Pharmaceutical Microbiology (PHT-226)

(Practical )
By
Elmutasim O. Ibnouf
Lecturer of Microbiology
Department of Pharmaceutics & Microbiology
College of Pharmacy

Salman bin Abdulaziz University

LABORATORY RULES
&
USING OF MICROSCOPE

LABORATORY RULES
Many of the microorganisms used in this course may be
pathogenic for humans.
Certain rules are necessary to insure safety for you.
1. The laboratory benches must be kept free of articles not
actually used.
2. Wear a coat during the laboratory period to protect your
clothes.
3. Smoking, eating, drinking and gum chewing are not
permitted.
4. A jar of disinfectant is provided on each bench for
holding the contaminated pipettes.
5. Please return all reagents, cultures and glassware in
their respective places.

6. A void unnecessary conversation, noise, or movement.

7. Laboratory notes should include results obtained from
the experiments, a drawing of each microscopic
observation, and explanation of results reported.
8. Close doors, and windows to prevent contamination.
9. Record the results of your experiments directly in your
laboratory notebook.
Do not record your results on a piece of paper with the idea
that they will
be transcribed later.
10. When you have finished using the microscope, clean
excess oil from the oil immersion lens with lens paper and
xylene.
11. At the end of laboratory period, turn off the Bunsen
burner, and re-arrange your staining rack before leaving
12. Each student should have a white coat and marker.

THE MICROSCOPE
Microbiology is the science that deals with the living organisms
very small to be seen with the naked eye, thus the advent of
microbiology dates from the invention of the microscope.
Types of Microscopy:
1. Light Microscopy:
a. Bright field microscopy
b. Dark field microscopy
The source of light set at the side of the specimen, and so the
light reaching the objective lens is the light which is
reflected from the specimen. The organism appears bright
in the dark background.
c. Florescent microscopy
The bacteria stained with florescent dye and U.V. light
is used.

d. Phase contrast microscopy

Here the object appears dark in bright background.
This can be used to illustrate the structure of the
specimen.
2. Electron Microscopy:
Has a very high resolution and very high
magnification power.
a. Transmission electron microscopy (TEM)
b. Scanning electron microscopy (SEM)

Light Microscope:
The compound light microscopy is composed of:
Two lens system of magnification :
a. Ocular lens:
Usually has a magnification of X10.
b. Objective lens:
The objective lenses are of two types:
1. Dry lenses:
a. Low power objective lens, magnification is X10.
b. High power objective lens, magnification is X40.
2. Oil immersion lens:
Magnification is X100.
Magnification:
The total magnification power = magnification of the ocular
lens X magnification of the objective lens.
Total magnification with low power = 10X10 = X100
Total magnification with high power = 10X40 = X400
Total magnification with oil immersion = 10X100 = X1000

2. Stage:
On which the slide will be placed.
In the center of the stage, there is a hole through which the
object can be illuminated from below.
Its movement is controlled by the coarse adjustment knob
and fine adjustment.
3. Illuminating system:
Its composed of:
a. Lamp.
b. Condenser
Lenses gather the light from lamp.
c. Iris of diaphragm
Regulate the amount of light passed through an opening in
the stage to the slide.

Importance of oil utilization:

Because the refractive index of air is less than that of
glass, light rays are refracted as they pass from the
microscope slide into the air. Thus many of the light rays
passed from the specimen are refracted as so great angle that
they completely miss the objective. Placing oil that has the
same refractive index as glass between the slide and the
objective lens will greatly decrease refraction so increase the
amount of light passed from the specimen to the objective.
Direction for using the microscope with oil immersion
lens:
Procedure:
1. Place the slide provided on the stage, specimen side up,
and center the section to be examined as accurately as
possible over the hole in the center of the stage.

2. With the low power objective in position, adjust the lamp

until it gives maximum amount of light through the specimen
with the aid condenser and iris of diaphragm.
3. Rotate the nosepiece until the oil immersion objective clicks
into position.
4. Place a drop of cider wood oil on the portion of the slide
directly under the objective.
5. Watching the objective from the side carefully lower it into
the oil. Dont allow the objective to touch the slide, look
through the ocular, and slowly focus with the coarse
adjustment. Then, fine adjustment until a good sharp image
will appear.
6. Record your results as shown below:
Name of stain:
Name of organism:
Description:
Shape of cell:
Arrangement of cells:
Colour reaction:
7. Each time after using the oil immersion lens, clean the oil
from the objective lens with lens paper moistened with xylene.

SOURCES OF CONTAMINATION
The purpose of this is to identify some of the sources of
contamination present in the laboratory in order to avoid
them.
Material:
You have been provided with 4 Nutrient Agar plates.
a. Contamination from hands:
1. Take a Nutrient Agar plate and divide it into 4 sections:
a. Unwashed.
b.Washed.
c. Disinfected.
d. Control.
2. Imprint your finger print on (a) unwashed area.
3. Wash your hands throughly, dry your hands in air and
imprint the same finger prints on (b) washed area.

