Micro biological Analysis Standard Operation Procedure

For Bottling Water

1 Aerobic plate count
1.1 Scope
 To describe how to Analysis aerobic plate count of bottling water

1.2 Abbreviation
 QCT- Quality Control Technician
 QC – Quality Control
 APC –Aerobic plate count
1.3 Definitions
 Aerobic plate count is used as an indicator of bacteria population
on a sample.
 Is used to signify an estimation of number of microorganism
[bacteria, fungi, moulds and yeast present per unit quantity of
food items, as enumerated from a sample under specific
condition; temperature of incubation, for fixed duration of time,
grown on defined microbiological media of defined PH.
 It also called as aerobic colony count, standard plate count,
mesophilic count or total plate count.
1.4 Responsibilities
 Quality Control Technician(micro biologist):
 Has overall responsibility for this work instruction.
 Ensure that QCs follow the work instruction. File the records.
 APC - Aerobic plate count
 Quality Control : QC follows the work instruction
Complete relevant forms Report out of
Specification result to the QCT
1.5 Frequencies / Sampling
 Once per a day
1.6 checking point
 Source may be( bore hole, spring, river ,ocean),raw water tanker, sand
filter, active carbon, ion exchanger, ultra filter(UF), ozone mixer, pure
tanker, filling machine, and finished product
  Notes that
 Before starting working on micro biological analysis;
 Clean microbiology laboratory with soap
 Mop the floor of the laboratory wit disfectant
 Mop also the hood with disfictant

 Wear clean gown

 Clean your hands with soap
 Disfect your hands with absolute alcohol
 Close microbiology laboratory

6 Materials / Equipment
 Pre-sterilized Petridis 60mm diameter and 15mm depth.
 Pre sterilized enlimenary flask 250mm or 500mm.
 Pre sterilized weighing pan
 Pre sterilized pipette(1mm ,0.1mm)
 Pre sterilized spatula
 Incubator –equipped to maintain constant temperature.
 Autoclave
 Hot air sterilizing oven that operate 115 degree Celsius
 Analytical balance
 Colony counter
 Measuring cylinder

7 Chemicals
 Plate count agar
 Alcohol

8 Media preparation
 Primarily bring sample with pre sterilized bottles or products from
production, line, raw water tanker, sand filter, active carbon , or a source
 Take pre sterilized weighing pan and weigh total plate count agar
according to the instruction of total plate count agar
 Pour it into conical flask
  Add the need amount of pre sterilized water and shake it well
 Pug the conical flask with pre sterilized cotton and aluminum foil and till
with foil with fiber.
 Heat the solution until it is look warm to mix very well
 Add a distilled water in to auto clave and put the media in to it for
sterilization.
 Close the lid of auto clave , and insure that the lid is properly seated, that
the pressure screw are correctly located in the centering provide for this

 purpose and that the pressure screw are uniformly tightened cross wise,
and pressure 1 bar.
 Adjust the whistling valves by its knurled screw to the required operating
temperature that is 121 degree Celsius.
 Open the pressure control valve by turning it clock wise until the stop
point is reached. The autoclave must be vented during the heating up
stage.
 Insert the plug in to the wall socket,
 Switch on the heating system
 When autoclave is adequately vented, when strong of stem is being
emitted by pressure control valve, close the pressure control valve. Thi is
done approximately at 100 degree Celsius.
 Observe the temperature and pressure rise.
 When the temperature reaches 121degree Celsius and pressure reaches 1
bar, wait for 15 minutes and switched down your autoclave heat.
 Wait the autoclave until it Cool down to 70 degree Celsius, then open the
autoclave.
CAUTION; DON’T OPEN THE LID OF AUTOCLAVE ABOVE 70 DEGREE CELSIUS, IT CAN
CAUSE DANGER
INOCULATION PROCEDURE
 Take out the sterilized total plate count agar and allow cooling to 41-
42degree Celsius
 Frist the pre sterilized petri-dishes marked with the marker based on
their type and dilution ratio.
 Then 1ml of sample added into each test tube from each sample. And add
pre –sterilized water added in to each test tube and shake gently each test
tube and 1ml water sample taken and inculated in to different petri dish
separately. These petre dishes marked as10-1 ratios. Each sample further
diluted to 10-2, 10-3 dilution ratio and 1ml of water taken and inculated
in topre sterilized petri dishes. 20ml of pre sterilized total plate count
 agar added in to each petri dish , all the petri dish swired for 7second
clock wise and 7 second anti clock wise before the agar solidified. Finally
the petri dish incubated at temperature 37degree Celsius.
 Take out the petri dish after 48 to 72 hours of incubation and count all
the colonies.
 Calculate the average number of bacteria;

