introduction to water bacteriology

7/27/2019 Introduction to Water Bacteriology

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Ruthie Jane P. Locayon, M.D., DPCP


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Is essential to support life No water, No Life


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Clear Colorless

Free from objectionable taste and odor

Must not contain: Any organisms

Chemicals

Radioactive materials


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Sewage Human or animal excreta

Other wastes Agricultural run-off water with pesticides


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Water is unsafe for human consumption whenit contains pathogenic and non-pathogenicorganism.

- WHO, 1993

Microbial contamination is still consideredthe most

critical risk factor in drinking waterquality.


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Food and waterborne diseases are amongthe most common causes of diarrhea.

Seventy percent of diarrhea due to food andwaterborne diseases have resulted fromingestion of contaminated food or water.

Diarrheal diseases for the past 20 years is thenumber one cause of morbidity and mortality,

incidence rate is high as 1,997 per 100,000population while mortality rate is 6.7 per100,000 population.


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Between 1988 and 1998, outbreak of FWBDsranks first (typhoid fever), second (cholera),fourth (food poisoning), sixth (hepatitis A),*and eight (diarrhea).


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1. Barrier approach involving treatment of

wastewater and raw water with disinfection,purification, etc.

2. Establishing allowable limits of indicators ofwater quality.


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Competence needed in the ability todemonstrate the presence and absence of aparticular organism/class of organism in agiven sample volume and provide an estimate

of their number.


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Outbreak investigation of waterborne disease Assessing safety, stability of water and water

products for public consumption.

Determining level of sanitation duringproduct preparation.

Regulatory compliance.

Incidence surveys


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Source water must be free from fecalcontamination.

Adequately treating fecal contaminated water.


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Bacteria Viruses

Protozoa

Helminths


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Salmonella Shigella

Escherichia coli

Vibrio cholera Campylobacter jejuni

Poliovirus, echovirus, Hepatitis Type A,Rotavirus, Norwalk virus

Giardia, Cryptosporidium, E. histolytica


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The methods of isolation and enumeration todetect the presence of many pathogens arecomplex and time-consuming

Detection of organisms normally present inthe feces of man and animals is used asindicator of excrement pollution in water.


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Indicates the presence of fecal materials. Possible presence of all relevant pathogens

Absence of fecal commensal organismsindicate that pathogens are probably alsoabsent.


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Indication of excrement pollution


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Always present when pathogenic organism ofconcern is present

And absent in clean uncontaminated water

Present in large numbers in the feces ofhumans and warm blooded animals

Easily isolated, identified and enumerated.


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Respond to environmental conditions andtreatment process similarly with othermicroorganisms

Ratio of indicator/pathogens should be high

Indicator and pathogens should come fromthe same source (gastrointestinal tract)

No organism fulfill all the criteria for anindicator organism butcoliform bacteriafulfill most.


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Easy to detect and enumerate in water Ability to ferment lactose in culture at 35C

Should not be detected in treated watersupply

Aerobic, facultative anaerobic

gram,-negative, non-spore forming

Rod-shaped


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E. coli most numerous Enterobacter

Klebsiella

Citrobacter False-positive results: coliforms from natural

aquatic flora and Aeromonas


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Subgroup of total coliform Ability to ferment lactose at 44C

Can be human or animal origin

Comprise the genus E. coli


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Normally found in human and animalintestines

Most reliable indicator of fecal contaminationin water.

Facultative anaerobe

Lactose-fermenting

Oxidase-negative

Gram-negative bacilli, motile


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Broad group which include: Pathogens and non-pathogens

Opportunistic microorganisms

Indicate general biological condition of

drinking water Supporting data on the significance of

coliform test results

Efficiency of various treatment process


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Multiple Tube Fermentation Technique Presumptive Test

Lauryl Tryptose Broth

Positive result gas formation within 24-48 hours


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Multiple Tube Fermentation Technique Confirmed Test

BGLB gas formation within 24-48 hours

EC gas formation within 24 hours or less


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Multiple Tube Fermentation Technique Completed Test

Single strength Lauryl Tryptose Broth

Completed result gas formation

LES Endo/MacConkey agar Gram staining gram-negative bacteria

(Lawrence Experimental Station )

(LES) formula


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The number of positive findings of coliformgroup organisms resulting from multiple-formation decimal dilution plantings

Estimates the density of coliforms in the

sample

Index of the number of coliform bacteria.


