GENERAL

MICROBIOLOGY
(MIC125)
LABORATORY MANUAL

NAME: _________________________________________________________________________________

CLASS: __________________________________________________________________________________

SEMESTER: ______________________________________________________________________________
LIST OF PRACTICALS

PRACTICAL TITLE PAGE

The usage of compound microscope and the examination of

1 3
cells.

2 Examination of living bacteria. 9

The staining of microorganisms

3 12
– Simple Staining and Gram Staining

The staining of microorganisms

4 17
– Capsule Staining and Spore Staining

5 Preparation of culture media 20

6 Pure culture Techniques 23

7 Viable count of bacterial culture 29

8 The use of ultraviolet radiation for sterilization 32

2
PRACTICAL 1: The Usage of Compound Microscope and
The Examination of Eukaryotic Cells.

Objectives:

i. To learn the structure and basic use of the compound microscope.

ii. To examine the structure of eukaryotic cells (Plantae and Animalia)

Overview:

The microscope is a biologist's basic tool. It has been developed to help explore the
world of living things too small to be seen with the naked eye. Early microscopes had
only one lens and were difficult to use. The biggest problem was magnification. The
more powerful the lens needed for greater magnification, meant the closer the
viewer's eye had to be to the lens. At very high magnification, the lens almost
touched the eye. The early microscope user had to be very steady. A major advance
in microscopes came with the invention of the compound microscope. It has two sets
of lenses, which magnify objects much greater than a single lens.

Plant and animal cells have several differences and similarities. For example, animal
cells do not have a cell wall or chloroplasts but plant cells do. Animal cells are mostly
round and irregular in shape while plant cells have fixed rectangular shapes. Plant
and animal cells are both eukaryotic cells, so they have several features in common,
such as the presence of a cell membrane, and cell organelles, like the nucleus,
mitochondria and endoplasmic reticulum.

3
Materials:

Compound microscope
Glass slides
Cover slips
Old newspaper (for letter 'e')
Scissors
Prepared slides – Plant and Animal Cell

Procedure:

A. Regular usage and care of compound microscope

i. Carry the microscope firmly with both hands; one on the arm and the other
under the base of the microscope. Carry it in upright position to your bench.
ii. Keep the microscope at least 6 inches from the edge of your work bench and
two feet from any open flame.
iii. Remove the dust cover and store it properly.
iv. Before switching the light on, turn the intensity dial to the lowest setting. This
will avoid the bulb being blown. If the microscope is not functioning properly,
report to the lab staff.
v. The position and lighting conditions of the condenser are correct and
appropriate.
vi. Wipe the lenses with lense paper before and after use, and do not touch any
lenses with your fingers.
vii. Check all the objective lens to make sure they are screwed properly. Do not
take anything apart.
viii. Ensure that all the optical sections are fully clean.
ix. Suitable oil must be used for all objective lenses requiring oil immersion.
x. 2 eyes and not one must be properly used for microscope with binocular
eyepieces, and adjusted to fit an individual's face width, giving a single
and properly aligned image field.
xi. The light rays should be avoided from direct contact with eyes, before
selecting the objectives and in any subsequent transition.
xii. After using the microscope, please ensure that:
a. Lens cleaning tissues are used to clean up the respective objective
lens immediately. A proper lens cleaning solution (eg: ether, ethyl
ether, ethanol etc..) may be used when necessary. This step is
particularly important in order to avoid any visible interference, as a
result of debris or oil particles left over during usage, which also enable
good storage and subsequent usage.
b. The microscopes are kept properly in the cupboards with their dust
cover fitted in properly.

4
B. Introducing the structure of microscope

The compound microscope has four basic parts: the lens system, the focusing
system, the stage, and the lighting system.

a) The Lens System

One of the two sets of lenses is the objective lenses. They work similarly to
the lens of the early, simple microscope. The objective lenses make the initial
or primary magnification. They are located in the nosepiece of the
microscope. Inscribed on each objective is the magnification or power of that
lens. This tells the number of times the lens magnifies the image. For
example, if you are looking at a strand of hair with a 4X lens, the hair will
appear four times its actual size. Your microscope probably has at least two
objective lenses. Some microscopes have as many as four objectives. Rotate
the lenses in the nosepiece until they click into position. The objective lens in
use is always the one directly under the body tube.

Usual powers for objective lenses are:

4X The scanning lens.

10X The low power lens.

40X The high power lens.

100X The oil immersion lens. (NOTE: This 100X lens should not
be used without special instructions from your instructor)

5
 The second kind of lens in the microscope is the ocular – sometimes called
the eyepiece. This lens is located at the top of the body tube. The ocular
serves as a small telescope, magnifying the image made by the objective
lens. This enlargement is called the secondary magnification. The
magnification of the ocular may be 5X, 10X, 15X, or 20X. The most common
power used in microscopes is the 10X ocular. Examine the ocular of your
microscope. Do not remove it from the body tube. If the power is not
stamped on the top portion of the ocular, you should assume that it is
10X. The total magnification of the microscope is determined by multiplying
the primary magnification (from the objective) by the secondary magnification
(from the ocular).

