The Presumptive Test In the presumptive test, a series of lactose broth tubes are

inoculated with measured amounts of the water sample to be tested. The series of
tubes may consist of three or four groups of three, five or more tubes. The more
tubes utilized, the more sensitive the test. Gas production in any one of the tubes is
presumptive evidence of the presence of coliforms. The most probable number
(MPN) of coliforms in 100 ml of the water sample can be estimated by the number
of positive tubes (see MPN Table).
PREPARATION OF STERILE DILUTION WATER
The dilution water used for making sample serial dilutions is prepared as follows:
1.

Prepare stock buffer solution:

Dissolve 34 g of potassium dihydrogen phosphate (KH2PO4) in 500 mL of distilled water. Adjust the
pH of the stock solution to 7.2 with 1 N sodium hydroxide (NaOH). Dilute to 1 L with distilled water.

2.

Prepare magnesium chloride solution:

Dissolve 38 g of magnesium chloride (MgCl2) in 1 L of distilled water.

3.

Prepare buffered dilution water by adding 1.25 mL of stock buffer solution and 5.0 mL of magnesium
chloride solution to each liter of distilled water. Dispense buffered dilution water into the dilution
bottles in large enough volumes to obtain 9 or 99 mL after the sterilization.

4.

Sterilize as noted above.

SERIAL DILUTION PROCEDURE

At times the density of the organisms in a sample makes it difficult to accurately determine the actual
number of organisms present. When this occurs, the sample size may need to be reduced to as little as
one millionth of a milliliter. In order to obtain such small volumes, a technique known as serial dilution has
been developed. In a serial dilution, successive volumes of diluted sample are further diluted until the
desired dilution range is obtained.
The following steps describe a serial dilution procedure:
1.

Prepare dilution bottles by placing sufficient volume of dilution water in each bottle to have 99 mL
after autoclaving.

2.

Pipette 11 mL of sample into dilution bottle #1 and gently swirl to mix.

3.

Pipette 11 mL from bottle #1 into bottle #2 and gently swirl to mix.

4.

Pipette 11 mL from bottle #2 into bottle #3 and swirl to mix.

5.

Pipette 11 mL from bottle #3 into bottle #4 and swirl to mix.

At this point, the sample has been diluted to such a degree that as little as 0.0001 mL of original
sample can be measured accurately by pipetting 1 mL of the dilution in bottle #4. Additional
reductions in sample size can be accomplished by further dilutions.
EQUIPMENT AND REAGENTS

EQUIPMENT
The following equipment and glassware will be needed to perform the MPN procedure. (Descriptions and
specifications for those items marked with an asterisk are given in previous sections.)
*Autoclave
*Dry heat sterilizer
*Incubator
*Water bath or heat sink incubator, 44.5C
*Triple beam balance, 0.1 g accuracy
*Fermentation tubes and shell vials
*Dilution bottles
*Serological pipettes, graduated at 1.0 and 1.1 mL
*Serological pipettes, graduated at 10.0 and 11.0 mL
*Transfer loops
*Corrosion resistant test tube racks
*Bunsen burner or alcohol lamp
PREPARATION OF STERILE MEDIA BROTHS
EC Broth
The EC broth can be prepared by dissolving 37.0 g of dehydrated EC media in 1 liter of distilled water.
PREPARATION OF FERMENTATION TUBES
After the broths are prepared, the fermentation tubes should be prepared by dispensing 10 mL of broth
into each fermentation tube. This volume should be sufficient to partially cover the inverted, inner test
tube after sterilization. Sterilization procedures for culture media are discussed above. After sterilization,
refrigerate the prepared fermentation tubes at 10C or less until they are needed. Incubate fermentation

tubes prepared and stored in this manner at 35 +/-0.5C for 24 hours prior to use. Discard any tubes in
which the inverted, inner test tube is not completely filled.

