After incubation, the growth of microorganisms is detected, followed by counting. There are several techniques involved in counting microbes. Direct counting techniques are used. The oldest of these is microscopy which involves magnification of individual cells to become visible to the naked eye. Direct counting techniques do not rely on cell population growth. A recent direct counting technique is the use of immunofluorescence and epifluorescence adaptations of cell labeling used in conjunction with cytometry. Total and viable counts of microorganisms are important practices in microbiology. The main difference between the two is that total count determines the count of all cells both dead and alive while viable count estimates the number of viable or live cells only capable of growing into distinct colonies.
Importance:
• Knowing how to count organisms and understanding their
growth cycles is often important in treating infections.
• By counting individual organisms and experimenting, we can
determine how many of them are needed to cause disease.
• Counting bacteria is also important in environmental
microbiology; to control environmental conditions or enhance growth to obtain
desired results.
Total Count
Total count is also termed as standard plate count or colony
count. It gives the total number of microbes both viable and non-viable. All
cells are counted. These include bacteria, yeasts, and molds. It is usually
done by the pour plate method. Total count generally requires a microscope.
Flowchart: Types of Total Count
Counting chamber: This method is easy, quick, and
inexpensive. It is used for both prokaryotes and eukaryotes. A hemocytometer is
used for cell counting. It is a microscope slide. The central part of this
slide has etched grids with precisely spaced lines to enable accurate counting.
In order to get an accurate count using this method, the cell number should range between 40 and 70 cells in a one-mm square (Fig). There is 1000 mm cubed per ml,
so the cell can calculate by a unit of cells/ml. Sometimes need to dilute a
cell suspension to get the cell density low enough for counting. In that case,
the final count is multiplied by the dilution factor.
Fig: Haemocytometer
Electronic counter: This method is known as the flow
cytometry method. This method is easy and fast but expensive. This machine
detects the difference in current as individual microorganisms pass through a
small orifice. This orifice connects two compartments of the counter which
contain conductive solution. In this instrument, two electrodes passing
constant current are placed on either side of a small hole, and due to that the
major resistance in the circuit is at the aperture, and when a non-conducting
particle is passing through the resistance is changed to an electrical pulse
(Fig). When each bacterium passes through the orifice, the electrical
resistance between the two compartments increases. The number of pulses is
equal to the number of cells counted and the strength of the signal is directly
proportional to the cell volume.
Fig: Coulter counter
Colter counter: It is a probe that measures variation
in conductivity of a solution as a bacteria passes through a narrow gap.
Advantage: Direct and indirect count processes are
automated.
Disadvantage: They cannot differentiate dead cells
from living ones.
Membrane filter method: This method is useful for
counting bacteria. Cells are stained with fluorescent dyes then cells are
filtered through a special membrane that provides a dark background for
observing cells. These filters have uniform porosity of predetermined size to
trap microorganisms. The membrane with trapped bacteria is then placed in a
special plate containing a pad saturated with a specific medium. During
incubation, the bacterial colonies grow which appear on the surface of the
membrane. This technique is mainly useful in analyzing aquatic samples.
Indirect count method: These depend on the effects of
the organisms to estimate their numbers.
Colorimetry: This method is also known as the turbidity
method. When the organisms are grown in the medium the nutrient broth becomes
turbid. The turbidity in the form of optical density is measured by the colorimetry
method. It consists of the light source, a filter that allows only a single
wavelength of light to pass through, the sample tube containing the bacterial
suspension, and a photocell that compares the amount of light coming through
the tube with the total light entering the tube. The ability of the culture to
block the light can be expressed as either percent of light transmitted through
the tube. The percent of light transmitted is inversely proportional to the
bacterial concentration. The optical density of the solution is high with the
maximum growth of the microorganism in the medium i.e. the absorbance is
directly proportional to the cell concentration.
McFarland standards are used as a reference to adjust the
turbidity of bacterial suspensions so that the number of bacteria will be
within a given range (Fig).
Fig: Turbidity method
Viable count
Viable count involves counting colonies produced by only
viable cells under favorable growth conditions. The number of living or dead
bacterial cells (viable count) in the culture is estimated and is expressed as
a Colony Forming Unit (CFU). This can be accomplished by techniques like pour
plating, spread plating, and most probable number with an assumption that each
and every viable cell gives rise to a pure colony. This method is useful in the
dairy industry and the food industry for quantitative analysis of milk and
spoilage of food products. It helps in the estimation of the shelf life of
processed food products as well as the evaluation of sanitary conditions under
which the products were manufactured. The efficiency of certain treatment
processes like pasteurization, sterilization, and cold storage done during
production is also evaluated by viable microbial count.
The advantage of viable count is that the method is very
sensitive and can able to count subsets of the population. The disadvantage is
that the method is time-consuming (counts require at least a few hours or
overnight for incubation) and sometimes the colony-forming units may
underestimate the number of cells because of clumping or chains of cells.
Flowchart: Types of viable count
Pour plating: In this method, the liquid media and
the diluted sample are poured together in Petri dishes while still in liquid
form and left to solidify. After solidifying, the Petri dishes are incubated at
the appropriate temperature for the required period of time during which the
growth is realized. The plates are then removed and distinct colonies are counted
and expressed in colony-forming units per ml (Fig).
Fig: Pour plating method
Spread plating: In this method, the media is prepared
separately and poured into Petri dishes while still in liquid form. It is then
left to solidify. After solidifying, a small known volume from the diluted
sample is put into each Petri dish, and with the help of a sterilized spreading
rod, the sample is evenly spread over the media. The plates are then incubated
at an appropriate temperature for a given period during which growth is
realized. The plates are then taken for counting of colonies using a magnifying
lens (Fig).
Fig: Spread plating method
Membrane filter technique: This method is used in the
laboratory during water testing. Bacteria from aquatic samples are trapped on
membranes by being placed on culture media. Bacterial colonies are grown on
membrane and then colony count determines the number of bacteria present in the
sample.
Turbidity for Most Probable Number: If the microbe
cannot be cultured on plate media then dilutions are made and added to suitable
media. After the growth of microorganisms, the turbidity was determined to
yield the most probable number. This method is useful where it is beneficial to
use broth other than solid media, especially for highly motile organisms that are
poor in forming colonies. It is mostly used when the organism is not able to
grow on solid agar or in situations where the microorganisms are too few to
give a reliable measure of population size by the standard plate count method.
Serial dilution: In this method, increments are made
in 1000, 100, or 10. The number of dilutions to be done depends on the
concentration of the original solution and the required concentration. If small
quantities of solutions are needed then more numbers of dilutions are required.
Serial dilution allows small aliquots to be diluted instead of unnecessary big
volumes of materials. During dilution, a small amount of the original sample is
removed to another container and its volume is adjusted to the original volume
using a suitable buffer or distilled water e.g. if 1 ml of the original
solution is taken and 10 µL removed and added in 990 µL of media or water then
the dilution is 1:100. If the original solution contained 5 × 106
cells/ml then the concentration is of 5 × 104 cells/ml because that
divided the concentration by 100.