WATER TESTING-Practical guidelines

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Dr. P.SREENIVASULU REDDY MD Professor of MICROBIOLOGY NARAYANA MEDICAL COLLEGE NELLORE-2 ANDHRA PRADESH

MICROBIOLOGY OF WATER & ANALYSIS FOR MICROBIOL AGENTS : 

MICROBIOLOGY OF WATER & ANALYSIS FOR MICROBIOL AGENTS

Safe :Wholesome:Potable:Contaminated / Polluted: Human and animal activity. Hazards of water pollution : Biological Chemical hazards : 

Safe :Wholesome:Potable:Contaminated / Polluted: Human and animal activity. Hazards of water pollution : Biological Chemical hazards

Bacterial flora in water: : 

Bacterial flora in water: Natural : Micococci Pseudomonas Serratia Flavobacterium Alkaligenes Acinetobacter With soil: B.anthracis Enterobacter aerogenes With sewage: E.coli Str.fecalis Cl.perfringes Salmonella typhi V.cholerae Sewage proper bacteria: Proteus vulgaris Cl.sporogenes Nocardia spp.

Fecal pollution: Introduces varieties of pathogens. : 

Fecal pollution: Introduces varieties of pathogens. Bacterial: Cholera Typhoid fever Shigellosis Diarrhoea E.coli Y.enterocolitica C.fetus Leptospirosis Viral: Hepatitis A , E Rota viral diarrhoea Poliomyelitis Helminthes: Round worm Thread worm Whip worm Hydatid disease Guinea worm disease Fish tape worm Schistosomiasis Protozoal: Amoebiasis Giardiaisis Balantidiasis

FACTORS AFFECTING NUMBER AND TYPE OF BACTERIA IN WATER : 

FACTORS AFFECTING NUMBER AND TYPE OF BACTERIA IN WATER Type of water: Surface or deep Sea water Mineral springs Presence of organic matter Temperature Light pH Dissolved oxygen Rainfall Season Storage Filtration

Microbiological examination of water : 

Microbiological examination of water For detection of recent and potentially pathogens. To provide a hygienic assessment of potable water. Water source should be tested by a simple test than sophisticated series of tests. Identification of individual organism is difficult. Indicator organisms of human/animal pollution are used.

Indicator organisms: Esch.coli. Faecal coliforms Faecal streptococci. Clostridium perfringens.Presence of fecal streptococci along with coliforms in the absence of E.coli is also confirmatory of fecal pollution. : 

Indicator organisms: Esch.coli. Faecal coliforms Faecal streptococci. Clostridium perfringens.Presence of fecal streptococci along with coliforms in the absence of E.coli is also confirmatory of fecal pollution.

Criteria for indicator organisms for water bacteriology: : 

Criteria for indicator organisms for water bacteriology: Present in feces in abundant number. Present in scanty number in other sources. Easy to isolate, identify and enumerate. Unable to grow in water. Able to survive in water than pathogens. More resistant to disinfectants like chlorine.

Water sampling for bacterial examination: : 

Water sampling for bacterial examination: Should be properly planned & ideally carried out based on seasonal variation. Collected, stored and transported in a suitable, sterilized bottles. Sample should be large enough & should be dispatchedas soon as possible. Sample details should be properly labelled. Testing should be done in authorized laboratories. 100 ml of bottle : 0.1 ml of 1.8% aqueous solution of Sodium thiosulphate

Sampling of Water:Three basic types : 1.From tap or fixed hand pump 2.From reservoir (River, Lake, Tank) 3.Water from dug well. : 

Sampling of Water:Three basic types : 1.From tap or fixed hand pump 2.From reservoir (River, Lake, Tank) 3.Water from dug well.

Sampling from Tap Water:Attachments of tap should be removed.Dirt has to be removed by using a sterile cloth.Water is allowed to flow for 2 minutes with maximum flow.Tap is sterilized with flame ( gas burner, lighter, cotton soaked in spirit).Open the tap for outflow.Fill the sterile bottle with water by leaving an air space. : 

Sampling from Tap Water:Attachments of tap should be removed.Dirt has to be removed by using a sterile cloth.Water is allowed to flow for 2 minutes with maximum flow.Tap is sterilized with flame ( gas burner, lighter, cotton soaked in spirit).Open the tap for outflow.Fill the sterile bottle with water by leaving an air space.

Sampling from reservoir:Take a sterile bottle.Hold it in its lower part.Submerge into the depth of 20 cm.Mouth should be slightly upwards.If there is current ,bottle should face the current.The bottle is packed in a brown paper. : 

Sampling from reservoir:Take a sterile bottle.Hold it in its lower part.Submerge into the depth of 20 cm.Mouth should be slightly upwards.If there is current ,bottle should face the current.The bottle is packed in a brown paper.