4. Wash your hands with soap, water and disinfectant, dry

your hands in air, do not use hand towel. Imprint the same
finger on the (c) disinfectant area, leave section (d) as
control.
5. Incubate your plates for 1 day at 370C and record the
appearance of the plate.
b. Contamination from breath:
1. Take a Nutrient Agar plate.
2. Hold it in front of your mouth.
3. Cough and breath vigorously.
4. Invert the plate and incubate for 1 day at 370C.
5. Record the appearance of the plate.
c. Contamination from air:
1. Expose a Nutrient Agar plate to the air on the bench for
one hour.
2. Invert the plate and incubate for 1 day at 370C.
3. Record the colonial appearance.

EXAMINATION OF THE MICROORGANISMS

UNDER THE MICROSCOPE
There are two ways in which the microorganisms may be
examined under a microscope, in the living state or in
the fixed state.
1.
In the living state:
a.
Wet Mount:
The purpose of this exercise is the examination of
living microorganisms in a liquid phase
I.
It is used to observe certain activity e.g. :

Reproduction

Motility
II.
Used in case where staining and other manipulations
affect the structure of microorganisms.

Materials:
Suspension of microorganisms in a mixed culture
bottle, marked (W).
Slides and cover slips.
Method:
1. Take a clean slide, pass through the flame of the
bunsen burner.
2. Add a loopful of culture on to the centre of the slide.
3. Carefully place a cover slip over the drop.
4. Look under the microscope under the dry lenses (X10
and X40).
5. Record the results.
b. Hanging Drop Preparation:
The object of this exercise is to observe the
microorganism and test of its motility.

Materials:
Culture (M) and (N) (One is motile and the other is
non-motile).
plastacine.
Slide and cover slip.
Method:
1. Take a clean slide.
2. Roll plasticine in between your palms to make a thin
elongated roll, and make a ring out of it.
3. Place it over the slide making a circle, smaller in size than
the cover slip.
4. Place drop of bacterial culture in the centre of cover slip,
by a loop.
5. Invert the slide over the cover slip and gently press the
slide against the plasticine ring.
6. Turnover the slide quickly, so that the cover slip is on top
and the slide at the bottom, allowing the drop to hang.
7. Look under the dry lens X10 and X40 later, record your
observations.

2. In the fixed state:

Staining:
Most microorganisms are transparent, so special
techniques are used to visualize microorganisms, one of
those techniques is staining.
1. Staining enhances the morphological appearance.
2. It is used to identify structural parts.
3. It is used to differentiate microorganisms (e.g. Gram +ve
, Gram -ve).
Smear preparation:
The purpose of this technique is to prepare the
microorganism for staining and preventing them from
being washed during the process of staining.
a. From liquid culture:
1. Take a clean slide, and pass through the flame
2. Place a loopful of culture in the centre of the slide and
spread evenly.

3.
4.
5.
b.
1.
2.
3.
4.
5.
6.

Allow it to dry in air.

Pass through the flame three times quickly to heat kill
and fix the organism to the slide.
Smear is ready to be stained.
From solid culture:
Take a clean slide and path through the flame.
Place a loopful of saline in the centre of the slide.
Take a little amount of solid culture on to the loop,
emulsify in the saline evenly.
Allow it to dry in air, and pass through the flame to
heat kill and fix the organism to the slide.
Smear is ready to be stained.
Stain the slide and record the results.

TYPES OF STAINING
Simple Staining:
The object of this exercise is to see the shape,
size of different microorganisms, protozoa and fungi,
by using a simple stain like Methylene Blue, a dye to
stain microorganisms.
Materials:
A basic dye (methylene blue).
Bacteria (Staphylococci) labled (S).
Fungus (Candida) labled (C).
Method:
1. Make a smear of the microorganism provided, as
mentioned previously.
2. Place the smear on to the staining rack.
3. Add methylene blue stain (for 2 minutes).
4. Wash the strain off the slide with water.
5. Blot the smear with filter paper.

6.
7.

Dry the slide, put the oil and look under the oil
immersion lens (X100).
Record your observations.

Negative staining:
Certain microorganisms are very difficult to
stain (e.g. Spirochetes), and can be visualized by
staining the background (negative staining) by certain
dye (India ink, nigrosine) leaving the cell transparent,
negative staining will not give any information about
the cell contents, however the cell shape and size are
easily determined.
Materials:
Culture of Bacillus species (marked B).
Nigrosine stain.

Method:
1. Take a clean slide and place a drop of Nigrosin at
one end of the slide.
2. Emulsify organisms in Nigrosin.
3. Using the edge of another clean slide spread the
drop out into a film
( like a blood film).
4. Allow it to dry in air and examine under oil
immersion objective.

Differential Staining:
It is used
More thato differentiate between microorganisms or parts of
same cell. n one dye is used in differential staining.
1. Gram staining:
Discovered by Christian Gram in 1884. This
staining process divides bacteria into two groups, Grampositive bacteria which retain the blue/violet color after
decolourization by alcohol and colored blue/violet.
Gram-negative bacteria loose their blue/violet color after
treatment with alcohol and color red by counter staining.
Material:
Bacteria labeled as (P) and (N).
Gram stain.

Method:
1. Prepare the smear as mentioned before.
2. Place the slide on the slide rack.
3. Add crystal violet for 1 minute, then wash with water.
4. Add iodine and leave on the slide for 1 minute, then
wash with water.
5. Decolorize with alcohol for 10 to 20 seconds, then wash
with water.
6. Add safranin on the slide for 30 seconds, then wash with
water, blot dry.
7. Examine under the oil immersion lens (X100).
8. Record your results.

2. Acid-Fast Staining (Ziehl-Neelsens Stain):

Acid-fast bacteria (Mycobacteria) are
difficult to stain by ordinary dye (due to high lipid
contents), so special techniques should be applied in
order to stain such bacteria. For example applying of
heat will facilitate the penetration of specific dye
(Carbol-fuchsin). Once the cells are stained they
retain the dye even after a very strong decolorizing
agent (acid alcohol) have been used. This treatment
decolorize other bacteria.
Materials:
Culture of Mycobacterium plate labeled as (Mb).
Ziehl-Neelsens stain.
Alcohol, saline.

Method:
1. Prepare smear of Mycobacterium.
2. Allow the film to dry in air. Heat fix the smear.
3. Cover the slide with concentrated carbol fuchsin.
4. Gently heat, till the steam rises.
5. Wash the slide with tap water.
6. Decolorize the smear with acid alcohol for 10 to 30
seconds.
7. Wash the slide with tap water.
8. Apply methylene blue for 30 to 45 seconds. Wash and
blot dry and examine under oil immersion objective.

Spore Staining:
Introduction:
Certain bacteria (e.g. Bacillus, Clostridium) form
endospores. Bacterial spores do not take up stains readily and
conventional techniques merely stain the vegetative portions
of the cell, leaving the spore as a clear one. By vigorous
treatment (e.g. strong stains and prolonged heat) it is possible
to stain the spore but, once introduce, the stain is resistant to
decolorizing agents. The latter bleach the vegetative parts of
the cell, which may then be counter-stained. The presence of
spores, their size, shape, position and whether they bulge the
walls of the parent cell are important characters in the
identification of bacteria.
Materials:
Culture of Bacillus species marked (B).
Malachite green.
Safranin.

Method:
1. Prepare and fix the smear with bacteria provided.
2. Cover the slide with malachite green stain.
3. Gently heat until you see steam rising (DO NOT BOIL
THE SMEAR).
4. Allow the stain to remain for 5 minutes.
5. Wash with tap water.
6. Counter-stain with safranin for 30 seconds.
7. Blot dry, and look under oil immersion objective.

Microbiological Culture Media

Cultivation of microorganisms in the laboratory is essential
for the study of their morphological and physiological
properties, also for their isolation in pure form and
identification. This cultivation is possible by using an
artificial medium.
The medium may be in liquid (broth) form, solid (containing
agar) form or semisolid (containing low concentration
of agar as solidifying agent) form.
Media may be classified on the basis of content into
complex, chemically defined (synthetic) and living
media.
1. Complex Media:
Contain extract or digest of animals (beef or meat extract),
plants (soya bean digest), or microbes (yeast extract)
that supply all the essential nutrients.

Chemically Defined (Synthetic) Media:

Essential nutrient is provided by a pure chemical of known
composition e.g. K2HPO4 , KH2HPO4 , (NH4)2SO4 ,
glucose, etc
3. Living Medium:
Designed specifically for obligate intracellular
microorganisms e.g. viruses.
Complex media may be classified as follow:
1. Ordinary or Basic Media support the growth of
ordinary microorganisms not a fastidious one:
a. Nutrient Broth is the basis for most media used for
growth of microorganisms, it contains: meat extract,
peptone and sodium chloride.

b. Nutrient Agar is nutrient broth solidified by

1.5-2% agar. It is the basis for other solid selective
or enriched media.

2.

a.

Enriched Media certain organisms e.g. gonococcus,

pneumococcus are so nutritionally that they cant grow
on the ordinary media. They require more complex
organic body fluids for their growth. Media containing
such substances are called enriched media, for example:
Blood Agar:
-It is a nutrient agar enriched with sterile blood (510%).
-Blood agar is very useful not only as an enriched
medium supporting
the growth of most fastidious
organisms, but also as indicator medium, differentiating
organisms according to their action on the blood.
-Some causes complete haemolysis and those are
called -haemolytic bacteria.

