Aquatic Microbiology X – Various laboratory tests for detection of indicator organisms in potable water

Take 3 sets of 10 ml of Lauryl Tryptose broth (LTB), each set coprising 6 test tubes
Inoculate 10 ml, 1 ml and 0.1 ml of the water sample in each of the sets. Incubate at 37°C/48 hrs
Check for gas formation in the Durham tubes. If there is gas production, confirmatory test is carried out
In this test, the water sample is inoculated BGLB broth (Brilliant Green Lactose Bile broth). It contains brilliant green dye which is inhibitory to gram positive bacteria. On incubation at 37°C/24 hrs, if there is gas formation then it is confirmed that coliforms are indeed present
Confirmatory test can also be carried out using EMB agar (Eosin Methylene blue agar). Coli-aerogenes organisms produce characteristic colonies. Faecal coliforms appear as small dark colonies with black centres and metallic sheen. Non-faecal coliforms show large, pink, mucoid colonies with dark centres and rarely show metallic sheen
It can also be carried out in Endo agar wherin lactose is fermented to acetaldehyde and sulphite, thus changing the colour of basic fuchsin to red
Completed test – If BGLB is used in confirmatory test, the broth culture is first streaked onto EMB agar to obtain colonies in order to characterize. If already EMB agar or Endo agar has been used, the typical colonies are selected and inoculated into LTB to check if coliforms produce gas and into agar slant and incubated. Gram stain is done. Coliforms will be observed as gram negative, non-sporulating bacilli

Differentiates between typical and atypical colonies

Indole test – Culture is inoculated in tryptone water. Contains xylol and Kovac’s reagent. Based on the ability to produce indole from tryptophan. Colour changes to red
Methyl Red test – Medium contains glucose and phosphate. Based on the amount of acidity produce in a special glucose – broth medium. Detected by pH indicator methyl red which turns red when acidic and is yellow under neutral conditions. Both organisms produce acid from glucose. However, E.coli produces a larger amount which lowers the pH and thus turns the indicator red whereas E.aerogenes does not produce as large a quantity which can lower the pH and hence, produces no colour change
Voges –Proskeur test (VP test) – Medium contains glucose and phosphate. Based on the ability to produce acetoin or acetylmethyl carbinol in a glucose-peptone medium. Detected using O’Meara’s reagent which contains naphthol and KOH. Indicator turns red when the compound is produced
Citrate test – Medium used is Simmon’s citrate agar. Based on the utilization of sodium citrate as the sole C source. Indicator used is bromothymol blue which turns yellow under acidic and blue under alkaline conditions

Stands for Membrane filter Faecal Coliforms medium
Selective and differential medium
Contains rosolic acid which inhibits the growth of bacteria except faecal coliforms
Bile salts inhibit the growth of non-enteric bacteria
Aniline blue is the indicator

Modification of MPN procedure
100 ml of water sample is inoculated in a single culture broth containing triple strength LTB
Indicator used in this case is bromocresol purple
It tests for the presence for the presence of coliforms
Positive result would yield production of acid and gas by fermenting lactose in the medium thereby turning the colour of the medium from purple to yellow

This test is used to indicate the presence of coliforms and coli. It enumerates E.coli and coliforms without the need of a confirmatory test
100 ml of the water sample is inoculated in a special medium containing only ONPG (ortho-nitrophenol galactopyranoside) and MUG (4-methyl-umbelliferyl glucoronide) as the nutrients
If coli is present, ONPG is metabolized to o-nitrophenyl by the beta galactosidase enzyme changing the colour of the medium to yellow.
It also converts the substrate MUG to 4-methyl-umbelliferone using beta glucoronidase. This product fluoresces when observed under UV light

Most accepted method for water quality monitoring
Highest number of positive samples is looked for and matched with McGrady table to obtain organism count in the water sample
Less frequent method and used under certain circumstances such as when membrane in MF technique gets choked before adequate water can be filtered and where deposited materials on the membrane could potentially inhibit the growth of organisms
Used for waters containing only small numbers of indicator organisms (ie., less turbid water)

Found in brackish and marine water as they die at a slower rate than the faecal coliforms and hence, used as an indicator of recent pollution
Enriched in Slanetz-Bartley medium that contains sodium azide and TTC (Triphenyl tetrazolium chloride)
Sodium azide is generally toxic to the ETS of aerobes, thus inhibiting their growth. Anaerobes that carry out fermentation such as lactic acid bacteria like faecalis are able to grow due to absence of ETC (electron transport chain)
These Enterococci reduce TTC to insoluble red dye called formazan that gives rise to dark red to maroon colonies

They are anaerobic spore-bearers
In litmus milk, they carry out stormy fermentation which produces a lot of acid and gas. The acid so formed causes milk to clot that can be broken down by normal fermentation
When grown in Differential Reinforced Clostridium medium, they produce black colonies

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