WATER ANALYSIS IN MICROBIOLOGY LABs.pptx

WATER ANALYSIS IN
MICROBIOLOGY
MODERATOR: DR. NAZEEMA TABASEERA
PRESENTER: DR. SANA BASHEER

LEARNING OBJECTIVES:
• Introduction: pathogens transmitted through drinking water
• What are biological indicators?
• Indicator microbes
• Water sample collection and transport
• Bacteriological examination of water
• Guidelines for drinking water

INTRODUCTION:
• The quality of water, whether used for drinking, domestic purposes, food production or
recreational purposes has an important impact on health.
• Water of poor quality can cause disease outbreaks and it can contribute to background
rates of disease manifesting themselves on different time scales.
• Access to clean, drinkable water is a fundamental human right, yet over a billion people
worldwide do not enjoy a reliable source of potable water. Water that is not adequately
monitored for purity can contain pathogens introduced from human and animal waste.
• According to the WHO, the mortality of water associated diseases exceeds 5 million
people per year. From these, more that 50% are microbial intestinal infections, with
cholera standing out in the first place.

INDICATOR ORGANISMS:
Although a wide range of viral, bacterial, and protozoan diseases result from the
contamination of water with human and other animal fecal wastes the detection of indicator
organisms as an index of possible water contamination by human pathogens has long been the
standard approach to monitoring drinking water safety.
• Among the suggested criteria for such an indicator microbe are:
1.The indicator bacterium should be suitable for analysis of all types of water: tap, river,
ground, impounded, recreational, estuary, sea, and waste.
2. It should be present in excess number than any pathogen so that they can be detected easily.
3. It should survive longer than the hardiest enteric pathogen.

4. It should not proliferate in the contaminated water, as this would produce an inflated
value.
5. Its level in contaminated water should have some direct relationship to the degree of
fecal pollution.
6. The assay procedure for the indicator should have great specificity; in other words,
other bacteria should not give positive results. In addition, the procedure should have high
sensitivity and detect low levels of the indicator.
7. The testing method should be easy to perform.

INDICATOR MICROBES:
Coliform (other than
Escherichia coli)
Remote contamination—either by fecal
(presumptive) or soil and vegetation
Fecal (thermotolerant)
Escherichia coli
Confirms recent fecal contamination of
water; most sensitive indicator
Fecal streptococci Confirms remote fecal pollution
Clostridium perfringens Remote contamination
Pseudomonas aeruginosa Least reliable
indicator
Useful in hospitals and food
establishments
Bacteriophages Phage specific for E.coli indicate fecal
pollution of water
Indirectly indicates viral pollution

COLIFORMS:
• Coliforms, including Escherichia coli, are members of the family Enterobacteriaceae These
bacteria are commonly found in the intestines of humans and other animals, and are used
widely as indicator organisms.
• Detection of coliforms in higher concentrations than pathogenic bacteria is used as an index of
the potential presence of enteric pathogens in water.
• Able to ferment lactose at 35-37°C with production of acid, gas, and aldehyde within 24-48
hours.
• Oxidase negative , non spore forming.
• Other coliforms such as Klebsiella, Citrobacter, Enterobacter are less abundant in feces than E.
coli, and survive longer than E. coli.

THERMOTOLERANT OR FECAL ESCHERICHIA COLI
• It is regarded as the most reliable indicator of fecal pollution of water.
• Fecal E.coli is the most abundant coliform in human and animal intestine and is derived
almost exclusively from these sources.
• Their presence indicates a potentially dangerous fecal pollution of water.
• Ferment lactose at 44-45°C with production of gas and acid.

• It does not survive in water for long time, and therefore is the best indicator of recent human
or animal fecal pollution of water
Table 27.1: Microbiological testing methods for water analysis.
FAECAL STREPTOCOCCI
• These are catalase negative, Gram positive cocci present in the intestinal tract of man
and animals.
• These organisms have the Lancefield group D antigen, hydrolyse aesculin and can
grow at 45°C, in the presence of azide and 40% bile.
• Less abundant in feces than E.coli, can survice longer than E.coli. Their presence
along with coliforms, despite absence of E.coli confirms fecal pollution of water.

CLOSTRIDIUM PERFRINGENS
• As the name represents, these are members of the genus Clostridium that can
reduce sulphite to sulphide.
• Least sensitive indicator.
• The presence of this organism in water in the absence of other indicators of
contamination of water implies remote or intermittent fecal pollution.

