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 bacterialnumbers 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 countCommingled Raw Milk …… 300,000/ml total countPasteurized 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 oC

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

colonies in the

plate 100

Number of

colonies in the

plate 10-1

Number of

colonies in the

plate 10-2

Number of

colonies in the

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.