Laboratory 6 Enumeration
One of the central questions a researcher asks when studying any bacterial system is
“how many bacteria are present in this sample?” This applies to environmental samples where
we want to learn about a native population as well as to lab cultures where we are measuring
physiological functions. There are many ways to count bacteria and each of them has advantages
Measuring the optical density (O.D.) of a sample is an indirect method of determining the
number of cells present. The amount of light of a specific wavelength that is absorbed by a
culture is related to the number of cells. This is a very fast and easy way to count cells when
possible. Unfortunately, in order to use this method, you first need to calculate a response factor
(the ratio of the number of cells to the change in absorbance) for the specific cells and media that
you are using. This requires use of an independent method for counting the cells.
Another common method for counting cells is to serially dilute the original sample and
spread-plate the resulting dilutions. This is known as the plate-count method. The colonies that
arise after incubation are counted to determine the original cell density. This method requires
more time and supplies than measuring the optical density but it does result in an absolute
number of cells rather than a relative value. The one important thing to remember when doing
plate-counts is that it only counts the viable cells (those cells that are able to grow under the
specific incubation conditions used).
If you want to count all of the cells present in a sample then a direct-counting method
should be chosen. This generally involves using a microscope to visualize the cells. Special
microscope slides that have surfaces calibrated into precise volumes can be used to determine
exactly how many cells there are in a milliliter of sample. Sometimes stains are used to make the
cells more visible. This method counts all cells whether they are alive or not. The decision as to
whether viable or total cell counts are more relevant will depend on the experimental question
In this lab you will be counting the number of bacteria in an overnight culture of E. coli
using three different methods: optical density, direct counts and plate counts. You will then be
able to calculate a response factor for optical density and compare the direct count and viable
Cultures Equipment - Escherichia coli (broth)
- spectrophotometer - microscopes Supplies - Petroff-Hauser chamber - Tryptic Soy Agar (TSA) plates - bunsen burners - Liquid TSB media - spreaders - Pasteur pipettes and bulbs
- Crystal violet stain
- 95% ethanol
Plate-Counts (Counting by Dilution Series)
1. Make a dilution series of the E. coli culture in liquid TSB media.
a. Aseptically transfer 1 ml of the culture into a tube containing 9 ml of TSB
b. Transfer 1 ml of the resulting tube into another tube with 9 ml of medium. -1-9 – 10). c. Repeat this process until you have produced 9 tubes. (10
2. Spread 0.1 ml of each dilution tube onto a TSA plate.
3. Incubate at 25?C for two days.
4. Count the number of colonies on plates that contain between 20 and 200 well-
5. Calculate the number of cells in the starting culture by multiplying the number of
colonies times the inverse of the dilution.
Optical Density (Counting by Spectrophotometer)
1. Turn on the spectrophotometer and set it to 525 nm.
2. Make a dilution series of the original E. coli culture in liquid TSB media.
a. Transfer 2 ml of the culture into a tube containing 2 ml of TSB medium.
b. Transfer 2 ml of the resulting tube into another tube with 2 ml of medium.
c. Repeat this process until you have produced 4 new tubes. (1:2 – 1:16).
3. Set the transmittance to 0 with no sample in the chamber.
4. Set the absorbance to 0 using un-inoculated TSB medium as a blank.
5. Measure the absorbance of each of the dilution tubes used in the dilution series.
6. Plot the absorbance against the dilution factor.
Direct Counting (Petroff-Hauser Chamber)
1. Use a pipette to transfer 0.9 ml of the starting culture to a clean test-tube.
2. Add 0.1 ml of crystal violet stain to the culture.
3. Use a Pasteur pipette to transfer a drop of the stained culture into the Petroff-Hauser
4. Carefully place the cover slip onto the calibrated surface of the counting chamber.
5. Place the chamber on the microscope stage and focus under the 20X objective to
locate the large counting grids.
6. Switch to high power and count the number of bacteria in at least 20 of the small
(If there are too many bacteria to count, make a 1:10 dilution of the culture and start
7. Average the number of cells and calculate the number per ml. (cubic centimeter).
Lab #6 20 points
Count the number of colonies on plates that contain between 20 and 200 well-separated
Calculate the number of cells in the starting culture by multiplying the number of colonies
times the inverse of the dilution.
Concentration of cells in starting culture = cells/ml
dilution tube absorbance
Plot the absorbance against the dilution factor. (Attach graph)
Average the number of cells/square and calculate the number per ml. (cubic centimeter). thththEach small square is 1/20 X 1/20 of one millimeter square and 1/50 of one millimeter
Average number of cells per square -
Cells per milliliter -
Cells per milliliter - (corrected for dilution by dye)
Use the absorbance graph, the plate-count data and the direct-count data to calculate two
separate response factors (# of cells / absorbance unit) for absorbance at 620nm for E. coli.
Plate count response factor cells/absorbance unit
Direct count response factor cells/absorbance unit
1). Which response factor is more accurate for correlating absorbance to cell numbers? Why?
2). Give an example of an experiment in which you would want to use a direct count and an
example of an experiment in which you would want to use a live count for quantifying bacteria.
Laboratory 7 Coliform Testing
The determination of coliform bacteria in water is one of the oldest and most commonly
used techniques in environmental microbiology. Coliform bacteria are used as indicators of
potential health threats. The presence of bacteria in the coliform group in any sample is
considered to be evidence of fecal contamination and therefore of the possibility that pathogenic
organisms are present. The coliform group is functionally defined according to the way they are detected as “all aerobic and facultative anaerobic, gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48hr at 35?C”. This definition was devised to detect E. coli but it also includes several other genera in the family
Enterobacteriaceae including Klebsiella, Enterobacter and Citrobacter. All of these organisms are coliforms and will give a positive result in these tests but not all of them are necessarily the
result of fecal contamination. In order to help clarify the results, and understand the implications
of a coliform test, it is helpful to identify the number of “fecal coliforms” or to identify the
species of bacteria detected. Fecal coliforms are defined as those coliforms that are capable of
growth at 44?C. The increased temperature is meant to select only those organisms adapted to
growth in the human body. Species typing is carried out by conducting several biochemical tests.
In this lab you will use two different methods two measure the number of coliforms
present in surface water samples. The first method (membrane-filtration) is a one-step process
where coliform colonies are counted directly from filters that have been incubated on m-Endo
media. This method is relatively quick and easy but it is more prone to errors than the alternative
“multiple tube fermentation” technique. Multiple tube fermentation is carried out in three
separate steps. The “presumptive” step involves testing for gas production from lactose fermentation in selective media. The number of coliforms is determined by using the MPN
method during this step. This is followed by the “confirmed” step where positive samples from
the presumptive test are transferred to a more selective media and re-tested for gas production.
Finally, the test is “completed” by isolating single colonies with a positive reaction on m-Endo
You will further characterize the isolates from the completed step by using a commercial
kit to determine the species.
Cultures Equipment - environmental water samples - incubator (35?C) - Escherichia coli - bunsen burners - Enterobacter aerogenes - inoculating loops - Pseudomonas sp.
- pipettes and bulbs
- sterile graduated cylinders
Media - 100 ml dilution bottles
- Durham tubes with lauryl tryptose - sterile filter holders and vacuum
broth (LTB) (2X and 1X) flasks
- Durham tubes with brilliant green
bile (BGB) broth
- m-Endo plates - Enterotube II tests - TSA plates and TSB media in tubes - sterile water - Tryptone broth - Kovac’s reagent
- 1% aqueous tetramethyl-p-Supplies and Reagents phenylenediamine HCl - membrane filters
Procedures (Membrane Filtration Technique)
