THE METHODS OF ENUMERATION IN MICROBES

Enumeration is counting of microorganisms present in a sample.

This is done to know the intense of presence of the spoilers in the spoiled food.
To detect which type of organism is responsible for the spoilage.
The methods of enumeration in microbes can be divided into four categories. Direct methods involve counting the
microbes, while indirect methods involve estimation. Viable methods only count cells that are metabolically active,
while total counts include dead and inactive cells.
Direct/Viable
A viable cell count allows one to identify the number of actively growing or dividing cells in a sample.
A direct/viable method involves a standard plate count, in which repeated dilutions of a sample are counted to
calculate the count in the original sample.
Indirect/Viable
Indirect/viable methods such as MPN (most probable number) involve making a statistical inference about the
microbe count based on patterns of growth.
Direct/Total
The microbes are counted with the aid of fluorescent stains and dyes, which make the microbes visible with the aid
of a fluorescent microscope.
Indirect/Total
Spectroscopy is a form of indirect/total enumeration, which involves estimating the amount of microbes based on the
amount of light passed through the culture by a spectrophotometer.

Counting Cultures
One direct measure for microbial enumeration is the standard plate count, also called a viable count. For this count
you culture a sample by diluting it, placing it on plates of culture medium and incubating them for a set amount of
time. You then count the numbers of colonies and use this number to deduce the original number of microbes in the
sample. Technically speaking, a plate count doesn't give the number of individual microbes, but rather of "colony-
forming units," because you can't know for sure whether each colony actually came from a single microbe or from a
tiny group of microbes. However, these counts are considered very accurate for estimating the number of microbes
in original samples. Drawbacks are that this test is time- and space-consuming and requires specialized equipment
that must be prepared correctly.
Individual Counts
Direct microscopic counts, also called total cell counts, are another form of direct enumeration. First you divide a
sample into a number of equally sized chambers. Then you determine the average number of microbes per chamber
by counting some or all under a microscope. Finally you use this average to calculate the number in the original unit.
The major drawback for direct microscopic counts is that it's difficult to distinguish living microbes from dead ones,
so this method may not give an accurate viable enumeration.
Rays of Light, Clouds of Microbes
Turbidity tests are forms of indirect enumeration. Turbidity is the cloudiness of a liquid. In turbidimetric measurement
you put a sample in solution, measure the new solution's cloudiness by shining light through it with a
spectrophotometer, then estimate the number of living microbes it would take to produce the observed cloudiness
level. The drawback here is that someone must have already done numerous standard plate counts of the microbe in
question in order to make sample solutions of varying turbidity, so that you have a standard to measure your current
sample against. You must also beware of overly concentrating your sample, because a turbidimetric count is only
accurate if no microbes in the sample are blocking any others. In a visual turbidity comparison you compare the
turbidity of your sample with the turbidity of a unit of the same size and known microbial count, and estimate an
enumeration based on this comparison.

PLATE COUNTING METHOD

The plate count is one of the most accurate means of enumeration of viable microbes because you get a visual indicator
for every cell in the specimen. The technique stems from Robert Koch's insight gained from viewing colonies growing
on the surface of a spoiling slice of potato. In practice, a small aliquot of a liquid suspension of microbes is spread on
the surface of solidified nutrient medium, which when incubated, leads to each cell 'developing' into a visible colony
through repeated fission. The pour plate technique can be used to determine the number of microbes/ml or
microbes/gram in a specimen. It has the advantage of not requiring previously prepared plates, and is often used to assay
bacterial contamination of foodstuffs. Each colony represents a "colony forming unit" (CFU). For optimum accuracy of
a count, the preferred range for total CFU/plate is between 30 to 300 colonies/plate.
A plate having 30-300 colonies is chosen because this range is considered statistically significant. If there are less than
30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic
effect on the final count. Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and
colonies will have grown together.
Procedure:

Step One: Diluti ng the sample

Depending on the source of the sample
used there might be thousands, millions or
even billions of microorganisms per
millilitre of sample. This is too many for
us to count so we dilute the sample.

1ml of sample is added to 9ml of a suitable

diluent (e.g. sterile buffer).

The sample and diluent are mixed together.

This new sample (Dilution One) has a

concentration (number of microorganisms
per ml) 1/10th that of the original sample.

1ml of Dilution One is added to another 9ml of diluent to make Dilution Two.

Dilution Two has a concentration 1/10th that of Dilution One and 1/100th that of the original sample.

This process is repeated until we have a series of dilutions.

Step Two: Plating the sample

To find out how many viable cells are in each of our dilutions we need to put some of the sample onto an agar plate.
The agar plate is prepared by mixing growth medium with agar and then autoclaving to sterilise. Once the agar has
cooled to ~50oC approximately 15ml is poured into a sterile Petri dish and left to set.

