GONO BISHWABIDYALAY

Nolam,Mirzanagar,savar,Dhaka-1344

DEPARTMENT OF MICROBIOLOGY
Practial Notebook
Course Title : Practical
Course Code : Microbiol. 1104

Submitted To Submitted By

Anwara Khatun Name : Md. Zahidul Kabir

Lecturer Exam Roll : 1432

Department Of Microbiology Batch No : 43rd

Gono Bishwabidyalay Reg No : G/Micro.-2264/23

Semester : B.sc. Hons 1st

Submission Date : 02-04-2023

 Index
Serial No Experiment Name Date Page No
01 Safety Rules & Regulations in 07-12-2022 01-02
Microbial Laboratory

02 Different Parts of Microscope 15-01-2023 03-05

03 Simple Staining 22-01-2023 06

04 Gram Staining 01-02-23 07-08

05 Negative Staining 08-02-2023 09

06 Spore Staining 05-03-2023 10

07 Acid Fast Staining 19-03-2023 11-12

08 Techniques for Isolation of Pure 22-03-2023 13-18

Culture
Date: 07-12-2022
Experiment No. 01
Experiment Name: Safety Rules & Regulations in Microbial Laboratory

These rules are for the safety of the students, instructors and support staff. Please read and follow
them. Failure to follow safety rules may result in removal from the class.

1. Wear a lab coat in lab. We will be working with a variety of materials that can cause permanent

stains on some fabrics. Also, a lab coat can help protect from accidental contamination by

microorganisms.

2. No eating or drinking during lab. Many pathogens spread by ingested food and drink. In

addition, food can carry microorganisms that might contaminate laboratory cultures.

3. Keep long or fluffy hair tied up and out of the way. Hair can contaminate and be contaminated

by microbial cultures.

4. Always wear shoes in lab.

5. Thoroughly wash your hands with soap and water before and after lab. Thorough and frequent

hand washing easily and effectively controls the spread of many pathogens.

6. Clean the lab bench with disinfectant before and after lab. This helps to prevent contamination

of cultures, books, clothing, etc.

7. Keep the lab bench free of unnecessary materials. Don't use the lab bench as a storage

area for coats, books, etc.

8. Do not take cultures from the lab area.

9. Dispose of all contaminated materials in autoclave bags. When in doubt, ask the instructor.

10. Immediately report all accidents and spills to the instructor. Cover spills with disinfectant

soaked paper towels for at least 15 minutes before disposing of them.

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11. Read all assigned materials before the lab session. Experiments will go smoother and have

greater chances of success when you know what you will be doing ahead of time.

12. Treat all microbial cultures as if they are pathogens. Better safe than sorry.

13. When in doubt, ask the instructor. The only stupid questions are those that are intended as

such.

14. Never pipette by mouth any broth cultures or chemical reagents. Doing so is strictly

prohibited. Pipetting is to be carried out with the aid of a mechanical pipetting device only.

15. Do not lick labels. Use only self-stick labels for the identification of experimental cultures.

16. Speak quietly and avoid unnecessary movement around the laboratory to prevent distractions

that may cause accidents.

17. Place test specimens and supplies in contact with these fluids into a container of disinfectant

prior to autoclaving.

NOTES:

1. Personal belongings are not to be stored in the laboratory.

2. You will be assigned to a group consisting of four students and you will work together in

a semester long project.

3. Please read the safety instructions posted in the lab.

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Date: 15-01-2023
Experiment No. 02
Experiment Name: Different Parts of Microscope

Microscope: The technology of making very small things visible to the naked eye.

Types of Microscope: There are mainly three types of Microscope

Microscope

Simple Compound Electron

1. Simple Microscope: to see plant cell

2. Compound microscope:

There are two types of microscope

A. Monocular: have single eyepiece
B. Binocular: have double eyepiece

3. Electron microscope: to see virus cell

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 Care and Use of a Microscope

1. Always carry a microscope using both hands

2. When not in use, a microscope should be protectected from dust moisture and direct
sunlight
3. Keep it standing in place ready for use, but protected
4. In humid climate, it is necessary to cover the microscope in a plastic bag with a drying
agent (silica gel) over night to avoid molds growing.
5. At the end of each day work, the surface of lenses, eyepiece and condenser should be
cleaned using lens tissue

Notes:
Never clean the lens and eyepiece with alcohol

Fig: Principal parts of compound light microscope

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 Parts and Function of a Microscope

1. Eyepiece: Where you place eyes. Its power 10x

2. Inclination Joint: An adjustment joint that lets the arm tilt th various angles
3. Course adjustment knob: A knob that makes large adjustment to the
focus

4. 9.10. High medium and power objective – high, medium and small lens with high,
medium and low magnifying power e.g – High power – 100×10 =1000X
Medium Power – 10×10 =100X
Low Power – 4×40 = 40X

5. Arm: This attaches the eyepiece and body tube to the base

6. Fine Adjustment knob: A knob that makes small adjustment to the focus
7. Body Tube: The body tube that supports the eyepiece
8. Revolving nosepiece: Rotating device that holds the objectives
11. Stage clip: Metal clips that hold a slide securely onto the stage
12.Stage: The platform on which a slide is placed
13.Diaphragm: An adjustable opening under the stage, allowing different amounts
of light onto the stage

14.Mirror: This directs light upward onto the slide

15. Basic: This Supports the microscope

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Date: 22-01-2023
Experiment No. 03
Experiment Name: Simple Staining

Learning Objective:
After completing this practical you will be able to:
1. Stain smears by simple staining method.
2. Compare various morphological forms and arrangement of bacterial cells.

Principle:
In simple stain use a single basic dye. Simple stain is done to identify the morphological shapes
and arrangement of bacteria.

Requirement:
Glass slide, Bunsen burner, Inoculating loop, Permanent glass marker, Sterilized loop, Immersion
oil, Microscope, sample

Reagents:
Basic dye (Crystal violet, Carbolfuchsin, safranin)

Procedure:
1. Take a clean grease free glass slide
2. By using a sterile loop, take a loopful organisms and make a smear
3. Air dry and heat fixed the slide by passing 2-3 times over a flame
4. Apply Crystal Violet for 1 minute and wash with running tap water
5. Observe the slide under microscope with oil immersion (Cederwood oil – It is used to increase
the intensity of light)

Results:

Figure: Crystal Violet stained smear of Staphylococcus aureus, 1000x.

Shape: Round shape organism

Arrangement: Cluster form arrangement

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Date: 01-02-2023
Experiment No. 04
Experiment Name: Gram Staining

Learning Objective:

After completing this practical you will be able to:

1. Stain smears by Gram’s staining method.
2. Differentiate between Gram positive and Gram negative bacteria.

Principle:

Some bacteria resist decolorization and stained dark blue or violet by gram staining due to thick
peptidoglycan layer. On the other hand some bacteria do not resist decolorization and stained red
or pink by gram staining due to thin peptidoglycan layer.

Equipment:

Glass slide, Bunsen burner, Permanent glass marker, Sterilized loop, Immersion oil, Microscope,
Sample (pus) .

Reagents:

1. Crystal violet stain. (purple)

2. Safranin stain. (red)
3. Gram's Iodine.
4. 95% Acetone Alcohol.

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Procedure:

1. Take a clean grease free glass slide.

2. Place a small drop of normal saline.
3. By using a sterile loop, take a loopful sample (pus) and make a smear.
4. Air dry and heat fixed the slide by passing 2-3 times over a flame.
5. Apply Crystal Violet (Primary dye - It is used to stain the all cells blue) For 1 minutes and
wash with running tap water.
6. Apply Grams Iodine (Used as Mordant interacts with CV+ and forms large complexes of
Crystal Violet and Iodine CV-I within the inner and outer layers of the cell) For 1 minutes and
wash running tap water.
7. Apply 95% Acetone Alcohol (Decolorization Agent - It removes the blue color of some
species but not others) Adding drop by drop For 10-15 sec and wash with running tap water
8. Apply Safranin (Secondary dye – It is used to stain red those cells that have been previously
decolorized) For 45 sec and wash with running tap water.
9. Observe the slide under microscope with oil immersion (Cederwood oil – It is used to
increase the intensity of light).

Results:

Figure: Gram Positive Blue or Purple color Figure: Gram Negative Pink or Red color

Examples of Gram-positive and Gram-negative bacteria:

Gram-positive bacteria: Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium

diptheriae
Gram-negative bacteria: Neisseria meningitidis, Escherichia coli, Pseudomonas aeruginosa

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Date: 08-02-2023
Experiment No. 05
Experiment Name: Negative Staining

Learning Objective:
After completing this practical you will be able to:
1. Perform a negative staining procedure for demonstration of bacteria.

Principle:
Negative stain or acidic stain is used to stain the background. In this stain bacterial cell wall do not
stain, because bacterial cell wall is negatively charged on the other hand the stain India ink or
Nigrosin is also negatively charged.
Requirement:
Sterilized loop, spirit lamp, Two glass slide, Permanent glass marker, Immersion oil, Microscope,
sample (organisms).
Reagents:
India ink, Nigrosin.
Procedure:
1. Take a clean grease free glass slide
2. Place a small drop of India ink to one end of a glass slide
3. By using sterile loop, place a loopful organisms onto India ink or Nigrosin and mixed well
4. By using another glass slide and the suspension at 45º angle
5. Then air dry (N.B. Do not heat fixed)
6. Observe the slide under microscope with immersion oil

Results:

Figure: Nigrosin-stained smear showing capsulated K. pneumoniae, 1000x.

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Date: 05-03-2023
Experiment No. 06
Experiment Name: Spore Staining

Learning Objective:
After completing this practical you will be able to:
1. Stain smears for bacterial endospores by malachite green stain.
2. Differentiate between bacterial spore and vegetative cell forms in the bacterial smear.
Principle:
Spore staining is used to determine the highly resistant spores of certain microorganisms (genus
Bacillus and Clostridium) within their vegetative cell.
Materials:
Sterilized loop, spirit lamp, glass slide, Hot plate with beaker, Permanent glass marker, Immersion
oil, Microscope, sample (organisms).
Reagents:
Malachite green, safranin.
Procedure
1. Take a clean grease free glass slide
2. Place a small drop of normal saline
3. By using a sterile loop, take a loopful sample (Sputum) and make a smear
4. Air dry and heat fixed the slide by passing 2-3 times over a flame
5. Apply heated with Malachite green (Primary stain) For 5 minutes and wash with running tap
water
6. Apply Safranin (Secondary dye) for 30 second with wash tap water
7. Observe the slide under microscope with oil immersion (Cederwood oil – It is used to increase
the intensity of light).
Results:

Figure: Spore staining

Green color organisms: spore forming bacteria
Red or pink color organisms: Non- spore forming bacteria

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Date: 19-03-2023
Experiment No. 07
Experiment Name: Acid-Fast Staining

Learning Objective:
After completing this practical you will be able to :
1. Stain smears by acid-fast staining method.
2. Become familiar with the chemical basis of the acid-fast stain

Principle:
The cell walls of acid fast organism (genus mycobacterium and Nocardia) contain a waxy like
lipid materials are called mycolic acid and it is impermeable to primary dye carbol fuchsin but
resist decolorization and stained red. Others organism are decolorized and stained with blue.

Equipment
Glass slide, Bunsen burner, Permanent glass marker, Sterilized loop, Immersion oil, Microscope,
Sample(sputum).

Reagents:
1. Carbolfuchsin stain
2. Methylene blue stain.
3. Acid-alcohol.

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Procedure:
1. Take a clean grease free glass slide
2. Place a small drop of normal saline
3. By using a sterile loop, take a loopful sample (Sputum) and make a smear
4. Air dry and heat fixed the slide by passing 2-3 times over a flame
5. Apply heated with Carbolfuchsin (Primary stain – It is used to stain all cells red) For 5 minutes
and wash with running tap water
6. Apply Acid Alcohol (Decolorization Agent - It removes red color from some species but not
others) For 1 minutes and wash with running tap water
7. Apply Methylene Blue (Secondary dye – It used to stain blue those cells that have been
previously decolorized) for 2 min with wash tap water
8. Observe the slide under microscope with oil immersion (Cederwood oil – It is used to increase
the intensity of light).

