MOST PROBABLE NUMBER (MPN) TEST FOR WATER QUALITY
AIM: To enumerate the number of
bacteria present in the water sample by the MPN method and to identify the
bacteria present in the drinking water sample.
Principle:
The most probable number (MPN) test is a statistical
method test based on the random dispersion of microorganisms per volume in a
given sample. In this
method, measured volumes of water are added to a series of tubes containing a
liquid indicator growth medium. The media receiving one or more indicator
bacteria show growth and a characteristic color change. The color change is
absent in those receiving only an inoculum of water without indicator bacteria.
From the number and distribution of positive and negative reactions, the MPN of
indicator organisms in the sample may be estimated by reference to statistical
tables.
MPN test is completed in three
steps:
1. Presumptive test
2. Confirmed test
3. Completed test
Presumptive test: This test, a specific
enrichment procedure for coliform bacteria, is conducted in fermentation tubes
filled with a selective growth medium (lactose broth), which contains inverted
Durham tubes for the detection of fermentation gas. 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 main selective factors found in the medium are lactose,
sometimes a surfactant such as Na-lauryl sulfate or Na-taurocholate (bile salt),
and often a pH indicator dye for facilitating detection of acid production,
such as bromcresol purple or brilliant green. The selective action of lactose
occurs because many bacteria cannot ferment this sugar, whereas coliform
bacteria and several other bacterial types can ferment it. The surfactant and
dye do not inhibit coliform bacteria, whereas many other bacteria, such as the
spore formers, are inhibited.
Confirmed test: This test serves to confirm
the presence of coliform bacteria when either a positive or doubtful
presumptive test is obtained.
1. A loopful of growth from such a
presumptive tube is transferred into a tube of brilliant green lactose bile
(BGLB) 2% broth (or other lactose broth) and incubated at 35°C for 48 hours.
This is a selective medium for detecting coliform bacteria in water, dairy, and
other food products. A selective agent in the medium is lactose. The broth tube
also contains a Durham tube to detect gas production.
2. A plate of Endo agar (or EMB
agar) is streaked with a loopful of growth from a positive tube and incubated
at 35°C for 18–24 hours. Typical coliform bacteria (E. coli and Enterobacter aerogenes) exhibit good growth on this
medium and form red to black colonies with dark centers or sheen. Salmonella typhi exhibits good growth but
the colonies are colorless. S. aureus growth is inhibited altogether.
Completed Test: A typical coliform colony from
an Endo agar plate is inoculated into a tube of brilliant green bile broth and
on the surface of a nutrient agar slant. They are then incubated at 35°C for 24
hours. After 24 hours, the broth is checked for the production of gas, and a
Gram stain is made from organisms on the nutrient agar slant. If the organism
is a Gram-negative, non-spore-forming rod and produces gas in the lactose tube,
then it is positive that coliforms are present in the water sample.
Materials required: Test tubes, Durham tubes,
Conical flask, Petriplates, Lactose broth, EMB Agar, Endo agar, BGLB Broth,
Gram’s staining kit, Glass slide, Inoculation loop, Microscope.
Procedure for Presumptive test:
1. Prepare Lactose broth of single
and double strength in test tubes with Durham’s tube and autoclave it.
2. Take three sets of test tubes
containing five tubes in each set; one set with 10 ml of double strength (DS)
other two containing 10 ml of single strength (SS) .
3. Using sterile pipettes,
transfer 10 ml of water to each of the DS broth tubes. Transfer 1 ml of water
sample to each of 5 tubes of one set of SS broth and transfer 0.1 ml water to
five tubes of remaining last set of SS broth tubes.
4. Incubate the tubes at 37°C for
24 hours.
5. After incubation, observe the
gas production in Durham’s tube and the color change of the media.
6. Record the number of positive
results from each set and compare with the standard chart to give presumptive
coliform count per 100 ml water sample.
Procedure for Confirmed test:
Some microorganisms other than coliforms also produce acid and gas
from lactose fermentation. In order to confirm the presence of coliform, a
confirmatory test is done. For this, a loopful of suspension from a positive
tube is inoculated into a 3 ml lactose-broth or brilliant green lactose
fermentation tube and to an agar plate (EMB agar or Endo Agar) or slant.
Gas formation
BGLB Broth
A.
Inoculation of the lactose-broth
1. Incubate the inoculated
lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2
hours.
2. If no gas production is seen,
further incubate up to a maximum of 48 ±3 hours to check gas production.
B.
Inoculation in media slants
1. Take a loopful of suspension
from a positive tube and inoculate it on the agar surface.
2. The agar slants should be
incubated at 37°C for 24± 2 hours.
3. Colonies must be examined
macroscopically.
Procedure
for Completed test:
1. Transform a typical coliform
colony from the agar plate into a tube of brilliant green bile broth with
placed Durham’s tube and on the surface of a nutrient agar slant.
2. Incubate at 35°C for 24 hours.
3. After 24 hours, check the broth
for the production of gas, and perform Gram staining for organisms on the
nutrient agar slant.
