Bacteriological Analysis Edited (1) صور 2

‫ درجة ثالثة‬Bacteriological Analysis of Water - ‫المسار الوظيفي لوظيفة كيميائي مياه‬
3. Estimation of Total Coliform by Multiple Tube Fermentation

Technique
1. Scope:
 Estimation of Total Coliforms (TC) by Multiple-Tube
Fermentation technique (MTF), also called Most Probable
Number (MPN) procedure, in 96 hours, or less, in water
samples on the basis of the production of gas and acid from
fermentation of lactose.
 This procedure can be applied for different types of water, in
microbiology laboratory it will be applied for the analysis of
surface raw water.
2. Principle:
 Volumes/dilutions of water sample to be tested are added to
tubes containing lauryl Tryptose broth as presumptive media (5
tubes per dilution) with inverted vials, or pH indicator. The
selectivity of media is due to sodium lauryl sulfate that acting as
inhibitor of bacteria other than coliforms. After incubation, the
tubes are examined for growth, gas, and/or acidic reaction
(shades of yellow color) if inner vial is omitted, growth with
acidity (yellow color) is signifies a positive presumptive reaction.
 An additional confirmatory test by brilliant green bile broth 2% is
required to confirm the result. The selectivity is due to presence
of both oxgall (bile) and brilliant green dye acting as inhibitor of
gram positive and selected gram negative bacteria. Organisms,
primarily coliform, which are resistant to those inhibitor and
ferment lactose with gas formation, indicated by Durham tube,
can replicate in this medium.
Lauryl
Brilliant Tryptose
green broth
31 ‫قطاع تنمية الموارد البشرية بالشركة القابضة – االدارة العامة لتخطيط المسار الوظيفي‬
 Total coliform bacteria (excluding E. coli) occur in both sewage

and natural waters. Some of these bacteria are excreted in the
faeces of humans and animals, but many coliforms are
heterotrophic and able to multiply in water and soil
environments.
 Total coliforms include organisms that can survive and grow in
water distribution system, particularly in the presence of
biofilms. They were, traditionally, regarded as belonging to the
genera Escherichia, Citrobacter, Klebsiella and Enterobacter,
but the group is more heterogeneous and includes a wider
range of genera, such as Serratia and Hafnia. Hence, they are
not useful as an index of faecal pathogens, but they can be
used as an indicator of treatment effectiveness and to assess
the cleanliness and integrity of distribution systems and the
potential presence of biofilms. However, there are better
indicators for these purposes.
 The presence of Total Coliform after disinfection indicates
inadequate treatment, and presence in distribution system or
stored water supplies can reveal regrowth and possible biofilm
formation or contamination through ingress of foreign material,
including soil or plants.
 The MTF procedure, in comparison with the membrane filter
(MF) procedure, is more difficult to perform, takes longer to
produce results, and lacks precision; the precision depends on
the number of tubes used. The most satisfactory information will
be obtained when the largest sample inoculum examined shows
acid and/or gas in some or all of the tubes and the smallest
sample inoculum shows no acid and/or gas in any or a majority
of the tubes. However, the MTF procedure is still of value when
conditions render the MF technique unusable e.g. with turbid,
colored, or grossly contaminated water
 Results are reported in terms of the Most Probable Number
(MPN/100ml) of organisms present. It can be estimated by the
formula given below (section 11) or from MPN tables (Appendix
2) using the number of positive tubes in the multiple dilutions.
This number, based on certain probability formulas, is an
estimate of the mean density of total coliforms in the sample.
3. Definitions:
 Total coliform bacteria in this SOP are those facultative
anaerobic, gram-negative, non-spore-forming, rod-shaped
bacteria that ferment lactose with gas and acid formation within
48 hours at 35°C.
4. Environmental Conditions:
 All sample analysis steps will be carried on disinfected working
bench inside cultivation room to ensure controlled
environmental conditions.
5. Interference:
 Since the MPN indexes are based on assumption of a Poisson
distribution (random distribution) that’s for if the sample is not
adequately shaken before portions are removed or if bacterial
cells clump, the MPN value will be an underestimate of actual
bacterial density.
 High densities of non-coliform bacteria and the inhibitory nature
of some MTF media, e.g. Lauryl Tryptose broth, may prevent
gas formation. It’s recommended to treat all tubes with turbidity,
indicating growth, regardless of gas production, as presumptive
total coliform-positive tubes in case if tubes form the next
dilution gives true positive reaction.
6. Equipment:
 16 × 100 mm tubes, borosilicate glass, with heat-resistant
plastic caps.
plastic caps.
 Durham tubes, borosilicate glass.
 Test tubes racks to hold sterile culture tubes.
 Automatic pipettes and associated sterile long tips capable of
delivering 1 ml.
 Dilution screwed cap bottles.
 Incubator: 35°C ± 0.5°C.
 Sterile disposable applicator stick.
 Sterile plastic petri dishes 90 mm.
 Indelible ink marker for labeling plates.
7. Chemicals and Reagents:

