MIC 461

GENERAL MICROBIOLOGY
LABORATORY MANUAL
 MIC461 GENERAL MICROBIOLOGY

CONTENTS

Practical 1 OPERATION OF THE MICROSCOPE

Practical 2 GRAM STAINING – METHOD AND IDENTIFICATION

Practical 3 THE UBIQUITY OF BACTERIA

Practical 4 THE PURIFICATION OF BACTERIAL CULTURES – PURE CULTURE

TECHNIQUES

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
 MIC461 GENERAL MICROBIOLOGY

PRACTICAL 1

OPERATION OF THE MICROSCOPE

The Use and Care of the Microscope

The compound microscope is a precision instrument composed of a series of lenses designed and
arranged to provide, under optimal conditions, maximum magnifications ranging from
approximately 1 – 2000 times (x) the diameter of the specimen being observed.

The ordinary compound microscopic consists of a series of optical lenses, mechanical adjustment
parts and supportive structures from various components. The optical lenses include the ocular or
eyepiece, usually three objectives with different magnifying powers, and the sub-stage
condenser. The coarse, fine and condenser adjustments knob together with the iris diaphragm
level comprise the major mechanical parts concerned with the operation of the instrument. The
various components of the scope are held in position by or contained within, supportive
structures such as the base, arm, pillar, body tube (barrel), and revolving nosepiece.

Successful operation of the microscope depends upon proper maintenance and correct use. Here
are some suggestions:

1. When carrying the instrument, grasp the arm with one hand, and place the other hand
under the base as a support. Keep the instrument in an upright position. In the event the
light source is not attached, take the scope to your laboratory table first, and return for the
light source. Do not carry both items at the same time.

2. Keep the microscope at least six inches from the edge of your laboratory table.

3. Do not handle the lenses with your fingers. Perspiration contains fatty acids and other
substances which can mar the lens glass. Always use the lens paper for the cleaning of the
optical system.

4. Wipe the lenses of the microscope before and after class use.

5. Do not temper with any components of the instrument. If the microscope does not seem
to be functioning, notify the instructor immediately. As other laboratory sections may use
the same instruments, any part previously damaged and subsequently reported may be
charged to you.

6. Do not allow chemicals to come in contact with any part of the instrument.

7. Remove immersion oil from the microscope with lens paper.

8. Always be certain that the low-power objective is in the working position, i.e. in line with
the body tube, before putting the microscope away.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
 MIC461 GENERAL MICROBIOLOGY

Operation of the microscope.

A. Components of Microscope

Materials:

Compound microscope and appropriate light source

Procedure:

1. Remove the microscope assigned to you from the cabinet by taking the arm with one
hand and supporting the instrument at the base with the other.

2. Place the microscope approximately six inches from the edge of the laboratory table
with the microscope arm facing you.

3. Examine the microscope and locate the components listed below:

(a) Ocular (h) Condenser

(b) Body tube (barrel) (i) Iris diaphragm knob
(c) Revolving nosepiece (j) Course adjustment knob
(d) Low-power objective lens (k) Fine adjustment knob
(e) High-dry objective lens (l) Mechanical stage
(f) Oil immersion (m) Pillar
(g) Stage

B. Use of the Microscope

Materials:

1. Glass slides and cover slips

2. Lens paper and xylene
3. Eye dropper
4. Newspaper
5. Scissors and inoculating needle

Procedure:

1. Clean a glass slide and cover slip as indicated by the instructor and place a drop of water
on the slide.

2. Next, cut a series of letters from the newspaper provided. Be certain that at least one of
the letters is a small “e”.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
 MIC461 GENERAL MICROBIOLOGY

3. Place the string of letters into the drop of water. Cover the preparation with a cover slip.
To limit the number of air bubbles in this preparation observe the following:
(a) Place the edge of the cover slip on the drop of water on the slide so that the
fluid completely wets the edge.
(b) Then place the inoculating needle under the cover slip and lower the cover slip
onto the slide.

4. Examine the preparation under the low-power and high-power objectives.

Procedure for microscope operation:

