Microbiological analysis of milk

Microbiological analysis of milk

Milk is the nutritious fluid secreted by mammals for the nourishment of their young ones.  Human beings use milk from various animals as a nutritious food.  milk contains 70-90% water in which several other molecules such as fat, proteins, carbohydrates, minerals, etc are either dissolved or suspended.  Milk is highly succeptible to microbial contamination and growth due to the various processes it undergoes from the moment of collection till the final consumption.  Due to these factors such as its complex composition and highly nutritive nature, milk requires high quality maintenance and testing standards. 

Various analyses of milk are performed to ensure its quality.  The main among them is the Platform tests or milk reception tests.  These are the tests commonly carried out by the persons who work at raw milk collection and/or reception. These are rapid quality control tests or organoleptic tests and are of crucial importance.  These tests enable the milks of inferior or questionable quality to be screened out before it is mixed with bulk milk during milk collection and/or reception. This is of crucial importance because one single lot of milk of poor quality can spoil the rest of the milk it is mixed with.

Generally, the platform tests do not directly involve laboratory analysis of raw milk samples, but in suspected cases of milk issues, a sample may be taken to the laboratory for further quality analysis. Such milk lot should be kept aside and should not be mixed with bulk milk till the laboratory results verifying their quality are available. If the milk sample does not pass the quality tests it should be rejected.

At milk reception sites - during milk collection and reception at milk plant - the platform tests generally performed are:Lactometer test, Organoleptic test, Clot on Boiling (COB), Alcohol Test, Ten-Minute Resazurin Test, etc.

Microbiological quality control test of milk can be divided into two, direct tests (Quantitative) and indirect tests (Qualitative).

The direct tests are helpful for assessment of the actual number of bacteria present in milk by microscopic examination, direct microscopic count (DMC) or by enumeration of the colonies formed by viable cells of bacteria, Standard plate count (SPC).

Qualitative or indirect methods assess the microbiological quality of milk based on the metabolic activity of the microorganisms. Examples are dye reduction methods such as MBRT, resazurin  test, etc. A correlation is made between the time required for the reduction of dye and probable number of bacteria in milk. 

1.      Lactometer test: If the milk appears too thin and watery during organoleptic inspections, it is suspected that milk contains added water. Lactometer test serves as a quick method for determination of adulteration of milk by adding water. It is based on the fact that the specific gravity of whole milk, skim milk and water differ.

2.    Organoleptic test - This includes judging the quality of milk by its taste and smell and this requires considerable skill which could be acquired by practice only. An experienced person can pick out bad milk samples accurately.

3.      Temperature - The temperature of milk can be measured using a standard thermometer.

4.      Determination of pH: The pH value or hydrogen ion concentration gives a measure of the acidity of milk. The pH of milk ranges from 6.6 to 6.8. Reduced pH value will be due to the development of acidity. Higher or alkaline pH indicates that the animal from which milk was obtained is suffering from mastitis. The pH of milk can be determined rapidly by using pH indicator strips.

5.    Clot on Boiling (COB): Clot on Boiling (COB) is used as rapid platform test for milk for testing increased acidity of milk. This quick test helps determining the quality of milk and to find out its suitability for pasteurization, boiling etc.

This test is performed by heating a small amount of milk in a test tube over a flame or immersed in boiling water for five minutes. Heating will precipitate proteins of milk if it is sour or acidic. The result can be observed immediately. If the side of the test tube and the film of milk shows any precipitated milk particles or clots it indicates positive (+ve) COB test. The '+ve' COB test indicates that milk has developed acidity above 0.17 percent.  Milk showing COB positive (+ve) test should be rejected or handled separately since such milk gets curdled during heat processing.

If no coagulation occurs, it is COB negative result and it indicates that milk can stand heating operations such as pasteurization.

This method is slower than alcohol test but is very useful where and when performing alcohol test is not possible.

6.    Alcohol Test: The Alcohol test is a rapid assessment test for the stability of milk to processing, particularly for condensing and sterilization. The alcohol test is useful as an indication of the mineral balance of milk. The test aids in detecting abnormal milk, such as colostrum, milk from animals in late lactation, milk from animals suffering from mastitis, etc.

