Bacteriology of Water, Milk and Air

In this article we will discuss about the Bacteriology of Water, Milk and Air.

Bacteriology of Water:

Introduction:

Safe water should be free from microorganisms and chemical substances and drinking water in parti­cular should not only be safe but also pleasant to drink, i.e. cool, clear, colourless and devoid of dis­agreeable taste or smell. Faecal pollution of water supplies may lead to introduction of a variety of intestinal pathogens that comprise water-borne diseases (Table 17.1).

Bacteriological indicators:

These are based on organisms indicative of water pollution by human/animal faeces, such as:

(i) Escherichia coli and coliform as a whole,

(ii) Faecal streptococci (S. faecalis) and

(iii) Clostridium perfringens.

While evaluating faecal pollution of water supplies, one has to keep in view the bacterial flora in water (Table 17.2) as natural water almost always contains a few harmless microorganisms.

1. Coliforms:

The coliforms ferment lactose, e.g. E. coli, Klebsiella spp, Enterobacter, Citrobacter and Edwardsiella spp. E. coli is the typical example of faecal group and Klebsiella aero-genes is an example of non-faecal group of coliform.

2. Faecal streptococci (Streptococcus faecalis) are regularly found in faeces though their number is much less than Escherichia coli. Their presence in water is regarded as confirmatory evidence of recent faecal contamination of water in doubtful cases.

3. Clostridium perfringens also occurs in faeces regularly. They are also excreted in much smaller number than Escherichia coli. Presence of spores of the organism in water supplies indicate faecal pollution.

Bacteriological examination of water:

1. Presumptive coliform test:

(a) Multiple-tube method:

By this test the most probable number (MPN) of coliform organisms are detected in 100 ml water.

Media:

Double-strength and single strength modified MacConkey’s fluid medium containing bromocresol purple sterilised in bottles/tubes conta­ining Durham’s tube (for indication of gas produ­ction).

Procedure:

Measured amounts of sample of water are added by sterile graduated pipettes as follows:

(i) 50 ml water to 50 ml double strength me­dium.

(ii) 10 ml water to 10 ml double strength me­dium.

(iii) Five 1 ml quantities each to 5 ml single strength medium.

(iv) 0.1 ml quantities of water to each 5 ml single strength medium.

The tubes/bottles are incubated at 37°C for 48 hours. An estimate of coliform count is made from the tubes showing acid and gas production by the help of a statistical table (Tables 17.4 and 17.5). The reaction may occasionally be due to combination of organisms or due to other organisms. Organism is identified by biochemical test (Table 17.3).

(b) Membrane-filter (MF) method:

A measured volume of water is filtered through a membrane specially made of cellulose ester. Bacteria are retained on the surface of membrane. The membrane is inoculated (face upwards) in suitable medium and incubated for 15-20 hours and the number of colo­nies are counted directly.

2. Detection of faecal streptococci and CI. welchii:

Their presence in water provides useful confir­matory evidence of the faecal pollution of water in doubtful cases. These are identified by subculture in solid media.

Colony count:

One ml test sample of water is placed in Petri dish (10 cm diameter) and then 10 ml melted yeast agar (45°-50°C) is poured on the water, mixed thoroughly and allowed to solidify. One more plate is prepared. One plate is incubated at 22°C and other at 37°C for about 18-24 hours.

Interpretation of total count:

(i) Growth at 22°C indicates the amount of decom­posing organic matter present in water.

(ii) Growth at 37°C is more important index of contamination of water sample.

3. Biological examination of water:

Sometimes water may contain microscopic organisms, such as algae, fungi, yeast, protozoa, minute worms, etc. which are collectively known as ‘plankton’. These plankton organisms are index of pollution and produce objectionable taste in water.

4. Interpretation of results:

On the basis of repeated tests of water, a standard has been suggested by pioneer workers in the field into class 1, class 2, class 3 and class 4 types of water based on presumptive coliform count per 100 ml sample (Table 17.6):

Bacteriological examination of sewage and sewage effluents:

1. Concentration of the organisms in the sample yields better result. Sample may be concentrated by membrane filter technique. Pathogenic bacteria retained on the surface of membrane are transferred into a suitable differential medium and incubated.

2. Dilution method:

As the number of patho­genic bacteria present may be scanty, larger volume of multiple samples may have to be examined for isolating the organisms.

A volume of 10 ml of en­richment and selective media (selenite broth for Salmonella spp., alkaline peptone water for Vibrio cholerae) is mixed with nine times its volume of water and incubated for 24 hours and then sub-cultured in suitable media. Isolated organisms are identified by biochemical test and serotyping.

Bacteriology of Milk:

Introduction:

A. Presence of bacteria in milk:

Milk always contain some bacteria derived from various sources:

(i) Milk ducts of udder:

Even when best possible precautions are taken, some bacteria are derived from the udders (Table 17.7). Their number is high­est in the fore-milk and lowest in milk obtained by stripping’s. Aseptically drawn raw milk may contain 10 bacteria to several thousand per ml.

(ii) Milking equipment:

Unsterile milking equip­ment is a major contributor of bacteria of milk.

(iii) The milker:

Milkers’ hands also contribute to bacteria in milk which can be avoided by proper cleaning of hands before milking the animal.

(iv) Unclean udders and dust in milking shed:

These are also sources of bacteria in milk.

