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Cell Counting and Serial Dilutions

Bacterial Cell Counting

There are many situations in which the number of bacterial cells in a population must be determined.
For example, microbiologists monitor the number of bacteria in our food and water in order to ensure
that it is safe for consumption. Water is also monitored to determine whether it is safe for swimming or
the harvesting of seafood. Healthcare providers need to monitor the concentration of bacteria in a
patients body fluid while the patient is undergoing antibiotic therapy for an infection. Because bacterial
populations may contain thousands to millions of individual cells, most methods of counting them are
based on direct or indirect counts of very small samples taken from the population.

I. Direct Count Methods

Direct Count Methods involve actually counting the cells in a population. In a direct microscope count, a
very small sample (such as 10microL of a cell suspension) of the population is placed into a special
microscope slide that contains a very small counting chamber that has a known volume. The number of
bacterial cells visible in the chamber is counted. Because the chamber has a specific volume, the
concentration (cells/volume) of bacteria in the population is known. Bacterial cells can also be counted
electronically in machines called Coulter counters. In this method, a sample from the population is
placed in the machine. The sample is passed between two electrodes. Every time a cell passes between
the electrodes it causes a disturbance in the electrical field, and the cell is counted. Both direct
microscope counts and electronic cell counts have the advantage that the population count is
determined immediately. However, they both have the disadvantage in that they do not distinguish
between living and dead cells.

In the dilution plate method, a sample from a liquid culture is inoculated and spread onto an agar plate.
To ensure uniformity of sample distribution on the plate spreading in this instance should be with a glass
rod and not by the heavy streak method. The plate is incubated to allow bacterial growth and colonies
are counted. Because every cell in the population will divide and produce a visible colony, the colonies
on the plate represent the number of cells that were present in the sample taken from the population.
Note that you should have between 30 and 300 colonies on a 100mm plate. More than 300 is termed
too numerous to count or TNTC, and fewer than 30 is not statistically significant.

The number of cells in a liquid culture is often very high, but low concentrations may also be
encountered. In a very dilute (low concentration) culture it may be necessary to pellet the cells and
resuspend in a smaller volume of media. Likewise, its usually not practical to take even a small volume
from a high concentration culture and place it directly on the platedoing so would produce more
colonies than could be reasonably counted and spreading may be uneven. In order to achieve a
countable number from a high concentration culture, the serial dilution technique is employed: a series
of dilutions of the original population is made, and samples from each dilution are spread onto agar
plates. The plate that has the appropriate number of colonies (30-300) is counted, and the count is
multiplied by the dilution factor of the plate in order to determine the number of bacteria in the original
population. Dilutions are always made into either sterile culture media or a sterile isotonic salt or buffer
solution

For example, assume you wish to know the number of bacteria in the flask below. If a 1 ml sample was
taken from the flask and placed in 9 ml of water in tube A, the contents of tube A would represent a 10-
fold dilution of the original sample. That is, the cell number per ml would be 1/10th of the original
concentration. If 1 ml was taken from Tube A and placed into 9 ml of H2O in Tube B, that is another 10-
fold dilution, and represents a total concentration decrease of 1/100 from the original. If 1 ml from Tube
B is placed in 9 ml of H20 in Tube C, that is another 10-fold dilution, now representing a 1/1000
concentration decrease of the original. Dilutions such as these can be prepared for many more tubes as
needed. If you spread 1 ml from each of tubes A-C onto culture plates and incubated overnight you
could count colonies the next day.

As shown in the figure only dilution D is acceptable (it has between 30-300 colonies). The other plates
cannot be used because they are either too thick with colonies (A-C) or too thin (E). The calculation of
cell concentration in the original culture is:
32 colonies on plate D x dilution factor of 10,000 = 320,000 cells/ml.

Note: if ml of solution was spread on plate D, youd have to multiply by 2 to get the concentration
in cells/ml. If 1/3 ml was spread, youd have to multiply by 3.

Advantages to the dilution plate method are that it systematically reduces cell density, thus producing at
least one dilution that will yield countable plates. Secondly, it provides a mathematical framework by
which to link the original cell density with the number of colonies on a plate.


Original Cell Density =

Another example you make 3 5-fold dilutions of an unknown concentration. You spread 1/3 ml of the
last dilution onto a plate, and 48 colonies grow after incubation. What was the original concentration of
bacteria?

Answer: Original cell density = = = 18,000 cells/ml. The dilution factor of 125

comes from our 3 5-fold dilutions. The first dilution is 1/5, the second is 1/5 * 1/5, and the third is 1/5 *
1/5 * 1/5 = 1/125.

There are two basic methods for preparing dilution plates. In the pour plate method, a serial dilution is
performed, and samples from each tube are aseptically pipetted into sterile, empty petri dishes. Agar
culture, melted and cooled to 50C, is then aseptically poured over the inoculum. Colonies will grow
throughout the agar and on the surface of the agar. Because of the differences in oxygen availability,
colonies that grow within the agar will have a different appearance than those found on the surface of
the agar but are nonetheless counted as colonies.

In the spread-plate technique, a sample is pipetted directly onto the surface of a solidified agar plate.
The liquid is then aseptically spread over the medium with a sterilized bent glass rod. In both methods,
the plates are incubated for 24-48 hours and the colonies are then counted. One disadvantage to these
techniques is that, because the cells must be given time to grow into visible colonies, the population
count is not determined immediately. Also, the accuracy of this method depends on the assumption
that each cell will grow into a single colony. This is not always truebacteria vary in their arrangements,
and it is possible that a colony may have arisen from more than one cell. Another potential disadvantage
is that these techniques are dependent on accurate dilution technique and thus more subject to human
error than direct cell counts. We used 10-fold dilutions in our example but one could easily do smaller
(or larger) dilutions to have more than one plate to count. Common dilutions are 1:3 and 1:5, for
example. One advantage to dilution plate techniques over direct microscopic or electronic counts,
however, is that these techniques do distinguish between living and dead cells.

II. Indirect Count Methods

Indirect methods do not count cells directly, but rather assess a property of the population, such as
mass or turbidity (cloudiness), that is proportional to the number of cells in the population. For bacterial
populations, increase in cell number is sometimes assessed indirectly by measuring the turbidity of the
sample. The greater the numbers of bacterial cells in a solution, the more the light rays entering the
solution are refracted (bent) by the cells, giving the solution a cloudy appearance. Turbidity can be
measured with an instrument called a spectrophotometer. These measure the amount of light that
passes from a light source to a light collector inside the machine. If a cloudy bacterial sample is placed
between the source and the collector, light will be refracted and will not pass into the collector. The
machine records the amount of light that passes into the collector, and thus relative turbidity of samples
can be determined. This turbidity is proportional to the number of bacterial cells in the population and
thus can be used to estimate the number of bacteria in the population.

III. Methods

This procedure is nonsterile.

Materials:

10 glass tubes in a test-tube rack


Beaker of water
Bottle of dye- safranin or crystal violet
Squeeze bulb
5ml pipet
Piece of parafilm

Procedure
1. Number your tubes 1-10 and place them into a test tube rack.
2. Add 6ml water to tube 1 (this will be your blank) and 6 ml to each of the remaining tubes.
3. Wipe all fingerprints off of the blank tube. Carefully place it in the spectrophotometer and turn
the spec on to let it warm up for 15 minutes. Note: Never leave the spectrophotometer on
without a test tube inserted.
4. Add 1 ml dye to tube two, cover with parafilm in invert 3 times to mix. You now have 7 ml
solution of the maximum concentration you will use.
5. By pipette transfer 1ml from tube 2 to tube 3, mix as above. This is your second concentration.
6. Transfer 1ml to the next tube, mix, and repeat this procedure for all remaining tubes. For the
last tube keep the entire contents and do not discard any liquid the last tube will have more
liquid than the others.
7. Set the wavelength on your spec to 550-600nm. Make sure the filter position is set correctly.
8. Take the blank tube out and set 0% transmittance. Then put the blank back in and set 100%
transmittance.
9. Your spec is now calibrated. Replace the blank with your first dilution. Read and record both
transmittance and absorbance of each of your tubes one by one (be sure to wipe fingerprints off
of your tubes each time). Follow up your last tube with your water blank: is the value zero? It
should be. When you are sure that all your data is correctly recorded, turn off the spec.
10. Plot the data on a computer using Microsoft Excel: dilution on the x-axis and absorbance or
transmittance on the y-axis. You should have a linear plot. You have a choice of either making 2
graphs (one for absorbance and one for transmittance) or plotting both on a single graph (only if
you know how to use 2 y-axes with 2 different scales).
11. Save your graph and e-mail it to your instructor as an attachment (at sredd@rtc.edu). For full
credit, your graph should include all of the following:
Descriptive titles (should describe what the graph is about)
Accurate labels and units on both axes (click your graph and click the Layout tab to
find axis label options)
The numbers on your graph should be realistic. I will be able to tell if you misread the
spectrophotometer.
Names of everyone in your group who worked on the graph