Also commonly known as "indicator organisms", coliform refers to a wide variety of bacteria that can be found throughout the environment. This means that these organisms can be found in soil, water surfaces, vegetations as well as on the skin or intestinal tract of warm-blooded organisms such as humans.
Although some are pathogenic (capable of causing diseases - mild to life threatening diseases) most of them are harmless. Regardless, detection of coliform (indicator organisms) indicates the presence of potential disease causing bacteria not only in water, but also in given foods and drinks (milk etc). Therefore, coliform are important because they help raise awareness and determine the source of the bacteria.
Divided into three main groups
This group is largely composed of harmless, closely related bacteria. Apart from human and animal waste, total coliform bacteria can be found in such environments as water, vegetation and soil where they live freely.
While they are generally harmless, the presence or detection of this group of bacteria in drinking water or water source that supply drinking water is important because they are indicative of possible contamination.
If detected in a water sample, this suggests that disease causing coliform may also be present and thus the need to treat the water source or determine the source of contamination (environmental contamination etc). Thermotolerant coliform are good examples of total coliform bacteria. These are coliform that are capable of fermenting lactose at 45 degrees.
* Detection of total coliform does not necessarily mean that disease causing bacteria are present in water
* Testing the presence of total coliform bacteria basically involves growing them in lactose media at about 35 degrees
Fecal coliform bacteria (FC) are a subgroup of the total coliform bacteria that can be found in the intestines and feces of warm blooded animals (human beings, pigs, cows, dogs, pigs etc). E. coli is an example that typically resides in the intestinal tract of warm-blooded animals and thus the animal's fecal matter.
When they are outside the host's body, these organisms cannot live for long because their survival is largely dependent on the host.
Compared to total coliform bacteria, which are largely harmless, the fecal are composed of both pathogenic and non-pathogenic bacteria. As such, their detection in a sample of drinking water is an indication that the water is contaminated by sewage.
The presence of these bacteria is also very important because
the source of the bacteria is well known compared to the source of total
coliform bacteria (TC). Here, therefore, it becomes easier to locate and fix
the source of the problem and treat the water more effectively in order to prevent possible diseases associated.
* Fecal coliform bacteria can also be found in such animals as shellfish. Therefore, people can get sick either by drinking water contaminated by the bacteria or from eating contaminated shellfish.
Some of the illnesses that can result range from mild stomach upsets to severe salmonellosis (salmonella poisoning) caused by salmonella bacteria.
E. coli is a sub-group of fecal coliform bacteria and is largely composed of E. coli (Escherichia coli). Compared to others, E. coli are almost exclusively found in the intestines of warm-blooded animals where they are able to live and reproduce.
Although they are mostly harmless in the host's intestines, there are strains of E. coli (e.g. E.coli 0157:H7) that can cause serious illnesses. Detection of these organisms in water is indicative of fecal contamination (recent contamination in most cases) as well as possible presence of other pathogenic organisms that may include viruses. In such cases, water is contaminated by sewage or animal feces.
* E. coli cannot live long outside the host, for this reason, their presence in water is evidence that water was recently contaminated.
* If contaminated animal meat (such as beef) is consumed (not cooked properly) it can cause the consumer to become ill
The following are some of the testing methods used to determine whether total coliform bacteria are present in a sample of water:
For this technique, the filter membrane is used to filter and thus retain any coliform bacteria that may be present in the sample.
After incubation, the bacteria (if present) will use the nutrients in the agar plate to grow.
If a blue color is observed, this indicates that the beta-glucuronidase enzyme of E. coli was involved in breaking down Indoxyl-beta-D-glucuronide (IBDG) in MI agar and thus indicates the presence of E. coli. On the other hand, a fluorescence appearance indicates that beta-galactosidase was involved in breaking down 4-methylumbel-liferyl-β-D-galactopyranoside (MUGal) which is also present in the agar.
* For this technique, absorbent pads with lauryl tryptose broth can also be used. These are transferred to either M-endo media or agar to grow the bacteria.
