Microbiology Laboratories
Report on Training Visit
In the framework of the project
No. SAMRS 2011/01/02
“Development of human resource capacity of Kabul Polytechnic
University”
Funded by
Bratislava 2013
Khalid “Nayab”
2
SLOVAK UNIVERSITY OF TECHNOLOGY IN BRATISLAVA
FACULTY OF CHEMICAL AND FOOD TECHNOLOGY
DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING
REPORT
ON MY ACADEMIC AND SCEINTIFIC ACTIVITIES IN TRAINING
COURSE AT THE SLOVAK UNIVERSITY OF TECHNOLOGY IN
BRATISLAVA
PREPARED BY: AHMAD KHALID “NAYAB”
Assistant Professor of Chemical Technology Faculty of Kabul Polytechnic University
GUIDENCE BY:
Ing. Barbora “Kaliňáková”
2013
3
Acknowledgements
First of all, I would like to express my appreciations for the scientific and
educational training to Slovak University of Technology in Bratislava
(STUBA) for organizing this training and SlovakAid for their financial
sponsor.
I would like to thank Ing. Barbora Kaliňáková, Ph.D for her guidance and
cooperation during all training period. I learned lots of new things from
her which would be useful in my professional life. I would also like to
thank Doc. Ing. Juma Haydary, Ph.D from bottom of my hearth for
coordinating this program.
Best Regards
Khalid Nayab
4
Table of Contents
Title
Page
Introduction
5
Laboratory Exercise 1
6
Laboratory Exercise 2
8
Laboratory Exercise 3
11
Laboratory Exercise 4
14
Laboratory Exercise 5
17
Laboratory Exercise 6
21
Laboratory Exercise 7
24
Laboratory Exercise 8
28
Laboratory Exercise 9
31
Laboratory Exercise 10
34
Laboratory Exercise 11
37
Laboratory Exercise 12
39
Laboratory Exercise 13
42
Laboratory Exercise 14
45
Laboratory Exercise 15
52
5
Introduction
This report contains theoretical and practical information about different
microbiology laboratory exercises.
These exercises are Preparation of culture media, Isolation of culture
from their natural sources, Isolation of pure culture, colony recognition of
bacteria; yeast; and fungi, different staining techniques; such as: wet
mount, negative, gram, and endospore staining; Influence of different
physical conditions on growth of microorganisms; such as: temperature,
pH, UV, and osmotic pressure; and quantification of cells.
Each exercise contains some brief theoretical information regarding
exercise, purpose of work, procedure, table of results (if necessary), and
questions answers about the exercise.
This was a brief information about this report, If you like to find more
about above mentioned topics, please turn page and read more what
are written in the text.
6
Laboratory Exercise 1
Preparation of Culture Media
For cultivating microorganisms, we need culture media. This media can be solid or
liquid. Difference between solid and liquid media is the agar (solidifying agent) which
is added in solid media.
PURPOSE OF WORK
A.
To prepare agar plates (NA, MEA)
B.
To prepare slant agars (MEA)
PROCEDURE
A.
The procedure of preparation of agar plates

weigh the ingredients as described

add the exact amount of distilled water by cylinder and dissolve (cultivation
flasks are filled up maximum 2/3 of the volume)

measure the pH of the nutrient medium (using pH paper) and adjust the pH to
the desired value using a solution of 3 mol / l KOH

add powdered agar (agar 2% w / vol)

close the cultivation flasks with a cotton stoppers, select the content and
cover with aluminium foil

autoclave at pressure of 120 kPa (about 121 ° C) for 20 minutes

after sterilization cool medium to 50 - 60 ° C under cold water

sterilize surface of laboratory table with disinfectant

prepare a sterile plastic Petri dishes and light up the gas burner

pour medium aseptically into Petri dishes (about 15 ml to 1 Petri dish)

allow Petri dishes to cool and solidify

store media turn upside down
B.
The procedure of preparation agar slant

weigh powder nutrient medium as described

add the exact amount of distilled water by cylinder and dissolve powder
(cultivation flasks are filled up 2/3 of the volume)
7

measure the pH of the nutrient medium (using pH paper) and adjust the pH to
the desired value using a solution of 3 mol / l KOH (pH 7.2 to 7.4 MPA, pH 6.4
6.6 for SLA)

add powdered agar (agar 2% w / vol)

close the cultivation flasks with a cotton stoppers

overcook medium in the microwave so that the agar in the medium is
completely dissolved

add 5 ml of overcook media to each tubes

close the tubes with a cotton stoppers, placed in a metal rack and cover with
aluminium foil

autoclave at pressure of 120 kPa (about 121 ° C) for 20 minutes

let solidify medium in tubes in an inclined position after sterilization
LABORATORY REPORT AND QUIZ
Questions:
1.
What is the function of culture medium?
2.
Define synthetic and natural medium
3.
Why are culture media sterilized before use?
4.
Why are Petri plates inverted after they cool?
5.
What is sterilization and what is its significance?
6.
How would you sterilize cultivation media if you do not have autoclave?
7.
How to sterilize thermo labile substances?
8.
Specify the conditions for sterilization of culture media prepared on laboratory
exercises.
Answers:
1- Providing nutrition and growth needy material for the microorganisms.
2- Synthetic medium is prepared from two or more compounds synthetically, but
natural medium are those natural compounds which would be suitable for the
cultivation of microorganisms.
3- To make sure it is not contaminated with any other microorganism and is
suitable for cultivation of selected microorganism.
4- To avoid destroying of colonies on the surface of the media from
accumulating of condensed water.
8
5- Sterilization is the process of destroying all life forms in a certain condition
and its significance is that it ensures the media and tools sterility and
decontamination.
6- If we don’t have autoclave, we can use Tyndallization.
7- For sterilizing thermo labile materials, we can mostly use filtration.
8- In laboratory exercises, sterilization is mostly done at 121 Degrees Celsius for
20 minutes; however it can be done at different conditions related to the
degree of sterility we expect.
Laboratory Exercise 2
Isolation of Microorganisms from their natural sources and their enumeration
by standard plate count method
Microorganisms are very diverse in nature, they can be found almost in every
environment. In this laboratory exercise, we isolate microorganisms from their
natural sources and try to find their concentration in the source.
THE PRINCIPLE
The principle of the method is the isolation (extraction) of microorganisms from the
environment, the dilution of MO in appropriate medium and smears the cell
suspension on surface of Petri dishes. Microorganisms growing on agar (Petri
dishes, Petri plates) media form masses of cells called colonies. A colony is a large
number of cells on solid medium, which is visible to the naked eye as a discrete
entity.
The assumption is that each viable bacterial cell is separate from all others and will
develop into a single discrete colony. However, organism normally forms multiple cell
arrangements, such as chains, the colony-forming unit may consist of a chain of
bacteria rather than a single bacterium. Therefore, generally we determine the
number of Colony-Forming Units (CFUs) in known dilution. The number of (CFU) on
plate can be used to calculate the number of CFU per millilitre or gram of original
sample. As the concentration of microorganisms in the sample is too high, original
sample must be serial diluted with sterile saline until the bacteria are dilute enough to
count accurately. It means that the final plates should have between 30 and 300
colonies. A plate having 30-300 colonies is chosen because this range is considered
statistically significant. If there are less than 30 colonies on the plate, small errors in
9
dilution technique or the presence of a few contaminants will have a drastic effect on
the final count. Likewise, if there are more than 300 colonies on the plate, there will
be poor isolation and colonies will have grown together. In Figure 3 is illustrated the
use of so-called decimal serial dilutions. Its principle is a gradual dilution of the
suspension always at 10 times lower concentration with pure saline. However, you
may use a combination of other arbitrary dilution (see laboratory report and quiz).
Figure 3- Principle of the standard plate count method.
PURPOSE OF WORK
C.
To prepare dilution series of the samples : 10 -1, 10-2, 10-3,10-4
D.
To calculate the number of colony forming unit per ml of original sample on the
base of number of grown colonies
EQUIPMENT FOR A COUPLE OF STUDENTS
-1
Sample of soil, sterile saline solution, sterile tubes labelled with wax pencil as 10 ,
-2 -3
-4
10 10 , 10
, sterile tips, micropipette, vortex, sterile spreader, agar plates
Comment [M1]: L_shape stick or
spreader
labelled with name and dilution as
-1
-2
-3
-4
10 , 10 , 10 , 10 , thermostated incubator
PROCEDURE
E.
Procedure of decimal dilutions of samples

aseptically pipette 4.5 ml of sterile saline solution into 4 sterile tubes

aseptically withdraw 0.5 gr of stock solution of soil into the first tube (you
-1
prepare dilution 10 ) and then tube homogenize with vortex mixer
Comment [M2]: 0,5 g of soil
10

aseptically withdraw 0.5 ml of suspension from 10-1 dilution into the second
tube (you prepare dilution 10-2) and then tube homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10 -2 dilution into third tube
(you prepare dilution 10-3) and then tube homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10 -3 dilution into the fourth
tube (you prepare dilution 10-4) and then tube homogenize with vortex mixer

aseptically dose 0.1 ml of suspension of stock solution as well as all prepared
dilutions on the surface of Petri dishes( from each dilution prepare 3 plate)

aseptically spread a suspension of microorganisms on the surface of Petri
dishes with a sterile stick and incubated in the inverted position for 48 hours at
30°C

at the end of the incubation period, record the number of CFU for each
dilution on a table in the laboratory report section

for the final calculation of CFU per ml of sample select only plate where the
number of colonies is in the range of 30-300
LABORATORY REPORT AND QUIZ
Comment [H3]: Explain, what does “-“
mean
Table of results
Dilution
Nutrition
Agar
Malt
Extract
Agar
Plate 1
Plate 2
Average
CFU/ml
Plate 1
Plate 2
Average
CFU/ml
Non
diluted
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
10-1
10-2
10-3
10-4
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
11
40
26
4
3
4
125
110
118
2
0
1
- TMTC
B)
Procedure of calculation of the number of colony forming unit per ml of
original sample
The final number of bacteria per 1 ml of drinking water or milk in the original sample
can be calculated as follows ( CFU / ml ):
CFU / ml  p * 10 * D
(3)
11
CFU / ml is the total number of colony forming units per 1 ml of original sample, p
is the average number of colonies on Petri dishes in tube with a given dilution, 10 is
recalculation of the number of colony units per 1 ml, D total dilution of the sample.
Questions:
1- Calulate the density of the original culture (CFU/ml), if a culture is diluted 2 x
105-fold, and 0.2 ml is plated and gives rise to 160 colonies ?
2- Original cell suspension contain 5 * 105 cells per 1ml but only 43.2% is live.
Cells suspension is diluted according to the scheme below and plated on Petri
dishes. Calculate how many colonies growth on plate D?
Answers:
1- The density of the original culture is
Colony Forming Unites per
Milliliter (CFU/ml).
2- The number of the cells which grow on plate (D) is 1500 Colonies.
Laboratory Exercise 3
Isolation of Pure Culture
Pure culture is called to that culture which is growth from a single cell. In order to
have pure culture, we must isolate them from a mixture of different microorganisms.
Here are some techniques which are suitable for isolation of pure culture.
Principle
A pure culture theoretically contains a single bacterial species. There are a number
of procedures available for the isolation of pure cultures from mixed populations. A
pure culture may be isolated by the use of special media with specific chemical or
physical agents that allow the enrichment or selection of one organism over another.
Easier methods for isolation of a pure culture include: spread appropriate diluted
culture on solid agar medium with a glass spreader or streak culture with a loop. The
purpose of spreading and streaking is to isolate individual cells (colony-forming units)
12
on a nutrient medium. The streaking patterns shown in the figure below result in
continuous dilution of the mix culture to give well separated surface colonies.
Purpose of work
Isolate pure culture from mix of microorganisms.
Equipment for a couple of students
Petri plates containing nutrient agar and/or malt extract agar, loop, and mix culture
Procedure
Flame loop, cool it and take a small among from the mix culture. Spread culture on
the surface of agar plate according to figure. Striate arrows show spreading lines (or
plate sectors) and curved arrows show turning plate between individual spreading.
There is necessary to flame and cool the loop between individual spreading. End of
previous sector and start of next sector must intersect, and first and last sectors must
be unlinked.
13
Laboratory report and quiz
Table of results
Group of
Cultivation
Microorganisms
Medium
Conditions of
Cultivation
Bacteria
Nutrition
Agar
Temperature:
25°C
Time: 3 days
Light mode:
normal
Yeast
Malt
Extract
Agar
Temperature:
25°C
Time: 3 days
Light mode:
normal
Count of
Colony
Types
26
18
Colony
Description
Diameter: 1mm
Appearance:
circular
Margin: entire
Elevation: flat
Color: white
Diameter: 1.5mm
Appearance:
irregular
Margin: undulate
Elevation: raised
Color: light yellow
Questions:
1. What is pure culture?
2. What is a principle of isolation of pure culture?
3. If you did not obtain isolated colonies, what changes should you make in your
technique to ensure isolated colonies?
Answers:
1- We can call pure for a culture which has only one species of microbes in it,
and it is not contaminated with any other microbes.
2- The name describes the main principle of the “Isolation of Pure Culture”, as
we isolate pure culture of one cell from a mixture of cells with different
species. The technical principle of this method is drawing lines and continuing
drawing another one from the end of the previous after flaming the loop. In
fact by drawing each line we gradually reduce concentration of the cells on
the surface of the media, in order to isolate a cell and grow an isolate colony
from one cell.
3- There are lots of parameters which can cause the isolation process. Maybe
we didn’t do our work in aseptic condition, or maybe we haven’t drawn the line
accurately, or we have forgot loop or flamed loop…..
14
Laboratory Exercise 4
Colony recognition of bacteria, yeast, and filamentous fungi
Colonies of different microorganisms have different properties. They can differ in
size, shape, arrangement, elevation, color, etc… from other, while some of them are
very similar. We can recognize some colonies macroscopically, without using any
microscope.
THE PRINCIPLE
Microbes are everywhere, they are found in the water we drink, the air we breathe,
and the earth we walk on. They live in and on our bodies. Microorganisms growing
on agar (Petri dishes, Petri plates) media form masses of cells called colonies. A
colony is a large number of cells on solid medium, which is visible to the naked eye
as a discrete entity. Colony morphology may be used as an aid to the identification of
microorganisms. Although colony morphology cannot be used as the sole identifying
criterion, it is used to recognize many common types of microorganisms. Six
parameters are normally used to describe microbial colonies. There are: overall
colony appearance, colony margin (edge), colony elevation, colony size, colony
pigmentation, and colony consistency. Most important parameters for practical
identification are:

size (useful characteristic for identification, the diameter of a representative
colony may be measured, prokaryotic cell colonies may be smaller than
eukaryotic cells colony - it is not always rule)

form (circular typical for bacteria, irregular-yeasts, filamentous-filamentous
fungai)

surface (bacterial colonies are frequently shiny and smooth, yeasts form
usually matt, rough, wrinkled or dull colonies, filamentous fungai are dry,
velvet)

consistency, texture (texture of bacterial colony is moist, mucoid, yeasts form
dry, hard, mealy colonies and fungal colonies are rigid)

color (consider the colour of the colony itself or pigment excretion to the
cultivation broth)
Bacteria and yeast colonies are usually white or cream colored. However for some
strains are characterized yellow, red or black pigments. Yellow pigments (flavin)
15
create yeasts Eremothecium, Ashby and some species of the genus Candida,
Cryptococcus. Red (carotenoid) pigment form yeasts of the genus Rhodosporidium,
Rhodotorula, Sporobolomyces and some species of bacteria (Mycobacterium phlei,
some species of the genus Micrococcus, Flavobacterium, Halobacterium).
Carotenoid pigments are formed in the genus Neurospora conidia micromycetes up
under the influence of light. Black (melanin) pigments are characteristic of some
yeast genus Cryptococcus and Aureobasidium pullulans. Light brown to black,
leathery colonies form diploid yeast genus Rhodosporidium (in haploid form is red)
and brown brown to black pigments are typical for Azotobacter chroococcum and
micromycets of the family Dematiaceae. Some microorganisms secrete colored
pigments into the soil, for example Pseudomonas bacteria secrete yellow, green,
blue and fluorescent pigments (fluorescein, pyocyanin). Some species of Fusarium
produce yellow, red, green, dark blue and black pigments into the medium. After
prolonged cultivation, the mycelium color themselves. The presence of red and black
pigments in microbial cells has a protective effect against lethal effects of UV
component of sunlight. Therefore, these microbes are frequent airy contamination
(eg Micrococcus, Rhodotorula, Alternaria, Cladosporium etc.).
Morphological characteristics of colonies can be summarized as follows:

filamentous fungi colonies of are hard, dry, mealy colonies, velvet. Colonies
are very distinctive and cannot be confused with the bacteria and yeast !!!

yeasts colonies are usually larger than bacterial colonies. Yeast colonies are
dull, pasty, characteristic smell of fermentation. But yeast colonies are very
similar colonies of bacteria and can therefore be distinguished to each other
only when viewed in the microscope.

bacteria form small, shiny, soft colonies often with unpleasant odour
PURPOSE OF WORK
A) Describe colony morphology using accepted descriptive terms
B) Recognize colony of bacteria, yeasts and filamentous fungi between them
EQUIPMENT FOR A COUPLE OF STUDENTS
Petri plates containing nutrient agar and malt extract agar
16
PROCEDURE

The purpose is to obtain sample of microorganism from environment on
surface of agar plate

Here are some suggestions: open agar plate to the air to 30 minutes, place a
hair on the agar or touch the plate with your fingers or cough on the plate,
wipe the surface of laboratory bench with a sterile cotton swab

Replace the lids of the Petri dishes, place the plates in an inverted position
(reducing the amount of condensation forming on the inside surface of the
Petri dish lid) and incubate the plates at laboratory temperature for a few
days.

Examine the colonies appearing on the plates. Describe at least three
different colony types using figure as a reference and fill in the following table
with description of the bacterial colonies.
LABORATORY REPORT AND QUIZ
Table of Results
Sample:
Colony Description
0.1ml
at: 25°C;
For: 7 days
Diameter
Appearance
Margin
Elevation
Color
#
Colony 1
1mm
Circular
Entire
Raised
White
22
Colony 2
1.5mm
Irregular
Undulate
Umbonate
Light Yellow
6
Colony 3
5mm
Circular
Entire
Flat
Green
1
Questions:
1.
How do bacterial colonies differ from fungal colonies?
2.
What is a bacterial colony?
3.
Are you able to distinguish bacteria from yeast only on the morphology of the
colonies?
4.
Are you able to distinguish filamentous fungi from yeast only on the
morphology of the colonies?
17
5.
What is the function of pigments in the microbial cell?
Answers:
1- Bacterial colonies are smaller and grow just on the surface of the media, but
fungi colonies are much larger than bacterial colonies. They look like fur and
can grow above and also inside the media.
2- A group of cells which are formed from growing of one individual cell of
bacteria are called a bacterial colony.
3- In some cases, we can distinguish between bacterial and yeast colony if they
are a typical bacterial or yeast colony, but, most of the times, we cannot do
so, because they are very similar.
4- In some cases we cannot distinguish between yeast colonies and fungi
colonies, but Most of the times, we can do, because they are very different in
their morphological properties.
5- Protection of the microbial cell.
Laboratory Exercise 5
Wet Mount
“Wet Mount” is a method of observing microorganisms microscopically. This method
is used for talking about size, shape, arrangement of the cells, and movement – the
presence of flagella
THE PRINCIPLE
Wet mounts are the most common preparations used to view living microorganisms.
A small drop of an sterile water is placed on a clean slide, then suspension MO is
added aseptically and clean cover slip is placed over liquid and press down lightly.
Although wet mounts can be made rapidly, they dry up after five or ten minutes and
so are not very useful if long-term observations are required. Wet mounts is useful
for determining cellular size, shape and arrangement which is sometimes destroyed
during the staining process. Also Brownian movement or true motility with flagella
can be observed in wet mounts. Brownian movement is not true motility but rather is
movement caused by continuous vibrating motion by invisible molecules striking the
bacteria. In Brownian movement the particles and microorganisms all vibrate at
about the same rate and maintain their relative positions. Only the bacteria are truly
18
motile, their movement will be over greater distances and will be multi-directional, not
just back and forth. The presence, number and arrangement of flagella are useful in
identifying bacterial species. True motile bacteria move from one position to another.
Their movement appears more directed than Brownian movement, and occasionally
the cells may roll or spin.
The dried preparation of cells is known as a smear. Smears can be prepared from
cells in a liquid culture or from agar plate or slant. When using a liquid suspension,
one loopfuls are smeared onto a glass slide and then allowed to air dry. However,
bacterial cells are small, virtually transparent and little or no detail in cell is
distinguishable when placed smear under the bright-field microscope. Hence the
smear stains in order easier viewing of cells. Stains with dyes (colouring,dyeing)
enhance the contrast and allow to view the cell more distinctly. The dye adheres to
the bacteria and morphology (shape, arrangement, structure, size) can be more
readily seen. More intense staining can be achieved by the extension of exposure
time, by increasing the temperature of dyeing or application of a mordant that
increases the interaction between the bacterial cell and the dye
In progressive staining color works as long as the preparation well stained. Then the
excess of dye is washed out with water. In regressive staining the preparation
intentionally stained intensely and then part of the dye washed out with dilute
alcohol.
Staining methods used in microbiology is divided into:
Simple stainning when a single stain or dye to create contrast between the cell and
the background is applied. Simple staining is often employed when information about
cell shape, size, and arrangement is desired.
At differential staining we apply the mixture of colors and different types of microbes
and their structures are different color. Different affinity of dyes can detect some
diagnostically significant features of microorganisms such as gram
positivity/negativity, presence of capsules and endospores, acid-fast staining
The cells in the dried smear are attached or fixed to the slide by briefly heating the
slide over a gas burner flame. This procedure is known as heat fixation. When using
colonies, a small drop of water is placed on the slide and very small amount of
19
material is mixed with water to separate and suspend the cells. The suspension is
then spread out, air dried, and heat fixed. These fixed smears then can be used for
staining.
PURPOSE OF WORK
A. To prepare and observe wet mount slides.
B. To distinguish between true motility and Brownian movement.
C. To make and fix a smear.
EQUIPMENT FOR A COUPLE OF STUDENTS
Slides, cover-slides, sterile water
Culture: Bacillus subtilis, Micrococcus luteus, Saccharomyces cerevisiae…
A)
PROCEDURE WET MOUNT TECHNIQUE
Place a small drop of sterile water on a clean slide using a pipette.
Suspend the small amount from the culture colony by stirring carefully.
Handle the cover slip carefully by its edge and place in on the drop.
Observe using microscope and record your observations to Table
B)
PROCEDURE FIX SMEAR
place a small drop of sterile water on a clean slide
aseptically add a small amount of culture colony, stir the suspension by
inoculation loop and spread over the slide so as to create a thin film.
let air dry
heat fixes the dried smear by passing the slide through the flame three times.
20
LABORATORY REPORT AND QUIZ
Comment [H4]: Saccharomyces
cerevisiae?
Table of observations
Appearance
Bacillus subtilis
Micrococcus
luteus
Saccharomyces
cerevisiae
Tetrads, oval
shaped, and raised
elevation
Oval
Brownian
movement, Fluid
movement
Same like m.l.
Draw the types
of
microorganism
s observed:
Shape and
arrangement:
Motility:
Rod shaped, convex
elevation, and entire
margin
True motility,
Brownian
movement, Fluid
movement
Questions:
1. How do you distinguish true motility from Brownian movement or motion of the
fluid?
2. Can you distinguish the prokaryotic organisms from the eukaryotic organisms?
3. Which of the bacteria exhibited true motility on the slides?
4. Why do you fix the smear before staining?
5. Can the light intensity of your microscope be regulated? Explain.
6. Why is oil necessary when using the 90 × to 100 × objective?
Answers:
1- Brownian movement is something like a regular shaking of the particles in the
liquid. Fluid movement is distinguished easily as it happens in a clear
direction, so the irregular movements which are not similar to Brownian
movement and fluid movement; are true motility from the microorganisms
which are movable. They have different types of motility.
2- Prokaryotic organisms have basic cell structure and much smaller cells as
eukaryotic organisms and Eukaryotic cell structures are much complicated
than prokaryotic cells.
3- Bacillus subtilis … those with flagellum.
21
4- For two reasons we do fix the smear. Not to lose the bacteria or yeast cells
from the surface of slide during staining and also fixing can kill the organisms
and help the microscopy process (means staining) as we can see better the
structures inside the cells.
5- Yes, intensity of the microscope light can be regulated with a related knob.
6- To make better the resolution of the view. - prevent loss of light conditions/sharp
vision through refraction of rays
Laboratory Exercise 6
Negative Staining
“Negative Staining” is another method of observing microorganisms microscopically.
Characteristic of this method is that, we do not color the microorganism, but the
background.
By coloring the background, it is much easier to observe the microorganisms,
because eyes can easily differentiate between the things which are different in
colors.
THE PRINCIPLE
The negative stain technique does not stain the bacteria but stains the background.
The bacteria will appear clear against a stained background. Since simple staining
procedures are rapid and easy to carry out, they are often used when information
about cell shape, size, and arrangement is desired.
Bacteria can generally be characterized as spheres (coccus, plural cocci), rods
(bacillus, plural bacilli), spirals (spirillum, plural spirilla), helices (spirochete, plural
spirochetes), or branched organisms. In addition, many organisms form very
distinctive arrangements that can be used to identify them. For example, bacteria
such as the streptococci form chains of cells, the staphylococci develop in grape-like
clumps, the neisseriae exist as pairs or diplococcic (diplo = pair of), and some
micrococci and sarcinae (sarcina – a package) are typically found in package of four
or eight.
22
Fig. 3- Common shapes and arrangements of bacteria.
PURPOSE OF WORK:
A) To prepare a negative stain.
EQUIPMENT FOR A COUPLE OF STUDENTS
Congo red (2% solution in ethyl alcohol), slide
Culture: Micrococcus luteus and Bacillus subtilis
PROCEDURE