 Lauryl Tryptose broth.
 Brilliant Green broth.
 Phosphate buffered dilution water.
 Nutrient agar.
 Gram Stain Reagent set.
 M-Endo LES agar.
 Bromcresol purple.
8. Precautions:
 Follow the normal safety procedures required in microbiology
laboratory.
 Mouth-pipetting is prohibited.
 Use a separate sterile tip for initial and subsequent transfers
from each tube, and different dilution.
 Replace the tip with a sterile one if becomes contaminated
before transfers are completed.
 Do not prepare dilutions in the direct of sunlight.
 Do not insert tips more than 2 to 3 cm below the surface of
sample or dilution when removing the sample volume.
 Avoid contamination when removing sterile tips from the
container by not dragging tip across lips and necks of dilution
bottles.
 Avoid picking up any membrane or scum on the needle while
transferring by inclining the fermentation tube.
 Insert end of loop or needle into the liquid in the tube to a depth
of approximately 0.5 cm.
 Decontaminate all the used tubes, plates, and materials at the
end of the analyses.
9. Procedure:
9. 1 Responsibility :
 Microbiologists are responsible for the implementation of the
test procedures inside the microbiology laboratory.
 Laboratory technician is responsible for preparation of the
media and reagents used in this procedure under supervision of
the microbiologist.
9. 2 Sample Handling:
 Analyze samples on day of receipt whenever possible. The time
between sample collection and the placement of samples in the
incubator must not exceed 30 hours.
 If arrival is too late for processing on same day, refrigerate
overnight as long as holding time conditions still be met i.e.
preservation temperature <8.0°C and time did not exceeded
30 h.
9. 3 Instrument Calibration:
 Calibrate and verify balances daily using reference weights.
 Calibrate pH meter prior to use, using at least two standards
points.
 Check temperature of incubators and refrigerators daily to
ensure operation within stated limits of method.
 Check sterilization procedure and efficiency to ensure media
sterility used in this SOP.
9. 4 Presumptive Phase:
9.4.1 Selection of sample size:
 Inoculate a series of tubes with at least three decimal dilutions
(see 10.4.3) of water sample (multiples and submultiples of 10
ml), based on the probable total coliform density.
9.4.2 Media Preparation:

 Prepare and handle media and reagents appropriately, and
carry out suitable performance test to ensure homogeneity,
sterility, and suitability for each prepared batch.
 Phosphate buffered dilution water:
 Stock phosphate buffer solution: Dissolve 34.0 g KH2PO4 in
a 500 ml reagent-grade distilled water, adjust the pH of the
solution to 7.2 with 1 N NaOH and bring volume to 1000 ml with
reagent-grade distilled water. Sterilize by autoclave for 15
minutes at 121°C, store in the refrigerator until using (If
evidence of mold or other contamination appears in the stock,
discard the solution, and prepare a fresh solution).
 MgCl2 solution: Dissolve 38 g anhydrous MgCl2 (or 81.1 g
MgCl2.6H2O) in one liter of reagent-grade distilled water.
Sterilize by autoclave for 15 minutes at 121°C, store in the
refrigerator until using (If evidence of mold or other
contamination appears in the stock, discard the solution, and
prepare a fresh solution).
 Working solution: Add 1.25 ml phosphate buffer stock and 5
ml MgCl2 stock for each liter of reagent-grade distilled water
prepared, mix well(Final pH 7.0 ± 0.2), and dispense in
appropriate amounts for dilutions in screw-cap dilution bottles or
culture tubes, and/or into larger containers for use as rinse
water. Autoclave at 121°C for 15 minutes.
 Lauryl Tryptose broth:
 Prepare and sterilize medium according to manufacturer
instructions.
 Dispense 10 ml medium, before sterilization, in fermentation
tubes with an inverted Durham tube.
 Alternatively, and only in case of unavailability of Durham tubes,
add 0.01 g/L bromcresol purple to medium to determine acid
production.
 Close tubes with heat-resistant plastic caps.
 Ensure, after sterilization, that media cover inverted tube with at
least one-half to two-thirds, and completely filled with media i.e.
no air space inside inverted Durham tubes.
 Use tubes within two weeks (if loose plastic cap used) and
within three months (for tight screw cap tubes).
9.4.3 Samples Dilutions:

 Mix the sample by vigorously shakes the bottle.
 Use a sterile tip to transfer 10 ml of sample to 90 ml, or 1 ml of
sample to 9 ml of sterile dilution water bottle, cap, and mix. 1 ml
of this dilution is considered 10-1 of the original sample.
 Repeat the previous step to prepare further dilutions.
9.4.4 Inoculation:
 Use lauryl tryptose broth for presumptive phase.
 Arrange fermentation tubes in rows.
 Prepare four sets of serial dilutions (1.0, 0.1, 0.01, 0.001 ml)
using five tubes per dilution.
 Shake sample or dilutions vigorously.
 Inoculate each tube in the set with sample or dilution volume.
 Mix test portions in the medium by gentle agitation.
9.4.5 Incubation:
 Incubate inoculated tubes at 35 ± 0.5°C.
 After 24 ± 2 h swirl each tube gently and examine it for growth,
gas, and acidic reaction (shades of yellow color). If inner vial is
omitted, growth with acidity (yellow color) is signifies a positive
presumptive reaction
 If no gas or acidic reaction is evident, reincubate and reexamine
at the end of 48 ± 3 h.
 Record presence or absence of growth, gas, and acid
production.
9.4.6 Interpretation:
 Production of an acidic reaction and/or gas in the tubes within
48 ±3 h constitutes a positive presumptive reaction.
 The absence of acidic reaction and/or gas formation at the end
of 48 ±3 h of incubation constitutes a negative test.
9. 5 Confirmed Phase:
 Brilliant green broth:
instructions.
within three months (for tight screw cap tubes) after preparation.
9.5.2 Inoculation:
 Submit tubes with a positive presumptive reaction to the
confirmed phase within 24 ± 2 h of incubation to the confirmed
phase.
 If additional presumptive tubes showed active fermentation or
acidic reaction at the end of 48 ± 3 h incubation period, submit
to the confirmed phase.
 Arrange brilliant green tubes rows in a similar manner to
positive presumptive tubes.
 Gently rotate positive presumptive tubes to resuspend the
organisms.
 Use a sterile loop 3.0 mm in diameter to transfer three loopfuls
from positive presumptive tube to a brilliant green tube
confirmation tube.
 Repeat for all other positive presumptive tubes.
9.5.3 Incubation:
 Incubate the inoculated tubes at 35 ± 0.5°C for 48 ± 3 h
 Formation of gas in any amount, in Durham tube, of the brilliant
green tubes, at any time, within 48 ± 3 h constitutes a positive
confirmed result.
 Calculate the MPN value (see section 11.0) of the number of
positive brilliant green lactose bile tubes from MPN index (see
A.1).
9. 6 Completed Phase:
9.6.1 Application:
 This phase is aiming to verify the presence of coliform bacteria
and to provide quality control data for non-potable water sample
analysis.
 Use the completed test on 10% of positive confirmed tubes on
seasonal basis. Analysis of samples for thermotolerant (fecal)
coliforms at elevated temperature (44.5±0.2°C) can be

considered as a completed test.
 Alternatively, the completed test for positive total coliforms may
be performed as follows:

 Use LES Endo agar plates and Nutrient agar slants for
completed phase.
 m-Endo LES agar:
instructions.
 Dispense in 5 to 7 ml quantities into lower section of 50 mm
Petri dishes, if dishes of any other size are used, adjust quantity
to give an equivalent depth of 4 to 5 mm.
 If plates are previously prepared and stored in the refrigerator,
allow them to warm at room temperature. The crystals that form
on agar plates after refrigeration will disappear as the plates
warm up. Use plates within two weeks after preparation.
 Nutrient agar:
instructions.
 Dispense 10 ml in screw-capped tubes before sterilization.
 Place tubes in an inclined position immediately after
sterilization, so that the agar will solidify with a sloped surface.
 Tighten screw caps after cooling and store in a protected, cool
storage area.
 If plates or tubes are made ahead of time and stored in the
refrigerator, remove them and allow warming at room
temperature before use.
9.6.3 Procedure:
 Use a sterile 3-mm-diam loop or an inoculating needle slightly
curved at the tip
 Tap and incline the fermentation tube to avoid picking up any
membrane or scum on the needle
 Insert the end of the loop or needle into the liquid in the tube to
a depth of approximately 0.5 cm
 Streak a plate for isolation with the curved section of the needle
in contact with the agar to avoid a scratched or torn surface
 Change loop between second and third quadrants to improve
colony isolation.
 Incubate plates (inverted) at 35 ± 0.5°C for 24 ± 2 h.
 Pick from each plate one or more typical (pink to dark red with a
green metallic surface sheen) well-isolated coliform colonies. If
no typical colonies are present, pick two or more colonies
considered most likely to consist of organisms of the coliform
group.
 Transfer growth from each isolate, by barely touching the
surface of colony to minimize the danger of transferring mixed
culture, to a single-strength lauryl tryptose broth fermentation
tube and onto a nutrient agar slant.
 Incubate nutrient agar slants at 35 ± 0.5°C for 24 ± 2 h.
 Incubate secondary broth tubes at 35 ± 0.5°C for 24 ± 2 h; if
gas is not produced within 24 ± 2 h reincubate and examine
again at 48 ± 3 h.
 Examine Gram stained preparations from nutrient agar slant
cultures corresponding to the secondary tubes that showed gas;
include gram positive and gram negative cultures as controls.
 Gram Stain technique :
 Prepare separate light emulsions of the test bacterial growth
using drops of distilled water on the slide.
 Prepare separate light emulsions of the positive and negative
control cultures on the same slide using drops of distilled water
on the slide.
 Fix the slides by air drying.
 Stain for 1 min with ammonium oxalate-crystal violet solution,
rinse slides in tap water and drain off excess.
 Apply Lugol’s solution for 1 min, rinse stained slides in tap
water.
 Decolorize for approximately 15 to 30 s with acetone alcohol by
holding slide between the fingers and letting acetone alcohol
flow across the stained smear until the solvent flows colorlessly
from the slide. Do not over-decolorize to not remove crystal
violet from gram positive bacteria.
 Counterstain with safranin for 15 s, rinse with tap water.
 Blot dry with absorbent paper or air dry, and examine

microscopically.
 Gram-positive organisms are blue (violet); gram-negative
organisms are red.
 Results are only acceptable when controls given proper
reactions.
Gram Stain technique
 Formation of gas in the secondary tube of lauryl tryptose broth
within 48 ± 3 h and demonstration of gram-negative, nonspore-
forming, rod-shaped bacteria from the agar culture constitute a
positive result for completed test, demonstrating the presence
of a member of the coliform group.
10. Calculations:
 The MPN values, for variety of positive and negative tubes
combinations, are given in appendix A.1.
 The samples volumes indicated in indexes A.1 illustrates MPN
values for combinations of positive results when five 10 ml, five
1 ml, and five 0.1 ml sample portion volumes are tested.
 If the sample portion volumes used are those found in the