For example, if the objective lens is 10X and the ocular is 10X,
the total magnification is:

10X x 10X = 100X.

b) The Stage

A specimen to be viewed through the microscope is mounted on a glass slide

and covered with a cover slip. The slide rests on the stage, the flat surface
beneath the body tube. Stage clips hold the slide in place. Also, they help in
making slight adjustments in the slide's position by holding the slide steady.
The stage should always be kept in a horizontal position. If you tilt the stage,
the specimen will slip to the bottom edge of the slide. Even with commercially
prepared slides, the stage should be kept horizontal. A commercial slide can
be ruined as the cover slip slowly slips downward on a tilted stage. Both the
slide and the stage are extremely smooth. Water between them acts like glue
and causes the slide to stick to the stage. If water gets on the stage, STOP,
and dry both the stage and the bottom of the slide with a paper towel before
proceeding.

c) The Lighting System

For you to see the specimen, light must pass through it and the lenses to your
eye. The lighting system is located under the stage of the microscope. There
are three different types of lighting systems. The simplest system uses a
concave mirror to focus a beam of light on the slide. Tilt the curved surface of
the mirror to face a light source: room lights, windows, or a desk lamp.
Another lighting system uses a lens under the stage to focus the light. If there
is a sub stage lens and a mirror on your microscope, use the flat side of the
mirror to reflect light through this lens.

A third lighting system uses a sub stage light instead of a mirror. If your
microscope has a light, turn it on only when you are actually looking at the
specimen. The light gets hot and can easily destroy your specimen. Under the

6
 stage you will also find the diaphragm. It is used to adjust the amount of light
that passes through the specimen. The diaphragm works like the aperture on
a camera. Practice opening and closing the diaphragm while looking through
the eyepiece. Notice how the amount of light increases and decreases.

d) The Focusing System

In order to bring the image of the specimen into proper focus, it is necessary
to change the distance between the slide and the objective lens. This can be
done in one of two ways, depending upon the microscope you are using.
Either the lenses can be moved or the stage upon which the slide rests can be
moved. Two knobs control the focus.

The coarse adjustment knob is for coarse focusing, and the fine adjustment
knob is for fine focusing. Locate these on your microscope. Turn the coarse
adjustment knob.

C. Using the compound microscope

i. Put the low power objective in place. Look through the ocular and adjust the
light so that you see a uniformly bright field of view. The field of view, also
called the field, is the area you see through the lens. If you see specks of dirt
in the field, clean your lenses with lens tissue.
ii. Now prepare a slide to view under the microscope. Cut a lowercase "e" from
an old newspaper and place it in the centre of a clean glass slide.
iii. Put a drop of water on top of the letter.
iv. Next, place the edge of a cover slip against the water, and gently lower the
cover slip over the "e." Placing the cover slip in this manner prevents bubbles
from forming.
v. Be sure that the bottom of the slide is dry. This type of slide is called a wet
mount.
vi. Place the slide under the stage clips, so that the "e" is right side up. You are
now ready to focus on the "e."
vii. Focusing always begins with the lower power (4X and 10X) objective. First,
click the low power objective into position in the nosepiece. Then, looking at
the side of the microscope, turn the coarse adjustment knob until the objective
is as close as possible to the slide without touching it.
viii. Now look through the ocular and turn the coarse adjustment knob in the
direction that will move the objective away from the stage. The "e" will come
into approximate focus. To sharpen the focus, turn the fine adjustment knob
back and forth.

7
 ix. Note the position of the letter through the microscope. The letter on the slide
is right side up.
x. Now, look at the "e" under high power. First, under low power, center the "e"
in the field of view. Switch to high power by turning the nosepiece until the
high power objective clicks into place.
xi. Sharpen the focus by turning the fine adjustment knob.
xii. If you cannot find the "e" under high power, try this. Look through the ocular
and move the slide slightly. If this does not bring the "e" into view, move the
slide in other directions.
xiii. Record your observation by both low and high power objective lenses.
xiv. Repeat the whole procedures with the prepared slides of plant cell and animal
cell.

Caution:

i. If the lens is dirty or you get water on it, gently wipe it with lens tissue. Never
use facial tissues. Lenses are made of soft glass and scratch easily.
ii. Be sure that the objective does not touch the slide; both the lens and slide can
be damaged. Do not look through the eyepiece while lowering the objective
toward the stage. It is difficult to judge through the eyepiece how far the
objective is moving.
iii. Never use the coarse adjustment knob in high power. The objective is very
close to the slide in high power, and coarse adjustment could cause the
objective to hit the slide.
iv. When you are finished using the microscope, remove the slide from the stage.
Rinse the slide and cover slip with water. Dry the slide with a paper towel and
not lens tissue.
v. Glass and cover slips should be air dried to prevent breakage. Return both to
their proper places. Finally, be sure the microscope is on low power and put it
away.

Question:

1. Do the lenses move up and down or does the stage move up and down?
2. Is the position of the letter viewed through the microscope the same as it is on
the stage?
3. What is the relationship between movement on the stage and movement seen
through the lenses?
4. Note the length of each lens. Is the higher power lens longer or shorter than
the lower power lens?

8
PRACTICAL 2: Examination of Living Bacteria

Objectives:

i. Prepare and observe wet-mount slides and hanging-drop slides.

ii. Distinguish between true motility and Brownian movement.

Overview:

Bacteria constitute a large domain of prokaryotic microorganisms. Typically a few

micrometres in length, bacteria have a number of shapes, ranging from spheres to
rods and spirals. Bacteria do not have a membrane-bound nucleus, and their genetic
material is typically a single circular bacterial chromosome of DNA located in the
cytoplasm in an irregularly shaped body called the nucleoid. The nucleoid contains
the chromosome with its associated proteins and RNA.

