Microbiology - 014 - Most Probable Number

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Water Quality: MPN Technique 

 

Measuring water quality is an important application of microbiology. For example, residential or agricultural sewage runoff could contaminate a source of water and make it unsafe to drink. One measure of water quality is the prevalence of coliform bacteria in the water. Coliforms are easy to identify and their presence in high numbers could indicate dangerous levels of fecal or other contamination, which is why they are important indicator organisms. 

 

A relatively easy water quality test uses the Most Probable Number, or MPN, method, and is composed of three phases: the presumptive, confirmed, and completed phases. While the MPN method does not exactly measure the number of coliforms present in a sample, it does give an estimate and can determine whether or not the water is below the safe threshold for potable water. 

 

  1. Presumptive phase 

 

The first phase is the presumptive phase. First, transfer one mL of the test sample to a 9 mL dilution blank and mix. Then, transfer one mL from that tube to another 9 mL dilution blank and mix. This will yield three different concentrations: the undiluted original sample, and both 10^-1 and 10^-2 serial dilutions. 

 

From each of the three different dilutions, inoculate five test tubes of lauryl tryptose broth with 1 mL of liquid to make fifteen total tubes. These tubes are not further dilutions, but instead replicate tests for the presence of coliforms at each dilution level. Incubate the tubes at 37 degrees Celsius. 

 

After incubation, tubes that are turbid with growth and have gas in their Durham tubes are presumed to have coliforms growing in them; these are positive tubes. Count the number of positive tubes at each dilution level; for example, here all five of the tubes from the undiluted sample are positive, while three are positive from the 10^-1 dilution, and one is positive from the 10^-2 dilution. Use these three numbers and the provided table to determine the MPN index, which is the estimated number of coliforms per 100mL of water. An MPN index below 2 is considered to be drinkable. Using the numbers from before, 5, 3, and 1, this water sample has an MPN index of 110. This sample isn’t safe to drink. 

 

 

  1. Confirmed phase 

 

The second phase is the confirmed phase, which confirms that coliforms are present. Select any one positive test tube from the presumptive phase, and use a sterile loop to transfer a loopful of liquid from it to a fresh Brilliant Green Lactose Bile Broth tube. Incubate the BGLB tube at 37 degrees Celsius. After incubation, turbid growth and a bubble in the Durham tube confirms that coliforms are actually present. If there is no growth and no bubble, the growth in the presumptive phase may have been a false positive. 

 

  1. Completed phase 

 

The third and final phase is the completed phase, which completes the test. Take a loopful from a positive BGLB tube and streak two plates of your choice from the following: MacConkey Agar, Eosin Methylene Blue Agar, or Violet Red Bile Agar. Incubate the plates at 37 degrees Celsius. All three media inhibit the growth of Gram + bacteria, but they have different ways of differentiating between bacteria that cannot ferment lactose and those that can, such as coliforms. 

 

MacConkey agar will turn pink when acid is produced from the fermentation of lactose, and the agar turns opaque or cloudy. Non-lactose fermenters will form colorless colonies, and turn the agar yellow. Notice the pink color of the agar around these coliform colonies, and the opaque nature of the agar. 

 

On Eosin Methylene Blue Agar, bacteria that cannot ferment lactose will form colorless colonies, but those that can and produce strong acids will form colonies with a metallic green sheen, as seen here. 

 

Finally, on Violet Red Bile Agar, coliform colonies will be red or pink and will be surrounded by a cloudy precipitate in the medium, while non-coliforms will form colorless colonies or fail to produce a cloudy precipitate.  This coliform formed red colonies and the medium has turned cloudy as well. 

 

Evaluate the results from all three phases. Did your water sample contain coliforms? Did it contain enough to be unsafe to drink? If the sample was from a river or lake, the presence of coliforms may not be surprising. However, the same results from a municipal water supply or a public water park would be a serious problem. These are the kinds of questions that microbiologists answer, and the MPN test is just one way to do so. 

Measuring water quality is an important application of microbiology. For example, residential or agricultural sewage runoff could contaminate a source of water and make it unsafe to drink. One measure of water quality is the prevalence of coliform bacteria in the water. Coliforms are easy to identify and their presence in high numbers could indicate dangerous levels of fecal or other contamination, which is why they are important indicator organisms.

A relatively easy water quality test uses the Most Probable Number, or MPN, method, and is composed of three phases: the presumptive, confirmed, and completed phases. While the MPN method does not exactly measure the number of coliforms present in a sample, it does give an estimate and can determine whether or not the water is below the safe threshold for potable water. 

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