Number of colony per 1ml of water sample ==

n
¿ ∑ colonies divide [(1*k1)+(1*k2)0.1+(1*k3)0.01+(1 *k4)0.001
k1

Figure 1.1 Colony developed

2 Total coliforms
2.1 Scope
  To describe how to Analysis Total coliform of bottling water

2.2 Abbreviation
 QCT- Quality Control Technician
 QC – Quality Control
 APC –Aerobic plate count

2.3 Definitions
 The term “total coliforms” refers to a large group of Gram-negative, rod-
shaped bacteria that share several characteristics.
 The group includes thermo tolerant coliforms and bacteria offaecal
origin, as well as some bacteria that may be isolated from environmental
sources.
 Thus the presence of total coliforms may or may not indicate faecal
contamination

2.4 Responsibilities
 Quality Control Technician (micro biologist):
- Has overall responsibility for this work instruction.
 Ensure that QCs follow the work instruction.
 File the records.
 Quality Control (micro biologist) : QC follows the work instruction

Complete relevant forms

Report out of specification result to the QCT

2.5 Frequencies / Sampling

 Once per a day And Once Per A Week.
2.6 checking point
 Source may be( bore hole, spring, river ,ocean),raw water tanker, sand
filter, active carbon, ion exchanger, ultra filter(UF), ozone mixer, pure
tanker, filling machine, and finished product

2.7 Materials / Equipment

 Incubator(s) or water-baths capable of maintaining a temperature to
within ± 0.5 ºC of 35and 37 ºC and to within ± 0.25 ºC of 44 and 44.5 ºC.
The choice of temperature depends on the indicator bacteria and the
medium.
 Autoclave for sterilizing glassware and culture media. The size required
 Depends on the volume of work to be undertaken. A capacity of 100-150
Liters would be required for a medium-size laboratory undertaking
work on a routine basis.
 Distillation apparatus, with storage capacity for at least 20 liters of
 Distilled water.
 Laboratory balance, accuracy ± 0.05 g, with weighing scoop. This may be
omitted if culture media and potassium dihydrogen phosphate are
available in pre-weighed packages of the proper size.
 Racks for tubes and bottles of prepared culture media and dilution
water. These must fitting to the autoclave.
 Pipettes, reusable, glass, 10ml capacity graduated in 0.1-ml divisions,
And 1-ml capacity graduated in 0.01-ml divisions.
 Test-tubes, 20 × 150 mm for 10 ml of sample + 10 ml of culture
Medium, with metal slip-on caps.
 Bottles, with loose-fitting caps, calibrated at 50 and 100 ml, for 50
ml of sample + 50 ml of culture medium.
 Measuring cylinders, unbreakable plastic or glass, capacity 100, 250,
500 and 1,000 ml.
 Test-tube racks to hold tubes in incubator and during storage.
 Thermometer for checking calibration of incubator or water-bath.
 Refrigerator for storage of prepared culture media.
 Hot-air sterilizer for sterilizing pipettes.
 Bunsen burner or alcohol lamp.
 Durham tubes, 6 × 30 mm.
 Pipette cans for sterilizing pipettes.
 Flasks for preparation of culture media.
 Wash-bottle.
 Pipette bulbs.
 Wire loops for inoculating media, and spare wire.
 Pre-sterilized Petridis 60mm diameter and 15mm depth
 Spatula
 Container for used pipettes.
 Brushes for cleaning glassware (several sizes).
 Fire extinguisher and first-aid kit.
 Miscellaneous tools.
 Waste bin.
 Pre-sterilized Petridis 60mm diameter and 15mm depth
 3.7 Chemicals
 Endo agar
 Alcohol
3.8 Procedure BY membrane filtration method
 Preparation and inoculation
 Preparation of the sample
 Place a volume of the test sample (or it dilution) not exceeding 2ml in
the Petri dish