Using a wet-mount technique, you will examine different environments to help you
become aware of the numbers and varieties of microorganisms found in nature. The
microorganisms will demonstrate either Brownian movement or true motility.
Brownian movement is not true motility, but rather movement caused by the
molecules in the liquid striking an object and causing the object to shake or bounce.
In Brownian movement, the particles and microorganisms all vibrate at the same rate
and maintain their positions. Motile microbes move from one position to another; we
call this “purposeful movement”. Their movement appears more directed than
Brownian movement, and occasionally the cells may spin or roll.

9
Materials:

Compound microscope
Cavity glass slides
Cover slips
Inoculating loop
Petroleum Jelly
Paper towels
Applicator sticks
Disposable pipettes
24-hours nutrient broth cultures of:
a. Staphylococcus aureus
b. E. coli

Procedure:

A. Wet Mount Techniques

i. Using one of the disposable pipettes, suction up some of the nutrient broth
that contain bacteria (use different pipette for different bacteria broth!) and
transfer a small drop to the center of a glass slide.
ii. Carefully place a coverslip on top of the drop of the fluid.
iii. Place the slide on the stage of your microscope and observe with low power.
iv. Adjust the iris diaphragm so that only a small amount of light is admitted.
v. Concentrate your observations on the larger, more rapidly moving organisms.
At this magnification, bacteria are barely discernible as tiny dots.
vi. Next, examine the slide with the 40x objective. Increase the light using the iris
diaphragm as needed. Some microbes are motile, whereas others only
exhibit Brownian movement.
vii. Record your observations.
viii. After recording your observations, examine the slide with the oil immersion
lens. Bacteria should now be magnified sufficiently to be seen.
ix. Record your observations, noting the relative size and shape of the
organisms.
x. Make a wet mount from the other bacterial broth, and observe it, using the low
and high power objectives. Record your observations.

10
B. Hanging Drops Techniques

i. Obtain a clean cavity glass slide.

ii. Pick up a small amount of petroleum jelly on an applicator stick.
iii. Using the technique demonstrated by the instructor, carefully touch the
petroleum jelly to all four edges of a coverslip to get a small rim of petroleum
jelly around the entire coverslip. Be careful not to get petroleum jelly on the
other side of the coverslip or smeared on the middle of the coverslip!
iv. Place the coverslip on a paper towel, with the petroleum jelly-side up.
v. By using inoculating loop, transfer a small drop of the bacterial broth onto the
coverslip.
vi. Place a cavity glass slide over the drop, scoot the paper towel to the edge of
the table, and carefully and quickly flip the slide over so that the drop of fluid is
suspended upside down from the coverslip into the space provided by the
cavity glass slide.
vii. Examine the drop under low power by locating the edge of the drop and
moving the slide so the edge of the drop crosses the center of the field.
viii. Reduce the light with the iris diaphragm and focus.
ix. Observe the different sizes, shapes, and types of movement.
x. Switch to high-power and record your observations. Do not attempt to use
the oil immersion objective!
xi. Using a new coverslip, repeat the procedure with the culture of another
bacterial broth. Record your observations.
xii. When completely finished, make sure your microscope has been cleaned and
return it to the proper cupboard.

Question:

1. How would you describe in words the size and shape of the microorganisms
that you see down the microscope?
2. Do you think the microorganisms are alive?
3. The movement of bacteria towards nutrients is known as ______________.

11
PRACTICAL 3: The Staining of Microorganisms -
Simple Staining and Gram Staining

Objectives:

i. Explain the rationale and procedure for the Simple Staining and Gram
Staining.
ii. Differentiate between Simple Staining and Gram Staining.

Overview:

Microbiologists study the characteristics of microorganisms such as algae, protozoa,

bacteria, fungi and viruses using a microscope. While some organisms such as
protozoa and yeast cells are easy to observe using a wet mount, bacterial cells
require staining. Scientists developed several methods such as Gram staining, acid-
fast staining and fluorescent staining for better visualization of bacterial cells and
cellular structures.

Using such staining methods, it is possible to identify structural features that help
classify bacteria. Bacterial organisms are so small that most of them are visible only
under a microscope with a magnification power of 1000X. However, mere
magnification of size does not provide a sufficient degree of clarity, so that bacteria
must therefore be stained before observation to provide the clarity needed for
visualization.

Staining bacteria to distinguish between bacterial types is known as differential

staining. The Gram stain is one such differential stain that distinguishes between
bacteria on the basis of their cell wall content. In this method, the bacterial cells react
with a crystal violet stain to take up a violet color. On adding a de-staining agent,
some bacterial cells lose color whereas others don't. On adding safranin stain, the
decolorized cells take up the stain to appear red while the bacterial cells that didn’t
lose color remain violet. The bacterial cells that take up the red color are called Gram
negative organisms and those that don’t take up the color are classified as Gram
positive organisms. Gram staining provides a rapid method for the initial identification
of bacteria involved in infections.

12
Materials:

Compound microscope
Student's saliva
24-hours cultures of:
- E. coli
- Staphylococcus aureus
- Bacillus subtillis
- Pseudomonas aeriginosa
- Unknown X
- Unknown Y
Glass slides
Cover slips
Inoculating loop
Crystal violet
Methylene Blue
Carbol fushsine
Test tubes
Paper towel
Gram’s Iodine
95% Ethanol
Safranin
Bunsen burner
Staining rack

Procedure:

A. Simple Staining Techniques

i. Clean and dry microscope slides thoroughly.

ii. Flame the slide's surface in which the smear is to be spread.
iii. Flame the inoculating loop.
iv. Transfer a loop full of tap water to the flamed slide surface.
v. Reflame the loop making sure the entire length of the wire that will enter the
tube has been heated to redness
vi. Remove the tube cap with the fingers of the hand holding the loop.
vii. Flame the tube mouth.
viii. Make sure the inoculating loop is not so hot that it will distort the
bacterial cells; then pick up a pinhead size sample of the bacterial growth.
ix. Reflame the tube mouth, replace the cap, and put the tube back in the holder.
x. Disperse the bacteria on the loop in the drop of water on the slide and spread
the drop over an area the size of a 10 cent coin. It should be a thin, even
smear.
xi. Reflame the inoculating loop to redness including the entire length that
entered the tube.
xii. Allow the smear to air dry thoroughly.

13
 xiii. Heat-fix the smear cautiously by passing the underside of the slide through
the burner flame two or three times. Test the temperature of the slide after
each pass against the back of the hand. It has been heated sufficiently when
it feels hot but can still be held against the skin for several seconds.
Overheating will distort the cells.
xiv. Stain the smear by flooding it with one of the staining solutions and allowing it
to remain covered with the stain for the time designated below.

Methylene blue - 1 minute

Crystal violet - 30 seconds
Carbol fuchsin - 20 seconds

xv. During the staining, the slide may be placed on the rack or held in the fingers.
xvi. At the end of the designated time rinse off the excess stain with gently running
tap water. Rinse thoroughly.
xvii. Wipe the back of the slide and blot the stained surface with paper towel.
xviii. Place the stained smear on the microscope stage, smear side up and focus
the smear using the low and high objectives lens.
xix. Choose an area of the smear in which the cells are well spread in a
monolayer. Put cover slips at the centre area to be studied, apply oil and
focus the smear with the 100X objective.
xx. Repeat all procedures with different type of bacteria and saliva (student have
to spit their saliva inside a test tube first!)
xxi. Record your observations as follow:

E. coli S. aureus B. subtillis

Methylene
Blue
P. aeriginosa Saliva

14
 E. coli S. aureus B. subtillis

Crystal
Violet
P. aeriginosa Saliva

E. coli S. aureus B. subtillis

Carbol
fuchsin
P. aeriginosa Saliva

15
B. Gram Staining Techniques

i. Prepare your smear. Gently agitate your E.coli culture broth tube to disperse
the bacteria.
i. Sterilize your inoculating loop and let it cool for 20-30 seconds.
ii. Place loop in the bacterial broth and put the loopful of the broth onto the glass
slide. Rub the drop into a 10 cent-sized smear.
iii. Sterilize the loop again to kill any remaining bacteria and let the smear to air
dry completely. Do not use heat to dry your smear!
iv. Fix the slide by using heat-fixing method; Hold the back-side of the slide
directly in front of the flame for 8 seconds. Be careful not to touch the front of
the flame. Place the slide against your hand. If the slide is warm, you may
proceed with staining. If the slide is not warm, repeat heat-fixing.
v. Stain your smear. Use a slide rack to hold your slide. Cover the smear with
crystal violet and let it sit for 1 minute. Rinse the slide carefully with tap water.
Do not turn the water on too high as it will wash the smear off your slide.
vi. Cover the smear with iodine and let it sit for 1 minute. Repeat rinse step.
vii. Pick up slide off of staining rack and tilt downward over the sink. Squirt
decolorizer (ethanol) onto the slide, directly ABOVE your smear.
viii. The decolorizer should run off of the slide. You should see purple running off
of your slide. Stop decolorizing when there is no longer any excess
purple running off. (5 – 10 seconds)
ix. IMMEDIATELY rinse smear with water and place smear back onto staining
rack to cover the smear with safranin and let it sit for 30 seconds.
x. Rinse the slide once last time with tap water and shake excess water off of
your slide and carefully blot between two layers of paper towel.
xi. Examine the stained slide microscopically; using the low, high and oil
immersion objectives to determine your sample is Gram Positive or Gram
Negative.
xii. Repeat procedures with Staphylococcus aureus, Bacillus subtillis, Unknown X
and Unknown Y cultures.

Question:

1. Are there any bacteria in your saliva sample?

2. Are they any differences between the cell wall of Gram positive and Gram
negative bacteria, which might explain differences in the rate of decoloration?
3. Based on your opinion, do you think gram staining is a simple procedure?
4. Can you add iodine before crystal violet when preparing a Gram stain?

16
PRACTICAL 4: The Staining of Microorganisms -
Capsule Staining and Spore Staining

Objectives:

i. Explain the rationale and procedure for the Capsule Staining and Spore
Staining.

Overview:

The cell capsule is a polysaccharide layer that lies outside the cell envelope of
bacteria, and is thus deemed part of the outer envelope of a bacterial cell. It is a well-
organized layer, not easily washed off, and it can be the cause of various diseases.

