Milk Examination

Methods of Examination

• Quantitative Method

• Qualitative Method

• Isolation and Identification

Quantitative Method

In this method exact quantity or number of microbes are determined on the basis of pathogens. We categorize the milk on the basis of population of pathogens by counting. There are three methods:

• Direct microscopic total bacterial count

• Plate count

• Most probable number count

 
Direct Microscopic Count (DMC)

It is also called:

• Breed count

• Total bacterial count (as to count the number of bacteria which are dead or live)

Formula:

TBC/ml = Avg. number of cells/microscopic field × MF × 100 × dilution factor

Microscopic Field:

A bright illuminating circle under microscope is called as microscopic field. Mark an area on the slide having 1 cm length and 1 cm width.

1 cm² = 10⁴µm

1 cm² = (10⁴)² µm² = 10⁸ µm²

This area is called breed area (A).

No. of microscopic fields in breed area = A ÷ a

In microscope we want to calculate the field area i.e.

Field (circle) area (a) = πr² = π (d/2)²

For diameter we use stage micrometer with divisions but nothing written. Distance between two smallest divisions is 10 µm

Diameter = total division × 10

Total divisions are supposing 38.

So diameter will be 380 µm

Now area of circle in microscope field will be calculated

a = 3.14 × (380/2)²

   = 113354 µm²

Total area of microscopic field is 1 cm² or 10⁸ µm²

No. of microscopic field in area = A ÷ a = 10⁸ µm² ÷113354 = 882

Microscopic Factor (MF):
Number of microscopic fields in 1 cm²  i.e. breed area.

Calculation for Determination of Total Number of Bacteria in 1 ml of Sample:

Select 10-15 microscopic fields from whole sample on slide. Count bacteria in each microscopic field one by one and then calculate average number of bacteria in one microscopic field.

Bacterial count from 10 microscopic fields is added. Say value is 110. Now divide it by

10 (number of microscopic fields selected), answer will be 11. It is average number of bacteria in one microscope field.

Average number of bacteria per microscopic field = 11

Total microscopic fields in 1 cm² (MF) = 601

No. of bacteria in 1 cm² in 0.01 ml   = 601× 11 = 6611

Standard inoculating loop takes 0.01 ml sample.

No. of bacteria in 1 ml = 6611 × 100 = 6.6 × 10⁵

Viable Count/ Plate Count

Only live bacteria are counted in this method.

Procedure:

1. Take 10 fold serial dilution of milk sample

2. Take 10 test tubes. 9 ml of any diluents is taken in each test tube e.g. normal saline.

3. 1 ml of milk sample is poured first in first test tube by a pipette and it is then mixed thoroughly. (dilution 1/10)

4. 1 ml is transferred from first test tube to second test tube by pipette. It is mixed again. (dilution 1/100)

5. In the same pattern dilution is done up to the last test tube (in this fashion as dilution is increasing, bacterial number is decreasing).

6. 10 Petri dishes are prepared now with general purpose nutrient agar in each.

7. Inoculation of 1 ml sample from each test tube is done in each respective plate by spread method with help of spreader or dispenser.

8. Incubate these plates at 37 ^oC for 24 hours. Dense colonies are formed in first two plates and then gradual decrease in intensity is seen.

9. Select only one plate having 30-300 colonies and count the number of colonies in it e.g. plate number 4 is giving 240 colonies. (As a colony is formed by a single living cell, so a colony represents a living cell).

Formula:

Viable count/ml = average no. of colonies × dilution factor

(If volume is 0.5 ml, then result is multiplied by 2 in order to make 1 ml, it is called volume factor).

Calculation:

Viable count/ml = 240 × 10⁴ = 2.4 × 10⁶ CFU

Disadvantages:

• Time consuming as 24 hours are required for incubation while TBC takes only 5 minutes

• More glassware is required

• It is laborious method

• There are more chances of error. There may be some bacteria whose requirement is not nutrient agar. So they will not grow. Similarly some bacteria may be sachrophilic or thermophilic. Anaerobic bacteria may also be there in milk. Some bacteria may require more incubation time.

• There may be aggregation of colonies and these count falsely as one colony

Most Probable Number Count (MPN)

• A statistically designed method

• Mostly done for Colliform bacteria which ferment lactose, so lactose broth is used in it

• Phenolphthalein indicator is used

• Durham’s tube is also used in order to detect the gas production

• Lactic acid production; low pH. Low pH converts the colour of phenol red to yellow

• If acid; change in color

• If gas; accumulation of gas in tube

 Procedure:

1. 75 ml lactose broth is prepared

2. 15 test tubes are taken which are divided into three groups/sets.

3. Medium is sterilized

4. 5 ml lactose broth in each tube + phenol red + Durham’s tube in inverted form

5. Similarly in second and third set

6. Now take milk sample

7. Take 10 ml milk in each test tube of first set

8. Take 1 ml milk in each test tube of second set.

9. Take 0.1 ml milk in each test tube of second set.

10. Incubate at 37 ^oC for 48 hours

Result:

If yellow color                            +ve

If yellow + Gas                          strongly +ve

If no color change                    -ve

Count positive and negative results.

