CHEE 229
Fall 2012
INSTRUCTORS:
Dr. Lauren Flynn and Dr. Juliana Ramsay
LABORATORY COORDINATOR:
Dorothy Agnew
agnewd@queensu.ca
TAs
Julian Dafoe, Dupuis B50, julian.dafoe@chee.queensu.ca
Bryen Turco, HMRC, bryen.turco@chee.queensu.ca
Course Timetable
Monday
Lecture
10:30 11:20
Dupuis 215
Tuesday
Lecture
9:30 - 10:20 Dupuis 215
Thursday
Lecture
8:30 - 9:20
Dupuis 215
Wednesday Lab ( Starts 1st Wednesday)
2:30 - 5:30 New Medical
Building
400B
Wednesday
2:30 - 3:30
Tutorial
(alternate Wed when there is no lab)
Dupuis 217
CHEE 229
Fall Schedule 2012
September
Week 1
Wed. 12th
Lab 2:30-5:30
LAB 1: General Microbiological Techniques
Week 2
Week 3
Wed. 26 th
Lab 2:30-5:30
LAB 2: Effects of Antibiotics on Growth
October
Week 4
Week 5
10th
Wed.
Lab 2:30-4:00
LAB 3: Phage Titration, Transduction
Week 6
Week 7
Wed. 24th
Lab 2:30-4:00
LAB 4: Bacteriological Examination of Water
and Histological Examination of Cells, Tissues,
and Organs
Week 8
November
Week 9
7th
Wed.
Lab 2:30-4:00
LAB 5: Enumeration of the Microbial Population
in Soil and Histological Examination of Cells,
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Tissues, and Organs
Week 10
Week 11
Wed.
21st
Lab 2:30-4:00
LAB 6: Lysozyme
Week 12
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POLICY ON ACADEMIC DISHONESTY
The Department of Microbiology and Immunology treats all cases of
academic dishonesty as serious matters. Students are advised to consult
the Faculty of Arts and Science calendar for descriptions of conduct, such
as plagiarism, which fall into the University's definition of academic
dishonesty. The policy in the Department of Microbiology and Immunology
states that the minimum penalty for students found guilty of academic
dishonesty on term tests, lab reports or other assignments, will be a mark
that is negative. In this course, a person found guilty of plagiarism of a
laboratory report or assignment will be assigned a 0 (zero) for the entire
laboratory component of their mark. In addition, a finding of academic
dishonesty is kept in the student's file in the Faculty Office. A student who
knowingly allows his or her work to be copied is considered equally guilty
and will receive the same penalty. Students should be aware that penalties
for academic dishonesty can include failure in a course or the requirement
to withdraw from the University.
I.
RULES FOR WORKING IN THE LABORATORY
1.
A lab coat must be worn at all times.
2.
Eating, drinking, and smoking are prohibited.
3.
Long hair must be tied back.
4.
Bare feet or open shoes and sandals are not allowed.
5.
It is advised that coats, bags, books, and other
materials not pertinent to the experiments be left
outside.
6.
Wash your hands before leaving the laboratory for
any reason.
The DEMONSTRATORS and TECHNICAL STAFF are
authorized to ask any individual not complying with these
regulations to LEAVE the laboratory.
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II.
-
LABORATORY PRACTICE
READ over the instructions BEFORE the lab period begins.
Plan your work accordingly and follow directions with care.
ASK YOUR DEMONSTRATOR if you don't understand something.
-
BEFORE starting your work, WET your work area with disinfectant
from the spray bottle and WIPE it down with a paper towel. Discard
the paper towel in the regular Garbage can.
-
LABEL all experimental material so that both you and your instructor
can readily identify it. Include your NAME and the DATE.
-
RECORD all observations and answer the questions pertaining to the
exercise.
-
Keep the microscopes CLEAN. NEVER place WET slides on the
stage. If stain gets on the underside of a slide, wipe it off with blotting
paper before attempting to look at the smear. If stain gets onto the
stage, clean it off before it dries.
-
At the END of the lab period:
1.
TURN OFF the microscope LAMP.
2.
REMOVE any SLIDES on the stage.
3.
CLEAN all IMMERSION OIL from the lens (use lenspaper).
4.
PUT the microscope into its CABINET under the bench.
-
DISCARD items into their DESIGNATED receptacles ONLY.
-
Place any experimental material that requires INCUBATION onto the
TRAYS as per instructions.
-
Before LEAVING the lab, WET your work area with disinfectant from
the squeeze bottle and WIPE it down with paper towels.
3
III.
GENERAL PRECAUTIONS
1. Treat EVERY bacterial culture as if it were potentially DANGEROUS.
2. REPORT ALL ACCIDENTS to an instructor immediately, especially
breakage or spillage of a culture.
3. WATCH OUT for lighted bunsen burners. Only ONE person at a time
should be working with a bunsen flame. Keep the burner away from
the shelf that runs down the centre of the bench.
4. TELL the instructor IMMEDIATELY about a fire in or near your
workspace.
5. Avoid putting pens, pencils, and your hands in or around your
MOUTH.
4
DISCARD USED ITEMS INTO THEIR DESIGNATED RECEPTACLES ONLY
CONTAINER LABEL
ITEMS IT IS MEANT FOR
CONTAINER TYPE
LOCATION
PASTEUR PIPETS/SLIDES
Pasteur Pipets, Slides
Blue Plastic Bin
Shelf Above Bench
PIPETS/GLASS BEADS
Glass Serological Pipets,
glass beads
Plastic Bucket
With Dilute Bleach
Bench Top
GLASS
Beakers, Flasks, etc.
Red Plastic Bin
Shelf Above Bench
PLASTIC
Swabs, Pipet Tips, Plastic
Test Tubes, Petri Dishes etc.
Yellow Plastic Bin
Shelf Above Bench
THE MICROSCOPE
The study of microbiology deals with living organisms too small to be seen with
the naked eye. Consequently, the compound microscope may be considered an
important tool in the study of structure, form and arrangement of microorganisms. A
thorough understanding of the use of the microscope is essential and will be of great
value in your laboratory work.
Each pair of students have for their use whenever required a compound
microscope with low power, high power and oil immersion lenses.
The compound microscope has two lens systems: that nearer the specimen is
called the objective and the other eye-piece system is referred to as the ocular. The
objective magnifies the real image and produces a virtual image which is seen by the
eye.
Objective lenses are usually designated by their focal length, i.e. low power
objective 16 mm, the high dry 4 mm and the oil immersion objective 1.8 mm. The
shorter the focal length the shorter is the working distance of the lens.
Total magnification is equal to the product of the ocular magnification and the
objective magnification. The initial magnification of an objective is engraved on the
objective mount and the magnification of the ocular is marked on the top of it. For
example, with a 1.8 mm oil immersion lens (magnification 100x) and the 10x eye-piece
you would get a total magnification of 1000x. Note the objective and eye-piece on your
microscope.
The coarse adjustment moves the body tube rather rapidly over a considerable
vertical distance while the fine adjustment moves the tube slowly over a smaller
distance for final focusing. Prior to using the microscope the fine adjustment should
always be set in the middle of its course of travel so you can utilize the maximum
distance in either direction.
Your microscope has a fixed source of illumination - a built-in lamp rather than a
mirror. The amount of light entering the lens system is controlled by the iris diaphragm
which is just below the substage condenser. When using the oil immersion objective the
iris diaphragm is generally used in an open position. Less light will be needed with lower
power objectives. Note the various parts of the microscope on the diagram and relate
these to your own microscope.
3
THE PROPER METHOD FOR FOCUSSING A MICROSCOPE IS AS FOLLOWS:
Low Power Objective.

