What is mlbb in microbiology?
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MPN is most commonly applied for quality testing of water i.e to ensure whether the water is safe or not in terms of bacteria present in it. A group of bacteria commonly referred to as fecal coliforms act as an indicator of fecal contamination of water. The presence of very few fecal coliform bacteria would indicate that water probably contains no disease‑causing organisms, while the presence of large numbers of fecal coliform bacteria would indicate a very high probability that the water could contain disease‑producing organisms making the water unsafe for consumption.
Water to be tested is diluted serially and inoculated in lactose broth, coliforms if present in water utilizes the lactose present in the medium to produce acid and gas. The presence of acid is indicated by the color change of the medium and the presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium. The number of total coliforms is determined by counting the number of tubes giving positive reaction (i.e both color change and gas production) and comparing the pattern of positive results (the number of tubes showing growth at each dilution) with standard statistical tables.
MPN test is performed in 3 steps
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 method of the presumptive test varies for treated and untreated water.
To view the full table download the PDF file from the link given in the reference.
Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to confirm the presence of coliform, a confirmatory test is done.
From each of the fermentation tubes with positive results transfer one loopful of medium to:
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 agar slants should be incubated at 37°C for 24± 2 hours and Gram-stained preparations made from the slants should be examined microscopically.
The formation of gas in lactose broth and the demonstration of Gram-negative, non-spore-forming bacilli in the corresponding agar indicates the presence of a member of the coliform group in the sample examined.
The absence of gas formation in lactose broth or the failure to demonstrate Gram-negative, non-spore-forming bacilli in the corresponding agar slant constitutes a negative test (absence of coliforms in the tested sample).
Tryptone Water Test
a. Confirmatory tests positive for indole, growth, and gas production show the presence of thermotolerant E. coli.b. Growth and gas production in the absence of indole confirm thermotolerant coliforms.
Since some of the positive results from the confirmatory test may be false, it is desirable to do completed tests. For this inoculum from each positive tube of the confirmatory test is streaked on a plate of EMB or Endo agar.
In this process, a loopful of a sample from each positive BGLB tube is streaked onto selective medium like Eosin Methylene Blue agar or Endo’s medium. One plate each is incubated at 37°C and another at 44.5± 0.2°C for 24 hours.
Following incubation, all plates are examined for the presence of typical colonies.
References and further reading
Image Source: Microbe Online and Scharlab.
This test, a specific enrichment procedure for coliform bacteria, is conducted in fermentation tubes filled with a selective growth medium (MacConkey lactose broth), which contains inverted Durham tubes for the detection of fermentation gas. A series of lactose broth tubes are inoculated with measured amounts of the water sample to be tested. The series of tubes may consist of three or four groups of three, five, or more tubes.
The main selective factors found in the medium are lactose, sometimes a surfactant such as Na-lauryl sulfate or Na-taurocholate (bile salt), and often a pH indicator dye for facilitating detection of acid production, such as bromcresol purple or brilliant green. The selective action of lactose occurs because many bacteria cannot ferment this sugar, whereas coliform bacteria and several other bacterial types can ferment it. The surfactant and dye do not inhibit coliform bacteria, whereas many other bacteria, such as the spore formers, are inhibited.
This test serves to confirm the presence of coliform bacteria when either a positive or doubtful presumptive test is obtained.
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.
Image Source: Microbe Online
Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to confirm the presence of coliform, a confirmatory test is done. For this, a loopful of suspension from a positive tube is inoculated into a 3 ml 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
B. Inoculation in media slants
Positive: The formation of 10% gas or more in the Durham tube within 24 to 48 hours, together with turbidity in the growth medium and the color change in the medium constitutes a positive presumptive test for coliform bacteria, and hence for the possibility of fecal pollution.
Negative: No growth or formation of gas in Durham’s tube.
Positive: Formation of gas in lactose broth and the demonstration of a coliform-like colony on the EMB agar indicate the presence of a member of the coliform group in the sample examined.
Coliforms produce colonies with a greenish metallic sheen which differentiates them from non-coliform colonies (show no sheen). The presence of typical colonies at high temperatures (44.5 ±0.2) indicates the presence of thermotolerant E.coli.
Negative: The absence of gas formation in lactose broth or the failure to demonstrate coliform-like colonies on the EMB agar.
