What is Most Probable Number (MPN) Test?

The Most Probable Number (MPN) test is a widely recognized method employed to determine the concentration of viable microorganisms in a given sample. This method involves the use of liquid broth growth in ten-fold dilutions to replicate and estimate microbial populations. It is especially beneficial for samples like soils, waters, and agricultural products, where particulate material might interfere with traditional plate count enumeration methods.

Water quality testing often employs the MPN method to ascertain the safety of the water in terms of bacterial presence. Specifically, a group of bacteria known as fecal coliforms serve as indicators of fecal contamination in water. Therefore, if only a few fecal coliform bacteria are detected, it suggests that the water likely does not contain disease-causing organisms. Conversely, a high concentration of fecal coliform bacteria indicates a significant risk that the water contains pathogens, rendering it unsafe for consumption.

The MPN method, also known as the multiple tube fermentation test, is both qualitative and quantitative. It aims to determine the presence of microorganisms, primarily coliforms, in water. The process involves a series of test tubes filled with fermentative broth. Water samples are then added to these tubes in specific proportions. The presence of coliforms in the water sample can be identified by observing the fermentation tube for acid production, evidenced by a color change in the fermentative broth from red to yellow. Additionally, the formation of gas bubbles in an inverted Durham tube signifies gas production in the fermentation tube.

Besides its primary function of detecting fecal coliforms, the MPN test also identifies potential fecal contamination in water, which poses a threat to human health. Then, it becomes imperative to test water quality to determine the concentration of microorganisms and assess whether the water is potable.

The MPN test operates on a statistical principle based on the random dispersion of microorganisms per volume in a sample. The procedure entails adding measured volumes of water to a series of tubes containing a liquid indicator growth medium. Tubes that receive one or more indicator bacteria will exhibit growth and a characteristic color change. In contrast, tubes that only receive an inoculum of water without the indicator bacteria will not show this color change. By analyzing the distribution of positive and negative reactions, one can estimate the MPN of indicator organisms in the sample using statistical tables.

In summary, the MPN test comprises three sequential steps:

1. The Presumptive test, which screens for potential presence.
2. The Confirmed test, which verifies the initial findings.
3. The Completed test, which finalizes the results.

In conclusion, the Most Probable Number test is a crucial tool in microbiology, especially in water quality testing. It offers a detailed and sequential method to estimate the concentration of viable microorganisms in a sample, ensuring the safety and health of consumers.

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 Test (MPN Test) is a methodical approach employed in microbiology to estimate the concentration of viable microorganisms, specifically coliforms, in a water sample. This test operates on a principle that revolves around the serial dilution of the water sample and its subsequent inoculation in lactose broth.

In the lactose broth medium, any coliforms present in the water sample will metabolize the lactose, leading to the production of acid and gas. The acid production manifests as a color change in the medium, while the gas production is evident through the accumulation of gas bubbles in the inverted Durham tube placed within the medium. Therefore, the detection of both a color change and gas bubbles serves as a positive indication of coliform presence.

To quantify the total coliforms, one must count the number of tubes exhibiting a positive reaction, which means both a color change and gas production. Then, the pattern of positive results, specifically the number of tubes showing growth at each dilution level, is compared with standard statistical tables. This comparison allows for an accurate estimation of the coliform concentration in the original water sample.

The MPN test is executed in a detailed and sequential manner, comprising three distinct steps:

1. The Presumptive test, which serves as an initial screening to detect potential coliform presence.
2. The Confirmatory test, which verifies the results of the presumptive test.
3. The Completed test, which finalizes and confirms the presence and concentration of coliforms.

Furthermore, the MPN test is fundamentally a statistical method. The results obtained from the test are juxtaposed with standard statistical tables to derive a precise estimation. The test involves three sets of dilutions, each containing fermentative broth and the water sample in question. A positive result, indicated by the formation of acid and gas, is then quantitatively analyzed. This involves counting the number of tubes that show a positive result and subsequently comparing this pattern of positive outcomes with established statistical data.

In conclusion, the Most Probable Number Test is an essential tool in microbiological analysis, particularly for water quality assessment. By leveraging a combination of biological reactions and statistical comparisons, the MPN test offers a reliable method to determine the presence and concentration of coliforms in water samples.