PRESUMPTIVE TEST
The first step of the MPN procedure for fecal coliform testing is called the presumptive test. In this test,
samples or serial sample dilutions are inoculated into a series of fermentation tubes. The fermentation
tubes are then incubated at 35 +/-0.5C. The tubes are observed at the end of 24 and 48 hours for gas
production. Any tube showing gas production during this test indicates the possible presence of coliform
group bacteria and is recorded as a positive presumptive tube. All positive presumptive tubes are
transferred to EC broth fermentation tubes to confirm the presence of fecal coliform bacteria.
Test Procedure
1.

Prepare a series of decimal dilutions of the sample to be tested for fecal coliform using the
procedure outlined in Section 13.

2.

For each dilution, inoculate 5 fermentation tubes containing lauryl sulfonate tryptose (LST) broth
(10 mL/tube). Mark each tube for identification using a non-water soluble marker or grease pencil.

NOTE 1: In steps 1 and 2, care should be taken to ensure that the sample or sample dilutions are well
mixed before the inoculum is withdrawn from the sample or dilution bottle.
NOTE 2: The volume of sample and the number and degree of serial dilution will vary with the nature of
the water being tested. In no case should less than 3 dilutions of 5 tubes each be used.
3.

Incubate the inoculated tubes at 35 +/-0.5C for 24 (+/-2) hours.

4.

Check each tube for the presence of gas in the inner shell vials. If gas production is not readily
apparent, shake the tubes gently and check for rising gas bubbles.

NOTE: DO NOT confuse gas production with possible air bubbles. Gas production should be
accompanied by a change in the appearance of the broth; it may become cloudy.
5.

Record positive presumptive results (gas produced) on the MPN test data sheet.

6.

Return all of the negative tubes to the incubator at 35C (+/-0.5C).

7.

Repeat steps 4 and 5 at the end of the remaining 24 (+/-2) hours.

8.

Discard any negative tubes left after step 7, using appropriate safety precautions.
All positive presumptive tubes should be carried into the fecal coliform confirming test procedure.
Transfers should be made as soon as the gas production is noted in a fermentation tube.

NOTE: DO NOT hold 24 hour positive presumptive tubes until the 48 hour total incubation time is
completed.
FECAL COLIFORM CONFIRMING TEST

In the confirming test procedure for fecal coliform bacteria, the positive presumptive cultures are
transferred to EC broth, which is specific for fecal coliform bacteria. Any presumptive tube transfer which

shows gas production after 24 (+/-2) hours incubation at 44.5C (+/-0.2C) confirms the presence of fecal
coliform bacteria in that tube and is recorded as a positive confirmed tube.
Test Procedure
1.

Pair each positive presumptive fermentation tube with a fermentation tube containing EC broth.
Mark each EC tube to match its paired presumptive tube.

2.

Using a sterile transfer loop, transfer a portion of the liquid from each presumptive tube to its paired
EC broth fermentation tube.

NOTE: Flame sterilize metal loops before each transfer or use individual pre-sterilized loops or wood
splints for each transfer.
3.

Discard the positive presumptive tubes after transferring using appropriate safety precautions.

4.

Place all of the inoculated EC broth tubes in a water bath incubator maintained at 44.5 +/-0.2C.

NOTE: The tubes should be placed in the water bath within 30 minutes of inoculation.
5.

Incubate the EC broth tubes for 24 (+/-2) hours.

6.

Remove the tubes from the water bath, shake gently and inspect for gas production.

7.

Record all fermentation tubes showing gas production as positive on the test data sheet.

8.

Calculate the test results and record as Most Probable Number (MPN)/100 mL.

9.

Discard the fermentation tube contents using appropriate safety precautions.

CALCULATION OF MOST PROBABLE NUMBER (MPN)/100 mL

The calculation of the MPN test results requires the selection of a valid series of 3 consecutive dilutions.
The number of positive tubes in each of the three selected dilution inoculations is used to determine the
MPN/100 mL. In selecting the dilutions to be used in the calculation, each dilution is expressed as a ratio
of positive tubes per tubes inoculated in the dilution, i.e. 3 positive/5 inoculated (3/5). There are several
rules to follow in determining the most valid series of dilutions. In the following examples, four dilutions
were used for the test.
1.