tables, report the value corresponding to the number of positive
and negative results in the series as the MPN/100ml.
 When the series of decimal dilutions is different from that in the
table, select the MPN value from index A.1 for the combination
of positive results and calculate according to the following
formula :
MPN / 100 ml = (Table MPN/ 100 ml) × 10/V
Where:
V = volume of sample portion at the lowest selected dilution.
 When more than three dilutions are used in a decimal series of
dilutions, select the three most appropriate dilutions and refer to
index A.1.
 Selected examples(A though C) illustrating correct selection are
listed below, more examples are available in Standard Methods
(see 16.0):
Volumes ml Combination MPN index

Example
10 1.0 0.1 0.01 0.001 of positive / 100 ml
A 5 5 1 0 0 5-1-0 330
B 4 5 1 0 0 4-5-1 48
C 4 3 0 1 1 4-3-2 39
 Example A: First, remove the highest dilution (smallest sample

volume) if it has all negative tubes and at least one remaining
dilution has a negative tube. Next, remove the lowest dilution
(largest sample volume) if it has all positive tube and at least
one remaining dilution has a positive tube. According to these
guidelines, the three dilutions in Example A are selected by
removal of the highest (0.001-ml) and the lowest (10-ml)
dilutions.
 Example B: If the lowest dilution does not have all positive
tubes, and several of the highest dilutions have all negative
tubes, then remove the highest negative dilutions.
 Example C: If no dilution has all positive tubes, select the lowest

two dilutions, corresponding to 10 and 1 ml sample. For the
third dilution, add the number of positive tubes in the remaining
dilutions (0.1, 0.01, and 0.001 ml sample), to yield a final
combination of 4-3-2
 If MPN value for combinations not appearing in the table, or for
other combinations of tubes or dilutions, estimate the result as
follows:
 First, select the lowest dilution that does not have all positive
results.
 Second, select the highest dilution with at least one positive
result.
 Select all dilutions between them.
 Use the selected dilutions in the following formula:
MPN / 100 ml (approx.) = 100 × P / (N × T) ½
Where:
P: number of positive results.
N: Volume of sample in all negative portions combined.
T: Total volume of sample in the selected dilutions.
 Examples :
 From (5/5, 10/10, 4/10, 1/10, 0/5) Use only (-, -, 4/10, 1/10. - ),
4/10 @ 0.1 ml sample/tube and 1/10 @ 0.01 ml sample/tube.
calculations will be:
MPN / 100 ml (approx.) = 100 × 5 / (0.69 ×1.1) ½ = 500/0.87 =
570/ 100 ml
 From (5/5, 10/10, 10/10, 0/10, 0/5) Use only (-, -, 10/10, 0/10. - )
MPN / 100 ml (approx.) = 100 × 10 / (0.1×1.1) ½ = 1000/0.332 =
3000/ 100 ml.
11. Quality Control:

11.1 Analytical Quality Control:
 Apply the presumptive-confirmed phase of the multiple-tube
procedure to all samples, if samples were analyzed for fecal
coliform using MTF technique there will be no need to apply
completed phase, otherwise proceed completed test (Section
10.6) to not less than 10% of all coliform-positive samples on a
seasonal basis.
 Plates with non-selective agar medium, labeled as
“Environmental Control”, will be opened along the samples
analysis run time to ensure adequate environmental conditions.
 Procedural Blank: Inoculate, under the same conditions as
sample, one additional tube for each dilution with sterile
phosphate buffered dilution water after inoculation of series of
10 samples. Absence of growth indicates absence of cross
contamination.
11.2 Non Conformance and Corrective Action:

 If the media performance and sterility assessment are
performed concurrently with the use of the media in testing and
the media quality is deemed unsatisfactory, the specific use of
the media in the test(s) should be investigated to determine the
potential impact on the test results and the necessity for
repeating the test(s).
 Testing must be repeated when unsatisfactory media and/or
reagents are used for critical aspects of testing.
 If procedural blank test gives unsatisfactory results, the samples
results should be investigated to determine the potential impact
of contamination on the test result, if test results deemed
unsatisfactory the test must be repeated.
 If dilution water gives positive result, the samples results should
be investigated to determine the potential impact of dilution
water on the test result, if test results deemed unsatisfactory the
test must be repeated.
 If one or more of environmental control plates gives
unsatisfactory results, the samples results should be
investigated to determine the potential impact on the test result,
if test results deemed unsatisfactory the test must be repeated.
 For all non conformance findings, trace all media, reagents,

equipment operations, and tools used in analysis to find out root
cause of non conformance.
 Take appropriate corrective action, if cause was temporary e.g.
if unsterile batch of media used accidently in the analysis;
discard all batch of prepared media, or preventive actions if
cause was permanent e.g. re-maintenance of autoclave if actual
temperature was less than adjusted value, to eliminate
reoccurrence of non conformance in the future.
12. Method Performance Data:

 A limited intralaboratory study was conducted. Four participated
microbiological analysts tested surface raw water matrix.
 Results of this intralaboratory study, table 1, may not be typical
of results for samples other than those studied, hence
secondary validation (cross validation) in form of long-term
study shall be carried out depending on routine samples results.
 Table 1
Criteria Surface Raw
Precision/ repeatability 0.461
Precision/Reproducibility 0.81
Bias % ±0.03
 Detection limit was originally established as 1.8 MPN/100 ml.