Objectives of Most Probable Number Test (MPN Test)

1. Enumeration of Bacteria in Drinking Water: The foremost objective of the MPN test is to enumerate, or count, the number of bacteria present in a drinking water sample. This is achieved through the MPN method, which involves serial dilution of the water sample and its subsequent inoculation in a specific growth medium. By observing the reactions in this medium, such as color changes and gas production, scientists can estimate the concentration of viable bacteria in the original sample. Therefore, this method provides a quantitative analysis of bacterial presence, ensuring that drinking water meets the safety standards set for microbial content.
2. Identification of Bacteria in the Drinking Water Sample: Besides quantifying the bacterial content, the MPN test also aims to identify the specific types of bacteria present in the drinking water sample. This is crucial because not all bacteria are harmful; some are benign or even beneficial. However, certain bacterial strains can pose health risks if consumed. By identifying the bacteria, one can ascertain whether the water is contaminated with potentially harmful microbial species. This identification is typically achieved through further tests and analyses, which might involve studying the morphology, biochemical characteristics, and genetic makeup of the isolated bacteria.

MPN Test Procedure

Also Read: MCQ on Most Probable Number Test (MPN Test)

MPN test is performed in 3 steps

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

1. Presumptive Test of MPN 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 a pivotal step in the Most Probable Number (MPN) Test, specifically designed to screen water samples for the presence of coliform bacteria. To conduct this test efficiently and accurately, certain requirements must be met. Here’s a detailed and sequential explanation of the requirements for the Presumptive Test:

1. Medium Requirements: The primary medium used for the Presumptive Test can be Lactose broth, MacConkey broth, or Lauryl tryptose (lactose) broth. These mediums are specifically formulated to promote the growth of coliform bacteria and facilitate their detection through observable changes.
2. Glassware Requirements: Various glasswares are essential for the test. This includes test tubes of different capacities, such as 20ml, 10ml, and 5ml. Additionally, Durham tubes, which are small inverted glass vials, are placed inside the test tubes to capture and detect gas produced during fermentation.
3. Other Requirements: Sterile pipettes are crucial for transferring the water sample and inoculating the medium without introducing any contaminants.
4. Preparation of the Medium:
   • Begin by preparing the chosen medium, be it MacConkey broth or lactose broth, in two concentrations: single strength and double strength.
   • For samples of untreated or polluted water: Dispense the double strength medium into 10 tubes, with each tube containing 10mL. Similarly, dispense the single strength medium into 5 tubes, with each tube holding 10 mL. Ensure that a Durham tube is placed in an inverted position within each test tube.
   • For samples of treated water: Dispense the double strength medium into 5 tubes, each containing 10mL. Additionally, pour 50 mL of single strength medium into a separate bottle. Again, place a Durham tube in an inverted position within each container.
   • After setting up the tubes and bottle, examine them to ensure that the Durham tubes are completely filled with the liquid medium, with no air bubbles present.
   • Finally, sterilize all the prepared tubes and bottle by autoclaving them at 15 lbs pressure (121°C) for a duration of 15 minutes. This step ensures that the medium is free from any contaminants before the test begins.

Procedure of Presumptive Test

The Presumptive Test is a preliminary step in the Most Probable Number (MPN) Test, designed to screen water samples for the presence of coliform bacteria. The procedure for this test varies based on the type of water being tested, whether it’s untreated (polluted) or treated (unpolluted). Here’s a detailed and sequential explanation of the procedure for the Presumptive Test:

For Untreated (Polluted) Water:

1. MPN Water Testing Setup: Begin by arranging 5 tubes containing double strength medium and 10 tubes containing single strength medium for each water sample that needs to be tested.
2. Inoculation of Tubes: Using a sterile pipette, transfer 10 mL of the water sample into the 5 tubes containing 10 mL of double strength medium. Next, add 1 mL of the water sample to 5 tubes containing 10 mL of single strength medium. For the remaining 5 tubes with single strength medium, add 0.1 mL of the water sample.
3. Incubation: Place all the inoculated tubes in an incubator set at 37°C. After 24 hours, check the tubes for positive reactions. If no tubes show positive reactions, continue incubating them for an additional 24 hours, totaling 48 hours.
4. Result Interpretation: After the incubation period, compare the number of tubes displaying a positive reaction to a standard chart. This will provide an estimate of the number of coliform bacteria present in the water sample. For instance, if a water sample yields a result pattern of 3–2–1, it translates to an MPN value of 17. This means the water sample contains an estimated 17 coliforms per 100 mL.