1. Assemble a sterile filtration apparatus with a membrane filter
2. Aseptically transfer 25 ml of the sample to 225 ml of sterile distilled water.
3. Mixed diluted sample well.
4. Filter 10 ml of diluted water.
5. Aseptically transfer the membrane filter onto the surface of an m-Endo plate.
6. Replace the membrane filter and filter 50 ml of diluted water.
7. Aseptically transfer the membrane filter onto the surface of an m-Endo plate.
8. Replace the membrane filter and filter 100 ml of diluted water.
9. Aseptically transfer the membrane filter onto the surface of an m-Endo plate.
10. Incubate the plates upright at 35?C for 24 hrs.
11. Count the number of dark red colonies with a metallic sheen.
12. Calculate the number of coliforms per 100 mL of the water sample.
Procedures (Multiple Fermentation Tube Technique)
Most Probable Number (MPN) Determination (Presumptive Test)
1. Inoculate 5 tubes of 2X lauryl tryptose broth with 10 ml of your water sample and 5
tubes of 1X broth with 1 ml.
2. Dilute your water sample 1:10 by adding 10 ml to 90 ml of sterile water.
3. Inoculate 5 tubes of 1X broth with 1 ml of the diluted sample and 5 tubes of 1X broth
with 0.1 ml.
4. Incubate the tubes at 35?C for two days.
5. Examine each tube for gas production.
6. Record the results for each tube and use the MPN Table to calculate the number of
coliforms per 100 mL of the water samples.
1. Inoculate BGB tubes with a loop full of culture from each of the most dilute positive
tubes from the presumptive test.
2. Incubate the tubes at 35?C for two days.
3. Examine each tube for gas production.
1. Streak two m-Endo plates from each positive BGB tube.
2. Incubate one plate at 35?C and the other at 44?C for 24 hrs.
3. Examine the plates for the presence of green, metallic colonies.
1. Select a positive E. coli colony from the m-Endo plates.
2. Streak a TSA plate with that colony from the m-Endo plate.
3. Incubate at 35?C for 24 hrs.
Procedures (Enterotube II kit)
1. Pick a colony from the TSA plate using a wooden applicator stick and smear it onto a
filter soaked in tetramethyl-p-phenylenediamine.
2. Development of a purple color indicates a positive reaction.
3. Record the result on the Data sheet.
Inoculating the Tube
1. Remove both caps from the Enterotube without touching the wire inside.
2. Use the tip of the inoculating wire to pick one large (2-3mm) colony from your TSA
plate (there should be a visible amount of cells on the wire).
3. Inoculate the tube by first twisting the wire then withdrawing it through all 12
compartments while slowly turning it.
4. Reinsert the wire back through all 12 compartments until the notch in the wire is even
with the opening of the tube.
5. Break off the handle end of the wire by bending it.
6. Use the handle of the wire to punch holes through the foil covering the air inlets of
the last eight compartments (adonitol, lactose, arabinose, sorbitol, Voges-Proskauer,
dulcitol/PA, urea and citrate).
7. Replace both caps.
8. Incubate at 37?C for 24 hrs.
Period 6 Reading the Results
1. First read the color reactions for all of the tests except indole and Voges-Proskauer
and record the results on the ID form.
2. Use a syringe and needle to pierce the plastic film and add one or two drops of
Kovac’s reagent to the HS / indole compartment. 2
3. Development of a red color in the compartment indicates a positive result.
4. Record the result on the ID form
5. Calculate the biotype number and determine the isolate’s identity from the code book.
6. If the code book indicates that it is necessary, perform the Voges-Proskauer test for
a. Add two drops of 20% KOH and three drops of alpha naphthol to the
b. Development of a red color within 20 min. indicates a positive reaction.
Lab #7 20 points
Water sample name:
Membrane filter test
# of coliform colonies:
10 ml 50 ml 100 ml
# of coliform cells per 100 mL
# of positive tubes
10 ml 1.0 ml 0.1 ml 0.01 ml
Most probable number
How many of the BGB tubes are positive? /
How does this change the estimate of how many coliforms were in the original sample?
How many of the EMB plates showed typical coliform colonies at 35?C?
How many of the EMB plates showed typical coliform colonies at 44?C?
Species typing (Attach Enterotube ID form)
Indole test Species identity