Coliform

Types, Characteristics, Tests, Techniques and Microscopy

What are Coliform Bacteria?

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.

Types of Coliform Bacteria

Low-temperature electron micrograph of E. coli bacteria, magnified 10,000 times. Photo by Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU. via Wikimedia Commons

Divided into three main groups which include:

• Total coliform bacteria
• Fecal coliform bacteria

• E. coli

Total Coliform Bacteria (TC)

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

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.

Escherichia Coli

E. Coli (Coliform)

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

Some Coliform Representatives

• Citrobacter - A genus in Enterobacteriaceae family that includes such bacteria as C. amalonaticus and C. freundii
• Enterobacter - A genus in class Gammaproteobacteria that includes Enterobacter cloacae and Enterobacter aerogenes
• Hafnia - Also belonging to the Enterobacteriaceae family, Hafnia includes such bacteria as Hafnia alvei
• Escherichia - Genus of Family Enterobacteriaceae and includes bacteria like E. coli

• Klebsiella

Some Coliform Characteristics

• Gram negative - Coliform bacteria have a thin peptidoglycan layer and thus are unable to retain the primary stain (gram staining) when washed with alcohol. Typically, they will stain red or pink during staining because they take up the counter stain. See Gram Stain

• Do not form spores - During extreme conditions, some bacteria form spores so that they can survive and germinate during favorable conditions. However, coliforms do not undergo this process

• They are facultative anaerobes - They can survive with very little or no oxygen by using anaerobic respiration.

• Rod shaped - Also known as bacillus, they are shaped like a rod (elongated)

• They ferment lactose - Fermentation of lactose by coliform results in the production of acid and gas, which helps in their identification during coliform testing

Indicator Tests

The following are some of the testing methods used to determine whether total coliform bacteria are present in a sample of water:

• Membrane
• Filtration (membrane filter technique)

Requirements

• Water sample (ground or waste water)
• Membrane filter - The filter (cellulose ester membrane) used for this technique has pores of 0.45 micrometers and measures about 47milimeters
• MI agar
• Incubator

Procedure

• Poor about 100 milliliters of the water sample through the filter
• Place the filter on the plate agar (on MI agar)
• Incubate for about 24 hours at 35 degrees (temperature)

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

Multiple Tube Fermentation Technique

(To determine the presence of rod shaped, facultative anaerobic, gram-negative coliform group of bacteria that do not form spores)

Requirements

• Lauryl tryptose broth
• Glass tubes

Procedure

For this technique, the procedure involves three main phases that include:

Phase 1: Presumptive stage

• Add lauryl trypsone broth into several fermentation tubes
• Inoculate varying quantities of the sample (water sample) in to the tubes
• Incubate the tubes for between 24 and 48 hours at about 35 degrees Celsius (check every 24 hours for gas formation)

* 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)

• For this step, only the samples that marked positive in the first phase (positive presumptive test) are used
• To the fermentation tube with the bile broth, inoculate a sample of the medium from the tubes that tested positive in the first phase (immediately after gas formation)
• Inoculate the tubes for about 48 hours at 35 degrees Celsius

* 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.

Procedure

• Using a wire loop, scoop and streak the methylene blue plates with the sample (this simply involved marking out lines of the sample on the methylene blue plate, violet red agar plate or MacConkey agar using a wire loop)
• Incubate the plates for about 24 hours at 35 degrees Celsius
• Obtain a colony of the bacteria from the plate and transfer it to the fermentation tube (lauryl tryptose broth) and nutrient agar slant
• Incubate the two (agar slants and fermentation tubes) for between  24 and 48 hours at 35 degrees Celsius to determine whether any gas is produced

• For the agar slant sample, staining is required (gram-staining) in order to view the sample under the microscope. As for the fermentation tube, check for presence of gas

* 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:

• MacConkey agar will turns pink and cloudy indicating the presence of coliforms that ferment lactose
• Eosin methylene blue agar will show a metallic green sheen in the presence of coliforms

• Violet red agar will turn red or pink in color in the presence of coliform bacteria

MPN (Most Probable Number) Technique

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.

MPN Index

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

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

Requirements

• Membrane filter (with 0.45 microns pores)
• A pair of forceps
• A low power microscope
• Sample (water, urine, wastewater)
• Collecting apparatus
• A funnel
• Prepared agar plate

* 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.

Procedure

• Place the funnel on the collecting apparatus (the collecting apparatus may be connected to a vacuum system)
• Place the filter below the funnel so that it is between the funnel and the collecting apparatus
• Pour the sample slowly into the funnel and apply a vacuum in the collecting apparatus until the funnel is empty
• If some sample is still in the funnel, pour some sterile buffered dilution water (20-30ml) to rinse it

• Apply the vacuum to empty the funnel

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)

• Cover the plate with a lid and invert the plate
• Incubate the plate for about 2 hours at about 35 decrees Celsius
• Using a low power microscope, observe the coliform colonies on the plate (using 10 to 15x magnification)
• Count the number of individual colonies on the filter

* 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.

Fluorescent In Situ Hybridization Technique (FISH)

DNA denatured via heat & formamide, PNA probe is allowed to hybridize,excess probe is washed away,reading is taken in flow cytometer - 3.0, https://en.wikipedia.org/w/index.php?curid=16016611

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.

Requirements

• 0.40-um
• black polyester membrane filter
• A pad (soaked in 80 percent ethanol)
• Petri dish
• Hybridization buffer (50ul) with respective probes
• Hybridization chamber
• 550 ul of washing
• buffer

Procedure

• Filter the sample using the 0.40 um membrane filter
• Transfer the filter the ethanol soaked pad and allow to stand for about 3minutes at room temperature in a Petri dish
• Dry the filter membrane and filter at room temperature for about 3 minutes
• Place the membrane filter on the hybridization buffer (50ul) in the hybridization chamber and incubate for about 90 minutes at 46 degree
• Place the membrane on the washing buffer soaked buffer (550ul)

* for this technique, epifluorescence microscopy is used to view the sample (with WIBA filter block)

Observation

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. 

See:  Microscope Experiments Page

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More info at Bacteria under the Microscope

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References

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.

Links

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2199584/?page=1

https://www.tandfonline.com/doi/pdf/10.1080/07438140609353891