Swing the low power objective into position and turn it down within a short
distance from your slide.

Be sure the light is well centred.

Adjust the condensor and iris diaphragm to produce a light intensity that yields
optimal illumination without glare.

Look through the eyepiece and focus upwards with the coarse adjustment until
the image is as clear as possible.

Sharpen the image by using the fine focus adjustment.

Swing the high dry objective into position and repeat the procedure just
described. In this case remember the working distance of the high dry is much
shorter so the lens must be closer to the specimen.
The High Dry Objective.

Swing the high dry objective into position.

Repeat the previous procedure but watch from the side of the microscope stage
when raising the stage.

Bring the objective close to the slide without touching it; the shorter focal length
makes this necessary.

Remember to always focus downward when using the High Dry Objective.

Sharpen the image by using the fine focus adjustment.
The Oil Immersion Objective.

Centre the area of interest as precisely as possible under low power otherwise
the reduced field width of the oil immersion lens will make it difficult to locate.

Put a drop of immersion oil on the slide directly under the immersion oil objective.

Rotate the oil immersion lens until it clicks into position.

Watching the objective from the side, raise the stage until the objective touches
the oil but do not let it touch the slide.

Look through the ocular and focus downward with the fine adjustment until the
image becomes clear.
Familiarize yourself with the parts of the microscope and their function before examining
specimens.
4
Some general rules of procedure as applied to microscopes are as follows:
1.
When moving a microscope always carry it in an upright position by grasping the
handle with one hand and supporting the base with the other.
2.
Avoid jarring the microscope suddenly.
3.
Always clean the oil immersion lens with lens paper at the end of the laboratory
period. Do not get the oil on the other objectives.
4.
When looking through a microscope, always focus downward until the object is
seen.
5.
Never grind the objectives against the slide or cover glass.
6.
When the microscope is not in use, keep the lower power in position and the light
switched off.
7.
Report any problems that you are having with your microscope to the
demonstrator.
5
LAB 1
GENERAL MICROBIOLOGICAL TECHNIQUES
The purpose of this lab is to familiarize the student with some of the basic
techniques used in the study of bacteria. Bacteria are ubiquitous in nature and can be
demonstrated from any environment. In today's exercise the presence of bacteria and
other microbes in a few environments will be demonstrated. Also a mixed bacteria
culture will be examined microscopically and the sample will be streaked onto an agar
plate so colonies arising from the growth of individual bacteria may be observed.
A common method of observing bacteria under the microscope is by staining
them with dyes. Living bacteria are almost colourless and do not present sufficient
contrast with the fluid in which they are suspended to be easily discernible. Staining the
organisms makes them contrast with their surroundings and they are more readily seen.
Stains may be simple, differential or structural. While simple stains stain all bacteria
on the basis of contrasting them with their environment, differential stains separate
bacteria into groups on the basis of differing chemical and physical properties. You will
employ the Gram Stain, perhaps the most widely used and useful of all bacterial stains.
Materials: (per pair)
1.
2.
3.
4.
5.
6.
1 tube of a mixed bacterial culture for streaking.
1 tube each of a Staphylococcus and Escherichia culture
Sterile cotton swabs and a tube of sterile water
4 Tryptic Soy Broth Agar plates
2 MacConkey agar plate
Inoculating loops, microscope slides and staining reagents
Method: DAY 1
GRAM STAIN
1.
Perform a gram stain on all broth cultures. Gently agitate the tube to resuspend
the bacteria.
2.
Sterilize the loop by gently inserting the loop into the heater element of the bacticinerator. Avoid scraping the sides of the heater element to ensure the longevity
of the heating element. The loop should be inserted toward the rear of the
heating element to avoid spattering. The loop should remain within the heater
element a minimum of five seconds. It is not necessary to obtain a glowing loop
to ensure sterility. Never leave the loop in for more than 10 seconds.
