microbiological examination of water2
TRANSCRIPT
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Water Significance and Contamination
Water is essential for all forms of life and certain human activities.
In the course of survival, certain living organisms may contaminate water required for use by others.
Human activities may also lead to contamination of water sources with toxic substances.
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Why control water standards?
Health and survival of man and other organisms depends on the purity of the water they use.
Different measures are used to access and control water quality with varying degrees of relevance and acceptability.
Hence the need to adopt generalized methods of assessment and arbitrary standards for water quality.
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Water Quality Assessment
Water Quality
Physical Chemical Microbial
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Water Quality Parameters• Turbidity
• Palatability
• Conductivity
• Total Dissolved Solutes
• Organoleptic Properties
• etc
Physical
• pH
• Dissolved Oxygen Concentration
• Residual Free Chlorine
• Radionuclides
• Organic/ inorganic Chemical Contents
• etc
Chemical
• Faecal Coliform Count
• Total Coliform Count
• Faecal Streptococci
• Cysts/ Ova of Parasites
Microbial
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Microbiological Analysis
Offers the most sensitive test for the detection of recent and potentially dangerous faecal pollution
Provides a hygienic assessment of water quality with high sensitivity and
specificity
It is important to examine water sources frequently by simple tests rather than infrequently by more complicated test or series of tests
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Indicator OrganismsInvestigation for individual specific
pathogens is rarely practical since they are fewer in numbers than the non-pathogenic organisms and their detection methods are too demanding.
Therefore, indicators of human/animal pollution e.g. coliforms are used to access water quality.
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Indicators
E. coli
Faecal Coliforms
Total Coliforms
Faecal Streptococci
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Indicator Organisms Cont’d
•Found in human and animal faeces; in sewage, treated
effluents, and natural waters subjected to
faecal contamination•Up to 109 per gram in
fresh faeces. •Routine identification is complex, but tests are
available for rapid reliable identification.
Escherichia coli
•Genus Escherichia and some spp of Klebsiella, Enterobacter and Citrobacter•May originate from organically enriched water or from decaying matter.•Rarely regrow in the distribution system
Faecal coliforms
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Indicator Organisms Cont’d
•Gram-ve, bacilli tolerant to bile salts.
•Not necessarily faecal bacteria.
•Should not be detected in treated water and, if found,
suggest inadequate treatment, post treatment
contamination, or excessive nutrients.
• Test used as an indicator both of treatment efficiency
and of the integrity of the distribution system.
Total Coliforms
• Present in the faeces human and animal faeces.
•Belong to the genera Enterococcus and
Streptococcus•Most spp are of faecal origin
and indicate faecal contamination.
•Rarely multiply in polluted water, and are more
persistent than the coliforms. •Indicators of treatment
efficiency.
Faecal streptococci
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SamplingSampling from a tap or pump outlet
Remove any attachments from tap that may cause splashing.
Wipe off the dirt from outside the tap.
Turn on the tap at maximum flow rate and let the water flow for 1-2 minutes.
Sterilize it for a minute with flame using gas burner, lighter or ignited cotton wool soaked in spirit.
Open the tap and allow water to flow at medium rate for 1-2 minutes.
Open the container for collecting the sample and fill the water by holding the bottle under the water jet. Leave a small airspace to facilitate shaking at thetime of inoculation prior to analysis.
Stopper the cap and label the container.
Sampling from reservoir Submerge the bottle in the
water. Open the bottle inside of the
water. Fill it by holding it by the lower
part, submerging it to a depth of about 30 centimetres, with the mouth facing slightly upwards. If there is a current, the bottle should face the current.
Pull it out when the bottle is filled.
Discard a little water to provide airspace
Stopper the bottle and label it.
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Methods for Microbial Analysis
Microbial Analysis
Membrane filtration method
Multiple-tube method
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Sampling cont’dSampling from a dug well
Attach a stone of suitable size to the sampling bottle with a piece of string.
Tie a 20 meter length of clean string on the bottle and to a stick.
Open the bottle as described above and lower into the well. Immerse the bottle completely in water without touching
the sides of the well and lower it down to the bottom of the well.
Pull it out when the bottle is filled. 6. Discard a little water to provide airspace. 7. Stopper and label the bottle.
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Membrane Filtration MethodAseptically introduce sample into sterile membrane filter assembly ( 0.2 or 0.45 μm)
Filter retains indicator organisms
Transfer to a suitable culture medium and incubate
Count colonies and report results in CFU /100 ml
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Membrane filtration machine
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Multiple-tube methodAdd different measured sample volumes of to tubes containing a suitable culture medium
Organisms reproduce and produce acid with or without gas
Statistically determine MPN of organisms in the original sample
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Principle
Separate analyses are usually conducted on five portions of each of three serial dilutions of a water sample.
The individual portions are used to inoculate tubes of culture medium that are then incubated at a standard temperature for a standard period of time.
The presence of coliforms is indicated by turbidity in the culture medium, by a pH change and/or by the presence of gas.
The MPN index is determined by comparing the pattern of positive results (the number of tubes showing growth at each dilution) with statistical tables.
The tabulated value is reported as MPN per 100 ml of sample.
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Culture media
Isolation media
Double strength broth: Dissolve 71.2 g of lauryl sulphate broth in 1 litre distilled water.
After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in Durhan-tubes.
Single strength broth: Dissolve 35.6 g of lauryl sulphate broth in 1 litre distilled water.
After solubilisation, dispense 10 ml into each test tube containing inverted Durhan-tubes.
Sterilise by autoclaving at 121°C for 15 minutes. Cool down slowly to prevent bubbles in Durhan-tubes.
Medium uses Incubation periods
Lactose broth Total or thermotolerantcoliforms
48 hours at 35 ± 0.5 °C or 37± 0.5 °C for total coliformsand 24 hours at 44 ± 0.25 °Cor 44.5 ± 0.25 °C forthermotolerant coliforms
MacConkeyBroth & Lauryl tryptose(lactose) broth
Total orthermotolerantcoliforms
48 hours at 35 ± 0.5 °C or 37± 0.5 °C for total coliformsand 24 hours at 44 ± 0.25 °Cor 44.5 ± 0.25 °C forthermotolerant coliforms
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Prepare the required number of tubes of culture medium. The volume and strength (single or double) of medium in the tubes will vary depending on the expected bacteriological density in the water and the dilution series planned.
Select and prepare a range of sample dilutions
Pipette the appropriate volumes of sample and diluted sample into the tubes of medium
Label the tubes with the sample reference number, the dilution and the volume of sample (or dilution) added to the tube.
Shake gently to mix the sample with the medium. Place the rack in an incubator or water-bath for 48 hours at 35 ± 0.5 ºC or 37 ± 0.5 ºC.
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Procedure cont’d After 18 or 24 hours, note which tubes show growth. Tubes that show turbidity and gas production, or a colour change
indicating the production of acid (if the medium contains a pH indicator), are regarded as positive
Return the tubes to the incubator and re-examine after a total of 48 hours of incubation. Continue with the next step of the procedure.
Confirmatory test Prepare the required number of tubes of confirmation culture medium
(BGLB broth for total coliforms and E. coli medium for faecalcoliforms).
Using a sterile wire loop, transfer inocula from positive tubes into the confirmation medium
Incubate them for 48 hours at 35 ± 0.5 °C or 37 ± 0.5 °C for total coliforms (BGLB broth) or for 24 hours at 44 ± 0.5 °C for faecalcoliforms (E. coli medium)
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Procedure cont’d
After the prescribed incubation time, note which tubes show growth with the production of gas,
and record the number of positives for each sample dilution
The test organism is cultured in a
medium which contains
tryptophane, kovac’s reagent
added to confirm indole
production
Compare the pattern of positive results with a most probable number table such as one of those given in Tables
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Sample 50ml 10ml 1ml 0.1 0.01ml
Treated drinking-water 1
1 5
Partially treateddrinking water
5 5 5
Recreational water
5 5 5
Protected source water
5 5 5
Typical sample volumes and number of tubes for multiple fermentation tube analysis
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Membrane Filtration...Method involves
comparatively simpler procedures.
Technique is inappropriate for highly turbid waters.
Filters may be expensive in some countries.
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MPN statistical table Combination of positive
MPN index /100ml 95% confidence levelUpper lower
0 0 0 < 2 - -
0 0 1 2 1 10
1 1 0 4 1 15
2 1 0 7 2 20
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Multiple-tube method...Method is applicable
to all kinds of water:
clear, coloured, or turbid
containing sewage, sludge,
mud or soil particles,
provided the contaminants
in the prepared test samples
are homogeneously
Distributed.
Method is comparatively complex.
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Water Quality StandardsRegulations on the purity of water used for various purposes.
Mandatory limits concerning constituents and contaminants of water known to be hazardous and/or cause inconveniencies.
Sets of procedures and practices required to meet the mandatory limits.
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Water Quality Standards
Water Quality Standards
Surface Water Quality
Standards
Drinking Water Quality
Standards
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Water Quality Parameters
Physical properties
Chemical / Inorganic Substances
Organic Constituents
Disinfectants and their by-products
Radionuclides
Microbiological Parameters.
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ISO Drinking Water Quality Standard
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Quality Standards of Water( Summary)
Physical parameter
Fresh water Communal water
Trout water High quality water
Turbidity 50/25 NTU 10 NTU < 10 NTU
Temperature RT RT RT RT
Hardness 100mg/l cacO3
Chemical parameter
Fresh water Communal water
Trout water High quality water
pH 6-9 6.8-8.5 7
Salinity N N N N
Chlorine 230mg/l 250mg/l N
Alluminum 87ug/l 6500ug/l
Dessolvedoxygen
Not less than 5.mg/l
Not less than 6mg/l
Not less than 6mg/l
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Quality Standards of Water cont’dBacteriological parameter
Fresh water Communal water High quality water
Enterococcus 35org. /100ml
Feacal coliforms 200org. /100ml
Key: NTU- Neplelometric Tubidity UnitRT- Room TemperatureN- Normal
Refer to Table 1. Microbial Examination of Water and Water Quality Standards (group 2) for more detail