STANDARD PLATE COUNT (SPC)

AIM:

To enumerate the viable number of microorganisms in a given milk sample by

standard plate count method.

INTRODUCTION:

American Public Health Association recommends SPC as the efficient method in its
milk ordinance and code. The presence of pathogenic bacteria may arise in milk due to
unsanitary handling of milk, a disease in the animal giving milk, improper storage conditions
or unhygienic milking conditions. Persistence of these microbes in milk may prove to be very
dangerous or even fatal sometimes. Hence it is necessary to estimate the viable number of
microorganisms present in the milk sample.

PRINCIPLE:

A small quantity of milk sample is mixed with standard plate agar medium and
poured into a Petri dish. After the agar has set, the plates are incubated at a specified
temperature for a definite period of time and the bacterial colonies that develop on the plate
are counted. Each colony is presumed to have grown from one bacterium or a clump of
bacterial cells present in the medium. Plates with 30-300 colonies are selected for counting to
obtain plate counts of colony forming units (CFU) per mL or gram of milk sample. In order
to calculate the total number of viable bacteria per gram or mL of the sample, the number of
colonies on each plate is multiplied by the dilution factor.

REQUIREMENTS:

Test tubes, saline water, graduated pipettes, petri plates, distilled water, milk sample.
REAGENTS:

Media for SPC

Tryptone – 5 g

Yeast extract – 5 g

K2HPO4 – 1g
 Glucose – 1 g

Standard Plate Agar – 20 g

Distilled water – 1000 mL

pH - 7

PROCEDURE:

1. The given milk sample was taken and shaken well for uniform distribution of microflora.

2. Five test tubes with saline water (0.2%) was labelled and arranged.

3. 1 mL of milk sample was pipetted out with a 10 mL pipette and transferred into a 9 mL
saline blank, which gives a 10-1 dilution.

4. The dilution was continued upto 10-5 .

5. The two sterile Petri plates were arranged and labelled with dilution factors.

6. 1 mL of sample was transferred to the respective labelled plates. Each sample were plated
in triplicates.

7. The melted standard plate agar was poured and cooled in each plate containing sample and
left for solidification.

8. After solidification, the plates were inverted and incubated at 37℃ for 48 hours.

9. the results were checked and the number of colonies were counted to get CFU.

RESULT:
INTERPRETATION:

REFERENCE:

Microbiology Practical Manual By R.C.DUBEY and D.K.MAHESWARI