Then perform the Gram stain as follows:
a)
Allow the slide to air dry.
b)
Fix the smear by placing on a hotplate for 3 minutes. (Method will be
demonstrated.)
c)
Flood the slide with Gentian violet and allow to act for 1 min.
Rinse with water.
6
d)
Flood the slide with Gram's iodine and allow to act for 1 min. Rinse off with
water.
e)
Decolorize the smear by the addition of alcohol-acetone solution until no
more colour is flowing off the slide (about 4 drops). Rinse off with water.
f)
Counterstain with safranin for 20 seconds. Rinse off with water and blot
dry with bilulous paper. Examine the slide under the microscope using the
oil immersion lens.
Cells that retain the primary stain (Gentian violet) after decolorization will appear
blue and are called Gram positive. Cells that are decolorized by the
alcohol-acetone treatment and accept the counterstain will appear red or pink
and are called Gram negative.
Observe the morphology and Gram reaction of as many organisms as possible.
Record your results. When finished, wipe off the lens with lens paper.
3.
Observe the demonstration slides showing Gram stains of pure cultures (a single
species of bacteria) that represent: Gram negative rods (bacilli), Gram negative
cocci, Gram positive rods (bacilli), and Gram positive cocci.
7
STREAKING FOR COLONY ISOLATION
Bacteria may be grown in liquid or solid medium. Addition of agar, a solidifying
substance, to a broth medium allows the bacteria to grow, to be immobilized instead of
floating free and as they multiply they produce a fixed colony that in most instances is
derived from the multiplication of only one organism. The observation of single colonies
is a necessary and useful tool in the isolation and identification of bacteria from any
environment.
The streak plate method of obtaining single colonies is based on dilution so that it is
possible to obtain individual colonies. There are a number of acceptable methods of
obtaining single colonies. A commonly used technique is diagrammed here.
Method:
DAY 1
1.
Sterilize the inoculating loop and streak one loopful of mixed culture suspension
onto the Tryptic soy broth agar (TSBA) plate.
Note illustration as well as having technique demonstrated to you. Repeat the
procedure for the MacConkey plates. Incubate these plates at 37 oC for 24 hours.
2.
Demonstrate the presence of bacteria in two of the following sites: air, laboratory
bench, human skin, and human respiratory tract (throat), by following the
appropriate directions given below (on Day 2 look at plates obtained by your
partner).
(i)
Air - Remove the lid from a TSBA plate and let it sit uncovered for one
hour.
(ii)
Bench, Skin (or other dry surface) - Moisten a sterile swab in the tube of
sterile water and squeeze it out against the inside wall of the tube. Cover
the tube. Rub the swab over about 1 sq. inch of the surface. Streak the
swab over a small area of a TSBA plate. Continue the streaking with a
sterilized loop.
8
(iii)
3.
Throat (or other moist surface) - Use a dry swab to obtain the bacteria.
Streak onto a plate as in (ii).
During the next lab period observe and record the various colonial morphologies
on both types of plates.
9
DAY 1. DEMONSTRATIONS
1. Slides:
a) Flagella
b) capsules
c) cell wall
d) spores
2. TSBA Plates:
a.
b.
c.
d.
e.
f.
g.
h.
Escherichia coli
Serratia marcescens
Pseudomonas aeruginosa
Chromobacterium violaceum
Staphylococcus aureus
Bacillus cereus var. mycoides
Micrococcus luteus
Klebsiella pneumoniae
10
The Inhibitory Action of Disinfectants and Antiseptics on Bacterial Growth
Antiseptics and disinfectants are chemical substances used to prevent
contamination and infection. Disinfectants are antimicrobial substances that kill or
prevent the growth of microorganisms and are used on inanimate objects. Disinfectants
must have high germicidal activity; rapidly kill a wide range of microorganisms; be
chemically stable and effective in the presence of organic compounds and metals and
be inexpensive and aesthetically acceptable. Concentration and contact time are critical
factors in determining their effectiveness. Antiseptics are antimicrobial agents with
relatively low toxicities to human tissues so they can be applied to the skin. The aim of
this laboratory exercise is to study the effectiveness of some commercially available
agents against two selected organisms. The agents have been diluted to mimic normal
use.
Material; (per group of 4)
6 TSB agar deeps
6 Sterile Petri plates
2 dishes of sterile well formers (12/dish)
Broth culture of Escherichia coli
Broth culture of Staphylococcus epidermidis
