Water Quality Analysis by Most Probable Number (MPN)

The presumptive test is the first step in the most probable number (MPN) analysis, which is a statistical method to estimate the concentration of viable microorganisms in a water sample. The presumptive test is used to screen the water sample for the presence of coliform bacteria, which are indicators of fecal contamination and potential pathogens. Coliform bacteria are Gram-negative, non-spore-forming rods that can ferment lactose with acid and gas production.

The presumptive test involves inoculating measured volumes of water into a series of tubes containing a selective growth medium, such as MacConkey lactose broth, Lauryl tryptose broth, or brilliant green lactose bile broth. The medium contains lactose as the sole carbohydrate source, a surfactant such as sodium lauryl sulfate or bile salt to inhibit non-coliform bacteria, and a pH indicator dye such as bromcresol purple or brilliant green to detect acid production. The tubes also contain inverted Durham tubes to collect gas bubbles.

The inoculated tubes are incubated at 35°C for 24 to 48 hours and observed for color change and gas production. A positive presumptive test is indicated by the formation of at least 10% gas in the Durham tube and a change in the color of the medium from purple or green to yellow, indicating acid production. A negative presumptive test is indicated by no gas formation and no color change. A doubtful presumptive test is indicated by gas formation without color change or vice versa.

The number and distribution of positive tubes in each set are recorded and compared with a standard MPN table to estimate the number of coliform bacteria per 100 ml of water sample. The MPN table gives a range of possible values with a 95% confidence interval. The presumptive test is only an estimation and not a definitive identification of coliform bacteria. Therefore, if any tube shows a positive or doubtful result, a confirmatory test is performed to verify the presence of coliform bacteria.

Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to confirm the presence of coliforms, a confirmatory test is done. For this, a loopful of suspension from a positive tube of the presumptive test is inoculated into a tube of brilliant green lactose bile (BGLB) 2% broth (or other lactose broth) and to an agar plate (EMB agar or Endo Agar) or slant.

A. Inoculation of the lactose-broth

• Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2 hours.
• If no gas production is seen, further incubate up to a maximum of 48 ±3 hours to check gas production.
• The formation of gas in the Durham tube indicates a positive confirmed test for coliforms.

B. Inoculation in media slants

• Take a loopful of suspension from a positive tube and inoculate it on the agar surface.
• The agar slants should be incubated at 37°C for 24± 2 hours.
• Colonies must be examined macroscopically.
• Coliforms produce colonies with a greenish metallic sheen on EMB agar or red colonies with dark centers on Endo agar, which differentiates them from non-coliform colonies (show no sheen or colorless).
• The presence of typical colonies at high temperatures (44.5 ±0.2) indicates the presence of thermotolerant E.coli.

This test helps to further confirm doubtful and, if desired, positive confirmed test results. A typical coliform colony from an LES Endo agar plate is inoculated into a tube of brilliant green bile broth and on the surface of a nutrient agar slant. They are then incubated at 35°C for 24 hours. After 24 hours, the broth is checked for the production of gas, and a Gram stain is made from organisms on the nutrient agar slant. If the organism is a Gram-negative, non-spore-forming rod and produces gas in the lactose tube, then it is positive that coliforms are present in the water sample.

The completed test is also known as the confirmed phase of the MPN test. It is used to verify that the bacteria isolated from the presumptive and confirmed tests are indeed coliforms and not other lactose-fermenting bacteria that may produce similar results. The completed test also provides information about the morphology and staining characteristics of the coliforms, which can help in their identification.

The completed test is important because it ensures that the MPN estimate is accurate and reliable. It also helps to differentiate between fecal coliforms (such as E. coli) and non-fecal coliforms (such as Enterobacter) based on their growth temperature and colony appearance. Fecal coliforms are more indicative of fecal contamination and potential health risks than non-fecal coliforms.

Therefore, the completed test is a crucial step in water quality analysis by MPN method. It confirms the presence and identity of coliform bacteria in water samples and provides an estimate of their number per 100 ml of water. The completed test also helps to assess the sanitary quality of water and its suitability for drinking or other purposes.

The main objectives of the MPN method are:

• To enumerate the number of bacteria present in the drinking water by the MPN method. This method provides an estimate of the concentration of viable microorganisms in a sample by means of replicating liquid broth growth in ten-fold dilutions.
• To identify the bacteria present in the drinking water sample. This method helps to detect the presence of coliform bacteria, which are indicators of fecal contamination and potential pathogens in water. The MPN method consists of three steps: presumptive test, confirmatory test, and completed test, which help to confirm the identity of the coliform bacteria by using different media and tests.

