fixed smears can be prepared according to instructions in Activity 6.1 in the
student guide. Staining racks can be made from two glass rods joined at each end
with rubber tubing (Fig.1 in teacher/lecturer guide).
Methylene blue stain (for dead organisms)
5g methylene blue
3g trisodium citrate
3.4g sodium chloride
500 cm3 distilled water
Mix well then filter.
Activity 6.3: Observation of the filamentous fungi, Mucor and Penicillium using
Lactophenol Blue.
Safety note
Lactophenol cotton blue contains phenol and is poisonous by absorption. Wear well
fitting nitrile gloves when preparing the stain. Disposable gloves need not be nitrile
when preparing slides.
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Plate cultures of Mucor and Penicillium
2 slides and coverslips
Lens tissue
Lactophenol blue
Forceps & mounted needle
Microscope
Bunsen Burner
Mat
Materials to be shared
Benchkote if necessary
Preparation of materials
The plate cultures should be 5- 7 days old to allow the observation of mycelium.
Lactophenol cotton blue
20g phenol
20 cm3 lactic acid
40 cm3 glycerol
0.05g cotton or methyl blue
20 cm3 distilled water
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Activity 6.4: Negative staining of yoghurt with nigrosin
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Live yoghurt
Small beaker
Nigrosin stain
Lens tissues
3 glass slides
Wire loop
Sterile water
Materials to be shared
Benchkote if necessary
Preparation of materials
Nigrosin should be made up 5g in 100 cm3 of distilled water. It must be stirred for
several hours. Correct preparation of nigrosin is critical to successful use.
Activity 6.5: Staining and observation of Rhizobium in root nodules
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
3 glass slides
Lens tissues
Wire loop
Forceps
Sterile distilled water
Root nodules on legume roots
Crystal violet stain
Staining rack and dish
Materials to be shared
Benchkote if necessary
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Preparation of materials
Root nodules can be obtained from pea seedlings grown in nitrogen rich soil.
Crystal violet
Dissolve 0.5g in 100 cm3 distilled water
6.6: Vital staining
Materials
Materials required by each student
Lab coat
Disposable gloves
Disinfectant and paper towels
Discard jar with freshly diluted clear phenolic disinfectant
Bunsen burner and mat
Lens tissues
Glass slide and coverslip
Wire loop
Sterile water
Plate culture of yeast
Neutral red
Blotting paper
Microscope
Materials to be shared
Benchkote if necessary
Preparation of materials
Neutral red
Dissolve 0.1g neutral red in 1 litre isotonic saline
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