Experiment No. 1 Good laboratory practice (GLP) in Microbiology and safety measures.

Experiment No. 2 Study of aseptic technique- preparation of cotton plug, wrapping of

glassware, transfer of media and Inoculum.

Preparation of Cotton Plug

Purpose:
Cotton plugs are used to close test tubes or flasks while allowing air exchange and preventing
microbial contamination.

Materials:

 Non-absorbent cotton
 Glass test tubes or flasks

Procedure:

1. Take a small piece of clean, non-absorbent cotton.

2. Roll it between fingers to form a cylindrical plug.
3. Insert the plug gently into the mouth of the test tube or flask.
4. The plug should be tight enough to prevent contamination but loose enough for
easy removal.
5. Ensure about 2–3 cm of cotton remains outside the mouth for handling.

Wrapping of Glassware

Purpose:
Wrapping protects glassware during sterilization and prevents contamination after
sterilization.

Materials:

 Test tubes, pipettes, Petri plates, flasks

 Kraft paper / aluminum foil
 Cotton plugs

Procedure:

1. Clean and dry the glassware thoroughly.

2. Plug test tubes or flasks with cotton.
3. Wrap the mouth or entire glassware using kraft paper or aluminum foil.
4. For pipettes, wrap individually or in bundles.
5. Label the wrapped items before sterilization.
6. Sterilize in a hot air oven (160–170°C for about 2 hours).

Transfer of Media
Purpose:
To transfer sterile culture media without introducing contaminants.

Procedure:

1. Work near a flame or inside a laminar airflow cabinet.

2. Flame the mouth of the media bottle or test tube.
3. Slightly open the container while keeping it near the flame.
4. Pour or pipette the media into sterile containers (e.g., Petri plates or tubes).
5. Flame the mouth again before closing.
6. Allow media in Petri plates to solidify if agar is used.

Transfer of Inoculum

Purpose:
To introduce microorganisms into sterile media for growth.

Materials:

 Inoculating loop/needle
 Culture medium
 Bunsen burner

Procedure:

1. Sterilize the inoculating loop by heating it in the flame until red hot.
2. Allow it to cool for a few seconds.
3. Flame the mouth of the culture tube.
4. Insert the loop and pick a small amount of microbial culture (inoculum).
5. Flame the mouth again and close the tube.
6. Open the sterile medium tube, flame its mouth, and transfer the inoculum.
7. Flame the mouth again and close it.
8. Re-sterilize the loop after use.

Conclusion:
Aseptic techniques such as cotton plugging, wrapping of glassware, and careful transfer of
media and inoculum help maintain sterility and prevent contamination, ensuring accurate
microbiological experiments
Experiments-3 To study the methods of cleaning and sterilization of laboratory
glassware and equipment used in microbiology.

Principle

In microbiology laboratories, glassware and equipment must be properly cleaned and

sterilized to remove dirt, chemical residues, and microorganisms. Cleaning removes visible
impurities, while sterilization destroys all microorganisms including spores. Common
sterilization methods include dry heat sterilization, moist heat sterilization, and flaming.

Materials Required

 Test tubes
 Conical flasks
 Pipettes
 Petri plates
 Beakers
 Detergent or laboratory cleaning solution
 Distilled water
 Cotton plugs
 Kraft paper / aluminum foil
 Hot air oven
 Autoclave
 Bunsen burner

Procedure
Cleaning of Glassware

1. Immediately after use, rinse the glassware with tap water to remove culture media or
chemicals.
2. Wash thoroughly using detergent solution and a brush to remove residues.
3. Rinse several times with tap water to remove detergent.
4. Finally rinse with distilled water to remove mineral deposits.
5. Allow the glassware to air dry or dry in a hot air oven.

2. Preparation for Sterilization

1. Plug test tubes and flasks with cotton plugs.

2. Wrap glassware such as pipettes and Petri plates with kraft paper or aluminum foil.
3. Arrange the wrapped glassware properly in sterilization equipment.

3. Sterilization Methods

a) Dry Heat Sterilization (Hot Air Oven)

  Used for glassware such as Petri plates, pipettes, flasks, and test tubes.
 Place glassware in a hot air oven at 160–170°C for about 2 hours.

b) Moist Heat Sterilization (Autoclave)

 Used for culture media and some equipment.

 Sterilize at 121°C, 15 psi pressure for 15–20 minutes.

c) Flaming

 Small instruments such as inoculating loops and needle tips are sterilized by direct
heating in a Bunsen burner flame until red hot.

Result

The glassware and equipment were properly cleaned and sterilized, making them suitable
for microbiological experiments without contamination.

Precautions

1. Glassware should be cleaned immediately after use.

2. Ensure complete removal of detergent during rinsing.
3. Do not overcrowd the hot air oven or autoclave.
4. Use proper temperature and time for sterilization.
5. Handle sterilized materials with aseptic precautions.
Experiments-4 Principle and applications of important laboratory instruments
Experiments-5 Preparation of different type culture media in Microbiology
Experiments -6 To prepare Potato Dextrose Agar (PDA) medium for the cultivation of fungi.

Principle

Potato Dextrose Agar (PDA) is a commonly used medium for the isolation and cultivation
of fungi such as molds and yeasts. It contains potato infusion, which provides nutrients,
dextrose as a carbohydrate source, and agar as a solidifying agent. The acidic nature of the
medium favors fungal growth and inhibits bacterial contamination.

Materials Required

 Potatoes (200 g)
 Dextrose (20 g)
 Agar (15–20 g)
 Distilled water (1000 ml)
 Beaker
 Conical flask
 Muslin cloth or filter paper
 Measuring cylinder
 Hot plate
 Petri plates or test tubes
 Cotton plugs
 Autoclave

Procedure

1. Peel and cut 200 g of potatoes into small pieces.

2. Boil the potato pieces in about 500 ml distilled water for 20–30 minutes.
3. Filter the extract through muslin cloth or filter paper to obtain potato infusion.
4. Add distilled water to make the volume up to 1000 ml.
5. Add 20 g dextrose and 15–20 g agar to the potato extract.
6. Heat the mixture gently with constant stirring until agar dissolves completely.
7. Transfer the medium into a conical flask and plug it with cotton.
8. Sterilize the medium in an autoclave at 121°C and 15 psi pressure for 15–20
minutes.
9. After sterilization, pour the molten medium into sterile Petri plates under aseptic
conditions.
10. Allow the medium to cool and solidify.

Result
Potato Dextrose Agar medium was successfully prepared and is ready for fungal culture
and growth.

Precautions
1. Use fresh potatoes for better nutrient content.
2. Ensure agar is completely dissolved before sterilization.
3. Maintain aseptic conditions while pouring plates.
4. Do not pour plates when the medium is too hot to avoid condensation.
5. Sterilize all glassware before use
Experiments -7 To obtain isolated colonies of bacteria/ fungi on an agar plate from a
consortium of cells by streak plate method
Experiments -8 To determine the Gram staining character of the given microorganism..
Experiments -9 To enumerate the bacteria in the given suspension using 1. Pour plate
method and 2. Surface spreading method