Viable and Total Bacterial Count

Viable Count: The number of viable microorganisms is counted in a given sample. The
following are the methods used for viable count:

(i) Pour plate method

(ii) Spread plate method
(iii) Membrane filtration method
(iv) Multiple tube dilution method (MPN method)

(i) Pour Plate Method: The sample is serially diluted and poured into liquefied agar medium.
Bacteria get embedded into the medium and after solidification and incubation develops colonies
within the medium. The numbers of colonies are counted and viable number of bacteria is
calculated for the given sample. Only viable cells develop colony and theoretically one cell
develops one colony. However bacteria present in different arrangements may develop only one
colony. So, here the viable count is reported as colony forming units per ml.

Steps involved in pour plate method:

(a) The given sample is serially diluted i.e. 1 ml of the sample diluted with 9 ml of
sterile normal saline solution (1 in 10 dilution). Similarly from 1 in 10 diluted sample
1 ml is further diluted with 9 ml of sterile normal saline solution. In the same way, the
give sample is diluted to 1 in 10 6 dilutions. Here bacterial cells get separated by
dilution.

(b) A small quantity of serially diluted sample is transferred into 15 ml of liquefied agar
medium at about 45oC. It is mixed well and poured into the plate. Such transferring
are carried out for each serially diluted sample and poured into the corresponding
plate.

(c) Plates are cooled so that media get solidified. In this step the bacterial cells get
seeded into the solid medium.

(d) Plates are incubated.

(e) After incubation period, colonies are observed and counted using colony counter
instrument. Then results are calculated for the actual given sample by considering the
dilution factor. Plates with distinct colonies are considered for counting purpose.
(ii) Spread plate method: The sample is serially diluted in ten-fold dilutions (as given above).
The diluted samples are spread on the surface of agar medium. Bacteria get adhered onto the
surface of the medium and after incubation develop colonies. The numbers of colonies are
counted and viable number of bacteria is calculated for the given sample.

Steps involved in spread plate method:

(a) The given sample is serially diluted i.e. 1 ml of the sample diluted with 9 ml of
sterile normal saline solution (1 in 10 dilution). Similarly from 1 in 10 diluted sample
1 ml is further diluted with 9 ml of sterile normal saline solution. In the same way, the
give sample is diluted to 1 in 106 dilutions. Here bacterial cells get separated by
dilution.

(b) A small quantity of serially diluted sample is transferred onto the agar medium. It is
spread well with the help of L-shaped glass rod. Such transferring are carried out for
each serially diluted sample and spread on the corresponding plate. In this step the
bacterial cells get adhered onto the solid medium. Spreading separates the bacterial
cells.

(c) Plates are incubated.

(d) After incubation period, colonies are observed and counted using colony counter
instrument. Then results are calculated for the actual given sample by considering the
dilution factor. Plates with distinct colonies are considered for counting purpose.

(iii) Membrane filtration method: The sample is filtered across bacteriological-proof

membrane filter. All the bacteria are obstructed by membrane filter and the liquid filtrate is
discarded. The membrane filter (containing bacteria) is transferred onto the surface of agar plate.
The plates are incubated. Colonies are developed on the surface of membrane filter by viable
bacteria. The numbers of colonies are counted and viable number of bacteria is reported for the
given sample. The method is suitable for large volumes of samples containing very few bacteria.
Here serial dilution is not required and the sample is just filtered as it is. Membrane filters with
grids are used, so that colony counting is easy.

(iv) Multiple tube dilution method (MPN method): Multiple tubes containing 9 ml of nutrient
medium are taken in 3 sets. Each set contains 3 tubes. First, add 1 ml of the test fluid to each of
three test tubes of first set and mix to make 10- times dilutions. Second, add 1 ml of each of the
10-times dilutions to each of another three test tubes (second set) and mix to make 100-times
dilutions. Third, add 1 ml of each of the 100-times dilutions to each of the remaining three test
tubes (third set) and mix to make 1,000- times dilutions. Maintain 3 tubes as controls without any
sample.
Incubate all 12 test tubes for at least 5 days at 30 - 35℃. No microbial growth should be
observed for the control test tubes. Calculate the most probable number of microorganisms per
ml or gram of the sample, using the MPN (most probable number) table given in standard
literature.

