MadSci Network: Microbiology

Re: What do Colony Forming units of aerobic bacteria in water mean?

Date: Wed Feb 18 15:49:48 2009
Posted By: Dean Cliver, Faculty, Food Safety Unit, Uiversity of California, Davis
Area of science: Microbiology
ID: 1233881312.Mi

First, let’s clarify the question: You say you tested samples of water, but don’t understand what results expressed in colony forming units mean. This tells me that you collected some water samples, but someone else did the testing. It would also have been helpful if you had mentioned what kind of water you were sampling. I am guessing that it was drinking water, but it might also have been well water or surface water (lakes, rivers, etc.) – probably not wastewater (sewage).

Water is the “blood” of the world; almost everything except oxygen that is essential to life on earth is carried by water. Our bodies are mostly water, and most of our foods are mostly water. Most water inside of living things has no bacteria in it, but most water out in the environment (including in water pipes) has at least some bacteria. We live with these all the time, and they generally do us no harm.

Bacteria are microbes (microorganisms), meaning that we need a microscope to see one. There are microscope methods for counting bacteria in water, but the microscope is seldom used for this purpose because it is slow. Also, many methods of counting bacteria with a microscope cannot determine whether the bacteria in the sample were alive or not. An advantage is that one can tell single cells from clumps of cells.

Colony-forming units are another way of counting bacteria. The sample is applied to a medium (soup that bacteria can use to multiply) that has been solidified into something like stiff Jell-O, usually with a substance called agar that comes from seaweed. The sample may be mixed with the agar before it solidifies, spread on top of the agar after it solidifies, or captured on a filter that is then carefully placed on top of the agar. If conditions are right, a bacterium from the sample will divide into two, and these into four, doubling in number perhaps twice per hour. Within a day or two, our little bacterium has made so many more like it that we can see a visible patch of growth called a colony. These range upward from the size of a pinhead. Whether the “seed” bacterium was just one cell or a clump cannot be determined by this method, so the “seed” is scored as a colony-forming unit. Many samples of water, food, etc. are tested by this method, using different media and different conditions of incubation.

Although you are in Canada, I will base my explanations on drinking water standards of the US Environmental Protection Agency (EPA) , with which I am more familiar. I believe that Canadian standards are quite similar. Most bacteriological testing of drinking water is not aimed at disease agents, but rather to get an idea of how well sanitation is being practiced. Interpretation depends on where the sample came from and how the test was done. In the field of water microbiology, the aerobic plate count (which is the name often used in food microbiology) used to be called the “standard plate count” and is now called the “heterotrophic plate count” (HPC). It’s pretty much all the same thing: aerobic means that the bacteria had access to air (oxygen) and heterotrophic means that the medium had complex nutrients in it. “Standard” meant that the test was done with the official medium, under official conditions. Obviously, heterotrophic sounds much more official than standard or aerobic.

Most of the methods used in water testing (chemical, microbiological, etc.) are compiled in Standard Methods for the Examination of Water and Wastewater (SMWW). This was published for many years in book form, but is now distributed as a CD. Its section on the HPC describes four methods of testing and three possible agar media. What they have in common is that incubation is 48 hours at 35°C. Colonies may form in the agar medium, on top of the agar medium, or on top of a filter, depending on the method used. Each colony that is counted is attributed to one colony-forming unit (CFU).

The EPA website has three things to say about the HPC:

1. The bacteria detected by heterotrophic plate count (HPC) are not necessarily harmful. HPC is simply an alternative method of determining disinfectant residual levels. The number of such bacteria is an indicator of whether there is enough disinfectant in the distribution system.
[A disinfectant is a chemical added to water to kill infectious bacteria and viruses. Community drinking water undergoes various treatments (depending on whether it came from a well or from the surface source), ending with disinfection. Disinfection at the treatment plant is often done with chlorine, but ozone and UV are common alternatives. Water in distribution (in mains and pipes under the ground) usually has some chlorine in it to keep bacteria from forming a slime inside the pipes. This is never entirely successful, so most community water systems flush out their pipes by opening fire hydrants from time to time. Water comes out of a fire hydrant so rapidly that the slime in the pipes is dislodged. Meanwhile, even if disinfection gets rid of all the disease agents, it won’t kill all the bacteria.]

2. Distribution system samples collected for the analysis of heterotrophic bacteria [measured as heterotrophic plate count (HPC)] are also required to be held at 10 deg.C during transit/storage. However, the analysis of heterotrophic bacteria is an optional substitute for maintaining a detectable disinfection residual.
[This means that if the community can show an active level of chlorine in samples of water in distribution, the HPC may not be necessary at all.]

3. EPA's surface water treatment rules require systems using surface water or ground water under the direct influence of surface water to (1) disinfect their water, and (2) filter their water or meet criteria for avoiding filtration so that the following contaminants are controlled at the following levels:
HPC: No more than 500 bacterial colonies per milliliter.
[This means that if the water comes from the surface source (river, lake, etc.) or a shallow well that receives water from the surface source, the community is required either to do a complete treatment series or show that the water was safe without complete treatment. There are other standards that I have not included, but this is the only instance in which the HPC has a maximum.].

Therefore, a sample that had an HPC of 0 CFU would not necessarily be free of all bacteria, and a sample that had 800 CFU would be regarded as a problem only if the source water was of the kind described. Many, but not all, kinds of bacteria can form colonies in the HPC test. As EPA says, these bacteria are assumed to be harmless. On the other hand, the greatest public health concern is with water contaminated with feces. A bacterial species that is present in the intestines of all warm-blooded animals is Escherichia coli. There are many varieties of this species, most of which are called “normal flora” that we live with all the time, but a few of which cause disease. For the sake of sanitation, one doesn’t want drinking water contaminated with feces, and E. coli is the standard indicator of fecal contamination in water. However, until fairly recently, testing for E. coli was complex and expensive, so a relatively easy (?) test was devised for bacteria called “coliforms,” meaning bacteria that resemble E. coli.

SMWW says that the coliform group comprises all facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48 hours at 35°C. If you really care what all that means, you can look up the various terms: I don’t think this is a good place to explain them all. The vast majority of coliforms are just environmental bacteria trying to make a living, but they should seldom be present if disinfection has been properly done. This is why they are accorded more sanitary significance than the bacteria that show up in the HPC.

This is only a small part of what there is to know about bacteria and other microbes in water and drinking water. Bacteriological tests are used for quality control on the processes used to produce and deliver safe drinking water. In most of North America, most of the time, we can safely drink water from a public supply rather than having to boil it or buy bottled water. There are certainly other countries that do as well, but unfortunately there are many other countries where one cannot safely drink water from a public supply. People who live in the countryside and manage their own water supplies must accept the responsibility for their water’s safety.

Dean O. Cliver

Standard Methods for the Examination of Water and Wastewater, 20th edition, American Public Health Association, Washington, DC, 1999.

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