For Treated (Unpolluted) Water:

1. MPN Water Testing Setup: Prepare 1 tube containing 50 mL of single strength medium and 5 tubes containing 10 mL of double strength medium for each water sample to be tested.
2. Inoculation of Tubes: With a sterile pipette, add 50 mL of the water sample to the tube containing 50 mL of single strength medium. For the 5 tubes with 10 mL of double strength medium, add 10 mL of the water sample to each.
3. Incubation: Incubate all the tubes at 37°C for 24 hours. If no positive reactions are observed after this period, continue incubation for an additional 24 hours, totaling 48 hours.
4. Result Interpretation: Post incubation, compare the number of tubes showing a positive reaction to a standard chart. This comparison will yield an estimate of the coliform count in the water sample. For example, if a water sample produces a result pattern of 1-4, it corresponds to an MPN value of 16. This indicates that the water sample contains an estimated 16 coliforms per 100 mL.

Presumptive Test Result

1. Positive Result:
   • Gas Formation: A positive result is primarily characterized by the formation of gas, amounting to 10% or more, in the Durham tube. This gas formation is observed within a time frame of 24 to 48 hours after the initiation of the test.
   • Turbidity and Color Change: Alongside gas formation, a positive result is also indicated by turbidity in the growth medium. Additionally, a noticeable color change in the medium further confirms a positive outcome.
   • Implication: The combined presence of gas, turbidity, and color change in the medium signifies a positive presumptive test for coliform bacteria. This result suggests the potential presence of fecal pollution in the water sample.
   • Nature of the Test: It’s essential to understand that the test is termed “presumptive” because, under the conditions set for this test, several other types of bacteria can produce results similar to coliforms. Therefore, while a positive result indicates the likelihood of coliform presence, it is not definitive.
2. Negative Result:
   • Absence of Growth and Gas: A negative result in the Presumptive Test is marked by the absence of any growth in the medium. Additionally, no gas formation is observed in the Durham’s tube.
   • Implication: A negative result suggests that the water sample does not contain coliform bacteria and is likely free from fecal pollution.

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 of MPN Test

The Confirmed Test is a crucial step in the Most Probable Number (MPN) Test, designed to validate the presence of coliform bacteria in water samples. This test is particularly significant because a positive result in the Presumptive Test does not necessarily confirm the presence of coliforms, as other microorganisms can produce similar results. Here’s a detailed and sequential explanation of the Confirmed Test:

1. Purpose of the Confirmed Test: The primary objective of the Confirmed Test is to ensure the presence of coliform bacteria by examining the tubes that showed positive results in the Presumptive Test. It’s essential to understand that the gas produced in the Presumptive Test does not guarantee the presence of coliforms in the water sample. Water contains various microorganisms, some of which, like certain yeasts and Clostridium species, can ferment lactose, producing both acid and gas. This can lead to false positive results in the Presumptive Test. Therefore, the Confirmed Test is vital to ascertain the presence of coliforms.
2. Methods of Confirmation: The Confirmed Test can be conducted in two primary ways:
   • Brilliant Green Lactose Bile Broth (BGLB): This method involves using BGLB, a selective medium designed to detect coliform bacteria in water, dairy products, and other food items. The selective agent in this medium is lactose. Additionally, the broth tube contains a Durham tube to capture and detect gas production.
   • Eosin Methylene Blue Agar Medium (EMB): This method involves using EMB agar, another selective medium. When a sample from a positive tube is streaked on this medium and incubated, typical coliform bacteria like E. coli and Enterobacter aerogenes exhibit growth and form distinctive red to black colonies with dark centers or a sheen. In contrast, other bacteria like Salmonella typhi might show growth but produce colorless colonies, while some, like S. aureus, might not grow at all.

Procedure of Confirmatory Test

Testing of positive presumptive in BGLB medium

The Brilliant Green Lactose Bile Broth (BGLB) medium is a specialized medium used to confirm the presence of coliform bacteria in water samples. The procedure for testing positive presumptive samples in BGLB medium is detailed and systematic, ensuring accurate results.

1. Preparation of BGLB Medium:
   • Begin by preparing a solution of BGLB medium. The components required are as follows:
     • Peptone: 10 g
     • Lactose: 10 g
     • Bile salt: 20 g
     • Brilliant green: 0.0133 g
     • Distilled water: 1 L
   • Once the components are combined, the next step is to sterilize the medium. This is achieved by autoclaving the BGLB medium for 15 minutes at a temperature of 121 degrees Celsius.
2. Inoculation of the Medium:
   • Before inoculation, it’s essential to shake the positive presumptive tubes gently to ensure an even distribution of the bacteria.
   • Using a sterile loop, transfer a loopful of culture from the positive presumptive tube into the BGLB fermentation tube. It’s crucial to note that the brilliant green dye present in the BGLB medium serves a specific function. This dye inhibits the growth of gram-positive bacteria, ensuring that only the desired coliform bacteria grow in the medium.
3. Incubation:
   • After inoculation, the test tubes are incubated at a consistent temperature of 35 degrees Celsius. This incubation period lasts for 48 hours, allowing sufficient time for the bacteria to grow and produce observable results.
4. Observation:
   • Post the incubation period, the test tubes containing the BGLB medium and the inoculum from the positive presumptive test are closely observed. The primary indicator of coliform presence is the production of gas in the inverted Durham tube. This gas production is a result of the fermentation process of the coliform bacteria.
5. Result Interpretation:
   • The results are interpreted based on the observations made. If gas production is observed in the BGLB medium, it indicates the presence of coliforms in the sample. This gas production is a definitive sign of coliform bacteria, as they are known to ferment lactose, producing gas in the process.

Therefore, the BGLB medium serves as an effective tool in the confirmation of coliform bacteria in water samples. The systematic procedure ensures that the results are both accurate and reliable, providing a clear indication of water quality.

Testing of positive presumptive in EMB medium

Eosin Methylene Blue (EMB) agar medium is a selective and differential medium primarily used to isolate and differentiate coliforms from other bacterial species. The procedure for testing positive presumptive samples in EMB medium is meticulously structured to ensure the accurate identification of coliform bacteria.

1. Preparation of EMB Medium:
   • Begin by formulating the EMB agar medium using the following components:
     • 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: 1L
   • After combining these components, the next step is sterilization. Sterilize the EMB agar by autoclaving it for 15 minutes at 121 degrees Celsius.
2. Pouring the Agar:
   • Once sterilized, pour the molten EMB agar onto sterilized Petri plates. Allow the medium to solidify, creating a firm surface suitable for bacterial growth.
3. Inoculation of the Medium:
   • Prior to inoculation, shake the positive presumptive tubes gently to ensure a uniform distribution of bacteria.
   • Using a sterile loop, streak a loopful of culture from the positive presumptive tube onto the solidified EMB agar surface.
4. Incubation:
   • Place the inoculated Petri plates in an incubator set at 35 degrees Celsius. Allow the plates to incubate for 48 hours, providing adequate time for bacterial growth and differentiation.
5. Observation:
   • After the incubation period, examine the Petri plates for bacterial colonies on the EMB medium. Typically, three distinct types of colonies can develop:
     • Typical colony: These colonies are characterized by their nucleated appearance and may exhibit a metallic sheen. This sheen is indicative of coliform bacteria, which ferment lactose, producing acid that reacts with the dyes in the medium.
     • Atypical colonies: These colonies appear non-nucleated, opaque, and mucoid. They represent bacterial species that do not produce the characteristic sheen.
     • Negative colonies: These colonies differ in appearance from both typical and atypical colonies and represent other bacterial species.
6. Result Interpretation:
   • The presence of typical colonies with a metallic sheen on the EMB agar is a clear indication of coliform bacteria. This sheen is a result of the interaction between the acid produced by lactose fermentation and the dyes in the EMB medium.

Therefore, the EMB agar medium serves as a crucial tool in the microbiological analysis of water samples. Its selective and differential properties ensure the accurate identification and differentiation of coliform bacteria from other microbial species.

Confirmed Test Result

1. Positive Result:
   • Gas Formation in Lactose Broth: One of the primary indicators of a positive result is the formation of gas in the lactose broth. This gas formation signifies the fermentation of lactose by bacteria, which is a characteristic trait of coliforms.
   • Observation on EMB Agar: On Eosin Methylene Blue (EMB) agar, coliform bacteria produce distinctive colonies that exhibit a greenish metallic sheen. This sheen is a hallmark of coliform bacteria and differentiates them from non-coliform bacteria, which do not produce such a sheen.
   • Presence of Thermotolerant E.coli: The growth of colonies at elevated temperatures, specifically around 44.5 ±0.2°C, indicates the presence of thermotolerant E.coli, a subset of coliforms. These bacteria can survive and thrive at higher temperatures, making their detection crucial for water safety assessments.
2. Negative Result:
   • Absence of Gas Formation: If there is no gas formation observed in the lactose broth, it suggests that the sample does not contain coliform bacteria or they are present in negligible amounts.
   • Observation on EMB Agar: A negative result is also indicated by the absence of coliform-like colonies on the EMB agar. Specifically, if there are no colonies exhibiting the characteristic greenish metallic sheen, it suggests that coliforms are not present in the 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 of MPN Test

The Completed Test is an integral part of the Most Probable Number (MPN) method, designed to provide a definitive confirmation of the presence of coliform bacteria in water samples. This test is particularly crucial when results from the Confirmatory Test are doubtful or when there’s a need for further validation of positive results. Here’s a detailed and sequential explanation of the Completed Test:

1. Purpose of the Completed Test:
   • The primary objective of the Completed Test is to reinforce the findings of the Confirmatory Test. It aims to eliminate any ambiguities and provide a clear indication of the presence or absence of coliform bacteria in the water sample.
2. Inoculation Process:
   • A representative colony, which is typical of coliforms, is selected from an LES Endo agar plate. This colony is then inoculated into two different mediums:
     • A tube containing brilliant green bile broth.
     • The surface of a nutrient agar slant.
3. Incubation:
   • Post-inoculation, both the brilliant green bile broth tube and the nutrient agar slant are incubated at a temperature of 35°C. This incubation period lasts for 24 hours, allowing sufficient time for the bacteria to grow and exhibit characteristic traits.
4. Observation and Analysis:
   • After the 24-hour incubation period, the brilliant green bile broth tube is meticulously examined for any signs of gas production. Gas formation in this medium is a characteristic trait of lactose fermentation by coliform bacteria.
   • Simultaneously, a Gram stain procedure is conducted using organisms from the nutrient agar slant. This staining process helps in visually differentiating between Gram-positive and Gram-negative bacteria.
5. Interpretation of Results:
   • If the observations reveal that the organism is a Gram-negative, non-spore-forming rod and there’s evident gas production in the lactose tube, then it conclusively indicates the presence of coliform bacteria in the water sample.

Procedure of Completed Test

1. Selection of a Typical Coliform Colony:
   • Begin by identifying a typical coliform colony on the agar plate. This colony would have been previously grown and identified during the confirmatory test.
2. Inoculation in Brilliant Green Bile Broth:
   • Using a sterile loop, transfer the selected coliform colony into a tube containing brilliant green bile broth. Ensure that a Durham’s tube is placed inside the broth tube. The Durham’s tube will serve to capture any gas produced during fermentation, providing a visual indication of coliform activity.
   • Besides the broth, streak the same colony onto the surface of a nutrient agar slant. This slant will be used for Gram staining, a technique that differentiates bacteria based on the structural characteristics of their cell walls.
3. Incubation:
   • Place both the brilliant green bile broth tube and the nutrient agar slant in an incubator set at 35°C.
   • Allow the samples to incubate for 24 hours. This duration provides adequate time for the bacteria to grow and exhibit characteristic behaviors.
4. Observation and Analysis:
   • After the 24-hour incubation period, inspect the brilliant green bile broth tube. Specifically, look for the production of gas in the Durham’s tube. The presence of gas indicates fermentation, a characteristic behavior of coliform bacteria.
   • Concurrently, perform a Gram stain on the organisms grown on the nutrient agar slant. This staining procedure will differentiate the bacteria based on their cell wall properties, further confirming their identity.
5. Further Inoculation (if required):
   • If additional validation is needed, take a loopful of culture from a tube that showed positive results in the confirmed test.
   • Inoculate this culture either in BGLB medium or by streaking it onto agar slants.
   • Again, incubate these test tubes for 24-48 hours at 35 degrees Celsius, observing for characteristic growth patterns and behaviors.

Completed Test Result

1. Positive Result:
   • Gas Production in Broth: One of the primary indicators of a positive result is the formation of gas in the brilliant green bile broth tube. This gas formation is a direct result of the fermentation process undertaken by coliform bacteria.
   • Gram Staining: Besides gas production, a positive result is further confirmed by the presence of Gram-negative, non-spore-forming rods when a Gram stain is performed on the nutrient agar slant. These specific characteristics are indicative of coliform bacteria.
   • Implications: A positive result in the completed test signifies the presence of coliform bacteria in the water sample. This, in turn, suggests potential fecal contamination, raising concerns about the water’s safety for consumption or use.
2. Negative Result:
   • Absence of Gas Production: A negative result is indicated by the lack of gas formation in the brilliant green bile broth tube. This absence suggests that the fermentation process, characteristic of coliform bacteria, did not occur.
   • Gram Staining: Additionally, the absence of Gram-negative, non-spore-forming rods on the nutrient agar slant, as revealed by Gram staining, further confirms a negative result.
   • Implications: A negative result in the completed test denotes the absence of coliform bacteria in the water sample. This suggests that the water is free from fecal contamination, making it safer for consumption or use.

