Most Probable Number (MPN) Test: Principle, Procedure, Results

What is Most Probable Number (MPN) Test?

The Most Probable Number (MPN) test is a widely used method in microbiology to estimate the concentration of microorganisms, specifically indicator organisms, in a given sample. It is commonly employed in water quality analysis to determine the presence of contaminants such as fecal coliforms, with Escherichia coli (E. coli) being a common indicator used to assess pollution levels.

The MPN test utilizes a statistical approach based on the concept of random dispersion of microorganisms within a given volume. The test involves a series of three steps: the presumptive test, the confirmed test, and the completed test.

1. Presumptive Test: The first step in the MPN test is the presumptive test. It involves inoculating multiple tubes of liquid growth medium with different volumes of the sample being tested. The growth medium used contains indicators that can detect the presence of indicator organisms, such as E. coli. These indicators promote the growth of indicator organisms and cause a noticeable color change in the medium. Tubes that show growth and a characteristic color change are considered positive, indicating the presence of indicator organisms.
2. Confirmed Test: Following the presumptive test, the tubes that yielded positive results are subjected to the confirmed test. In this step, a portion of the positive tubes from the presumptive test is transferred to a secondary growth medium specifically designed to confirm the presence of indicator organisms. The confirmed test helps eliminate false positives that may have occurred during the presumptive test, ensuring more accurate results.
3. Completed Test: The final step is the completed test. It involves observing the growth and color change in the tubes from the confirmed test. The number of tubes showing positive results is recorded. Based on the statistical distribution of positive and negative reactions, the MPN of indicator organisms in the sample can be estimated by referring to MPN tables or using statistical software.

The MPN method provides a quantifiable measure of the concentration of indicator organisms in a sample, allowing for the assessment of water quality and the presence of potential pathogens. The advantage of using the MPN test lies in its ability to estimate the concentration of microorganisms even when direct counting methods are impractical or time-consuming. However, it is important to note that the MPN test provides an estimation rather than an exact count of microorganisms.

In conclusion, the Most Probable Number (MPN) test is a statistical method used to estimate the concentration of microorganisms, particularly indicator organisms, in a given sample. By employing a series of tests and analyzing positive and negative reactions, the MPN test provides valuable information for assessing water quality and detecting the presence of contaminants, ensuring the safety of water sources for various purposes.

Definition of Most Probable Number (MPN) Test

The Most Probable Number (MPN) test is a statistical method used to estimate the concentration of microorganisms, particularly indicator organisms, in a sample by analyzing positive and negative reactions in a series of tests. It is commonly employed in water quality analysis to assess contamination levels.

Principle of Most Probable Number Test (MPN Test)

The Most Probable Number (MPN) test is based on the principle of specific enrichment and selective growth of coliform bacteria in a fermentation medium. The test is performed using fermentation tubes filled with a selective growth medium, typically MacConkey lactose broth, which includes inverted Durham tubes to detect fermentation gas production. The process involves inoculating a series of lactose broth tubes with measured volumes of the water sample being tested.

The selective growth medium contains certain components that promote the growth of coliform bacteria while inhibiting the growth of other bacterial types. Lactose, a sugar present in the medium, acts as a selective factor since coliform bacteria and some other bacterial strains can ferment lactose, while many other bacteria cannot. Additionally, the medium may include a surfactant, such as Na-lauryl sulfate or Na-taurocholate (bile salt), and a pH indicator dye, such as bromcresol purple or brilliant green. The surfactant and dye do not inhibit coliform bacteria but hinder the growth of other bacteria, such as spore-formers.

In the MPN test, a statistical approach is used to determine the most probable number of coliform bacteria present in the water sample. It involves three sets of dilutions, each containing a fermentative broth mixed with the water sample. The presence of coliform bacteria leads to the production of acid and gas in the fermentation tubes, indicating a positive result. The quantitative analysis of coliform bacteria is determined by counting the number of tubes that exhibit positive results and comparing the pattern of positive results with statistical data.

By comparing the observed results with standard statistical tables, the MPN test provides an estimation of the concentration of coliform bacteria in the water sample. This method allows for the assessment of water quality and the presence of potential pathogens, particularly those associated with fecal contamination. The MPN test offers a practical and reliable approach to evaluate the microbiological safety of water sources.

