MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
1
Unit IV: Estimating Cell Density of Laboratory Cultures
Activities:
7.1 Viable Plate Counts (Ex 11)
7.2 Estimating Cell Density by Turbidity (Ex 12)
Appendix 4.1: Performing Quantitative Dilutions
p 1
p 4
p 6
Introductions:
In many situations microbiologist need to determine the number of viable (live) organisms in a
sample population. The enumeration of cells in a sample can be accomplished by a number of
methods. Some of the most commonly used are: Viable (standard) Plate Counts, Turbidity, and Total
Direct Counts. Viable plate counts rely on quantitative dilution and cultivation of the sample on agar
media. The colony count on a plate is related back to the volume of initial culture used as inoculum,
and reported as colony forming units (CFU) per milliliter (/mL). This assay only determines the
density of viable and culturable bacteria in the sample. Turbidity, as we saw in Exercise 12, is an
easy way to assess cell density of a culture; however, its values must be calibrated to some other
measure of cell density. The optical distortion causing a turbid culture is due to both live and dead
bacteria; so turbidity values may at times be unreliable when intending to relate them to live cell
abundance. Microscopy can be used for direct counting of bacterial cells (live and dead) in a known
volume of sample. The simplest approach uses a phase contrast-microscopy and a special counting
chamber. The number of cells counted within a known area of the chamber is related back to cell
density. Other microscopy methods for directly counting bacteria use special stains to enhance
visualization of cells; some stains can even differentiate between live (metabolizing) and dead cells.
Often these stains are fluorescent and require a special fluorescent microscope. We will use the later
approach in future lab unit on water quality, but for the next few periods we’ll determine cell density
by viable plate counts and use this value to calibrate turbidity measurements.
7.1 Viable Plate Counts: (Ex 11)
In this method a sample or appropriate dilutions (usually 0.1–1 ml) of it are plated on or in an agar
medium which will support the growth of some, if not all the bacteria in the sample. The method
assumes that, after the incubation period, one cell gives rise to a homogeneous population or colony.
In reality, it is possible that some of the colonies arise not from one individual cell but from chains and
clumps that were not broken apart before plating. Because there is no certainty that all colonies come
from a single cell, the count of colonies is reported as colony forming units (CFU’s) rather than
number of viable cells. The method produces a good measure of the viable cells in the population and
it is much more sensitive than the turbidity method described below. On the negative side, the Viable
Plate Count Method is time consuming, labor intensive, and consumes large amounts of media and
supplies.
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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A sample may contain more bacteria than can be counted as colony-forming units on one plate.
In order to obtain plates with countable numbers of colonies, between 30 and 300 per plate (this
number range produce the most statistically reliable results), a series of dilutions are made of the
sample and plated. This assures that one of the plates will be in the right range to count colonies, and
the dilution factor for that diluted sample can be used to calculate back to the CFU/mL in the original
sample.
Plating the samples may be done in one of two ways: (a) pour plates: the sample is mixed
with molten agar so colonies grow within the agar medium; (b) spread plates: a small sample (usually
0.1 ml) is spread over the surface with a sterile spreader, so colonies grow on the surface of the agar.
We will use the later today (more on the method below).
EXERCISE 11:
Viable (standard) Plate Count:
Materials:
TSB culture of your assigned bacterium
7x tubes with 4.5 mL sterile water
4x 1 mL disposable pipettes
blue pipette pump
6x nutrient agar plates
250 mL beaker with 75% ethanol
Glass spreading rod (“hockey stick”)
Procedures:
Serial Dilution of Culture:
1. Label seven tubes containing 4.5 ml sterile water with a ten-fold dilution value from 101 to 107.
2. Make sure that the cells in the 24h stock culture are suspended homogeneously, with no pellet
remaining on the tube bottom. Do this by tapping the bottom of the tube with your index finger
while holding the tube near the top with the other hand (be very careful not to spill the tube
contents). Alternatively, pipette the culture up and down a few times with a 1 mL pipette.
3. Pipette 0.5 ml of your bacterium culture into the first dilution tube to make the 101 dilution. MIX
VERY WELL by pipetting up and down. This not only mixes your dilution, but also allows the
use of the same pipette in the next transfer.
4. Using the same pipette, continue making dilutions up to 107. Remember to mix very well by
pipetting up and down before making each subsequent dilution. You are now ready to spread your
plates.
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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Spread Plate Technique:
This is an aseptic technique used to spread a volume of liquid bacterial suspension onto the
agar surface of a pre-poured plate using a “hockey-stick” shaped sterile glass spreading rod. Usually,
an amount between 0.1 ml – 1 ml of liquid culture is spread. By convention, when a 0.1 ml of a
dilution is spread this counts as an additional 10 fold dilution from the original source. When a 1 ml of
a dilution is spread this is still counted as the original dilution (this is because 0.1 ml is 1/10 of a ml ).
How is the technique performed?
The well mixed bacterial solution is aseptically pipetted onto the agar surface and spread
evenly on the agar surface with the sterile glass spreading rod. The glass rod is sterilized by dipping it
in a beaker with 75% ethanol and then the alcohol is removed by briefly flaming the glass rod to ignite
and burn-off the ethanol. Do remember that after flaming the rod will be hot and it should be allowed
to cool down before using it to spread the bacteria. The technique will be demonstrated by your
instructor.
5. Label the bottom of six nutrient agar plates from 102 mL to 108 mL. These values represent the
product of the dilution factor times a 0.1 mL inoculum (e.g. 0.1 mL of the 10-2 dilution tube will
inoculate your 10-3 mL plate). Also write your initials and initials of the bacterium you work with.
6. Start at the highest dilution and with a new pipette (you will keep using this one from now on).
Pipette 0.1 ml of the 10-7 dilution onto the plate labeled 10-8 mL. (work fast and move to step 7
right away)
7.
Spread the inoculum with alcohol sterilized glass spreader.
8. Repeat steps 6 and 7 for the other dilution, progress from high dilution to low dilution tubes.
9. When all plates have been spread, invert them and incubate for 48 hours at 37 C.
<<NEXT PERIOD>>
Analysis and Computation:
10. Examine your plates and count the number of colonies on the plates that have between 30-300
colonies. If all of your plates have too many or two few talk to your instructor.
11. Proceed to calculate the number of colony forming units (CFUs) per ml that were in the original
24h bacterial culture using only plates with between 30 and 300 CFUs. The original sample value
will be the CFUs divided by the volume of original culture used as inoculum that is written on the
plate.
For example, if you inoculated the plate with 0.1 mL of the 10-5 dilution, then this means
you had inoculated the plate with 10-6 mL of the original (undiluted) culture. If 50 CFUs
were counted on this plate, labeled 10-6 mL, then the original culture was 50 CFUs / 10-6
mL = 5 x 107 CFU/mL.
Do the same calculations for each of the plates that can be counted. Calculate the average (the
numbers should be relatively close) and report the average as the CFU’s for the original culture.
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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7.2 Estimating Cell Density by Turbidity: (Ex 12)
You may have notice that growth of bacteria in liquid culture turns the broth cloudy, or turbid.
This turbidity is due to the presence of tens of millions to billions of bacteria in the culture. The
spectrophotometer is a device that can be used to quickly measure turbidity, which can be quickly
converted to an estimate cell density using a calibration factor specific for the bacterium and media.
A spectrophotometer quantifies the amount of light from a known internal source that passes,
or is transmitted, through a sample to a detector (photocell). Without any sample in place, the
instrument will read 100% transmittance, or zero absorbance of light. When a test tube containing a
suspension of bacterial cells is inserted in the instrument, some of the source light will be absorbed and
scattered as it passes through the cells. The reduction in the % transmittance will be proportionate to
the cell density; more cells will scatter and absorb more light, so less transmittance. However, one
should know that the relationship between transmittance and absorbance by the sample is not linear. A
sample’s ability to absorb light is referred to as its optical density (O.D.), which equates to %
transmittance according to the following function:
O.D. = 2 – log of percent transmittance
We will make readings off the spectrophotometer in units of absorbance, i.e. optical density.
The most reliable values for bacterial culture turbidity are between 0.1 and 0.8 absorbance. A
minimum number of cells (about 107 cells/mL) are required for an accurate reading at the lower end.
The is a loss in linearity between culture density and absorbance at values greater than 0.8, due to
source light reflection by the very large mass of cells. Furthermore, at these cell densities some of the
cells will effectively shield others cells from the light. Both reflection and self shading will result in an
underestimate the actual cell count. It is important to remember that the turbidity method will only
produce a (rough) estimate of viable cells per ml of culture because both living and dead cells are
measured. However, the method is useful to produce a quick estimate of the cell population as long as
a standard curve is available to calibrate turbidity to cell numbers.
A standard curve is made by plotting the Absorbance values against the CFUs obtained when the same
sample is evaluated by the viable plate count method . This graph can be used to rapidly estimate a
bacterial population in a new sample of the same species. Standard curves are very important for
research concerning bacterial physiological properties, setting up immunological and molecular assays,
etc. In all these lab procedures, known amounts of cells are required to standardize assay conditions;
turbidity allows us to make real-time estimates.
EXERCISE 12:
Turbidity Assay:
Materials:
6 Tubes containing 3 ml of tryptic soy broth (TSB)
24 h old culture of E. coli
5 ml pipettes
green pipette pump
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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Spectronic 20
Kim Wipes
Procedures:
Turbidity of Diluted Culture:
1) Prepare the spectrophotometers as instructed previously (see Appendix 3.1 from last week).
Again use a tube with 3 mL TSB to zero the instrument.
2) While waiting for the spectrophotometer to warm up, prepare a series of two-fold dilutions
(1/2, 1/4, 1/8, 1/16, 1/32) from the 24 h culture of E. coli used in Exercise 14. Proceed as
follows:
a. Label 5 tubes containing 3 ml TSB with the dilution value (1/2, 1/4, 1/8, 1/16, 1/32).
b. Make sure that the cells in the 24h stock culture are suspended homogeneously, with no
pellet remaining on the tube bottom. Do this by tapping the bottom of the tube with your
index finger while holding the tube near the top with the other hand (be very careful not to
spill the tube contents). Alternatively, pipette the culture up and down a few times with a 5
mL pipette.
c. Transfer 3 ml of the culture suspension into the tube labeled ½ and MIX WELL. This is
the first two-fold dilution of your stock solution.
d. Transfer 3 ml of the ½ dilution into the tube labeled ¼ and mix well. What is the
bacterium content of this dilution with respect to the stock culture?
e. Keep repeating this serial procedure to make 1/ 8, 1/ 16, and 1/ 32 dilutions of the stock
culture
3) Read the Absorbance of each of the 5 dilutions made and record your results in the lab
notebook.
<< NEXT WEEK>>
Standardize Turbidity to Viable Plate Count:
4) Using the estimate of CFU/mL in the original culture, calculate the CFU/mL for each dilution
you measured turbidity on. Enter these data in an Excel spreadsheet for plotting.
5) Plot CFU/mL (y-axis) versus turbidity (x – axis); label axes well, including units. Fit a linear
regression to your data and place the equation on your plot. Attach this graph in your lab
notebook, and write a proper figure legend underneath the plot.
6) Compare your result with a lab colleague working with a different bacterium. Was the
turbidity to CFU/mL relationship similar? Think how you would test this statistically.
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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Appendix 4.1
Performing Quantitative Dilutions
What does it mean to do a quantitative dilution?
It simply means to thin down a culture by mixing it with another liquid, called the diluent. The actual
dilution is most commonly calculated by dividing the initial sample volume by the total volume
obtained after mixing with the diluent.
Why do we quantitative dilute? There are many reasons, including diluting a sample for analyzes or
diluting a stock solution to working solutions. For example one may need to prepare a small amount
of a solution at a very low concentration. Say, you need to prepare 1ml of 0.05% NaCl w/v; this will
require you to weight 0.0005 g of NaCl which is not possible. A way to get around it is to make a
stock solution of say 0.1% NaCl and dilute a portion of it to make your 0.05% dilution.
In microbiology we may have a culture containing millions of bacteria per ml of culture. We cannot
count millions but we can count a small sample of the population and relate back to the original
population. Two things are needed to accomplish this:
a) Need to get a sample of the population that is small enough but manageable
b) Need to keep track of what size is this fraction of the population so you can figure out what the
total is.
These are accomplished by using the appropriate quantitative dilution techniques
Dilution = volume of sample / total volume (i.e. sample + diluent)
Division of the numerator (volume of original sample) by the denominator (total final volume of the
dilution) will give you the amount of material (sample) per milliliter of dilution. So, if you mix 1 ml
of bacterial sample with 2 ml of saline solution you end with a total volume of 3 ml of diluted sample
1 ml / 3 ml = 0.33 (0.33 means that in your dilute sample every ml has 0.33 ml of the original bacteria
culture)
Another question that you may ask is: how many times has the sample being diluted?
To figure this out we simply divide the initial concentration IC (which for the original sample is
assigned the value of 1) by the final concentration FC. The resulting value is called the dilution
factor DF.
For our working example this would be: DF = IC/FC 1 / 0.33 = 3 the Dilution Factor (DF)for
this dilution is 3 and it means that your original sample was diluted 3X
You can use the dilution factor to relate back to the original sample. In the example above; the
dilution factor indicates to us that in the original sample of bacteria there are 3 times as many
bacteria/ml as in the diluted sample
In some books you will find that they refer to The Dilution Factor as the number or factor used to
multiply to obtain the original concentration of a sample. It is generally is calculated by the inverse
or reciprocal of the dilution, DF = 1 / Dilution . In the example above DF = 1 / 0.33 = 3. THE
SAME THING! But you may find that this way is easier for calculating DF.
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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Dilutions can be expressed as a exponent or as a ratio. An exponent is usually used only when
working on dilutions dealing with factors of 10. When using ratios you should bear in mind that
according to the American Society for Microbiology Style Manual , dilution ratios may be reported
with either colons ( : ) or slash ( / ). It is very important that you be aware that there is a difference
among them. For example, 1 / 2 and 1 : 2 ARE NOT THE SAME THING !
A slash ( / ) indicates that the ratio is part of a whole; so, 1 / 2 means 1 of 2 parts
A colon ( : ) indicates the ratio is 1 part to 2 parts with a total of 3 parts
So, How would you express the ratio 1 / 2 using a colon ( : ) ?
Answer
1: 1
More Examples :
a) If 1 ml of bacterial culture is mixed with 9 ml of water the dilution of the bacterial sample would be
Dilution = 1ml/ 1+ 9 ml = 0.1
This dilution expressed as ratio would be 1:9 (for a total of 10 ml) or 1 / 10
Or, expressed as exponent 101
What is the dilution factor? DF = 1 / 0.1 = 10
b) If 0.5 ml of bacterial suspension is mixed with 9.5ml of water the dilution of the bacterial sample
would be
Dilution = 0.5 ml / 0.5 + 9.5 ml = 0.05, or 1 / 20, or 1:19
What is the dilution factor?
DF = 1 / 0.05 = 20
c) 1 ml of bacterial suspension is mixed with 3 ml of diluent
Dilution = 1 / 1+ 3 = 0.25, or 1 / 4 or 1: 3
DF = 1/ 0.25 = 4
Types of Dilution:
In Biology and chemistry working with dilutions is a very important skills and there are several types
of dilutions depending on what is to be accomplished.
1) Dilution of a solution to specified volume in one step : Usually it is used when there is a stock
solution and from it a specific volume of the lesser concentration is required
For example you have a stock solution of 30 % sugar and want to prepare 50 ml a solution containing
8% sugar. All you need is to calculate how much volume of the original stock solution you will need
to mix with the diluent to end with 50 ml of 8%
Use the formula C1 V1 = C2 V2
V1 = C2 x V2 / C1 = 8% x 50 ml / 30% = 13 ml you will need to take 13 ml of the 30% stock
solution and mix it with the diluent to 50 ml
2) Dilution of a Solution to specified volume in several steps
MICRB 202: Introductory Microbiology Lab
Unit IV: Estimating Cell Density of Laboratory Cultures
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This is needed for large dilutions. Sometimes the dilution have to be done in two or more steps.
Example:
You need to make 100 ml of a media containing 1 g/ml of the antibiotic ampicillin, you cannot
weight 100 g but there is a stock solution of ampicillin containing 0.5 g /ml
Still, this stock solution is 500,000 X (0.5 g x106 g/g = 500,000 /ml ) more concentrated than the
required final concentration. This can be easily handled if the dilution is done in two steps.
a) make a 10 ml of a stock with a concentration of 0.05 % and
b) use a dilution from this 0.05% stock to make your media (in both cases you use the formula
shown above.
3) Serial dilutions
In serial dilutions a certain volume of material is being transferred from one vessel to another with the
purpose of increasing the dilution of the material by certain increments. For example in a two fold
serial dilutions the dilution factor is doubled each time
Example:
Make 3 two-fold serial dilutions from a bacterial culture. In addition, you are told to aim for a final
volume for each dilution of 6 ml. This means that you start with the original culture 1/1 and from this,
dilute down to produce 1 /2 , 1 /4, 1 /8 . You may use C1 V1 = C2 V2
i) to make the ½ from the stock culture
C1 = 1/1
V1 = ?
C2 = 1 /2 = 0.5
V2 = 6ml
V1 = C2 x V2 / C1 0.5 x 6 ml / 1 = 3 ml
ii) to make the ¼ from the ½ dilution
C1 = 1/ 2
V1 = ?
C2 = 1 / 4 = 0.25
V2 = 6ml
V1 = C2 x V2 / C1 0.25 x 6 ml / 0.5 = 3 ml
iii) to make the 1/8 from the ¼ dilution
C1 = 1/ 4 = 0.25
V1 = ?
C2 = 1 / 8 = 0.125
V2 = 6ml
V1 = C2 x V2 / C1 0.125 x 6 ml / 0.25 = 3 ml