Results:

Figure:Acid fast stained M. tuberculosis Figure:Acid fast stained M. leprae

Acid Fast Bacteria appear bright red color

Non-Acid Fast Bacteria appear blue color

Examples of acid fast and non-acid fast bacteria:

Acid fast bacteria: Mycobacterium tuberculosis, Mycobacterium avium
Non-acid fast bacteria: Escherichia coli, Staphylococcus aureus,

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 Techniques for Isolation of Pure Culture

Date: 22-03-2023
Experiment No. 08
Experiment Name: Pour Plate Method

Learning Objective:
After completing this practical you will be able to:
1. To isolate the microorganisms from the liquid specimen(or suspension)
2. Perform the pour plate inoculation procedure

Principle:
In this Method, serial dilutions of the inoculum (serially diluting the primary specimen) are added
within sterile Petri plates to which is poured melted and cooled (42-45°C) agar medium and
completely mixed by revolving the plates which are then left to solidify.
After incubation, the plates are observed for the appearance of individual colonies growing
everywhere in the medium. The pure colonies which are of varying size, shape and color may be
isolated/transported into test tube culture media to prepare pure cultures.

Requirements:
 24 hours old nutrient broth culture of two different organisms.
 Nutrient Agar Medium
 Test tube
 Six 9 ml Sterile Distilled Water Blanks
 Sterile Petri plates
 Marker
 Micro pipette

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 Figure: Pour Plate method

Procedure:
1.Sterile the media by autoclaving
2. Pour the media into sterile test tube
3.Miexed diluted sample in test tube at 45° C temperature
4. Pour the sample mixing media in petridish
5. Incubate 37° C for 24 hours
6. Observe the colony
7. Subculture the colony

Observation and Results:

Study the plates for the appearance of individual colonies growing throughout the agar medium. It
will be observed that progressively poured plates will have a lesser and lesser number of colonies
which will be distributed more or less sparsely in the plates which may be transferred (subcultured)
to other media (fresh plates) or agar slants for further study.

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Date: 22-03-2023
Experiment No. 09
Experiment Name: Spread Plate Method

Learning Objective:
After completing this practical you will be able to:
1. To count the individual bacteria present in a diluted sample counting a mixture of different
species.
2. Perform the Spread plate inoculation procedure.

Principle:
The spread plate technique involves using a sterilized spreader with a smooth surface made of
metal or glass to apply a small amount of bacteria suspended in a solution over a plate. The plate
needs to be dry and at room temperature so that the agar can absorb the bacteria more readily. A
successful spread plate will have a countable number of isolated bacterial colonies evenly
distributed on the plate.

Requirements:
 Sterile loop
 spirit lamp
 Sterile petridish
 Culture media
 Diluted sample
 Incubator
 Test tube
 Micro pipette
 Glass bent rod.
 Distilled water

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 Figure: Spread plate method

Procedure:

1. Take 0.1 ml of diluted sample over the media

2. Spread the sample with the help of sterile glass bent rod
3. Incubate at 37 C temperature for 24 hour
4. Then separate the colony by observing the same size, shape, colony
5. Then we can obtain a subculture or pure culture

Observation and Results:

After incubation observes the colonies on the agar plate. Some of the colonies will be free from
each other. Select any colony from the plate and record the elevation, pigmentation, and size.

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Date: 22-03-2023
Experiment No. 10
Experiment Name: Streak Plate Method

Learning Objective:
After completing this practical you will be able to:
1. To produce isolated colonies of an organism (mostly bacteria) on an agar plate.
2. Perform the Streak plate inoculation procedure.

Principle:
The sample/inoculum is diluted by streaking it across the surface of the agar plate. While streaking
in successive areas of the plate, the inoculum is diluted to the point where there is only one bacterial
cell deposited every few millimeters on the surface of the agar plate. When these lone bacterial
cells divide and give rise to thousands and thousands of new bacterial cells, an isolated colony is
formed. Pure cultures can be obtained by picking well-isolated colonies and re-streaking these on
fresh agar plates.

Requirements:
 Sterile loop
 spirit lamp
 Sterile petridish
 Culture media
 Diluted sample
 Incubator
 Microscope

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 Figure: Streak plate method

Procedure:

1. Take a sample with the help of sterile inoculating loop

2. Touch the loop at one edge of the sterile media
3. Streaking forward and backward upto1-4th of the media
4. Then rotate the petridish and then sterile the loop and cool
5. Then the loop touch at last streaking forward and backward without touching the previous
streaks.
6. Streak four time by rotating the petridish by similar method
7. Incubate overnight and identify texture, color and shape of colony
8. We can obtain subculture or pure culture

Observation and Results:

The streaked plate is incubated at 37°C for 24 hours. Examine the colonies grown in the plate
carefully. All colonies should have the same general appearance. If there is more than one type of
colony, each type should be streaked again on a separate plate to obtain a pure culture.

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