Slides must be clean and grease-free. Place a small drop of Congo red at the
end of the slide.

Mix a small amount of the culture on solid media in the Congo red dropusing loop. Using the loop spread the drop out to produce a smear.

Let the smear air dry.
23

Dip the completely dried smear in to the solution of chloride acid (1%) for a
few seconds (Congo red changes colour from red to blue).

Let the smear air dry again.

Examine the stained slides microscopically using the low, high-dry, and oil
immersion objectives.
LABORATORY REPORT AND QUIZ
Draw a representative field of your microscopic observation of negative staining
Appearance
*Bacillus subtilis
Micrococcus luteus
Magnification
40X
40X
Morphology and
arrangement
Rod shaped, chains
Spherical shaped, Tetrads
Questions:
1.
When is negative staining used?
2.
What is an advantage of negative staining?
3.
Why is negative staining also called either indirect or background staining
Answers:
1- When we need information about the size, shape, and arrangement of the
cells, we can use negative staining.
2- It stains the background, not the bacteria itself and it would be easier to talk
about the colony description.
3- Because it stains the background, not the bacteria itself.
Comment [H5]: Only objective, total is
400x?
24
Laboratory Exercise 7
Gram Staining
According to their cell wall composition, bacteria can be gram positive or gram
negative. Gram positive bacteria have much thicker peptidoglycan layer than gram
negative bacteria. That is why these two categories of bacteria react differently to the
gram staining which shows the gram negativity or gram positivity of them.
PRINCIPLE
The Gram stain is a very useful stain for identifying and classifying bacteria. The
Gram stain is a differential stain that allows you to classify bacteria as either grampositive or gram-negative. Bacteria stain differently because of chemical and
physical differences in their cell walls. The Gram-positive bacteria have a thick cell
wall that consists primarily of peptidoglycan (see figure 4). Many Gram-positive
bacteria, however, have polymers called teichoic acid in their cell walls, which may
account for as much as 50% of the wall’s weight. The peptidoglycan layer of the cell
wall consists of polysaccharides, made up of alternating N-acetylglucosamine and
N-acetylmuramic acid units, cross-linked by short peptides, while teichoic acids are
long polymers of alternating phosphates and carbohydrates (e.g., ribitol or glycerol).
The cell wall of Gram-positive bacteria is generally between 20 and 80 nanometers
thick. The walls of Gram-negative bacteria have much less peptidoglycan than those
of Gram-positive bacteria. The peptidoglycan layer, usually about 2 nanometers
thick, is surrounded by a complex lipid bilayer called the outer membrane. No
teichoic acids are associated with the cell wall of Gram-negative bacteria. The Gram
stain is most consistent when done on young culture of bacteria (less than 24 hours
old). The staining technique consists of the following steps:
Apply primary stain – crystal violet. All bacteria are stained purple by this basic dye.
Apply mordant – Gram’s iodine. The iodine combines with the crystal violet in the cell
to form a crystal violet-iodine complex.
Apply decolorizing agent – ethyl alcohol or acetone. The crystal violet is washed out
(decolorized) of some bacteria, while others are unaffected. The crystal violet –
iodine complex is larger than the crystal violet or iodine molecules that initially
entered the cell and cannot pass through thick peptidoglycan in Gram-positive
25
bacteria. In Gram-negative cells, the alcohol dissolves the outer lipopolysaccharide
layer, and the complex washes out through the thin layer of peptidoglycan.
Apply secondary stain – safranin. This basic stains the decolorized bacteria red.
Fig. 4- Gram positive and Gram negative cell wall.
PURPOSE
A) Perform and interpret Gram stains.
EQUIPMENT FOR A COUPLE OF STUDENTS
Gram staining reagents: crystal violet, Gram’s iodine, ethyl alcohol, safranin, Wash
bottle of distilled water, slide, Culture: Escherichia coli and Bacillus subtilis
PROCEDURE

Prepare and fix smears.

Prepare a Gram stain. Use a clothespin or pincette to hold the slide.

Cover the smear with crystal violet and leave for 60 seconds.


Cover the smear with Gram’s iodine for 30 seconds.
Decolorize with 95% ethyl alcohol. Let the alcohol run through the smear
(usually 5 to 10 seconds). This is a critical step. Do not over decolorize.

Immediately wash gently with distilled water by tilting the slide and squirting
water above the smear so that the water runs over the smear.
26

Add safranin for 60 seconds.

Wash with distilled water and let dry the slide freely in air.

Examine the staining slide microscopically using the low and oil immersion
objectives.
LABORATORY REPORT AND QUIZ
Draw a representative field of your microscopic observation of Gram staining
Bacillus subtilis
Escherichia coli
Micrococcus
luteus
Morphology,
arrangement,
relative size
Rod Shaped
Rod Shaped
Tetrads
Color
Dark blue
Red
Dark blue
Gram reaction
Gram Positive
Gram Negative
Gram Positive
Appearance
Sketch a few
bacteria.
Questions:
1.
If you leave the alcohol on too long, what would you expect to see after Gram
staining a mixture of Escherichia coli and Bacillus subtilis?
2.
When you counter stain with safranin, why do the Gram-positive bacteria not
pick up safranin and stain red?
3.
Why might a physician perform a gram stain on a sample before prescribing an
antibiotic?
4.
Explain the principle of Gram staining.
27
Answers:
1- If we leave alcohol for too long time than usual, in our results we will have to say
that both are gram negative bacteria as both of them take red color during gram
staining.
2- Because they are already colored purple.
3- To determine if the bacteria are gram positive or negative to prescribe broadspectrum antibiotics.
4- Bacteria are classified according to their cell wall structure to two main groups
which are gram positive and gram negative bacteria. The peptidoglycan layers of
the gram positive bacteria are much thicker than gram negative.
After fixing the smear, first we add crystal violet for 30 to 60 seconds, then add
iodine for 30 to 60 seconds, and then throw both away and wash it wish alcohol
for 5 to 10 seconds and then cover the smear with safranin.
Crystal violet color both gram positive and negative bacteria purple, but when we
wash them with alcohol gram negative become colorless because losing crystal
violet, but gram positive bacteria have thicker layer and keep the crystal violet.
After adding safranin gram negative bacteria which are now colorless, get red
color, so this way we can determine which bacteria are gram positive and which
are gram negative.
28
Laboratory Exercise 8
Endospore Staining
Endospore staining is a method of observing microorganisms microscopically. This
method is used to determine the endospores of the endospore forming bacteria.
As we know, endospores are much stronger in physical and chemical properties, as
they are used to protect bacteria in unfavorable conditions, that is why endospore
staining is much difficult process than other types of staining, and needs special
requirements and conditions.
THE PRINCIPLE
An endospore is a heat- and chemical-resistant resting form produced by member of
certain bacteria genera in response to adverse environmental condition. The only
bacteria known to produce endospores belong to the following genera: Bacillus,
Clostridium, Desulfotomaculum, Sporolactobacillus, Thermoactinomyces, and
Sporosarcina. Because of endospore’s outer coat is an effective barrier to chemicals,
endospores generally stained poorly. Endospores can be stained by using very hot
dyes. Figure represents typical life cycle of spore forming bacterium.
Fig. 5- The development cycle of Endospores.
29
PURPOSE
A) Prepare and interpret endospore stain.
EQUIPMENT FOR A COUPLE OF STUDENTS:
Endospore staining reagents: malachite green, safranin, Wash bottle of distilled water,
slide, and heater
Culture: Bacillus subtilis (older than 48 hours)
PROCEDURE

Prepare and fix smears.

