Experiment No: 01

Experiment Name: Plate Preparation for Total Viable Count with

Nutrient Agar Media
Introduction
The total viable count (TVC) is a microbiological technique used to
estimate the number of viable bacteria or fungi present in a sample. This
information is valuable for assessing the quality of food, water, and
other products, and for monitoring the effectiveness of sanitation
procedures.
Objective
The primary objective of this lab experiment is to prepare nutrient agar
plates for the enumeration of total viable counts in a given sample.
Requirement
i. Nutrient agar powder or prepared nutrient agar plates
ii. Distilled water
iii. Autoclave or pressure cooker
iv. Sterile Petri dishes
v. Sterile pipettes or spreaders
vi. Incubator set to the appropriate temperature for the
microorganisms you are testing (typically 30-37°C for bacteria)
Procedure
1. Preparation of Nutrient Agar: First dissolve the agar in distilled water
and autoclaving the solution to sterilize it. After autoclaving, cool the
agar to around 45-50°C.
2. Pouring Agar Plates: Sterilize the Petri dishes by autoclaving or using
a sterilization method of your choice. In aseptic conditions (such as
within a laminar flow hood or using a Bunsen burner), pour the molten
agar into the sterile Petri dishes. Allow the agar to solidify by letting the
plates cool and solidify on a flat surface.
3. Inoculation: Using aseptic techniques, transfer a small sample of the
microorganism-containing solution onto the surface of the solidified
agar. Spread the inoculum evenly over the surface of the agar using a
sterile pipette or spreader.
4. Incubation: Place the inoculated plates in an incubator at the
appropriate temperature for the microorganisms you are testing.
Common temperatures are 30-37°C for bacteria. Incubate the plates for a
suitable period (usually 24-48 hours) to allow the microorganisms to
grow.
5. Counting Colonies: After incubation, observe the plates for the
presence of colonies. Each visible colony represents a viable
microorganism. Count the colonies on each plate. It's common to count
colonies on plates with 30-300 colonies to ensure accuracy.
6. Calculation: Calculate the Total Viable Count based on the number of
colonies and the dilution factor used in the procedure.
Result
The prepared nutrient agar plates were clear and transparent with a
smooth, even surface. No bubbles or cracks were observed on the agar
surface. The plates were ready for use after solidifying at room
temperature.
Discussion
Proper preparation of nutrient agar plates is essential for obtaining
accurate and reliable results in the TVC procedure. The use of sterile
materials, correct temperature control, and proper sterilization
techniques are crucial to ensure that the plates are free from
contamination and support the growth of viable bacteria or fungi.
Precautions
i. Sterilize sampling tools to prevent cross-contamination between
samples.
ii. Avoid touching the agar surface or the inner rim of the Petri dish to
prevent contamination.