The capsule—which can be found in both Gram-negative bacteria and Gram-positive

bacteria—should not be confused with the second lipid membrane, which contains
lipopolysaccharides and lipoproteins and is found only in Gram-negative bacteria.
Because most capsules are so tightly packed, they are difficult to stain using
standard stains because most stains cannot adhere to the capsule. For examination
under the microscope, the bacteria and their background are stained darker than the
capsule, which doesn't stain. When viewed, bacterial cells as well as the surface
they are on, are stained dark, while the capsule remains pale or colorless and
appears as a ring around the cell.

Bacterial spores are highly resistant, dormant structures (i.e. no metabolic activity)
formed in response to adverse environmental conditions. They help in the survival of
the organisms during adverse environmental conditions; they do not have a role in
reproduction. Spore formation (sporulation) occurs when nutrients, such as sources
of carbon and nitrogen are depleted. Bacterial spores are highly resistant to heat,
dehydration, radiation and chemicals.

The shape and the position of spores vary in different species and can be useful for
classification and identification purposes. Endospores may be located in the middle
of the bacterium (central), at the end of the bacterium (terminal) and near the end of
the bacteria (subterminal) and may be spherical or elliptical.

17
Materials:

Compound microscope
24-hours nutrient broth culture of:
- Klebsiella sp.
- Unknown cultures
48-hours nutrient agar slant culture of:
- Bacillus subtillis
- Staphylococcus aureus
- Unknown cultures
Indian ink
Schaeffer-Fulton staining set:
- Malachite green
- Safranin
Glass slides
Paper towel
Bunsen burner

Procedure:

A. Capsule Staining

i. Place several loopful of the Klebsiella sp. culture onto a clean glass slide very
near to the edge.
ii. Place an equal amount of Indian ink near the bacterial culture on the slide.
iii. Mix well by using a loop, the spread the mixture along the slide by using the
blood smear technique as shown in the figure below:

BLOOD SMEAR TECHNIQUE

18
 iv. Allow the smear to air dry.
v. Cover the smear with safranin for 10 seconds, rinse carefully and blot dry with
paper towel.
vi. Observe the smear by using low and high objectives lens.
vii. Record your observation.
viii. Repeat the procedures with the unknown culture provided.

B. Spore Staining

i. Place several loopful of the Bacillus subtillis culture onto a clean glass slide
near to the edge and prepare a smear of the culture by using the blood smear
technique as shown in page 18.
ii. Cover the smear with malachite green, then heat steaming for 5 minutes.
iii. Allow the slide to cool for 5 minutes.
iv. Rinse carefully under slowly running tap water.
v. Counter stain the slide with safranin for 1 minute, then rinse carefully again
under slowly running tap water.
vi. Blot dry with paper towel.
vii. Observe by using low, high and oil-immersion objective lens.
viii. Record your observation.
ix. Repeat the procedures with Staphylococcus aureus culture and the 48-hours
unknown culture provided.

Question:

1. What is the importance of the extra cellular slime in the food industry?
2. What role do capsules play in the infectivity towards organisms?
3. What is the difference between spore and endospore?

19
PRACTICAL 5: Preparation of Culture Media

Objectives:

i. Learn to prepare a culture media such as agar plates, slants and deep tube.

Overview:

Microbiological culture media could be classified according to; Consistency, which

could be adjusted by changing the concentration of solidifying or gelling agents such
as agar and gelatine. Cultures in liquid media (or broth) are usually handled in tubes
or flasks and incubated under static or shaken conditions. This way, homogenous
conditions are generated for growth and metabolism studies. Semisolid media are
usually used in fermentation and cell mobility studies, and are also suitable for
promoting anaerobic growth. Solid media are prepared in test tubes or in Petri
dishes, in the latter case; the solid medium is called agar plate.

In the case of tubes, medium is solidified in a slanted position, which is called agar
slant, or in an upright position, which is called agar deep tube. Solid media are used
to determine colony morphology, isolate cultures, enumerate and isolate bacteria
and for the detection of specific biochemical reactions.

Composition such chemically-defined (or synthetic) media are composed only of

pure chemicals with defined quantity and quality. As for complex (or non-synthetic)
media are composed of complex materials, like yeast extract, beef extract and
peptone (partially digested protein), therefore their chemical composition is poorly
defined. On the other hand, these materials are rich in nutrients and vitamins.

As for function, all-purpose media do not contain any special additives and they aim
to support the growth of most bacteria. Selective media enhance the growth of
certain organisms while inhibit others due to the inclusion of particular substrate(s).
Differential media allow identification of microorganisms usually through their unique
(and visible) physiological reactions. In the detection of common pathogens, most
practical media are both selective and differential. Enrichment media contain specific
growth factors that allow the growth of metabolically fastidious microorganisms. An
enrichment culture is obtained with selected media and incubation conditions to
isolate the microorganisms of interest.

20
 Materials:

1L sterile molten agar (prepared by lab staff)

Sterile petri dishes
Bunsen burner
500ml blue cap bottles
Tripod and wire gauze
Sterile 10ml pipettes
Standard culture tubes and rack
Dehydrated nutrient agar
Cotton wool
Distilled water
Autoclave
Autoclave bucket
Autoclave tape

Procedure:

A. Agar Plates

i. Collect one bottle of sterile molten agar from the water bath.
ii. Hold the bottle in the right hand; remove the cap with the little finger of
the left hand.
iii. Flame the neck of the bottle.
iv. Lift the lid of the Petri dish slightly with the left hand and pour the
sterile molten agar into the Petri dish and replace the lid.
v. Flame the neck of the bottle and replace the cap.
vi. Repeat the procedure for another 7 sterile petri dishes.
vii. Gently rotate the dish to ensure that the medium covers the plate evenly.
viii. Allow the plate to solidify.
ix. The base of the plate must be covered, agar must not touch the lid of the plate
and the surface must be smooth with no bubbles.
x. The plates should be stored inside sealed plastic bags to prevent the agar from
drying out and to be kept in the chiller, to be used in Practical 6.

