Microbiological Examination of Dairy products

LAKSILU PEIRIS
GS/MSc/FOOD/3630/08
2008/2010
3.0 Microbiological Examination of Dairy products

Title: Microbiological Examination of Dairy products

Date: 21.02.2010
Experiment No :03
3.1 Introduction:
Raw Milk: Milk, when synthesized in the udder of a healthy cow is virtually sterile.
As milk passes through the teat cistern and teat channel, it may be contaminated with
low levels of bacteria (<1000/ml), which are generally not significant to milk quality
and safety. Milk from a cow with mastitis (infection of the mammary gland) however,
may harbor large numbers of the infectious bacteria. After it leaves the cow, milk may
be contaminated from the exterior of the cow (dirty cows), the environment and/or
poorly cleaned equipment. Poor cooling allows faster growth rates and can result in
rapid increases in bacterial numbers in raw milk before it is processed. While the legal
limit for bacteria in raw milk is 100,000/ml, the production of milk with bacteria
counts less than 10,000/ml should be easily achievable for most farms

Pasteurized Milk: Pasteurization, while initially designed to destroy potential

pathogens in raw milk, substantially reduces the total numbers of bacteria present,
increasing the shelf-life potential of the milk. Unless gross recontamination has
occurred, bacterial numbers in freshly pasteurized milk generally reflect the
organisms that survive pasteurization (thermoduric). The legal limit for bacterial
numbers in pasteurized milk is 20,000/ml, although bacteria counts for most freshly
pasteurized milks are generally less than 1,000/ml. Under proper refrigeration, the
bacteria that become significant in the shelf-life and spoilage of milk are
psychrotrophic in nature. These types of organisms generally occur as post-
pasteurization contaminants, although a few thermoduric bacteria may be
psychrotrophs.

Sources of Bacteria in Processed Milk:

1) Survive pasteurization (thermoduric).
2) Post-Pasteurization Contamination:
a) Insufficient cleaning/sanitizing - valves, pipelines, gaskets, pasteurized milk
tanks, fillers.
b) Personnel - hands, clothing, sneezes, coughs.
c)Environmental-air,dust,water,condensate.

Bacteriological Standards: (For Grade “A” Milk)

Raw Producer Milk ……….. 100,000/milliter (ml) total count
Commingled Raw Milk …… 300,000/ml total count
Pasteurized Milk……....... 20,000/ml total count, 10 coliform
3.2 Materials
• Raw milk
• Pasteurized milk
• Sterile test tubes
• Water bath at 37°C,
• Sterile Pipettes

3.3 Method

Methylene Blue Reduction Test

 All glass ware and stoppers were sterilized.

 10 ml of sterilized milk was transferred to labeled test tubes.

 1 ml of Methylene Blue solution was added to each tube containing

milk and closed - using sterilized stoppers.

 Test tubes were inverted gently for about four times.

 Test tubes were placed in water bath of 37°C.

 Blank sample was kept to observe the colour change.

 Discolourations were observed in every 30 minutes time.

 Time taken for Methylene Blue to get discoloured was recorded.

(ii) The Resazurin Test

1 ml of Resazurin solution was placed in a test tube.

10 ml of sample was added to the test tube and placed stopper kept in incubator at 36
o
C

Tubes were observed at the end of one hour and recorded.

(iii) Plate count method

 Dilution series of the two milk samples were prepared using Ringer solution.
  1 ml of each dilution was transferred to labeled petri dishes and 15 ml of
sterilized molten agar to each of them.
 Kept the petri dishes to solidify for 15 to 20 minutes and incubated at 37°C for
2 days.
 After 2 days the plates with colonies in between 30 to 300 in number were
selected and count the number of colonies present.

3.4 Results
3.4.1 Methylene blue test

Classification of milk is as follows.

Class 1 : Excellent, not decolorized in 8 hours.
Class 2 : Good, decolorized in less than 8 hours
Class 3 : Fair, decolorized in less than 6 hours. But not less than 2 hours
Class 4 : Poor, decolorized in less than 2 hours.

Time required approximate no of microorganisms present

20 in or less 2 x 107
20 min to 2 hrs 4 x 106 – 2 x 107
2 hrs to 5 ½ hrs 5 x 105 – 4 x 106
5½ hrs – 6 ½ hrs 2 x 105- 5 x 105
6 ½ hrs – 8 hrs < 1 x 105

3.4.1.1 Observations and comments

We observed the samples only up to 6 1/2 hours and both the samples were not
decolorized at that time.

Time Taken for Sterilized milk - colour changed in 6 hours

Time Taken for raw milk - Colour changed in 2 hours.
CFU values sterilize milk - 2 x 105- 5 x 105 /ml

3.4.2 The Resazurin Test

Classification of milk is as follows

1. Blue (no color change): Excellent

2, Blue to deep mauve: Good
3. Deep mauve to deep pink: Fair
4. Deep pink to whitish pink: Poor
5. White: Bad

3.4.2.1 Observations and comments

Colour change in Sterilized milk -No color change
Colour change in raw milk - It turned in to pinkish white color within 15
minutes
So the quality of the sterilized milk was excellent.
The quality of the raw milk was poor.

3.4.3 Plate count method

No of colonies present in each plate

Sample Number of Number of Number of Number of

colonies in the colonies in the colonies in the colonies in the
plate 100 plate 10-1 plate 10-2 plate 10-3
Sterilized Milk 290 29 2 -
Raw Milk TNTC TNTC 3X102 1X 102

TNTC-Too Numerous to count

3.4.3.1 Calculation- According to the results we obtained, the colonies should be

counted in the plates that the number of colonies lies between 30-300.

So for sterilized milk we can take the count in any plate, and the count was
290CFU/ml (CFU-Colony Forming Units)

For raw milk we have to select the highest dilution; ie 103 for the calculation. So the
count in raw milk was 1X102X 103(dilution) = 1X 105 CFU/ml
Discussion

Several factors affect for the Methylene blue reduction test. Therefore the steps of
operation should be done uniformly.

Any exploitation that increases oxygen is affecting the experiment since the oxygen
content must be used up before the color disappears. Cold milk can holds more
oxygen than warm milk. Also when pouring milk back and forth from one container
to another increases the amount of oxygen and at milking time much oxygen may be
absorbed.

The kind of organisms present affects the rate of reduction. The coliforms appear to
be the most rapidly reducing organisms, closely followed by Streptococcus lactis,
some of the faecal Streptococci, and certain Micrococci. Thermoduric and
psychrotrophic bacteria reduce Methylene blue very slowly if at all. A large number
of leucocytes affect the reduction time materially.

Light speed up reduction and therefore the tests should be kept covered. The
concentration of the dye should be uniform as an increased concentration lengthens
the time of reduction. Increasing the incubation temperature enhance the activity of
the bacteria and therefore shortens the reduction time.