Result
for Presumptive test:
Positive: The formation of 10% gas or
more in the Durham tube within 24 to 48 hours, together with turbidity in the
growth medium and the color change in the medium constitutes a positive
presumptive test for coliform bacteria, and hence for the possibility of fecal
pollution.
·
The
test is presumptive only because under these conditions several other types of
bacteria can produce similar results.
Negative: No growth or formation of gas in
Durham’s tube.
Result
for Confirmed test:
Positive: Formation of gas in
lactose broth and the demonstration of a coliform-like colony on the EMB agar
indicate the presence of a member of the coliform group in the
sample examined.
Coliforms
produce colonies with a greenish metallic sheen which differentiates them from
non-coliform colonies (show no sheen). The presence of typical colonies at high
temperatures (44.5 ±0.2) indicates the presence of thermo tolerant E.coli.
Negative: The absence of gas
formation in lactose broth or the failure to demonstrate coliform-like colonies
on the EMB agar.
Result
for Completed test:
Positive: The presence of gas in
the brilliant green bile broth tube and Gram-negative, non-spore-forming rods on
NA slant constitutes a positive completed test for the presence of coliform
bacteria, which, in turn, infers possible contamination of the water sample
with fecal matter.
Negative: Absence of growth and gas
formation in the broth. Absence of gram-negative, non-sporing rods on Gram’s
staining.
STREAKING METHODS
INTRODUCTION:
Streaking is a common method used to isolate
a particular bacterial colony from a mixture of bacteria that is pure culture.
In the streak plate method, the concentration of bacterial colonies is more at
the starting point and it goes on decrease toward the last point of the streak.
It helps to isolate individual colonies from other colonies and each colony is
considered a pure colony.
During the
identification of a microorganism, the first and important step is to isolate
the individual species from a mixed sample. This is mainly done by the streak
plate method; therefore streak plate method is
an isolation technique.
In this method a
sterile inoculating loop is first dipped into a diluted bacterial culture; then
the culture-containing loop is streaked on the surface of a solidified agar
plate to make a series of parallel, non-overlapping streaks.
As the culture
is diluted before streaking on solid agar, the organism number will decrease by
the third or fourth quadrant. Therefore only a few bacterial cells are
transferred on the solidified agar medium as a result it will give discrete colony
forming units (CFUs).
During the
streaking an agar plate different patterns are used, depends on the source of
inoculum and the microbiologist’s preference. The patterns of streaking
patterns range from simple to more complex, these are designed to separate
deposited cells (CFUs) on the agar surface so individual cells (CFUs) grow into
isolated colonies.
A quadrant
streak pattern is used for samples suspected of high cell density, while a
simple zigzag pattern used for samples containing lower cell densities.
AIM: To
perform the Streak plate method on different media
REQUIREMENT:
General
Procedure:
Three Sector Streak (t- streak) & Zig - Zag Streak Method:
Four Quadrant Streak method:
Observation: Examine the growth of isolated colonies on the
surface of the nutrient
agar plate.
Result: Few numbers of isolated colonies appear along with
the points of the streak.
STERILIZATION
AND MEDIA PREPARATION
Introduction:
Sterilization: Autoclaving is a process that
use moist heat and pressure so that all parts of the material to be sterilized
reach 121 degree Celsius for 15 minutes. An autoclave is, in essence, a large
pressure cooker; a chamber which may be sealed off against surrounding air.
Materials
for sterilization are placed in the chamber, the door is sealed, and
pressurized steam is forced into the chamber. The incoming steam displaces
cooler air through an exhaust valve; this valve closes when the cell cooler air
has been vented.
Steam
is continually forced into the chamber until the pressure reaches 103 kPa above
atmospheric pressure; at sea level, this pushes the temperature in the chamber
to 121 degree Celsius. The high pressure prevents solutions from boiling over
at this temperature. Larger volumes require longer than 15 minutes to heat up
to 121 degree Celsius throughout. After sterilization, the steam pressure is
slowly decreased to atmospheric pressure. The sterilized objects can then be
removed.
Media
Preparation: Microorganisms
depend on a number of factors such as nutrients, oxygen, moisture and
temperature to grow and divide. In the laboratory, except for the above
factors, the culture medium should be sterile and contamination of a culture
with other organisms should be prevented.
The properties of agar which make
it ideal in bacteriology are
1) Solid agar melts (dissolves)
at 100 °C,
2) Remains solid at all incubation
temperatures,
3) Transparent,
4) It is not heat-labile and
therefore easily sterilized, and
5) It is unaffected by almost all bacteria.
Liquid agar solidifies at 42-44
°C which is useful because sterile, heat-labile components such as antibiotics,
blood, serum, carbohydrates and even bacterial cultures may be added before
allowing the medium to solidify. Solid media generally contain agar at a
concentration of 1.5%. Semi-solid media contain 0.05-0.3% agar and are useful
in culturing anaerobic and microaerophilic organisms because such media form an
oxygen gradient in test tubes, allowing all degrees of oxygen tension to exist
in the culture vessels.
