ASEPTIC TECHNIQUE PROCEDURE OF WATER SAMPLE...

WORK INSTRUCTION MANUAL LABORATORY SECTION

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ASEPTIC TECHNIQUE PROCEDURE OF WATER SAMPLE COLLECTION

1. VOLUME OF SAMPLE

The volume of sample should be sufficient to carry out all tests required,

preferably not less than 100mL

When the sample is collected, leave ample air space (at least 2.5 cm) to

facilitate mixing by shaking, before examination.

2. IDENTIFICATION OF SAMPLES

Sampling bottles must be tagged with complete and accurate identification and

description. Samples should be clearly labeled with the site, date, time, nature

of the analysis to be done, and other relevant information.

The information about the samples can be recorded in a request form for

analysis of water quality.

3. SAMPLE CONTAINER

Collect samples for microbiological examination in nonreactive 120-mL clear bottles

that have been cleansed and rinsed carefully, given a final rinse with distilled water,

and sterilized as directed.

Use glass bottles capable of holding a sufficient volume of sample for all required

tests and an adequate air space, permitting proper washing, and maintaining samples

uncontaminated until examinations are completed.

Plastic autoclaveable bottles of suitable size, wide-mouthed and made of non-toxic

materials polypropylene that can be sterilized repeatedly are also suitable for sample

bottles.

Sampling bottles should be provided with either ground glass stoppers or metal/plastic

screw caps and ensure that the use of these caps produces no toxic compounds during

sterilization.

Cover tops and necks of sample bottles with Kraft paper or thin aluminum foil before

sterilization.

Presterilized plastic bottles, with or without dechlorinating agent , are also available

commercially and may be used. Plastic containers eliminate the possibility of

breakage during shipment.


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4. DECHLORINATION

If the water to be examined is treated likely with chlorine, chloramines, chlorine

dioxide, ozone, or other disinfectants, then sodium thiosulfate should be added to the

sampling bottle before sterilization/autoclaving the sample bottle, so as to neutralize

any residual disinfectant of water sample obtained, in which neutralization takes place

inside the sample bottle.

For drinking water samples, in a 120-ml sample bottle, add 0.1 ml of a

3% solution of sodium thiosulfate before sterilization. This will

neutralize up to 5mg/l residual chlorine.

5. PRESERVATION AND STORAGE

Holding Time and Temperature

a.) General.

i. Start microbiological analysis of water as soon as possible after

collection to after sampling and certainly within 24 hours. Avoid

unpredictable changes in the microbial population

ii. The changes may occur in the bacterial content of water on storage can

be reduced to a minimum by ensuring that samples are not exposed to

light and are kept cool. preferably between 4⁰C and 10⁰C, but not

frozen.

iii. For most accurate results ice samples during transport to the

laboratory, if they cannot be processed within one (1) hour after

collection.

iv. If samples cannot be cooled, they must be examined within 6 hours of

sampling.

v. If neither condition can be met, the sample should not be analyzed.

vi. The time elapsed between collections and processing should not

exceed 24 hours

vii. The box used to carry samples should be cleaned and disinfected after

use to avoid contaminating the surfaces of bottles and the sampler's

hand.

b.) Drinking water for compliance purposes .

Preferably hold samples at <10⁰C during transit to the laboratory .

Analyze samples on day of receipt whenever possible and refrigerate

overnight if arrival is too late for the processing time on the same day.


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Do not exceed 30 hour holding time from collection to analysis for coliform

bacteria.

Do not exceed 8 hour holding time for heterotrophic plate count (HPC).

6. GENERAL GUIDELINES DURING SAMPLING

1. Care must be taken to ensure that samples are representative of the water to be

examined and that no accidental contamination occurs during sampling.

2. When a number of samples are to be taken for various purposes (e.g. physico-chem,

heavy metals) from the same location, the sample for bacteriological examination

should be taken first to avoid contamination of the sampling point.

3. Collect samples that are representative of the water being tested, flush or disinfect

sample ports, and use aseptic techniques to avoid sample contamination.

4. Keep sampling bottle closed until it is to be filled.

5. Remove stopper and cap as a unit; Do not contaminate inner surface of stopper or cap

and neck of bottle.

6. Fill container without rinsing, replace stopper or cap immediately, and if used, secure

hood around neck of bottle.

7. SAMPLING PROCEDURES

a.) Potable Water.

A1) Sample taken from distribution-system tap without attachments.

a) Samples must be taken from different parts of the distribution

system to ensure that all parts of the system are tested.

b) Select a tap that is supplying water from a service pipe directly

connected with the main, and is not served from cistern or

storage tank.

c) Open tap fully and let water run to waste for 2-3 minutes, or for

a time sufficient to permit clearing the service line.

d) Reduce water flow to permit filling the bottle without

splashing.

e) If tap cleanliness is questionable, choose another tap.


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f) If a questionable tap is required for special sampling purposes,

disinfect the faucet (inside and outside) by applying a solution

of sodium hypochlorite (100mg NaOCl/L) to faucet before

sampling; let water run for additional 2 to 3 minutes after

treatment.

g) Do not sample from leaking taps that allow water to flow over

the outside of the tap

h) In sampling from a mixing faucet attachments such as screen or

splash guard, run hot water first for 2 minutes, then cold water

for 2 to 3 minutes and then, collect the sample aseptically.

B.2) Sample taken from a Well Fitted With Hand Pump

a) Pump water to waste for about 5 to 10 minutes or until

temperature has stabilized before collecting sample.

b) If an outdoor sampling location must be used, avoid collecting

samples from frost-proof hydrants.

c) If there is no pumping machinery, collect a sample directly

from the well by means of as sterilized bottle fitted with a

weight at the base; take care to avoid contaminating samples by

any surface scum.

b.) Raw water supply.

In collecting sample directly from flowing or underdeveloped spring,

stream, or shallow well, obtain samples representative of the water that

is the source of supply to consumers. It is undesirable to take samples

too near or too far from the point of draw-off, or at a depth above

or below the point of draw-off of the raw water source.


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8. SAMPLE COLLECTION PROCEDURE FROM TAP WATER

a. If treated water is being sampled, sterilized sample bottle is added with neutralizing

agent before bottle sterilization.

b. When water sample is treated with chlorine; at the time of collection get:

1. The concentration of residual chlorine at the sampling point;

2. water pH

Procedures:

1. Clean the tap. Remove from the tap any attachments that may cause splashing and, using a clean cloth, wipe the outlet in order to remove any dirt.

2. Open the tap. Turn on the tap at maximum flow rate and let the water flow for 1-2 minutes.

3. Sterilize the tap. Sterilize the tap for a minute with the flame from an ignited cottonwool swab soaked in alcohol: alternatively, a gas burner or cigarette lighter may be used.

4. Open the tap prior to sampling. Carefully turn on the tap and allow the water to flow for 1-2 minutes at a medium flow rate.

5. Open a sterilized bottle. Unscrew the stopper or cover of the bottle with the cover facing downwards (so as to prevent entry of dust that might carry microorganisms).

6. Fill the bottle. Immediately hold the bottle under the water jet, and fill. 7. Small air space. A small air space be left to facilitate shaking or mixing at the

time of inoculation prior to analysis. 8. Stopper or cap the bottle. Place the stopper in the bottle or screw on the cap

with its protective cover in place. 9. Label & Submit immediately. Samples collected should be submitted

immediately to the laboratory within six hours in a cool condition (using ice chest or cooler.)

***Only with sterilized sample bottle should be used in this process.


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9. FREQUENCY OF SAMPLING

The minimum number of samples to be collected and examined periodically must be based

on:

The mode and source of water supply.

Past frequency of records yielding unsatisfactory results

the quality of raw water treated

the number of raw water resources

the adequacy of treatment and capacity of the treatment plant

risks of contamination at the source and in the distribution system

the size and complexity of the distribution system

the risk of an epidemic

the practice of disinfection (with disinfectants, e.g. chlorine)

Table 1. Minimum Frequency of sampling for Drinking -Water Supply Systems for Microbiological

Examination

SOURCE AND MODE OF SUPPLY POPULATION SERVED MINIMUM FREQUENCY OF SAMPLING

Water District Level I 90-150 households Once in every three (3) months

Water District Level II 600 households Once in every two (2) months

Water District Level III

Less Than 5.000 One (1) sample only

5,000-10,000 households

One (1) sample per 5,000 population monthly

More than 10,000 households

Twenty (20) samples and additional one (1) per 10,000 population

monthly

Emergency Supplies of Drinking Water

Before delivery to users

Water Refilling Stations (Product water/Bottled water)

One (1) sample monthly

Water Vending Machines (Product Water)

One (1) sample monthly


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10. LOCATION OF SAMPLING POINTS

Adherence to the set guidelines for sampling point selection must be observed. These

guidelines cover zoning of piped water supply as well as sampling from point of source.

GUIDELINES FOR SELECTING THE LOCATION OF SAMPLING POINTS.

10.1 Piped Water supply zoning

A zone can be considered as coverage area per source, service reservoir

supplies specific area, an area where different parts of distribution system

operates at different pressures and elevations and an area where leakages or

reliability differs in different parts of the system.

Zoning must be adequately covered for water quality sampling.

10.2 Point Source

Samples should be taken from the point source from the principal outlet-

handpump or spring outlet.

For routine monitoring boreholes or deepwells generally requires less frequent

sampling as they are usually of better quality than shallow groundwater given

the depths of water abstraction.

I t is also important to undertake an extended assessment of point source

quality in order to develop an understanding of the process causing water

quality failure and thus the appropriate interventions required to improves.

10.3 Selection Of Sampling Sites

10.3.1 Sample locations and frequencies of sampling visits must be calculated

first.

10.3.2 The final stage then, is the selection of sampling sites.

Sample sites can be:

Fixed - i.e. every time sampling is carried out in the area, a

sample is always picked from the sample point.

Random- with the exact location of the sample point in zone or

areas varying between sample rounds.