 Upper working limit was previously established for this method
as 1600 MPN/100ml.
 Range of reliable estimation was previously established for this
method between 1.8-1600 MPN/ 100ml.
13. Reporting:
 Report coliform concentration as the Most Probable Number
(MPN)/100 ml.
 If sample was delayed more than permissible time, write
“Delayed” in report comment.
14. References:
 Eugene W. Rice, Rodger B. Baired, Andrew D. Eaton, Lenore
S. Clesceri eds.2012 Standard Methods for the Examination of
Water and Wastewater. Method 9221B#, 22nd edition (on-line
edition). American Public Health Association, American Water
Works Association, and Water Environment Federation
 World Health Organization. 2011, Guidelines for Drinking-
Water Quality, Fourth Edition. Geneva
 Guidelines for Canadian Drinking water: Guideline Technical
Document. 2006. Total Coliform. Health Canada. Ottawa.
Ontario.
A.1 MPN index and 95% confidence limits for various

combinations of positive results when five tubes are used per
dilution (10 ml, 1.0 ml, 0.1 ml).
4. Estimation of Fecal Coliform by Multiple Tube Fermentation

Technique
1. Scope:
 This is a detailed procedure for estimation of Fecal Coliforms
(FC) by Multiple-Tube Fermentation technique (MTF), also
called Most Probable Number (MPN) procedure, in 72 hours, or
less, in water samples on the basis of the production of gas and
acid from fermentation of lactose.
 This procedure can be applied for different types of water, in
microbiology laboratory it will be applied for the analysis of
surface raw water.
2. Principle:
 Volumes/dilutions of water sample to be tested are added to
tubes containing lauryl Tryptose broth as presumptive media (5
tubes per dilution) with inverted vials, or pH indicator. The
selectivity of media is due to sodium lauryl sulfate that acting as
inhibitor of bacteria other than coliforms. After incubation, the
tubes are examined for, gas, and/or acidic reaction (shades of
yellow color). If inner vial is omitted, growth with acidity (yellow
color) is signifies a positive presumptive reaction.
 An additional confirmatory test by EC broth is required to
confirm the result. The selectivity is due to presence of bile salt
that acting as inhibitor of gram positive bacteria, particularly
bacilli and fecal streptococci. Incubation at elevated incubation
temperature (44.5±0.2°C) inhibits other coliform bacteria, which
is not thermo-tolerant, from growing up.
 Total coliform bacteria that are able to ferment lactose at 44.5°C
are known as thermo-tolerant coliforms. Thermo-tolerant
coliforms were traditionally called fecal coliforms, but they also
have been documented in organically rich waters or tropical
climates in the absence of recent fecal contamination. So,
testing for E. coli, a specific indicator of fecal contamination, is
recommended.
 In most waters, the predominant genus is Escherichia, but
some types of Citrobacter, Klebsiella and Enterobacter are
also thermo-tolerant. They are usually found in sewage and
water recently subjected to fecal pollution.
 Populations of thermo-tolerant coliforms are composed

predominantly of E. coli; as a result, this group is regarded as a
less reliable but acceptable index of faecal pollution. The
presence of thermo-tolerant coliform (fecal coliform) provides
evidence of recent fecal pollution
 The MTF procedure, in comparison with the membrane filter
(MF) procedure, is more difficult to perform, takes longer to
produce results, and lacks precision; the precision depends on
the number of tubes used. The most satisfactory information will
be obtained when the largest sample inoculum examined shows
acid and/or gas in some or all of the tubes and the smallest
sample inoculum shows no acid and/or gas in any or a majority
of the tubes. However, the MTF procedure is still of value when
conditions render the MF technique unusable e.g. with turbid,
colored, or grossly contaminated water
 Results are reported in terms of the Most Probable Number
(MPN/100ml) of organisms present. It can be estimated by the
formula given below (section 11) or from MPN tables
(Appendices) using the number of positive tubes in the multiple
dilutions. This number, based on certain probability formulas, is
an estimate of the mean density of total coliforms in the sample.
3. Definitions:
 Fecal coliform bacteria in this SOP are those facultative
anaerobic, gram-negative, non-spore-forming, rod-shaped
bacteria that ferment lactose with gas and acid formation within
24 hours at 44.5°C.
4. Environmental Conditions:
 All sample analysis steps will be carried on disinfected working
bench inside cultivation room to ensure controlled
environmental conditions.
5. Interference:
 Since the MPN indexes are based on assumption of a Poisson
distribution (random distribution) that’s for if the sample is not
adequately shaken before portions are removed or if bacterial
cells clump, the MPN value will be an underestimate of actual

bacterial density.
 High densities of non-coliform bacteria and the inhibitory nature
of some MTF media, e.g. Lauryl Tryptose broth, may prevent
gas formation. It’s recommended to treat all tubes with turbidity,
indicating growth, regardless of gas production, as presumptive
fecal coliform-positive tubes in case if tubes form the next
dilution gives true positive reaction.
6. Equipment:
 16 × 100 mm tubes, borosilicate glass, heat-resistant plastic
caps.
plastic caps.
 Durham tubes, borosilicate glass.
 Test tubes racks to hold sterile culture tubes.
 Automatic pipettes and associated sterile long tips capable of
delivering 1 ml and up to10 ml.
 Dilution screwed cap bottles.
 Incubator: 35°C ± 0.5°C.
 Incubator: 44.5°C ± 0.2°C.
 Sterile disposable applicator stick.
 Indelible ink marker for labeling plates.
7. Chemicals and Reagents:

 Lauryl Tryptose broth.
 EC broth.
 Phosphate buffered dilution water.
 Bromcresol purple.
8. Precautions:
 Follow the normal safety procedures required in microbiology
laboratory.
 Mouth-pipetting is prohibited.
 Use a separate sterile tip for initial and subsequent transfers

from each tube, and different dilution.
 Replace the tip with a sterile one if becomes contaminated
before transfers are completed.
 Do not prepare dilutions in the direct of sunlight.
 Do not insert tips more than 2.5 cm below the surface of sample
or dilution when removing the sample volume.
 Avoid contamination when removing sterile tips from the
container by not dragging tip across lips and necks of dilution
bottles.
 Avoid picking up any membrane or scum on the needle while
transferring by inclining the fermentation tube.
 Insert end of loop or needle into the liquid in the tube to a depth
of approximately 0.5 cm.
 Decontaminate all the used tubes, plates, and materials at the
end of the analyses.
9. Procedure:
9. 1 Responsibility:
 Microbiologists are responsible for the implementation of the
test procedures inside the microbiology laboratory.
 Laboratory technician is responsible for preparation of the
media and reagents used in this procedure under supervision of
the microbiologist(s),.
 Microbiologists and technician are well trained in the
implementation and performance of these procedures.
9. 2 Sample Handling:
 Analyze samples on day of receipt whenever possible. The time
between sample collection and the placement of samples in the
incubator must not exceed 30 hours.
 If arrival is too late for processing on same day, refrigerate
overnight as long as holding time conditions still be met i.e.
preservation temperature <8.0°C and time did not exceeded 30
hr.
9. 3 Instrument Calibration:
 Calibrate and verify balances daily using reference weights.
 Calibrate pH meter prior to use, using at least two standards

points.
 Check temperature of incubators and refrigerators daily to
ensure operation within stated limits of method.
 Check sterilization procedure and efficiency to ensure media
sterility used in this SOP.
9. 4 Presumptive Phase:
9.4.1 Selection of sample size:
 Inoculate a series of tubes with at least three decimal dilutions
(see 10.4.3) of the water (multiples and submultiples of 10 ml),
based on the probable fecal coliform density.

 Prepare and handle media and reagents appropriately, and
carry out suitable performance test to ensure homogeneity,
sterility, and suitability for each prepared batch.
- Phosphate buffered dilution water:
 Stock phosphate buffer solution: Dissolve 34.0 g KH2PO4 in
a 500 ml reagent-grade distilled water, adjust the pH of the
solution to 7.2 with 1 N NaOH and bring volume to 1000 ml with
reagent-grade distilled water. Sterilize by autoclave for 15
minutes at 121°C, store in the refrigerator until using (If
evidence of mold or other contamination appears in the stock,
discard the solution, and prepare a fresh solution).
 MgCl2 solution: Dissolve 38 g anhydrous MgCl2 (or 81.1 g
MgCl2.6H2O) in one liter of reagent-grade distilled water.
Sterilize by autoclave for 15 minutes at 121°C, store in the
refrigerator until using (If evidence of mold or other
contamination appears in the stock, discard the solution, and
prepare a fresh solution).
 Working solution: Add 1.25 ml phosphate buffer stock and 5
ml MgCl2 stock for each liter of reagent-grade distilled water
prepared, mix well(Final pH 7.0 ± 0.2), and dispense in
appropriate amounts for dilutions in screw-cap dilution bottles or
culture tubes, and/or into larger containers for use as rinse
water. Autoclave at 121°C for 15 minutes.
- Lauryl Tryptose broth:

instructions.
 Alternatively, and only in case of unavailability of Durham tubes,
add 0.01 g/L bromo-cresol purple to medium to determine acid
production.
9.4.3 Samples Dilutions:

 Mix the sample by vigorously shakes the bottle.
 Use a sterile tip to transfer 10 ml of sample to 90 ml, or 1 ml to
9 ml, of sterile dilution water bottle, cap, and mix. 1 ml of this
dilution is considered 10-1 of the original sample.
 Repeat the previous step to prepare further dilutions.
9.4.4 Inoculation:
 Use lauryl tryptose broth for presumptive phase.
 Arrange fermentation tubes in rows.
 Prepare four sets of serial dilutions (1, 0.1, 0.01, 0.001 ml)
using five tubes per dilution.
 Shake sample or dilutions vigorously.
 Inoculate each tube in the set with sample or dilution volume.
 Mix test portions in the medium by gentle agitation.
9.4.5 Incubation:
 Incubate inoculated tubes at 35 ± 0.5°C.
 After 24 ± 2 h swirl each tube gently and examine it for growth,
gas, and acidic reaction (shades of yellow color) If inner vial is
omitted, growth with acidity (yellow color) is signifies a positive
presumptive reaction.
 If no gas or acidic reaction is evident, re-incubate and