 Add 15 ml to 20 ml of the molten medium, allow to cool and maintain

it at (45±1)ºC in the Petri dish
  Time between addition of the test sample (or it dilution) and addition
of the molten medium shall not exceed 15min.
 The inoculated doublets for incubation at each temperature
 Mix carefully by gentle rotation
 Leave the Petri dish on a flat surface aprox. 10 minutes until the gel
completely solidifies
 Invert the plates and incubate one set at (36±2)ºC for
(44±4)h;incubate the other set at (22±2)ºC for (68±4)h.
 Examine the plates as soon as they are removed from the incubators.
 Counting of colonies
o Count all colonies (from the surface and interior of the culture
medium).
o A counter of colonies with a magnifying glass helps with
counting small colonies grown in culture medium.
 Calculate the results in accordance with ISO 8199
 Expression of results
Calculate the numbers of E.coli and coliform bacteria present in 100 ml
of the sample in accordance with EN ISO 8199.
The count of coliform bacteria is the sum all oxidase negative pink to red
colonies plus all dark –blue to violet colonies.
E. coli are all dark-blue to violet colonies
 Calculation of results
o Since each colony is assumed to have arisen from on micro-
organism or from a single aggregate of micro-organism, the
results is expressed as the number of colony-forming units(cfu)or
colony-forming particles(cfp) in a specified reference volume of
the sample by Equation:

o
Where:
 Cs = estimate of the number of cfu or cfp in the reference volume
Vs.
Z = is the sum of colonies counted on plates or on membranes
derived from dilutions d1, d2, …di, or derived from separate
volume of the test portion (sample or dilution)
Vs = is the reference volume
Vtot = is the calculated total volume of original sample included in
the plates enumerated.
Vtot = (n1V1d1) + (n2V2d2) ….+(niVidi), where:
n1, n2, ni = is the number of plates counted for dilution d1, d2,… di
V1, V2, Vi = is the test volume use with dilution d1, d2,… di
d1, d2,di =is the dilution used for the test volume V1,V2,Vi (d=1
for an undilute sample, d= 0,1, for a ten-fold dilution, etc)

Case after identification or confirmation

o After identification or confirmation, calculate for each of the
plates the confirmed results as the proportion of presumptive
colonies complying with the identification or confirmation criteria
 usig Equation

Where:
x = is the estimated number of confirmed colonies per plate;
k = is the number of colonies complying with identification or
confirmation criteria among the inoculated colonies n;
n = is the number of presumptive positive colonies inoculated
from a plate for confirmation;
z= is the total number of presumptive positive colonies counted
on the plate

3 Escherichia coli(E. coli )

3.1 Scope
 To describe how to Analysis E.Coli of bottling water
 3.2 Abbreviation
o QCT- Quality Control Technician
o QC – Quality Control
o APC –Aerobic plate count
3.3 Definitions
 E. coli are found in intestine, their ability to survive for brief periods
outside the body makes them an ideal indicator organism to test
environmental samples for fecal contamination.
 Indicator organisms indicate that water received contamination of
intestinal origin.

 E. coli are Gram negative bacterium that is commonly found in the lower
intestine of warm-blooded animals,
while other coliforms( Enterobacter, Klebsiella) can be found on plants and
in soil.
1.4 Responsibilities
 Quality Control Technician (micro biologist):
- Has overall responsibility for this work instruction.
 Ensure that QCs follow the work instruction.
 File the records.
 Quality Control (micro biologist) : QC follows the work instruction
Complete relevant forms
Report out of specification result to the QCT
3.5 Frequencies / Sampling
 Once per a day/one per a week
3.6 checking point
 Source may be( bore hole, spring, river ,ocean),raw water tanker,
sand filter, active carbon, ion exchanger, ultra filter(UF), ozone
mixer, pure tanker, filling machine, and finished product
2 .7 Materials / Equipment
 Incubator(s) or water-baths capable of maintaining a temperature
to within ± 0.5 ºC of 35and 37 ºC and to within ± 0.25 ºC of 44 and
44.5 ºC. The choice of temperature depends on the indicator
bacteria and the medium.
 Autoclave for sterilizing glassware and culture media. The size
required
  Depends on the volume of work to be undertaken. A capacity of
100-150 Liters would be required for a medium-size laboratory
undertaking work on a routine basis
 Distillation apparatus, with storage capacity for at least 20 liters of
 Distilled water.
 Laboratory balance, accuracy ± 0.05 g, with weighing scoop. This may
be Omitted if culture media and potassium dihydrogen phosphate are
available in pre-weighed packages of the proper size.
 Racks for tubes and bottles of prepared culture media and dilution
water. These must fitting to the autoclave.
 Pipettes, reusable, glass, 10ml capacity graduated in 0.1-ml divisions,

And 1-ml capacity graduated in 0.01-ml divisions.