0.1ml of sample is pipetted onto the agar surface and spread around using a sterile glass rod. We usually put 0.1ml of
one of our sample dilutions onto a plate – if we use more than this it can make the plates very wet and if we use less
than this it is difficult to spread evenly.

This is repeated so that we have 2 or 3 repiclate plates for our original sample and for each of our dilutions.

Step 3: Incubating the plates

Once all of the plates have been prepared they are left to dry and then moved to an incubator at a suitable growth
temperature for the microorganism being studied. The incubation time depends on the organism and the growth
medium but during the incubation, each viable cell that was spread to a discrete position on the agar surface will grow
and divide many times to form a visible colony of microorganisms.

After the incubation period we can count the colonies to determine how many microorganisms were present in the
original sample.

Step 4: Counting t he colonies

The plates will have different numbers of colonies depending on the dilution of the sample.

If there are too many colonies it can be impossible or very difficult to count them.

If there is only a small number of colonies it is easy to count them but the results are prone to error.

As a compromise we always aim to count plates with between 30 and 300 colonies.

We record our results noting the dilutions that had between 30 and 300 colonies and how many colonies there were on
these plates.

Calculation:

CFU / 100 mL = Number of colonies developed on the plate * 100/ mL of sample * Dilution factor
MOST PROBABLE NUMBER METHOD / MEMBRANE TUBE FERMENTATION TECHNIQUE

Definition of MPN

MPN stands for a most probable number. It refers to the qualitative and quantitative analysis of water, which can
enumerate the presence of faecal coliforms. E.coli is a common faecal contaminant present in water, and it can cause
serious illness if it goes into the body. Thus, in most probable number method, E.coli is used as “Pollution indicator”
to analyse the water quality. The most probable number includes three sets of methods like presumptive, confirmative
and completed-test.

Principle of Most Probable Number

A most probable number is a statistical method, in which the results are compared with the standard statistical tables.
It involves three sets of dilutions containing fermentative broth and the water sample. The formation of acid and gas
indicates a positive result. The quantitative analysis of coliform is determined by counting the number of tubes giving a
positive result and comparing the pattern of positive results with the statistical data.

Method of Most Probable Number

MPN is a method, which involves three consecutive tests, namely presumptive, confirmatory and completed-test.

Presumptive Test

The preliminary or screening test uses a

series of fermentation tube
containing lactose broth of known
concentration. The negative presumptive
analysis indicates that the water is
microbiologically safe and does not
require further testing. But in the case of
positive results, one needs to confirm the
presence of coliforms through the
confirmed MPN-test.
The production of gas requires 40-390
million per ml of coliforms according to
the Chambers. The gas formation
depends upon the ratio of coliform to the
non-coliforms. If the ratio of non-
coliform bacteria is high, it will also
reduce the production of gas. The
presumptive test involves the following steps:
Procedure of Presumptive Test

1. In a sterile conical flask add 40 g of the MacConkey broth in 1000 ml of distilled water to prepare a single
strength medium. In the other flask, add 80 g of the MacConkey broth in 1000 ml of distilled water to
prepare a double strength medium.
2. Then, sterilize the flasks in an autoclave for 15 minutes at 121 degrees Celsius.
3. After autoclaving, take water samples in a series of test tubes. Add the known concentration of water into
the three sets like 10 ml in first five tubes, 1ml in the other five tubes and 0.1 ml in the last five tubes. To
test 10 ml of a water sample, add 10 ml of double strength MacConkey medium. Conversely, to check 1 ml
and 0.1 ml of a water sample, add 10 ml of single strength MacConkey medium.
4. Incubate the tubes for 24-48 hours at 35 degrees Celsius.
Observation: After incubation, the results are made based on any colour change or gas production in the MacConkey
broth.
Result interpretation: The colour change of MacConkey broth from red to yellow and the gas production inside the
Durham tube will indicate a positive result for the presumptive test. If the broth colour remains the same, i.e. red and no
gas forms, it will give a negative result for the presumptive test.
Confirmed Test

It is the confirmatory test ensuring the

presence of coliform by testing the
positive tubes of the presumptive test. The
gas produced in the presumptive test does
not mean that there must be coliform in
the water sample. Many other
microorganisms are also present in water,
which can give a false presumptive test.

Some yeasts and Clostridium species are

present in the water, which can ferment
lactose by producing both acid and gas. Therefore, it becomes necessary to confirm the presence of coliform in water.