Total Bacterial Count: The numbers of total microorganisms are counted in a given sample.
Here dead cells are also counted along with viable cells. The following are the methods used for
total count:

(i) Direct microscopic method

(ii) Turbidity method
(iii) Electron particle counter method
(iv) Dry weight method
(v) Flow cytometer

(i) Direct microscopic method: Direct microscopic counts are possible using special slides
known as counting chambers, consisting of a ruled slide and a cover slip. A known volume of
liquid is taken in a specified area of the slide. It is dried and stained by stains like methylene
blue. It is constructed in such a manner that the cover slip, slide, and ruled lines delimit a known
volume. The number of bacteria in a small known volume is directly counted microscopically
and the number of bacteria in the larger original sample is determined by extrapolation. Dead
cells cannot be distinguished from living ones. Only dense suspensions can be counted.

Bacteria can be counted easily and accurately with the petroff-Hausser counting chamber. This is
a special slide accurately ruled into squares that are 1/400 mm2 in area; a glass cover slip rests
1/50 mm above the slide, so that the volume over a square is 1/20,000 mm3 i.e. 1/20, 000, 000
cm3 . If for example, an average of five bacteria is present in each ruled square, there is 5 x
20,000,000 or 108, bacteria per milliliter. A suspension of unstained bacteria can be counted in
the chamber, using a phase-contrast microscope

(ii) Turbidity method: This technique depends on the fact that as the number of cells in a
solution increases, the solution becomes increasingly turbid (cloudy). The solution looks turbid
because light passing through it is scattered by the microorganisms present. Turbidity is
proportional to the number of microorganisms in the solution. The turbidity of a culture can be
measured using a photometer or a spectrophotometer.

Absorbance increases in a linear fashion as the cell number increases. When measuring growth
of a culture the term optical density (OD) is normally used to more correctly represent the light
scattering that is occurring. If a precise cell number for a given OD is desired, a standard curve
can be generated, where viable plate count or cell mass is plotted as a function of OD. After the
standard curve is made, it is then possible to simply measure the OD of the culture and read the
cell number from the curve.

Turbidity readings are species-specific and cannot be compared between different microbes or
even between different strains of the same species. Also both living and dead cells scatter light
and are therefore counted. However, the method is very rapid and simple to perform and
provides reliable results when used with care.

(iii) Electron particle counter method (Coulter counter method): A Coulter counter is an
apparatus for counting and sizing particles suspended in electrolytes. It is used
for cells, bacteria, prokaryotic cells and virus particles.

A typical Coulter counter has one or more microchannels that separate two chambers containing
electrolyte solutions. As fluid containing particles or cells is drawn through each microchannel,
each particle causes a brief change to the electrical resistance of the liquid. The counter detects
these changes in electrical resistance.
Cells, being poorly conductive particles, alter the effective cross-section of the conductive
microchannel. If these particles are less conductive than the surrounding liquid medium, the
electrical resistance across the channel increases, causing the electric current passing across the
channel to briefly decrease. By monitoring such pulses in electric current, the number of particles
for a given volume of fluid can be counted.

(iv) Dry weight method: A membrane filter of known diameter and porosity (usually O.45 μm)
is taken and dried in oven. Weight of the membrane filter is taken (W1). Then given sample is
filtered across the membrane filter. Bacteria are collected on the membrane filter. Dry the
membrane filter along with bacteria without any residual moisture. Now weigh the membrane
filter (W2). The difference in the two weights (i.e. W2-W1) gives us the dry weight of the
bacteria. This parameter gives us the biomass of the given sample.

(v) Flow cytometry: Flow cytometry is by far the most sophisticated and expensive method for
cell counting. In a flow cytometer the cells flow in a narrow stream in front of a laser beam. The
beam hits them one by one, and a light detector picks up the light that is reflected from the cells.
Flow cytometers have many other abilities, such as analyzing the shape of cells and their internal
and external structures, as well as measuring the amount of specific proteins and
other biochemicals in the cells.
Image of Pour plate method:
Membrane Filtration method:

Direct Microscopic method:

Turbidity Method:

Coulter current method: