A Simplified Guide

to

Bacteria in Water

Part 1

By

D. Roy Cullimore, Ph.D., R.M.

copyright © 2000, D. Roy Cullimore

All Rights Strictly Reserved

Copying for personal use only

COLIFORMS

The name "coliform" is given to a whole group of bacteria which can occur in water and indicate potential health problems. They are divided into two groups: TOTAL coliform (TC), which are all of the coliform bacteria, and FECAL coliforms (FC), which are a portion of the TC. Both of these bacteria are closely related inside a large family of bacteria known as the ENTERICS. However, most of the enterics do belong to the TC group, but very few belong to the FC group, which is considered much more serious from the hygiene viewpoint.

FECAL COLIFORMS

Fecal coliforms are pretty specialized types of bacteria and are dominated by Escherichia coli (Esh-her-ick-ee-ah co-lie), also known as E. coli. This bacterium thrives in the healthy human intestine and passes out in high numbers in the fecal material. These can be counted in water by using the fecal coliform tests and the counts are usually given as FC cells in 100 ml of water.

Counting the FC is done by growing the bacteria under very specialized conditions, at higher temperatures (44.5'C) which discourages the total coliforms from growing at all or, from growing in a peculiar (atypical) way which the technician will ignore when counting the typical colonies. Human feces tend to have much more FC than Fecal Streptococci (FS), which some

people look for as well as or instead of FC. These FS tend to be more common in animal feces and so comparing the numbers of FC to FS (FC:FS ratio) is handy for getting an ideal as to whether the water has been polluted with human fecal wastes (>2:1 ratio) or animal wastes (<1:1).

Escherichia coli is common in the human intestine, but it is not usually harmful. (However, there are some strains which can cause infections.) What is important to remember is that the presence of FC is a widely accepted indicator of the potential pollution of water with fecal material. If that material is present, then there is a much greater risk of infectious microorganisms occurring in numbers large enough to cause an infection to break out if the water is consumed. These organisms include viruses, other bacteria, protozoa and a variety of worms.

TOTAL COLIFORMS

The total coliform (TC) group contains a wider variety of bacteria including Escherichia coli and a broad spectrum of the enteric bacteria. These enteric bacteria are able to grow frequently in the intestine, but can also grow to a variable extent in the environment. In consequence, the TC count does no necessarily relate specifically to fecal pollution, but to the bacterial loading of enterics within the water system. Since the count is dominated by the bacteria which can occur in the intestine, it is used as a more broad spectrum test for fecal pollution in a water system. Frequently, these bacteria like to grow in muds, septic wastes and organic rich wastes which can distort the numbers and findings. For example, when swimmers and bathers kick up the sediment and muds around a bathing beach, large numbers of TC can enter the water to give higher counts. The natural conclusion is that the daytime unsanitary habits of the bathers caused the elevation of the TC bacterial count when it could have been at least in part from these other sources.

COLIFORM TEST METHODS

How can bacteria as small as one thousandth of a millimeter, or a micron, be counted and at the same time identified? Good question! Bacteria are like big bags of reactive chemicals (enzymes) which have the habit of dividing into two bags when the bag gets too full or things get too stressful! The counting procedures take advantage of these features and there are two principal methods used. These are known as the Membrane Filtration (MF) and the Most Probable Number (MPN) methods. You got it, one method filters the water to trap the coliforms (MF) while the other makes a hopefully good guess (probability) at the number of coliforms present (MPN). Let's look at the two methods separately.

Membrane Filtration Coliform Test Method

There are special filters which can trap bacteria in water by having pore sizes too small to allow the bacteria to pass through (0.45 or 0.22 microns). The bacteria are therefore trapped on the upper surface of the filter. For example, if there were only one bacterial cell in 100 ml of water filtered through the membrane filter, it would become entrapped into the top of the filter as the water passed through.

Now, to see these entrapped bacteria, we could run a microscope over the whole field but most of the bacteria are colorless and would not be seen unless stained! What is better is to grow the single bacterial cells into large colonies containing millions of its offspring in a single heap of growth called a colony.

Bacteria, like human beings, prefer particular foods and are repelled by other foods. When the "menu" is properly adjusted, specific groups of bacteria can be made to grow while being stained (if stains are added) at the same time. Even when a colony grows into either a dome, pancake, spreading slime, a feathery rhizoid or some other pattern, those characteristics are typical.

In the coliform MF tests, very specific foods and stains are made available in either a gel (agar) or in liquid added to an absorbent pad. The nutrients and stains pass up into the filter pad placed upon the bacterial colony and, at the right temperature (35 - 37^oC for TC, 44.5^oC for FC) , colonies for the TC or FC bacteria grow within 24 hours and can be counted visually. If you know how much water you filtered and count the number of typical colonies, then you can calculate the numbers present in 100 ml. Other bacteria may also grow, but will not produce the characteristic (typical) colonies, but instead produce atypical growths which are usually not recorded.

