Coliform Bacteria Sampling and m-Coli Blue Analysis Methodologies

Rob Turner – 2018

Overview:
This document provides guidance and protocols to follow for the collection and analysis of water
samples for the sake of enumerating fecal coliform bacteria. The analysis procedure consists of
filtering, incubation, and counting. Filtering of coliform bacteria samples should take place as
soon as possible after collection. The samples are unpreserved so the microbial ecology will be
changing in the bottle! Accordingly, filtering should be done the same day as sample collection.
If this is not possible, samples can be stored in the refrigerator (not the freezer) for up to one day.
After each sample is filtered, the filter paper is placed in a petri dish with growth media then
incubated at 35° C for 24 hours (Figures 1-3). Immediately after the incubation period ends, you
count how many “other” coliform and E. coli colonies have grown on the filter paper and
calculate the density of colonies per sample filtered (see Figure 4). FYI – our method is one of
several EPA approved methods for enumerating fecal coliform bacteria in environmental
samples. Specifically, it is known as EPA Method 10029 – m-ColiBlue24.1,2, 3

Sampling Equipment:
Cooler, ice packs, nitrile gloves, at least one liter of de-ionized (DI) water, 120ml clear plastic
bottles, labels, GPS, Sharpie, in situ water testing meters as desired, Van Dorn bottle or swing
arm sampler if needed to get samples at depth, in a catch basin, under a bridge or pier, or away
from the shoreline.

Sampling Procedures:
In the lab
1. Make sure all sample bottles are new (sealed) or have been sterilized with a 10% HCL
acid wash.
2. Gather a cooler, nitrile gloves, ice packs, labels, a Sharpie, and at least a liter of DI water.
3. Also gather the in situ water quality measuring meters you’ll want in the field (dissolved
oxygen meter, turbidity meter, pH meter, etc.). Don’t forget to calibrate!
4. Gather three of the clear plastic 120 ml bottles and lids to be used for field blanks.

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 5. Gather three of the clear plastic 120 ml bottles and lids for each site you will collect
samples.
6. Throw in at least 6 extra bottles in case bottles get contaminated, or you lose lids, or you
come upon another site or two where you want to collect samples.

In the field
7. When you get to the site, turn on the dissolved oxygen meter. It needs to warm up.
8. If this is your first time collecting samples at this site, take a GPS reading and make a
sketch of the site in your notebook.
9. Record the environmental characteristics of the site, along with weather information, in
your notebook. What is unique about this site? Why sample here? Where, exactly, are
you taking samples? On a shoreline? Off a bridge? Surface or depth? Any signs of
contamination? Do ducks or geese or crows reside here? Does the water come from a
residential area? A forest? Farms? City streets?
10. Put on nitrile gloves.
11. Take measurements with any sensors you have (e.g., temperature, conductivity, dissolved
oxygen, turbidity, etc.). Rinse the probes with DI water.
12. Label three bottles with the character and location of the site (e.g., UWB runoff, Site
RS1), the date, and either A, B, or C (indicating which of the triplicate samples this bottle
represents).
13. Triple rinse each bottle with the sample water, then fill them with sample water, close
them, and place them in the cooler.
14. Repeat steps 5-10 until you have collected samples at all sites.
15. While still in the field, label three bottles as field blanks, along with the date and either A,
B, or C (indicating which of the triplicate samples the bottle represents).
16. Triple rinse each of the three blank bottles with DI water, then fill them up with DI water,
close them, and place them in the cooler.
17. Take your samples to the lab as soon as possible for analysis. Place them in the
refrigerator if you cannot filter them right away. You can wait as long as a day to analyze
the samples, but it is MUCH BETTER to filter them on the same day. The microbial
populations in the sample can/will change over time.

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Analysis Equipment:
Lab coat, vacuum pump, side-arm Erlenmyer flask, filtering stage, filtering stage funnel, 47mm
white gridded membrane filter paper, petri pad dishes, m-ColiBlue broth, forceps, sharpie, 10%
hydrochloric acid solution (200ml), 70% solution of ethanol, plastic bins for holding the acid
baths and the ethanol bath, UV Light “oven”, de-ionized (DI) water, baking soda, paper towels.

Analysis Procedures:
1. Put on a lab coat, nitrile gloves, and goggles.
2. Set up an acid wash station (see the Acid Washing and Nutrient Filtering Procedures) and
a separate 70% ethanol bath in the fume hood. Acid washing of empty sample bottles can
take place during the filtering procedure or afterwards.
3. Ethanol wash the forceps, filter stage, and funnel cap. Then rinse them with DI water
over the sink. Let them dry in the fume hood.
4. Place some paper towels on the lab bench and set up the vacuum air pump and the
sidearm flask for filtering (see Figure 1).

filtering stage

side-arm Erlenmyer flask

forceps

Vacuum pump
Figure 1. Filtering apparatus for preparing filter paper for coliform bacteria analysis.

5. Place the forceps, filter stage, and funnel cap in the UV oven for 3 minutes.

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6. Organize your samples by site on the lab bench and place a petri pad dish next to each
sample bottle. Label each petri pad dish with the I.D. of the site, the date, and sample
replicate letter on the side that says Millipore.
7. Once the petri dish is labeled, INVERT the petri dish so what you have written is upside
down in contact with the lab bench.
8. Using the forceps, remove the filter paper from its individual package (Figure 2), discard
the blue backing paper, then place the filter paper with the GRID SIDE UP on the filter
stage, and then place the funnel on top. Do not touch the filter paper with your hands.

Figure 2. 47 mm white gridded membrane filter paper Figure 3. Ampules of m-Coli blue broth

9. Turn on the vacuum air pump.

10. Starting with a field blank sample, slowly pour 100ml of water on to the filter paper
within the funnel. Minimize the amount of water that touches the funnel. You don’t
want bacteria adsorbing to the funnel.
11. After the sample has been filtered and the vacuum has made the filter paper look dry, turn
off the vacuum.
12. Open up the petri dish for this sample, then snap open an ampule of m-ColiBlue 24 broth
(Figure 3) and squeeze it steadily onto the white pad in the petri dish. Spread it out
evenly and make sure there are no air bubbles on the pad in the petri dish. You can pop
and suck up bubbles with the tip of the empty ampule. Don’t touch the pad or broth with
anything else.
13. Carefully pull up the filter paper off the filtering stage with the sterile forceps and place
the filter paper GRID SIDE UP onto the media of the petri pad dish. Make sure all of
the filter is in contact and remove any trapped air. Bubbles under the filter paper or warps

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 in the filter paper will prevent the m-ColiBlue broth from saturating it. You will not get
any growth anywhere sections of filter paper are not in contact with the blue broth. This
will confound your counts.
14. Rinse the funnel and filter stage with D.I. water from a squeeze bottle. Turn on the
Should I dilute my sample?

vacuum pump to dry out the filter stage.

15. Repeat steps 8-14 for the other two field blanks that you should have. As an alternative,
you can hold off on filtering other blanks to see if your filtering apparatus is becoming
contaminated.

16. Time to stop and think. Before you follow the procedure above for your environmental
samples, you need to consider the degree of coliform bacteria contamination in your
samples. If you have a sample that is highly “enriched” in bacteria and you filter too high of a
volume, your filter paper will be completely overgrown in 24 hours and you won’t be able to
count individual colonies. If your samples are relatively devoid of coliform bacteria and you
filter too low of a volume, then you may get no results. See Figure 4 at the end of this guidance
document to see how much the growth can vary and how that can make counting a challenge. If
you are clueless, it is best to filter the same sample twice at different volumes until you get a feel
for what will produce good results. 100 ml of sample run through the filter paper can be
considered a standard amount. You might want to filter as much as 200 ml if the samples are
quite clean (such as a “pristine” mountain stream). If you know there are many critters pooping
within or near your sample site, you should dilute your sample with deionized water by 50%. If it
is grossly contaminated, as in the crow roost zone of our wetland, make a 10% solution (90ml of
deionized water + 10 ml of sample). Some of our sites are so bad (i.e., SW8) that we need to
make a 1% solution.

17. If you need to do dilutions of your samples, use 100 ml glass graduated cylinders and pipettes
that have been rinsed with the ethanol solution, rinsed with de-ionized water, and placed in the
UV oven for 3 minutes. Change out or clean your graduated cylinders between every sample (not
just between sites, but between each sample). Dispose of pipette tips after every sample dilution.
18. When your sample, diluted or not is ready for filtering, follow steps 8-14 above.
19. Then filter samples B and C from the same site following steps 8-14 and 17 (if necessary).

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20. When you want to filter samples collected from a different site you must swap out or sterilize the
filter stage, funnel, and forceps. Swish the dirty gear around in the ethanol bath, allow each piece
to dry in the fume hood (it goes fast), then give them a thorough rinse with deionized water over
the sink. The swishing in ethanol solution should kill bacteria from the last filtering. But you
don’t want the ethanol on the gear to kill bacteria during the next filtering, thus the drying and
rinsing with DI water. Then place the gear in the UV oven for 3 minutes. If you have another
clean filter stage and funnel ready for action, you can use it immediately while your first filtering
apparatus is cleaned.
21. Filter all of your samples following steps 8-14, 17, and 20, as well as steps 22-24 below.
22. After you have successfully filtered a sample, you can dump any water remaining in the sample
bottle into the sink. The bottle can be placed in a container for acid washing.
23. Keep an eye on the level of sample water collecting in the Erlenmyer flask. DO NOT let it fill
up to the side arm part of the flask. If it gets sucked up into the vacuum pump, you will
ruin the pump. After it is filled up ~ halfway, empty the sample water into a large beaker
or down the sink.
24. Once all the samples are filtered, incubate all the petri pad dishes in the incubator at 35° C
for 24 hours. When you place them in the incubator, put them in UPSIDE DOWN (your
labeling facing up) so the condensation that will form on the upside will not obscure your
view of the filter paper and colonies.
25. Clean up the lab. This includes:
a. ethanol washing the filtering gear one more time, including the Erlenmyer flask,
leaving the gear on the rack in the fume hood to dry;
b. pouring the ethanol rinse solution back in the ethanol bottle with the funnel;
c. putting the vacuum pump away;
d. neutralizing the acid bath water with baking soda and dumping it in the sink;
e. recording the volume of neutralized acid bath water you have dumped on the
sheet over the sink;
f. wiping the counters; and
g. giving the sink that has received your sample water a good rinse.
26. Remove your lab coat, gloves, and goggles. Wash your hands with soap and water.
27. Turn out the lights, go away, and have a beverage.

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28. Come back 24 hours later and remove the Petri dishes from the incubator. This timing is
very important! If you leave the petri dishes and filter papers in longer, you will get more
growth. To make your results comparable to all the coliform bacteria results ever
reported, you need to stop the incubation after this universally agreed-upon duration.
29. Turn off the incubator.
30. It is best to count the colonies right away. If this is not possible, place the Petri dishes,
upside down, in the refrigerator. They can stay in the refrigerator prior to counting for up
to one day. [Well, to be truthful, they are stable for longer, but it is best to do your counts
as soon as possible. Things can happen to your petri dishes if left in the refrigerator.
Terrible things…

31. To count your colonies…

a. Log into one of the computers in the lab and download from the course Canvas
site the Excel spreadsheet titled Coliform Bacteria Counting Spreadsheet. It will
help you record your raw colony counts and calculate your colonies per 100 ml of
sample, taking into account dilution factors. It will also calculate mean values
and standard deviations around the means of replicate samples.
b. Get a counter out of the drawer marked lab supplies.
c. Take your petri dishes and counter to the big microscope by the window.
d. Set up the lamps on the flexible arms so they point at the microscope stage.
e. Turn on the lamp and place a petri dish on the microscope stage and have a look.
f. If there is little growth of colonies (few dots), simply count all the blue (E. coli
and red (other coliform) dots on the filter paper, enter that data in your
spreadsheet – taking care to indicate the site location and other information
requested - and skip to step 32. See Figure 4 on page 9.
g. If there is too much growth of either red or blue colonies so that the colonies
have grown together into indistinct masses (i.e., are confluent), record “Too
numerous to count” (or TNTC) in your notebook and spreadsheet.
h. If the growth is abundant (lots of dots), but not confluent and colonies are
more or less uniformly distributed, follow steps 31 i-q. If the growth is
abundant but not uniformly distributed (nor confluent), skip to step 31 r.