B. Slant Agar and Deep Tube

i. Suspend the required amount of dehydrated nutrient agar in the required

amount of distilled water (as shown on the nutrient agar bottle) inside the 500ml
blue cap bottle.
ii. Heat the suspension to boiling, to completely dissolve the dehydrated powder.
iii. The solution will be clear when the agar is completely dissolved. Be careful
not to heat the agar too rapidly as it will char the nutrient agar and the
bottom of the bottle. When the medium approaches boiling point, lower
the flame to prevent excessive foaming.

21
 iv. For agar deep tube, pipette 10ml of the melted nutrient agar into each 5 culture
tubes, and plug them with the cotton wool.
v. For slant agar, pipette 5ml of melted nutrient agar into each 5 culture tubes.
Replace the culture tubes cap loosely.
vi. Then, put all the culture tubes into autoclave bucket. Don't forget to apply the
autoclave tape and label your bottles. The lab staff will bring you to the
autoclave machine to explain about how to operate it, and will autoclave those
media at 15lbs-pressure for 15 minutes at 121○C.
vii. After the sterilization process completed, the deep tubes will be allowed to
solidify in the upright position while the slant agar will be place at an angle so
that they solidify slanted.

Question:

1. Why are petri plates inverted after they cool?

2. What are the three types of sterilization?
3. What are the two types of media used to culture microorganisms?

22
PRACTICAL 6: Pure Culture Techniques

Objectives:

i. To apply the aseptic techniques during culturing bacteria process

ii. To obtain the pure cultures of E. coli and Serratia marcescens

Overview:

Most environments carry a mixed microbial population. To fully appreciate the

contribution of each group of organisms to the ecology of the mass, one must first
dissect this mixed culture to obtain single colonies. The single colony is transferred
(picked) to a fresh medium to obtain a larger, homogeneous culture that may be
studied and characterized by a variety of techniques. One such technique is called
aseptic technique. Microbiologists and health workers use this technique to prevent
contamination of cultures from outside sources and to prevent the introduction of
potential disease agents into the human body (infection can occur through
contamination of your hands and clothing with material from your bacterial cultures).

Aseptic techniques (also called sterile techniques) are defined as the processes
required for transferring a culture from one vessel to another without introducing any
additional organisms to the culture or contaminating the environment with the
culture. The following conditions must exist for aseptic technique to be successful;
The work area must be wiped with an antiseptic to reduce the number of potential
contaminants. The transfer instruments must be sterile. The work must be
accomplished quickly and efficiently to minimize the time of exposure during which
contamination of the culture or laboratory worker can occur.

Developing a thorough understanding and knowledge of aseptic techniques and

culture transfer procedures is a prerequisite to working with microbiological cultures.
You will save yourself a lot of time and energy and avoid erroneous result if a few
simple and common sense rules are observed when working with cultures.

Always sterilize your inoculating loop by flaming before using it to enter any culture
material. Always flame the lip of the culture tube before inserting your sterile loop
into the culture. This destroy s any contaminating cells that may have been
inadvertently deposited near the lip of the tube during previous transfers or by other
means.

23
Keep all culture materials covered with their respective caps and lids when not
making transfers. Do not lay tube caps or petri dish lids on the table top, thereby
exposing cultures to possible contamination. When transferring colonies from petri
plates, use the lid as a shield by slightly raising it enough so that your loop can be
inserted but the agar surface is still protected from contaminants falling upon it. Do
not allow tube closures and petri dish lids to touch anything except their respective
culture containers. This will prevent contamination of closures and therefore of
cultures.

Aseptic Techniques

24
Materials:

Inoculating loop
Bunsen burner
Nutrient agar plates
Nutrient broth of mixed bacterial culture
Nutrient broth culture of E. coli
Nutrient broth culture of Serratia marcescens
Incubator

Procedure:

i. Obtain the nutrient agar plates. Turn these culture media dishes bottom side up
and label the perimeter of the dishes with your name, date, group number and
class, temperature of incubation, type of medium and specimen.
ii. Draw two perpendicular lines with a marker on bottom of the plate to divide the
circle into 4 quadrants. Note: After you become proficient in streaking, you
could visualize each petri dish divided into quarters instead of actually
drawing lines.
iii. Holding an inoculating loop between your thumb and index finger, insert the wire
portion into the Bunsen burner flame, heating the entire length of the wire until it is
red and glowing. Allow the wire to cool before doing the next step. Do not wave
the loop in the air. Note: The wires of your loops are made of special alloy
that makes them heat fast and cool fast. Still, the loop takes about a minute
to get down to room temperature after being in the flame. If your loop is not
sufficiently cooled down, it may kill the organisms that it comes in contact
with and you may observe no growth on your plates.
iv. Using your free hand, pick up the tube containing the mixed culture and gently
shake it to disperse the culture. Remove the tube cap or plug with free fingers of
the hand holding the sterile inoculating loop and carefully flame the mouth of the
tube in the Bunsen burner flame.
v. Tilt the tube to bring the broth culture within 1 inch from the lip of the tube. Insert
the sterile loop and remove a small amount of growth; a loopful is usually
sufficient. Try not to touch the sides of the tube with the loop.