Prepare an endospore stain. Use a clothespin or pincette to hold the slide.

Cover the smear with malachite green.

Heat the stain. The malachite green must heat for at least 5 minutes.

Cool the slide for about 1 minute before continuing.

Wash gently with distilled water by tilting the slide and squirting water above the
smear so that the water runs over the smear.

Add safranin for 60 seconds.

Wash with distilled water and let dry the slide freely in air.

Examine the staining slide microscopically using the low and oil immersion
objectives. Label the vegetative cells and endospores.
30
LABORATORY REPORT AND QUIZ
Draw a representative field of your microscopic observation of endospore staining
Appearance
Bacillus subtilis
Sketch a few bacteria
(1000X)
Questions:
1.
How did the appearance of the 24-hour and 72-hour Bacillus subtilis culture differ?
2.
Which organism (genus and species) is responsible for each of the following
diseases: anthrax, tetanus, botulism, and gas gangrene?
3.
Why is heat necessary in order to stain endospores?
4.
What is the function of an endospore?
5.
Why are endospores so difficult to stain?
Answers:
1- 72 hours old culture has more spores than 24 hours old culture.
2- Anthrax – Bacillus anthracis
Tetanus – Clostridium tetani
Botulism – Clostridium botulinum
Gas gangrene – Clostridium perfringens
3- Because endospores are much stronger against laboratory temperatures.
4- Endospores protect bacteria from abnormal physical and chemical conditions.
5- Because of theirs strong physical structure.
31
Laboratory Exercise 9
Fungi (yeast and molds)
Fungi can be yeast or molds. Yeast and mold are two different types of fungi. Both
yeast and fungi are eukaryotic organisms. Yeasts are unicellular, microscopic
microorganism, while molds are multicellular macroscopic organisms.
PURPOSE
A. Characterize and classify fungi.
B. Identify common saprophytic molds and yeasts.
C. Explain dimorphisms.
THE PRINCIPLE
Fungi possess eukaryotic cells and can exist as unicellular or multicellular organisms.
They are heterotrophic and . Fungi are aerobic, facultative anaerobic and anaerobic.
Unicellular yeasts and multicellular molds are included in the Kingdom Fungi.
Yeasts are nonfilamentous, unicellular fungi that are typically spherical or oval in shape.
They reproduce asexually by budding. In some instances, when buds fail to detach
themselves, a short chain of cells called a pseudohypha forms. Depending on the strain
and the external conditions that prevail in the culture yeast form or (pseudo)hypha form,
that is called dimorphism.
A macroscopic mold colony is called a thallus and is composed of a mass.
PURPOSE
D. Characterize and classify fungi.
E. Identify common saprophytic molds and yeasts.
F. Explain dimorphisms.
EQUIPMENT FOR A COUPLE OF STUDENTS
Petri plates containing Sabouraud agar, Methylene blue, Lactophenol Cotton Blue
32
Culture: Rhodotorula glutinis, Candida utilis, Saccharomyces cerevisiae, Geotrichum
candidum
Rhizopus oryzae, Aspergillus niger, Penicillium purpurogenum
PROCEDURE
Yeasts
Make a wet mount of each culture by using a small drop methylene blue. Record your
observations.
Molds
Make tease mount wet hyphae for observing fungi and record.
LABORATORY REPORT AND QUIZ
Draw a representative field of your microscopic observation
Organism
Saccharomyces cerevisiae
Color: cream
Draw a Typical Colony
Wet Mount
33
Rhodotorula glutinis
Color: red
Candida utilis
Color: cream
Rhizopus oryzae
Colony appearance:
filamentous
Macroscopic hyphae
color: Light green
Spore color: Light green
Underside color: Light
yellow
Aspergillus niger
Colony appearance:
filamentous
Macroscopic hyphae
color: Black
Spore color: Black
Underside color: White
34
Penicillium
purpurogenom
colony appearance:
filamentous
Macroscopic hyphae
color: Light blue
Spore color: green
Underside color: red
Geotrichum candidum
Colony appearance: f
Macroscopic hyphae
color: Light blue
Spore color: NA
Underside color: White
Laboratory Exercise 10
Rapid determination of lethal temperatures
Temperature is one of the most important physical conditions which can influence the
growth of microorganisms. There are three different groups of microorganisms
according to their reaction to the temperature, which we will discuss it later. Lethal
temperature is the lowest temperature at which microorganism cannot survive more
than 10 minutes.
THE PRINCIPLE
Cells in addition to nutrients and enough available energy must also have optimal
physical, chemical and biological conditions. Microorganisms are able to at least
partially adapt to external physical agents. Ambient temperature is the most important
factor that affects the growth of microorganisms. Temperatures that allow growth of
microorganisms are from 0 °C to 85-90 °C, although described even higher
temperatures at which certain types of bacteria grow and live. Then to evaluate the
35
dependence of the growth on temperature, it is possible to determine three
temperatures: minimum is the lowest temperature at which micro-organism yet
reproduce measurable speed, optimum at which achieves the highest growth rate (μ
max) and maximum is the highest temperature at which even leads to cell division.
Psychrophiles organisms have optimum growth temperature at 15 to 20 ˚ C. No
pathogenic microorganisms are not among psychrophiles. Most microorganisms are
mesophiles and have optimum temperature 37˚C. These include, for example
Escherichia coli, Bacillus, Saccharomyces, and all pathogens. Thermophiles will grow
best at 55 to 60 ˚ C, we find here the representatives of cyanobacteria, streptococci,
lactobacilli. Usually, it is important to determine the optimum growth temperature or
lethal temperature, less frequent temperature range of growth. Lethal temperature is the
lowest temperature that kills within a certain time microorganism under precisely defined
conditions. Usually lethal temperature means the temperature at which kills the cells in
suspension for 10 minutes. Lethal temperature for different organisms varies and is
influenced by cell concentration, culture physiological state (age) composition of media
and pH. Determination of lethal time is important from a technological point of view.
Lethal time is the time required to kill the microorganisms in a given temperature and
the defined conditions. Relationship between lethal temperature and the time needed to
death organism is especially important in the canning industry.
PURPOSE OF WORK

To determine lethal temperature of various microorganisms
EQUIPMENT FOR A COUPLE OF STUDENTS
Suspension of microorganism, 8 sterile tubes with cup, agar plate, micropipette, sterile
tips, water thermostat.
PROCEDURE OF LETHAL TEMPERATURE DETERMINATION

to 7 sterile tubes aseptically add 2 ml of tested suspension
36

the bottoms of the Petri dishes with agar divide into 8 pieces and marked them
from1 to 8.