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 i. Count the number of red dots (other coliform colonies) in 6 squares. Record this
value in your spreadsheet.
j. Count the number of blue dots (E. coli colonies) in 6 squares. Record this value
in your spreadsheet.
k. Move the petri dish on the microscope stage so you are looking at a different area.
l. Do steps i and j again.
m. Move the petri dish on the microscope stage so you are looking at yet another
area.
n. Do steps i and j again.
o. The spreadsheet will calculate the average other coliform colonies per square and
the average E. coli colonies per square for each sample.
p. The spreadsheet will multiply your average number of red colonies per square and
number of blue colonies per square by the total number of squares in the growth
circle (100) to get an estimate of the total number of other coliform colonies and
E. coli colonies on your filter paper.
q. Once you have typed in the volume of environmental sample you filtered (thus
taking into account any dilution you might have done), the spreadsheet will
calculate the colony forming units/100 ml for both other coliform and E. coli for
each sample.
r. If the growth is abundant but not uniformly distributed, do your best to get a
representative estimation of colonies per square. Do steps 30 i-l above at least 5
times, but don’t look (i.e., randomize) when you move the petri dish between
counts.
s. Go through steps 31 o-q above.
32. You should have at least two other petri pad dishes with filter paper that filtered sample
water collected at the same site on the same day. Those were your replicate samples.
Follow the various elements of step 31 for those replicate samples.
33. When you have entered your data for replicate samples in the spreadsheet, it will
calculate an average of the other E. coli and coliform CFU/100ml results for all the
replicate samples you filtered for each site, as well as a standard deviation around the
means.

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 34. Follow steps 31-33 for the rest of your petri dishes.
35. When you have counted the colonies on all of your filtered samples and blanks, place the
petri pad dishes in the refrigerator. Keep the lids on so you don’t stink up the lab or
refrigerator.
36. Turn off the lamp adjacent to the microscope and cover the microscope.
37. Save your spreadsheet and mail it to yourself, or download it onto a portable USB drive.
38. Log off the computer.
39. You are done. Do a happy dance.

A B

Photo by Paul Parker, 2013 Photo by Anne Power, 2013

Figure 4. Examples of filter papers with colonies of coliform bacteria produced by running a water
sample though the filter paper and feeding the bacteria with m-ColiBlue Broth. The blue blobs are
colonies of E. coli bacteria. Red blobs are colonies of “other” coliform bacteria.
When filtering water samples for coliform bacteria counting, you want to shoot for producing filter
papers with the kind of growth you see in photo A. Clearly the sample that produced the filter paper in
photo B should have been diluted prior to filtering. Imagine counting that one! Sometimes, only the
center of a colony will show color. A colony with any amount of red color should be counted towards
your other coliform counts. A colony with any amount of blue color should be counted towards your
E. coli counts.

References

1 – Hach Company (2012). Coliforms Total and E. Coli – USEPA Membrane Filtration Method.
http://www.hach.com/asset-get.download-en.jsa?code=56438
2 – National Water Quality Monitoring Council (n.d.). Hach Co.: 10029:  E. coli by m-ColiBlue24 Broth Procedure
for Membrane Filtration, in National Environmental Methods Index.
https://www.nemi.gov/methods/method_summary/5577/

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3 – US Environmental Protection Agency (2008). Analytical Methods Approved for Drinking Water Compliance
Monitoring under the Total Coliform Rule. National Environmental Publications Information System
http://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=P1008J4A.txt

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