1. Wipe all lenses with lens paper.

2. Move the low-power objective into position under the body tube. You will feel a click
when it is correctly in place.
3. Connect your light source and turn it on.
4. Place the slide preparation to be examined over the central opening in the stage.
5. Arrange the portion of the preparation to be examined over the central opening in the
stage.
6. Lower the low-power objective with the aid of the coarse adjustment knob to
approximately one quarter of an inch above the cover slip of your preparation.
7. Raise the condenser as far as it will go with the aid of the condenser adjustment knob.
8. Look through the ocular. Next, move the iris diaphragm lever so that the diaphragm is
open to its maximum limit. This is determined by looking through the eyepiece and
noting changes in the intensity of the light while the iris lever is being manipulated. If the
light is too bright, make the necessary adjustments.
9. Next, focus upward with the fine adjustment knob, until the specimen comes clearly into
view. With a microscope having the body tube in a fixed position, the respective
adjustment knob will control the stage. Focusing in this case will be downward until the
specimen comes into view. In other words, this is opposite to the procedure described in 6
and 9 because the objectives will remain fixed while the stage lowers the specimen.
10. To change objectives (switch from low-power to high-power) do not raise the body tube,
just simply swing the objective desired into place. The microscope should stay in focus
with any objective. If it does, it is said to possess the property of being par focal.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
 MIC461 GENERAL MICROBIOLOGY

QUESTIONS

1. (a) What is the total magnification obtainable with the low-power objective?
(b) What is the total magnification obtainable with the oil immersion lens?

2. What happened to the original orientation of the letter “e” when the slide was moved:
(a) side-to-side
(b) up and down

3. Complete the following table:

No. Microscope Parts Function (s)

1. Ocular Lens
2. Objective Lens
3. Condenser
4. Iris Diaphragm Lever
5. Coarse Adjustment Knob
6. Condenser Adjustment knob
7. Fine Adjustment Knob

C. Examination of Stained Cells

The numbers and types of organisms seen under the microscope are limited by the
magnification of the objectives used. Because of their extreme small size bacteria are not
generally studied with the low or high dry objectives but instead they are stained with the
oil immersion objective. This practical will give you the experience in the use of oil
immersion and also serves as a comparative study of the stained cells between bacterial
cells and typical plant and animal cells.

Materials:

1. Prepared slide of plant cell (dicot)

2. Prepared slide of animal cell (alveoli)

Procedure:

1. Examine all the slides under the oil immersion objectives.

2. Observe the size and shape and any visible structures of these organisms.

3. Make illustrations/drawings of all observations.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
 MIC461 GENERAL MICROBIOLOGY

QUESTIONS

1. How would a stained preparation appear under the oil immersion objective without the oil
between the slide and the objective lens?

2. What are the structural differences between the animal and plant cell?
Could you observe them in this exercise?

3. Could you detect the presence or absence of the structures in Question 2 in the bacterial
cell?

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

PRACTICAL 2

GRAM STAINING – METHOD AND IDENTIFICATION

The objective is to study the Gram staining and identify the Gram type of bacteria.

DIFFERENTIAL STAINS

Differential stains are so named because they do not stain all kinds of bacteria equally.
Microorganisms differ from one another chemically and physically and thus may react
differently to the given stains. For example, the Gram stain differentiates between the two
cell wall types in prokaryotic cells. The Spore stain, stain the spore and the vegetative cell in
different colors.

A. THE GRAM STAIN

The Gram staining technique is the most widely used differential staining technique in
bacteriology, and was first developed by Christian Gram in the 1880’s. It is used primarily to
divide bacteria into two broad groupings:
Gram-positive and Gram-negative cells.

This procedure is of considerable importance in bacterial taxonomy and it also indicates

fundamental differences in cell wall structures in bacteria.

The Gram stain requires four different solutions: a basic dye, a mordent, a decolorizing agent
and a counterstain.

Cells are first stained with crystal violet, a basic dye, followed by iodine which forms a
complex inside the cells. A mordant is a substance which increases the affinity or attraction
between the cell and the dye. Examples of mordent are acids, bases, metallic salts and iodine.
Under the action of a mordant, a cell is more strongly stained, and it is more difficult to wash
out the stain after the application of the mordent.

Decolorization is then performed with alcohol. A decolorization agent is a substance which

removes the dye from a stained cell. The cells which retain the basic dye after decolorization
are called Gram-positive and those that lose the stain are called Gram-negative. After
decolorization, a counterstain safranine is used to make visible the now decolorized Gram-
negative bacteria.

Gram positive cells stain blue/purple

Gram negative cells stain red/pink

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

Preparation of smears.

Before bacteria can be stained, a smear must made, and heat fixed. A smear is made by
spreading a bacterial suspension on a clean slide and allowing it to air dry. The smear is
passed through a Bunsen burner flame to heat fix the bacteria. Heat fixing denatures bacterial
enzymes, preventing autolysis. The heat also adherence of the bacterial cells to the
microscope slide.

Clean slides are essential for the preparation of microbial smears. Grease or oil from fingers
on slides must be removed.

I) Cultures from a broth

1. Sterilise an inoculating loop, allow it to cool and remove one drop of bacterial
suspension from the tube.

2. Place the drop in the center of a clean, grease-free microscope slide and spread the
drop out to form a thin film towards the top end of the slide. Do not go too near the
edges of the slide.