set temperature of the thermostat to 40C. After temperature stabilization insert
first tube in the thermostat. After 11 minutes take tube and cools quickly for 5
minutes in an ice solution

raise the temperature of the thermostat gradually to 45, 50, 55,60,65,70,75,85C
and proceed as in the previous paragraph

inoculate 50 ml of microbial suspension from each tube to the appropriate section
of agar

for each studied microorganism determine the lowest temperature (lethal
temperature) at which no cells grow
LABORATORY REPORT AND QUIZ
1 Day old Bacillus subtilis
Temperature 20
40
Cells
++
+++
14 Days old Bacillus subtilis
Temperature 20
40
Cells
+++
+++
Escherichia coli
Temperature 20
40
Cells
++
+++
50
+
60
+
70
-
80
-
90
-
100
-
50
++
60
++
70
+
80
+
90
-
100
-
50
+++
60
++
70
++
80
+
90
+
100
-
Questions:
For each studied microorganism determine the lowest temperature (lethal temperature)
at which no cells grow.
1.
What is the minimum, optimum and maximum temperature for growth of
microorganisms?
2.
Define the lethal temperature
3.
How can you determine experimentally whether a bacterium is a psychrophiles
or a mesophiles?
37
Answers:
Lethal temperature for one day old Bacillus subtilis is 70 degrees Celsius, for 14
days old Bacillus subtilis is 90 degrees Celsius, and for E. coli it is 100 degrees Celsius.
1- Minimum growth temperature is the lowest temperature that a microorganism can
survive.
Optimum growth temperature is the temperature in which the microorganism has
the highest growth speed.
Maximum growth temperature is the highest temperature that a microorganism
can survive.
2- Lethal temperature is the lowest temperature that a microorganism can survive
at, for more than 10 minutes and in a specific condition.
3- Psychrophiles have lower lethal and optimal temperature than mesophiles.
Laboratory Exercise 11
Influence of pH on growth
Microorganisms, like other organisms can survive and grow at a range of pH. This
range consists of minimum pH, maximum pH, and optimal pH, which is important for a
microorganism to grow.
THE PRINCIPLE
The content of hydrogen ions (pH) in the environment strongly influences the growth of
microorganisms. The concentrations of H + ions change charge of cell membrane and
thus permeability and nutrient intake. Most bacteria grow in neutral or slightly alkaline
environment. For yeast is optimal acidic environment (pH 4.8 to 5.5). The optimum pH
for most molds is near the neutral value, but generally the molds reproduce a very wide
range of p H, i.e. from 1.2 to 11.0. In many cases, the micro-organisms themselves
affect the pH of its environment using metabolism, such as the acidifying bacteria, fungi
and yeast produce organic acids and reduce the pH of the medium. Biological buffers
are used to absorb fluctuations in pH of the culture medium. In industrial bioreactors pH
was maintained by addition of concentrated solutions of alkali or acids.
38
PURPOSE OF WORK
A) To determine optimum pH for the tested microorganisms
EQUIPMENT FOR A COUPLE OF STUDENTS
Suspension of microorganism (inoculum), liquid cultivation media, Na2 HPO4 *2H2O,
citric acid, pH meter, volumetric flasks 0,5 l, tubes with metal plug, spectrophotometer,
cuvettes, vortex
PROCEDURE OF OPTIMAL pH DETERMINATION

Prepare a stock solution of 0.5 l Na2 HPO4 *2H2O with s concentration of 0.2
mol/l

Prepare a stock solution of 0.5 l citric acid with concentration of 0.1 mol/l

Mix using a pH meter buffers with different pH from stock solutions of Na2 HPO4
*2H2O and citric acid

Mix the nutrient media with different pH buffers in ratio 1:1 (final concentration of
nutritives in solutions are correct) as reported in Table:

prepared culture media (different pH) sterilized in an autoclave

after sterilization, aseptically add 5 ml culture media with different pH to the
empty tubes


incubate the bacteria at 37 ° C for 24 hours and yeast at 25 ° C 48 hours

measure the absorbance of all samples against blank
LABORATORY REPORT AND QUIZ
PH
2
3
4
5
6
7
8
9
10
MO
Bacillus subtilis 0.06 0.01 0.05 0.03 0.19 0.72 1.66 1.76 1.73
Esherichia coli
0.07 0.04 0.04 0.08 0.10 0.42 1.64 1.71 1.66
Saccharomyces
0.42 2.00 2.00 1.55 1.22 0.42 0.14 0.09 0.08
cerevisiae
Rhudutorula
0.79 0.07 0.68 0.49 1.05 1.22 0.81 0.79 0.73
glutinis
39
For each studied microorganism determine optimal pH for growth
1. What is the optimal pH for growth of yeasts, bacteria and fungi?
2. Why are buffers added to culture media?
3. What is the pH tolerance of bacteria compared to yeasts?
4. How do microorganisms change the pH of their own environment?
Bacillus subtilis and E. coli can grow better in alkaline environment, but
Saccharomyces cerevisae need an acidic environment for growth, while Rodutorulla
glutinis prefer neutral pH.
1- Bacteria 6-8
Yeast 4-4.5
Fungi 4-8.5
2- To stabilize the pH and keep pH in an exact value during cultivation.
3- Yeast can tolerate more acidic environment than bacteria.
4- Microorganisms can change the pH of their environment by producing some
special compounds or interacting with the existing compounds.
Laboratory Exercise 12
Influence of osmotic pressure on cell growth
If osmotic pressures inside the cell wall and outside the cell wall are the same, the cell
is in isotonic condition and can grow normally, but these pressures can differ because of
higher or lower concentrations of the salts in two sides of the cell wall. Higher osmotic
pressures will lead to drying of cell and lower osmotic pressures will lead to the lyses of
the cell.
THE PRINCIPLE
Microorganisms grow best in an environment that has the same or only slightly
lower osmotic pressure than the cytoplasm. Osmotic pressure is the force developed
when a membrane that is permeable only to the solvent separates two solutions of
different solute concentrations. In an isotonic solution, the concentration of solutes is the
same (means equal) outside and inside the bacterium. The bacterium is in osmotic
40
equilibrium with its environment and does not change volume. In hypotonic environment
(low solute, high-water content) higher osmotic pressure of the cell is to be
compensated so that the water enters through the cytoplasm membrane into the cell
and cause it to burst. On the other hand, in the hypertonic environment (high solute,
lower water content), the water leaves from cell and the drainage of cells occurs thereby
reduces the metabolic activity of cells. This effect is used for preserving food with salt or
sugar. Microorganisms that are able to grow at high salt concentration are halophiles
organisms. The so-called osmotolerant yeast are growing well in the presence of
60%sucrose, some halophiles bacteria need to his growth NaCl concentrations of 520%, other so-called extreme halophiles need 30% of NaCl.
PURPOSE
Perform an experiment that relates bacterial growth to osmotic
pressure
EQUIPMENT FOR A COUPLE OF STUDENTS
24- to 48-hour broth cultures of different bacteria, wax pencil, inoculation loop, Bunsen
burner
1 Petri plate nutrient agar with 0% NaCl
1 Petri plate nutrient agar with 0.5% NaCl
1 Petri plate nutrient agar with 5% NaCl
1 Petri plate nutrient agar with 10% NaCl
1 Petri plate nutrient agar with 20% NaCl
1 Petri plate nutrient agar with 25% NaCl
PROCEDURE

With a wax pencil, divide the bottom of each of the five Petri plates into half

Place the name of the bacterium to be inoculated in each section and salt
concentration

Streak the respective bacteria onto the six different Petri plates.
41

Incubate the plates, inverted, for 48 hours

Observe the relative amount of growth in each section at each salt concentration.
Record this

