WORK INSTRUCTION MANUAL LABORATORY SECTION 1 | Page 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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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