Automatic pipettes and tips
Tubes containing:
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
Javex 10% (concentration used in lab.)
Lysol 1.5% (general cleaning concentration)
Mr. Clean 1.5% (general cleaning concentration)
Betadine 10% (veterinary surgical scrub) dated 2009
TeraSeptic 5%
Biolife dish Soap 5%
PalmoliveUltra 5%
Palmolive Antibacterial 5%
Lysol Healthy Touch Antibacterial Soap 5%
Laboratory Hand Soap 5%
Dial Antibacterial Hand Soap 5%
Water
11
Procedure:
1.
Take the 6 petri dishes and draw 4 quadrants on the bottom of the plate. Label
three plates E. coli and number the quadrants 1 to 12. Label the other three
plates S. epidermidis and number the quadrants 1 to 12.
2.
Heat the forceps for 5 seconds and using sterile technique place one sterile well
former in the center of each quadrant.
3.
Take an agar deep from the waterbath and add 0.1ml for E. coli broth culture to
the tube. Carefully pour the agar into a petri dish labeled E. coli. Repeat for the
other two E. coli plates.
4.
Repeat step 4 with the S. epidermidis culture.
5.
Allow the agar to harden (about 20 minutes). Remove the well formers with
flamed forceps. If there is an agar plug left after the well former is removed, then
remove the plug with a Pasteur pipet. Replace the well formers in the glass petri
dish and place in a red disposal bin.
6.
Place 0.1 ml of the appropriate disinfectant into each numbered well. Do not
invert the agar plates.
7.
Leave the plates on the bench. The TAs will place the plates at 37oC for
incubation overnight.
8.
In the next laboratory period, measure (in mm.) the diameter of the zones of
clearing for each of the disinfectants. Compare the results for the different agents
and the different organisms.
12
Student#______________________
Questions (Hand in)
1.
Demonstrator’s name _____________________________________
Email_____________________________________
2.
If the cell wall is removed from a gram positive bacterium, what colour would the
cell appear in a Gram stain?
3.
The composition of the __________ is the basis of the differential reaction of
bacteria to the Gram-staining procedure.
a.
chromosome
b.
cytoplasmic membrane
c.
cell wall
d.
slime layer
4. Define:
Pasteurization:
Disinfectant:
Sterilization:
13
5. True or False
In order to stain flagella so that they may be readily observed by
light microscopy, it is usually necessary to increase their thickness.
6.
Which of the following environmental factors generally do(es) not have an impact
on the efficiency of an antimicrobial agent?
a.
pH
b.
viscosity
c.
concentration of organic matter
d.
refractive index
7.
When antiseptics and disinfectants are compared, antiseptics are generally
a.
less toxic.
b.
more toxic.
c.
equally as toxic.
d.
unpredictable in toxicity.
8.
In a spore stain that employs hot malachite green and safranin, what color are
the spores?
9.
What color are the demonstration cultures of:
Serratia marcescens
Pseudomonas aeruginosa
Chromobacterium violaceum
10. True or False
Agar is used as a gelling agent for microbiological media because it
is not readily digested by most microorganisms.
14
DEMONSTRATION 1
1. DIRECT STAINING OF BACTERIAL CELLS WITH METHYLENE BLUE
Dyes are usually salts which have a coloured ion; for example, methylene blue
chloride dissociates into methylene blue+ and chloride- . The colour in this instance
resides in the methylene blue cation.
When this dye is mixed with bacterial cells near neutral pH the positively charged
cation combines with negatively charged cells resulting in stained organisms.
Dyes with colour in the cation are called basic dyes while dyes with colour in the
anion are called acidic dyes. For the most part basic dyes are employed in bacterial
staining procedures.
2. CELL WALL STAIN
The bacterial cell wall is not seen in most stained preparations of bacteria as it is
very thin and resistant to many stains. The use of a mordant makes it possible to
observe the cell wall. In the procedure we use today, the cell wall which normally is
negatively charged becomes positively charged by treatment with a cationic surface
active agent. The cationic surface active agent, cetyl pyridinium chloride dissociates
in water. A positive charged cetyl pyridinium cation and a negatively charged
chloride is formed. The cations neutralize the negative charge at the cell surface and
it becomes positively charged due to adsorption of cetyl pyridinium cations. The cell
wall is stained red with an acid dye (Congo red). The cytoplasm takes on the colour
of the basic dye, methylene blue.
3. BACTERIAL FLAGELLA
