Importance of Coliform Testing in Drinking Water

Testing drinking water for every possible pathogen that could be transmitted through its consumption is impractical. But how do we test the microbial quality of water then? Coliform bacteria holds the answer.

Pipette dropping chemicals into test tubes

What is Coliform?

Coliform is a collective term for bacteria that are present in plant material, water, soil, and, most importantly, digestive tracts and feces of both man and animal. Most coliforms are harmless bacteria with some important exceptions.1

The most important properties of the coliform group are the following:1

They are either aerobic or facultatively anaerobic

Aerobic bacteria are those bacteria that need oxygen so that they could carry out their metabolic processes and grow. There are different kinds of aerobic bacteria, those that cannot survive without oxygen are obligate aerobes.8
Facultative anaerobic bacteria are, like aerobic bacteria, needs oxygen to carry out their metabolic processes. The major difference between them is that facultative anaerobic bacteria can survive even without oxygen. They are capable of doing so by reducing other compounds, a process that acts as a substitute for oxygen.7

They are Gram-negative, non-spore-forming, and rod-shaped

Gram-negative refers to bacteria that cannot retain the crystal violet stain due to the thinness of their peptidoglycan, sugar, and amino acid polymer, cell wall.
During the Gram staining procedure, bacteria are stained with the violet-colored primary stain crystal violet. The bacteria are allowed several seconds to take up the stain. To make sure that the thick peptidoglycan cell wall of the Gram-positive bacteria would bond properly with the crystal violet stain, an iodine solution is added. Afterward, a decolorizer such as ethyl alcohol or acetone is added. Gram-positive bacteria would retain the crystal violet stain due to the thickness of their peptidoglycan cell wall and are colored violet or purple. At this point, the Gram-positive bacteria is resistant to staining and would not take up the secondary stain. Gram-negative bacteria, on the other hand, are decolorized because their cell wall is thin. When the secondary stain is added, they would take up the secondary stain, the pink-colored safranin.6
Bacterial spores are made up of keratin they act like a tough protein coat that is resistant to stains. Due to this property, spores cannot be stained through the Gram-staining method and would appear colorless under the microscope. non-spore-forming bacteria are without spores, meaning the entire bacterial cell is always stained.10
There are a lot of bacterial shapes but the well-known ones are the cocci and the bacilli. The cocci are spherical-shaped and the bacilli are rod-shaped.

They ferment glucose and produce gas within 48 hours at an incubation temperature of 35 degrees Celsius

All clinically significant bacteria need glucose as a source of energy. Some of them possess internal processes that are capable of breaking down glucose and converting it to energy in the absence of oxygen-producing gas as a byproduct, a process called fermentation. Coliforms are more efficient in performing this process when they are incubated around 35 degrees Celsius. Usually, they are given 48 hours to exhibit signs of fermentation and gas production.

These are the properties that microbiologists or laboratorian would take advantage of when it comes to isolating bacteria from the coliform group. More on this later on.

Coliforms vs Fecal Coliforms

Fecal coliform is another bacterial group with its own characteristics:2

Gram-negative, rod-shaped, and non-spore-forming
Ferments lactose within 24 hours at 44.5oC
Is able to grow with or without oxygen, a Facultative Anaerobe

Technically, we could say that Fecal Coliforms are Coliforms. However, not all Coliforms are Fecal Coliforms.

Magnifying glass displaying bacteria in a glass of drinking water

Indicator organisms

Coliforms and fecal coliforms are rarely pathogenic. Take note that they are ‘rarely pathogenic’, meaning that some of them are. E. coli O157:H7 is one strain that is pathogenic but this strain is rare. The reason why we would look for such organisms is because of the fact that they are referred to as ‘Indicator organisms’, they indicate the presence of fecal contamination.2,3

As mentioned previously, testing for all possible waterborne pathogens is impractical. This is no longer needed because we could test for Indicator organisms. Drinking water which would test positive for coliforms or fecal coliforms is highly likely to contain water-borne pathogens that are spread through the fecal-oral route. In other words, if drinking water is contaminated with fecal matter from humans or animals, it is highly likely that pathogens that may reside in the gut of humans and or animals are present in the said drinking water.3

Detection of coliform and fecal coliform through the Multiple-tube fermentation technique

The multiple-tube fermentation technique is a three-stage water testing procedure. The first stage is called the presumptive stage because you’re still uncertain whether the sample contains coliforms or not. The second stage is called the confirmed stage, at this stage, you know that the sample contains coliforms. However, just to be sure that the water is actually contaminated with fecal matter, you’re going to look for fecal coliforms. The last stage is the completed test, at this stage, you are going to look for E. coli which is a bacteria that is a great indicator of fecal contamination. At the end of the entire procedure, the number of coliforms present in the sample is approximated statistically through the ‘Most Probable Number’.1

