MICROBIOLOGY (XBMB3104)

LAB MANUAL

Faculty of Technology and Applied Sciences (FTAS)

&
Research and Project Management Unit (RPMU)
Students are expected to have read the module, identified the scope of the course, comprehend the
content of the course and identify the practical skills required in this course. Before beginning the
laboratory exercise, you must read this manual and understand the safety guidelines you ought to
obey.

Safety Guidelines:

As a responsible individual you must be aware of the importance of strict obedience of the rules and
regulations especially in the laboratory. The following are the safety guidelines to be observed by all.

1. Make initial preparations before coming into a laboratory to conduct experiments. Comply
with all oral and written instructions. If in doubt, do not hesitate to forward your enquiries to
the person in charge.
2. Never play or fool around in the laboratory. Concentrate on the task at hand at all times.
3. Report all accidents, wounds or damaged instruments immediately to the person in charge.
4. Dress appropriately. Avoid wearing loose fitting-clothes and jewellery. Pin or tie up long hair.
5. Wear or use recommended safety gears.
6. Use the apparatus carefully.
7. Do not conduct an experiment without supervision. Obtain permission from the instructor
before attempting something new.
8. Exercise caution when dealing with hot apparatus. Use a wet towel or tongs to move the hot
apparatus when contact is required.
9. In any case of equipment damage or accident, report immediately to the instructor. You must
be aware of potential dangers and how to manage such situations.
10. Ensure any electric circuit is wired up correctly with all safety precautions in place before
activating it.
11. Turn off the electric supply to an electric circuit before making any changes/modifications.
12. Return all used apparatus to its original positions in a clean and tidy state.
Assessment Guideline:

The assessment contributes 10% of the total assessment percentage for this course. The
experiments are to be conducted based on the instructions given for each session. Print and bring
along the worksheet before you attend the laboratory session. All worksheets must be verified by
the demonstrator and submitted by the end of the practical session.
 MICROBIOLOGY (XBMB3104)

Subject Microbiology
Code XBMB3104
Semester JAN / MAY / SEPTEMBER 2023

Information on student
Name of student HARTINI BINTI MOHAMMED DAHALAN
Matric number 870530025096001
I/C number 870530-02-5096
Learning centre (PPU) SEBERANG JAYA LEARNING CENTRE

Laboratory session
Date 02 DECEMBER 2023
Venue Universiti Teknologi MARA, Cawangan Pulau Pinang Kampus Bertam
Fakulti Sains Kesihatan
Time 8.30 A.M – 4.30 P.M
Name of demonstrator Dr. SHAMSUL BAHRIN GULAM ALI
Dr. NURHIDAYAH AB. RAHIM

List of experiments
Experiment 1 Blood smear
Experiment 2 Aseptic technique
Experiment 3 Streaking technique
Experiment 4 Gram staining
Experiment 5 Media culture
 EXPERIMENT 1

Title:

Blood smear

Objectives:

1. To prepare blood smear

2. To observe the blood smear under microscope
3. To interpret the peripheral blood film

Material/apparatus:

Gloves, 90% alcohol, lancets, alcohol swab, slides, absolute methanol, hair dryer, immersion oil,
microscope.

Procedure:

1. Wear gloves.
2. Clean slides with 90% alcohol and allow to dry. Do not touch the surface of the slide where
the blood smear will be made.
3. Select the finger to puncture, usually the middle or ring finger.
4. Clean the area to be punctured with 70% alcohol; allow drying.
5. Puncture the ball of the finger.
6. Wipe away the first drop of blood with clean gauze.
7. Touch the next drop of blood with a clean slide. Repeat with another slides. If blood does
not well up, gently squeeze the finger.
8. Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the specimen
slide.
9. Wait until the blood spreads along the entire width of the spreader slide.
10.While holding the spreader slide at the same angle, push it forward rapidly and smoothly.
11.Properly air dried the smear.
12.Fix the dried smear with 2 to 3 drops of absolute methanol (100%) for ½ - 1 minute.
13.Remove the methanol and dry the slide using hair dryer (not too hot).
14.Observe the slide under the microscope. Identify the blood cells on the slide.
Results:

Observe under 40X microscope Observe under 10X microscope

Questions:

1. Describe the observation would be made if the person has contracted malaria.

Malaria is a mosquito-borne infectious disease caused by parasites of the Plasmodium

genus. The symptoms of malaria can vary, and they often manifest in cycles corresponding
to the life cycle of the parasite within the human body. Common symptoms include fever,
chills, sweats, fatigue, weakness, headache, nausea and vomiting are often not specific.