* Once bacteria are cultures, the colonies are then counted under the microscope
(To determine the presence of rod shaped, facultative
anaerobic, gram-negative coliform group of bacteria that do not form spores)
For this technique, the procedure involves three main phases that include:
Phase 1: Presumptive stage
* Gas formation (bubbles) in the tubes is marked as a positive presumptive test
Phase 2: Confirmed state (using fermentation
tubes with brilliant green lactose bile broth)
* If gas is formed during this phase, it is an indication of positive confirmed test
Phase 3: Completed test
For this phase, requirements include samples with positive confirmed test, eosin methylene blue plate, incubator, lauryl tryptose broth fermentation tube as well as nutrient agar slant.
* The presence of gas in fermentation tubes indicates the presence of bacteria and this is marked satisfactory completed test. If gram-negative, rod shaped (without spores) bacteria are identified through microscopy, this is also indicative of the presence of the bacteria (total coliform group)
For the agar plates, the media used (culture media) inhibit the growth of gram positive bacteria and only allow the test to determine their presence of gram-negative bacteria capable of fermenting lactose.
Depending on the media used, the color of the agar plate will help indicate whether coliform are present in the sample:
Essentially, MPN is similar in principle to
multiple tube fermentation technique. However, rather than simply being used to
determine the presence of the bacteria (particularly fecal coliform) MPN is
used to estimate bacterial concentration in water in order to determine whether
the water is safe for use in homes.
As with multiple tube fermentation technique, the technique involves three main steps (presumptive test, confirmatory test and completed test). The sample is diluted in different tubes of different sample concentration and inoculated in lactose broth. This technique makes it possible to determine the amount of bacteria in different dilutions of the sample after they are cultured.
The presence of the bacteria in the sample is indicated by the production of either gas or acid (change in the color of the media or presence of bubbles). For instance, all the tubes with the highest concentration of the sample may test positive for the bacteria while a few of the less concentrated tubes (less sample concentration) may prove positive following the test.
The MPN index is used to show the number of bacteria in the water and thus help determine whether the water is safe to drink.
This involves the following steps:
For instance, if there were three sets of broth tubes each with 5 tubes. Each set would be of different concentration. The first set (with 5 tubes) may be the original, undiluted sample, the second set (with 5 tubes) may be 10 to the negative 1 dilution (half the concentration of the original) while the third set (also with 5 tubes) may be 10 to the negative 2 dilutions (half the concentration of the second set).
Assuming that all of the tubes in set 1 are positive of the bacteria, 3 in the second set are positive and only 1 in the third set is positive, then these results can be compared to the MPN table to determine the MPN index (estimated number of coliforms in 100mL of water) and therefore determine whether the water is safe for use.
* MPN index below 2 is considered safe for drinking
Microscopy can be used to view E.coli coliform in wastewater, ground water or urine. Here, microscopy can be used to view bacteria colonies or count individual bacterial cells.
Observing bacterial cells on a membrane
* Before any step is taken, it is important to ensure that all apparatus used are sterile. This helps prevent contamination that can result in false results.
Using a pair of forceps, carefully remove the membrane filter and place it on the agar plate in a Petri dish (MacConkey agar, Eosin methylene blue agar and Violet red agar)
* To determine the number of colonies in 100ml of the sample, divide the number of colonies counted with the milliliters of the sample used in the procedure and multiply the results with a 100. This will give the percentage of the coliform in the water/urine or wastewater.
FISH (fluorescent in situ hybridization) refers to a technique that uses fluorescent probes for the purposes of detecting and comparing DNA sequences.
When it comes to detecting E.coli bacteria,
this technique has the advantage of saving time compared to the other
techniques commonly used for detecting the presence of coliform.
* for this technique, epifluorescence microscopy is used to view the sample (with WIBA filter block)
Depending on the type of coliform present in the
sample, microscopy will show weak or high fluorescence intensity from the
fluorescent probes attached to the bacteria. This makes it possible to count
the number of individual bacteria in the specimen.
More info at Bacteria under the Microscope
Cara Gleeson and Nick Gray (1996) The Coliform
Index and Waterborne Disease: Problems of microbial drinking water assessment.
Cliff Treyens (2009) Bacteria and Private Wells. Information Every Well Owner Should Know. Cliff Treyens, Director of Public Awareness, National Ground Water Association.
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