Presumptive stage

In the presumptive stage, samples are taken out aseptically, ensuring that contamination does not happen, and inoculated into a series of large test tubes which contain lauryl tryptose broth and a Durham tube. The tubes are then incubated at 35oC for 48 hours. After 24 hours of incubation, the tubes are checked for turbidity and gas formation. If there are none, they are incubated for another 24 hours and checked again.1

The lauryl tryptose broth contains the sugar lactose which the bacteria would utilize as a food source. This abundance of food would encourage the growth of lactose-fermenting bacteria such as coliforms and fecal coliforms. To inhibit the growth of other bacteria besides coliforms, sodium lauryl sulfate is added to the broth. This substance discourages the growth of Gram-positive bacteria. The Durham tube is a small tube that is inverted but is free of air pockets. When gas formation occurs, the gas bubbles would enter the Durham tube which would slowly create an air pocket inside of it. Gas formation is actually hard to detect because the bubbles that the bacteria would produce are hard to see. With the use of a Durham tube, gas formation is now easily detectable.5,6

Confirmed stage

The tubes that would yield a positive result, which is turbidity and gas formation, in the presumptive stage are aseptically inoculated to another set of large test tubes. The test tubes, this time, contain Brilliant Green Lactose Bile Broth instead of lauryl tryptose broth. Just like the presumptive test, a Durham tube is also placed inside. The tubes are incubated at 35oC for 48 hours. The tubes are checked for turbidity and gas formation after 24 hours of incubation. If there are none present, they are incubated further for another 24 hours.1

The most important component of the Brilliant Green Lactose Bile Broth when it comes to differentiating fecal-coliform from coliform is the ox-bile and the brilliant green. These inhibit the growth of other bacteria except those that can be found in the gut, the fecal coliforms. Turbidity and gas formation would indicate a positive result, thus a Durham tube is also needed.4

Completed Test

Whichever tubes would yield a positive result in the confirmed stage are then inoculated onto an Eosin-Methylene blue agar through streaking. The plated-agars are then incubated at 35oC for 24 hours. After incubating the agars, the colonies that would grow are assessed. If a colony exhibits a dark purple color with a greenish metallic sheen, then that colony is an E. coli colony.1

The primary component of the Eosin methylene blue agar that aids in the identification of E. coli colonies is the stains incorporated in it, the Eosin and methylene blue which are kept at a 6:1 ratio. The reason as to why E. coli colonies would exhibit a characteristic greenish-metallic sheen when grown on an Eosin methylene blue agar has to do with their capability to produce strong acids. Eosin and Methylene blue reacts to E. coli’s rapid production of strong acids by producing a greenish-metallic appearance.9

Importance of the Aseptic Technique

Most of the time, bacterial contamination of drinking water is minute. This makes bacterial identification difficult. The presumptive stage encourages the growth of whatever bacteria is present in the water while inhibiting other bacteria to grow to make bacterial identification later on easier. Despite this, extra care must be taken to avoid unintentional contamination of the water samples.

One bacteria that may remain throughout the three stages is the Enterobacter aerogenes bacteria. However, they are easily differentiated from E. coli in the last stage. While E. coli would exhibit colonies with a greenish-metallic sheen, Enterobacter aerogenes, on the other hand, would exhibit no greenish-metallic sheen.11

If there was a situation wherein the aseptic technique was not followed and the water samples were accidentally contaminated with the Enterobacter aerogenes bacteria, the microbiologist or the laboratorian might conclude that the samples are contaminated with fecal matter. When in fact the samples were contaminated by accident due to the fact that aseptic techniques were not followed.

To ensure that the bacteria present in the water samples, actually comes from the water sample and nothing else. The following guidelines are followed:

Glasswares used for the entire process must be sterilized by autoclaving them. This includes the tubes that would hold the broth, the pipette used to transfer the samples, the glass bottles used to hold the samples, etc. They may still be wet after the autoclaving process. Something we do not want because the moisture might encourage bacteria growth. To remedy this problem, the glasswares are oven-dried.
The entire Water Quality Testing Process must be done inside a biosafety cabinet to avoid untoward contamination of the samples.

Wrap Up

The advent of the usage of indicator organisms in water quality testing has made microbial quality testing feasible. The multiple fermentation tube method is one of many water quality testing methodologies that rely on the detection of indicator organisms. It involves a labor-intensive three-stage process. The first stage is called the presumptive stage where we encourage the growth of coliforms in the sample while inhibiting the growth of other bacteria. The second stage is called the confirmatory stage where we encourage the growth of fecal coliforms while inhibiting the growth of other bacteria. The third stage is the completed test where we would grow the bacteria on an EMB agar to confirm whether it is E. coli or not. The entire process may take around 5 days to complete. Fortunately, advances in technology have afforded us the means of detecting E. coli in water faster.

Coliform - Why Are They Important For Drinking Water Testing?

What is Coliform?

Coliforms vs Fecal Coliforms

Indicator organisms