ENUMERATION OF BACTERIA
Instructor St.Peters Institute of Pharmaceutical Sciences Vidyanagar, Hanamkonda, India
Introduction
To be specific, the count actually represents the
number of Colony Forming Units (cfu) per g ( or per ml) of the sample.
Direct
Indirect
Viable count
Total count
Turbidity Metabolic activity
Dry mass
Plate counts
measurement of cell quantification is achieved. The basis of a direct count is the actual counting of every organism (or every living organism---often limited to a single type of microorganism ) present in a sub-sample of a population.
DIRECT COUNT
Dilutions of samples are observed under a
microscope the number of bacterial cells from a given volume of sample are counted
DIRECT COUNT
DIRECT COUNT
Dilution factor of 107 Number of bacteria in square = 5 5 X 107 = 50,000,000 bacteria per ml
Merits
1)The technique is sensitive and has the advantage of only
counting living bacteria, 2) Any concentration of microorganism can be easily counted, if the appropriate dilution is plated 3) It is even possible to concentrate a solution before counting, as is often done in water analysis, where bacterial populations are usually at low density. 4) The equipment necessary for performing viable plate counts is readily available in any microbiology lab and is cheap in comparison to other methods. 5) Finally, by using a selective medium it is possible to determine the number of bacteria of a certain class, even in mixed populations.
Demerits
One major disadvantage of the viable plate count is the
assumption that each colony arises from one cell. In species where cells grow together in clusters, a gross underestimation of the true population results. One example of this are species of Staphylococcus, which is known to form clumps of microorganisms in solution. 2)t he temperature of incubation and medium conditions must also be optimized to achieve the largest colonies possible so that they are easily counted. Finally, this technique takes time. Depending on the organism, one day to several weeks might be necessary to determine the number of CFUs that were present when the experiment started. Such information may no longer be useful for many experiments.
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viable cells are counted. Viable counts can be accomplished by such techniques as:
Total count A total count is a direct counting method in which all cells
are counted, whether dead or alive . Generally taking total counts requires the employment of microscopes.
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Note
A sample to be counted is diluted in a solution that will not harm the microbe, yet does not support its growth (so they do not grow during the analysis).
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Spread-plate technique
In the spread-plate technique some of the highest dilutions (lowest bacterial density) are then taken and spread with a sterile glass rod onto a solid medium that will support the growth of the microbe. It is important that the liquid spread onto the plate soaks into the agar. This prevents left over liquid on the surface from causing colonies to run together and the need for dry plates restricts the volume to 0.1 ml or less.
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TNTC
if the number of colonies is too great (over 300) the
limitation of viable plate count selective as to the bacterial types that will grow given the incubation temperature and nutrient type
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may be used to determine the count of viable microorganisms . Plate counts assume that every colony is founded by a single cell. That cell must have been alive in order to grow and form a colony .
Caveats:
Problems with plate counts are:
they require lengthy incubation for colonies to become visible cell clumping can lead to an undercount of viable cells it is very easy to have too many or too few colonies on a plate to accurately measure viable count prevention of crowding often requires serial dilution* too few cells requires concentrating, e.g., by centrifugation or filtration** *Depending on your organism, and other circumstances, you will want to have no more than 300 to 500 colonies per plate (e.g., for big colonies 300 will be many, for small colonies 500 may still work).
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of extinction
dilution level at which no cells are deposited
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dilution tube has bacteria present the pattern of negative results compared statistically results
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that show growth (brown) and those that do not (orange) are compared with a statistical table to calculate MPN
diluting cultures then growing the dilution cultures in broth tubes. At the dilution at which broth becomes turbid or not with similar likelihood, the culture has been dilution to the point that the broth tubes were inoculated with on the order of only a single microorganism (turbid ) or fewer (not turbid ). The concentration of the culture is then taken to be equal to the amount of dilution necessary to have reached this point.
Advantages
MPN is especially useful in situations where there is an advantage to using broth over solid
medium . For example, many organisms are not good at forming colonies, such as highly motile organisms.
"When samples contain too few organisms to give reliable measures of population size by the standard plate count method, as in food and water
sanitation studies, or when organisms will not grow on agar, the most probable number method is used."
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Total count
Direct microscopic count Direct microscopic count is a determination of the number
of microorganisms found within a demarcated region of a slide known to hold a certain volume of culture . This total count method of cell quantification is very rapid but has the problem of requiring high cell concentrations (e.g., 107 / ml) as well as potentially counting dead and living cells with equal probability.
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Indirect methods
Estimation of cell number Correlates to cell number:
Estimates of cell number use various correlates of cell number
rather than direct counting . Such methods are often preferable either for convenience or because direct counting is difficult or even impossible in many situations (for example, when quantifying filamentous organisms ).
must be concentrated prior to determining its plate count. Filtration, which sieves microorganisms out of the medium, is one method of concentrating microorganisms .
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turbidity of a culture is to be used to estimate cell count . This standardization usually works best for cultures growing in exponential phase .
Metabolic activity The metabolic output or input of a culture may be used to estimate viable count. This method has the same caveats as turbidity methods. Examples include: "The rate at which metabolic products such as gases and/or acids are formed by culture reflects the mass of bacteria present. . . The rate at
which a substrate such as glucose or oxygen is used up also refelcts cell mass.
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activity with the added problem of having to dry cultures before employing it. "To calculate the dry weight of cells, they must be separated from the medium by some physical means such as filtration or centrifugation. The cells are then dried, and the resulting mass is weighed
compared to the other methods listed that dry mass determination is employed.
When clumped both or more cells are counted as only a single cell in a viable
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