10.3.3 Key fixed points that should always be included in the surveillance

include:

Water leaving treatment works (usually the first Tap)

The inlets and outlets of service reservoirs

The critical points in the distribution system- (e.g. low-

pressure area or parts of the system prone to frequent

discontinuity.


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Pumping Stations (fixed sampling sites)

Service Connections - random sampling

Service Connections as per concessionaire's request.

Service Connections - during service interruptions and

pipeline repairs.

10.3.4 Regular sampling points will include public taps in high density areas or in places such as markets where large number of people congregate.


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Water Analysis Methods On Enumerating and

Detecting E.coli & Coliform Bacteria

Definition

Coliform Bacteria are aerobic and facultative aerobic, gram negative, non- spore-forming

lactose-fermenting bacteria that produce gas within 48 hours at 35⁰C-37⁰C. Escherichia coli (E.coli),

is a coliform bacteria which consistently inhabits the intestinal tract(enteric) of humans and other

warm blooded animals is generally regarded as the primary indicator of fecal contamination in

drinking water.

The production of ß-D-glucuronidase is unique to E.coli among the Escherichia spp.

However, this enzyme is also found in some Salmonella, Shigella and Yersenia. ß-D-glucuronidase

breaks down the substrate 4-methylumbelliferyl-β-D-glucuronidase (MUG) to release 4-

methylumbelliferone which fluoresces under long wave ultraviolet (UV) rays. There are three (3)

widely accepted practices done in bacteriological examination of potable water. These are:

1. Conventional Method - this established method widely used in laboratory and academe

worldwide and had established strict method of determining E. coli and coliform bacteria as

primary indicator of water drinking contamination. This involves 3 processes such as

Multiple Tube Fermentation Technique. (MTFT), Heterotrophic Plate Count (HPC), and

Streak Plate Method on selective agar.

2. Chromogenic Substrate Method - The Chromogenic Substrate Method for water analysis is

granted NF (French Norm) Validation by Association Française de Normalisation (AFNOR)

Certification as an alternative method to the standard ISO 9308-1 for enumeration of

Escherichia coli ß-D-glucuronidase positive and coliform bacteria ß-D-galactosidase positive

in drinking water. It is now being allowed in the Philippines to be used as an alternative

method aside from conventional method, granted by the Philippine National Standards for

Drinking Water (PNSDW) and the Department of Health (DOH).

3. Primary Health Care (PHC) Method – This is chemical test used as an alternative method for

water analysis. This screening test used to detect the presence and the absence of coliform

group of bacteria rather than enumerating it and useful in urban water supplies where

resources, time and manpower are limited in which no available bacteriological laboratory.


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MULTIPLE TUBE FERMENTATION TECHNIQUE (MTFT)

Water Sample

20 mL portions in five (5) Triple Strength Lauryl Sulfate Broth (LSB) fermentation tubes and incubate at 35⁰C for 24-48 hours

1.0 mL portion into petri dish + 10 mL Plate Count Agar (PCA) and incubate at 35⁰C for 24-48 hours

Production of gas Report as Positive (+)

Presumptive Test

No gas production Report as Negative (-)

Count Colonies by Quebec Colony Counter

Determined number of colonies Report as Heterotrophic Plate

Count (HPC)

B. Confirmatory Test (MTFT)

Positive (+) Presumptive Test Tubes

Transfer 0.1 mL from each tube to BGLB fermentation

tube and incubate at 35⁰C for 24-48 hours

Transfer 0.1 to E. C. Broth Medium and incubate at

44.5⁰C +/- 2⁰C for 24 hours in water bath

Streak EMB plates from each tube and incubate

at 35⁰C for 24 hours

A. Presumptive Test (MTFT) HPC Method of Examination

Typical coliform nucleated colonies report as (+) confirmed test for coliform. Metallic sheen

colonies report as positive (+) confirmed test for E. coli

No gas production, report as negative (-) confirmed test

No typical nucleated colonies found and/or no metallic sheen observe report as negative (-) confirmed test

Production of gas, report as positive (+)

confirmed test for Total Coliform

Gas production, report as positive (+) confirmed test

for Fecal Coliform

No gas production, report as negative (-) confirmed test

Schematic Diagram on conventional method of bacteriological examination on potable water that involves three

processes: MTFT, HPC (Pour Plate Method), and Streak Plate Method on selective (EMB) agar.


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Multiple Tube Fermentation Technique (MTFT)

One of the standard methods used in Water Microbiology which aids in the detection of

coliform group of organisms (including Thermotolerant/Fecal Coliform) in the drinking water. This is

a tube-dilution method using a nutrient rich medium. Which involves two (2) PHASES:

A. ) Presumptive Phase - using LSB (Lauryl Sulfate Broth) as culture medium;

B.) Confirmed Phase - using BGLB (Brilliant Green Lactose Bile Broth) particular for Total

coliform, EC Broth and EMB agar for fecal coliform group.

During presumptive phase, for every one (1) water sample is coupled with one (1) plate of

Petri dish undergoing heterotrophic plate count (HPC) using pour plate method; and when a

particular sample being subjected to confirmed phase using EC broth, it will also undergo streak

plate method using EMB selective agar to correlate results from EC enrichment broth specific for

fecal coliform group detection and quantifying processes.

Thus, one sample will undergo MTFT, HPC, and streak plate selective enrichment (EMB) agar

method as a complete package for bacteriological analysis of water. This is also true when using

rapid (chromogenic /enzyme substrate) test but with the absence of correlation with EMB agar

plates because rapid test is more precise and specific on quantifying E.coli instead of fecal coliform

group.

The results are reported in terms of Most Probable Number (MPN) of organism present.

Most Probable Number is based on certain probability formulas and is an estimate of the mean

density of coliforms in the sample. On the other hand, colonies on agar plates will be counted and

reported as colony forming units cfu/mL.


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Equipments/Materials/ Paraphernalia/Apparatus:

Autoclave

Water Bath

Refrigerator

Hot Plate with magnetic Stirrer

Pressure cooker in 121⁰C with 0.15 MPa

Alcohol Lamp

Aluminum Foil

Mechanical Pipettor,

Balance, analytical and top-loading

Vortex Shaker

Reagent Dispenser (10ml)

Fermentation Tubes, 20x150mm

Thermometer

Beaker, 500mL, 1000ml, 2000ml

Erlenmeyer Flasks, 250ml, 500ml

Incubator

Durham Tubes, 8x45 mm

Inoculating Loop (Wire Loop, Michrome Loop)

Petri Dishes (15x100mm)

Reagent Bottles (Clear, Amber Color)

Culture of E. coli, Enterobacter aerogens, Klebsiella pneumonia and Bacillus spizizenil (ATC6633)

Cotton

Biosafety Cabinet

Test Tube rack

Disinfectants: Isopropyl OH 70%,

Ethyl OH 70%,

Lysol (O-Benzyl-Chlorophenol)

Zonrox (Sodium Hypochlorite)


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WASHING AND STERILIZATION

1. All glasswares should be thoroughly washed with liquid detergent and Sodium Hypochlorite)

Zonrox for disinfection.

2. To remove all traces of residual washing compounds, glasswares should be sterilize for not

less than one hour at 170⁰C in dry-sterilization oven by putting the glasswares in one (1)

craft paper pouch in a bundle of 35 pcs with Durham tubes for fermentation tubes, and 10

pcs in one (1) craft paper pouch for Petri dish plates.

3. Place a foil cap in the beaker mouth for the beakers, stirring rods in a aluminum foil, cap the

mouth of the Erlin Meyer flasks with aluminum foil, etc. when autoclaving.

4. Sample bottles, autocleavable plastics, etc., should sterilized in an autoclave for 15 minutes

in 0.15 MPa/121⁰C.


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Media Preparations

For each Batch of Culture Media, Record the following information

for each component in a media dispensing logbook:

Date of Preparation

Name of Reagent

Amount of Reagent (in grams)

Amount of Water (in mL)

A. PREPARATION OF DEHYDRATED LACTOSE SULFATE BROTH (LSB) in Triple Strength

1. Weigh 106.8 grams of dehydrated Lactose Sulfate Broth (LSB) powder

2. Dissolved into the beaker with 1000 ml of distilled water.

3. Warm slightly by placing the beaker in hot plate with magnetic stirrer to dissolve

completely

4. Dispense (10 ml each) portion into fermentation test tubes (20x150mm) fitted with

Durham tubes (8x45mm) using media dispenser . That would produce 105

fermentation test tubes in all or equivalent to 21 samples in 5 tubes.

5. Sterilize for 15 minutes at 0.15 MPa/ 121⁰C in an autoclave. pH= 6.8+/-0.2 at 25⁰C

after sterilizing.

6. After Sterilizing, let it cool overnight to be used for tomorrow's water analysis or put

these prepared culture media broth in refrigerator. These culture media broths are

potent up to 2 weeks when refrigerated. Unused broth will be discarded after 2

weeks.

7. Make sure to put the date prepared on the test tube rack where these test tubes are

kept. Older date prepared should be used first, in first made first to be used

principle guide or in "first in first out" basis.

* Refer to MPT (Media Preparation Table) for desired volume of culture media reagent to be

prepared.


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B. PREPARATION OF DEHYDRATED BRILLIANT GREEN LACTOSE BILE (BGLB) BROTH

1. Weigh 40 grams of dehydrated Brilliant Green Lactose Bile (BGLB) broth powder .

2. Dissolved into the beaker with 1000ml of distilled water.


completely.


Durham tube (8x45mm) using media dispenser. That would produce 100


5. Sterilize for 15 minutes at 0.15 MPa/121⁰C in an autoclave. pH= 7.2+/-0.2 at 25⁰c

after sterilizing.


these prepared culture media broth in a refrigerator. These culture media broths are


weeks.

7. Make sure to put the date prepared on the test tube rack where these test tubes are

keep. Older date prepared should be used first, in first made first to be used



prepared.


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C. PREPARATION OF DEHYDRATED EC BROTH

1. Weigh 37 grams of dehydrated EC broth powder .

2. Dissolved into the beaker with 1000ml of distilled water.


completely


Durham tube (8x45mm). using media dispenser. That would produce 100


5. Sterilize for 15 minutes at 0.15 MPa/121⁰C in an autoclave. pH= 6.9+/-0.2 at 25⁰C

after sterilizing.


these prepared culture media broth in a refrigerator. These culture media broths are


weeks.

7. Make sure to put the date prepared on the test tube rack where these test tubes

are keep. Older date prepared should be used first, in first made first to be used



prepared.


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D. PREPARATION OF DEHYDRATED PLATE COUNT AGAR (PCA) Plates

1. Weigh 22.5 grams of dehydrated PCA (Casein-peptone glucose yeast extract) agar

powder.

2. Dissolved into the Erlenmeyer flask with 1000ml of distilled water.

3. Mix thoroughly. Heat with frequent agitation by placing it into hot plate with

magnetic stirrer for 1 minute to dissolve powder. Note that the mixture will become

clear eventually after the agar is thoroughly mixed.

4. Sterilize for 15 minutes at 0.15MPa/121⁰C in an autoclave. pH= 7.0+/-0.2 at 25⁰C

after sterilizing.

5. After sterilization, let it cool overnight reserve for tomorrow's water analysis or put

in refrigerator to prolong preservation up to 2 weeks potency.

6. During Heterotrophic Plate Count (HPC) Method, the solidified PCA agar will be

reheated again into hot plate with magnetic stirrer. After the agar thoroughly

melted again with lukewarm heat, distribute/pour into sterile Petri dishes (10ml

each) portion with 1ml water sample in each Petri dish in pour plate method. The

1000ml PCA agar sample will produce 100 prepared agar in Petri dishes.


prepared.


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E. PREPARATION OF DEHYDRATED EOSIN METHYLENE BLUE (EMB) AGAR Plates

1. Weigh 36 grams of dehydrated EMB agar powder.




clear eventually after the agar is thoroughly mixed with no undissolve powder is

visible.


after sterilizing.

5. Distribute or pour 10ml portion into sterile Petri dishes. Let it cool and solidified. The

1000 ml EMB agar will produce up to 100 prepared agar in Petri dishes.

6. Place into incubator overnight in an inverted stacked position and observe growth of

bacterial colonies. If no visible colonies observed, that means that no bacteria in the

surrounding air contaminates the Petri dishes and is ready for use, or place it in

refrigerator in inverted position for future use. Potency is up to 2 weeks to 1 month.

7. The prepared EMB agar in Petri dish should be place in a close container inside the

refrigerator so that no occurrence of dehydration of prepared agar in a dish.


prepared.


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F. PREPARATION OF DEHYDRATED CETRIMIDE (Pseudomonas selective) AGAR Plates

1. Weigh 45.3 grams of dehydrated EMB agar powder.


3. Add 10 mL glycerol into the mixture in Erlenmeyer flask.




visible.


after sterilizing.


1000 ml cetrimid agar will produce up to 100 prepared agar in Petri dishes.





8. The prepared CETRIMID agar in Petri dish should be place in a close container inside

the refrigerator so that no occurrence of dehydration of prepared agar in a dish.


prepared.


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G. PREPARATION OF DEHYDRATED NUTRIENT AGAR (NA) Plates

1. Weigh 20 grams of dehydrated NA agar powder.





visible.


after sterilizing.


1000 ml cetrimid agar will produce up to 100 prepared agar in Petri dishes.





7. The prepared NA agar in Petri dish should be place in a close container inside the

refrigerator so that no occurrence of dehydration of prepared agar in a dish.


prepared.


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Multiple Tube Fermentation Technique (MTFT) Test Procedure

A. Presumptive Test Procedure

1. Shake water sample 25 times by making up and down of back and forth movements of

about 0.3 (1ft) in 7 seconds.

2. Pipet 10 mL of the water sample into each 5 tubes of primary pre-enrichment LSB

fermentation tubes.

3. Incubate at 35⁰C-36⁰C for 24 hours.

4. Observe at the end of 24 hours and record the presence or absence of gas.

5. Separate the gas positive tubes (with gas formation trapped in Durham tubes and the

sample is turbid) to undergo confirmatory test. While the clear negative tubes record as

or interpret as negative sample result.

6. Incubate for another 24 hours if the sample tube may fall in the category of doubtful

observation.

7. If no gas has developed after 48 hours, record as or interpret as negative sample result.

8. If gas formation observes after 48 hours, then proceed to confirmatory method.

*Examination for gas formation after 48 hours has an arbitrary limit of 48 hours for doubtless

observation excludes occasional members of the coliform group which form gas slowly, but

for the purpose of a standard test based on definition of coliform, exclusion of occasional

slow-gas-forming is satisfactory.


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B. Confirmatory Test Procedure

1. Test Procedure for Brilliant Green Lactose Bile (BGLB) Broth

a) Gently shake or rotate (hand using shake or using Vortex shaker) gas positive

primary fermentation tube.

b) Transfer 0.1mL using micropipette of gas positive primary LSB medium broth to

a BGLB tube, and/ or transfer 3 loopfuls (using sterile inoculating loop in 3mm

diameter) from gas positive primary LSB medium broth to a BGLB tube.

c) Mix by gently shaking.

d) Incubate the inoculated tubes for 24 hours-48 hours +/- 3 hours at 35⁰C +/-

0.5⁰C

Interpretation:

1. The formation of gas in any amount in the inoculated tube at any time

within 24-48 hours constitutes a confirmed positive test for Total

Coliform.

2. The absence of gas formation after 24-48 hours interpreted as

confirmed negative test for Total Coliform.

e.) Refer to Most Probable Number (MPN) Table for BGLB for the equivalent

quantification number of positive tubes.

MOST PROBABLE NUMBER (MPN) FOR TOTAL COLIFORM

Number of Positive Tubes MPN

0 <1.1

1 1.1

2 2.6

3 4.6

4 8.0

5 >8.0


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2. Test Procedure for EC Thermo-tolerant (Fecal) coliform bacteria in EC Broth

a. Gently shake or rotate (hand using shake or using Vortex shaker) gas

positive presumptive tube.

b. Transfer 0.1mL using micropipette of gas positive primary LSB medium broth

to a EC broth tube, and/ or transfer 3 loopfuls (using sterile inoculating loop

in 3mm diameter) from gas positive primary LSB medium broth to an EC

broth tube.

c. Mix by gently shaking.

d. Incubate the inoculated tubes in water bath for 24 hours +/- 2 hours at

44.5⁰C +/- 0.2⁰C within 30 minutes after planting. The water depth in the

incubator should be sufficient to immerse tubes to the upper level of the

medium.

Interpretation:

1. Gas production in an EC fermentation tube within 24 hours or less is

considered as fecal coliform in origin. Record and interpret as

confirmed positive for Fecal Coliform.

2. Failure to produce gas (growth sometimes occur; the medium is

turbid) constitute a negative reaction indicating a source other than

the intestinal tract of warm-blooded animal. Record and interpret as

confirmed negative for Fecal Coliform.

3. Absence of gas production and the medium is clear; interpret as

confirmed negative for Fecal Coliform.

e.) Refer to the Most Probable Number (MPN) Table for equivalent quantification

MOST PROBABLE NUMBER (MPN) FOR FECAL COLIFORM

Number of Positive Tubes MPN

0 <1.1

1 1.1

2 2.6

3 4.6

4 8.0

5 >8.0


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3. Test Procedure for Eosin Methylene Blue (EMB) Agar Plates

a. Gently shake or rotate presumptive positive tubes.

b. Streak each positive presumptive tube into EMB agar plates with a straight

inoculating needle slightly bent at the tip to prevent scratch and produce

well isolated colonies.

c. Incubate the plates in inverted position at 35⁰C +/- 0.5⁰C for 24 hours.

Interpretation:

The colonies developing on EMB agar plates maybe divided as:

1. Typical colonies- nucleated colony with metallic sheen color in

reflected light record as confirmed positive for E.coli.

2. Atypical- colonies are larger than those of E. coli, opaque, pink

mucoid, confluent, unnucleated record as confirmed positive for

Total Coliform

3. Translucent, amber colored, clear watery colonies or swarming

4. colonies record as confirmed negative for Total and Fecal Coliform.

5. Clear, no growth or no colonies observed record as confirmed

negative for Total and Fecal Coliform.


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HETEROTROPHIC PLATE COUNT

Procedure: Pour Plate Method

a. Mix sample by making 25 complete up and down or back and forth movements, of

about 0.3 (1ft.) in 7 seconds.

b. Pipet 1mL or 0.1 mL of the sample into a sterile petri dish

c. Pour aseptically 10 mL melted PCA agar medium which has been allowed to cool as

like lukewarm heat.

d. Rotate the plate over a smooth surface first in one direction, then in the opposite

direction or by rotating it so that the melted agar and the water sample will mix

thoroughly. Take care not to splash mixture over the edge of the dish.

e. Allow the plates to solidify (within 10 minutes) on a level surface.

f. Invert the plates and incubate at 35⁰C+/- 0.5⁰C for 24-48 hours +/- 3 hours.

g. Check sterility of medium by having control plates for each series of samples.

h. After incubation, count no. of growth per petri dish

i. if growth is less than 30 colonies per plate, count as is

j. if growth is more than 30 colonies per plate, count growth by means of colony

counter.

k. Report results as colony forming units.


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HETEROTROPHIC PLATE COUNT (HPC) COLONY COUNTING

GUIDELINES

ACTUAL READING ON PLATE

COMPUTATION/ INTERPRETATION

0-30 colonies/plate

Count colonies as is

>30 Colonies or <10 colonies/cmᵌ

Count colonies in 13 squares in an Quebec Colony

Counter

(7 Horizontal & 6 vertical squares)

Total colonies multiply by 5 = cfu/mL ( glass plate)

Total colonies multiply by 4.38 = cfu/mL disposable plate

11- 100 colonies

or

>10 colonies cmᵌ

Count 4 representative squares then take the average.