 Test-tubes, 20 × 150 mm for 10 ml of sample + 10 ml of culture

Medium, with metal slip-on caps.

 Bottles, with loose-fitting caps, calibrated at 50 and 100 ml, for 50

ml of sample + 50 ml of culture medium.

 Measuring cylinders, unbreakable plastic or glass, capacity 100, 250, 500

and 1,000 ml.
 Test-tube racks to hold tubes in incubator and during storage.
 Thermometer for checking calibration of incubator or water-bath.
 Refrigerator for storage of prepared culture media.
 Hot-air sterilizer for sterilizing pipettes.
 Bunsen burner or alcohol lamp.
 Durham tubes, 6 × 30 mm.
 Pipette cans for sterilizing pipettes.
 Flasks for preparation of culture media.
 Wash-bottle.
 Pipette bulbs.
 Wire loops for inoculating media, and spare wire.
 Pre-sterilized Petridis 60mm diameter and 15mm depth
 Spatula
 Container for used pipettes.
 Brushes for cleaning glassware (several sizes).
 Fire extinguisher and first-aid kit.
 Miscellaneous tools.
 Waste bin.
3.7 Chemicals
 Lactose broth
 EC medium
 Alcohol
 MacConkey broth
3.8 procedures for Multiple Tube Fermentation
Technique
Note: Aseptic technique must be used.
1. Prepare the required number of tubes of culture medium. The volume
and strength (single or double) of medium in the tubes will vary depending
on the expected bacteriological density in the water and the dilution series
planned. For most surface waters, 10 ml volumes of single-strength
medium are appropriate.

2. Select and prepare a range of sample dilutions; these will normally be

suggested by experience. To prepare a 1/10 dilution series, mix the
sample bottle well. Pipette 10 ml of sample into a dilution bottle containing
90 ml of phosphate-buffered dilution water. To prepare a 1/100 dilution,
mix the 1/10 dilution bottle well and pipette 10 ml of its contents into a
bottle containing 90 ml of dilution water. Subsequent dilutions are made in
a similar way. Alternatively, 1 ml of sample may be added to a bottle
containing 9 ml of dilution water.

3. Pipette the appropriate volumes of sample and diluted sample into the
tubes of medium,

4. Label the tubes with the sample reference number, the dilution and the
volume of sample (or dilution) added to the tube. Shake gently to mix the
sample with the medium. Place the rack in an incubator or water-bath for
48 hours at 35 ± 0.5 ºC or 37 ± 0.5 ºC.

5. After 18 or 24 hours, note which tubes show growth.

Tubes that show turbidity and gas production, or a color change indicating
the production of acid (if the medium contains a pH indicator), are
regarded as positive. Record the number of positive tubes at each dilution.

Return the tubes to the incubator and re-examine after a total of 48 hours
of incubation. Continue with the next step of the procedure.

6. Prepare the required number of tubes of confirmation culture medium

(BGLB broth for total coliforms and E. coli medium for faecal coliforms).
Using a sterile wire loop, transfer inocula from positive tubes into the
confirmation medium. Sterilize the loop between successive transfers by
heating in a flame until it is red hot. Allow it to cool before use. If
confirmation of both total and faecal coliforms is required, a BGLB and an
E. coli medium tube should be inoculated from each presumptive positive.
Label these tubes carefully with the same code used in the presumptive
test and incubate them for 48 hours at 35 ± 0.5 °C or 37 ± 0.5 °C for total
coliforms (BGLB broth) or for 24 hours at 44 ± 0.5 °C for faecal coliforms
(E. coli medium).

7. After the prescribed incubation time, note which tubes show growth
with the production of gas, and record the number of positives for each
sample dilution.

