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MANUAL FOR BACTERIOLOGICAL ANALYSIS

OF NATURAL WATER SUPPLY SOURCES
IN DISASTER SITUATIONS
M e m b r a n e Filter M e t h o d Portable Equipment

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Carmen V a r g a s de M a y o
Biologist-Microbiologist

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MANUAL FOR BACTERIOLOGICAL ANALYSIS OF NATURAL WATER SUPPLY

SOURCES IN DISASTER SITUATIONS

MEMBRANE FILTER METHOD

PORTABLE EQUIPMENT

Carmen Vargas de Mayo

Biologist-Microbiologist

Lima, Peru, January 1984

Pan American Health Organization

Environmental Health Program

Emergency Preparedness and Disaster Relief Coordination Program
 CONTENTS
Pa
ge

1. Introduction 1

2. Definition of Total Coliforra Group 2

3. Membrane Filter Method Using Portable Equipment 2

3.1 Summary of Method 3

3.2 Application 4

3.2.1 Advantages 4

3.2.2 Drawbacks 4

3.3 Equipment, Supplies, and Culture Medium 6

3.3.1 Equipment and Supplies 6

3.3.2 Culture Medium 11

3.4 Sampling 12

3.5 Procedure 13

3.6 Results 17

3.7 Water Monitoring Recommendations 19

3.8 Logging the Data 19

4. References 21

5. Text for Slides 22

1. INTRODUCTION

Among the environmental health measures that must be considered in

the wake of a natural disaster is the bacteriological analysis of water
supplies. Water contamination is one of the principal public health
hazards associated with disasters, sinci contaminated water can set off
an increase in gastroenteritis, diarrhea, and other water-borne
diseases. Such contamination may take place at the source, at the
treatment plant, or in the distribution system.

Appropriate techniques and portable, sensitive, easy-to-operate

equipment are required in order to be able to analyze the
bacteriological properties of water supplies in disaster situations.

The use of membrane filters in the bacteriological examination of

water (bacterial count, coliform determination, isolation of pathogens)
was first considered prior to World War II. Germany and the Soviet
Union were the first countries to apply this technique, particularly
Germany after many of its laboratories were destroyed during the war.

In 1955 the 10th edition of Standard Methods for Examination of

Water, Sewage and Industrial Wastes included the membrane filter
technique as a tentatively approved method for the determination of
coliforms. In the 11th and 12th editions of the same publication, the
method was officially sanctioned for coliform determination. At the
present time the membrane filter test is being used as a standard
method for evaluating the sanitary properties of water and determining
its potability.

This manual is designed to provide a practical guide for

bacteriological analysis using the membrane filter technique. It was
prepared as an activity of PAHO's Emergency Preparedness and Disaster
Relief Coordination Program as part of its component on the training of
environmental sanitation professionals for disaster emergencies.
 - 2 -

The manual and the set of slides intended to illustrate it are

available upon request from:

- Emergency Preparedness and Disaster Relief

Coordination Program
Pan American Health Organization
525 Twenty-third Street, N. W.
Washington, D.C. 20037, United States of America
or from;
- Eng. Robert Swart
Pan Caribbean Disaster Preparedness and
Prevention Project (PCDPPP)
P. 0. Box 1207
St. John's, Antigua (W.l.)

Portable equipment using a membrane filter is available from at

least two firms. A field kit manufactured by Millipore is used as a
practical model in this manual. This, however, does not imply that
PAH0/WH0 approves that equipment or recommends it over others of a
similar kind.

2. DEFINITION OF TOTAL COLIFORM GROUP

When the membrane filter (MF) technique is used in coliform

determination, the coliforms may be defined, according to the Standard
Methods, as nonsporulated gram-negative bacilli, which produce a pink
to dark red colony with a golden metallic or yellowish green shine in
18-24 hours when grown in the MF-Endo medium at 37 C+.

3. MEMBRANE FILTER METHOD USING PORTABLE EQUIPMENT

This technique has several strong advantages: it can be used

anywhere, it makes it possible to examine a wide variety of water
volumes, and it provides a direct reading of the total concentration of
coliform bacteria instead of a statistical estimate as is common with
the multiple tube technique.
 - 3 -

Certain types of samples cannot be filtered because the water is

turbid or because of the presence of unusually high populations of
nonconform bacteria or heavy metallic compounds. These difficulties
may be encountered in the examination of samples of water from certain
wells, reservoirs, small ponds, industrial affluents, and low-quality
chlorinated affluents. In turbid samples with a low concentration of
coliform bacteria, the use of the multiple tube procedure is
recommended.

3.1 Summary of the Method

The filtering procedure consists of using vacuum pressure to pass

the water through a 0.45 micron-thick cellulose membrane. The volume
that can be filtered also depends on the presence of turbidity.
Samples of contaminated water must be diluted before filtering.

In diluting a sample it should be taken into account that in order

to determine the total coliform count, the ideal number of colonies on
the membrane filter should be from 20-80. With the help of a two-way
vacuum valve, the sample is passed through the membrane filter, which
is properly placed on the filter stand.

The filter is then placed in a Petri dish containing a medium with

agar or a pad impregnated with MF-Endo culture medium.

The inoculated Petri dish is placed in the incubator, reticulated

side down, at a temperature of 37 C for 18-24 hours. No more than 30
minutes should elapse between filtration and incubation.

All pink to dark red colonies with a golden or yellowish green

metallic sheen are total coliforms.
 - 4 -

3.2 Application

Most water samples can be analyzed by the membrane filter method

using portable equipment.

3.2.1 Advantages

- A quicker reading is obtained, particularly for coliform group

bacteria.

- Larger volume of samples can be examined.

- The readings are more precise than those to be expected with

the multiple tube technique.

- The required equipment and supplies take up less space than

those for the multiple tube technique.

- The method is ideal for the performance of bacteriological

water quality analysis in rural areas lacking electric power or
suitably equipped laboratories.

3.2.2 Drawbacks

- Samples with a high number of nonconform bacteria and a low

number of coliform bacteria cannot be examined by this method
because of possible suppression of the coliform group bacteria.

- In samples with a low coliform count and a realtively large

amount of suspended solids, the bacterial growth may at times
produce a continuous film on the surface of the membrane,
precluding the possibility of a count.

- Some samples with copper or zinc quantities greater than 1 ag/£

yield irregular results for coliforms.
 - 5 -

The quality of membrane filters may involve such problems as:

Presence of toxic wastes from the manufacturing process

. Irregular porosity
. Hydrophobic areas
The use of ink in drawing the grid lines that is too thick,
impeding the growth of the colonies
Grid lines drawn in toxid ink
. Presence of Glycerol.

Problems with respect to absorbent pads;

. Presence of sulfite wastes or other substances will inhibit

bacterial growth.

Problems with respect to sterilization of membrane filters;

When a filter is sterilized with ethylene oxide, a residue

of this gas, which is toxic to bacteria, may be left on the
filter.

When a filter is sterilized in an autoclave, uneven porosity

areas may result unless the time and temperature have been
properly controlled.

In regard to culture medium:

. These may at times contain nutrients and stains of

inadequate quality.
 - 6 -
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 - 7 -

INCUBATOR

Adaptor
for battery
operation

PORTABLE INCUBATOR (Figure 2)

Stainless Steel Stand and Filter Case

The stand and the filter case should be sterilized in an

autoclave at 12l°C for 15 minutes or boiled. (See Figure 3)
 STAND, FILTER CASE, AND SYRINGE (Figure 3)

Membrane Filters

The filter's bacterial retention capacity should be

certified by the manufacturer, and its filtration speed
should be satisfactory. (The pore diameters, for
bacteriological use, are 0.22 mi crones for sterilizing
liquids and 0.45 microns for retaining bacteria.)

If the filters have not been previously sterilized, they

should be placed in an autoclave at 121 C for 10 minutes.
 - 9 -

Absorbent Pads for Culture Medium

The pads should be made of filter paper and should be

free of inhibiting substancss that interfere with the growth
of colonies. They should be uniform in thickness to allow
the absorption of 1.8-2.2 mt of the culture medium. They
should be sterilized prior to use in an autoclave at 121 C
for 10 minutes.

As an alternative, these pads may be replaced by culture

broth to which 1.5% agar has been added. This preparation
should be placed in the Petri dishes with care to prevent
bubbles from forming; the surface should remain smooth and
damp.

Forceps

Forceps with round ends and a smooth straight point

should preferably be used. Forceps with rough surfaces may
damage the membranes. When not in use they should be kept
in alcohol, and they should be flamed before use in an
analysis.

Petri dishes

Petri dishes 50-60 mm in diameter and 12 mm high are used

in the membrane filter technique. Plastic dishes that can
be sealed hermetically are preferred.
 - 10 -

PETRI DISHES (Figure 4)

Precipitation Cups

Cups containing 95% ethyl alcohol for sterilizing the

forceps.