Sampling from a dugwell: A suitable size stone has to be attached to the sampling bottle. A 20 meter length of clean string is tied on the bottle and a stick. Bottle is immersed completely in the water and lowered down to the bottom of the well. Once the bottle is filled ,it is pulled out. Little water is discarded to provide air space. : 

Sampling from a dugwell: A suitable size stone has to be attached to the sampling bottle. A 20 meter length of clean string is tied on the bottle and a stick. Bottle is immersed completely in the water and lowered down to the bottom of the well. Once the bottle is filled ,it is pulled out. Little water is discarded to provide air space.

Processing of the sample: Should be processed within 6 hours. In case of delay ,water can be filtered by using a membrane filter. Filter paper has to be transported on an absorbent pad saturated with transport medium. : 

Processing of the sample: Should be processed within 6 hours. In case of delay ,water can be filtered by using a membrane filter. Filter paper has to be transported on an absorbent pad saturated with transport medium.

Methods of analysis:1.Multiple tube method. Water to be tested are added to the tubes containing a suitable culture medium. Useful for: Clean, colored or turbid water containing sewage or sewage sludge , mud and soil particles. Medium : Double strength MacConkeys medium with bromocresol purple indicator and inverted Durham’s tube. : 

Methods of analysis:1.Multiple tube method. Water to be tested are added to the tubes containing a suitable culture medium. Useful for: Clean, colored or turbid water containing sewage or sewage sludge , mud and soil particles. Medium : Double strength MacConkeys medium with bromocresol purple indicator and inverted Durham’s tube.

One 50ml of water to 50 ml of Double strength medium.Five 10 ml quantities each to 10 ml Double strength medium.Five 1 ml quantities each to 5 ml Single strength medium.Five 0.1 ml quantities each to 5 ml Single strength medium.Bottles are inoculated at 370C and examined after 24 hrs for acid and gas production.From the number and distribution of positive (presumptive coliforms) and negative reactions, Most Probable Number (MPN) of indicator organisms in the sample may be identifiedAccording to McCrady tables. : 

One 50ml of water to 50 ml of Double strength medium.Five 10 ml quantities each to 10 ml Double strength medium.Five 1 ml quantities each to 5 ml Single strength medium.Five 0.1 ml quantities each to 5 ml Single strength medium.Bottles are inoculated at 370C and examined after 24 hrs for acid and gas production.From the number and distribution of positive (presumptive coliforms) and negative reactions, Most Probable Number (MPN) of indicator organisms in the sample may be identifiedAccording to McCrady tables.

Eijkman test (Differential coliform test or confirmed Esch.coli count): To confirm the presence of typical coliforms in the presumptive test ( E.coli or spore bearing bacilli). Subcultures to be done from positive presumptive test samples in a single strength medium. Incubate at 440C for 24 hours. Samples with gas & acid confirms Esch.coli. (Confirmed by positive Indole and Citrate utilization test.) : 

Eijkman test (Differential coliform test or confirmed Esch.coli count): To confirm the presence of typical coliforms in the presumptive test ( E.coli or spore bearing bacilli). Subcultures to be done from positive presumptive test samples in a single strength medium. Incubate at 440C for 24 hours. Samples with gas & acid confirms Esch.coli. (Confirmed by positive Indole and Citrate utilization test.)

Detection of Fecal Streptococci: Subcultures are done from positive bottles in the presumptive coliform test. Use the Sodium azide broth (toxic). Incubate for 18 hours at 450C Positive test indicated by gas production. Confirmed by culturing on MacConkey’s agar for typical colonies.

Detection of Clostridium perfringens: 1.Water should be inoculated in Litmus milk medium. Inoculate for 5 days at 370C. Typical Stormy clot reaction with acidity -- positive. 2.Sulphate reducing medium : Black precipitate (sulphite  sulphide ) : 

Detection of Clostridium perfringens: 1.Water should be inoculated in Litmus milk medium. Inoculate for 5 days at 370C. Typical Stormy clot reaction with acidity -- positive. 2.Sulphate reducing medium : Black precipitate (sulphite  sulphide )

Membrane –Filter method: : 

Membrane –Filter method: Advantages: Rapid. Easy & Economical. Gives direct result. Useful in rural areas. Samples can be tested in the field Disadvantages: Turbid water interferes with bacterial growth. Noncoliforms interferes with counting of coliforms. Filters have to be procured. Toxic substances in the water may be absorbed by filter and interferes with bacterial growth.

Classification of drinking water: : 

Classification of drinking water: