water testing for coliforms

Bacteriology 102:
A General Overview of Coliforms

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COLIFORMS AND THE CONCEPT OF INDICATOR ORGANISMS

Due to the wide variety of intestinal pathogens (bacteria, viruses, protozoa) that could be found in feces, sewage and (ultimately) water, it would not be cost-effective to test specifically for the presence of each different pathogen. This is where the concept of the indicator organism applies. Generally, an "innocent" organism which often finds itself associated with (i.e., in the same natural habitat as) problem-causing organisms can be, when isolated from a site, an indication that the problem organisms may also be present at that site. Such an associated organism is therefore an indicator of the possible problem. Specifically, if pollution of a water resource by sewage or other fecal pollution (and the associated intestinal pathogens) is suspected, a desirable indicator organism for such pollution would follow these criteria:

It is always present in the feces from both normal and infected individuals.

It is not found anywhere in the environment except where there is contamination by fecal pollution.

It survives longer in the environment than pathogens, but not so long as to indicate "historical" contamination.

It is easy to detect in the laboratory in that its characteristics can be exploited to make it easily enriched for and isolated (according to principles we learned in Exp. 11).

It should not cause a "problem" itself and pose a health risk for laboratory workers.
In the United States, significant indicator organisms for certain kinds of contamination of water and food are the coliforms. The usual definition for coliforms that one may encounter is that they are gram-negative rods that ferment lactose rapidly with the production of gas (insoluble gas that is detectable in a Durham tube) in Lactose Lauryl Tryptose Broth and Brilliant Green Bile Broth at 35°C. Those which additionally do so in EC Broth at 44.5°C belong to the subset of coliforms called "fecal coliforms." Coliforms happen to be easy to detect with the appropriate selective-differential media. These media tend to inhibit gram-positive bacteria, and the presence of coliforms is suggested by gas from lactose fermentation in the Durham tube of the enrichment media and by acidic colonies on EMB Agar which is the first step in the actual isolation process. Naturally any isolate must at least show gram-negativity and the ability to ferment lactose to acid and gas – two hallmarks of identification as a coliform. Most often a coliform isolate is ultimately identified as a species of Escherichia, Enterobacter, Klebsiella or Citrobacter, and one that has come through the EC Broth enrichment at 44.5°C is usually identified as Escherichia coli.

The presence of E. coli is considered a definitive indication of fecal pollution (with the possibility of the associated intestinal pathogens), as the natural habitat of these organisms is the intestinal tract of humans and higher animals. Coliforms identified otherwise may be found throughout the environment such as in soil or on plants. Finding these "non-fecal coliforms" may not indicate fecal contamination, but their presence in drinking water may indicate contamination by organisms from soil where there could be significant chemical or biological contamination. So, coliforms find their utility in being indicator organisms – and not just for fecal contamination.

Coliforms do not constitute a discrete taxonomic group. Many strains of the aforementioned genera may not ferment lactose at all and would therefore not be called coliforms. For example, some pathogenic E. coli strains do not ferment lactose, and it is sometimes very difficult to differentiate them from Shigella. Also, most strains of Citrobacter (such as the one we use in Experiments 14 and 17) very weakly attack lactose and may be initially confused with Salmonella when isolations are made from clinical or natural samples. So, considering the absolute definition of this non-taxonomic term, there can be no such thing as a non-lactose-fermenting coliform.

TESTING FOR COLIFORMS

From the foregoing it can be seen that in order to enrich for coliforms selectively, one can formulate media which (1) inhibit gram-positive bacteria as much as possible and (2) allow for the enrichment and detection of those organisms which ferment lactose to acid and gas, the latter being detectable in a Durham tube.

In the traditional coliform testing procedure, a water sample is inoculated into a tube of Lactose Lauryl Tryptose Broth (LLTB) to set up the selective enrichment for coliforms which is termed the Presumptive Test. If growth and gas are seen in the tube after incubation for up to 2 days at 35°C, one can presume that at least one coliform was originally inoculated into the medium, multiplying into a large population of cells while fermenting lactose with the production of acid and gas. Hydrogen is the insoluble gas that collects in the Durham tube; carbon dioxide tends to be soluble although some can be found in the gas bubble. (No pH indicator is included in the medium as detection of acid would be unnecessary.) The culture in the tube is certainly not a pure culture. Many non-coliform organisms such as Pseudomonas can also grow in the medium. In order to achieve maximum recovery of coliforms – possibly "damaged" by deleterious agents in the environment and thereby sensitive to selective agents in media – LLTB was made a medium of low-selectivity. It may not inhibit totally the gram-positive organisms in the sample, as strains of Bacillus and Clostridium which ferment lactose to acid and gas may grow.

Each positive tube (i.e., showing growth and gas) is then inoculated (by loop) into a tube each of two media for the Confirmatory Test. These media are strongly selective for gram-negative organisms and may even inhibit some enterics. Growth and gas constitute a positive result as for LLTB.

For the selective enrichment (and detection) of the "true" (gram-negative) coliforms, Brilliant Green Lactose Bile (BGLB) Broth is used and incubated for up to 2 days at 35°C. Thus, if any cells in the inoculum were coliforms, they should multiply, fermenting lactose and producing gas as in LLTB. Any of the above-mentioned lactose-positive strains of Bacillus and Clostridium which had contributed to a positive result in the Presumptive Test are inhibited in this medium. One should not expect only coliforms in BGLB, however, as Pseudomonas and many other gram-negative organisms can grow in the medium.

For the selective enrichment (and detection) of the fecal coliforms, a subset of the coliforms (not a separate group!), EC Broth is inoculated and incubated for up to 2 days at 44.5°C. Growth and gas together indicate a probability of E. coli and associated fecal contamination being present.
For quantitative coliform analysis, the Most Probable Number (MPN) method is applied with the dilution of the sample and inoculation of the LLTB Broth from the dilutions for the Presumptive Test, with each positive tube then being "confirmed" in the next step of the procedure.

A summary table of the aforementioned selective enrichment media follows. Note that these media increase in selectivity for the desired organisms from left to right. Of course, we would not expect pure cultures of anything in any enrichment medium (selective or otherwise), so the subsequent isolation is important as in any enrichment-isolation procedure as we have learned in Experiment 11. For a tube which shows growth and gas, suggesting the presence of coliforms, all it might have taken for a population of coliforms to develop in the tube could have been just one coliform cell in the inoculum. One way or another in the enrichment and isolation process, the non-coliforms will be "sorted out."

Chemoheterotrophic
Organisms in Water Growth Response of Organisms in Coliform Enrichment Media
LLTB BGLB EC Broth at 44.5°C
I. The true coliforms: "fecal" and others growth & gas growth & gas growth & gas for fecal coliforms (no growth for non-fecal coliforms)
II. Gram-negative bacteria other than coliforms growth
growth
little or no growth
III. Lactose-fermenting (to acid & gas) strains of Bacillus and Clostridium ("false coliforms") growth & gas no growth no growth
IV. Gram-positive bacteria other than those in the row above possible growth
no growth no growth

Now it is desired to obtain pure cultures of any coliforms present and identify them to some degree. Positive tubes from the Confirmatory Tests are streaked onto Eosin Methylene Blue (EMB) Agar to begin the Completed Test (actually a series of tests). Dark colonies on EMB Agar indicate probable coliforms (see explanation of the medium in Period 4). Representative colonies may be inoculated into Nutrient Agar slants and Lactose Fermentation Broth to check for basic coliform characteristics (gram-negative rods, fermentation of lactose to acid and gas). Traditionally the IMViC tests are done for each isolate; the four major letters of this acronym stand for the indole, methyl red, Voges-Proskauer and citrate tests. A certain combination of results usually leads to a probable identification, although additional tests must be run if definitive identification is required.

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Page last modified on 6/5/09 at 8:00 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison

It's official: Men are obsessed with sex, hide their emotions, and cheat - Yahoo! India News

ANI
It's official: Men are obsessed with sex, hide their emotions, and cheat

Thu, Apr 1 12:25 PM
London, Apr 1 (ANI): Expert Dr Louann Brizendine has dived inside a man's mind and confirmed what most women long suspected: men are obsessed with sex, hide their emotions, and cheat.
According to Brizendine, testosterone causes the "man trance", where blokes have to stare at boobs, reports The Daily Star.
She says: "The best advice I have for women is make peace with the male brain. Let men be men."
Some of the other findings in the expert's new book Male Brain: A Breakthrough Understanding Of How Men And Boys Think are:
1 Men really are sex-crazed
The brain's part inked to sexual pursuit is two-and-a-half times larger in males than females.
2 They're programmed to perv
The testosterone drives what Louann calls the "man trance" - a glazed-eye stare at breasts. She says: "I wish I could say that men can stop themselves from entering this trance. But the truth is, they can't."
3 Men want more partners
According to the book, men want an average of 14 sexual partners in their lifetime. Women want one or two.
Louann says: "It's postcoital narcolepsy. During orgasm, males release a huge amount of oxytocin in their brains, and it is very sedating. It's not that he doesn't love you."
4 Men lie more about sex
Biologically speaking, men are more comfortable lying to the opposite sex.
5 Foreplay round the clock
In case of women, foreplay is everything that happens in the 24 hours before intercourse. For men it's what happens three minutes before entry
Louann says: "The male brain's initial emotional reaction can be stronger than the female. But within 2.5 seconds his face changes to hide the emotion, or even reverse it."
The expert doesn't reckon her book justifies bad behaviour. She says: "This is not giving men an excuse to rape and pillage. But men do have a right to give voice to their biological predisposition and have it come in to the dialogue." (ANI)