This test is based on fact that the proteins in milk, which has become sour, e.g. due to lactic acid formation by bacteria undergo precipitation in presence of alcohol. If the mixing of equal quantities of milk and 68% alcohol in a test tube results in coagulation of proteins it indicates that milk is sour. This milk is not fit for any processes where heating is involved such as pasteurization.  This is due to the fact that the proteins in milks having increased acidity are not stable at temperatures used for pasteurization.

For this test 5ml of milk and equal quantity of alcohol are mixed in a test tube by inverting several times. The presence of a flake or clot denotes a positive test should be rejected. A negative test indicates low acidity and good heat stability of the milk sample.

7.    Acidity of milk: The titrable acidity of milk is estimated to ascertain its keeping quality and heat stability. It measures the amount of alkali (NaOH) required to change pH of milk from its initial value of pH (6.6 - 6.8) to pH 8.33. using this titration, Phenolphthalein indicator is added to milk which changes to pink colour at the end point, that is pH-8.33.  This method does not measure the true acidity but measures the buffering capacity of milk.

Titrable acidity (as lactic acid per 100 ml of milk) = 0.9VN.

Where: V = Volume in ml of the standard sodium hydroxide required for titration of 10ml of milk sample and N= exact normality of the standard sodium hydroxide solution.

The normal range of acidity of milk varies from 0.10 to 0.17 per cent lactic acid. Any value in excess of 0.17 percent is considered as developed lactic acid.

8.      Sediment Test: Sediment test reveals the extent to which visible insoluble matter is present in the milk and the extent to which such materials were not removed from milk by strainers. The sediment test is a simple, rapid and a quantitative measure that indicate the cleanliness of milk with respect to visible dirt. The test is carried out by allowing a measured quantity of milk to pass through a filter disc and observing the sediment left in the filter by comparing it with standards.

For this test a milk sample will be filtered through a properly adjusted firm cotton disc held in the sediment tester so that a filtration area of 28mm in diameter is exposed. The sediment disc will be compared with prepared sediment standard discs to record the sediment score.

The presence of appreciable sediment in unprocessed milk indicates careless or insanitary dairy farm practice. However, the lack of sediment is not always indicative of ideal conditions since visible sediment may be readily removed by straining at the dairy farm.

9.    Ten-Minute Resazurin Test: This is a rapid method of detecting milk of poor keeping quality. Resazurin is an oxidation-reduction indicator which is blue in the oxidised stage and upon reduction it will first turn irreversibly into a pink compound "resorufin" and then reversibly into the colourless 'dihydroresorufin'.  The reduction may be due to bacterial activity or other causes

During the first reduction from resazurin (blue) to resorufin (pink) in milk sample, the developed colour shades can be matched with standard colour discs in a comparator. During the second stage the pink colour fades out at a fast rate and the milk eventually turns white with a narrow pink band on the surface. The rate of reduction of resazurin is governed by the extent of bacterial activity in milk. This principle forms the basis of the ten-minute resazurin reduction tests or one hour resazurin reduction tests for judging the bacteriological quality of milk. Resazurin is also susceptible to the reducing action of leucocytic cells.

Take 10 ml milk sample in a sterilized test tube to which  l ml resazurin solution (0.05%) will be added and mixed by inverting.  Then it will be placed in water bath maintained at 37^o C for 10 minutes.  After removing the tube from water bath it will be placed in the right section of the comparator and Control milk tube will be kept in the left section of the comparator to compensate for the natural colour.  Using the standard resazurin disc the corresponding disc reading of the sample will be noted. When the colour falls between two disc numbers it will be recorded as half value.

Standards Resazurin disc reading values

Resazurin Disc Reading	Keeping quality	Remarks
6, 5  or 4	Satisfactory	Accept the milk
3.5 to 1	Doubtful	Requires further examination
0.5 to 0	Unsatisfactory	Reject the milk

10.  Alizarin-Alcohol Test: The stability of milk to alcohol or high temperature is affected by

a.       developed acidity or sweet curdling as results of bacterial growth

b.      disturbance in normal salt balance

c.       abnormal chemical composition (e.g. colostrum, late lactation and mastitis milks)

The alcohol test is used to assess the stability of milk to heat processing. Addition of alizarin along with alcohol helps to find out whether milk is acidic or alkaline.