Objectives of Most Probable Number Test (MPN Test)

The Most Probable Number (MPN) test has several objectives aimed at providing valuable information about the concentration of microorganisms in a sample, particularly indicator organisms such as coliform bacteria. Here are the key objectives of the MPN test:

1. Sensitivity: The MPN test aims to detect the presence of discrete entities, such as microorganisms, enzymes, or catalytic reactions, with high sensitivity. It allows for the detection of even low concentrations of these entities in a sample.
2. Quantification: While easy quantification may be challenging due to amplification or catalysis reactions, the MPN test seeks to provide an estimation of the original concentration of the entities under investigation. By subdividing the original sample by orders of magnitude and assessing presence or absence in multiple subdivisions, the MPN test allows for quantification within the appropriate order of magnitude.
3. Dilution Assessment: The MPN test involves diluting the original solution or sample to the point where absence begins to appear. This indicates that the entities have been diluted to a degree where subsamples may contain none of the entities. By determining the dilution at which absence is observed, the MPN test provides insights into the concentration of entities in the original sample.
4. Resolving Concentration: Through the use of replicates at various dilutions, the MPN test enables finer resolution and estimation of the original concentration of the entities. By comparing the number of positive and negative samples, the test allows for estimating the concentration within the appropriate order of magnitude.

Overall, the objectives of the MPN test revolve around sensitivity, quantification, dilution assessment, and resolving the concentration of discrete entities present in a sample. By employing statistical methods and subdivision techniques, the test provides valuable information for assessing the presence and concentration of indicator organisms, particularly in the context of water quality analysis.

MPN Test Procedure

MPN test is performed in 3 steps

1. Presumptive test
2. Confirmatory test
3. Completed test

1. Presumptive Test

The presumptive test is a preliminary screening method used to detect the presence of coliform organisms in water samples. It involves utilizing a series of fermentation tubes containing lactose broth with known concentrations. The purpose of this test is to quickly assess whether the water source may be contaminated with coliform bacteria, which can indicate potential fecal contamination and the presence of harmful pathogens.

When conducting the presumptive test, multiple fermentation tubes are inoculated with the water sample and incubated at an appropriate temperature. If the test yields negative results, it indicates that the water is likely free from coliform bacteria and may be considered microbiologically safe. In such cases, no further testing is typically required.

However, if any of the fermentation tubes show signs of acid and gas production, it suggests the potential presence of coliform bacteria. The production of gas is an important indicator and requires a specific concentration of coliforms to occur. According to Chambers, the formation of gas typically requires a range of 40 to 390 million coliforms per milliliter. It is worth noting that the amount of gas produced also depends on the ratio of coliform to non-coliform bacteria. Higher levels of non-coliform bacteria can reduce gas production, affecting the overall test results.

In the event of a positive presumptive test, further confirmation is necessary through a confirmed Most Probable Number (MPN) test. The confirmed MPN test provides a more accurate determination of coliform levels in the water sample, helping to validate the initial presumptive test results. This confirmation step is essential for ensuring accurate detection and to rule out any false positives or negatives.

It’s important to mention that the method of conducting the presumptive test may vary depending on whether the water being tested is treated or untreated. Different protocols and procedures may be employed for each case to account for variations in water sources and potential contaminants.

In summary, the presumptive test is an initial screening test used to sample water for the presence of coliform organisms. If the results are negative, indicating the absence of coliform bacteria, no further testing is typically required, and the water source can be considered microbiologically safe. However, if the test yields positive results, further confirmation through the confirmed MPN test is necessary to accurately determine the presence and concentration of coliforms in the water sample.

Requirements for Presumptive Test

The presumptive test is an important step in microbiological analysis, particularly in detecting the presence of coliform organisms in water samples. To conduct an effective presumptive test, several requirements must be fulfilled. These requirements encompass the medium used, the necessary glassware, and the use of sterile pipettes.

1. Medium: The choice of medium is crucial for facilitating the growth and detection of coliform organisms. Commonly used media for the presumptive test include lactose broth, MacConkey broth, and lauryl tryptose (lactose) broth. These media contain nutrients necessary for the growth of bacteria and facilitate the differentiation of coliforms based on their ability to ferment lactose.
2. Glassware: Various types of test tubes with different capacities are required for conducting the presumptive test. Test tubes of 20ml, 10ml, and 5ml capacities are commonly used, depending on the volume of the water sample being tested. These test tubes serve as containers for the medium and the water sample during incubation.
3. Additionally, Durham tubes are essential components of the test setup. These small inverted tubes are placed within the test tubes to capture and detect the production of gas during bacterial growth. Gas production, indicated by the presence of a bubble in the Durham tube, is an important criterion for determining the presence of coliform bacteria.
4. Sterile Pipettes: Sterile pipettes play a crucial role in transferring and measuring precise volumes of water samples and media during the presumptive test. These pipettes ensure the integrity of the test by minimizing the risk of contamination and providing accurate measurements for analysis.

To maintain sterility, it is important to use disposable or properly sterilized pipettes for each sample. Contamination from external sources could lead to inaccurate test results and compromise the reliability of the presumptive test.

