ENVIRONMENTAL BIOTECHNOLOGY

Experiment 7

Aim of the Experiment : STANDARD PLATE COUNT (SPC)

Introduction

It is a cantitative bacteriological analysis which enumerate total viable population capable of growing under a given set of conditions. Plate counts are useful in determining the effe ciency of water/waste water treatment. It is assumed that water of a good quality should give counts less than 100 colonies per milliliter.

Principle

TVC / SPC is based on the assumption that each viable bacterium de colony. Hence, original number of microorganism in the sample can be calculated number of colonies and then multiplying it with aliquot factor.

Materials required

• • • Water Sample

    • Petri plates

    • Pipettes

    • Molten Nutrient Agar

Procedure:

Prepare 10^-1 10^-2 and 10^-3……dilution of the water sample.

From each of the dilution transfer a fixed amount (e.g 0.2 ml) into sterile melted nutrient agar tube (previously cooled at 50^oC),mix it well and pour immediately in sterile petridishes.

Label plates (e.g as 10^-1/0.2 or as the case may be) ,clearly indicating the dilution and the volume plated respectively. Incubate all plates at 37^oC FOR 24 hours.

Count total number of colonies that has developed on each of the plate. If necessary, use colony counter to help counting of colonies.

Calculate final number of oraganisms present in the water sample as follows. Calculations & interpretation of results

1. For accuracy of results,countable plates are those which have colonies in between 30-300.Fewer than 30 colonies are not acceptable for statistical reasons,and more than 300 colonies on a plate is likely to produce colonies too close to each other to be distinguished as individual CFUs.

2. Lower dilution (e.g. 10^-1 , 10^-2) at time may show confluent (lawn)growth,due to high load of organisms

in the sample , these results are represented as too numerous to count (TNTC)

3. Final CFUs/ml can be calculated by multiplying the average number of colonies per countable, plate by the reciprocal of the dilution and the reciprocal of the volume plated .

CFUs/ml = Average number of the colonies /Dilution X Volume plated