For this test 5ml of milk is mixed with equal quantity of alizarin-alcohol solution in a test tube by inverting several times. The colour of the mixture and presence of flakes or clots and the size of the flakes will be observed

The presence of flakes or clots indicates poor heat stability and unsatisfactory quality of milk.

Presence of flakes or clots	Colour of mixture	Reference regarding heat stability	Quality
Negative	Lilac or pale red	Good - Low acidity	Satisfactory
Positive	Lilac pale red	Poor - sweet curdling	Unsatisfactory
Positive	Violet (Alkaline)	Poor – late lactation or Mastitis	Unsatisfactory
Positive	Brown (Acidic)	Poor – Developed acidity 0.1-0.2%	Unsatisfactory
Large flakes	Yellow  (Highly acidic)	Poor -Developed acidity more than 0.2%	Unsatisfactory

11.  Direct Microscopic Count: Direct Microscopic Count (DMC) is a quantitative test to assess the actual number of bacteria present in milk. It is a platform test also knows as breeds count used to assess the microbiological quality of milk.  It consists of examining stained films of a measured volume of milk under a compound microscope.  Generally, 0.01ml milk will be spread on glass slides over one sq.cm area. Each microscopic field observed represents a quantitative aliquot of the milk sample. The number of microscopic fields occurring in one square centimeter area of the milk smear will vary with different microscope as the diameter of the microscopic field varies. So a calculation using microscopic factor is done.

The microscopic factor or MF is calculated as follows, where r is radius of the microscopic field, which varies with different microscopes and lenses

This method is not suitable for examination of pasteurised milk since where dead cells are also counted.  Due the inability to discriminate between live and dead cells, this method gives a higher microbial count than the plate count (which gives estimate about live cells only) and the ratio of the standard plate count to direct microscopic count has been reported to be 1:4.

The microscopic appearance (types and arrangement of cells) of the milk is possible which will give indication about the source of contamination such as whether udder infection or utensil contamination or inadequate cooling, etc.

For this test, 0.01 ml milk will be spread evenly over 1 cm2 marked area on a grease free slide, it will be dried on a warm surface at 40 – 45^oC and stained using Newman's stain for 1 minute.   Newman's stain removes the milk fat, fixes the smear and stains the bacteria. The tetrachloroethane in the stain dissolve the milk fat globules, ethyl alcohol fixes the smear and methylene blue stains the microbial cells.  the smear will be observed under oil immersion objective. Any isolated single cell, pair of cells or clump of cells is treated as a ·clump'. The field for counting are selected at random. If the average number of clumps per field is under 0.5, 0.5 to 1, 1 to 10 and 10 to 30, the number of fields to be counted will be 50, 25, 10 and 5 respectively. If the number of clumps per field is over 30, then the milk will have to be diluted and staining procedure is to be repeated.  The average number of clumps per field will be multiplied by the microscopic factor (MF) to obtain the Direct Microscopic count per milliliter of milk.

Grading of milk based on DMC

Direct Microscopic Clump Counts per ml.	Bacteriological quality of milk
Less than 500000	Good
5,000,01 to 4000,000	Fair
4,000,000 to 20,000,000	Poor
Over 20,000,000	Very Poor

Detection of source of contamination of milk using DMC

Types of Organisms	Probable causes of high counts or poor quality of milk
Many cocci and rods in clumps and patches	Improperly cleaned milk utensils.
Excessive numbers of rod shaped bacteria and spores	Exposure of milk to dust and dirt
Large number of Cocci in pairs and short chains	Improperly cooling of milk
Large number of leucocyte cells (over 50,00,00 per ml) together with long chains of cocci	Mastitis infection

12.  Methylene Blue Reduction Test (MBRT): This test is based on the principle that methylene blue which is blue in its oxidised state, is reduced to a colorless compound as a result of the metabolic activities of bacteria in milk. Methylene blue is an oxidation-reduction dye or indicator.  MBRT is a rapid, sensitive, low cost, simple quantification method to evaluate microbial load of milk sample. This test involves the addition of methylene blue to milk sample and measuring the time required for decoloration. The disappearance of color is due to the removal of oxygen from milk and formation of reducing substances during bacterial metabolism. The time taken for the reduction of methylene blue depend on the number and types of bacteria growing in milk. The greater the number of organisms and greater their activity the more rapidly dye will be reduced and decolourised.