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Inoculate 20 ml water sample to 5 LTB

35C for 24-48 H

Presumptive testRecord no. of (+) tubes

44C water bath also for 24 hours

35C water bath also for 24-48H

Confirmatory test

Record no. of (+) tubes

EC(+) and BGLB(+) =(+) completed test

Completed test

Gram stain


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No. of positivetubes MPNindex/100ml Lower limit Upper limit0 8.0


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Total Coliform


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Sample container Made of glass or plastic (pre-sterilized)

Wide-mouthed

Use 120 ml clear bottle

Add 0.1 ml 3% sodium thiosulfate for chlorinatedwater sample

Volume of sample: Required volume should not be less than 100 ml


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Clean the tap Open the tap/pump outlet

Wipe using clean cloth or cotton swab

After cleaning the tap, flush for 1-2 minutes


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Sterilize the tap Close the tap or pump outlet and sterilize by

flaming with ignited cotton swab.

NOTE: For plastic tap, sterilize with cotton soaked

with cotton swab soaked in chlorox or 100 mg/Lsodium hypochlorite solution


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Flush the tap Open the tap and allow water to flow for 1-2

minutes.

Maintain normal pumping of water when sampling

hand pumps.


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Open the sterilized sampling bottle Untie the string of the protective paper cover and

unscrew cap.

Hold the cap with the protective paper cover facing

downward to avoid contamination.


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Fill the bottle Fill the bottle, avoid touching the mouth of the

bottle to the tap during filling.

Fill the bottle up the shoulder or leave at least 2.5

cm air space to facilitate mixing by shaking prior toanalysis.


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Cap the bottle Tightly screw the cap together with the protective

paper cover on the bottle.

Secure cover with string.


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Label the bottle Write the data and time of sampling, name of

collector, water sampling point code on a maskingtape.


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Store the bottle Place the sampling bottle in a cooler with ice or ice

gel pack.

Do not submerge the bottle in ice water during

storage and transport.


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Transport the water sample to the examiningwater laboratory immediately within 6 hoursafter collection or within 24 hours aftercollection under proper storage condition

(stored in an ice chest/box with sufficient iceor ice gel pack.)


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Heterotrophic plate count Formerly Standard Plate count

Procedure for estimating the number of liveheterotrophic bacteria in water.

Measures changes during water treatment anddistribution or in swimming pools

Employed as indicators of the proper functioning ofprocess and thereby as indirect indicators of watersafety.


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Heterotrophic plate count As a measure of numbers of re-growth (growth

following drinking water treatment)that may or maynot have sanitary significance.

Measure of possible interference with coliformmeasurements in lactose-based culture methods.


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Heterotrophic plate count Pour Plate Method

Simple to perform

Can accommodate volumes of sample or diluted

sample ranging from 0.1 to 2.0 ml. Colonies produced are relatively small and compact.

Media used R2A and NWRI agar.


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Heterotrophic plate count Quebec ColonyCounter used Spread Plate Method

Limited by the small volume of sample or diluted

sample that can be absorbed by the agar. To use this procedure, maintain a supply of suitable

pre-dried absorbent agar plates.

Media used R2A and NWRI agar.


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Membrane filter technique added expense ofthe membrane filter Benefits:

Concentration of larger samples on a membrane filter

is a key benefit over the MPN and Pour Plate andSpread Plate Technique.

Highly reproducible

Usually yields numerical results more rapidly thanMTFT.


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Membrane filter technique Uses:

Used by water treatment plants to monitor drinking,waste and surface water for the presence of coliformbacteria and E.coli

Used for microbial monitoring to the followingindustries.

Pharmaceutical

Cosmetics

Electronics

Food and beverage


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Membrane filter technique Advantages:

Permits testing of large sample volumes (of low turbiditywater)

Reduces preparation time as compared to many traditional

methods. Allows isolation and enumeration of discreet colonies of

bacteria.