2. Discuss the common clinical indication from peripheral blood film analysis.

Clinical indication:

 Cytopenia, anemia, leukopenia/thrombocytopenia

 Unexplained leucocytosis, lymphocytosis/ monocytosis
 Unexplained jaundice/haemolysis
 Congenital homolytic amenia

3. Discuss the laboratory safety precaution for any blood related procedure.
 Wear PPE (gloves and mask)
 Dispose needle in sharp bin
 Hand Hygiene
 Biohazardous Waste Disposal
 Training and Education
 EXPERIMENT 2

Title:

Aseptic technique

Objectives:

1. To apply the aseptic technique

2. To observe the growth found on the petri dish.

Material/apparatus:

Gloves, incubator, soap for handwashing, tissues, nutrient agar, 70% alcohol

Procedure:

1. Wipe off the bench with 70% alcohol.

2. Draw a line down the middle of the petri dish to divide the plate in half.
3. Label each halves with A and B.
4. Press your unwashed thumb onto the agar at column A.
5. Apply proper handwashing technique.
6. Put the same thumb after washing onto agar at the column B.
7. Incubate the petri dish at 37°C for 24 hours or overnight.
8. Observe and note down the colonies.
Results:

Bacteria
Colony

Column A (Unwashed Hand) Column B( washed Hand)

Questions:

1. Compare the growth on the plate in column A and B.

Column A (Unwashed Hand) Column B( washed Hand)

 More colonies bacteria growth  Less colonies bacteria growth
 Margin undulate & irregular  Margin entire
 Elevation raised  Elevation flat
 Forms irregular and spreading  Forms round with raised margin

2. State the importance of handwashing.

 To prevention of Infections and bacteria from growth.
 Reduction of Respiratory Illnesses
 Breaking the Chain of Transmission

3. In your opinion, is handwashing necessary when medical and surgical personnel wear gloves
during surgery or examining patients?
Yes, handwashing remains necessary even when medical and surgical personnel wear gloves
during surgery or when examining patients. While gloves provide an additional barrier against
the transmission of microorganisms, they are not foolproof and can develop punctures or
tears. Moreover, the process of putting on and removing gloves, known as donning and
doffing, can present opportunities for contamination.
 EXPERIMENT 3

Title:

Streaking technique

Objectives:

1. To perform streaking technique

2. To isolate the bacteria
3. To apply the aseptic technique

Material/apparatus:

Gloves, nutrient agar, Bunsen burner, inoculation loop, incubator, culture (S. aureus, E. coli, etc.)

Procedure:

1. Disinfect the surface of bench and your hand with disinfectant.

2. Label the bottom of petri dish with your name, and date.
3. Work near the flame of Bunsen burner in aseptic zone.
4. Heat the inoculating loop until it red hot.
5. Cool the inoculating loop and take a loopful of mixed culture aseptically.
6. Streak the mixed culture on the solid surface of medium culture by lifting the cover of petri
dish from one edge.
7. Streak over the first area near the edge of the plate.
8. Flame the loop and cool it. Turn the plate slightly so that the second area is on the top. Make
several streaks from first area through second area.
9. Flame the loop, cool it, turn the plate and streak from second area through third area.
10. Flame the loop, cool it, turn the plate and streak from third area through fourth area.
11. Flame the loop before keep it away.
12. Incubate the plate in inverted position at 25°C for 24 to 48 hours.
13. Observe and record your observation.
Results:

Escherichia Coli Staphylococcus Aureus

Questions:

1. Select a discrete colony. Describe the appearance of the colony.

Colony Size Colour Texture Elevation Margin Forms

Escherichia Moderat Milky Dry Raise undulate Round with
Coli e scalloped
margin
Staphylococcus Moderat Yellowish Mucous Raise Irregular Round with
Aureus e raised
margin
2. Describe the aseptic technique applied in this experiment.
 Disinfection of Bench and Hands:

- Before starting the experiment, the bench surface and hands are disinfected with a disinfectant.
This step helps eliminate potential contaminants from the working area and the hands of the
experimenter.