MBRT is used for

(i) Judging the hygienic quality of milk and grading raw milk supplies

(ii) For assessing the probable quality of milk

(iii) for detecting post pasteurization contamination in milk

One ml. of Methylene blue solution is added to 10 ml of milk in a test tube and mixed by inverting the tubes twice. the tube will be kept in the water bath at 37^oC. and observed after every 30 minutes for colour.  Continue the observations until the complete reduction of the dye or complete decolourisation occurs.  Two control tubes, one containing 10 ml of milk and 1 ml. of the methylene blue solution, after heating it in boiling water for 3 minutes and another with 10 ml. of milk plus 1 ml of tap water are also kept in water bath to compare the colour changes in experiment tubes.

Grading of milk based on MBRT

MBR Time (Hours)	Quality of Milk
5 and above	Very good
3 and 4	Good
1 and 2	Fair
0.5 and below	Poor

13.  Alkaline Phosphatase test: Phosphatase enzyme is present in milk and is destroyed during pasteurization. Phosphatase test is performed ·to determine the efficacy of pasteurization.  this enzyme converts the substrate P-nitrophenyl phosphate to p-nitrophenol, which is yellow coloured in alkaline solution.  This reaction occurs at pH 9.5 and 37^oC.

For phosphatase test, 10 ml buffer solution containing the substrate disodium P-nitrophenyl phosphate will be taken in two test tubes and kept at 37^oC.  2ml milk will be added to one of the test tubes (test) and to the other tube 2 ml of boiled and cooled milk will be added (boiled milk control).  The tubes will be closed and mixed and incubated at 37 ^oC for 30 minutes in a water bath.  After 30 minutes, the colour of the test is compared with the boiled milk control. The intensity of colour should be same in both the test and the boiled milk control. Any excess yellow colour in the test than the boiled milk control, indicates improper pasteurization.

14.  One-Hour Resazurin Reduction Test for Raw Milk: As discussed previously Resazurin is an oxidation-reduction indicator which undergoes reduction in two stages, first to a pink compound, resorufin and then to a colourless compound, dihydroresorufin as a result of bacterial activity. The first stage of reduction is irreversible and the second is reversible.  The reduction of resazurin in milk occur through  a series of colour changes, from blue to lilac, mauve, purple, pink, and finally colourless and these can be compared with standard colour discs in a lovibond comparator and expressed in terms of standard resazurin disc numbers (6 to 0). The time taken for the reduction of resazurin in milk to any particular stage or colour change is used as a criterion of bacterial activity in milk.

In the one-hour modification of the test the milk sample containing resazurin solution is incubated at 37^oC and the colour changes recorded at the end of one hour are used for grading milk. Rapid reduction of resazurin to the pink and colourless stages indicates high bacterial content and poor keeping quality. Resazurin test also helps to detect abnormal milk samples since leucocyte cells present in mastitis and late lactation milk also reduce resazurin.

One hour Resazurin Disc No.	Quality of Milk
4 or higher	Good
3.5 to 1	Fair
0.5 to 0	Poor

15.  Standard Plate Count (SPC) of Milk: Classically SPC procedures are used to determine the Total Plate Count (TPC) or Aerobic Plate Count (APC) or Total Viable Count (TVC).

Agar media containing Tryptone, Yeast extracts and Glucose of pH 7.0 .± 0.2 is used. Milk sample will be thoroughly agitated so that microorganisms are distributed as evenly as possible by rapidly inverting the container 25 times.  Appropriate dilutions of the sample will be prepared, 10^-3 dilution sufficient for pasteurized milk while upto 10^-7 dilution might be required for raw milk etc. The preparation of dilutions and the inoculation into media should not take more than 15 minutes.  Pour plate technique is generally employed, 1ml of dilution of the test sample will be mixed with 15ml of the medium at 45^oC. After complete solidification the plates will be inverted and incubated in the incubator at 37^oC for 48h . After the incubation the colonies developed will be counted using the colony counter and number colony forming unit perr ml of milk is calculated.

A standard plate count of lower than 30,000 cfu per ml. of pasteurized milk is indicative of satisfactory quality. Presence of many pinpoint colonies on the plates indicates thermophilic contamination

Microbiological Standards for Grading of Raw Milk

SPC/ ml	Grade
Not exceeding 2,00,000	Very good
2,00,000 - 10,00000	Good
10,00000 - 50,00000	Fair
Over 50,00000	Poor

Advantages of SPC

·         Enumeration of viable microbes only.

·         Cultural and morphological differentiation is possible based on colony characteristics.

·         Suitable for determination of quality of milk samples with low bacterial number, such as pasteurized milk.

Disadvantages of SPC

·      Gives only a rough estimate of microbial counts

·      Time consuming, laborious and cumbersome.

·      Not capable to grow all the species of bacteria present in milk, temperature of incubation may not be optimum for all types of bacteria, pathogens like Mycobacterium tuberculosis cannot grow easily

·      Amount of sample taken may not be representative.

16.  Coliform Count: Coliforms are aerobic and facultative anaerobic, gram-negative, non-spore forming rods able to ferment lactose with the production of acid and gas at 35^oC in 48 hrs. They grow in the presence of bile salts. These organisms are present in the intestinal tract of warm-blooded animals. Typically, these bacteria are the genera Escherichia, Enterobacter, Citrobacter and Kebsiella. The presence of these coliforms in milk indicates unsanitary conditions or practices during production, processing or storage of milk. Absence of coliforms in 1:100 dilutions in raw milk and in 1:10 dilution of pasteurized milk is accepted as a satisfactory quality.

Following protocol is used for determining the presence of coliforms:

·         Presumptive test

·         Confirmatory test

·         Completed test

·         Test for fecal coliforms

·         Most probable number (MPN) for enumeration of low counts

·         Differentiation of Escherichia coli and Enterobacter aerogenes

Presumptive test: This is commonly used for the detection of coliforms in milk. Here a sample of milk is inoculated into MacConkeys broth or Bile salt lactose peptone broth, and incubated at 37^oC, the production of acid and gas within 24-48 hour is regarded as presumptive evidence of coliforms in milk.

Confirmatory test: The positive presumptive test tubes showing acid and gas production is selected for confirmatory test.

A loop full of inoculum from the positive presumptive tube is streaked on the surface of Eosin Methylene Blue or Endo agar plates which are then incubated at 37^oC for 24-48 hours. Coliforms will grow as pink colonies with or without dark center and green metallic sheen.

A loop full of inoculum is also transferred to Brilliant Green Lactose Bile broth tubes that are incubated at 37^oC for 48 hours and the formation of gas in the tubes gives confirmatory result for coliforms.

Completed test: Broth tubes showing gas production in confirmatory test or typical colonies from agar plates will be used to inoculate MacConkeys broth tubes and nutrient agar slants. Acid and gas production in MacConkeys broth after 24 - 48 hours at 37^oC indicates positive completed test. Gram stain preparation from the nutrient agar slants showing Gram negative, non-spore forming cocco-bacillary rods also indicates the presence of coliforms.

Test for fecal coliforms (Eijkman’s test): In this test, inoculums from positive presumptive tubes are transferred to Brilliant Green Lactose Bile broth or MacConkeys broth tubes which are then incubated at 44.5^oC for 24 hours. Gas production in the inoculated tubes indicate the presence of fecal coliforms. 

Most probable number (MPN): A count of the number of tubes showing acid and gas production in the presumptive test give an idea about the probable number of bacteria present in milk by referring to MPN table.

Differentiation of Escherichia coli and Enterbacter aerogenes: Escherichia coli and Enterobacter aerogenes which are two types of coliforms in milk can be differentiated on the basis of biochemical tests, known as IMViC test (Indole test, Methyl red test, Voges-Proskaur test and Citrate test)

17.  Enumeration of other types of bacteria

Counting Proteolytic bacteria: Most proteolytic bacteria belong to psychrotrophs that grow at 7^oC. Different methods for isolation and identification of proteolytic microorganisms in milk are

Proteolytic microbial count on casein agar plates on incubation for 6 days. The number of colonies will be counted after flooding the plates with dilute acid

By using milk agar plates prepared by adding 10 percent of sterile milk to nutrient agar. If there is a clear zone around colony, it is considered proteolytic. The clear zone is due to dilute lactic or other acid.

Using an improved medium containing caseinate, citrate and Ca²⁺ . Casein breakdown can be observed as a white zone of precipitation.

Counting Lipolytic bacteria: Lipolysis results in the development of free fatty acids that cause a bitter taste. Psychrotrophs are generally responsible for lipolysis.   

Tributyrin agar is used for the estimation of psychrotrophic lipolytic count by incubating at 6.5^oC for 10 days.  Colonies appears with clear lytic zone.

Lipolytic activity can also be tested in tributyrin agar by incubating at 37^oC for 3 days.

Spore Count: Spore formers withstand pasteurization of milk.  Spore-forming bacteria from raw milk are predominantly Bacillus spp., such as Bacillus licheniformis, Bacillus cereus Bacillus subtilis, and Bacillus megaterium.

For their isolation, samples are kept at water bath maintained at 80^oC and then cooled to room temperature slowly. During slow cooling, the spores will germinate.  The sample will be then plated and incubated at 37^oC for 48 hours for enumeration of mesophilic spore forming bacteria and at 55^oC for 48 hour for thermophilic spore forming bacteria

Thermophilic Count: The thermophilic microorganisms such as Bacillus spp and Clostridium spp are enumerated using standard plate count by incubating at 45^oC or 55^oC for 48 h.

Thermoduric Count: Thermodurics such as Micrococcus, Microbacterium, Bacillus, etc are enumerated as follows. The milk samples are placed in a water bath maintained at 63^oC for 30 min and then cooled down to 5^oC.  Standard plate count is performed by incubating the plates at 37^oC for 48 h.

Psychrotrophic Count: Psychrotrophs are primarily aerobic Gram-negative rods of family Pseudomonadaceae, Neisseriaceae and Flavobacterium spp. and Alcaligenes spp. The standard plate count is used by incubating in a refrigerator (7-10^oC) for 7-10 days or kept in an incubator at 15^oC for 3 days.

18.  Enumeration of yeast and mould: The most common yeasts present in dairy products arc Kluyveromyces marxianus, Debaromyces hansenii, Candida famata, Rhodotorula spp, and Torulospora spp. The most common moulds belong to Penicillium spp, Cladosporium spp, Fusarium spp, Asperigillus spp, Rhizopus spp, Trichoderma spp, Geotrichum spp. and Mucor spp.

Yeasts and molds may be enumerated by plating milk sample in Potato dextrose agar or Malt extract agar and incubating at 30-32^oC for 3 -5 days.

Microscopic method -- Mould mycelia count: Mould mycelia count is made as direct microscopic count

Macroscopic method -- Methylene blue borax test - Five ml of warm milk is mixed with hot methylene blue and borax solution in a test tube, shaken well and the agglutinated mold  mass are gathered by a scalpel into a circular disc.  The diameter of disc is measured and its area in square mm is calculated.

Visual mould test or modified methylene blue borax test: A small amount of milk is mixed with methylene blue and alkaline salts, it is stirred and heated. The mixture is filtered and the mould mycelium retained is measured visually.

 

References

• ·   Chemical & Microbiological Analysis of Milk & Milk Products, Ramakant Sharma, International Book Distributing Co., First Edition 2006
• ·    Manual by Dr.Sunitha Gover,Profand Head,NDRI,Karnal
• ·    http://ecoursesonline.iasri.res.in/mod/page/view.php?id=65171