Sampling Containers

Made of stainless steel.

Sampling Bottles

100 m£ capacity, sterile, made of neutral glass or

autoclavable plastic, nontoxic.

Alcohol Burner
 - 11 -

Pipettes

Sterile glass or plastic.

Supplies for Preparing Culture Medium

Glass or stainless steel container.

3.3.2 Culture Medium

MF-Endo Broth

Formula

Polypeptone 10.00 g
Triopectone 5.00 g
Trypticase 5.00 g
Yeast e x t r a c t 1.50 g
La ctose 12.50 g
Sodium c h l o r i d e 5.00 g
Potassium dihydrogen phoi sphate 1.375 g
Dipotassium hydrogen phosphate A.375 g
Sodium l a u r y l sulphalt e 0.05 g
Sodium deoxycholate 0.10 g
Sodium s u l f i t e 2.10 g
Gasic fuchsin 1.05 g
D i s t i l l e d water 1 I
Ethyl a l c o h o l , 95% 20 m£

Preparation

Dissolve 4.8 graais of the medium in 100 m£ of distilled

water containing 2ra£of 95% ethyl alcohol. Mix and heat
the medium to the boiling point, withdrawing it frequently
from the flame. Cool to 45°C. Do not sterilize.

Use 1.8-2.0 ml of medium for each absorbent pad. It is

recommended that the medium be prepared on the same day it
I-s to be analyzed. It should be kept refrigerated; even so,
It can be stored for no more than a maximum of 4 days.
 - 12 -

CULTURE MEDIUM ( F i g u r e 5)

3.4 Sampling

Sterile 125 n£ bottles should be used for taking bacteriological

samples. Samples for bacteriological water analysis in disaster
situations should be collected at the source (well or spring), at the
municipal supply facilities and private systems, and at water mains and
storage tanks. (For further information on the taking of samples, see
Vigilancia de la Calidad del Agua Potable, World Health Organization.
Geneva, 1977.)

In lakes, reservoirs, and rivers, the abundance of bacteria varies

and depends on the depth and hour at which the sample is taken.
 - 13 -

To collect the sample, dip the bottle some 15-20 cm below the
surface of the water. This should be done quickly to avoid collecting
any floating material. The mouth of the bottle should be pointed
against the flow of the current to prevent hand contact with the water
sample.

To collect samples from a well, plump water from the well for 5-10
minutes and take a sample in a sterile bottle.

To collect a sample from a faucet, select one with no leaks and

let the water run 2-5 minutes.

Since the bacterial population is strongly influenced by

temperature, it is Lraportant to keep the samples refrigerated at 4 C
even after they reach the laboratory, or to begin the analysis
immediately with the help of portable equipment.

3.5 Procedure

The filtering equipment should be sterilized before beginning each

series of filtrations. If it Is necessary to sterilize the equipment
several times during the day, it is advisable to expose the funnel and
the stand to ultraviolet light for 2 minutes. The equipment may also
he sterilized in boiling water for 5 minutes.

An alcohol burner is attached to the stand. This is used to

sterilize the filtering cup.

Table 1: RANGE OF SAMPLE VOLUMES FOR TOTAL C0HF0RM

USING THE MEMBRANE FILTER METHOD

Volume Required (m£)

Type of Sample 100 50 10 1 0.1

Lakes, r e s e r v o i r s X X X
Springs, wells X x x

Surface s o u r c e s for water

treatment plants X X X
 - 14 -

Place the filter case aseptlcally on the stand and set the
filter in place with the help of sterile forceps.
(See Figure 6)

FILTER ON STAND (Figure 6)

Carefully set the upper part or funnel on the base of the

filter case and secure it firmly by turning the blue neck
clockwise.

Connect the syringe to the filtering equipment with a plastic

tube.

To safeguard against contamination, some 50 m£ of sterile water

should be filtered through the equipment prior to beginning
filtering the samples.

Pour the samples into the funnel or filtration receptacle,

pumping the syringe to start the filtration. (See Figure 7)
 - 15 -

PUMPING THE SAMPLE THROUGH THE FILTER ( F i g u r e 7)

- F i l t e r the sample through t h e 0.45 micron-thick, c e l l u l o s e

membrane.

- After the samples have been f i l t e r e d , wash the funnel 3 times

with 20-30 m£ of s t e r i l e w a t e r . T)etach the f u n n e l .