The MPN method consists of three steps: presumptive test, confirmed test, and completed test. Each step involves inoculating measured volumes of water into tubes or plates containing a selective medium for coliform bacteria and observing the growth and gas production.

I. Presumptive Test

The presumptive test is a screening test to sample water for the presence of coliform organisms. If the presumptive test is negative, no further testing is performed, and the water source is considered microbiologically safe. If, however, any tube in the series shows acid and gas, the water is considered unsafe and the confirmed test is performed on the tube displaying a positive reaction.

The procedure of the presumptive test varies for treated and untreated water.

For untreated (polluted) water

• Prepare MacConkey purple media of single and double strength in test tubes with Durham’s tube and autoclave it.
• Take three sets of test tubes containing five tubes in each set; one set with 10 ml of double strength (DS) other two containing 10 ml of single strength (SS).
• Using sterile pipettes, transfer 10 ml of water to each of the DS broth tubes. Transfer 1 ml of water sample to each of 5 tubes of one set of SS broth and transfer 0.1 ml water to five tubes of remaining last set of SS broth tubes.
• Incubate the tubes at 37°C for 24 hours.
• After incubation, observe the gas production in Durham’s tube and the color change of the media.
• Record the number of positive results from each set and compare with the standard chart to give presumptive coliform count per 100 ml water sample.

For treated (unpolluted) water

• Prepare lauryl tryptose broth (LTB) media in test tubes with Durham’s tube and autoclave it.
• Take five sets of test tubes containing five tubes in each set; all containing 10 ml of LTB media.
• Using sterile pipettes, transfer 100 ml, 10 ml, 1 ml, 0.1 ml and 0.01 ml of water sample to each set respectively.
• Incubate the tubes at 35°C for 24 hours.
• After incubation, observe the gas production in Durham’s tube and the turbidity of the media.
• Record the number of positive results from each set and compare with the standard chart to give presumptive coliform count per 100 ml water sample.

II. Confirmed Test

The confirmed test serves to confirm the presence of coliform bacteria when either a positive or doubtful presumptive test is obtained. For this, a loopful of suspension from a positive tube is inoculated into a lactose-broth or brilliant green lactose fermentation tube and to an agar plate (EMB agar or Endo Agar) or slant.

A. Inoculation of the lactose-broth

• Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2 hours.
• If no gas production is seen, further incubate up to a maximum of 48 ±3 hours to check gas production.

B. Inoculation in media slants

• Take a loopful of suspension from a positive tube and inoculate it on the agar surface.
• The agar slants should be incubated at 37°C for 24± 2 hours.
• Colonies must be examined macroscopically.

III. Completed Test

The completed test helps to further confirm doubtful and, if desired, positive confirmed test results. A typical coliform colony from an LES Endo agar plate is inoculated into a tube of brilliant green bile broth and on the surface of a nutrient agar slant. They are then incubated at 35°C for 24 hours. After 24 hours, the broth is checked for the production of gas, and a Gram stain is made from organisms on the nutrient agar slant. If the organism is a Gram-negative, non-spore-forming rod and produces gas in the lactose tube, then it is positive that coliforms are present in the water sample.

The results of the MPN test are based on the number and distribution of positive tubes in each set of lactose broth tubes inoculated with different volumes of water sample. A positive tube is one that shows both acid production (indicated by color change of the medium) and gas production (indicated by gas bubbles in the inverted Durham tube) within 24 to 48 hours of incubation. A negative tube is one that shows no growth or no acid and gas production.

The number of positive tubes in each set is recorded and compared with a standard MPN table that gives the most probable number of coliform bacteria per 100 ml of water sample for different combinations of positive tubes. The MPN table also gives a 95% confidence interval for the estimated number, which indicates the range of possible values with a 95% probability.

For example, if the results of the presumptive test are as follows:

The MPN table shows that the most probable number of coliform bacteria per 100 ml of water sample is 110, with a 95% confidence interval of 41 to 280.

