Quantifying Bacteria
review, understanding results, conclusion expectations
Review: excerpt from Ch3 of J. Kirk Brown’s text
Quantifying Bacteria
Serial Dilution and Plate Counts
It is often necessary to determine the number of bacteria that are present in a culture. Inoculating an agar plate with a
portion the culture and counting the colonies is one way to quantify the bacteria in the culture. However, the entire
culture cannot be directly inoculated onto the agar plate, because even 100 microliters of bacterial culture may contain
billions of bacteria that would overcrowd the agar plate. To determine the number of bacteria, the culture must first be
diluted. The culture must be diluted at least one million times or 1ml into 1,000 L to obtain an agar plate with single
colonies. Not only is it unlikely you have a container that could hold 1,000L, but the cost in broth needed to dilute the
bacteria would be prohibitive! Serial dilutions are used instead. See Figure 3.19. A serial dilution means diluting the
culture several times by the same dilution factor. For example, 10 times and then diluting that culture another 10
times, and so on. By making seven serial dilutions, a 10 million fold dilution or a 10-7 dilution is made. To perform a
serial dilution, 100 µl of culture is added to 900 µl of broth to make a 100X dilution, and so on, until the original
culture has been diluted 10 million times. The culture from each tube is then plated on agar plates. Each individual
bacterium spread on the plate will grow into a colony and is referred to as a colony forming unit (CFU). The number
of bacteria in the original culture can then be calculated by multiplying the final number of colonies on the plate by the
dilution factor. For example, from Figure
3.19, there are 133 colonies on the plate
diluted 103 times indicating there were 133
viable bacteria (or 133 CFU) in 100 µl that
was plated. To calculate the number of CFU
in the undiluted culture, the number of CFU
must by multiplied by the same dilution
factor that is 133 X 103 or 1.33 X 105 in
scientific notation. This is the number of
CFU present in 100 µl of original culture. To
convert the answer to CFU per ml rather than
CFU per 100 µl of culture, the CFU per 100
µl must be multiplied by a unit conversion to
convert µl to ml.
Calculating Concentration of bacteria (
(
)
)X (dilution factor) X conversion factor
Ex: (
) X (103) X
=
or
Sample problem/ food for thought: Calculate the CFU/ml for…
If you had 34 Colonies growing on your 105 dilution plate.
And you had 2 Colonies growing on your 106 dilution plate.
In an ideal world the CFU/ml calculations would be the same, what might account for the difference?
Activity 3.5 Results analysis (excerpt from book)
Retrieve the culture plates from the refrigerator (or incubator if you just did the lab yesterday) and arrange
them on the laboratory bench from least to most dilute. Record your observations including the number of
CFU across all the plates. Do not open plates!


Plates that have bacterial growth so dense that no single colonies can be seen should be recorded as
having a “lawn” of bacteria.
Plates where the single colonies can be seen but are too desne to actually count (more than 200
colonies per plate) should be recorded as “too many to count, “ or “TMTC”
To count colonies, use a skiny sharpie and place a dot on the BOTTOM of the plate in the middle of where
each colony lies as it is counted. If you lose your place, you can wipe off the dots with 70% alcohol (spray
located in the hood) and start again.
To accurately calculate the number of bacteria per ml in the original culture, count each plate that has
between 200 CFU (not too many colonies) and 30 CFU (not too few colonies), and multiply the number of
CFU by the dilution factor for the plate. You also need to adjust for the fraction of the dilution that was
actually inoculated. (The actual CFU in the dilution would have been 10 times higher if the entire 1,000 µl
had been spread and allowed to grow on the petri dish.)
Notebook guidelines
Results:
Record # figure: diagrams for each petri dish….don’t forget the caption
 Draw your petri dish results as accurately as possible
 Include a colony description/ count…Lawn (solid bacterial cover), Too many to count (selfexplanatory), or number of bacterial colonies grown on plate
 Optional: You can glue in printed photographs as additional figures in your results…don’t forget they
need #/Figure/caption…how you add them in… side by side the drawings, or below the drawings is
up to you (Warning: photography does NOT replace hand drawings!)
 With so many figures you might consider a summary description of results!
Record Observations: too!
FYI:
If 2 or more plates with countable colonies look the same there was a problem
Either w/ the plating or the dilution
Calculations
Include the general equation for how to calculate the CFU/ml of original bacterial solution.
Show actual calculations for one of the plates that contain 200 CFU or fewer. You could include a
summary description of each sample calculation.
If more than 1 plate is countable, summarize the results of all calculations in a processed data table.
Conclusion
So what was the original concentration of E.coli HB101 per ml of solution? How confident are you of this
number what is your best estimate/ or estimate range, any thoughts about sig figs? How could you change the experiment to more
accurately determine the number of CFU/ml in the original culture?