By adhering to these requirements, laboratories can establish a standardized protocol for conducting the presumptive test. Following proper techniques and using the appropriate materials and equipment ensures reliable and accurate results, which are vital for assessing the microbiological safety of water sources.

Preparation of the Medium

To conduct the presumptive test, it is essential to prepare the medium correctly. The medium, either MacConkey broth or lactose broth, needs to be prepared in single and double concentrations, depending on the type of water being tested. Here is a step-by-step guide on the preparation of the medium:

1. Gather the required materials:
   • MacConkey broth or lactose broth (depending on the test chosen)
   • Sterile test tubes (10 mL capacity)
   • Sterile bottles (50 mL capacity for treated water)
   • Inverted Durham tubes
   • Autoclave or pressure sterilization equipment
2. Prepare the medium for untreated or polluted water:
   • For double strength medium: Dispense the double concentration media into 10 sterile test tubes, with 10 mL per tube.
   • For single strength medium: Dispense the single concentration media into 5 sterile test tubes, with 10 mL per tube.
   • Add an inverted Durham tube to each test tube.
3. Prepare the medium for treated water:
   • For double strength medium: Dispense the double concentration media into 5 sterile test tubes, with 10 mL per tube.
   • For single strength medium: Dispense the single concentration media into a sterile bottle, with a capacity of 50 mL.
   • Add an inverted Durham tube to each test tube or the bottle.
4. Check the tubes:
   • Ensure that the inverted Durham tubes are filled with liquid and do not contain any air bubbles. The presence of liquid in the Durham tube is necessary to detect gas production during incubation.
5. Autoclave sterilization:
   • Place the prepared tubes or bottle in the autoclave, ensuring that they are properly sealed or capped.
   • Set the autoclave to 15 pounds of pressure and a temperature of 121 degrees Celsius.
   • Autoclave the tubes or bottle at these settings for 15 minutes to achieve proper sterilization.

Autoclaving the medium at the specified temperature and pressure ensures the elimination of any potential contaminants. Sterilization is crucial to maintain the integrity of the medium and prevent interference with the test results.

Once the medium is prepared and sterilized, it is ready to be used for the presumptive test. The prepared tubes or bottle can be stored appropriately until needed for analysis.

Procedure of Presumptive Test

a. For untreated (polluted) water

The presumptive test is a crucial step in assessing the microbiological quality of untreated or polluted water. Here is a step-by-step procedure for conducting the presumptive test on untreated water:

1. Prepare the required materials:
   • 5 tubes of double strength medium
   • 10 tubes of single strength medium
   • Sterile pipettes
   • Water samples to be tested
   • Standard chart for MPN interpretation
   • Incubator set at 37°C
2. Label the tubes:
   • Clearly label the tubes to indicate the strength of the medium (double or single) and the volume of water to be added.
3. Add water to the tubes:
   • Using a sterile pipette, add 10 mL of the water sample to 5 tubes containing 10 mL of double strength medium.
   • Similarly, add 1 mL of the water sample to 5 tubes containing 10 mL of single strength medium.
   • Add 0.1 mL of the water sample to the remaining 5 tubes containing 10 mL of single strength medium.
4. Incubation:
   • Place all the labeled tubes in the incubator set at 37°C.
   • Incubate the tubes for 24 hours. If no tubes show positive reactions, continue incubation for up to 48 hours.
5. Interpretation:
   • After the incubation period, observe the tubes for positive reactions. A positive reaction is indicated by the presence of gas production, usually identified by the presence of a bubble in the inverted Durham tube.
   • Record the number of tubes showing positive reactions for each dilution (double, 1 mL, and 0.1 mL) of the water sample.
6. Compare to the standard chart:
   • Refer to a standard chart that correlates the number of positive tubes with the most probable number (MPN) of coliform organisms present.
   • Identify the pattern of positive reactions observed in the tubes and determine the corresponding MPN value based on the chart.

For example, if the water sample tested shows a result of 3-2-1 (3 tubes of 10 mL positive, 2 tubes of 1 mL positive, and 1 tube of 0.1 mL positive), the MPN value would be determined as 17, indicating an estimated presence of 17 coliform organisms per 100 mL of the water sample.

The presumptive test’s results help assess the level of coliform contamination in the untreated water sample, providing valuable information for further analysis and treatment if necessary.

2. For treated (unpolluted) water

The presumptive test for treated (unpolluted) water follows a similar procedure to that of untreated water. Here is a step-by-step guide for conducting the presumptive test on treated water:

1. Prepare the required materials:
   • 1 tube of single strength medium (50 mL)
   • 5 tubes of double strength medium (10 mL)
   • Sterile pipettes
   • Treated water samples to be tested
   • Standard chart for MPN interpretation
   • Incubator set at 37°C
2. Label the tubes:
   • Clearly label the tubes to indicate the strength of the medium (single or double) and the volume of water to be added.
3. Add water to the tubes:
   • Using a sterile pipette, add 50 mL of the treated water sample to the tube containing 50 mL of single strength medium.
   • Similarly, add 10 mL of the treated water sample to each of the 5 tubes containing 10 mL of double strength medium.
4. Incubation:
   • Place all the labeled tubes in the incubator set at 37°C.
   • Incubate the tubes for 24 hours. If no tubes show positive reactions, continue incubation for up to 48 hours.
5. Interpretation:
   • After the incubation period, observe the tubes for positive reactions. A positive reaction is indicated by the presence of gas production, typically identified by the presence of a bubble in the inverted Durham tube.
   • Record the number of tubes showing positive reactions for each dilution (single and double strength) of the treated water sample.
6. Compare to the standard chart:
   • Refer to a standard chart that correlates the number of positive tubes with the most probable number (MPN) of coliform organisms present.
   • Identify the pattern of positive reactions observed in the tubes and determine the corresponding MPN value based on the chart.

For example, if the treated water sample tested shows a result of 1-4 (1 tube of 50 mL positive and 4 tubes of 10 mL positive), the MPN value would be determined as 16, indicating an estimated presence of 16 coliform organisms per 100 mL of the water sample.

The results of the presumptive test for treated water help assess the effectiveness of the treatment process in removing coliform contamination. It provides valuable information about the microbiological quality of the treated water and can guide further analysis and actions if necessary.

Presumptive Test Result

The presumptive test result provides important information regarding the presence or absence of coliform bacteria, indicating the potential for fecal pollution. Here is a description of positive and negative results:

1. Positive Result: A positive presumptive test result indicates the presence of coliform bacteria, suggesting the possibility of fecal pollution. The following criteria are observed for a positive result:

• Gas Formation: The formation of 10% gas or more in the Durham tube within 24 to 48 hours is considered positive. Gas production is typically indicated by the presence of a bubble in the inverted Durham tube.
• Turbidity: The growth medium in the test tube shows turbidity, indicating bacterial growth.
• Color Change: The medium may undergo a color change, which can be indicative of the metabolic activity of coliform bacteria.

It’s important to note that the positive result obtained from the presumptive test is considered presumptive because other types of bacteria can produce similar results under these test conditions. Therefore, further confirmatory tests, such as the confirmed MPN test, are necessary to confirm the presence of coliform bacteria definitively.

2. Negative Result: A negative presumptive test result indicates the absence of coliform bacteria in the tested water sample. The characteristics of a negative result include:

• No Growth: There is no visible growth observed in the growth medium of the test tubes.
• No Gas Formation: No gas is produced in the Durham tube, and no bubble is present.

A negative result suggests that the water sample does not contain coliform bacteria and implies a lower likelihood of fecal pollution. However, it’s important to note that the absence of coliform bacteria does not guarantee complete absence of other potentially harmful microorganisms, and further comprehensive testing may be necessary to assess the overall microbiological safety of the water sample.

Interpreting the results of the presumptive test is a crucial step in determining the microbiological quality of the water sample and making informed decisions regarding its suitability for various purposes, such as drinking, recreational, or industrial use.

Result	Description
Positive	– Formation of 10% gas or more in the Durham tube within 24 to 48 hours. – Turbidity in the growth medium. – Color change in the medium. – Indicates the presence of coliform bacteria and suggests the possibility of fecal pollution.
Negative	– No visible growth observed in the growth medium. – No gas formation in the Durham tube. – Indicates the absence of coliform bacteria.<br>- Suggests a lower likelihood of fecal pollution. – Note: Does not guarantee absence of other microorganisms.

2. Confirmatory Test

The confirmatory test is a crucial step in validating the presence of coliform bacteria in water samples, following a positive result in the presumptive test. Although gas production in the presumptive test indicates potential coliform presence, it is necessary to confirm the presence of coliforms due to the possibility of false-positive results caused by other microorganisms present in water.

