Bacteriology of Milk: An Overview

The below mentioned article provides an overview on Bacteriology of Milk:- 1. Introduction to Bacteriology of Milk 2. Bacteria in Human Milk 3. Pasteurization of Milk 4. Biological Standards of Milk 5. Bacteriological Examination of Milk 6. Sampling of Milk 7. Bacteriology of Air 8. Bacteriological Examination 9. Interpretation.

Contents:

1. Introduction to Bacteriology of Milk
2. Bacteria in Human Milk
3. Pasteurization of Milk
4. Biological Standards of Milk
5. Bacteriological Examination of Milk
6. Sampling of Milk
7. Bacteriology of Air
8. Bacteriological Examination
9. Interpretation

---

1. Introduction to Bacteriology of Milk:

Milk always contain some bacteria derived from:

(i) Milk ducts of udder: Even when best possible precautions are taken, some bacteria are derived from the udder. The number is highest in the pore and lowest in milk obtained by striping. An aseptically drawn raw milk may contain 10 bacteria to several thousand per ml.

(ii) Milk equipment: Unsterile milking equipment is a major contribution of bacteria of milk.

(iii) The milker’s hands also contribute to bacteria in milk, which can be avoided by proper cleaning of hands before milking the animal.

(iv) Unclean milker and dust in milking shed : These are also sources of bacteria in milk.

(v) Water used for cleaning udders and for adulteration also partly contribute to bacteria in milk.

(vi) Diseased animals: Pathogenic organisms causing infections in animals (e.g. mastitis, brucellosis, tuberculosis) may be excreted in milk.

(vii) Carriers: Carriers of infectious disease (e.g. typhoid, paratyphoid, dysentery, food poisoning bacilli, coagulase positive staphylococci, or haemolytic streptococci) who milk the animal sometimes act as sources of pathogenic bacteria of milk.

---

2. Bacteria in Human Milk:

(a) Prior to Infant Feeding:

Staph, epidermidis (100% samples), str. mitis (69%), Gaffkya tetragena (19%), Staph, aureus (13%)

(b) After Breastfeeding:

Some organisms mentioned above are seen but in large numbers derived from maternal skin and infant’s mouth.

---

3. Pasteurization of Milk:

Pasteurization of milk eliminates the risk of most infections (90%) including the more heat- resistant tubercle bacilli and Q fever organisms but it does not destroy thermophilic bacteria and bacteria spores.

Pasteurization is the process in which the milk is heated at a particular temperature for a fixed period required to destroy any pathogens but may be present in the milk while causing minimal changes in the composition, taste and nutritive value.

There are several methods of pasteurization:

(a) Holder Method:

Temperature employed is 63-66°C for 30 minutes followed by rapid cooling to 5°C. It is useful in smaller set ups.

(b) Flash Method:

Temperature employed is 72°C for 15 seconds and then rapidly cooled to 4°C. It is used for larger quantities of milk.

(c) Ultra-High Temperature (UHT) Method:

Rapidly heated usually in two stages between 125°-132°C for a few seconds (2-3) only and then rapidly cooled.

---

ADVERTISEMENTS:

4. Biological Standards of Milk:

Standards of milk is laid down under Milk Regulation Acts of 1965 and 1972 in England and Wales:

(i) Untreated milk: Raw milk under most favourable conditions may contain at least 500 bacteria per ml. But under bad conditions, the number may be several million.

(ii) Pasteurized milk should not contain coliform in 0.1 ml milk, and on submission to phosphatase test, must give a reading not exceeding 10 µg of p-nitro-phenol per ml of milk.

(iii) Sterilised milk should satisfy the turbidity.

(iv) Ultra-heated milk must contain less than 10 bacteria per 0.01 ml. (i.e. 1000 bacteria per ml).

---

ADVERTISEMENTS:

5. Bacteriological Examination of Milk:

It consists of:

(a) Enumeration of viable bacteria

(b) Test of coliform bacilli

(c) Chemical tests

(d) Examination of specific pathogens.

---

ADVERTISEMENTS:

6. Sampling of Milk:

(i) If the milk is contained in retail milk bottles, one unopened is to be sent to the laboratory.

(ii) When the milk is in a large container, sample is collected by a sterile dipper from the depth of milk by a sterile 4 oz. screw-capped bottle.

(1) Viable Count:

The plate count method of test is done by serial dilutions of milk sample (1;10,1:100; 1:1000 in sterile Ringer’ solution) incorporating in yeast, extract milk agar in 10 cm. petri dish and then incubated at 30-31°C for 72 hours. Number of colonies multiplied by dilution factor gives colon)) count in the fixed amount of milk.

Coliform Test:

Varying dilutions of milk are inoculated in 3 tubes of McConkey’s fluid medium with Durham’s tube and incubated at 37°C for 48 hours. The production of acid and gas is noted. Contamination of milk by coliform bacilli usually occurs from dairy utensils, dust and dairy workers.

Chemical Test:

Methylene Blue Test:

This simple test is an economical substitute of viable count. Viable bacteria reduce the dye in milk when kept in cool dark place. Solution of methylene blue used gives a final concentration of 1/300,000. The milk is decolorized after reduction of methylene blue and rate of reduction is related to the degree of bacterial contamination.

Raw or untreated milk is considered satisfactory if it fails to decolorize methylene blue in 30 minutes under standard conditions.

Resazurin test is also dye reduction test similar to methylene blue test. Unlike methylene blue test, reduction of resazurin by bacteria passes through a series of colour changes — blue, lilac, marve, pink to the final colourless state of complete reduction.

