Microbiology of Water, Milk and Air

APPLIED MICROBIOLOGY Microbiology of Water, Milk and Air Basic Concepts 9 9 9

9 9 9

Water is the most important vehicle for a variety of pathogens causing intestinal or systemic infections, particularly in developing countries because of its contamination with sewage or other excreted material Water is said to be contaminated (polluted) when it contains infective and parasitic agents, poisonous chemical substances, industrial or other wastes or sewage The hazards of water pollution are of two types: • Biological hazards - hazards due to infective agents, which may lead to water borne diseases • Chemical hazards - hazards due to chemical poisonous substances Faecal contamination of water supplies may lead to water borne-diseases

Safe water (potable water) is one that cannot harm the consumer, even when ingested over a prolonged period. Drinking water should not only be safe but also pleasant to drink, i.e. clear, colorless and devoid of unpleasant taste, smell or appearance.

Bacterial flora of water

Type of Water Natural

With soil washed in With sewage

Sewage proper bacteria

Flora • • • • • •

Micrococcus Pseudomonas Serratia Flavobacterium Alkaligenes Acinetobacter

• • • •

Bacillus subtilis Bacillus megaterium Bacillus mycoides Enterobacter aerogenes

• • • • • • • •

Escherichia coli Streptococcus faecalis Clostridium perfringens Salmonella typhi Vibrio cholerae Proteus vulgaris Clostridium sporogenes Nocardia species

Water born diseases

Bacterial Cholera Typhoid fever Shigellosis Diarrhoea due to: Esch. coli Yersinia enterocolitica Campylobacter fetus

Helminthic Roundworm Threadworm Whipworm Hydatid disease Guineaworm disease Fish tapeworm Schistosomiasis

Viral Hepatitis A Hepatitis E Rotavirus diarrhoea Poliomyelitis

Protozoal Amoebiasis Giardiasis Balantidiasis

Leptospirosis

Collection of samples: is done in sterilized containers of 230 ml with ground glass stoppers protected by kraft paper. It is to be ensured that contamination of sample does not take place whatever may be source of sample. Microbiological Examination of Water: Microbiological examination offers the most sensitive test for the detection of recent and potentially dangerous faecal pollution, thereby providing a hygienic assessment of water quality with high sensitivity and specificity. Indicators of human/ animal pollution are used. The organisms which have thus been used as indicators are: o Esch. coli o Faecal coliforms o Faecal streptococci (Strep. faecalis) o Sulfite reducing clostridia (especially Clostridium perfringens ) Anaerobic bacteria such as bifidobacteria and Bacteroides are more abundant than coliform organisms in faeces but routine methods for their detection and enumeration are not as yet available hence these are not used. Faecal streptococci are regularly present in the faeces in varying numbers but their number is fewer than Esch. coli and they probably die and disappear at the same rate. Presence of faecal streptococci along with coliforms in absence of Esch. coli is also confirmatory of faecal pollution. Cl. perfringens is also present in faeces in small numbers. Their spores which can resist chlorination survive for longer times. Thus, the presence of Cl. perfringens in natural water suggests that faecal contamination has occurred and in the absence of coliform organisms suggests that it occurred quite sometimes ago. Methods of Analysis Detection of Faecal Streptococci and Detection of Clostridium welchii provides confirmatory evidence of feacal contamination. Subcultures from presumptive positive bottles in coliform test are made into tubes containing 5cc of glucose azide broth and incubated at 45*C. Production of acid in the medium indicates the presence of Stretococci faecalis. When water sample is inoculated in litmus milk and incubated anaerobically at 37*C for 5 days, stormy fermentation confirms the presence of Clostridium welchii. Multiple Tube Method In this method, the most probable number (MPN) of coliform bacilli present in the water sample can be determined statistically. Double strength and single strength MacConkey's broth with inverted Durham's tube for indication of gas production are used as media.

An estimate of colifonn count per 100 ml is made from the tubes/bottles showing positive results using the probability tables of McCrady. The presumptive coliform count is reported as follows: Presumptive coliform count

Class

O 1-3 4-10 More than 10

Excellent Satisfactory Suspicious Unsatisfactory

Membrane-Filter (MF) Method In this method a measured volume of water is filtered through a membrane which retains the bacteria on its surface. The membrane is then incubated on a suitable selective medium, allowing the bacteria to reproduce and to form colonies. The number of colonies counted is directly related to the bacteriological content of the water sample being analyzed. This method has not been as extensively used as multiple-tube method. It is not suitable for turbid water but may otherwise have several advantages

Types of bacteria found in milk: Milk supports the growth of a variety of bacteria including pathogenic one The different types of bacteria present in milk are, as follows: 1. Acid-fonning bacteria, such as o Streptococcus lactis o Str. faecalis o Lactobacilli These ferment lactose, forming lactic acid, and lead to the formation of curd. 2. Alkali-fonning bacteria, such as o Alkaligenes sp. o Achromobacter o Aerobic spore-forming bacilli These render the milk alkaline. 3. Gas-forming bacteria, such as o Coliform bacteria o Cl. peifringens o Cl. butyricum These produce acid and gas. 4. Proteolytic bacteria, such as o Bacillus subtilis o B. cereus o Proteus vulgaris o Staphylococci o Micrococci These bacteria are responsible for proteolytic activity. 5. Inert bacteria, such as o Achromobacter o Pathogenic bacteria o Cocci They do not produce any visible change.

Diseases and infections transmitted through milk are: I. Infections of animals transmitted to man o Tuberculosis o Brucellosis o Streptococcal and staphylococcal infections o Salmonellosis o Q fever

o Anthrax o Leptospirosis o Cowpox and milker's nodes o Foot and mouth disease o Tickborne o viral encephalitis 2. Infection due to ingestion of milk contaminated with excreta of small mammals o Streptobacillus moniliformis o C. jejuni o Y. enterocolitica 3. Infections primarily of man transmitted through milk o S. typhi o Paratyphoid bacilli o holera vibrio o Shigella o E. coli o Streptococcal and staphylococcal infections o Tubercle bacilli o Hepatitis virus o Diphtheria bacilli

The tests used for examining bacteria present in milk are: 1. Viable count · It is detected by plate counts with serial dilutions of the milk samples 2. Coliform count o Dilutions of milk are inoculated into MacConkey's liquid medium o The production of acid and gas is noted after incubation-positive test indicates presence of coliform bacilli (in two tubes out of three) o All coliforms are killed by pasteurization o The presence of coliforms in pasteurized milk indicates improper pasteurization or postpasteurization contamination 3. Methylene blue reduction test o An indirect method for detection of microorganisms in milk o An economical substitute for viable count o Procedure: 1 ml of methylene blue is mixed with 10 ml of milk in a test tube and o incubated in dark at 37°C o Milk is considered satisfactory if it fails to decolourize methylene blue III 30 minutes 4. Resazurin test It is also a dye reduction test similar to methylene blue dye test. Reduction of resazurin by bacteria passes through a series of color changes-blue to pink to colorless, which depends upon degree of contamination 5. Phosphatase test Phosphatase is an enzyme normally present in milk, which gets inactivated by pas teurization of milk. Its presence in milk after pasteurization indicates improper/incomplete pasteurization. Its presence is detected by adding disodium phenyl phosphate to 1 ml of milk in a test tube and incubating in a water bath at 37°C for 2 hours. Development of yellow color indicates presence of phosphatase 6. Turbidity test This test is used to check sterilization of milk. When milk is sterilized properly by heating at 100°C

for

5 minutes, all coagulable proteins are precipitated and when ammonium sulphate is added to this milk, no turbidity results because of denaturation of proteins. This indicates that milk has been heated at 100°C for 5 minutes

Specific tests include: 1. Test for tubercle bacilli The milk is centrifuged at 3000 rpm for 30 minutes and sediment is inoculated into two guinea pigs and two L-J media. L-J media are observed for growth and guinea pigs are observed for a period of 3 months for tuberculosis. Confirmation is done by using biochemical tests. 2. Tests for Brucella bacilli Isolation of brucellae is attempted by inoculating cream from the milk sample on serum glucose agar and then injecting centrifuged deposit of the milk sample intramuscularly in guinea pigs. Guinea pig can be sacrificed after 6 weeks and the spleen used for culture of brucella and serum used for demonstration of Abs by milk ring test. Milk ring test is the highly sensitive test for demonstration of brucella Abs in the milk of infected cows. The test is used for diagnosis of brucellosis in animals. The positive milk ring test is confirmed by Whey's agglutination test In case of suspected food poisoning, the sediment of centrifuged milk should also be examined for: o o o o

Staph. aureus Salmonella sp. Campylobacter sp. Y. enterocolitica

Bacteriology of air Air always contains foreign substances including microorganisms and it is never pure. The microbes are present in air in all places at all times. The level of bacterial contamination is expressed as the number of bacteria carrying particles per m3 or per ft3. Since an adult male inhales about 15 m3 and a baby about 1 m3 of air in a day, the bacterial content of air is important, particularly when the air contains pathogenic microorgamsms. Possibility of a person to acquire infection will be more if he is exposed to high concentration of airborne pathogens Humans contribute significantly in spreading microbes in the environment, e.g. during talking, coughing and sneezing The bacterial content of air depends on • Increased density of human and animal population • Nature of soil • Amount of vegetation • Atmospheric conditions such as humidity, temperature, wind, rainfall and sunlight • Location, Outdoor air contains usually nonpathogenic organisms, however indoor air contains droplets of organisms disseminated by humans and animals, which may include pathogens Bacteriological examination of air is required in: • Surgical operation theatres • Hospital wards • Storehouses or premises of food articles or pharmaceutical preparations

Bacteriological Examination of Air

Settle Plate Method (Sedimentation Method) Petri plates containing culture media are exposed to air for 30 to 60 minutes Nutrient agar or blood agar (for streptococci and staphylococci) or selective media for special pathogens can be us bacteria carrying dust particles settle onto the medium The plates are incubated at 37°C for 24 hours

The number of colonies is counted and the number of bacteria present in air is calculated It is simple but measures only the rate of deposition of large particles from air but not the total number of large bacteria carrying particles in air. Uses: Testing air in surgical operation theatres and hospital wards Slit Sampler Method · It determines the number of bacteria in a measured volume of air · It is better than settle plate method, as it is efficient in detection of very small particles also. Air Contamination Standards: Bacterial count should not exceed the upper limit of: · 10/ft3 in operation theatre · 1/ft3 in operation theatres for neurosurgery and burns · 50/ft3 in factories, offices and homes