3. Allow the film to dry by wafting the slide gently above a Bunsen flame. Do not blow
on the slide or wave it in the air.

4. Pass the dried smear, film uppermost, quickly once or twice through the Bunsen
flame. This will fix the bacteria i.e. kill them and stick them firmly to the slide.

5. Place the slide on a staining rack and apply the stains.

II) Cultures from a solid/agar medium

1. Place a drop of sterile water in the center of a slide.

2. Sterilize the loop and remove a minute quantity of the bacterial culture.
DO NOT scrape the loop across the culture but simply touch the loop to the culture.

3. Mix the cells in the drop of water and proceed as in the case of broth cultures. (Steps
2-5).

Materials

1. Unknown cultures
2. Microscope slides
3. Gram stain reagent sets
4. Immersion oil
5. Nutrient broth cultures of the following microorganisms:
Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

a. Escherichia coli
b. Staphylococcus aureus
c. Bacillus subtilis
Procedures
1. Clean and label the slides. Prepare a fixed smear of the bacterial cells. Please note that
the quality of the smear (too heavy or too light cell concentration) will affect the Gram
Stain results.
2. Flood the smear with crystal violet for 1 minute. Wash slide in a gentle and indirect
stream of water.
3. Flood slide with the mordant (iodine). Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water. Do not splash water directly
onto the smear.
5. Flood slide with decolorizing agent (alcohol). Wait 15 seconds or add drop by
drop to slide until decolorizing agent running from the slide runs clear.
Please note that to prevent excess decolorization in the gram-positive cells, stop
adding decolorizer as soon as the solvent is not colored as it flows over the slide.
6. Wash slide in a gentle and indirect stream of tap water.
7. Flood slide with counterstain (safranin) for 30 seconds to 1 minute.
8. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and
excess water is blotted with the blotting paper (Be careful not to wipe the cells off the slide).
The slide can also be air-dried after shaking off excess water.
9. Observe the results of the staining procedure under oil immersion using a microscope.
10. Record your observations.

COMMENTS

Certain species of bacteria do not give a consistent reaction to the Gram stain - sometimes
reacting positively and sometimes negatively. These bacteria are said to be Gram variable.
Older or dead Gram-positive cell sometimes lose their Gram positiveness and may also
appear Gram-negative. The best Gram stains are therefore given by fresh cultures.

QUESTIONS

1. Are there any chemical differences between the cell wall of Gram-positive and Gram-
negative bacteria which might explain differences in the rate of decolorization?

2. Does the age of culture affect the Gram stain, and why?

3. Based on your experiences in the laboratory, do you feel that the Gram stain is a
simple procedure, and why?

4. What are the influences of pH on the Gram stain reaction?

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

PRACTICAL 3

THE UBIQUITY OF BACTERIA

The fact that large number of microorganisms exist in the environment is well established. In
the production of consumable products, it is important to limit the exposure of these products
from contact with the environment. Hence in all consumable industries Good Manufacturing
Practice GMP is observed. The types of organisms which maybe isolated vary greatly with
the material sampled and the methods employed for demonstrating their presents.

An appreciation of the ubiquity of bacteria in the environment contributes to our

understanding of the necessity for aseptic techniques in microbiology.

Materials:

per group

1. 4 nutrients agar plates

2. Sterile petri dishes
3. Vial containing approximately 3 ml of lake water
4. Vial containing approximately 2 grams of garden soil.
5. 4 sterile swabs
6. Lake water
7. Saline water
Procedure:

1. AGAR PLATE 1 (Random objects) – Divide the agar plate into 4 sections. Expose
each section on a nutrient agar plate to some object such as a coin, key, finger, or any
other objects of choice. Incubate the plate in an inverted position at room temperature
for 24 hours.

2. AGAR PLATE 2 (Lake water) - Place 500 ul of lake water onto the surface of
prepared agar. Spread it by using a sterile swab around the surface of the nutrient
agar.

3. AGAR PLATE 3 (Soil) - Place a small amount of garden soil into a tube of saline
water and mix the suspension. Place 2 loopfuls of the mixture onto an agar plate and
spread it by using a sterile swab around the surface of the nutrient agar.