 Labeling of Petri Dish:

- The bottom of the petri dish is labeled with the experimenter's name and the date. Proper
labeling ensures traceability and documentation of the experiment.

 Working Near the Bunsen Burner Flame in Aseptic Zone:

- The experiment is conducted near the flame of a Bunsen burner, creating an aseptic zone. The
heat from the flame helps reduce airborne contaminants, creating a more sterile environment for
the experiment.

 Heating the Inoculating Loop:

- The inoculating loop is heated until it becomes red hot. This step is essential for sterilizing the
loop, ensuring that any microorganisms from previous use are eliminated.

 Cooling the Inoculating Loop Aseptically:

- After heating, the inoculating loop is cooled aseptically before taking a loopful of the mixed
culture. This prevents thermal damage to the microbial sample.

3. Explain the reason streaking technique can isolate colonies.

 Dilution:
- In the initial steps of streaking, the inoculating loop picks up a small amount of the
microbial sample. By streaking the loop over a small area of the agar surface, the
microorganisms are spread out thinly. This dilution effect is crucial because it reduces the
number of microorganisms in subsequent streaks.
 Spatial Separation:
- As the streaking process continues, each new streak covers a different section of the agar
surface. The inoculum from the previous streak is spread out further, resulting in spatial
separation of individual microbial cells. This separation is essential because it allows for the
growth of individual cells into visible colonies.
 Isolation of Colonies:
- The dilution and spatial separation achieved through streaking enable the isolation of
individual microbial cells. As the streaking pattern progresses, the number of microorganisms
in each subsequent section decreases. Eventually, individual cells or small clusters of cells are
deposited onto the agar surface, and these cells have sufficient space to grow and form
discrete colonies.
 EXPERIMENT 4

Title:

Gram staining

Objectives:

1. To prepare culture smear on slide.

2. To conduct gram staining.
3. To observe the stained culture under microscope.

Material/apparatus:

Gloves, clean slides, culture (S. aureus, E. coli), crystal violet, iodine, acetone, safranin, inoculating
loop, Bunsen burner, microscope.

Procedure:

1. Prepare culture smear on slides for both S. aureus, and E. coli. Air dry and heat fix.
2. Pour crystal violet on the smears and allow to stand for one minute.
3. Wash gently with running water.
4. Apply iodine on the smears for one minute.
5. Wash gently with running water.
6. Apply acetone drop wise for three seconds.
7. Wash the slides immediately with tap water.
8. Apply safranin and keep for one minute.
9. Wash the slides gently with running water, blot dry the slides.
10. Observe and record your observation.
Results:

Escherichia Coli Staphylococcus Aureus

Questions:

1. Discuss the result. Explain for each bacteria their shape, arrangement and whether Gram
positive and negative.

Gram Positive Gram Negative

Shape Cocci - Spherical or round Bacilli - Rod-shaped
Arrangement Staphylococci: Clustered Diplobacilli: Pairs.
arrangement. Coccobacilli: Intermediate
Streptococci: Chain between cocci and bacilli.
arrangement. Streptobacilli: Chains.
Diplococci: Pair arrangement
Colour Purple Red or Pink
Culture Staphylococcus Aureus Escherichia Coli

2. Explain the differences between Gram positive and negative.

Gram-positive and Gram-negative bacteria differ primarily in the structure and composition of them

cell walls, which results in distinct reactions during the Gram staining procedure. Here are the key
differences between Gram-positive and Gram-negative bacteria:

 Cell Wall Composition:

- Gram-Positive:

- Thick Peptidoglycan Layer: Gram-positive bacteria have a thick layer of peptidoglycan in their cell
walls, which is responsible for retaining the crystal violet stain during Gram staining.

- Teichoic Acids: Some Gram-positive bacteria possess teichoic acids, which are polymers that
contribute to the overall structure of the cell wall.

- Gram-Negative:

- Thin Peptidoglycan Layer: Gram-negative bacteria have a thinner peptidoglycan layer compared
to Gram-positives.

- Outer Membrane: Gram-negative bacteria have an outer membrane external to the

peptidoglycan layer. This outer membrane contains lipopolysaccharides (LPS) and porins,
contributing to the unique characteristics of Gram-negative cell walls.