There are two common methods used for performing the confirmatory test:

1. Brilliant Green Lactose Bile Broth (BGLB):
   • This method involves inoculating the positive tubes from the presumptive test into brilliant green lactose bile broth.
   • The BGLB medium contains nutrients that specifically support the growth of coliform bacteria, suppressing the growth of non-coliform organisms.
   • Incubate the BGLB tubes at a suitable temperature, usually around 35-37°C, for a specified period, typically 24-48 hours.
   • Positive confirmation is determined by the presence of gas production and turbidity in the BGLB tubes, indicating the growth of coliform bacteria.
2. Eosin Methylene Blue Agar Medium (EMB):
   • Another method for confirming the presence of coliform bacteria is by using eosin methylene blue agar medium.
   • Inoculate the positive tubes from the presumptive test onto EMB plates or agar slants.
   • EMB contains specific indicators and selective agents that inhibit the growth of non-coliform bacteria while supporting the growth of coliforms.
   • Incubate the EMB plates or agar slants at an appropriate temperature, typically around 35-37°C, for a specified period, usually 24-48 hours.
   • Positive confirmation is determined by the appearance of characteristic colonies on the EMB medium, which may exhibit a metallic green sheen or dark purple coloration.

Both the BGLB and EMB methods are commonly used to confirm the presence of coliform bacteria. These confirmatory tests help to accurately identify the presence of coliforms and differentiate them from other microorganisms that can produce false presumptive test results.

Confirmatory testing is crucial for ensuring the reliability of the initial presumptive test and providing a more accurate assessment of the microbiological quality of the water sample. It allows for a better understanding of the potential risks associated with coliform contamination and aids in determining appropriate remedial actions, if necessary.

Procedure of Confirmatory Test

Testing of positive presumptive in BGLB medium

1. Preparation of BGLB Medium:
   • Prepare a solution of Brilliant Green Lactose Bile Broth medium using the following components:
     • Peptone: 10 g
     • Lactose: 10 g
     • Bile salt: 20 g
     • Brilliant green: 0.0133 g
     • Distilled water: 1 L
   • Mix the components thoroughly until they are completely dissolved.
   • Sterilize the BGLB medium in an autoclave for 15 minutes at 121 degrees Celsius.
   • Allow the medium to cool down before use.
2. Gently Shake the Positive Presumptive Tubes:
   • Take the positive tubes obtained from the presumptive test.
   • Gently shake the tubes to ensure even distribution of the bacteria throughout the liquid medium.
3. Inoculation:
   • Using a sterilized inoculating loop or pipette, transfer a loopful or a suitable volume of the culture from the positive presumptive tube into the BGLB fermentation tube.
   • The Brilliant Green dye present in the BGLB medium acts as a selective agent, inhibiting the growth of gram-positive bacteria while allowing coliform bacteria to grow.
4. Incubation:
   • Seal the BGLB fermentation tubes to create a suitable anaerobic environment.
   • Incubate the tubes at the specified temperature of 35 degrees Celsius.
   • Allow the tubes to incubate for 48 hours, providing sufficient time for the growth and metabolic activity of coliform bacteria.
5. Observation and Interpretation:
   • After the incubation period, examine the BGLB fermentation tubes for any changes or indications of bacterial growth.
   • Positive confirmation is determined by the presence of gas production (indicated by a bubble in the Durham tube) and turbidity in the BGLB medium.
   • The absence of gas production and turbidity indicates a negative result.

It is important to adhere to proper aseptic techniques throughout the procedure to avoid contamination. Following these steps, the confirmatory test using the BGLB method helps confirm the presence of coliform bacteria and provides additional insights into the microbiological quality of the water sample.

Testing of positive presumptive in EMB medium

To test positive presumptive tubes in Eosin Methylene Blue (EMB) medium, the following procedure is followed:

1. Prepare EMB Agar Medium:
   • Combine the following components to prepare the EMB agar medium:
     • Peptone: 10 g
     • Agar: 15 g
     • Lactose: 10 g
     • Eosin Y: 0.4 g
     • Methylene blue: 0.065 g
     • Dipotassium hydrogen phosphate: 2 g
     • Distilled water: 1 L
   • Autoclave the EMB agar medium at 121 degrees Celsius for 15 minutes.
   • Pour the sterilized EMB medium into Petri plates and allow it to solidify.
2. Shake and Streak:
   • Gently shake the positive presumptive tubes to ensure proper mixing of the culture.
   • Using an inoculating loop, streak a loopful of the culture onto the surface of the solidified EMB agar medium.
3. Incubation:
   • Incubate the plates at a temperature of 35 degrees Celsius.
   • Allow the plates to incubate for 48 hours.
4. Observation and Interpretation:
   • After incubation, observe the EMB plates for the appearance of bacterial colonies.
   • Three types of colonies may develop:
     • Typical Colonies: These colonies exhibit a nucleated appearance and may display a metallic sheen.
     • Atypical Colonies: These colonies are non-nucleated, opaque, and mucoid in nature.
     • Negative Colonies: These colonies differ from the typical and atypical colonies in their appearance.
5. Result Interpretation:
   • The presence of typical colonies, characterized by nucleated colonies and the potential formation of a metallic sheen, indicates the presence of coliform bacteria in the tested sample.