4. AGAR PLATE 4 (Buccal/Cheek swab) – Using a sterile cotton swab, press firmly
and twirl the swab against the inside of the inner cheek using an up and down motion
from front to back and back to front. Use a stopwatch to time the collection for at least
30 seconds per swab to collect cells. It is important to collect samples from maximum
Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

mucosal surfaces. Spread the collected cells around the surface of the nutrient agar.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

5. Incubate the plates in an inverted position at 37 ᵒC for 16-18hours.

The following day:

1. Examine the various cultures provided and record all your observations (notice the
variety off colony types that are present).
2. Also comment upon the number and size of the colonies.
3. All results should include the following observations.

 colony description
o estimated diameter
o whole colony appearance
o margin characteristic (elevation properties)
o pigmentation if any

CARE! – Aseptic technique is important here because of the potential pathogenicity

of your isolates: TRY TO KEEP THE LIDS ON YOUR PETRI DISHES DURING
YOUR EXAMINATIONS.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
BIO461 MICROBIOLOGY

Results and Observations:

Agar Plate Photo of agar plate (label Brief description of variety of colony
isolated colonies with types that are present on the plate
different morphologies)

QUESTION:

1. List and briefly discuss some of the practical methods by which the number of
airborne microorganisms can be reduced to a relatively low level.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
MIC461 GENERAL MICROBIOLOGY

PRACTICAL 4

THE PURIFICATION OF BACTERIAL CULTURES – PURE CULTURE

TECHNIQUES

It is essential that pure cultures be utilized in order to identify and characterize bacteria. A
pure culture is one in which all cells are descendants of a single cell.

Bacteria can be separated form each other in a mixture of bacteria by progressively diluting
the mixture until single bacteria cells are so widely spaced that they will grow, divide and
produce separate colonies. Two dilution methods are generally used to achieve this aim:

1. Streak plate method

2. Pour plate method.

A. STREAK PLATE METHOD

Materials

1. Nutrient broth culture of E. coli

2. Nutrient broth culture of Serratia marcescens

3. Eight (8) nutrient agar plates

4. One (1) tube containing 10ml of sterile saline

5. Four (4) tubes containing 12-15ml of nutrient agar (melted and in the water bath at
45˚C-50˚C)

6. Four (4) sterile Petri dishes

Procedure

1. Prepare 4 plates of Nutrient Agar (NA).

2. Mark the plates on the underside into 4 sectors A, B, C, D.

3. Sterilise an inoculating loop and transfer a drop of E. coli to sector A. Draw the loop
over the surface of the agar (DO NOT CUT INTO THE AGAR) in a series of parallel
streaks.

4. Resterilise the loop, allow it to cool and draw a series of parallel streaks in sector B
cutting across the ends of the first streak in sector A.

5. Resterilise the loop, allow it to cool and draw a series of parallel streaks in sector C
cutting across the end of the streaks in sector B.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
MIC461 GENERAL MICROBIOLOGY

6. Resterilise the loop, allow it to cool and draw a series of parallel streaks in sector D
cutting across the streaks in sector C BUT NOT touching the original streaks in sector
A.

7. Do the same with Serratia marcescens culture.

8. After streaking the plates above, carefully pour the culture of E. coli into the culture
of Serratia marcescens to obtain a mixed suspension.

9. Prepare a streak plate of the mixed culture following the procedures above.

10. Incubate all plates at 37˚C for 2 days and then at 30˚C to allow Serratia marcescens to
develop its characteristic red pigment.

11. After 2 days, examine the cultures which you have inoculated and evaluate your
streaking effectiveness in producing pure colonies.

B. POUR PLATE METHOD

Each student will prepare the pour plates with the mixed culture of E. coli and Serratia
marcescens.

Procedure

1. Transfer one loopful of the mixed culture to the tube of sterile saline. Mix the tube
well by rotating the tube between the hands.

2. Transfer two loopfuls from the saline to a tube of melted agar (label it as tube 1).

3. Mix thoroughly and transfer two loopfuls from tube 1 to a second tube of melted agar
(label it as 2). Quickly pour the contents of tube 1 into an identically labeled Petri
dish.

4. Repeat the procedure for tubes 2 and 3.

5. Do not inoculate the fourth tube but pour the agar from this tube into a Petri dish as a
control.

6. Invert the Petri dishes after the agar hardens.

7. Incubate all plates and tubes at room temperature.

The following day

1. Examine the cultures and evaluate your plate techniques in producing pure colonies.

2. Compare the appearance of sub-surface and surface colonies on the pour plates.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023
MIC461 GENERAL MICROBIOLOGY

QUESTIONS

1. At what temperature does the agar dissolve? At what temperature does it solidify?

2. Why is the agar cooled to approximately 45˚C before preparing pour plates?

3. Why are the Petri dishes inverted during incubation?

4. How can you prevent splattering when flaming a loop which has just been used to
transfer a culture?

5. Comment upon these two techniques.

Prepared by:
Norashirene Mohamad Jamil
Siti Hajar Sadiran
2022
Revised: 2023