Select the highest dilution showing all positive results (no lower dilution showing less than all
positive) and the next two higher dilutions.

November 18, 2015

This is the first day of our On The Job Training (OJT)in DENR-emb 12 we are
assigned on our respective areas such as BOD, ph & color, Oil & grease and
Microbiology. I am assigned in microbiology my superior here is maam Merly Dagum
she teach me how to prepare the Laurels Sulfate, Dillution water, sterilization of
glasswares and the flow of works in water analysis. Because of the sample present in
my assigned area. Maam Merly teach me that in each of the sample there should be a
three (3) bottles of dilution water. Then she also teach me that in each sample there is
fifteen (15) Testube that contains the 20ml Laurel Sulfate after maam Merly teaching
me I do it independently, I distribute the 100ml dilution water in a bottle. I used the
pippette to get 10ml of sample then I placed in the first bottle of dilution water after that I
get another 10ml from the first bottle of dilution to the second and from the second to
the third bottle. In the first bottle I get 10ml then I placed it in the testube that contains
the 20ml Laurel Sulfate. But before placing the10ml in the testube I need to heat the
mouth of the testube in the alcohol lamp to avoid contamination. Then I placed the
laurel sulphate with 10ml sample in the incubator.

November 19
This day maam Merly teach me how to record the result after 24 hours of
incubation.

Then I prepared a Brilliant Green Lactose Broth and EC broth for the

confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose

Broth in the 37o Celsius and the EC broth is 45 o Celsius. Then I assist Mona in BOD
room.

November 20, 2015

This day I record all of the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 24 hours of incubation. Then I placed it
again the respective incubator.
November 23, 2015
This day we have our flag raising at the main office then we go back to the
laboratory. Then I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.
November 24, 2015
Because there is no sample present I prepared, the lauryl sulfonate tryptose broth firstly I

dissolved 35.6 grams of dehydrated media in 1 liter of distilled water. If the volume of sample
being tested is greater than 1 mL per fermentation tube, the strength of the broth must be
increased to maintain the correct proportions.

November 25, 2015

This day there is only two sample then I get the testube that contain the Laurel
Sulphate then I use the pipette to get the exact 10ml of sample for each of the first,
second and third dilution water. Then I get another 10ml from the dilution to the laurel
sulphate of testube. After all of the procedures placed I in to the incubator.
November 26, 2015
This day I record the result after 24 hours of incubation and confirm the two
sample Then I prepared a Brilliant Green Lactose Broth and EC broth for the
confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose
Broth in the 37o Celsius and the EC broth is 45o Celsius.
November 27, 2015
This day I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.
November 30, 2015
I only help Mona in BOD by distributing the sample in the BOD Bottles and I
record the sources of the sample present in the BOD logbook.
December 1, 2015

There is no available sample present in my assigned room. That is why I help

Mona in BOD to distribute the sample in the BOD bottles and I operated the Aqua meter
to measure the ph, conductivity, salinity, turbidity and temperature.
December 2, 2015
I prepared the BGLB and EC broth firstly I weigh the 40g of powder of BGLB and
37g for EC broth then I put a 20ml testtube and pour 10ml of BGLB the same as EC
broth in another small testube. Then I put it in the autoclave for 15mins.
December 3, 2015
This day there is the sample from the Beaches then I get the testube that
contain the Laurel Sulphate then I use the pipette to get the exact 10ml of sample for
each of the first, second and third dilution water. Then I get another 10ml from the
dilution to the laurel sulphate of testube. After all of the procedures placed I in to the
incubator.
December 3, 2015
This day I record the result after 24 hours of incubation and confirm the two
sample Then I prepared a Brilliant Green Lactose Broth and EC broth for the
confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose
Broth in the 37o Celsius and the EC broth is 45o Celsius.
December 4, 2015