The testing of positive presumptive tubes in EMB medium helps identify the presence of coliform bacteria based on the appearance of typical colonies. It is important to carefully observe and interpret the results to draw accurate conclusions regarding the presence of coliform bacteria in the tested water sample.

Confirmed Test Result

The confirmed test result provides a definitive assessment of the presence or absence of coliform bacteria in the tested sample. The following descriptions outline the outcomes of the confirmed test:

Positive Result: A positive confirmed test result indicates the presence of coliform bacteria or a member of the coliform group in the water sample. The positive result is determined by the following observations:

• Gas Formation: The formation of gas in the lactose broth confirms the metabolic activity of coliform bacteria.
• Colony Appearance: On the Eosin Methylene Blue (EMB) agar medium, the presence of colonies exhibiting a greenish metallic sheen is indicative of coliform bacteria. This distinct sheen differentiates them from non-coliform colonies that do not display this characteristic.
• Thermotolerant E.coli: The presence of typical colonies with a metallic sheen, specifically at elevated temperatures (44.5 ± 0.2 degrees Celsius), suggests the presence of thermotolerant Escherichia coli (E.coli), a subset of the coliform group.

Negative Result: A negative confirmed test result indicates the absence of coliform bacteria in the water sample. The absence is determined by the following observations:

• No Gas Formation: The lactose broth does not show any gas production, indicating the absence of coliform bacteria.
• Colony Appearance: On the EMB agar medium, there is no demonstration of coliform-like colonies with a greenish metallic sheen.

Interpreting the confirmed test result is critical in determining the presence or absence of coliform bacteria, which is indicative of potential fecal contamination. A positive result highlights the need for further investigation and potential remediation measures, while a negative result suggests a lower likelihood of fecal pollution. It is important to consider these results in conjunction with other relevant tests and guidelines to assess the overall microbiological safety of the water sample.

Result	Description
Positive	– Formation of gas in lactose broth medium. – Presence of colonies with a greenish metallic sheen on Eosin Methylene Blue (EMB) agar medium. – Indicates the presence of coliform bacteria or a member of the coliform group in the water sample. – High-temperature presence of typical colonies suggests the presence of thermotolerant Escherichia coli (E.coli).
Negative	– Absence of gas formation in lactose broth medium. – Failure to demonstrate coliform-like colonies (no greenish metallic sheen) on EMB agar medium. – Indicates the absence of coliform bacteria in the water sample.

Tryptone Water Test 

The Tryptone Water Test is a diagnostic procedure used to detect the presence of indole, which is an enzymatic byproduct produced by certain bacteria. Specifically, this test helps identify the presence of thermotolerant Escherichia coli (E. coli) or other thermotolerant coliforms in a water sample. The following steps outline the procedure:

1. Incubation:
   • Incubate the tryptone water sample at a specific temperature of 44.5 ± 0.2 degrees Celsius.
   • Allow the sample to incubate for a duration of 18 to 24 hours.
2. Addition of Kovacs Reagent:
   • After the incubation period, add approximately 0.1 mL of Kovacs reagent to the tryptone water sample.
   • Ensure thorough mixing of the reagent with the sample.
3. Observation and Interpretation:
   • The presence or absence of indole is determined by the observation of a color change in the Kovacs reagent.
   • Positive Result:
     • If a red hue appears in the Kovacs reagent and forms a film over the aqueous phase of the medium, it indicates the presence of indole.
     • Additionally, positive confirmatory tests for indole, along with growth and gas generation, signify the existence of thermotolerant E. coli.
   • Negative Result:
     • If there is no change in color or absence of a red hue in the Kovacs reagent, it indicates the absence of indole.
     • However, if there is still growth and gas production observed, it confirms the presence of thermotolerant coliforms.

The Tryptone Water Test, by detecting the presence or absence of indole, provides valuable information about the presence of specific bacteria in a water sample. Positive results for indole, growth, and gas generation are indicative of thermotolerant E. coli, while the absence of indole but the presence of growth and gas production suggests the presence of other thermotolerant coliforms. It is important to carefully interpret the results of this test in conjunction with other relevant tests and guidelines to assess the microbiological safety of the water sample.

3. Completed Test

This test helps confirm dubious and, if required, confirmed positive test results. A typical coliform colony from an LES Endo agar plate is injected into a tube of vivid green bile broth and a nutrition agar slant. They are then incubated for 24 hours at 35°C. After 24 hours, the broth is examined for the formation of gas, and the organisms on the nutrient agar slant are stained with a Gram stain. If the organism is a Gram-negative, non-spore-forming rod that creates gas in the lactose tube, then the presence of coliforms in the water sample is confirmed.