 
Qualitative Method

Methylene Blue Reduction Test

This test tells about bacterial load in milk sample.

Principle:
Reducing agent is produced when bacteria multiply which cause the reduction of methylene blue and color is changed from blue to white.

Procedure:

1. 10 ml milk sample + 1 ml methylene blue

2. Place in a water bath at 37 ^oC for 2-8 hours

3. Observe the results after each 30 minutes within 8 hours

Result:

On the basis of time of reduction. Note the change in color

Time is inversely proportional to bacterial population

Observation:

Observed the sample after 30 minutes and there was no change in color.

 
Resazurine Test

Principle:
Reducing agent is produced when bacteria multiply and this will cause reduction of resazurine dye. There is change of color from blue to white.

Procedure:

Take 10 ml milk sample + 1 ml of resezurine dye

Incubate it at 37 ^oC for 1 hour.

Result:

On the base of change in color.

Observation in Lab:

Sample + dye .........  white in 1 hour i.e. bad

Advantage:

• Less time is required.

Disadvantages:

• Dye should be kept on refrigerator at 4 ^oC

• Dye is light sensitive

• Interpretation of result is difficult.

Antibiotic Residual Evaluation in Milk and Milk Sample

STAF Test (Swab Test on Animal Foods)

Apparatus:

• Spore suspension of Bacillus subtilis (non pathogenic bacteria) with a ratio 10⁶ spores/ml,

• Soft agar which has agar agar 0.5-0.7 %. Nutrient agar has 2-3 % agar agar. Soft agar is jelly like.

• Petri dishes

• Sterilized cotton swabs

• Standardize antibiotic disc e.g. Kenamycin (5 µg)

• Milk or milk product i.e. sample

Procedure:

1. Put soft agar in test tube

2. Put 0.2 ml spore suspension

3. Mix it and pour the whole material in a Petri dish. Pouring as whole is called as over loading.

4. Wait for solidification

5. Now dip the sterilized cotton swab in milk for 20-30 seconds and place on the surface of nutrient agar plate and press it gently with the help of forceps.

6. Arrange all samples in the similar way. There should be distance of 1.5 cm among swabs.

7. Take the disk of kenamycin with sterilized forceps and place it above on the surface. Also press the disk. Distance between disk and swab should be 1.5 cm.

8. Then incubate for 20-24 hours at 37 ^oC.

Observations:

• Clear zone of inhibition is seen around the kenamycin disk i.e. 14-16 mm or less. it is control

• Now look at any other such zone around the cotton swabs

• If it is more than 2 mm, antibiotics residues are more than the standard.

Interpretation:

• If zone of inhibition around antibiotic disc is 14-16 mm, it means the test is valid or complete.

• If zone of inhibition is less than it then repeat the whole procedure.

• If swab zone is equal or more than 2 mm then the antibiotic residues are present and greater than permissible limits.

• If swab zone is less  than 2 mm then the antibiotic residues are of permissible level.

• If no zone around swab, no antibiotic residue.

Note: Simple boiling can break down the antibiotic residues.

Isolation of Indigenous Micro Flora of Mouth and Hands

There are two methods of examination

Direct Microscopic Examination:

• Take sample from the space between teeth with sterilized toothpick.

• Make smear on slide

• Fix with flame

• Stain with gram staining

• Examine under microscope and observe either G +ve, G –ve, rods, or cocci.

Cultivation of Bacteria:

• Sample is taken as in above method

• Spread on the surface of culture medium in the form of streaks

• Incubation for 24-48 hours

• Study the morphology of colonies

• Prepare the smear from colonies

• Stain and examine under microscope.

From Hands:

• Take sample from hand with cotton swab. Rub cotton swab on back, palm and between fingers of the hands.

• Also see the effect of washing hand simply or with disinfectant.

 
Microscopic Examination of M. tuberculosis

There are three methods:

Direct Method:
Make smear, fix it and do acid fast staining. If red or pink colored, bacilli are observed, M. tuberculosis is positive. Negative in case of blue color.

Cultivation Method:

• Stonebriks (for bovine type)

• Levestin Jenson Medium (Human type)

• Streak sample on medium

• Incubate at 37 ^oC for 2-3 days.

• Make the smear of colonies

• Stain with acid fast

Animal Incubation Method:

• Inject milk sample in guinea pigs in thigh region subcutaneously

• Keep the animals under observation

• If dies before four weeks then post mortem is performed. If not died then kill one and see postmortem lesions on lungs.

• Make smear on slide and observe under microscope.

• If lesions are not observed, slaughter the other pig after eight weeks and same observation is done.
  
   

Bacteriological Examination of Ice Cream/Milk Product

For general examination: blood agar or nutrient agar are used

For specific examination: specific medium are used e.g.

Staphylococcus                         Staph 110 medium

Streptococcus                           blood agar

Mycobacterium                          stonebriks

E. coli                                        Mackonkey’s agar

Salmonella and Shegella           SS agar

Fungus                                      Sabaroud’s agar

Streaks are made on medium and incubated at 37 ^oC for 48 hours

Colonies slide stain is applied accordingly.

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