(v) Water:

Water used for cleaning the udders and for adulteration also partly contribute to bacteria in milk.

(vi) Diseased animals:

Pathogenic organism cau­sing infection in animals (e.g. mastitis, brucellosis, tuberculosis) may be excreted in milk.

(vii) Carriers:

Carriers of infectious diseases (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.

B. Types of bacteria in milk:

The various types of bacteria that may be encoun­tered in milk are given in Table 17.7. Milk, even under best precautions, contains some bacteria. Fresh milk is bactericidal as well as bacteriostatic but this activity disappears a few hours after the milk has been withdrawn.

C. Types of bacteria in human milk:

(a) Prior to infant feeding:

S. epidermidis (100% sam­ples), S. mitis (69%), Gaffkya tetragena (19%) and S. aureus (13%). Other organisms are found in about 10% samples.

(b) After breast-feeding:

Same organisms as men­tioned above are seen but most bacteria are derived from maternal skin and infant’s mouth.

D. Milk-borne diseases (Table 17.8):

Milk is a good medium for bacteria and also a good vehicle for many organisms and may result from infection of animals, contaminated milk and infections of man.

Pasteurization of milk:

Pasteurization of milk — heating to 72°C/161°F for 15 seconds followed by rapid cooling to 10°C/ 50°F—eliminates the risk of most infections (90%) including the more heat-resistant tubercle bacillus and Q fever organisms, but it does not destroy ther­mophilic bacteria and bacterial spores.

E. Biological standards of milk:

Standards of milk as laid down under Milk Regu­lations Act of 1965 and 1972 in England and Wales is as follows:

(i) Untreated milk: Raw milk under most favour­able conditions, may contain at least 500 bacteria per ml.

(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 must satisfy the turbidity test.

(iv) Ultra heated milk must contain less than 10 bacteria per 0.01 ml (i.e. 1,000 bacteria per ml).

Milk analysis:

Sample is collected aseptically.

Sampling:

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

A. Bacteriological tests:

1. Viable count:

This plate count method of test is done by serial dilutions of milk sample (1:10, 1: 100, 1 :1,000 in sterile Ringer’s 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 the colony count in the fixed amount of milk.

2. Coliform test:

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

B. Chemical test:

This test includes methylene blue test, phospha­tase and turbidity tests.

1. Methylene blue test:

This simple test is an economical substitute of viable count. Viable bacte­ria reduce the dye in milk when kept in a 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 the 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 condition.

Resazurin test is also a 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, mauve, pink to the final colourless state of complete reduction.

The “Ten Minute Resazurin Test’ is usually done in creameries, in which change of colour of milk containing the dye is noted after 10 minutes and compared with a set of colour standards in a Lovibond comparator.

2. Phosphatase test:

This is statutory test for pasteurized milk in England. The test is done by adding 1 ml of milk to be tested to 5 ml 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 phenol is produced if the milk contains phosphatase. The colour is compared with a standard solution by a comparator or colorimeter. In a properly pasteurized milk, the concentration of p-nitro phenol is less than 10 μg per ml of milk.

C. Turbidity test:

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

D. Detection of specific pathogens:

(a) Tubercle bacilli:

The milk is thoroughly mixed and 50 ml amounts are centrifuged in each of two tubes at 300 rpm for 30 minutes. Part of the deposit is used for AFB-film and part is used for inoculation into guinea pigs.

(b) 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, serum is examined by Br. abortus suspension and spleen is used for culture of brucellae.

Brucellosis in animals can be detected by demon­strating antibodies against brucellae in milk by milk ring test and whey agglutination test.

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 of air in a day, the content of air is important particularly so when the air con­tains pathogenic organisms.

I. Sources of air pollution:

(i) Human sources:

Man is an important source of spreading bacteria in the environment in droplet during coughing and sneezing. The air-borne bac­teria may be derived by evaporation of droplets or 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 droplet include tuberculosis, Q fever, psittacosis and coccidioido­mycosis, etc.

(ii) Environmental sources:

Soil and vegetation’s contain non-pathogenic organisms such as Achromobacter, Sarcina, Micrococci, sometimes coliform bacilli, spores and fragments of moulds.

II. Pollution in different types of air:

Bacterial content in air depends on location whether it is outdoor or indoor.

(i) Outdoor air:

The bacterial content depends on type of soil and vegetation, humidity of air and density of population. The air above 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, spores and frag­ments 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 disseminated by man and animals. Hospital air may contain droplet nuclei or skin scales carrying infective organisms.

III. Bacteriological examination:

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

There are two methods:

(i) Sediment method:

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

(ii) Slit sampler method:

The number of bacteria in a measured volume of air is determined by this method. A known volume of air is directed onto 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 evenly in the medium. One cubic foot of air is made to pass through these slits. 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.

IV. Interpretation:

(i) 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.

Staphylococcus aureus is the predominant air-borne organism, about 0.1 to 10 organisms are present in one cu. ft. air. Streptococcus pyogenes may be found in larger numbers in the air (10/cu. ft. air) in rooms occupied by patients with scarlet fever and streptococcal tonsillitis.

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

It has been found in guinea pig that as little as 0.00008 tubercle bacilli per cu. ft. air is sufficient to cause infection. Thus, a man may be infected even when he inhales only a single patho­gen in 500 or so cu. ft. air that he respires during 24 hours.

(iii) 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.