reexamine at the end of 48 ± 3 h.
 Record presence or absence of growth, gas, and acid
production.
 If the inner vial is omitted, growth with acidity signifies a positive
presumptive reaction.
 Production of an acidic reaction and/or gas in the tubes within
48 ±3 h constitutes a positive presumptive reaction.
 The absence of acidic reaction and/or gas formation at the end
of 48 ±3 h of incubation constitutes a negative test.
9. 5 Confirmed Phase:
- EC broth.
instructions.
9.5.2 Inoculation:
 Submit tubes with a positive presumptive reaction to the
confirmed phase within 24 ± 2 h of incubation to the confirmed
phase.
 If additional presumptive tubes showed active fermentation or
acidic reaction at the end of 48 ± 3 h incubation period, submit
to the confirmed phase.
 Arrange EC tubes rows in a manner similar to the positive
presumptive tubes.
 Gently shake or rotate positive presumptive tubes to re-suspend
the organisms.
 Use a sterile loop 3.0 mm in diameter to transfer three loopfuls

from positive presumptive tube to EC tube confirmation tube.
 Repeat for all other positive presumptive tubes.
9.5.3 Incubation:
 Incubate the inoculated tubes at 44.5 ± 0.2°C for 24 ± 2 h.
 Formation of gas in any amount, in Durham tube, of the EC
tubes, at any time, within 24 ± 2 h constitutes a positive
confirmed result.
 Calculate the MPN value (see section 11.0) of the number of
positive EC broth tubes from MPN index (see A.1).
10. Calculations:
 The MPN values, for variety of positive and negative tubes
combinations, are given in appendix A.1.
 The samples volumes indicated in indexes A.1 illustrates MPN
values for combinations of positive results when five 10 ml, five
1 ml, and five 0.1 ml sample portion volumes are tested.
 If the sample portion volumes used are those found in the
tables, report the value corresponding to the number of positive
and negative results in the series as the MPN/100ml.
 When the series of decimal dilutions is different from that in the
table, select the MPN value from index A.1 for the combination
of positive results and calculate according to the following
formula :
MPN / 100 ml = (Table MPN/ 100 ml) × 10/V
Where:
V = volume of sample portion at the lowest selected dilution.
 When more than three dilutions are used in a decimal series of
dilutions, select the three most appropriate dilutions and refer to
index A.1.
 Selected examples(A though C) illustrating correct selection are
listed below, more examples are available in Standard Methods
(see 16.0):
Volumes ml
Combination MPN index /
Example
of positive 100 ml
10 1.0 0.1 0.01 0.001
A 5 5 1 0 0 5-1-0 330
B 4 5 1 0 0 4-5-1 48
C 4 3 0 1 1 4-3-2 39
 Example A: First, remove the highest dilution (smallest sample

volume) if it has all negative tubes and at least one remaining
dilution has a negative tube. Next, remove the lowest dilution
(largest sample volume) if it has all positive tube and at least
one remaining dilution has a positive tube. According to these
guidelines, the three dilutions in Example A are selected by
removal of the highest (0.001 ml) and the lowest (10 ml)
dilutions.
 Example B: If the lowest dilution does not have all positive
tubes, and several of the highest dilutions have all negative
tubes, then remove the highest negative dilutions.
 Example C: If no dilution has all positive tubes, select the lowest
two dilutions, corresponding to 10 and 1 ml sample. For the
third dilution, add the number of positive tubes in the remaining
dilutions (0.1, 0.01, and 0.001 ml sample), to yield a final
combination of 4-3-2If MPN value for combinations not
appearing in the table, or for other combinations of tubes or
dilutions, estimate the result as follows:
 First, select the lowest dilution that does not have all positive
results.
 Second, select the highest dilution with at least one positive
result.
 Select all dilutions between them.
 Use the selected dilutions in the following formula:
MPN / 100 ml (approx.) = 100 × P / (N × T) ½
Where:
P: number of positive results.
N: Volume of sample in all negative portions combined.
T: Total volume of sample in the selected dilutions.
 Examples :
 From (5/5, 10/10, 4/10, 1/10, 0/5) Use only (-, -, 4/10, 1/10. - ),
MPN / 100 ml (approx.) = 100 × 5 / (0.69 ×1.1) ½ =
500/0.87 = 570/ 100 ml
 From (5/5, 10/10, 10/10, 0/10, 0/5) Use only (-, -, 10/10, 0/10. -),
MPN / 100 ml (approx.) = 100 × 10 / (0.1×1.1) ½ =
1000/0.332 = 3000/ 100 ml.
11. Quality Control:

11.1 Analytical Quality Control Batch:
 Apply the presumptive-confirmed phase of the multiple-tube
procedure to all samples.
 Plates with non-selective agar medium, labeled as
“Environmental Control”, will be opened along the samples
analysis run time to ensure adequate environmental conditions.
 Procedural Blank: perform, under the same conditions as
sample, sterile phosphate buffered dilution water after
inoculation of samples series. Absence of growth indicates
absence of cross contamination.
 Procedural Blank: Inoculate, under the same conditions as
sample, one additional tube for each dilution with sterile
phosphate buffered dilution water after inoculation of series of
10 samples. Absence of growth indicates absence of cross
contamination.
11.2 Non Conformance and Corrective Action:

 If the media performance and sterility assessment are
performed concurrently with the use of the media in testing and
the media quality is deemed unsatisfactory, the specific use of
the media in the test(s) should be investigated to determine the
potential impact on the test results and the necessity for
repeating the test(s).
 Testing must be repeated when unsatisfactory media and/or
reagents are used for critical aspects of testing.
 If procedural blank test gives unsatisfactory results, the samples

results should be investigated to determine the potential impact
of contamination on the test result, if test results deemed
unsatisfactory the test must be repeated.
 If dilution water gives positive result, the samples results should
be investigated to determine the potential impact of dilution
water on the test result, if test results deemed unsatisfactory the
test must be repeated.
 If one or more of environmental control plate gives
unsatisfactory results, the samples results should be
investigated to determine the potential impact on the test result,
if test results deemed unsatisfactory the test must be repeated.
 For all non-conformance findings, trace all media, reagents,
equipment operations, and tools used in analysis to find out root
cause of non-conformance.
 Take appropriate corrective action, if cause was temporary e.g.
if unsterile batch of media used accidently in the analysis;
discard all batch of prepared media, or preventive actions if
cause was permanent e.g. re-maintenance of autoclave if actual
temperature was less than adjusted value, to eliminate
reoccurrence of non-conformance in the future.
12. Method Performance Data:

 A limited intra-laboratory study was conducted. Four
participated microbiological analysts tested surface raw water
matrix.
 Results of this intra-laboratory study, table 1, may not be typical
of results for samples other than those studied, hence
secondary validation (cross validation) in form of long-term
study shall be carried out depending on routine samples results.
 For complete validation procedure and calculation refer to
validation report for Detection and Enumeration of Total
Coliform by Membrane Filter Technique.
 Table 1
Criteria Surface Raw
Precision/ repeatability 0.515
Precision/Reproducibility 0.59
Bias % ±0.59
 Detection limit was originally established as 1.8 MPN/100 ml.

 Upper working limit was previously established for this method
as 1600 MPN/100ml.
 Range of reliable estimation was previously established for this
method between 1.8-1600 MPN / 100ml.
13. Reporting:
 Report fecal coliform concentration as the Most Probable
Number (MPN)/100 ml.
 If sample was delayed more than permissible time, write
“Delayed” in report comment.
14. References:
 Eugene W. Rice, Rodger B. Baired, Andrew D. Eaton, Lenore
S. Clesceri eds.2012 Standard Methods for the Examination of
Water and Wastewater. Method 9221E#, 22nd edition (on-line
edition). American Public Health Association, American Water
Works Association, and Water Environment Federation
 World Health Organization. 2011, Guidelines for Drinking-
Water Quality, Fourth Edition. Geneva.
 Guidelines for Canadian Drinking water: Guideline Technical
Document. 2006. Total Coliform. Health Canada. Ottawa.
Ontario.
Appendices:
A.1 MPN index and 95% confidence limits for various
combinations of positive results when five tubes are used per
dilution (10 ml, 1.0 ml, 0.1 ml).

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‫ درجة ثالثة‬Bacteriological Analysis of Water - ‫المسار الوظيفي لوظيفة كيميائي مياه‬

3. Estimation of Total Coliform by Multiple Tube Fermentation

 Total coliform bacteria (excluding E. coli) occur in both sewage

 Indelible ink marker for labeling plates.

7. Chemicals and Reagents:

9.4.2 Media Preparation:

9.4.3 Samples Dilutions:

coliforms at elevated temperature (44.5±0.2°C) can be

9.6.2 Media Preparation:

 Blot dry with absorbent paper or air dry, and examine

Gram Stain technique

 If the sample portion volumes used are those found in the

Volumes ml Combination MPN index

 Example A: First, remove the highest dilution (smallest sample

 Example C: If no dilution has all positive tubes, select the lowest

11. Quality Control:

11.2 Non Conformance and Corrective Action:

 For all non conformance findings, trace all media, reagents,

12. Method Performance Data:

Criteria Surface Raw

Precision/ repeatability 0.461

 Detection limit was originally established as 1.8 MPN/100 ml.

A.1 MPN index and 95% confidence limits for various

4. Estimation of Fecal Coliform by Multiple Tube Fermentation

 Populations of thermo-tolerant coliforms are composed

cells clump, the MPN value will be an underestimate of actual

7. Chemicals and Reagents:

 Use a separate sterile tip for initial and subsequent transfers

 Calibrate pH meter prior to use, using at least two standards

9.4.2 Media Preparation:

 Prepare and sterilize medium according to manufacturer

9.4.3 Samples Dilutions:

 If no gas or acidic reaction is evident, re-incubate and

 Use a sterile loop 3.0 mm in diameter to transfer three loopfuls

 Example A: First, remove the highest dilution (smallest sample

11. Quality Control:

11.2 Non Conformance and Corrective Action:

 If procedural blank test gives unsatisfactory results, the samples

12. Method Performance Data:

Criteria Surface Raw

Precision/ repeatability 0.515

 Detection limit was originally established as 1.8 MPN/100 ml.

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