Most Probable Numbers Coliform Test Method

All bacteria are like bags of enzymes and they selectively use different foods, particularly sugars. One of the favorite sugars for the coliform group is lactose. Strangely, that is the main sugar in mother's milk and, therefore, the first one that the bacteria in the baby's intestine come across after the baby has been born and is suckling the milk. Bile is pretty lethal to bacteria, but the coliform bacteria can tolerate it. Therefore, it was found that a soup rich in lactose and bile encouraged the coliforms to grow while discouraging competition (that cannot use lactose or tolerate the bile salts). Not only that, but the coliform. bacteria produce much gas when feeding on the lactose.

It was Durham who first trapped this gas in an upside down test which was then named after him. When acids with or without gases are produced by bacteria this is referred to as fermentation.

It is possible to use this fermentation to detect coliforms. For example, if you took 100 ml of water and found that gas was produced from 2 ml sample of water when the lactose bile broth was added, but none was produced from 1 ml, then that would suggest that the water contained 50 coliforms per 100 ml (ie. enough for just 1 cell in every 2 ml of water. As you are probably thinking, there is an element of chance in that happening and so the statisticians took over and developed a fractionating series of tests to apply to the water sample that would allow a population to be predicted. This prediction is based upon which dilutions produced gas and is called the most probable number of coliforms per 100 ml. If you're very lucky and the lab very honest, there will be a second number which will give the range of error. This number usually represents the range around the MPN number within which two-thirds of the predictions of populations would in fact be accurate.

At the moment, there is an argument as to which method is better, since both affect the ability of coliforms to grow during the test. The MF method has been reported to sometimes give false negatives since the coliforms become damaged by chlorination, while the MPN test sometimes gives false results when non-coliforms occasionally produce gas to give a positive.

IRON RELATED BACTERIA

Rusty slimes, plugging, yellow to orange waters, production losses from wells and blackened corrosive slimes have all been connected to the iron bacteria. These iron bacteria are usually found in waters having relatively high concentrations of iron and manganese in the water (greater than 0.5 to 1.5 parts per million for iron and ten times less than that for the manganese). When they grow, the slime can plug pipes, well screens, pumps (particularly the impellers and screens) and can even grow back into the groundwater of an aquifer! Because they grow better when iron is present, they are often called iron related bacteria (IRB). There are three major groups of iron bacteria: Gallionella, sheathed IRB and heterotrophic IRB groups.

Sheathed Iron Related Bacteria

There are a whole group of IRB's which are pretty specialized and relatively easily recognized by their shape and the ways they grow. In almost all cases, the bacterial cells grow with a tube-like coating or within a envelope where iron and manganese oxides have been deposited to give the growths an orange to brown to black color.

The cells are often able to escape from these tubes or envelopes and swim off into the water to set up new colonies or slimes elsewhere. Some waters are dominated by one or more of these types of iron bacteria, but not enough is known to diagnose why the different dominance would occur. Common genera include Leptothrix, Crenothrix and Sphaerotilus.

Heterotropic IRB group

There are a great deal of bacteria which grow or survive in water and will take up iron or manganese into their cell walls. These bacteria all live on organic materials that are present dissolved or suspended in the water even when the amounts are only present in parts per million or even parts per billion! These bacteria often play key roles in the degradation of even very toxic and/or carcinogenic chemicals. One could almost visualize these bacteria as being a very important part of Nature's Vanguard in cleaning up of the many organic pollution spills that occur from time to time. Their activities are utilized as a part of the bioremediation processes designed to contain and then remediate spill sites. They commonly grow in biofilms, slimes, nodules, foams in soils, waters, muds and all types of surfaces. Slimes tend to grow where there is very little oxygen present, but some oxygen is essential for growth. If the oxygen is taken out, the slime organisms in part pack up their bags and move to the nearest place where there is a low level of oxygen present. Being very innovative, some cells can substitute nitrate for oxygen and are still able to respire and grow. That means that in the absence of oxygen, slime can still form if nitrates are present - a common occurrence where there has been a pollution of the groundwater with organic wastes. Remember that in a well with many slimy growths on the screen, on pumps and in the aquifer, the chemistry of the water would have been changed considerably by the slime. These slimes fall into four major groups based upon their colors (which are interchangeable). These colors are grey, brown, black, and red with black dominating under oxygen free (anoxic) conditions while the grey is common where there are very low concentrations of iron or manganese.