25
vi. Flame the tube mouth again, carefully replace the tube cap or plug, and return
the culture tube to the test tube rack.
vii. Expose the agar surface of each plate for inoculation by raising the lid at an
angle over the agar, thus keeping the plate surface protected from aerial
contamination.
viii. Apply the mixed culture on the loop onto the first quadrant by sweeping the
area of this quadrant.
ix. Spread the specimen out well.
x. Flame the loop and allow it to cool. You may cool the loop in an uninoculated
area of the medium. DO NOT wave it in the air to cool!

xi. Now streak the inoculum from quadrant 1 into quadrant 2. Use smooth, non-
overlapping strokes. Utilize the entire quadrant 2 as shown in the figure
above. Flame the loop when done.
xii. Let the loop cool. Now streak the inoculum from quadrant 2 into quadrant 3 by
smooth, non-overlooking strokes again.
xiii. Flame your loop one more time and let it cool. Now bring some inoculum from
quadrant 3 into quadrant 4 in the same manner as for other previous
quadrants. Note: In this procedure, the number of times you enter back
into the preceding quadrant depends on how heavy the initial inoculum
is. If the initial inoculum comes from a plate, slant or a heavy broth
culture, enter the preceding quadrant only once (as shown in the above
figure). However, if the inoculum is obtained from food material, very
light broths or any other source where you expect to have few bacteria,
you may need to bring the inoculum from the previous quadrant to a
new quadrant a few times.
xiv.Flame your loop and cool.
xv. Invert the plates and incubate the plate at 30°C. The reason the plate is
inverted is the fact that the air space between the dish lid and the agar surface
is saturated with moisture; during incubation the moisture condenses on the
upper lid as droplets.
26
 Note: Plates are always incubated inverted, even (especially) in the
refrigerator. As these droplets collect into a large drop, the water drips
onto the agar surface causing the spread and mixing of colonies.
Inversion of the plate eliminates this problem.

xvi.Repeat the procedures with E.coli and Serratia marcescens on different

nutrient agar plate with the same techniques.
xvii. After 24hours of incubation, observe and record the condition of your plates
and ensure to look at these characteristics as below:

Noting the Observable Characteristics of Bacteria

Here are the more common culture characteristics of bacteria on agar plates:

1. Odor Production: Some bacteria produce a unique aroma (gas) as a result

of their metabolic processes. An astute microbiologist will always make note
of the specific aroma of the organism under study. Examples of aroma are
ammonia (NH), rotten egg (H2S), alcoholic (-OH), fecal, etc.

2. Pigment Production: Pigments produced by bacteria are either cell bound

(limited to cells and the colony), extracellular (pigment is released into the
medium) or both. The color of the pigment should be noted, as this
characteristic could be very helpful in the identification of the organism.

3. Colonial Morphology: This category includes the colony density, texture,

elevation, cohesiveness, shape and margin.

a. Colony density: Hold the plate in front of a light source to determine

if the colonies are clear, opaque (no light passes through the colony) or
translucent.

b. Colony texture: This is best observed when direct light is reflected

off the colonies. The texture is usually either smooth (even surface) or
rough (irregular, nonsmooth surface)

c. Colony elevation: Colony elevation can be observed under direct

light. It can be either:

27
 d. Colony cohesiveness: This can be grouped into stringy, creamy,
mucoid and dry. The dry colonies can be lifted off the plate entirely and
they are hard to suspend in liquid.

e. Colony shape: If viewed from above, the colony shape could be

categorized as:

f. Colony margin: Colony edge or margins could likewise be described

as entire:

4. Colony size: The relative diameter (in mm) of average individual colonies
growing on a certain medium can be a useful characteristic, especially when
one is comparing different species.

Question:

1. How does a single environmental factor, temperature, affect the number and
type of colonies found in the culture?
2. What is the difference between mixed bacterial culture with E.coli and S.
marcescens growth on the agar?
3. Why aseptic techniques are important to be applied while performing the
streaking method?

28
 PRACTICAL 7: Viable Count of Bacterial Culture

Objectives:

i. To make a serial dilution of bacteria culture

ii. To transfer the bacteria to petri plates
iii. To compare the number of bacteria presents in petri plates

Overview:

There are a variety of ways to enumerate the number of bacteria in a sample. A

viable cell count allows one to identify the number of actively growing/dividing cells in
a sample. The plate count method or spread plate relies on bacteria growing a
colony on a nutrient medium. The colony becomes visible to the naked eye and the
number of colonies on a plate can be counted. To be effective, the dilution of the
original sample must be arranged so that on average between 30 and 300 colonies
of the target bacterium are grown. Fewer than 30 colonies makes the interpretation
statistically unsound and greater than 300 colonies often results in overlapping
colonies and imprecision in the count. To ensure that an appropriate number of
colonies will be generated several dilutions are normally cultured. The laboratory
procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc. ) in
sterile water and cultivating these on nutrient agar in a dish that is sealed and
incubated. Typical media include Plate count agar for a general count or MacConkey
agar to count gram-negative bacteria such as E. coli.