-Other causing greenish coloration or partial haemolysis

and those are called - haemolytic bacteria.
-Others producing no change, e.g. gamma-haemolytic
bacteria.

b.

Chocolate Agar: (or heated blood agar):

- Prepared from blood agar by heating of the melted
blood agar at 1000C for 2 minutes.
-Used particularly for the culture of the haemophilus
group of organisms.

3. Enrichment Media:
-They are fluid media
-They contain some substances which support the
growth of most microorganisms e.g. Thioglycolate
medium, or substances which inhibit the growth of
unwanted organisms. For example:
* Fluid Thioglycolate Medium:
-This medium supports the growth of both aerobic,
microaerophilic and anaerobic bacteria.
-It contains sod.Thioglycolate, cysteine and dextrose
as reducing agent.
-Aerobic bacteria will grow near the top whereas
anaerobic grow at the bottom and microaerophilic
grows in between.

* Selenite Broth: used for isolation of shigella and

salmonella groups from faeces.
Tetrathionate Broth: used for isolation of salmonella
causing food poisoning.

4.
a.
-

Selective Media:
Mannitol Salt Agar (MSA):
This medium is a selective medium for pathogenic
staphylococci.
It contains salt (7.5%) which will inhibit the growth
of other microorganisms except staphylococci.
Also contain mannitol as a test sugar and phenol red
as indicator.
Staphylococcus aureus ferments mannitol, producing
yellow colonies.

b.
-

Sabouraud Dextrose Agar (SDA):

It is a selective media for fungi, it contains high
concentration of dextrose, chloramphenicol to inhibit the
growth of bacteria and has pH of 5.5 6.
5. Selective and Differential Media:
-Selective media contain some dye or other chemical that
inhibits the growth of certain organisms, and allow the
growth of others.
-Differential media usually contain an indicator which
changes its color with certain organisms and not with others.
MacConkeys Medium:
- It is used in the detection and isolation of all types of
enterobacteriaceae from stools.
-It contains bile salts which will inhibit the growth of all other
microorganisms except that of enterobacteriaceae.

Also contains lactose as a test sugar and neutral red as

indicator. On this medium the lactose fermenters produce a
rose pink colony, and the non-lactose fermenters produce a
pale yellow colony.

6. Media for Anaerobic Bacteria:

Cooked meat medium.

Fluid thioglycolate medium.

Materials:
A. Liquid culture of :
1. E.coli
2. B.subtilis
3. C.albicans
4. C.perfringens
5. S.aureus
B. Media:
1. Nutrient broth
2. Nutrient agar
3. Thioglycolate broth
4. Blood agar
5. Sabouraud agar
6. Mannitol salt agar

Procedure:
1. Examine each medium for content and colour.
2. Inoculate the media with the exact microorganism
following the table.
3. Incubate for 24 hours at 370C.
4. Record your results in the table.
5. Make a wet mount preparation from the cell culture and
examine it under microscope, draw the shapes of these
cells.

No

Media

Nutrient broth

Nutrient agar

Thioglycolate broth

Blood agar

Sabouraud dextrose agar

Mannitol salt agar

E.coli S.aureus B.subtilis C.albicans

CULTIVATION OF MICROORGANISMS
Inoculation with the loop:
The object to illustrate how to handle the loop in
inoculation and the effectiveness of loop flaming.
Materials:
- 3 tubes containing 5 ml of media.
- Culture of Bacillus subtilis lablled B.
Procedure:
1. Lable the tubes 1, 2, 3 .
2. Pick up the culture (B) in your left hand between your
fore and middle fingers.
3. Pick up the loop in your right hand.
4. Sterilize the loop by flaming until it becomes red hot.
5. Remove the cap of the culture by the little finger of your
right hand.
6. Introduce the loop into the tube containing the culture
(B) withdraw.

7.
8.
9.

Flame the mouth of the tube and cap.

Transfer a loopful of the culture to tube (1).
Take another loopful from the culture, flame the
loop,and inoculate into tube (2).
10. Leave tube 3 as control.
11. Incubate all tubes at 370C for 1 day.
12. Report your results.

Isolation of bacteria by streaking technique:

This method may be used for checking the purity of a bacterial
culture, and may also be used for isolating individual
species from a mixture. Culture with single type of colony
is regarded as a pure culture.
Materials:
Nutrient Agar plates.
Bacterial culture marked (B).
Procedure:
1. With a sterile loop, transfer a loopful on to the surface of a
nutrient agar plate. Place the drop near the edge.
2. With the trailing edge draw the loop tightly over surface in
parallel line.
3. Flame the loop.
4. Turn the plate at a 900 angle and repeat the streaking across.