This day I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.
December 7, 2015
I am assigned in the main office I make paper works such as the updating the
industries for the year of 2013 until 2015 then I had been fax the letter from different
Beaches owner for their meeting.
December 8, 2015
I dont have a paper works because my superiors here are busy doing their
monthly report.
December 9, 2015
Same as yesterday I dont have a paper works because my superiors here are
busy doing their monthly report.
December 10, 2015
Same as the following day I dont have a paper works because my superiors
here are busy doing their monthly report.
December 11, 2015

Almost a week I dont have a paper works because my superiors here are busy
doing their monthly report.
December 14, 2015
We are practicing our dance step in the laboratory for the Christmas party
because the receiving of samples are cut off.
December 15, 2015
Practicing our dance step in the laboratory for the Christmas party because the
receiving of samples are cut off.
December 16, 2015
Practicing our dance step in the laboratory for the Christmas party because the
receiving of samples are cut off.
December 17, 2015
Practicing our dance step in the laboratory for the Christmas party because the
receiving of samples are cut off.
December 18, 2015
This the day of our Christmas party at the Ramona Hotel. We perform our
prepared danced
December 21
I am assigned again in the main office but then the superior are busy.

December 22, 2015

I am assigned again in the main office but then the superior are busy.
January 4, 2015
I am now assigned in the laboratory, I clean the laboratory and inventorying
the chemicals present in the BOD room.
January 5, 2015
This day I assigned in the oil and grease./
January 6, 2015
Same as yesterday
January 7, 2015
Oil and grease
January 8, 2015
Oil and grease
January 12, 2015
This day I am assigned in the check meter such as to record the ph, color,
turbidity salinity, conductivity temperature and dissolve oxygen. If the samples are water
bodies there should be a ph, color, turbidity salinity, conductivity temperature and
dissolve oxygen. If the sample are from the industries there is only ph, color, DO and
turbidity that should be recorded in the log book.

January 14, 2015

I record the result in the check meter because there is the sample present
that came from the industries I only record ph, color, DO and turbidity.
January 15, 2015
The same sample that came from the industries, I only record ph, color, DO
and turbidity in the logbook.
January 18, 2015
The same sample that came from the industries, I only record ph, color, DO
and turbidity in the logbook.
January 19, 2015
The same sample that came from the industries, I only record ph, color, DO
and turbidity in the logbook.
January 20, 2015
The same sample that came from the industries, I only record ph, color, DO
and turbidity in the logbook.
January 21, 2015
The same sample that came from the industries, I only record ph, color, DO
and turbidity in the logbook. Then I help Mona in the BOD for distributing the sample in
the BOD bottles.

January 22, 2015

This they I record the sample that we received in the BOD room in the
logbook then I put the code for each sample.
January 25, 2016
This day we have our flag raising in the main office then we go back into the
laboratory, in the laboratory we prepared our things because tomorrow is our sampling
in Beaches suh as the water checker, galloons, packing tape, pentle pen and Camera
for documentations
January 26, 2016
This the day of our sampling in the Beaches in Saranggani and Gensan.
January 27, 2016
This day I analyse the sample that we bring from the Beaches from water
checker, BOD, and water faecal, because my fellow trainees have the sampling in Lake
Sebu.
January 28, 2016
This day there is a lot of sample from Lake Sebu that is why all Of my fellow
trainees are busy I assist maam Merly in Water Fecal. then I get the testube that
contain the Laurel Sulphate then I use the pipette to get the exact 10ml of sample for
each of the first, second and third dilution water. Then I get another 10ml from the

dilution to the laurel sulphate of testube. After all of the procedures placed I in to the
incubator.
January 29, 2016
This day I record the result after 24 hours of incubation and confirm the two
sample Then I prepared a Brilliant Green Lactose Broth and EC broth for the
confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose
Broth in the 37o Celsius and the EC broth is 45o Celsius.
February 1, 2016
This day I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.

