LPT 311: Milk and Milk Products Technology (1+1)

STANDARD PLATE COUNT

This method is used for determining the total number of viable bacteria per ml of milk and consists of mixing appropriate quantity of milk with suitable nutrient agar medium in a petridish and counting the bacterial colonies developed after incubation at a specified temperature for a definite period of time. This method is time consuming and expensive.

Apparatus required

• 1.1 ml pipettes, Petridishes, petridish can, pipette can, conical flask, incubator, refrigerator, autoclave, hotair oven, test tubes.

Reagents required

• Standard plate count agar and dilution blank

Composition of agar / litre

• Tryptone - 5 g
• Yeast extract - 2 g
• Dextrose - 1 g
• Agar - 15 g
• pH - 7 ± 0.2 (approx)

Identifying plates

• Each plate is identified with samples number and dilution to be used. Plating time as well as the date is to be noted.

Shaking sample and dilution

• The sample and diluetent has to be thoroughly mixed by shaking before drawing out the sample or dilution blank. Mechanical shaker can also be used to shake the blank for 15 seconds.

Selecting dilutions

• Dilutions are set in such a way that the total number of colonies in a plate is between 30 and 300.

Measuring the sample portions

• While removing the sterile pipette from the containers it should not be dragged over the exposed exterior surface of the can. While transferring milk or dilution blank sufficient time should be given for the column to drain from the graduation mark to the tip of the pipette. The pipette should not be rinsed with dilution blank as it may increase the count.

Transfer of dilution blank to petridish

• While transferring, the pipette should be held at an angle of 120⁰ and the petridish cover should be opened just enough to insert the pipette. Complete draining of the pipette should be allowed.

Plating

• Melt the required amount of sterilized media in boiling water. Cool it to 40-45⁰ C. Transfer the serially diluted milk sample in the petridish. Then approximately 10-15 ml of the media is poured into the petridish, time taken should not be more than 20 minutes is allowed before diluting the first sample and pouring the last plate. The serially diluted sample of milk and medium should be thoroughly mixed by rotating 5 times clockwise and 5 times anticlockwise and 5 times up and down and 5 times sideways, and the plate is kept undisturbed for 10 minutes for solidification of media. Plates are inverted and kept in the incubator maintained at 37⁰ ± 0.5⁰ C for 48 hrs.

Counting the plates

• Plates should be counted in such a way that the number of colonies does not exceed 300 per plate. All the colonies irrespective of size, shape and colour should be counted.

Interpretation

• The number of colonies is multiplied by the dilution factor gives the result and recorded as colony forming units per ml of milk.

Standard for grading

Type of milk	Count in cfu per ml	Grade
Pasteurized Milk Raw Milk	Not more than 30,000/ml < 200,000/ml 200,000-1 Million  1 – 5 Million  > 5 Million	Very good Good  Fair Poor

Result

 

Comment

 

Questions

1. What are the sources of error in SPC method? 
2. What are the limitations of the standard plate count?
3. Why are the petridishes kept inverted in the incubator?
4. Why are the plates incubated at 37⁰C?