The results of the confirmed test and the completed test are based on the presence or absence of gas production in lactose broth tubes and the growth and morphology of colonies on agar plates or slants. A positive confirmed test indicates that the lactose broth tube inoculated with a loopful of growth from a positive presumptive tube shows gas production within 48 hours. A positive completed test indicates that the lactose broth tube inoculated with a typical coliform colony from an agar plate shows gas production within 24 hours, and that the Gram stain of the organism from a nutrient agar slant shows Gram-negative, non-spore-forming rods.

The results of the MPN test can be interpreted as follows:

• If all three tests (presumptive, confirmed, and completed) are negative, then the water sample is considered safe and free from coliform contamination.
• If the presumptive test is positive but the confirmed and completed tests are negative, then the water sample may contain non-coliform bacteria that can ferment lactose and produce gas, but are not indicative of fecal pollution. The water sample may be safe for consumption, but further testing may be required to identify the non-coliform bacteria present.
• If the presumptive and confirmed tests are positive but the completed test is negative, then the water sample may contain coliform bacteria that are not fecal coliforms, such as Enterobacter or Klebsiella. These bacteria may be present in soil or vegetation and may not indicate fecal pollution. The water sample may be safe for consumption, but further testing may be required to differentiate between fecal and non-fecal coliforms.
• If all three tests (presumptive, confirmed, and completed) are positive, then the water sample contains fecal coliform bacteria, such as E. coli, that indicate fecal pollution and potential presence of pathogens. The water sample is unsafe for consumption and requires treatment or disinfection.

The MPN method is widely used to estimate the concentration of viable microorganisms in various samples, such as water, food, soil, and agricultural products. The technique is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods. The MPN method is also used to detect the presence of coliform bacteria, which are indicators of fecal contamination and potential pathogens in water sources. The MPN method can also be applied to count bacteria that grow poorly or reluctantly on agar plates or membrane filters, but grow readily in liquid media. The MPN method has also been suggested as an alternative method to trend environmental monitoring studies.

The MPN method has some advantages over other methods of enumerating bacteria, such as plate count or membrane filtration. Some of the advantages are:

• Ease of interpretation: The MPN method does not require counting colonies or interpreting growth patterns on agar plates. The presence or absence of gas and acid production in the broth tubes is a simple and clear indicator of bacterial growth.
• Sample dilution: The MPN method involves serial dilutions of the water sample, which reduces the effect of any toxins or inhibitors that may be present in the sample. This also allows for the detection of bacteria that are present in low numbers or are difficult to grow on solid media.
• Effective for turbid samples: The MPN method can be used to analyze samples that contain particulate matter, such as sediments, sludge, mud, etc. These samples may interfere with other methods that rely on visual inspection of agar plates or membrane filters.
• Detection and enumeration of bacteria in various samples: The MPN method can be used to estimate the number of bacteria in water, food, soil, and other environmental samples. It can also be used to detect and enumerate specific groups of bacteria, such as coliforms, fecal coliforms, E. coli, etc., by using selective and differential media.

Although MPN is a widely used method for estimating the number of bacteria in water samples, it has some limitations that should be considered before applying it. Some of the limitations are:

• Poor accuracy and precision: MPN is based on statistical probabilities and assumptions that may not always hold true in practice. For example, MPN assumes that each bacterium in the sample can grow and produce gas in the medium, but some bacteria may be injured or dormant and not show any growth. MPN also assumes that each tube contains a single type of bacterium, but some tubes may have mixed cultures or contaminants that affect the results. Therefore, MPN counts may not reflect the true number of bacteria in the sample and may vary widely depending on the sample size, dilution scheme, and number of tubes used.
• Labor-intensive and expensive: MPN requires a large number of tubes, media, and incubator space to perform the test. It also involves multiple steps of inoculation, incubation, observation, and confirmation that may take several days to complete. Moreover, MPN requires skilled personnel to interpret the results and identify the bacteria using Gram staining and other tests. Therefore, MPN is not a cost-effective or time-efficient method for routine analysis of water samples.
• Large margin of error: MPN relies on statistical tables that provide a range of possible values for the number of bacteria in the sample based on the number of positive tubes observed. However, these tables are derived from mathematical models that may not account for all the factors that influence bacterial growth and gas production in the tubes. Furthermore, these tables have wide confidence intervals that indicate a high degree of uncertainty in the estimates. Therefore, MPN results may have a large margin of error and may not be reliable for comparing different samples or detecting small changes in bacterial populations.

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