Testing of positive presumptive in BGLB medium

Procedure
1. First, prepare a solution of brilliant green lactose bile broth
2. Sterilize the BGLB medium in an autoclave for 15 minutes at 121 degrees Celsius.
3. Shake the positive presumptive tubes gently.
4. Then transfer a loopful of culture into the BGLB fermentation tube. The brilliant green dye in the BGLB
medium inhibits the growth of gram-positive bacteria.
5. Incubate the test tubes for 48 hours at 35 degrees Celsius.
Observation: Observe the test tubes containing BGLB medium and inoculum of positive presumptive test for the
production of gas in the inverted Durham tube.
Result interpretation: The production of gas in the BGLB medium indicates the presence of coliforms

Completed Test

It is the final test to ascertain the presence of coliforms. A completed test involves the following steps:

Procedure of Completed Test

• Take a loopful of culture from positive
confirmed tube.
• Inoculate a culture either in BGLB medium or
by streaking the culture onto the agar slants.
• Incubate the test tubes for 24-48 hours at 35
degrees Celsius.
Observation: Observe the test tubes for gas
production in BGLB medium or the growth of a
bacterial colony in the agar slants.
Result interpretation: The production of gas in the
Durham tube of the BGLB fermentation tube
confirms the presence of coliform bacteria. The
growth of gram-negative, non-sporing rods in the agar slants confirms the presence of coliforms.

Advantages
1. Through the MPN method, turbid water containing sediments, sludge, mud etc. can be analysed.
2. The result’s interpretation is easy by observing the production of gas or by the growth of gram-negative
rod-shaped coliforms.
3. Most Probable Number test is applicable for all the types of water, which is an advantage over the membrane
filtration method.
Disadvantages

1. To confirm coliforms’ presence, it includes three tests that take a long time to get the results.
 2. MPN is a very sensitive method, which sometimes can give false results.
3. Most probable number method requires multiple test tubes and many glasswares for the media preparation.
In the MPN method, a number of tubes showing positive results will estimate the coliform count per 100ml.

MEMBRANE FILTER TECHNIQUE

1. Collect the sample and make any necessary

dilutions.
2. Select the appropriate nutrient or culture medium.
Dispense the broth into a sterile Petri dish, evenly
saturating the absorbent pad.
3. Flame the forceps, and remove the membrane
from the sterile package.
4. Place the membrane filter into the funnel
assembly.
5. Flame the pouring lip of the sample container and
pour the sample into the funnel.
6. Turn on the vacuum and allow the sample to draw
completely through the filter.
7. Rinse funnel with sterile buffered water. Turn on
the vacuum and allow the liquid to draw
completely through the filter.
8. Flame the forceps and remove the membrane filter
from the funnel.
9. Place the membrane filter into the prepared Petri dish.
10. Incubate at the proper temperature and for the appropriate time period.
11. Count and confirm the colonies and report the results.

Advantage of Membrane Filter Technique

• Permits testing of large sample volumes. Theoretically, almost any volume of non-turbid water could be filtered
through the disk, the organisms from any given volume being deposited in the disk.
• The membrane can be transferred from one medium to another for purposes of selection or differentiation of
organisms thus allowing isolation and enumeration of discrete colonies of bacteria.
• Results can be obtained more rapidly than by the conventional MPN standard methods. It provides presence or
absence information within 24 hours.
Comparison of Most Probable Number (MPN) and Membrane Filtration Technique for analysis of coliform
bacteria

Most Probable Number (MPN) technique Membrane filter technique

More rapid: quantitative results in or presumptive

Slower: requires 48 hours for a positive
positive about 18 hours

More labor-intensive Less labor-intensive

Requires more culture medium Requires less culture medium

 Requires more glassware Requires less glassware

More sensitive Less sensitive

Result obtained indirectly by statistical approximation Results obtained directly by colony count (high
(low precision) precision)

Not readily adaptable for use in the field Readily adapted for use in the field

Applicable to all types of water Not applicable to turbid waters

Consumables readily available in most countries Cost of consumables is high in many countries

May give better recovery of stressed or damaged

No such difference observed
organisms in some circumstances

PERSISTENT POLLUTANTS
Persistent organic pollutants are toxic chemicals that are slow to break down. When released, they stay in the
environment for a long time and accumulate in the food chain and living organisms. That’s why they are also sometimes
referred to as forever chemicals.
The most commonly encountered POPs are organochlorine pesticides, such as DDT, industrial chemicals,
polychlorinated biphenyls (PCB) as well as unintentional by-products of many industrial processes, especially
polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF), commonly known as dioxins.
Bioconcentration
The direct uptake of organic toxicants from aqueous phase by mass transport through gills and epithelial tissues of
aquatic organisms is called bio-concentration.
Bioaccumulation
The process of accumulating toxic chemicals such as pollutants, pesticides and other toxins directly into the human body
either through the air, water, food intake, or directly through the skin is termed Bioaccumulation. As this toxic compound
accumulates within the human body, it increases the risk of chronic poisoning and other severe health disorders.
Biomagnification
The process of a buildup of certain chemical substances or toxins at the higher trophic levels of a food chain is termed
Biomagnification. This is also referred to as biological magnification. The chemical substances include certain toxins,
heavy metals, mercury and other harmful products at a higher concentration. As these substances increase and
accumulate, it moves up in the food chain. When these contaminated substances are consumed by different levels of
organisms in a food chain, it results in severe health hazards.