Multiply the result by:

65 = cfu/mL (glass plate)

57 = cfu/mL (disposable Petri dish)

>100colonies cmᵌ

Report as >6,500 cfu/mL


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Water Analysis Methods On Enumerating E. coli and

Coliform Group of Bacteria

2. CHROMOGENIC SUBSTRATE METHOD (Rapid Test)

Summary

All materials and reagents had been sterilized to ensure high quality standards of these factory

prepared reagents for rapid test/enumeration and detection of fecal contamination in the water

sample. The Quanti-Tray had been sterilized with ethylene oxide and designed to give quantified

bacterial counts of 100ml samples using prepared sterile reagent product. The sterile clear bottle

had been added with sodium thiosulfate as a neutralizing agent for those chlorine-treated water

samples. Treated water sample has an incubation period of three (3) weeks. But with the addition of

sodium thiosulfate, the incubation time is 18-22 hours only. The 100mL water sample will be poured

out to the sterile clear bottle and the sterile prepared reagent in a snap pack will be added. Then,

the reagent/sample mixture will be poured into Quanti-Tray, to be sealed in a Sealer and will be

incubated per the reagent instructions. After incubation, positive wells will be counted using Most

Probable Number (MPN) Table to determine the MPN of coliforms while positive wells for E. coli on

the other hand, would be determined with the aid of UV Lamp as to make the E. coli fluoresce under

specific range of UV light and then positive wells will be referred to the MPN Table to determine the

MPN of the E. coli.


28 | P a g e

Quantification Materials/Paraphernalia:

Sterile Quanti-Tray

Quanti-Tray Comparator

Sterile 100ml clear bottle

Sterile prepared reagent in a snap pack for 100ml water sample

Quanti-Tray Sealer

UV Lamp housed inside viewing box

MPN Table specific only for Chromogenic Substrate Method

Incubator

Quantification Test Procedure:

1. Carefully remove snap pack from the

strip, open and add the reagent to a

100ml water sample in a sterile vessel.

Aseptically cap and seal the vessel and

shake until dissolved. Caution: Do not

touch the opening of the pack or vessel.

2. Use one hand to hold a Quanti-Tray upright with the well

side facing the palm.


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3. Squeeze the upper part of the Quanti-Tray so that it bends

toward the palm.

4. Open the Quanti-Tray by pulling the foil tab away from the

well side. Avoid touching the inside of the foil or tray.

5. Pour the reagent/sample mixture directly into the

Quanti-Tray, avoiding contact with the foil tab. Allow

foam to settle.

6. Place the sample-filled Quanti-Tray onto the rubber

tray carrier of the Quanti-Tray Sealer with the well side

(plastic) of the Quanti-Tray facing down to fit into the

carrier and sealed.

7. Incubate the sealed sample-filled Quanti-Tray into the incubator for 18 hours with specific

temperature of 35⁰C. After 18 hours pull out the sample-filled quanti-tray from the

incubator.


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8. Read quanti-tray results. Yellow Wells = confirmed Total Coliforms. Count number of

positive wells (including overflow well) and refer to MPN table.

9. Yellowish-fluorescent wells = confirmed E. coli. Count number of positive wells (including

overflow well) and refer to MPN table.

10. Read the results at 18 hours (but no later than 22 hours) compare each result to a

comparator.

* If no yellow color is observed, the sample is negative for coliforms and E. coli.

Overflow Well

Positive wells

Positive wells


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* If the sample has a yellow color equivalent to the comparator, the sample

confirmed negative for coliforms.

* If the sample has a yellow color ≥ comparator, the sample is confirmed positive for

coliforms.

* If yellow color is observed, check quanti-tray for fluorescence by placing a 6 watt

365nm UV light 5 inches (13cm) from the samples in a dark environment. If

the fluorescence is ≥ to the comparator the sample is confirmed positive for

E. coli. Caution: Be sure the light is facing away from eyes.

11. Put the used bacterial culture media quanti-trays into autoclavable bag.

12. Put the autoclavable bag in the autoclave to be decontaminated for 30 minutes under 127⁰C

temperature with pressure of 0.15 MPa.

13. After decontamination, pull out the bag from the autoclave.

14. Discard the decontaminated liquid media in the sink.

15. Dispose the plastic quanti-trays into non-biodegradable waste bin.


32 | P a g e

Water Analysis Method on Detecting Coliform Group Of

Bacteria

3. Primary Health Care

This is a chemical test used to detect the presence of H₂S in water which is associated with E.

coli and other organisms which indicate the presence of fecal contamination in drinking water

supplies. H₂S producing microorganisms are consistently associated with the presence of coliform in

water. Enteric bacteria such as Citrobacter, Salmonella, Proteus and certain species of Klebsiella also

produce H₂S in which further studies also reported H₂S producing variants of E.coli of fecal in origin.

PRINCIPLE:

The test is based on the ability of the coliform bacteria to produce H₂S (Hydrogen Sulfide)

PURPOSE:

To obtain rapid testing result (24hours) in the absence of modern laboratory facilities

MEDIA PREPARATION FOR PHC (WHICH MAKES APPROXIMATELY 120 BOTTLES):

Ingredients:

*Peptone - 40 grams

Dipotassium Hydrogen Phosphate - 3 grams

Ferric Ammonium Citrate - 1.5 grams

**Sodium Thiosulfate, anhydrous - 2 grams

Teepol (dishwashing liquid) - 2 grams

Distilled Water - 100 grams

Fine Facial Tissue (white)


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*If Peptone is not available, 30 grams of casamino acid maybe used.