Some bacteria are motile by means of organs of locomotion called flagella which are
very fine, whip-like appendages, composed mainly of protein and originating from a
granular body in the cytoplasm. They are too small to be seen with the ordinary light
microscope. Brownian movement exhibited by all microorganisms is a dancing
movement in one general vicinity; it is caused by collisions of the relatively small
microorganisms with the molecules of the suspending fluid (water) which are in
constant motion and thus the indeterminate bounding movement results. True
motility is a purposeful gliding movement in which the organism may even be
observed to leave the field being examined. Motility of bacteria due to flagella may
be observed in wet-mount preparations of living bacteria.
Special staining techniques have been developed by which it is possible to bring the
flagella within the limits of resolution of the light microscope. This is accomplished by
applying the stain in conjunction with a tannic acid mordant that layers the stain on
the flagella in successive coats and thus the apparent diameter of the flagella is
increased so that they become visible by ordinary microscopy. Flagella are very
fragile and are easily detached from the cells during the staining procedure so the
flagella stain requires a meticulous technique. You will be shown a demonstration
15
slide of a flagella stain, as the technique requires considerable practice and is
therefore not applicable to the student laboratory.
4. NEGATIVE STAINING (for demonstration of capsule)
As the term implies, negative staining is not true staining of the bacterial capsule as
it stains the background and leaves the capsule colourless. One advantage of
negative staining is that a better idea is gained of the true size and shape of the cell.
This method shows up structures, which, because of their chemical nature, are
difficult to stain by most routine staining procedures. Your negative stain will show
grey cells, colourless capsules and a black background.
5. THE SPORE STAIN
Certain bacteria, for example, species of the genera Bacillus and Clostridium,
develop an endospore that is highly resistant to many physical and chemical agents.
It survives for much longer than the vegetative cell and withstands extremes of
temperature and chemical agents which would kill the vegetative cell.
Spores are not reproductive structures, and are not necessarily produced in
response to adverse conditions, although their production may be influenced by
temperature, media, etc. In those species which do produce spores, the spores are
not found in young cultures.
The endospore is very resistant to staining but, once stained by a special technique,
is equally resistant to decolourization and counterstaining.
The method you will use employs hot malachite green as the first stain and
safranin as a counterstain. Thus the spores stain green and the vegetative part of
the cell stains a light red.
6. GRAM STAIN
Bacteria may differ from one another chemically and physically therefore they may
react differently to any given staining procedure. This principle is used in
microbiology in the technique of differential staining which distinguishes between
types of bacteria. The gram stain is perhaps the most widely used differential
staining procedure employed in microbiology.
"There have been a number of theories to explain the Gram reaction and most of
these have been discarded. The current theory is that Gram differentiation is
primarily due to the permeability differences in the cell walls of different bacteria
during treatment with 95% ethanol (or similar solvent). The cell walls of Gramnegative bacteria contain a high percentage of lipid and a low percentage of the
muramic acid complex; they are also thinner than Gram-positive cells. As a result of
the treatment with concentrated alcohol the wall of a Gram-negative bacterium is
dissolved or disorganized allowing the alcohol soluble Gentian violet-iodine complex
to escape from the cell. The difference between Gram-positive and Gram-negative
16
cells is not the presence of some unique compound within the cells of Gram-positive
bacteria, but merely the greater permeability of Gram-negative cell walls when
treated with high concentrations of ethanol. If this theory is correct, then all soluble
molecules would also escape from these cells and any of these could be used as an
indicator of the altered permeability. This has been found to be true." (Levy,
Campbell and Blackburn, Introductory Microbiology, 1973, J. Wiley & Sons, Inc.)
17