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Enumeration of Bacteria

Background and Introduction

Sometimes it is necessary to determine how many bacteria are present in a liquid culture, that is to determine the concentration or titer of the solution.  This task can seem a bit daunting when one considers that there can be as many as 1,000,000,000 bacteria per milliliter.  There are two basic method types, direct and indirect, that are used to assist in this task. 

 

Indirect Methods 

  1. Spectrophotometer – The more concentrated a liquid culture, the more turbid that culture and the more light that it scatters.  A spectrophotometer can be used to measure the light scattering/turbidity (generally termed the Optical Density (OD)) of a culture and that can be equated with the number of cells per milliliter.  This is a very rapid way to estimate culture titer.  Most frequently the OD is measured at 600 nm and is termed the OD600.  A concentration (bacteria/mL) versus OD600 curve is then used to estimate the titer. 

  2. Measurement of the dry weight of the bacterial cells 

  3. Measurement of metabolic products produced by the bacterial cells 

 

Direct Methods 

  1. Microscopic counts – A set volume of a liquid culture is placed onto a microscope slide and one actually counts the number of cells.  The advantage of this method is that no incubation time is required to obtain results.  However, it can take a great deal of time to count that many cells and there is no way to distinguish between living and dead cells. 

  2. MPN (Most Probable Number) – This is a statistical estimation that will be discussed in greater detail later in the semester. 

  3. Standard Plate Count (SPC) – In this method, a liquid culture undergoes a series of dilutions.  Set aliquots of the final, dilute solution are spread onto an agar plate and the plates are incubated overnight.  Every cell that was spread onto the plate develops into a visible colony.  These colonies can be counted and since they originated from a single cell they represent the number of cells in the final aliquot that was spread onto the plate.  The dilutions made from the original culture can then be taken into account and one can work backwards to determine the concentration (titer) of the original culture. 

Today in lab, we will use the Standard Plate Count.  In order to do a standard plate count, one must understand how to figure dilutions.  Before beginning the dilution section, review "exponents" in the Appendix.

Dilutions

Background and Introduction

Remember that the SPC method requires one to figure backwards to the original culture concentration (titer) by taking into account the number of colonies on the plates and the dilutions that were made before an aliquot was spread onto a plate.  Following is an example of a set of dilutions that could be made on a culture before plating. 

Determining Titer from Spread Plate Count (SPC) Procedure 

Method 1 dilution scheme

First, consider that the number of colonies on each plate indicates the number of bacterial cells that were in the final 0.1 mL aliquot that was spread on the plate.  Each of the three plates shown above were spread with the same volume of liquid (0.1 mL) from bottle 3 so we can average the number of colonies on the three plates: 

90 + 106 + 98 = 294 colonies 

294 / 3 = 98 colonies 

Remember, this is also the average number of cells per 0.1 mL of fluid taken from bottle 3.  Thus the concentration (titer) of bottle 3 can be calculated: 

98 cells / 0.1 mL = 980 cells/mL in bottle 3 

In order to figure the titer of bottle 2, we must recall General Chemistry and the equation C1V1 = C2V2, where C1 is the concentration of the more concentrated solution, V1 is the volume taken out of that solution, C2 is the concentration of the more dilute solution and V2 is the final total volume of the more dilute solution.  Let’s manipulate this equation to apply to our current situation and allow us to calculate the concentration of bottle 2: 

Cbottle2Vremoved from bottle2 = Cbottle3Vfinal in bottle3 

Cbottle2 (11 mL) = 980 cells/mL (110 mL) 

Cbottle2 = 9.8 X 103 cells/mL 

We can used this same equation to determine the titer in bottle 1 and finally in culture A: 

Cbottle1Vremoved from bottle1 = Cbottle2Vfinal in bottle2 

Cbottle1 (1 mL) = 9.8 X 103 cells/mL (100 mL) 

Cbottle1 = 9.8 X 105 cells/mL 

CcultureAVremoved from cultureA = Cbottle1Vfinal in bottle1 

CcultureA (1 mL) = 9.8 X 105 cells/mL (100 mL) 

CcultureA = 9.8 X 107 cells/mL 

Method 2 

This method uses more of a pictorial approach.  The first step of this method is to write each dilution as a fraction with the volume transferred out of the more concentrated solution over the total volume of the less concentrated solution.  In the final transfer, 0.1 mL is transferred out of the more concentration solution and put directly on the plate.  This is a 1/10 dilution. dilution scheme

In the second step of this method, the fractions are converted to scientific notation:dilution scheme with fractions

In step 3, the product of the individual dilution factors is calculated to give the final dilution factor: 

Step 3: 1 X 10-2 * 1 X 10-2 * 1 X 10-1 * 1 X 10-1 = 1 X 10-6 (final dilution factor) 

In microbiology, the reciprocal of the final dilution factor is called the plating factor.  In step 4, the plating factor is calculated. 

Step 4: plating factor (p.f.) = 1/(1 X 10-6

Plating factor (p. f.) = 1 X 10

Finally, to determine the concentration of culture A, the average number of colonies (as calculated in method 1) is multiplied by the plating factor: 

Step 5: p. f. * average # of colonies = titer of original culture 

(1 X 106) * 98 = 9.8 X 107 bacteria/mL 

**As can be seen, this is the same number that was determined using method 1!** 

Please feel free to use either method 1 or 2.  Method 1 is more concise and more clearly tracks units.  Unfortunately, in microbiology method 2 is commonly used and even if you select method 1 it will be necessary to be familiar with the plating factor term.  

* Significant figures: The greatest number of significant figures that can be known is three, as it is impossible to count halves of colonies.  However, less than three significant figures may be retained depending on the number of colonies and the accuracy of the measuring devices selected. 

 

Procedure

Results

Count the isolated colonies on each plate. Remember that each plate must have between 30-300 colonies to be used to calculate the titer.

isolated colonies on a spread plateisolated colonies on a spread plate

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