BIFIDOBACTERIA:
• Obligately anaerobic, non-acid-fast, non-spore-forming, non-motile, Gram-positive bacilli
which are highly pleomorphic and may exhibit branching bulbs (bifids), clubs, coccoid,
coryneform, Y and V forms.
• They are all catalase-negative and ferment lactose (except the three insect species; B.
asteroides, B. indicum and B. coryneforme) and one of the most numerous groups of
bacteria in the faeces of warm blooded animals.
BACTERIOPHAGES (PHAGES):
• The phages that infect E.coli can be used as indicator of fecal pollution of water.
• They may also be used as indicator for viral pollution as their occurrence and resistance in
environment and to chlorine is similar to that of enteroviruses.

COLLECTION OF WATER SAMPLES:
Water specimen should be collected in a screw-capped wide sterile borosilicate glass or
plastic bottles of 500ml holding capacity that have been cleansed, rinsed with deionized or
distilled water, and then heat sterilized.
• Volume: At least 150–200 mL of water should be collected
• Neutralizer: Sodium thiosulfate is added to neutralize the bactericidal effect of residual
chlorine present in water if any. (0.1-0.2ml/200ml water)
• Sampling method from the tap: Care should be taken while collecting water to
minimize extraneous contamination. Hand washing should be performed and gloves
should be worn before collection. Tap should be sterilized for a minute with flame from
an ignited cotton wool swab soaked in alcohol: or by a gas burner.

• Water from streams or lake or swimming pool: The bottle should be opened only
after immersed at a depth of 30 cm with its mouth facing the current.
• Tap swabs: Sterile swab is inserted into the nozzle of the tap carefully, without touching
the outer tap surface. The swab is then rubbed around– that is, moved backwards and
forwards and up and down, as much as possible, on the inside surface of the tap outlet or
flow straightener.
There are two methods of sample collection from tap.
1. Pre-flush technique
2. Post-flush technique

BACTERIAL COUNT INTREPRETATION
PRE-FLUSH SAMPLES has substantially
higher bacterial count compared to post-
flush.
Indicates that contamination is near tap and
associated pipeworks and fittings near to tap
outlet.
POST-FLUSH SAMPLES has substantially
higher count compared to pre-flush
Indicates contamination is remote from the
point of delivery and suggest stagnation in
water system and inadequate flushing
Similar bacterial count in pre-flush and
post-flush samples
Indicates attention to whole water supply,
storage and distribution system.

LABELLING
Label the specimen + details
• Reason for examination
• Source of water
• Whether filtered / chlorinated
• Date & time of collection
• Possible sources of contamination

If the delay between sample
collection and bacteriological
analysis will be less than 2 hours,
samples should simply be kept in a
cool, dark place.
When more than 2 hours will
elapse, samples should be kept in
ice box, protected from light and
refrigerated (2-8).

BACTERIOLOGICAL EXAMINATION INDICATIONS
Multiple-tube method • Extensively used for drinking water analysis
• For highly turbid samples
• For semisolid such as sediments or sludges in
water to be analyzed
Membrane filtration method • For testing dialysis water
• For testing clean water, where the bacterial
count in water is expected to be low
• For testing large volume of water
Presence/absence method To monitor quality of drinking water where
contamination is likely to be rare
Medium containing sodium azide Used for detection of streptococci
Differential reinforced Clostridium medium Used for detection of Clostridium perfringens

MULTIPLE-TUBE METHOD:
• Used for the estimation of presumptive coliform count expressed as the most probable
number (MPN) of coliform organisms in 100ml water.
• Positive presumptive test is subsequently subjected to a confirmation test to find out the
presence of thermotolerant E.coli.
• Presumptive or Total Coliform Count
Culture media for multiple-tube method include:
For presumptive coliform count:
- MacConkey purple broth - Lactose broth
- Formate lactose glutamate medium - Lauryl tryptose (lactose) broth

Confirmatory media for detection of thermotolerant e.coli include:
• Brilliant green bile lactose broth
• E. coli medium
• Tryptone water
• Lauryl tryptose mannitol broth with tryptophan.

MACCONKEY PURPLE BROTH:
• Standard medium of choice for water surveillance and widely used across the
globe.
• used as both single strength and double strength media and dispensed in tubes or
bottles.
• When 10 mL of the sample is inoculated in 10 mL of single strength broth, the
media ingredient will get diluted to half, which may not support a positive growth
of the organisms that may give inaccurate results.
• Bromocresol purple is used as indicator.
• Each tube or bottle should contain an inverted durrham tube to detect gas
production.

PRINCIPLE:
• Water to be tested is diluted serially
and inoculated in MacConkey purple
broth, coliforms if present in water
utilize the lactose present in the
medium to produce acid and gas.
• The presence of acid is indicated by
the color change of medium and
presence of gas is indicated by gas
bubbles in Durham tube.

DETERMINATION OF MPN:
• The number of tubes giving positive reaction is
compared with McCrady statistical to determine the
most probable number (MPN) of coliform count
present per 100 mLof water.
• This is called as presumptive coliform count.
• Quality of water supply: Depending upon the
MPN/100mL, the quality of the water specimen can
be interpreted as excellent, satisfactory, intermediate
or unsatisfactory.

DIFFERENTIAL COLIFORM COUNT (EIJKMAN
TEST)
• To confirm the coliform bacilli detected in presumptive test are fecal E.coli.
• S/C from positive tubes to fresh tubes of
lauryl tryptose /brilliant-green lactose bile broth (inhibitory to spore froming
bacteria)
• Incubated at 440
C for 24 hrs
• GAS in Durham’s tube & indole formation– E.coli

MEMBRANE FILTRATION TEST
Minimum vol. of 10mL
introduced aseptically into
sterile filtration apparatus
Resuscitation: 37C for 2-4hrs
allowed for bacteria to
acclimatize to new
conditions before
transferring to incubator.

• Reading : taken by counting the visually identified colonies, and the results expressed in
numbers of “colony forming units(CFU)” per 100ml of original sample.
• Confirmation: For colifrom, colony subcultured onto tubes of suitable media such as
lactose peptone water with Durham’s tube and incubated at 37°C for 48hrs.
• For thermotolerant coliform - The colonies are processed for detection of lactose
fermentation with the production of acid and gas at 44°C to check for thermotolerant E.
coli. A positive indole confirms thermotolerant E.coli.

FOR STREPTOCOCCI AND CLOSTRIDIA:
• Faecal streptococci - GLUCOSE AZIDE broth.
1. Incubate tubes containing 5ml sterile glucose azide broth in a water bath thermostatically
controlled at 44-45°C.
2. When tubes are warmed to incubation temperature, seed them with heavy inocula from all
tubes in presumptive coliform test .
3. Re-incubate at 44-45°C
4. Confirm presence by subculturing positive glucose azide onto a plate of bile aesculin azide
agar.
5. Examine after a few hours for a brown-black coloration around inoculum.
• Examination for C. Perfringens –
- Water is incubated in litmus milk medium at 37°C for 5 days, if positive, stormy fermentation
occurs.
- Differential reinforced clostridial medium (DRCM) – black ppt

POUR PLATE METHOD SPREAD PLATE METHOD
• Spreading approx. 0.1mL of sample
over a premade agar plate.
• Allow to Incubate
• This method gives three-dimensional
colonies than pour plate method.
• Water sample added to molten agar
• Poured into petri dishes
• Incubated at different temperatures
• Result expressed as number of colony
forming unit per mL of water.
• Not routinely followed.

PRESENCE-ABSENCE METHOD:
• Qualitative method
• Detects the presence or absence of organisms in water
• Methods: commercial kits such as
1.) Manja’s method – H2S coated strips used for detection of Salmonella
2.) Presence-absence broth with MUG (4-methylumbelliferyl-beta-D glucuronide) – water
under test fluoresces under UV light if E.coli is present.

.Growth of E. coli in EC-MUG broth observed
(A) under UV light and (B) without UV light.
Photo courtesy: Cheeptham N, Lal A. Use of EC-MUG media to
confirm Escherichia coli contamination in water samples protocol.

PORTABLE BACTRALERT H2S BACTERIOLOGICAL WATER TEST KIT:

• The test is based on the readily observable formation of an iron sulfide precipitate on a
paper strip in a bottle, as a result of the reaction of H2S with iron.
• The test is intended to detect bacteria associated with fecal contamination due to the
activity of these microorganisms in reducing organic sulfur to the sulfide oxidation state
(as H2S gas) which then reacts rapidly with iron to form a black, iron sulfide precipitate.

Method for detection of legionella ( health care
associated pathogen) :
• Recommended for high-risk settings such as transplant units.
• Sampling: one litre preferred; large volumes are required to detect legionella.
• Can be isolated from water samples by methods like centrifugation or membrane
filtration.
• Culture media:
 BCYE ( buffer charcoal yeast extract) with added antibiotics to suppress background
envt flora.
MWY (Wadowsky and yee medium)
• Incubation – 36C in a humidified candle jar for 72-96 hrs , if no growth until 7 days.

NEWER METHODS
1. LUMINOMETER: Detects microbial ATP
present when swabs soaked in water
are dipped into the luminometer system.
2. MOLECULAR METHODS: to detect and quantitate the microbial DNA: PCR, real-time
PCR, Fluorescence in situ hybridization (FISH),etc.
3. IMMUNOLOGICAL METHODS: ELISA, lateral flow assay
PC: LuminUltra Technologies Ltd.

4. CHROMOGENIC MEDIA
• The addition of chromogenic-fluorogenic substrates to selective isolation media offers
new perspectives for direct detection and identification on a single medium without
confirmation.
• These substrates can be applied in the MPN technique, in media used for membrane
filtration, as well as in presence/absence tests.
• Cleavage by the target enzyme transforms the initially uncolored or nonfluorescent
substrate into a colored or fluorescent reaction product.
• The chromo-genic-fluorogenic substrates must of course not be toxic for the cell and must
be metabolized in the same way as the normal substrates.

WHO GUIDELINES FOR DRINKING WATER QUALITY:
I. PHYSICAL PARAMETER
GUIDELINE VALUES COMMENTS
TURBIDITY Ideally, no visible turbidity but
permissible standard is <1.0
NTU(nephelometric turbidity
units) for household drinking
water.
• >4 NTU -cloudiness visible
to naked eye.
• <4 NTU -measured only by
instruments.
COLOR Ideally, no visible color but
permissible standard is <15TCU
(true color units).
>15 TCU is detectable by the
naked eye.
TASTE, ODOR AND
TEMPERATURE
No health based guideline values It may be indicative of
contamination and pollution.
High water temperature may
enhance growth of
microorganisms and influence
taste, odor and color.

II. CHEMICAL PARAMETER:
GUIDELINE VALUE
AMMONIA NO HEALTH BASED GUIDELINE VALUE
Natural levels in surface and ground water is 0.2
mg/l.Anaerobic groundwater may have up to 3
mg/l.
ALUMINIUM up to 0.9 mg/l
ARSENIC up to 0.01 mg/l
CHLORIDE 250 mg/l, as above this a detectable taste appears.
Maximum permissible level is 600 mg/l. Sodium
thiosulphate can be used to dechlorinate drinking
water or tap water.
COPPER up to 2 mg/l.
Excess copper in water can have gastrointestinal
effects.

FLUORIDE up to 1.5 mg/l. Above this, there is risk of
dental and skeletal Fluorosis.
HYDROGEN SULPHIDE Taste and odor threshold is between 0.05 and 0.1
mg/litre.A rotten egg smell of water indicates
hydrogen sulphide.
NITRATES 50 mg/l as nitrate ion, to prevent
methaemoglobinaemia and thyroid effects
pH Acceptable pH is in the range of 6.5 - 8.5.
LEAD up to 0.01 mg/lThe concentration of lead must be
kept as low as possible. It is the most undesirable
metal in drinking water
MERCURY 0.006 mg/l for inorganic mercury. It is nephrotoxic
SODIUM Above 200 mg/l may give an unacceptable taste.

III. BIOLOGIC INDICATORS:
• Total coliform bacteria:
• They are the primary bacterial indicators for drinking water.
• Total coliforms are good indicators of water disinfection because they are abundant in the human
intestine and are foreign to potable water.
• E. coli is considered the best indicator of fecal contamination of water when resources for
supplementary microbiological examination are limited.
• Guidelines state that coliforms must not be detectable in 100ml of sample water.
IV. RADIOLOGICAL ASPECTS:
The proposed guidelines for gross alpha activity is 0.5Bq/litre and for gross beta activity, it is 1.0 Bq/litre
of drinking water.

Water Quality Monitoring and Surveillance Program (WQMSP)
• The Department of Rural Drinking Water and Sanitation is
predominantly focusing on supplying clean and safe drinking
water to Rural Karnataka. Drinking water to rural areas will be
supplied only after the water quality is assured.
• In this regards, the department is working on the chemical and
bacteriological testing of all drinking water sources in rural areas.
• In the first phase, at Gram Panchayat level, with the help of
Village Water and Sanitation Committee (VWSC), chemical
(fluoride, arsenic, nitrate, iron, pH, chloride) and bacteria
(coliform, E.coli) samples of all drinking water sources in rural
areas are being tested.
• Drinking water quality testing laboratories have been established
by the department in each district and in 47 Taluk centers across
the state to test water quality through Field Test Kit & H2S Vials
and labs.

REFERENCES
• Mackie and McCartney, Practical medical Microbiology, 14th
edition, Haryana, India:
Relx India Pvt.Ltd (2020).
• Sastry AS, Bhat S. Essentials of medical microbiology. JP Medical Ltd; 2018 Oct 3
• Water quality monitoring: A practical guide to the design and implementation of
freshwater quality studies and monitoring programs 14 January 1996 Guideline.
• Sastry SA, Deepashree R. Essentials of hospital infection control. Jaypee Brothers
Medical Publishers; 2019 Feb 8.
• Willey JM, Sherwood LM, Woolverton CJ. Prescott's microbiology. McGraw-Hill; 2014.
• Guidelines for drinking-water quality: fourth edition incorporating the first addendum.
Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO

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