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

1. Estimation in Diverse Samples:
   • Soils: The MPN test is instrumental in determining microbial populations in soil samples. This is crucial for understanding soil health, fertility, and its ability to support plant growth.
   • Waters: Water sources, whether natural or treated, can be assessed for microbial contamination using the MPN test. This is vital for ensuring water safety and quality.
   • Agricultural Products: The test is also employed to gauge the microbial load in agricultural products, ensuring they meet safety standards before reaching consumers.
2. Handling Particulate Material:
   • The MPN technique stands out, especially when dealing with samples laden with particulate matter. Such samples often pose challenges for traditional plate count enumeration methods. Therefore, the MPN test offers a more effective alternative, circumventing the issues posed by particulates.
3. Environmental Monitoring:
   • In the realm of environmental studies, monitoring microbial populations is paramount. The MPN test has been proposed as an alternative method to trend environmental monitoring studies, providing insights into microbial dynamics in various environments.
4. Counting Reluctant Bacteria:
   • Not all bacteria readily form colonies on standard agar plates or membrane filters. Some might be reluctant or slow to colonize these mediums. In such scenarios, the MPN test proves invaluable. It facilitates the counting of such bacteria, as they tend to grow more willingly in liquid media.

Advantages of MPN Test

1. Ease of Interpretation:
   • One of the primary advantages of the MPN test is its straightforward interpretation. The results can be discerned either by direct observation of the sample or by detecting gas emission. This simplicity in interpretation ensures that even those with minimal training can effectively utilize the test.
2. Dilution of Sample Toxins:
   • The MPN method inherently involves dilution, which means any toxins present in the sample are diluted. This not only ensures safety during testing but also minimizes the potential impact of toxins on the test results.
3. Analyzing Turbid Samples:
   • The MPN test is particularly adept at analyzing samples with high turbidity. Such samples, which may include sediments, sludge, and mud, often pose challenges for other testing methods. However, the MPN test can handle these with ease, ensuring accurate results even in challenging conditions.
4. Versatility in Sample Types:
   • Another significant advantage of the MPN test is its ability to analyze a wide variety of water samples. This versatility is especially notable when compared to the membrane filtration method, which might not be suitable for all water types. Therefore, whether the water is clear or turbid, the MPN test remains a reliable choice.
5. Applicability to Non-filterable Samples:
   • Some samples, due to their composition or consistency, cannot be analyzed using membrane filtration. In such cases, the MPN test comes to the rescue, permitting the analysis of these challenging samples and ensuring that no sample is left untested.

Limitations of MPN Test

1. Accuracy and Precision Concerns:
   • One of the primary limitations of the MPN test is its questionable accuracy and precision. The inherent variability in the method often means that it is considered a last-resort option, especially when other counting methods are available and more appropriate.
2. Labor-Intensive and Expensive:
   • The MPN test can be laborious, requiring meticulous attention to detail. Besides, it demands a significant amount of materials, glassware, and incubator space, making it a more expensive option compared to some other methods.
3. Large Margin of Error:
   • The results obtained from the MPN test come with a relatively large margin of error. This can pose challenges, especially when precise counts are essential for decision-making or further analysis.
4. Extended Duration for Results:
   • To confirm the presence of coliforms, the MPN test involves a series of three tests. This sequential testing means that obtaining definitive results can be time-consuming.
5. Sensitivity Issues:
   • While sensitivity is often seen as a strength, in the case of the MPN test, it can sometimes lead to false results. This heightened sensitivity can occasionally detect contaminants that are not of primary concern, leading to false positives.
6. Requirement of Extensive Hardware:
   • The MPN method necessitates the use of multiple test tubes and a significant amount of glassware for media preparation. This not only increases the cost but also demands more storage and handling.
7. Potential for False Positives:
   • The probability of obtaining false positives is another limitation of the MPN test. This can lead to unnecessary interventions or further testing, increasing both time and cost.