February 2, 2016
Another sample that we receives from the water bodies to the water fecal I
prepared the samples and . then I get the testube that contain the Laurel Sulphate then
I use the pipette to get the exact 10ml of sample for each of the first, second and third

dilution water. Then I get another 10ml from the dilution to the laurel sulphate of testube.
After all of the procedures placed I in to the incubator.
February 3, 2016
After 24 hours of incubation and confirm the two sample Then I prepared a
Brilliant Green Lactose Broth and EC broth for the confirmation of the positive result. I
separate the positive result for the confirmation. Each of the Brilliant Green Lactose
Broth and EC broth must contain the loopful bacteria from the positive result in the
presumptive test then I place the Brilliant Green Lactose Broth in the 37 o Celsius and
the EC broth is 45o Celsius.
February 4, 2016
I

record again the result in the confirmation of the bacterial present in

Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.
February 5, 2016
This they I record the sample that we received in the BOD room in the
logbook then I put the code for each sample.
February 8, 2016
This day there is only two sample then I get the testube that contain the Laurel
Sulphate then I use the pipette to get the exact 10ml of sample for each of the first,

second and third dilution water. Then I get another 10ml from the dilution to the laurel
sulphate of testube. After all of the procedures placed I in to the incubator.
February 9, 2016
This day I record the result after 24 hours of incubation and confirm the two
sample Then I prepared a Brilliant Green Lactose Broth and EC broth for the
confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose
Broth in the 37o Celsius and the EC broth is 45o Celsius.
February 10, 2016
This day I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.

February 11, 2016

This the day of our sampling in the Beaches in Saranggani and Gensan.

February 12, 2016

We analyse our sample yesterday in the water fecal. In which I get the testube
that contain the Laurel Sulphate then I use the pipette to get the exact 10ml of sample
for each of the first, second and third dilution water. Then I get another 10ml from the
dilution to the laurel sulphate of testube. After all of the procedures placed I in to the
incubator.
February 13, 2016
This day I record the result after 24 hours of incubation and confirm the two
sample Then I prepared a Brilliant Green Lactose Broth and EC broth for the
confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose
Broth in the 37o Celsius and the EC broth is 45o Celsius.
February 14, 2016
This day I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.

February 15, 2016

There is the sample that came from the industries, I only record ph, color,
DO and turbidity in the logbook.

February 16, 2016

There is the sample that came from the industries, I only record ph, color,
DO and turbidity in the logbook.

February 17, 2016

There is only four sample then I get the testube that contain the Laurel Sulphate
then I use the pipette to get the exact 10ml of sample for each of the first, second and
third dilution water. Then I get another 10ml from the dilution to the laurel sulphate of
testube. After all of the procedures placed I in to the incubator.
February 18, 2016
This day I record the result after 24 hours of incubation and confirm the two
sample Then I prepared a Brilliant Green Lactose Broth and EC broth for the
confirmation of the positive result. I separate the positive result for the confirmation.
Each of the Brilliant Green Lactose Broth and EC broth must contain the loopful bacteria
from the positive result in the presumptive test then I place the Brilliant Green Lactose
Broth in the 37o Celsius and the EC broth is 45o Celsius.
February 19, 2016

This day I record again the result in the confirmation of the bacterial present in
Brilliant Green Lactose Broth and EC broth after 48 hours of incubation. Then I wash the
glasswares that I used during the incubation like the test tube. The final washing of
glasswares are distilled water then I placed in to the tray and ready for autoclave in 15
minutes.

Autoclaves
Autoclaves must be of sufficient size to prevent internal crowding. They should be constructed to provide
uniform temperatures within the chamber (up to and including the sterilizing temperature of 121C) and
equipped with an accurate thermometer. The thermometer bulb must be located on the exhaust line to
record the minimum temperature within the sterilizing chamber. Autoclaves should be equipped with a
pressure gauge and properly adjusted safety valves connected directly to the saturated-steam power
lines or steam generator. Autoclaves should be capable of reaching the desired temperature within 30
minutes. Pressure cookers may be substituted for autoclaves, provided they are equipped with efficient
pressure gauges and thermometers, the bulbs of which are 2.5 cm (1 in.) above the water level.