Provides presence or absence information within 24 hours

Effective and acceptable technique. Used to monitor drinking

water in government laboratories. Useful for bacterial monitoring in the pharmaceutical,

cosmetics, electronics and food beverage industries.


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Membrane filter technique Procedure:

Collect the sample and make any necessary dilutions.

Flame the forceps and remove the membrane filter intothe funnel assembly.

Flame the pouring lip of the sample container and pourthe sample into the tunnel.

Turn on the vacuum and allow the sample to drawcompletely through the filter.


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Membrane filter technique Procedure:

Rinse funnel with sterile buffered water. Turn onvacuum and allow the liquid to draw completelythrough the filter.

Flame the forceps and remove the membrane filterfrom the funnel.

Place the membrane filter into the prepared Petri dish.

Incubate the proper temperature and for the

appropriate time period. Count the colonies under 10-15x magnification.

Confirm the colonies. (ID)


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Enzyme substrate coliform test (Chromogenicsubstrate test)

Principle: Total coliform bacteria

Chromogenic substrate Ortho-nitrophenyl--D-galactopyranoside (ONPG) or chlorophenol-red -D-galactopyrasonide (CPRG) detects enyme -D-galactosidaseproduced by coliforms

Fecal coliform (E. coli)

Fluorogenic substrate 4 methylumbelliferyl--D-

glucoronide (MUG) detects enyme -glucoronidase producedby fecal coliforms


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Enzyme substrate coliform test (Chromogenicsubstrate test)

Principle: Colorless: negative

Yellow: Total Coliforms Yellow Fluorescence: E. coli

May be quantified in Quanti Tray.


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A. MULTIPLE TUBE FERMENTATION TECHNIQUEInoculate 5 tubes of 10ml LSB triplestrength, 10 ml with 20 ml water sample

Incubate at 35C for 24 hours


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B. AQUA HETEROTROPHIC COUNT PLATE


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A. MULTIPLE TUBE FERMENTATION TECHNIQUE1. Look for gas production in LSB

if + inoculate BGLB incubate at 35C for 24hrs

inoculate EC incubate at 44C for 24hrs

if - re-incubate for another 24hrs


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B. AQUA HETEROTROPHIC COUNT PLATE1. Count number of coloniesinterpret

2. End of exercise


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A. MULTIPLE TUBE FERMENTATION TECHNIQUE1. Read LSB that were negative after 24 hrs and is + after

48hrs

if + inoculate BGLB and EC

if - discard end of procedure

2. Read BGLB ( from + LSB on Day 2 after 24 hrs incubation)

if + inoculate Mac Conkey incubate at 35C for 24hrs

if - reincubate

3. Read EC (from + LSB on Day 2 after 24 hrs incubation)

if + positive for fecal coliformsif - absence of fecal coliforms

Discard EC after reading


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1. Read MacConkey for colored colonies ( from BGLB after24hrs)

if + inoculate LSB single strength and incubate for

24-48hrs

inoculate TSA slant and incubate

if - discard


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2. Those inoculated from LSB after 48hrsRead BGLB ( after 48 hrs incubation)

if + inoculate Mac Conkey incubate at 35C for 24 hrs

if - discard

Read EC

if + positive for fecal coliforms

if - absence of fecal coliforms

if BGLB and EC are negative END OF PROCEDURE


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1. From MacConkey (from BGLB after 24hrs)Read LSB single strength and incubate for another 24hrsif negative

if + positive for coliforms

if - no coliforms

From TSA SlantDo gram stain and look for gram negative bacilli2. Read MacConkey for colored colonies ( from BGLB after48hrs)

if + inoculate LSB single strength and incubate for

24-48hrs

inoculate TSA slant and incubate

if - discard


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1. From MacConkey (from BGLB after 48hrs)Read LSB single strength and incubate for another 24hrsif negative

if + positive for coliforms

if - no coliforms

From TSA SlantDo gram stain and look for gram negative bacilli


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Group WaterSource Presumptivetestof+tubes

Confirmatorytest of+ tubesMPN Completed Test Remarks

BGLB

EC BGLB EC MC LTB

1 1 5 5 0 >8.0 + + Unsafe

introduction to water bacteriology

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