Then Ten Minute Resazurin test is usually done in Creameries, in which changes of colour of milk containing the dye is noted after 10 minutes and compared with a set of colour standards in a Lovibond comparator.

(b) Phosphatase Test:

This is statutory test for pasteurized milk. The enzyme phosphatase, normally present in cow’s milk, is inactivated during pasteurization of milk. The standard technique of detecting the presence of enzyme is based on its ability to breakdown disodium p-nitro-phenyl phosphate and liberate p-nitro-phenyl.

The test is done by adding 1ml of milk to be tested to 5ml buffer substrate solution (buffer + disodium phenyl phosphate) in a test tube. The mixture is incubated in water bath at 37°C, for 2 hours. A yellow coloured solution of p. nitro-phenyl is produced if the milk contains phosphatase.

The colour is compared with standard solution by a comparator or colorimeter. In a properly pasteurized milk, the concentration of p. nitro-phenyl is less than 10 µg per ml of milk.

(c) Turbidity Test:

The test for sterilised milk distinguishes between pasteurized and sterilised milk. When milk is heated to at least 100°C for 5 minutes, the soluble protein in milk get denatured and that cannot be precipitated by ammonium sulphate.

4. Detection of specific pathogens:

Tubercle Bacilli:

The milk is thoroughly mixed and 50 ml amounts are centrifuged in each of two tubes at 300 r.p.m. for 30 minutes. Part of the deposit is used for ACID FAST BACILLI (AFB) and part is used for inoculation into guinea pigs.

Brucella:

Isolation of Br. abortus is attempted by inoculating cream on serum, dextrose agar. The cream and centrifuged deposit of milk may also be injected intramuscularly into guinea pigs. The animals are killed after six weeks, the serum is examined by Br. abortus suspension and spleen is used for culture of brucella.

Brucella in animals can be detected by demonstrating antibodies against brucellae in milk by Milk Ring Test (MRT) and other agglutination tests.

---

7. Bacteriology of Air:

The immediate environment of man comprises of air on which depends all forms of life. Since a man respires about 500 cft in a day, the content of air is important; particularly so when the air contains pathogenic organisms.

Sources of Air Pollution:

(i) Human Sources:

Man is an important source of spreading bacteria in the environment in the droplet during coughing and sneezing. The air-borne bacteria may be derived by evaporation of droplet by aerosols. The bacterial content in air increases with increased density of human and animal population.

Pathogenic organisms do not multiply in air and are seldom carried from more than short distance infections that spread by droplets include tuberculosis, Q fever, psittacosis and coccidioidomycosis etc.

(ii) Environmental Sources:

Soil and vegetation’s contains organisms which also become air­borne. Majority of these organisms are non-pathogenic such as Achromobacter, Sarcina, Micrococci, sometimes coliform bacilli, spores and fragments of moulds.

Pollution of Different Types of Air:

(i) Outdoor Air Pollution:

Outdoor air bacteria depends on types of soil and vegetation, humidity of air and density of population. The air in ocean is almost bacteria-free. However, outdoor air contains much less bacteria than indoor air and the organisms present are non-pathogenic e.g. Achromobacter, Sarcina, Micrococcus, Bacillus subtilis and spores and segments of moulds.

The upper air contains much less bacteria. Pathogenic bacteria usually do not survive in outdoor air.

(ii) Indoor Air:

Indoor air also contains droplets of organisms dissimilated by man and animals or skin scales carrying infective organisms.

Hospital air may contain droplet nuclei or skin scales carrying infectious organisms.

---

8. Bacteriological Examination:

Bacteriological examination is necessary for operation theatre, some store house of food, pharmacy, and hospital wards.

There are two methods:

1. Sediments Method:

Petri-dish (9-10 cm diam) containing nutrient agar and blood agar is exposed to air for half to one hour and the plates are then incubated at 37°C for 24 hours colonies are then counted.

2. Slit sampler Method:

The number of bacteria in a measured amount of air is determined by this method. A known volume of air is directed into a plate of culture medium through a slit of 0.25 mm wide. The plate is rotated mechanically so as to allow the organisms to spread out in the medium.

One cubic foot of air is made to pass through the slit. In the same way, 10 cubic feet is tested. The culture media are incubated and colonies counted which gives the number of bacteria present in the air.

---

9. Interpretation:

Most bacteria found in the air are harmless saprophytes, or commensals and even in the wards of hospital and other closed rooms occupied by the patients and carriers.

Approximately 1% of the air-borne organisms in the wards or closed rooms are pathogenic. Staph, aureus is predominant air-borne organisms., about 0.1 to 10 organisms are present in one cubic foot air.

Streptococcus pyogenes may be found in large in the air (10/cu ft. air) in rooms occupied by patient with scarlet fever and streptococcal tonsillitis.

1. It is not definitely known what should be the minimum size dose of organisms that may cause infection following inhalation. This depends upon the type of pathogenic organisms and the best immune response.

2. It has been found that as little as 0.000008 tubercle bacilli per cubic foot air is sufficient to cause infections. Thus, a man may be infected even when the inhalation is only a single pathogen in 500 or so cu ft. air that he respires during 24 hours.

3. The following limits of air pollution may be acceptable:

(a) Factories, offices, homes etc. 50 per cu ft.;

(b) Operation theatre 10 per cu ft.

(c) Dressing room operation theatre for neurosurgery-1 per cu ft.