Positive: The presence of gas in the brilliant green bile broth tube and Gram-negative, non-spore-forming rods on NA slant constitutes a positive completed test for the presence of coliform bacteria, which, in turn, infers possible contamination of the water sample with fecal matter.
Negative: Absence of growth and gas formation in the broth. Absence of gram-negative, non-sporing rods on Gram staining.
MPN test involves a series of a test tube containing fermentative broth, in which the water samples are added in a definite proportion. We can detect coliform in the water sample by observing the fermentation tube for the production of acid and gas. A colour change in the fermentative broth (from red to yellow) indicates the production of acid.
Formation of gas bubbles in an inverted Durham tube indicates the production of gas in the fermentation tube. A most probable number is extensively used to estimate or determine the number of microorganisms from the large samples like water, soil etc. MPN test detects the presence or absence of faecal coliforms, which can cause faecal contamination in water and making it unsafe for human consumption.
Therefore, the quality testing of water becomes necessary to know the concentration of microorganisms or water quality, i.e. whether water is potable or not. In this lesson, we will discuss the definition. principle, methods, advantages and disadvantages of the most probable number test.
MPN stands for a most probable number. It refers to the qualitative and quantitative analysis of water, which can enumerate the presence of faecal coliforms. E.coli is a common faecal contaminant present in water, and it can cause serious illness if it goes into the body. Thus, in most probable number method, E.coli is used as “Pollution indicator” to analyse the water quality. The most probable number includes three sets of methods like presumptive, confirmative and completed-test.
A most probable number is a statistical method, in which the results are compared with the standard statistical tables. It involves three sets of dilutions containing fermentative broth and the water sample. The formation of acid and gas indicates a positive result. The quantitative analysis of coliform is determined by counting the number of tubes giving a positive result and comparing the pattern of positive results with the statistical data.
MPN is a method, which involves three consecutive tests, namely presumptive, confirmatory and completed-test.
The preliminary or screening test uses a series of fermentation tube containing lactose broth of known concentration. The negative presumptive analysis indicates that the water is microbiologically safe and does not require further testing. But in the case of positive results, one needs to confirm the presence of coliforms through the confirmed MPN-test.
The production of gas requires 40-390 million per ml of coliforms according to the Chambers. The gas formation depends upon the ratio of coliform to the non-coliforms. If the ratio of non-coliform bacteria is high, it will also reduce the production of gas. The presumptive test involves the following steps:
Observation: After incubation, the results are made based on any colour change or gas production in the MacConkey broth.
Result interpretation: The colour change of MacConkey broth from red to yellow and the gas production inside the Durham tube will indicate a positive result for the presumptive test. If the broth colour remains the same, i.e. red and no gas forms, it will give a negative result for the presumptive test.
It is the confirmatory test ensuring the presence of coliform by testing the positive tubes of the presumptive test. The gas produced in the presumptive test does not mean that there must be coliform in the water sample. Many other microorganisms are also present in water, which can give a false presumptive test.
Some yeasts and Clostridium species are present in the water, which can ferment lactose by producing both acid and gas. Therefore, it becomes necessary to confirm the presence of coliform in water. A confirmed test can be performed usually in two ways:
Testing of positive presumptive in BGLB medium
Observation: Observe the test tubes containing BGLB medium and inoculum of positive presumptive test for the production of gas in the inverted Durham tube.
Result interpretation: The production of gas in the BGLB medium indicates the presence of coliforms
Testing of positive presumptive in EMB medium
Observation: Observe the plates for the appearance of bacterial colonies on the EMB media. Generally, three types of colony develops:
Result interpretation: The production of a typical colony indicates the presence of coliform bacteria.
It is the final test to ascertain the presence of coliforms. A completed test involves the following steps:
Observation: Observe the test tubes for gas production in BGLB medium or the growth of a bacterial colony in the agar slants.
Result interpretation: The production of gas in the Durham tube of the BGLB fermentation tube confirms the presence of coliform bacteria. The growth of gram-negative, non-sporing rods in the agar slants confirms the presence of coliforms.
In the MPN method, a number of tubes showing positive results will estimate the coliform count per 100ml. The numbers are compared with the statistical data that Maccardy gave.
