Milk and Milk Products Technology

COLIFORM COUNT

The presence of coliform groups shows the unsanitary condition prevailing in the areas or may be used to find out the faecal contamination. The unsanitary condition in the place of production and at the processing site may be due to

1. Improper cleaning of dairy utensils
2. Improper cleaning of animals
3. Contaminated water supply to the dairy farms.

Presence of coliform causes souring, taints, gassiness and unclean flavour in milk and milk products.Estimation of coliforms in milk is carried out in similar manner as that of standard plate count. Violet red bile agar is used as the selective media for coliform count.

Apparatus required

• Petridishes, Petridish can, 1.1 ml pipettes, Pipette can, conical flask, test tubes, incubator, hot air oven, autoclave, refrigerator.

Reagents required

• Violet red bile agar, dilution blank

Composition of media

• Peptone 20 g
• Lactose 10 g
• Bile salts 5 g
• Nacl 1.5 g
• Neutral red 2.25g
• Agar 15 g
• Distilled water to make up 1 litre pH adjusted to 7.4

As the bile salts inhibit the growth of other type of micro organisms and only coliform groups grow well in this medium. Coliform group of organisms are present only in 1/100 and hence the dilution can be restricted upto 1/100. Coliform group will form subsurface colonies.

Procedure

• 1 ml of milk sample is taken and serially diluted. The serially diluted sample is then transferred to a sterile petridish and about 15 ml of media is poured. Petridish is left undisturbed for solidification and is then followed by addition of 5 ml of agar over the solidified medium which is known as over laying. Then the petridishes are incubated at 37⁰ C ± 0.5⁰ C for 20 hours. Colonies are pink in colour in the centre surrounded by an area of discolouration or zone of precipitation which is due to precipitation of bile salts by coliforms.
• Colonies more than 0.5 mm in diameter should be counted. Sometimes the diameter of the colony may be less than 0.5 mm when the violet red bile agar is over heated or if the plates are incubated more than 24 hrs. In ice cream samples also the colonies will be less than 0.5 mm in dia. In such cases, confirmatory tests have to be carried out by using broths like Brilliant green lactose peptone bile broth or Formate ricinoleate lactose peptone broth. Durham’s tubes have to be inserted into the broth for detecting gas production.
• Brilliant green lactose peptone bile broth helps to differentiate lactose and non- lactose fermenting organisms. Brilliant green and sodium ricinoleate inhibit gram positive organisms and favour the growth of gram negative organisms where as formate aids in the increased gas production.

BIS standard

• Raw milk: Coliform should be absent in 1/100 dilution
• Pasteurized Milk: Coliform should be absent in 1/10 dilution

Result

Comment

Question

1. What is the significance of finding coliform organisms in milk and water?
2. What type of fermentation is brought about by coliform bacteria in milk?