*8 If sodium thiosulfate, anhydrous (Na₂S₂O₃) is not available, 3.14 grams of sodium thiosulfate,

Hypo (Na₂S₂O₃5H₂O) maybe used.

DIRECTION:

1. Carefully weigh the preceding chemicals in top loading balance and place in a glass

beaker (400mL)

2. 2. Add 100 mL distilled water

3. Place the beaker in hot plate with magnetic stirrer so as to mix the chemicals

thoroughly with moderate heat. Set Aside the dissolved medium after mixed

thoroughly for a while

4. Fold facial tissue several times

5. Place 1/2 folded tissue in 30 mL bottle capacity and the other 1/2 portion in another

30 mL bottle capacity. Put a 1/2 portion of the folded tissue paper in all 120 pcs 30

mL bottle capacity

6. Pipette 1 mL of dissolved medium that has been set aside for a while into each

bottle and let it absorb on the folded tissue paper inside the bottom of the bottle

7. Cap the bottle loosely

8. Sterilize bottles with medium at 0.15MPa for 18 minutes in an autoclave

9. Pull out the bottles from the autoclave, let it cool, then incubate at about 37⁰C

overnight

10. Remove the bottles and cool. Attach 1 inch masking tape on one side for labeling/

identification of the sample.


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WATER TESTING PROCEDURE USING PHC BOTTLE:

1. Open the cap and fill the sterilized PHC bottle with water sample (water to be tested) up to

the neck of the bottle

2. Cap the bottle tightly and taking care not to contaminate the top or the inside of the bottle

with your hands

3. The water inside the bottle then turns yellowish, do not be alarmed, this is normal

4. Allow to stand at room temperature away from direct sunlight for 24 hours (If an incubator

is available, incubate sample bottles at 30-37⁰C for 12-18 hours)

5. Observe the appearance of the water sample after 24 hours

Interpretation:

a. If the tissue paper inside the bottom of the bottle turns black and the water is

turbid, it indicates presence of H₂S producing bacteria (coliform organisms)

commonly found in animal and human waste. Thus, the water is contaminated;

interpret as positive for contamination.

*There are some instances that the contamination may vary in color, you may categorize the

result by:

(+++) = Very black - highly contaminated;

(++) = Moderately Black - moderately contaminated;

(+) = Slightly Black - slightly contaminated

nil = clear yellowish water - no contamination


35 | P a g e

b. If the tissue paper inside the bottle is still white and the water is still yellowish, it

indicates the water is negative for coliform group of organisms and interpret as

negative for contamination.

6. Disinfect used PHC bottles by placing it in a container with disinfectant or about an

overnight prior to washing and/or disposal, or:

7. Decontaminate by autoclaving at 126⁰C/ 0.15 MPa for 30 minutes.


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DISINFECTION AND DECONTAMINATION

1.) In case of culture spills:

a.) Pour contaminated area with 70% isopropyl/ethyl alcohol:

b.) Allow to react for 30 minutes.

c.) Soak tissue paper on the spill area and wipe off the disinfectant.

d.) Discard contaminated tissue paper in any autoclaveable bag and decontaminate by

autoclaving at 126⁰C/ 0.15 MPa for 30 minutes.

e.) After decontamination, pull out the bag from the autoclave.

f.) Discard the decontaminated liquid media in the sink.

g.) Dispose the tissue paper in biodegradable waste bin.

2.) Disinfect/decontaminate contaminated materials/paraphernalia (e.g. slides, pipettes, plastic

wares, glasswares, pipette tips, disposable inoculating loops) by placing it in a container with

disinfectant or about an overnight prior to washing and/or disposal.

3.) Autoclave contaminated used glasswares and autoclaveable plastic wares containing live

microbial cultures for 30 minutes under temperature 126⁰C with pressure of 0.15 MPa.

Discard decontaminated culture media in the sink and wash glasswares/plastic wares to let

it dry ready for glassware sterilization for the next usage.


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REFERENCES:

GSCWD Quality System Manual

Philippine National Standards for Drinking Water (PNSDW) 2007

Standard Method of Examination of Water and Wastewater (SMEWW) 22nd Edition

Manufacturer's Manual

Department of Health- National Reference Laboratory (DOH-NRL)

Training Course on QC of Culture Media & Culture Preservation Technique

Philippine National Collection of Microorganisms,

National Institute of Molecular Biology and Biotechnology,

UPLB College Laguna, July 30- August 1, 2014

Seminar/ Workshop on "Microbiological Analysis for EU & FDA",

National Institute of Molecular Biology and Biotechnology

UPLB College Laguna, November 21-22, 2013

Natural Sciences Research Institute, UP Diliman, Quezon City

Cover picture:

Courtesy of Gen Medica

http://www.genmedica.eu/blog/tag/Lab%20Chemicals

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