 Compare the pattern of positive results with a most probable number

MPN Determination from Multiple Tube Test

 Most Probable Numbers (MPN) Index for Various Combinations of

Positive and Negative Results When Three 10-ml Portions, Three 1-ml
Portions, and Three 0.1-ml Portions Are Used
Number of tubes giving positive MPN Index per 95% Confidence Limits
reaction out of 100 ml
3 of 10 ml 3 of 1 ml 3 of 0.1 ml Lower Upper
each each each
 0 0 0 <3 <0.5
0 0 1 3 <0.5 9
0 1 0 3 <0.5 13
1 0 0 4 <0.5 20
1 0 1 7 1 21
1 1 0 7 1 23
1 1 1 11 3 36
1 2 0 11 3 36
2 0 0 9 1 36
2 0 1 14 3 37
2 1 0 15 3 44
2 1 1 20 7 89
2 2 0 21 4 47
2 2 1 28 10 150
3 0 0 23 4 120
3 0 1 39 7 130
3 0 2 64 15 380
3 1 0 43 7 210
3 1 1 75 14 230
3 1 2 120 30 380
3 2 0 93 15 380
3 2 1 150 30 440
3 2 2 210 35 470
3 3 0 240 36 1,300
3 3 1 460 71 2,400
3 3 2 1,100 150 4,800
3 3 3 >2,400

Sources:

 Standard Methods for the Examination of Water and Wastewater, 13th

Edition New York.
 American Public Health Association, 1971.
 Standard Methods for the Examination of Water and Wastewater, 12th
Edition New York.
 American Public Health Association, 1967, p. 608.

Medium USES Incubation Remarks

temperature
Isolation media
 Lactose broth Total or 48 hours at 35 ± 0.5 °C or Prepare single strength
thermotolerant 37± 0.5 °C for total medium
coliforms coliforms and 24 hours at by diluting double strength
44 ± 0.25 °C or 44.5 ± 0.25 medium with distilled
°C for thermotolerant water.
coliforms Each tube or bottle should
contain an inverted
fermentation
(Durham) tube
MacConkey Total or 48 hours at 35 ± 0.5 °C or
broth thermotolerant 37± 0.5 °C for total
coliforms coliforms and 24 hours at
44 ± 0.25 °C
or 44.5 ± 0.25 °C for
thermotolerant coliform
Improved Total or 48 hours at 35 ± 0.5 °C or Available commercially in
formate lactose thermotolerant 37 ± 0.5 °C for total dehydrated form as
glutamate medium coliforms coliforms and 24hours at Minerals Modified
44 ± 0.25 °C or 44.5 ± 0.25 Glutamate Medium
°C for thermotolerant
coliforms
Lauryl tryptose Total or 48 hours at 35 ± 0.5 °C or
(lactose) broth thermotolerant 37± 0.5 °C for total
coliforms coliforms and 24hours at
44 ± 0.25 °C or 44.5 ± 0.25
°C for thermotolerant
coliforms
Confirmatory media
Brilliant green Total or 44.5 ± 0.25 °C for
lactose bile thermotolerant thermotolerant
broth coliforms (gas coliforms
production)
EC medium Thermotolerant 44.5 ± 0.25 °C for Addition of 1 % (m/m) L- or
coliforms (indole thermotolerant DLtryptophan
production) coliforms may improve
performance of the medium
Tryptone water Thermotolerant 44.5 ± 0.25 °C for
coliforms (gas + thermotolerant
indole production coliforms
Lauryl tryptose Thermotolerant 44.5 ± 0.25 °C for
mannitol broth coliforms (gas + thermotolerant
with tryptophan indole production) coliforms

CHART Culture media for most probable number (MPN) analyses

Source: Adapted from ISO, 1990b

4 Analysis of Salmonella in Bottling water

 4 .1 Scope

 To describe how to of Analysis of Salmonella bottling water

4.2 Abbreviation

QCT- Quality Control Technician

QC – Quality Control

4.3 definition
 Salmonella is one of the leading causes of intestinal illness all over the
world as well as the etiological agent of more severe systemic diseases
such as typhoid and paratyphoid fevers. While water is known to be a
common vehicle for the transmission of typhoidal Salmonella serovars,
non-typhoidal salmonellae are mainly known as foodborne pathogens

4.4 Responsibilities

 Quality Control Technician (micro biologist):

- Has overall responsibility for this work instruction.

 Ensure that QCs follow the work instruction.