Gallionella group of IRB

Gallionella is the most distinctive group of iron bacteria in many ways. For the scientist looking into the water, this organism can be easily recognized by its long ribbon-like tail which is produced out of one side of its rod- or bean-shaped cell. It appears to be a very cunning little bacterium since it extracts energy out of the iron and manganese and deposits the spent iron and manganese as oxide pellets in the ribbon-like tail. Thus, it produced a super long tail which is discarded and then the cell can swim off through the water to start all over again. This explains why water can be seen with so many tails and yet few heads! These bacteria do appear to need oxygen to grow, but very little (even parts per billion) appears to be enough and they can easily be over-dosed when there is much oxygen present. Because of their huge distinctive tail, water is often diagnosed as having a principally Gallionella problem when in practice the slimy "gloop" around them contains far more bacteria which could be the actual cause.

SLIMES

Why do bacteria produce slimy growths which interfere in many ways with the production performance of a system? The slime consists of a massive amount of long stringy molecules called polymers which hold much water and protect the bacterial cells growing in the slime.

This polymeric slime is a super sponge and will take up and concentrate much of the nutrients from the water and into the slime where it cycles between the cells and the outside slime. When a cell dies in the slime, all of its components are fed upon by the neighboring cells so that nothing is lost in this super-efficient albeit cannibalistic community.

TOTAL BACTERIA

Like the majority of plants and animals, most bacteria are not dangerous to humans. They occur widely in soils and waters, reaching populations sometimes in the million per gram or per milliliter. They come in all sorts of shapes and sizes and yet each group has somewhat different preferences for habitat, foods and the level or needs for oxygen.

This makes it very difficult to accurately quantify all of the bacteria in water. Bacteria can make the water go cloudy (it can take as many as 100,000 to 10 million cells per milliliter to do this!), taste peculiar, generate odd odors and the population can harbor some potentially dangerous organisms to human health. Traditionally, the coliform test has been used to determine whether there are any dangerous bacteria present, since the coliforms are thought to be a good indicator of fecal pollution.

In general, when looking at the total number of bacteria in water, the idea is to determine what level of bacterial presence is occurring in the water. These activities can include corrosion, degradation, putrefaction, plugging and self-purification. The last function is a phenomenon wherein the waters can undergo a gradual improvement as a direct result of pollutants being degraded or removed from the water. Oxygen is a major driving force, along with temperature, in helping to speed this process. Because there are so many different functions occurring concurrently as a result of the presence of the various bacterial groups, it is very difficult to monitor all of the groups in one simple test. As a result, a somewhat confusing range of tests has been generated, but predominant amongst the methods are the standard plate count (SPC) and the total plate count (TPC).

Standard Plate Count (SPC) Method

This test is reasonable fast but tends to indicate only the bacteria which grow rapidly at higher temperatures (30-40^oC) in two days (these are common times and temperatures that many laboratories use). These bacteria are more likely to have originated from warm blooded animals (including humans) rather than from the environment where temperatures are commonly at less than 30⁰C. The test originally used to be a pour plate technique and it is still used sometimes. In this test, the water is diluted and then mixed with molten agar at >45^oC so that colonies are mixed into the molten agar before it sets. This means that they can grow throughout the agar and be easily counted. To many bacteria growing naturally in the environment, this exposure to the 45+^oC temperatures can create a shock which can be lethal. As a result, many of these shocked bacteria will not grow in two days when the counting of the colonies is often done. All colonies are counted and weighted by the dilution factor to give the bacterial population as colony forming units (cfu) per ml.

Each colony is presumed to have been formed from one bacterial unit (which can consist of one or more cells in a clump, possibly of diverse but compatible types) to form a colony forming unit which grows distinctly within or upon the agar. To eliminate the temperature shock effect, another technique can be used whereby the diluted water is streaked across the surface of the agar. The food medium commonly used to grow bacteria for this test is the standard plate count agar medium which also tends to be too rich in nutrients for many of the environmental bacteria! The total plate count was introduced to obtain a better handle on the bacterial population.

Total Plate Count (TPC) Method

There is a much greater flexibility in the way that the spread plate technique can be used. The medium has not rigidly been laid down and there are seven or eight different media are in widespread use. Paralleling this, there is a flexibility in the temperatures and incubating times used. Common temperatures used include 8 to 10, 20 to 22, 25 to 28, 30, 35 to 37 and 45 ^oC. The selected temperature reacts directly on the incubation times, particularly at the lower end of the scale. At temperatures below 25^oC, a prolonged incubation allows more time for the bacteria to grow. For example, at 8 to 10^oC, bacteria can take up to six weeks or longer before forming a visible and countable colony and so the plates should be incubated to the longest term. In requesting a TPC, questions should be raised as to the medium, temperatures and times that are going to be used. one of the frustrations in requesting bacterial counting of a water sample is the long delay before the numbers return, perhaps long after the treatments have been implemented. A number of new techniques are being examined to cut down this response time. These include particle counting, epiflorescent microscopy sophisticated staining and biochemical methods which search individual enzymes or marker chemicals.