AIM:
To
understand the sterilization process using an autoclave and to learn the
procedures used in preparing media needed for culturing microorganisms.
MATERIALS
REQUIREMENT:
Commercial nutrient agar, Balance,
Distilled water, Scott bottles, Measuring cylinder Beaker, Forceps, Universal bottles
PROCEDURE: STERILIZATION
The usual procedure for sterilization
using the autoclave is as follows:
1. Open door, and place items to
be sterilized into the autoclave chamber.
2. Close door. Push down door
lock lever until door studs are completely in place.
3. Turn hatch wheel clockwise
until it is secured tightly.
4. The temperature of the
autoclave is set at 121°C.
5. Set timer by turning the large
knob just below the hands to the desired setting.
6. Crank operating handle around
to the Sterilize position till the red steam light goes on.
7. Wait and be sure the chamber
reaches the proper temperature and pressure.
9. The Slow Exhaust and Fast
Exhaust & Dry cycles both take 12-15 minutes longer than the time set to
finish.
10. At the end of the run the
white STERILE light will go on, and a loud, obnoxious buzzer will come on:
a. Rotate the operating handle
all the way to OFF. Check that the chamber pressure is zero, and the temperature
is below 100 °C.
b. Turn hatch wheel
counterclockwise, push up door lock lever and slowly open door.
c. Use heatproof gloves to remove
materials.
11. Allow liquid materials to
cool before tightening caps.
PROCEDURE MEDIA
PREPARATION:
1.
Measure
approximately 250 ml of distilled water in a 1 L graduated cylinder and pour
into a 1 L flask.
2.
Weigh
out 1.5 g beef extract and 2.5 g peptone and add into the flask. Use
approximately 100 ml of the water to rinse any powder stuck to the side of the
flask down into the mixture.
3.
Stir
over gentle heat from a bunsen burner to dissolve completely.
4.
Pour
the mixture into the 1 L graduated cylinder and add warm water to the 500 ml
mark. Pour back into the flask.
5.
Check
the pH of the medium and adjust to pH 7.
6.
Autoclave
the flask and the petriplates for 15 minutes at 121 °C and 15 lb/in2 pressure
at the slow exhaust mode.
7.
After
removing the media from the autoclave, allow the media to cool, and store for
later use.
8.
Lay
your petri dishes on the bench. The cover should be on top. Light your Bunsen
burner, and then remove the NA flask from the water bath.
9.
Remove
the tapes and cotton plug from the flask. Carefully flame the neck of the
flask, open the plate cover about half way and pour the media in to petri plate
about 30 ml.
10. Flame the neck of the flask
between each plate. Allow plates to solidify completely, which will take 15
minutes. Then invert, label and incubate at 37 °C overnight to dry off excess
moisture and check for contamination.
CONCLUSION:
Different types of agar are needed for
the cultivation of different types of microorganisms. Agar of the same
composition with the commercial agar can be made by following the correct
procedures. Preparation and sterilization of culture media should be done with
great care to avoid contamination of unwanted microorganisms. We had learnt the
preparation and sterilization of culture media via autoclaving process and the
precaution that we need to take into consideration when handling this
experiment.
PREPARATION OF CLEANING SOLUTION: ETHANOL
INTRODUCTION
Ethanol is a flammable
chemical. If not stored and handled properly, this can pose a serious threat to
the health and safety of laboratory personnel, emergency responders and
chemical waste handlers. Hence, it is important to follow safety protocols to handle
this chemical. Ethanol is mainly used as solvent. It is miscible with water and
with many organic solvents, including acetic acid, acetone, benzene, carbon
tetrachloride, chloroform, diethyl ether, ethylene glycol, glycerol,
nitromethane, pyridine, and toluene. It is also miscible with light aliphatic
hydrocarbons, such as pentane and hexane, and with chlorinated organic such as
trichloroethane and tetrachloroethylene.
AIM:
To prepare the cleaning solution for disinfect the
surface of working table and glassware.
MATERIALS REQUIRED:
Measuring jar, 0.2 micron filter,
Distilled water, 99.9% ethanol, Filtration apparatus, Glass bottle
PROCEDURE:
1.
Assemble the filtration unit to achieve filter sterilization
to the water and the 99.9% lab ethanol. All the steps shall be performed in to
the LAF to avoid contamination.
2.
Pour the water in to the 0.2 micron filter containing
filtration cup and turn on the pumb.
3.
Repeat the procedure for sterilizing the lab ethanol.
4. To
prepare 1 liter of 70% Ethanol using 95% lab Ethanol add 740ml of lab ethanol
to 260ml water.
5. Label
the container and store in a safe place.
RESULT:
Ethanol cleaning
solution has been prepared as followed the procedure for the disinfection
purpose and stored in a secure place. The effectiveness of the cleaning
solution will be checked periodically.