- Add 1.8 t o 2.0 m£ of c u l t u r e medium to the absorbent pad using

a sterile pipette. (See F i g u r e 8)
 - 16 -

ADDING THE CULTURE MEDIUM ( F i g u r e 8)

With s t e r i l e f o r c e p s remove t h e membrane from t h e filtering

u n i t and p l a c e i t very c a r e f u l l y on the P e t r i d i s h with MF-Endo
agar o r pad with MF-Endo b r o t h . (See Figure 9)

FILTER ON PAD ( F i g u r e 9)
 - 17 -

- It is important to keep air bubbles from forming in the

membrane. Any bubbles that are formed can be eliminated by
pressing down gently along the edges of the membrane with
sterile forceps.

Incubate the samples with the grid face down for 18-24 hours at
37°C.

- For analysis in rural areas, the equipment should be connected

first to be battery of the vehicle so that the incubator can be
kept at a constant temperature. This equipment operates on 6
and 12 volts.

- No more than 30 minutes should elapse between filtration and

the start of incubation.

- All pink or red colonies with a metallic sheen are total

coitforms.

3.6 Results

- S e l e c t t h o s e membranes t h a t have 20-80 pink o r red colonies

with a m e t a l l i c sheen.

- Count the c o l o n i e s using a s t e r e o s c o p i c microscope with 10-15x

m a g n i f i c a t i o n and f l u o r e s c e n t light.

- The following formula i s used for c a l c u l a t i n g the total

coliform:

No
No. of t o t a l coliform counted/100 ml - " o f c o 1 ^ " " " c o l o n i e s counted x 1 Q 0 m£
Volume of o r i g i n a l sample f i l t e r e d
 - 18 -

DIAGRAM FOR THE DETECTION OF TOTAL COLIFORM

MEMBRANE FILTER TECHNIQUE (kigure 10)

Water sample

Filter volumes with a growth

of 20-80 colonies

MF-Endo b r o t h
18-24 hours a t 37°C+ 0.5°

• V
Pink or red colonies with Other colonies
metallic sheen — Total coliform
Total coliform present absent
 - 19 -

7 Water Monitoring Recommendations

The monitoring of bacteriological water quality should begin

immediately after a disaster occurs. The following are among the
measures to be considered for maintaining the sanitary quality of
the water:

- Daily monitoring of residual chlorine in the water in the

public supply system.

- Increasing the pressure of the water to counter contamination.

Protecting the supply system by cleaning and disinfecting water

mains, storage tanks and wells.

8 Logging the Data

The following items should be considered in logging the data;

- Sample number

- Date and hour it was taken

- Date and hour it was analyzed
- Sampling location
- Volume filtered
- Number of total coliform colonies counted.

The "volume filtered" data make it possible to calculate the

number of total coliform colonies counted per 100 aZ. Table 2 can
be used to register a number of samples.
 - 20 -

Table 2: DETERMINATION OF TOTAL-COLIFORM/MEMBRANE FILTER METHOD

PAGE N o . OF

PROJECT ANALYST

No. of total coliform counted/100 m£ = No. of coliform colonies counted Y 10o ml

Volume of original sample filtered

Sample Date and Date and Sampling Volume Total- Number of

Number Hour Hour Location Filtered Coliform Total-Coliform/
Taken Analyzed Counted 100 ml
 - 21 -

i5FERENCES

Assar, W. F. Guide to sanitation in natural disasters. World Health

Organization, Geneva, 1971.

American Public Health Association et.al. Methods for the examination

of water and wastewater. 15 ed. APHA, New York, 1981.

CETESB. Anailsis microbiologicos de aguas. Normalizacao Tecnica

Saneamiento Anbiental, NT-08. Sao Paulo, 1978.

Environmental Protection Agency. Microbiological methods for

monitoring the environment: water and wastewater. EPA. Cincinnati,
1978.

Geldrich, Edwin E. Handbook for evaluating water bacteriological

laboratories. Municipal Environmental Research Laboratory,
EPA-670/9-75-606. Cincinnati, 1975.

Pan American Health Organization. Scientific Publication No. 430,

Environmental health management after natural disasters. Washington,
D.C., 1982.

World Health Organization. Monograph Series No. 63. Vigilancia de la

calidad del agua potable. Geneva, 197 7.

Pan American Health Organization/WorLd Health Organization. Manual del

curso sobre abastecimiento de agua potable en situaciones de desastre.
CEPIS, Lima, 1982.

Vargas de Mayo, Carmen A. Metodos simplificados de analisis

microbiologicos de aguas reslduales. Technical Document No. 12.
CEPIS/PAHO/WHO, 1983.
 - 22 -

TEXT FOR SLIDES

PROCEDURE FOR DETERMINATION OF TOTAL COLIFORM

Membrane Filter Method Using Portable Equipment

The environmental health measures to be considered in the wake of a

natural disaster include, among others, bacteriological analysis of the water
supplies.

The classical method to investigate coliform bacteria is with the use

of multiple fermentation tubes. A newer procedure based on the use of
membrane filters is being employed as a standard method for evaluating the
sanitary quality of water and determining its potability.

In disaster situations and In the absence of adequate laboratory

facilities, portable membrane filter equipment is used in performing the
bacteriological analysis of water.

00 PAHO Logo

01 Title frame

02 Portable membrane filter kit

The portable membrane filter kit for bacteriological analysis of water

consists of an incubator with a capacity for 27 samples that can be
operated on either current or battery, a stainless steel stand and
filter case, a syringe for producing a vacuum, and a stainless steel
sampling container.

03 Other basic components of the kit

In addition to this equipment, other basic components are Petri dishes,

pipettes, culture medium, membrane filters, forceps, and absorbent pads.
 - 23 -

04 Necessary items to prepare culture medium

A scale, sterile distilled water, 95% ethyl alcohol, a test tube, a

beaker, and sterile spatulas are needed for preparing the culture
medium. The culture medium for total colifonn is MF-Endo Broth.

05 Weighing amount of culture medium

Weigh the necessary amount of the medium (dehydrated MF-Endo broth).

06 Adding sterile distilled water to the medium

Add sterile distilled water containing 95% ethyl alcohol. It is

advisable to prepare only the amount of medium that is to be used,
si ice it can only be stored for a maximum of 96 hours.

07 Dissolving the medium

Dissolve the culture medium by heating it gradually, bringing it close

to the fire, and then withdrawing it, until the boiling point is
reached.

08 Assembling the filtering unit

Begin to assemble the filtering unit by inserting the forceps in the

slot.

09 Feeding alcohol into the burner

Using the lid of the bottle of ethyl alcohol, feed alcohol into the
burner attached to the base of the filtering unit.

10 Sterilizing the filtering unit

Use the burner to sterilize the filtering unit before performing the
analysis. Both the filter case and the sampling container have to be
properly sterilized. Sterilization time is 15 minutes.
 - 24 -

11 Placing the filtering unit ewer the sampling containter

After the system is sterilized, continue assembling the filtering

unit. Set the base of the unit over the sampling container.

12 Setting the filter on filter case

Place the membrane filter on the filter case using sterile forceps.

13 Securing filter case

Then carefully set the upper part or funnel on the base of the filter
case, secure firmly, and turn the blue neck clockwise.

14 Placing absorbent pad in the Petri dish

Place the absorbent pad in the Petri dish with the pad distributor.

15 Adding the medium to pad

Add 1 . 8 - 2 . 0 at of the c u l t u r e medium t o the pad.

16 Pouring distilled water through the filter prior to filtering sample

As a safeguard against contamination, about 50 ra£ of sterile water

should be pumped through the filter before starting the analysis.

17 Filtering sample

Homogenize the sample and then filter it.

18 Pumping sample with syringe

Begin pumping the sample with the syringe through the 0.45 micron-thick
cellulose membrane.
 - 25 -

19 Placing membrane in Petri dish

Detach the funnel and with sterile forceps remove the membrane from the
filtering unit and place it very carefully on the pad with M-Endo
medium.

20 Placing sample in the incubator

Incubate the sample with the grid face down for 18-24 hours at 37°C.

21 View of equipment in the laboratory

Here, the equipment is installed in the laboratory. The samples are in

the incubator that forms part of the portable kit.

22 Pluging incubator into extension cord

When analyses are to be performed in a rural area, begin by connecting

the equipment to the battery of the mobile unit.

23 Connecting clamps to battery

For doing this, the system includes a set of clamps that can be
connected to the poles of the battery.

24 Equipment inside the movile unit

Having connected the equipment to the mobile unit, continue with the
incubation of the samples.

25 Incubation in process
 - 26 -

26 Typical coliform colonies

After 18-24 hours observe the typical total coliform colonies. These
are pink or red with a metallic sheen. To calculate the results,
select those membranes that have 20-80 pink colonies with a metallic
sheen.

27 Formula to calculate total coliform count

The findings may be expressed as follows:

No. of total coliform counted/100 m£ = No. of coliform colonies counted Y 100 ^

Volume of original samples filtered

28 Credits

*«