To complete the test after obtaining a positive confirmed tube, the following steps are performed:

1. Inoculation:
   • Using a sterile inoculating loop, transfer a loopful of culture from the positive confirmed tube.
   • Inoculate the culture by either adding it to Brilliant Green Lactose Bile (BGLB) medium or streaking the culture onto agar slants.
2. Incubation:
   • Place the inoculated test tubes or agar slants in the incubator.
   • Incubate them at a temperature of 35 degrees Celsius.
   • Allow the cultures to incubate for a period of 24 to 48 hours.
3. Observation and Interpretation:
   • After the incubation period, observe the test tubes or agar slants for any visible growth or changes in the medium.
   • Look for the presence of bacterial colonies, color changes, or any other relevant indicators.

The completion of the test by inoculating the culture and incubating it in the appropriate conditions allows for further growth and observation of the bacterial culture. The incubation period of 24 to 48 hours provides sufficient time for the bacteria to proliferate and exhibit characteristic traits or reactions.

The observation of the cultures after incubation helps in interpreting the results and drawing conclusions regarding the presence of specific bacteria or their characteristics. The exact interpretation of the completed test will depend on the specific parameters, medium used, and any specific indicators or reactions being observed.

Completed Test Result

The completed test result for the presence of coliform bacteria can be interpreted as follows:

Positive Result:

• Gas Formation: The presence of gas in the brilliant green bile broth tube indicates a positive result.
• Gram Staining: Observation of Gram-negative, non-spore-forming rods on the nutrient agar (NA) slant further confirms the positive result.
• Implication: A positive completed test suggests the presence of coliform bacteria, which indicates the potential contamination of the water sample with fecal matter.

Negative Result:

• No Gas Formation: The absence of gas formation in the brilliant green bile broth tube indicates a negative result.
• Gram Staining: The absence of Gram-negative, non-spore-forming rods on the Gram staining also supports the negative result.
• Implication: A negative completed test implies the absence of coliform bacteria, suggesting that the water sample is not contaminated with fecal matter.

Interpretation of the completed test result is based on the presence or absence of gas formation in the brilliant green bile broth tube and the observation of Gram-negative, non-spore-forming rods on the NA slant. These indicators help determine the presence or absence of coliform bacteria, which serves as an indication of potential fecal contamination in the water sample.

Test Result	Gas Formation in Brilliant Green Bile Broth	Gram Staining (NA Slant)
Positive	Presence	Gram-negative, non-spore-forming rods
Negative	Absence	Absence

Uses/Applications of MPN Test

The Most Probable Number (MPN) test has a wide range of applications across various fields. Here are some key uses and applications of the MPN test:

1. Environmental Microbiology: The MPN test is commonly employed in estimating microbial populations in soils, waters, and agricultural products. It provides a practical method for quantifying microorganisms present in these samples, particularly when plate count enumeration methods are hindered by the presence of particulate material or when bacteria are reluctant to form colonies on agar plates or membrane filters.
2. Environmental Monitoring: The MPN test has been suggested as an alternative method for trend environmental monitoring studies. It allows for the assessment of microbial populations and changes over time, providing valuable insights into the environmental conditions and the presence of potentially harmful microorganisms.
3. Microbiological Cultures: In microbiology, the MPN test finds application in counting bacteria that do not readily form colonies on agar plates or membrane filters but grow well in liquid media. This method bypasses the need for tedious colony counting or expensive microscopic counts. The cultures are incubated and visually assessed, making the MPN test a convenient and efficient approach for microbial enumeration.
4. Molecular Biology: The MPN test has applications in molecular biology, particularly in quantitative PCR (Polymerase Chain Reaction). It involves diluting DNA templates into PCR reactions, where reactions only proceed in the presence of the template. This allows for quantitative assessment of the original concentration of template molecules.
5. Enzyme and Antigen Quantification: The MPN test can be utilized to measure the original concentration of enzymes or antigens in solutions. Enzyme stocks can be diluted into a solution containing a chromogenic substrate, while antigens can be diluted for assays such as ELISA (Enzyme-Linked ImmunoSorbent Assay) or other antibody cascade detection reactions. The MPN test provides a means to quantify the concentration of enzymes or antigens accurately.

Overall, the MPN test finds applications in diverse areas such as environmental microbiology, environmental monitoring, microbiological cultures, and molecular biology. It serves as a valuable tool for quantifying microorganisms, assessing changes over time, and quantifying concentrations of various entities in samples, providing crucial information for research, quality control, and environmental assessment.

Advantages of MPN Test

The Most Probable Number (MPN) test offers several advantages that make it a valuable method for microbial analysis. Here are the key advantages of the MPN test:

1. Ease of Interpretation: The MPN test provides straightforward interpretation of results. The presence of gas emissions or the growth of gram-negative rod-shaped coliforms can be easily observed, allowing for quick determination of positive or negative results. This simplicity makes the test user-friendly and accessible to a wide range of individuals.
2. Dilution of Sample Toxins: The MPN test involves diluting the sample, which can be advantageous when dealing with samples containing toxins or inhibitory substances. Dilution helps to mitigate the effects of these substances and ensures a more accurate assessment of microbial populations.
3. Analysis of Turbid Samples: One of the notable advantages of the MPN test is its effectiveness in analyzing highly turbid samples, such as sediments, sludge, mud, and other particulate-rich matrices. These samples can interfere with other microbial analysis methods, such as membrane filtration. The MPN test allows for accurate assessment even in the presence of turbidity, ensuring reliable results.
4. Applicability to Various Water Types: The MPN test is applicable to all types of water, which is a significant advantage over other methods like membrane filtration. It can be used to analyze different water sources, including freshwater, marine water, wastewater, and more. This versatility makes the MPN test widely applicable in various settings and industries.
5. Cost-Effectiveness: The MPN test is often considered a cost-effective method for microbial analysis. It eliminates the need for specialized equipment, such as filtration systems, and can be performed with basic laboratory supplies. This affordability makes it accessible to a broader range of users and facilitates routine monitoring of microbial populations.

In summary, the Most Probable Number (MPN) test offers advantages such as ease of interpretation, dilution of sample toxins, suitability for turbid samples, applicability to various water types, and cost-effectiveness. These benefits contribute to its widespread use in microbial analysis, particularly in assessing water quality and detecting the presence of indicator organisms.

Limitations of MPN Test

The Most Probable Number (MPN) test, despite its usefulness, has certain limitations that should be considered. Here are the key limitations of the MPN test:

1. Accuracy and Precision: The MPN test is associated with poor accuracy and precision compared to other counting methods. This limitation makes it a method of last resort, to be considered only when other counting methods are not suitable or feasible. The results obtained through the MPN test may have a higher degree of variability and may not provide highly precise quantification.
2. Laborious and Expensive: The MPN test can be laborious and expensive in terms of the required materials, glassware, and incubator space. It involves multiple test tubes and necessitates the preparation of specialized growth media. This can increase the workload and resource requirements in a laboratory setting.
3. Margin of Error: The MPN test has a relatively large margin of error compared to more precise quantification methods. The statistical estimation used in the MPN method introduces inherent uncertainties and variability, which can affect the accuracy of the results. The margin of error should be taken into account when interpreting and relying on the MPN test results.
4. Time-Consuming: To confirm the presence of coliforms, the MPN test involves multiple tests, including presumptive, confirmative, and completed tests. These tests require incubation periods and can take a considerable amount of time to obtain the final results. This extended timeframe may limit its applicability in situations where timely results are crucial.
5. Potential for False Results: The MPN test is highly sensitive, which means it can be susceptible to false results. Factors such as contamination, interference from substances in the sample, or technical errors during the test procedure can lead to inaccurate or misleading results. Careful attention to proper techniques, controls, and quality assurance measures is necessary to minimize the occurrence of false results.
6. Requirement for Multiple Glassware: The MPN method requires multiple test tubes and various glassware for media preparation. This can increase the workload for laboratory personnel, as well as the need for proper sterilization and maintenance of the glassware inventory.

While the MPN test provides valuable insights into microbial populations and is widely used, it is essential to consider these limitations when selecting and interpreting the results. Alternative methods with higher accuracy and precision may be preferred in certain situations where these limitations pose significant concerns.

References

• Williams, M. G., & Busta, F. F. (1999). TOTAL VIABLE COUNTS | Most Probable Number (MPN). Encyclopedia of Food Microbiology, 2166–2168. doi:10.1006/rwfm.1999.4000
• Rowe R, Todd R, Waide J. Microtechnique for most-probable-number analysis. Appl Environ Microbiol. 1977 Mar;33(3):675-80. doi: 10.1128/aem.33.3.675-680.1977. PMID: 16345226; PMCID: PMC170744.
• Sui Sien, Leong & Ismail, Johan & Denil, N.A. & Sarbini, Shahrul & Wasli, Wafri & Lingoh, Arlene. (2018). Microbiological and Physicochemical Water Quality Assessments of River Water in an Industrial Region of the Northwest Coast of Borneo. Water. 10. 1648. 10.3390/w10111648. 
• Chandrapati, S., & Williams, M. G. (2014). TOTAL VIABLE COUNTS | Most Probable Number (MPN). Encyclopedia of Food Microbiology, 621–624. doi:10.1016/b978-0-12-384730-0.00333-5 
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