 Retention of Stains:

- Gram-Positive:

- Crystal Violet Retention: After the initial crystal violet stain, Gram-positive bacteria retain the
stain even after the addition of iodine and alcohol (decolorization step). This is due to the thick
peptidoglycan layer.
 - Gram-Negative:

- Crystal Violet Loss: Gram-negative bacteria lose the crystal violet stain during the decolorization
step, primarily because of the thinner peptidoglycan layer and the presence of the outer
membrane.

 Staining Reaction:

- Gram-Positive:

- Purple or Blue Stain: After the entire Gram staining process, Gram-positive bacteria appear
purple or blue under the microscope due to the retention of the crystal violet stain.

- Gram-Negative:

- Pink or Red Stain: After the decolorization step, Gram-negative bacteria take up the safranin
counterstain and appear pink or red under the microscope.

3. State the principal of Gram staining.

- The primary factor determining Gram staining results is the thickness of the peptidoglycan layer in
the cell wall.

- Gram-positive bacteria have a thick peptidoglycan layer and retain the crystal violet-iodine
complex, appearing purple or blue.

- Gram-negative bacteria have a thinner peptidoglycan layer and lose the crystal violet stain during
decolorization, taking up the safranin counterstain and appearing pink or red.

Gram staining is a valuable tool in microbiology for initial bacterial classification and is an essential
step in the identification of bacterial species. It provides valuable information about the bacterial cell
wall structure and aids in the selection of appropriate antibiotics for treatment.
 EXPERIMENT 5

Title:

Media culture

Objectives:

1. To apply aseptic technique.

2. To observe the appearance of different bacteria in different media agar.
3. To explain on the physiology of microbes.

Material/apparatus:

Gloves, Bunsen burner, inoculation loop, incubator, MSA agar, EMB agar, MacConkey agar, blood
agar, E. coli, E. aerogenes, S. epidermidis, and S. aureus.

Procedure:

1. Label the bottom of petri dish with your name, date and type of media culture.
2. Divide each petri dish into 4 quadrants, by marking the bottom of the dish. Label each
section with the name of bacteria to be inoculated.
3. Using aseptic technique, inoculate all plates except blood agar, with the designated bacteria
by making a single line of inoculation of each bacterium in its appropriate section. Ensure
that the petri dishes are close and the inoculation loop flamed between the inoculations of
different bacteria.
4. Using aseptic technique, inoculate blood agar, with the designated bacteria by making a
single line of inoculation of each bacterium in its appropriate section like in step 3. Upon
completion of each single line of inoculation, use the inoculation loop and make three of
four stabs at a 45° angle across the streak.
5. Incubate the petri dishes in inverted position for 24 to 48 hours at 37°C.
6. Observe and record your observation
Results:

EMB Agar

MSA Agar
Mac Conkey Agar

Blood Agar
Questions:

1. Discuss the appearance of media; with the appearance of bacteria grow on the media.

Agar S. Aureus S. Epidermidis E. Coli

MSA Yellow Colony Pink Colony Gram Negative
Yellow Zone (No yellow Zone) Inhibited Growth
(Fermentation Stock tolerance
mannitol)
Mac Conkey Gram Positive No Gram Positive No Pink Colony
Growth Growth Lactose fermenting
species will grow
pink colonies
EMB Gram Positive No Gram Positive No Green Metallic
Growth Growth sheen
Blood Growth with clear Growth with no Growth with beta
zone of beta hemolysis zone or hemolysis
hemolysis gamma hemolyses

2. Indicate the specific selective and/or differential purpose of MSA agar, EMB agar, and
MacConkey agar.

MSA Selective and

differential
Mac Conkey Selective and
differential
EMB Differential
Blood Agar Selective

3. Explain the purpose of blood in blood agar.

Blood agar is a type of differential and enriched medium used in microbiology for the cultivation and
identification of various bacteria. The main purpose of adding blood to agar is to provide nutrients
for bacterial growth and to create a medium that supports the differentiation of bacteria based on
their hemolytic activity. There are three types of hemolysis that can be observed on blood agar:
alpha, beta, and gamma.

In summary, the addition of blood to agar in blood agar serves a dual purpose: it provides a nutrient-
rich medium for bacterial growth and facilitates the differentiation of bacteria based on their
hemolytic activity, which can aid in the identification and classification of bacterial species.

APPENDICES – If relevant

[Relevant Equipment and Techniques to described in Appendix]