 File the records.
 Quality Control (micro biologist) : QC follows the work instruction
 Complete relevant forms
 Report out of specification result to the QCT

4.5 Frequencies / Sampling

 Once per a day

2.6 checking point

 Source may be( bore hole, spring, river ,ocean),raw water tanker, sand
filter, active carbon, ion exchanger, ultra filter(UF), ozone mixer, pure
tanker, filling machine, and finished product
Membrane filter technique
 The membrane filter technique can be used to test relatively large
numbers of samples and yields results more rapidly than the multiple
fermentation tube technique.
 It was originally designed for use in the laboratory but portable
equipment is now available that permits use of the technique in the field.
 Principle
 The membrane filter method gives a direct count of total coliforms and
faecal coliforms present in a given sample of water. A measured volume
of water is filtered, under vacuum, through a cellulose acetate
membrane of uniform pore diameter, usually 0.45 μm.
 Bacteria are retained on the surface of the membrane which is placed on
a suitable selective medium in a sterile container and incubated at an
appropriate temperature. If coliforms and/or faecal coliforms are
present in the water sample, characteristic colonies form that can be
counted directly.
 Membrane filtration and colony count techniques assume that each
bacterium, clump of bacteria, or particle with bacteria attached, will give
rise to a single visible colony. Each of these clumps or particles is,
therefore, a colony forming unit (cfu) and the results are expressed as
colony forming units per unit volume. In the case of thermotolerant
coliform bacteria the result should be reported as thermotolerant
coliforms [No.] cfu per 100 ml.
4.6Chemicals
 Brilliant green Agar
 alcohol

4.7 Apparatus and Procedure

4.7.1 Apparatus
 Incubator(s) or water-bath(s) capable of maintaining a temperature
to within ± 0.5 °C of 35 and 37 °C and to within ± 0.25 °C of 44 and
44.5 °C. Choice of temperature depends on the indicator bacteria and
the medium.
 Membrane filtration apparatus complete with vacuum source
(electrically operated pump, hand-pump or aspirator) and suction
flask.
 Autoclave for sterilizing prepared culture media. A pressure-cooker,
heated on a hot-plate or over a Bunsen burner, may be substituted in
some circumstances.
 Boiling-pan or bath (if filtration apparatus is to be disinfected in
boiling water between uses).
 Laboratory balance, accurate to ± 0.05 g, and with weighing scoop.
This may be omitted if media and potassium dihydrogen phosphate
are available in pre-weighed packages of the correct size.
  Racks for bottles of prepared culture media and dilution water.
These must fit into the autoclave or pressure-cooker.
 Distilling apparatus with storage capacity for at least 5 litres of
distilled water.
 Refrigerator for storage of prepared culture media.
 Hot-air sterilizer for sterilizing pipettes and glass or metal Petri
dishes.
 Thermometer for checking calibration of incubator or water-bath.
 Pipette cans for sterilising pipettes.
 Boxes for Petri dishes for use in hot-air sterilizer.
 Reusable bottles for culture media.
 Measuring cylinders, capacity 100 ml and 250 ml.
 Reusable pipettes, glass, capacity 1 ml and 10 ml.
 Bottles to contain 9-ml volumes of buffered dilution water.
 √ Flasks for preparation of culture media.
 Wash-bottle.
 Blunt-edged forceps.
 Pipette bulbs.
 Spatula.
 Container for used pipettes.
 Brushes for cleaning glassware (several sizes).
 Fire extinguisher and first-aid kit.
 Miscellaneous tools.
 Waste bin.

Filtration apparatus should be disinfected between analyses of consecutive

samples and sterilized at intervals. The choice of method will depend on
the type of apparatus, where it is being used, the equipment available and
cost considerations. Sterilizing is generally achieved by autoclaving but any
of the following methods is suitable for disinfection between analyses of
consecutive samples:

- Immersion of components in boiling water for at least 1 minute,

- rinsing in methanol followed by rinsing in distilled water,

- Flaming with methanol, or

- Exposure to formaldehyde gas generated by burning methanol in the

absence of oxygen (Generally preferred as a field technique).
Consumables

 Methanol for disinfecting filtration apparatus using formaldehyde

gas (unnecessary in the laboratory, but essential if analyses are done
in the field). It is essential to use methanol.

Ethanol or methylated spirits cannot be substituted.

 Membrane filters, 0.45 μm pore size and of diameter appropriate for

the filtration apparatus being used and complete with absorbent
pads.
 Disinfectant for cleaning laboratory surfaces and a container for
discarded pipettes.
 Culture media (options are listed in the section on media).
 Phosphate-buffered dilution water.
 Petri dishes, glass or aluminum (reusable) or plastic (disposable).
 Polyethylene bags for wrapping Petri dishes if dry incubator is used.
 Magnifying lens (as an aid to counting colonies after filters are
incubated).
 Wax pencils for labeling Petri dishes.
 Autoclave tape.
 Detergent for cleaning glassware and equipment.

General Steps in the membrane filter technique

a. Add absorbant pad to Petri dish
b. Soak pad in nutrient medium
c. Disinfect tips of blunt-ended forceps and cool
d. Remove membrane filter from sterile packet
e. Place membrane filter in filtration apparatus
f. Add sample to filtration apparatus
g. Apply vacuum to suction flask
h. Remove filter with sterile forceps
i. Place filter in prepared Petri dish
j. Label Petri dish
k. . Leave to resuscitate and then incubate
l. Count colonies after full incubation

4.7.2 Procedure
  Add absorbent pads to sterile Petri dishes for the number of samples to
be processed. Sterile pads may be placed in the Petri dishes with sterile
forceps or with an automatic dispenser
 Soak the pads with nutrient medium. Nutrient medium may be
dispensed with a sterile pipette or by carefully pouring from an ampoule
or bottle, cases, a slight excess of medium should be added (e.g. about
2.5 ml). Immediately before processing a sample, drain off most of the
excess medium, but always ensure that a slight excess remains to
prevent the pad drying during incubation.

Note: Absorbent pads soaked in liquid medium may be replaced by

medium solidified by agar. In this case, Petri dishes should be prepared
in advance and stored in a refrigerator.
 Sterilize the tips of the blunt-ended forceps in a flame and allow them to
cool.
 Carefully remove a sterile membrane filter from its package, holding it
only by its edge
 Place the membrane filter in the filter apparatus, and clamp it in place. If
the apparatus has been disinfected by boiling, ensure that it has cooled
down before inserting the membrane filter.
 Mix the sample by inverting its container several times. Pour or pipette
the desired volume of sample into the filter funnel .This volume should
normally be chosen in the light of previous experience If the volume to
be filtered is less than 10 ml, it should be made up to at least 10 ml with
sterile diluent so that the sample will be distributed evenly across the
filter during filtration.
 Apply a vacuum to the suction flask and draw the sample through the
filter; disconnect vacuum.
 Dismantle the filtration apparatus and remove the membrane filter using
the sterile forceps, taking care to touch only the edge of the filter.
 Remove the lid of a previously prepared Petri dish and place the
membrane, grid side uppermost, onto the pad (or agar). Lower the
membrane, starting at one edge in order to avoid trapping air bubbles.
 Replace the lid of the Petri dish and mark it with the sample number or
other identification. The sample volume should also be recorded. Use a
wax pencil or waterproof pen when writing on Petri dishes.
 If membranes are going to be incubated at 44 or 44.5 °C, the bacteria on
them may first require time to acclimatize to the nutrient medium. After
processing samples from areas of temperate climate, leave each Petri dish
at environmental temperature for 2 hours before placing it in the
 incubator. Samples from areas of tropical climate may be incubated
immediately.
 Maintain the Petri dish in a humid atmosphere (e.g. in a plastic bag or in a
small container with a moist pad in the base) and incubate it either in an
incubator or in a weighed canister in a water bath. This ensures that the
pad does not dry out during the incubation period.
 The incubation periods and temperatures required for each culture
medium are listed in.
 After incubation, count the colonies. Express the results as number of
colonies per 100 ml of sample. Where smaller volumes have been used,
results are calculated from the following formula:
No. of colonies per 100 ml = [(No. of colonies)/(volume
filtered)] × 100
The colonies counted at this stage are presumed to be coliform bacteria
(presumptive Results).

Medium Colony characteristics

Total coliforms at 35 or 37 °C Thermo tolerant coliforms
at 44 or 44.5 °C
Lactose TTCagar Yellow, orange or brick red coloration with Same as total coliforms at
with Tergitol 7 yellow 35 or 37 °C
central halo in the medium under the
membrane
Lactose agar with Yellow central halo in the medium under Same as total coliforms at
Tergitol 7 the membrane 35 or 37 °C
Membrane enriched Yellow color extending on to the Same as total coliforms at
Teepol broth membrane 35 or 37 °C
Membrane lauryl Yellow color extending on to the Same as total coliforms at
sulphate broth membrane 35 or 37 °C
Endo agar or broth Dark red color with golden-green metallic (not applicable
sheen
LES Endo agar Dark red color with golden-green metallic (not applicable
sheen
MFC medium not applicable) (not applicable
Brilliant green Agar Greenish brown to orange brown coloured, (not applicable)
clear to slight opalescent gel.

Chart Colony characteristics following analysis by the membrane filtration method