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 SEPTEMBER 2022

Information on student
Name of student
Matric number
I/C number
Learning centre (PPU)

Laboratory session
Date
Venue
Time
Name of demonstrator Puan Nursalfarina binti Abdul Samat
Submission Date 15 DECEMBER 2022 (THURSDAY)
Submission Link https://forms.gle/1PF5Fjo8QSXAmHkx6

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:
Questions:

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

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

3. Discuss the laboratory safety precaution for any blood related procedure.

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:
Questions:

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

2. State the importance of handwashing.

3. In your opinion, is handwashing necessary when medical and surgical personnel wear gloves
during surgery or examining patients?

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:
Questions:

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

2. Describe the aseptic technique applied in this experiment.

3. Explain the reason streaking technique can isolate 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:
Questions:
1. Discuss the result. Explain for each bacteria their shape, arrangement and whether Gram
positive and negative.

2. Explain the differences between Gram positive and negative.

3. State the principal of Gram staining.

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

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

3. Explain the purpose of blood in blood agar.

APPENDICES – If relevant
[Relevant Equipment and Techniques to described in Appendix]