5.
6.
7.
8.
9.

Flame the loop.

Repeat (4) & (5) again at right angle.
Label at the back of the plate.
Invert the plate and incubate for 1 day at 370C .
Examine and describe the appearance of the growth of
the colonies on the plate.

Bacterial Count
Measurement of the bacterial growth in liquid medium can be
done by:
I.
Measuring the cell number.
II.
Measuring the cell mass.
III.
Measuring the bacterial metabolic activity.
(I)
Measuring the cell number: (Direct count)
This can be done by the following methods:
1.
Measuring the total cell number by:
a.
Using the counting chamber:
The counting chamber is divided by lines into
small squares by counting the number of organisms in all
squares, then the number of organisms per milliliter can
be calculated by multiplying the counted number by
dilution factor. This is the total count of live and dead
organisms.

b.

Using electronic cell counter (Coulter counter):

The electrical resistance of the fluid in a small
hall is measured, as each cell passes through the hole,
the resistance increases highly and the increase is
recorded. Also this is the total count of both live and
dead cells, but t is faster method than the previous
method.
2.
Measuring only the live cell (Viable count):
This method is used to determine the number of
living bacterial cells. The principle of this method is
based on the ability of each cell to give rise to a colony
if it is allowed to grow over solid medium.
Three methods are used in this determination of viable
bacteria:
A. Spread plate method.
B. Pour plate method.
C.
Dropping on Agar surface.

(A)

Spread plate method:

In this method a given volume of sample is
serially diluted and the small amount from highest
dilution is pipetted on to surface of hardened Nutrient
Agar. Then spread with glass spreader.
(B) Pour plate method:
From the sample diluted as mentioned above, a
constant amount is added from each dilution into a
sterile plate and molten Agar is added and mixed well in
the plate and Agar allowed to set.
(C) Dropping on Agar surface:
Miles and Misras technique:
Here also the sample should be serially diluted,
with a standard 50 pipette add 5 drops from each
dilution on the well dried nutrient agar plate. Allow the
drops to dry. After each experiment incubate the plates at
370C for 24 hours. Count the number of colonies and
multiply by the dilution to give you the organism / ml in
original culture.

Determination of viable count:

Materials:
1. Bacterial culture (E.coli).
2. 7 bottles each containing 9 ml Ringer solution.
3. 1ml pipettes (7).
4. Petri dish plates (3).
5. 50 l dropper.
6. Nutrient Agar plates (3).
7. 319 ml molten nutrient agar.
Method:
1. Label the bottles from 1-7 .
2. Make a serial dilution as follows:
a.
Discard the used pipette, then by using a sterile
pipette mix the contents of bottle # 1 and remove 1 ml
into bottle # 2 .
b.
Repeat this step up to bottle # 7 .

3.
A.
I.
II.

III.
IV.

From the last 3 dilutions (10-5, 10-6, 10-7( do the

following:
Pour plate:
Label 3 petri dishes matching the 10-5, 10-6, 10-7
dilution.
From each dilution transfer 1 ml of the diluted culture
to petri dish then add 19 ml of melted Agar and mix
well.
Wait until the Agar solidify, then incubate at 370C for
24 hours. Count the No. of colonies / dilution.
Calculate the # of CFU / ml.

B.
I.
II.

III.
IV.
V.

Surface viable count:

Label 3 nutrient agar plates of the dilution (10-5, 106, 10-7(.
Using the dropper (50) l starting from (10-7(
dilution transfer 5 drops to each Plate, then go to the
next dilution (10-6( using the same dropper. Repeat
that with final dilution (10-5( .
Wait till the drops dry. Incubate at 370C for 24 hours.
Count the colonies in each drop for average
concentration. Take the average of 5 drops.
Calculate the # of CFU / ml.

(II)
A.

B.

C.

Measuring the cell mass:

Turbidimetric method:
This method depends on the change in the
optical density that occurs due to the increase in the
number of bacterial cells. The number of cells is
directly proportional to the amount of light
absorbed.
Determination of net weight:
This is done by centrifuging the cells and
weighing the pellet of cells obtained.
Determination of dry weight:
It is done by drying the centrifuged cells
mass before weighing by placing it overnight in an
oven at 100-1050C.

(III) Measuring the bacterial metabolic activity:

By measuring either the metabolic products
e.g., acids or the decrease in the substrates that are
consumed in the metabolic activity e.g. sugars.

EFFECT OF ENVIRONMENTAL CONDITIONS ON

BACTERIAL GROWTH
A.

Effect of temperature on bacterial growth:

Temperature is one of the most important factors
influencing growth of bacteria since temperature effect
enzymes activities. In this exercise the effect of different
temperatures on growth will be examined.
Materials:
- Culture of Echerichia coli and Bacillus subtilis.
- Eight Nutrient Broth.
Methods:
1. Label the tubes as follow:
- E.coli (1-4).
- B.subtilis (5-8).
2. Inoculate tubes (1-4) with E.coli.
3. Inoculate tubes (5-8) with B.subtilis.

4. Incubate the tubes as follow:

Tubes No.

Temp.

1,5
2,6
3,7

50C
250C
370C

4,8

450C

5. Record your results.

B. Effect of pH on bacterial growth:

pH effects bacterial growth since it limits the activity
of enzymes required for bacterial growth.
Materials:
- Culture of E.coli , S.aureus and C.albicans.
- Nine Nutrient Broth at pH 3, pH 7, and pH 9 .
Method:
1. Label tubes with the proper bacterial name at different
pH.
2. Inoculate each tube with the labeled bacteria.
3. Incubate at 370C for 24 hours.
4. Report your results and comment.

C. Effect of osmotic pressure on bacterial growth:

Cell walls protect prokaryotes against changes in
osmotic pressure over a wide range. However, sufficiently
hypertonic media at concentrations greater than those inside
the cell (such as 20% sucrose) cause water loss from the cell
by osmosis. Fluid leaves the bacteria causing the cell to
contract, which, in turn, causes the cell membrane to separate
from the overlying cell wall. This process of cell shrinkage is
called plasmolysis.
When bacteria are placed in hypotonic media with
concentrations weaker than the inside of the cell, water tends to
enter by osmosis. The accumulation of this water causes the
cell to swell and then to burst, a process called osmotic lysis.
Materials:
Culture of E.coli and S.aureus .
Nutrient broth at 0.9% NaCl and 5% NaCl (2 each).

Methods:
1. Label tubes with proper bacterial name at different
salt concentration
2. Inoculate each tube with the labeled bacteria.
3. Incubate at 370C for 24 hours.
4. Report your results.

D. Effect of heat:
Destruction by heat:
Non-spore forming bacteria (vegetative cells) can not
withstand temperature at 600C within 30-60 min.
Spore forming bacilli can withstand this temperature and
can withstand boiling from few minutes to 3 hours but
will kill at 1200C at 2 atmosphere steam pressure within
20-30 minutes.
In this experiment you will compare the heat resistance
of Bacillus as example of spore forming bacteria and
S.aureus as example of vegetative cells.
Materials:
1. Nutrient broth 15 ml each (6 tubes).
2. Culture of S.aureus (S) and of Bacillus subtilis (B).
3. Nutrient broth 5 ml each (30 tubes).
4. 3 water baths set at 600C, 750C and 1000C.

Procedure:
1. Divide the six large tubes into 2 groups each containing
3 tubes.
2. 0.5 ml of the culture (S) is transferred to each tube of
one group, and 0.5 ml of culture (B) is transferred to
each tube of the other group.
3. A tube of each group is taken and placed in water bath
set at 600C, 750C and 1000C respectively.
4. On specific time interval 0, 5, 15, 30 and 60 min., one
loopful of each tube is transferred to a labeled tube of 5
ml Nutrient broth.
5. The tubes of 5 ml Nutrient Broth are collected and
incubated at 370C overnight.
6. Read the results and tabulate as follows:

Effect of Heat on Bacteria at 600C

Time
Organism
S.aureus
B.subtilis

0 Min.

5 Min.

15 Min.

30 Min.

60 Min.

Effect of Heat on Bacteria at 750C

Time
Organism
S.aureus
B.subtilis

0 Min.

5 Min.

15 Min. 30 Min. 60 Min.

Effect of Heat on Bacteria at 1000C

Time

Organism

0 Min.

5 Min.

15 Min.

30 Min.

60 Min.

CONTROL OF MICROBIAL GROWTH

Microbial growth can be controlled either by:
-Complete killing of all M.O (sterilization)
-Or inhibiting the growth of M.O.
Sterilization is an absolute term. This means that the sterile
substance is completely free from any M.O.
The methods used for sterilization are classified into:
-Physical methods.
-Chemical methods.
Physical methods of sterilization:
1. Heat (Dry & Moist) 2. Radiation (U.V. light) 3. Filtration

1.

Dry Heat:
The thermal death of M.O. takes place due to
inactivation of essential cellular proteins or enzymes
through oxidation.
Methods:
a. Incineration e.g. platinum loop.
b. Flaming e.g. mouth of culture tubes.
c. Hot air oven e.g. 1600C for 1 hr.
Sterilization of dry glass wares as test tubes, flasks
pipette, petri dishes ..etc.
Sterilization of fixed oils and powders.

2. Moist Heat:
The thermal death takes place through coagulation.
Methods:
A.
At temperature below 1000C:
- Pasteurization of milk (630C for 30 min.[holder
method] or 720C for 20
sec.[flash method]).
Such temperature is enough to kill any pathogenic
M.O.that can be transmitted by milk e.g.
Mycobacteria, Salmonella and Brucella.
- Tyndalization 560C for 1 hr on several successive
days. This method is used only for thermolabile
substances in which bacterial spores can germinate
between the first and second heating as milk, serum
and body fluids.
B.
At temperature of 1000C:
1.
Boiling at 1000C for killing of non-spore forming
M.O.
2.
Steaming at 1000C e.g. sterilization of sugar media
and gelatin media either by:
I.
Steaming for 90 min.

II.
3.

Steaming for 20-40 min on three successive days.

Heating with bactericide e.g. 0.2% chlorocresol or
0.002%phenyl mercuric nitrate
C.
At temperature above 1000C e.g. Autoclaving:
Autoclaves are widely used in sterilization of culture
media, surgical supplies and many other instruments.
A complete sterilization can occur at 15 Ib per sq. in
guage pressure at 1210C for 15 min.
Sterilization by Radiation:
1.
Ultraviolet light:
The ability of sun light to kill bacteria is mainly due
to the ultraviolet rays.
The germicidal activity of ultraviolet light increased
as it is wavelength () decrease and intensity increase.

Ultraviolet radiations can be produced by mercury

vapour lamps
The lethal effect of U.V. rays is due to it is effect on
the DNA of the cell through the formation of
pyrimidine dimer on the same strand.
Ultraviolet rays are used for sterilization of air (e.g.
operating theatres), fluids and punches.
2. Ionizing radiation:
They are very lethal to cells, and their lethal effect is
due to either direct ionizing of the cellular DNA or
by the chemical effect of ionized water, e.g. -rays
which have a very high penetration power, and
widely used for sterilization of plastics (e.g.
disposable syringes, pipettes and dishes) foodstuffs
and drugs.

Sterilization by filtration:
Fluids can be rendered free from bacteria by passing
through special filters.
This method is used in making sterile preparations of
the soluble products of bacterial growth such as
toxins, and for sterilization of liquids that would be
damaged by heat such as serum and antibiotic
solutions also for oil damage by heat.

CHEMICAL METHODS USED FOR

MICROBIAL CONTROL
The microbial control can be achieved by using chemical
antimicrobial agents.
Definition of some terms:
Bactericide: An agent which kills bacteria.
Bacteriostat: An agent which inhibits the microbial growth.
Fungicide: An agent which kills fungi.
Fungistat: An agent which which inhibits the growth of
fungi.
Disinfectant: Is a substance which has the ability to kill M.O
when applied to the surface of an inanimate object.

Antiseptic: Is a substance which has the ability to kill M.O.

on living tissues.
Preservatives: Are agents which are used in food stuffs and
many pharmaceutical preparations to prevent microbial
spoilage of the product and to minimized the risk of consumer
acquiring infection when the preparation is administered.
Evaluation of the antimicrobial agents:
A. Determination of MIC:
The principle of evaluation is based on determining
the minimum inhibitory concentration MIC of the agent.
MIC: Is the lowest concentration of the agent which inhibits
the growth of a given microorganism in a given time.

Methods which can be applied to determined MIC:

1. Serial dilution in broth (Broth dilution method):
A serial dilution of the agent is made in broth,
and the tubes are inoculated with the test organism
and incubated. The lowest concentration at which no
growth occurs in taken as the minimum inhibitory
concentration (MIC).
2. Serial dilution in agar (Agar dilution method):
In this method the dilutions of the substance
under test are made in agar instead of broth. The agar
containing the substance under test is subsequently
poured onto petri dish. This method has the
advantage over the previous one in which on a single
concentration of tested substance, several organisms
may be tested.

Determination of MIC by broth dilution method:

Materials:
A standard solution of disinfectant / antibiotic .
9 ml Mueller-Hinton broth tubes (5).
(E.coli) culture labeled as (E).
Procedure:
1. Label the 5 broth tubes from 1-5 with marker.
2. Add 1 ml of disinfectant to tube 1and mix .
3. Add 1 ml from tube 1to tube 2 and mix .
4. Continue dilutions in this manner to tube 5 .
5. Add 50 l E.coli broth culture in each tube with a
dropper.
6. Shake well the tubes and incubate at 370C for 24
hours.
7. Record the results.

Susceptibility by disk diffusion method:

The susceptibility of tested microorganism to
the discs of filter paper impregnated in different
antimicrobial agents is tested, and the zone of
inhibition are observed.
Materials:
Mueller-Hinton agar plates.
Antibiotic discs.
E.coli culture labeled (E).
Procedure:
1. Using sterile cotton swab inoculate the surface of
Mueller-Hinton agar plate with bacterial culture (E).
2. With sterile forceps apply the different discs of
provided antibiotics to the surface of the agar plate.

3.
4.

5.

Incubate at 370C for 24 hours..

Measure the zone of inhibition in mm of each disc
using the bottom of the plate.
Record the results.

Evaluation of disinfectants (Determination of Phenol

Coefficient):
This is a method used to measure the bactericidal
power of disinfectants. Phenol is used as the standard and
the agents activity is compared to phenol ability that kills
standard culture of bacteria at specific time.
The Rideal-Walker test:
Materials:
-Phenol solution 10% .
-Disinfectant solution 1% .
-Culture of E.coli .
-Nutrient broth tubes (40).
-Sterile test tubes (20).

Method:
1. Label 5 tubes and make 5 dilutions of phenol with
distilled water as follows:
1/95 , 1/100 , 1/105 , 1/110 , 1/115 .
2. Take an other set of test tubes (5 tubes) and dilute
the disinfectant with distilled water to give the
following dilution:
1/100 , 1/200 , 1/300 , 1/400 , 1/450 .
3. Label 5 sterile test tubes A, B, C, D and E . From
each phenol dilution add 5 ml to each tube.
4. Take an other set of sterile test tubes (5 tubes) and
label them K, L, M, N and P . To each tube add 5
ml of the different concentration of disinfectant.

5.

For each set of tubes (phenol disinfectant) the

following steps are followed:
6. Take 20 tubes of nutrient broth and arrange them in a
rack in four rows of five. Label them as below:
A 1- 4
K 1-4
B 1- 4
L 1-4
C 1- 4
M 1-4
D 1- 4
N 1-4
E 1- 4
P 1-4
7.

Take 1 ml from the culture add 0.2 ml to tube (A) then

at 30 second interval add another 0.2 ml to tube (B).
Continue the addition every 30 seconds until tube (E).
(Avoid touching the wall of the tube).

8.
9.
10.
11.
12.
13.
14.
15.

After 30 seconds from the previous step, using a loop,

transfer a loopful from tube (A) to (A1).
Repeat this step with tube (B), then (C), (D) and (E).
Then transfer another loop from (A) to (A2) and
repeat for the other.
Continue this treatment until tube (E4).
The same procedure is done for (K) - (P) tubes.
Incubate all broth tubes for 42-48 hrs at 320C.
Examine the tubes for growth and record the result as
positive and negative in the provided table.
Phenol coefficient is the dilution which shows growth
in 2.5 minutes and 5minutes but not in 7.5 minutes
and 10 minutes, divided by the phenol concentration
which shows the same results.

2.5

A1

B1

C1

D1

E1

A2

B2

C2

D2

E2

7.5

A3

B3

C3

D3

E3

10

A4

B4

C4

D4

E4

Exposure time (minutes)

Bacteria

Dilution
2.5

Phenol

Tested Disinfectant

7.5

10

1/95
1/100
1/105
1/110
1/115

+
+
+
+

+
+
+

+
+

1/100
1/200
1/300
1/400
1/450

+
+
+
+

+
+
+

+
+

R-W Coefficient:
tD
dilution of disinfectant kill M.O. in 7.5 min. not at 5 min.
300
__ = _________________________________________
= ____ = 2.9
pD dilution of phenol which kill M.O. in 7.5 min. not at 5 min. 105

Sterility Testing
Sterile object is an object that is completely free from all
living organisms e.g. bacteria, fungi, virus, etc. The term
sterile is an absolute term, i.e., the material is sterile or not.
Sterility test is used to insure the sterility of the object like
liquids that will be injected into the body, ophthalmic
preparations, sutures, and surgical dressings.
Sterility tests are carried out on statistically significant
random samples of the batch and carried out for bacteria
and fungi only.

Media for bacteria:

For both aerobic, anaerobic and microaerophilic bacteria
fluid thioglycolate broth is used. Incubated at 35 37 C
up to 10 days, checked daily.
Media for Fungi:
Sabouraud dextrose broth (SDB) is used. Incubate at 22
25 C, checked daily.
Positive control:
o Fluid thioglycolate media are inoculated with
staphylococcus aureus as aerobic bacteria and
Clostridium histolyticum as anaerobic bacteria.
o Sabouraud dextrose broth is inoculated with Candida
albicans.

Negative control:
o Both of the media used above with out inoculation
are incubated as negative control.
Materials:
Ampoule of 2 ml distilled sterile water.
Sterile Sabouraud dextrose broth.
Sterile fluid thioglycolate.
Procedure:
I. From the ampoule of the sterile distilled water 1 ml
is transferred to the Sabouraud dextrose broth and the
other 1 ml is added to the fluid thioglycolate medium.
ii. Positive and negative controls are also made.
iii. Inoculate and check the result.