growth as – (none), +, ++, +++, and ++++ (the most) in the report
LABORATORY REPORT AND QUIZ
Table of observation
Media
E. coli
0% NaCl
0.5% NaCl
5% NaCl
10% NaCl
20% NaCl
25% NaCl
++++
++++
+++
+
-
S.
marcescence
++++
++++
-
B. subtilis
M. luteus
++++
++++
+++
+
+
-
+++
++
+
-
Question:
1.
Which of these bacteria tolerates the most salt?
2.
Which of these bacteria tolerates a broad range of salt?
3.
What foods can you think of that are protected from microbial destruction by
salting?
4.
What is:
a. osmosis
b. osmotic pressure
c. plasmolysis
d. halophiles
Answers:
1234-
Bacillus subtilis
Bacillus subtilis
Foods with higher salt concentrations.
Osmosis – the process of transferring of small molecules through semipermeable
membrane and not transferring larger molecules.
42
Osmotic Pressure – the pressure which is produced in the both sides of a
semipermeable membrane because of the difference of the concentrations
between the both sides.
Plasmolysis – Process of losing of water from inside the cell and cell drying.
Halophiles – Organisms especially microorganisms that grow in a saline
conditions.
Laboratory Exercise 13
Influence of UV
UV radiation is also one of the most important physical conditions which can influence
the growth of microorganisms. UV can stop the growth of the microorganisms or also
can kill them. In this laboratory exercise, we use different materials for protecting
microorganisms against UV radiation and compare, which ones can do protect
microorganisms from UV, and ones cannot.
Principle
Radiant energy comes to Earth from Sun and other extraterrestrial sources, and some
is generated on Earth from natural and man-made sources. Radiation differs in
wavelength and energy. The shorter wavelengths have more energy. X rays and
gamma rays are forms of ionizing radiation. Their principal effect is to ionize water into
highly reactive free radicals that can break strands of DNA. Some no ionizing
wavelengths are essential for biochemical processes, e.g. photosynthesis. No ionizing
radiation between 15 and 390 nm is called ultraviolet (UV). The most lethal
wavelengths, called germicidal, are in the range of 200 – 290 nm. These wavelengths
correspond to the optimal absorption wavelengths of DNA.
Ultraviolet light induces pyrimidine dimmers in the nucleic acid, which results in a
mutation. Mutations in critical genes result in the death of the cell unless the damage is
repaired. When pyrimidine dimmers are exposed to visible light, the enzyme pyrimidine
dimmerase is active and splits the dimmers. This is called light repair or
photoreactivation. Another repair mechanism, called dark repair, is independent of light.
Dimmers are removed by endonuclease, DNA polymerase replaces the bases, and
DNA ligase seals the sugar-phosphate backbone.
43
Purpose of work
Examine the effects of ultraviolet radiation on bacteria.
Ascertain protection properties of material against ultraviolet radiation.
Equipment for a couple of students
Petri plates containing nutrient agar, sterile cotton swabs, and covers (choose three):
gauze, paper, aluminum foil, glass; ultraviolet lamp, suspension of Serratia marcescens
in saline solution
Procedure
Swab the surface of each plate with Serratia marcescens, to ensure complete covering,
swab the surface in two directions.
Remove the lid of an inoculated plate and cover one-half of the plate with one of the
covering materials.
Place each plate directly under the ultraviolet light about 30 cm from the light agar side
up.
Expose plate for 30 seconds, 60 seconds, 90 seconds, and 120 seconds and incubate
in dark at 25°C.
Laboratory report and quiz
Sketch your results, note any pigmentation.
Material
Used for
Covering
Paper
0 sec
30 sec
60 sec
90 sec
120 sec
44
Glass
Guaze
Al foil
Questions:
1. If you use Bacillus like biological model, would it be any difference between 24 hour
and 72 hour old culture?
2. What is the practical meaning of radiation treatment?
3. Why are plates incubated in dark?
4. What was the color of Serratia colonies? Were any of them white? If yes, try to
explain it.
5. Which cover material was the best for protection before radiation?
Answers:
1- As 72 hours old Bacillus has more spores than 24 hours Bacillus and spores are
much stronger against UV than vegetative cells, then older one can survive more
in UV conditions.
2- In microbiology laboratories, the term “Radiation Treatment” refer the treatment
of the air of the environment of the laboratory with the radiations especially UV
radiation.
3- After exposing the plates (microorganisms) to the UV radiation, we incubate them
in dark, because we let the their auto-regulatory system to eliminate the
destructive influence of UV on them.
45
4- Normally the color of Serratia is red, but in some cases of exposing them to the
UV; their color change to white, and it is because a mutation changes in their
structure. It doesn’t happen usually, but sometimes.
5- The materials which we used, all were good for protecting the microbes from UV
except “guaze”. Aluminum foil is the best material for protecting microbes against
UV radiation; comparing to paper, guaze, and glass.
Laboratory Exercise 14
Quantification of cell
In microbiology is often necessary to determine the number of microbial cells in a given
environment.
To quantify the number of cells is important:

in food raw materials, the finished products, the air, or water

for the control of technological processes of fermentation

in search of the optimal growing conditions for a particular type of microorganism

in monitoring the effectiveness of disinfectants, chemotherapeutic agents,
antibiotics
Direct microscopic counting in chamber is a quick and easy method, but it is impossible
to distinguish between live and dead cells. The counting chamber (Burker chamber) is a
specimen slide that is used to determine the concentration of cells in a liquid sample. It
is frequently used to determine the concentration of blood cells but also the
concentration of yeasts or bacteria in a sample. The cover glass, which is placed on the
sample, does not simply float on the liquid, but is held in place at a specified height
(usually 0.1mm). Additionally, a grid is etched into the glass of the chamber. This grid,
an arrangement of squares of different sizes, allows for an easy counting of cells.
46
Fig.6- Burker chamber
This way it is possible to determine the number of cells in a specified volume. Usually
we count the number of cells on the area of 1/25 mm. Then the total number of cells per
1 ml (P/ml) of the suspension is then determined according to the following formula:
P  p * 250 000 * D
(4)
where p is the average number of cells per area of 1/25 mm2, 250 000 the calculation
to 1 cm3 (i.e. 1 ml, 1/25 · 1/10 · 1/1, 000 mm3), D is overall suspense dilution.
Cultivation method is based on dilution of MO in appropriate medium and smears the
cell suspension on surface of agar. Microorganisms growing on agar form colonies and
the number of bacteria (CFU) per millilitre or gram of sample can be calculated on the
base of number of visible colony. Assuming a very small number of cells, samples were
concentrated by filtration.
Conversely, at high concentration samples are diluted 10 or 100 times. If we do not
know the approximate number of cells, we cultivate several different dilutions in order to
achieve the required number of colony i.e. 30-300. In liquid culture, the medium
appears more and cloudier as the bacteria increase in number by division. A tube of
bacteria will tend to reflect light so that less light is transmitted through the tube. A
spectrophotometer can measure the amount of light passing through the tube, or
47
conversely the amount of light absorbed. These measurements of turbidity or optical
density (OD) are not direct measurements of bacterial numbers, but an indirect
measurement of cell biomass that includes both living and dead cells. As the bacterial
cell population increases, the amount of transmitted light decreases, increasing the
absorbance reading on the spectrophotometer. Based on the calibration line, which is
dependence of absorbance on cell number, the total number of cells in the unknown
sample can be calculated. Because a value of absorbed light depends on the
wavelength of the light used, cell turbidity should be measured in the range 500-600nm
when cells exhibit absorption maximum.
PURPOSE
A) To measure absorbance a bacterial culture by spectrophotometer and using a
calibration line to calculate the number of cells in suspension
B) To determine the concentration of viable cell by cultivation method on agar
plates
C) To determine the number of cells by direct microscopic counting in Burker
chamber
D) To determine the concentration of dry cell per millilitre
EQUIPMENT FOR A COUPLE OF STUDENTS
analytical balance, sterile saline solution, sterile tips, micropipette, vortex, sterile sticks,
agar plate, incubator, spectrophotometer, cuvettes, agar plate, tips, micropipette,
moisture analyser, cellulose acetate membrane 0.2 µm
A) PROCEDURE OF SPECTROPHOTOMETRIC DETERMINATION OF CELL
NUMBER

aseptically pipette 5 ml of sterile saline solution into 6 tubes
aseptically withdraw 5 ml of the original cell suspension into the first tube (you prepare
dilution 2-1) and then homogenize with vortex mixer
48

aseptically withdraw 5 ml of suspension from 2-1 dilution into the second tube
(you prepare dilution 2-2) and then homogenize with vortex mixer

aseptically withdraw 5 ml of suspension from 2-2 dilution into third tube (you
prepare dilution 2-3)

and then homogenize with vortex mixer

aseptically withdraw5 ml of suspension from 2-3 dilution into the fourth tube
(prepared by diluting your 2-4) and then homogenize with vortex mixer

aseptically withdraw 5 ml of suspension from 2-4 dilution into the fourth tube
(prepared by diluting your 2-5) and then homogenize with vortex mixer

1 tube with pure cultivation media is blank

measure the absorbance of the all dilutions against blank at 550 nm by
spectrophotometer and prepare analytical line A = f (dilution)
B) PROCEDURE OF DETERMINATION OF VIABLE CELL BY CULTIVATION
METHOD

aseptically pipette 4.5 ml of sterile saline solution into 4 tubes

aseptically withdraw 0.5 ml of the cell suspension into the first tube (you prepare
dilution 10-1) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-1 dilution into the second tube
(you prepare dilution 10-2) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-2 dilution into third tube (you
prepare dilution 10-3) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-3 dilution into the fourth tube
(you prepare dilution your 10-4) and then homogenize with vortex mixer

aseptically withdraw 0.5 ml of suspension from 10-4 dilution into the fourth tube
(you prepare dilution 10-5) and then homogenize with vortex mixer

aseptically add 0.2 ml of suspension of all the dilutions on the surface of Petri
dishes

spread a suspension on the surface of Petri dishes with a sterile stick and
incubated in the inverted position for 48 hours at 30C
49

at the end of the incubation period, select all of the Petri plates containing
between 30 and 300 colonies.
The number of yeasts per 1 ml in the original sample can be calculated as follows
( CFU / ml ):
CFU / ml  p * 5 * D
(5)
CFU / ml is the total number of colony forming units per 1 ml of original sample, p is
the average number of colonies on Petri dishes in tube with a given dilution, 5 is
recalculation of the number of colony units per 1 ml, D total dilution of the sample.
C) PROCEDURE OF DIRECT COUNTING CELL IN BURKER CHAMBER

Attach the cover slide on a clean chamber Burker.

Stir thoroughly test tube with dilution of 2-5 and transfer a drop in the centre of
computing
chamber. Check that the number of yeasts on surface of a one small square
(1/25 mm 2) is in the range 8-12. If necessary, take a cell suspension with more
(2-6) or less dilution (2-4).

count least the cells in 8 squares at a magnification of 400 ×. Cells lying or
touching the borderline squares are counted only if they are on the top or right
side of the square.

Calculate the total number of cells per 1 ml (P/ml) of original suspension
according to
the formula (4)
D) PROCEDURE OF DRY CELL MASS DETRMINATION

insert empty-cellulose acetate membrane on aluminium weighing pan

dry empty membrane to constant weight at 105C

after drying marks the mass of the empty membrane shown on display

Filter the 15 ml of the cell suspension using a vacuum pump
50

wash filter cake on the membrane several times with saline solution

insert wet cellulose acetate membrane with filtration cake on aluminium weighing
pan

dry wet membrane with filtration cake to constant weight at 105 °C
The concentration of yeasts dry mass per 1 ml (mg/ml) of the original sample can be
calculated as follows:
c
m1  m0
V
(6)
where m1 , m0 , V are weight of membrane with cell, empty membrane and volume of
filtered suspension.
LABORATORY REPORT AND QUIZ
Process the results of determining the cell number in tables:
Dilution
Spectrophotometry
Absorbance
20
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2.00
1.57
0.82
0.62
0.34
0.13
0.10
0.03
10-6
10-7
10-8
46
5
-
58
6
-
52
5.5
Dilution
Plate Cultivation Method
10-3
Number of Colonies
(Plate A)
Number of Colonies
(Plate B)
Number of Colonies
(Average)
CFU/ml
10-4
10-5
TMTC
TMTC TMTC
TMTC
TMTC TMTC
51
Dilution
Burker Chamber
Method
Number of Cells per
square (1/25 mm2) (Plate
A)
Number of Cells per
square (1/25 mm2) (Plate
A)
Number of Cells per
square (1/25 mm2)
(Average)
CFU/ml (in original
sample)
Questions:
123456-
10-1
10-2
2-5
2-5
39
2
13
9
37
5
11
16
38
3.5
12
12.5
In excel plot the calibration line as dependence A550 = f (number of cells)
How can you determine the number of microbial cells in the environment?
Which of above mentioned methods is the fastest?
Which methods are suitable for determining number of living cells?
Why we evaluate only nutrient agar plates containing 30 -300 colonies?
When and how to perform gravimetric determination of dry cells?
Answers:
1- Calibration Line in Excel
Absorbance
Absorbance-Number of Cells
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
10000000 20000000 30000000 40000000 50000000 60000000
Number of Clls (Cells/ml)
52
2- By using different methods such as spectrophotometer, dilution and macroscopic
counting, and cell dry mass.
3- Spectrophotometer
4- Counting colony forming unit (CFU/ml).
5- Because if the number of colonies are less than 30, then the counting mistake
will be more and if the number of colonies are more than 300, then its
macroscopic counting would be difficult.
6- Mostly when we are working with filamentous fungi and need to measure the
quantity of its growth, we use this method. Counting filamentous fungi colonies is
a very difficult work, as they do not grow in such colonies as bacteria and yeast.
Laboratory Exercise 15
The effect of chemical agents on bacteria
Chemical agents can influence the growth of bacteria. Chemicals can have cidal or
static effect on microorganisms. Cidal effect of the chemicals can kill the
microorganisms, but chemicals with static effect can only stop growth and will not kill the
microorganisms.
It can happen that chemicals which are microbicidal, can have microbistatic effect if it is
used in low concentrations. We are using disk diffusion method for measuring the
activity of different chemicals.
THE PRINCIPLE
We know of a number of antimicrobial agents. They are used as disinfectants,
preservatives or as medicines. Some of them are produced by microorganisms such as
filamentous fungi or streptomycetes, we call them antibiotics. Others are synthesized in
the laboratory and we call them chemotherapeutics. They may have microbicidal or
microbiostatic effects. Many microorganisms are resistant to antimicrobial compounds,
either primary or secondary (due to e.g. mutations). Methods for testing the
susceptibility of microorganisms to antimicrobial compounds can be divided into two
categories.
In the disk- diffusion method a Petri plate containing an agar growth medium is
inoculated uniformly over its entire surface. Antibiotics are impregnated onto paper
53
disks and then placed on a seeded agar. The plate is then incubated and the diameter
of the zone of inhibition around the disk is measured to the nearest millimetre. The
inhibition zone diameter that is produced will indicate the susceptibility or resistance of a
cell to the antibiotic. For example, a zone of a certain size indicates susceptibility, zones
of a smaller diameter or no zone at all shows that the bacterium is resistant to the
antibiotic. Size of zone depends on size of the inoculum, distribution of the inoculum,
incubation period, and depth of the agar, diffusion rate of the antibiotic, concentration of
antibiotic in the disk, and growth rate of the bacterium. This method is qualitative and
may also be used to measure the sensitivity of any microorganism to a variety of
antimicrobial agents. Diffusion tests are primarily qualitative methods that normally
should only be used to report whether a bacterium is resistant or not. Dilution method is
quantitative and involves subjecting the cell to a series of concentration of antimicrobial
agents in broth environment. The lowest concentration at which the cell is completely
inhibited (as evidenced by the absence of visible bacterial growth) is recorded as the
minimal inhibitory concentration or MIC. The MIC is thus the minimum concentration of
the antibiotic that will inhibit cells. Growth (or growth inhibition) depends on the
concentration of antimicrobial compound in the medium, so there is a dependency,
called curve toxicity.
EQUIPMENT FOR A COUPLE OF STUDENTS
Petri plate containing nutrient agar, cotton swab, antibiotic disks, tweezers, ruler, tips
and pipette
Culture: Bacillus subtilis, Escherichia coli, Micrococcus luteus, Serratia marcescens
PROCEDURE

Aseptically swab the assigned cultured onto the appropriate plate. Swab in three
directions to ensure complete plate coverage. Let stand at least 5 minutes.

Sterilize forceps by dipping in alcohol and burning off the alcohol. Obtain a disk
impregnated with a chemotherapeutic agent and place it on the surface of the
agar. Gently tap the disk to ensure better contact with the agar.
54

Incubate the plate inverted until the next period. Measure the zones inhibition in
millimetres using a ruler on the underside of the plate.
LABORATORY REPORT AND QUIZ
Process the results in table below.
Antimicrobial
Agent
Saccharomyc
es
Candid
a utilis
0
0
0
0
0
0
0
0
Bacillu
Micrococcu Escherichi
s
s luteus
a coli
subtilis
Serratia
marcescen cerevisiae
s
Giseofulvin
(Anti-fungal)
0
0
0
0
Ampicillin
500mg (Antibacterial)
2
0
2.5
3.0
(size of
inhibition
zone in mm)
Betnesol-N
Eye
Ointment
(Antibacterial
0.5
Clotrimazolu
m 100mg
2.5
(Anti-Fungal)
0.1
1
0
0
2.5
0.2
Questions:
1. In which growth phase is an organism most sensitive to an antibiotic?
2. What is the difference between microbicidal and microbiostatic?
3. Why can not compare the effect of antibiotics by diffusion method?
4. Which bacteria are more sensitive to antimicrobial compounds and why?
5. Think about how resistance can develop.
55
Answers:
1- In the cell division phase
2- Microbicial kills the microorganisms and microbistatic just stop their growth and
after removing the effect of the microbistatic agent, the microbe will be able to
continue its growth again.
3- Because in this method we do not use antibiotics with different concentrations to
be able to compare the antibiotics to each other.
4- Gram positive bacteria are the most sensitive bacteria against antimicrobial
agents and chemicals.
5- If we apply antibiotic to microorganisms in lower concentrations than their
microbicidal dose, a number of them will die, and some will survive. The once
who survived are much stronger than before, so it can be a method for
strengthening of useful microorganisms in industries.