Typically one set of plates is incubated at 22°C and

for 24 hours and a second set at 37°C for 24 hours.
The composition of the nutrient usually includes
reagents that resist the growth of non-target
organisms and make the target organism easily
identified, often by a color change in the medium.
Some recent methods include a fluorescent agent so
that counting of the colonies can be automated. At the
end of the incubation period the colonies are counted
by eye, a procedure that takes a few moments and
does not require a microscope as the colonies are
typically a few millimeters across. In the pour plate
method a diluted bacterial sample is mixed with
melted agar and then that mixture is poured into a
petri dish. Again the colonies would be counted and
the viable cell count calculated.

29
Materials:

Vitagens drinks
25 Tryptone glucose yeast agar plates (prepared by lab assistant)
5 200ml beaker
4L distilled water
5 1ml micropippete with tip
25 test tubes (autoclaved)
5 test tube racks
5 spreading glass (hockey stick)
70% alcohol (for dipping hockey stick)
Parafilm
Colony counter

Procedure:

i. Label 5 test tubes as T1 to T5.

ii. Label the base of each Tryptone glucose yeast agar plates as P1 to P5.
iii. Transfer aseptically 9ml of sterile distilled water into each of the test tubes, T1
to T5.
iv. Transfer aseptically 1ml of vitagen drinks into T1. This gives 10X dilution.
Shake T1 gently to ensure thorough mixing.
v. Using fresh tip, transfer 1ml from T1 into T2, shake gently, and repeat this step
from T2 into T3, then T3 into T4, Then T4 into T5.
vi. Using fresh tip, transfer 1ml from T1 into plate labelled P1, lifting the lid by a
minimal amount.
vii. Dip the glass spreader in 70% alcohol, allow alcohol excess to drip off and hold
the spreader vertically in a burner flame.
viii. Allow the spreader to cool off for few seconds.
ix. Spread the sample over the surface of the plate. Re-sterile the sreader.
x. Repeat previous step with T2 – T5, into P2 – P5, respectively.
xi. Tape the lid of the plates to avoid contamination.
xii. Incubate plates upside down at 37°C for 24 hours.
xiii. Examine the plate for bacterial growth and count the number of individual
colonies on each plate.
xiv. Use colony counter to estimate the total number of bacteria on the plate in
CFU/ml.

30
Question:

1. “Fewer than 30 colonies makes the interpretation statistically unsound and

greater than 300 colonies often results in overlapping colonies and
imprecision in the count”. Discuss.

31
PRACTICAL 8: The Use of Ultraviolet Radiation for
Sterilization

Objectives:

i. To study the effect of UV radiation towards growth of bacteria

Overview:

Nonionizing radiation has a wavelength longer than that or ionizing radiation, usually
greater than about 1 nm. The best example of non ionizing radiation is ultraviolet
(UV) light. When microorganisms are subjected to UV light, cellular DNA absorbs the
energy by purines and pyrimidine bases, and adjacent thymine molecules link
together. Linked thymine molecules are unable to encode adenine on messenger
RNA molecules during the process of protein synthesis. Moreover, replication of the
chromosome in binary fission is impaired. The damaged organism can no longer
produce critical proteins or reproduce, and it quickly dies. Ultraviolet light is
especially effective in inactivating viruses.

Ultraviolet (UV) light consists of light of wavelengths between 40 to 390 nm, but
wavelength in the 200 nm range are most effective in killing microorganisms.
Ultraviolet light effectively reduces the microbial population where direct exposure
takes place. It is used to limit airborne or surface contamination in a hospital room,
morgue, pharmacy, toilet facility, or food service operation. In some communities,
ultraviolet light is replacing chlorine in sewage treatment. Running the sewage
effluent under ultraviolet light before discharging it can destroy microorganism
without altering the odor, pH, or chemical composition of the water and without
forming carcinogenic compounds.

A major disadvantage of UV light as a disinfectant is that the radiation is not very

penetrating, so the organism to be killed must be directly exposed to the rays. It is
noteworthy microorganisms in the air and upper layers of the soil, but it may not the
effective against all bacterial spores. Organisms protected by solids and such
coverings as paper, glass, and textiles are not affected. Another potential problem is
that UV light can damage human eyes, and prolonged exposure can cause burns
and skin cancer in humans. And it may cause damage in human skin cells and
permanent damage the eyes.

32
Materials:

48-hours nutrient broth cultures of:

- Serratia marcescens
- Bacillus subtillis
Nutrient agar plates
Ultraviolet lamp / Laminar Flow
Inoculating loop
Bunsen burner
Incubator

Procedure:

i. Obtain the nutrient agar plates, 2 plates for each culture.

ii. Label any details and place a streak of each bacterial culture on the agar
surface of the plate by using the techniques as in Practical 6. Refer to the
procedures first!
iii. Expose the plates to the ultraviolet radiation for 10 minutes, with one plate of
the same bacterial cultures totally uncovered while the second plate is half
covered with the petri dish lid.
iv. The distance should be around 10 – 12 inches depending to the intensity of
the radiation. Do not look directly at the radiation source / lamp as severe eye
damage can result.
v. Cover the plates, and then incubate them at room temperature for 24hours.
vi. Compare and record your observations as follows:

Exposed plate Partially Exposed plate

Serratia
marcescens

33
 Exposed plate Partially Exposed plate

Bacillus
subtillis

Question:

1. Would you expect the Bacillus subtillis culture to be more resistant to

radiation? Why?
2. List several clinical application of UV irradiation.
3. Describe other methods of radiation sterilization besides Ultraviolet.

34
NOTES

35