# 1.8: Total Coliform Rule

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Learning Objectives

• Explain biofilms
• Explain common microbiological techniques
• Outline and describe coliform bacterial analysis

## ​​​​​​Common Microbiological Techniques

### Biofilms

In nature, microorganisms do not live in isolation as a rule. They live in communities called biofilms. Today, using confocal microscopy, three-dimensional structures of biofilms are visible. Biofilms reside in a matrix of primarily polysaccharides, containing DNA and proteins, called slime. A biofilm is considered to be a hydrogel, which is a complex polymer containing many times its dry weight in water. Cell-to-cell chemical communication, or quorum sensing, allows bacteria to coordinate their activities and to group together into communities that provide benefits like a multicellular organism. Biofilms are bacterial slime layers and biological systems. The bacteria are organized into a coordinated functional community. Biofilms are attached to surfaces. The bacterial community may be a single species or a diverse group of microorganisms. Biofilms can appear in other forms. An example would be the floc that forms in sewage treatment facilities. In fast-flowing streams, biofilms can form filamentous streamers. Within a biofilm community, the bacteria are able to share nutrients and be sheltered from harmful factors in the environment. The close proximity of microorganisms within a biofilm have the advantage of facilitating the transfer of genetic information by conjugation.

A biofilm begins to form when free-swimming bacteria, planktonic, attaches to a surface. If the bacteria grew in a uniformly thick monolayer, they would become overcrowded, nutrients would not be available in lower depths, and toxic wastes would accumulate. Microorganism in biofilm communities avoid these problems by forming pillar-like structures with channels between them, so that water can carry incoming nutrients to the cells and carry outgoing wastes away from the cells. This constitutes a primitive circulatory system. Individual microbes and clumps of slime occasionally leave the established biofilm and move to a new location where the biofilm becomes extended. Such biofilm is generally composed of a surface layer about 10 microns thick, with pillars that extent 200 microns above the surface.

The microorganisms in biofilms can work cooperatively to carry out complex tasks. Biofilms are essential elements in sewage treatment systems, and they can be a problem in pipes and tubing where their accumulations impede the flow of water.

## Culture Media

A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture medium. Some bacteria can grow well on any culture medium, and other bacteria require special media. Other bacterial cannot grow on any nonliving medium. Microbes that are introduced into a culture medium to initiate growth are called an inoculum. The microbes that grow and multiply in or on a culture medium are referred to as a culture.

A wide variety of media are available for the growth of microorganisms in the laboratory. These media have premixed components and require only the addition of water and then sterilization. Medial are constantly being developed or revised for use in the isolation and identification of bacteria that are of interest to researchers in such fields as food, water, and clinical microbiology.

When it is desirable to grow bacteria on a solid medium, a solidifying agent such as agar is added to the medium. A complex polysaccharide derived from a marine alga, agar has long been used as a thickener in foods such as jellies and ice cream.

Agar medial are contained in test tubes or Petri dishes. The test tubes are called slants when their contents are allowed to solidify within the tube being held at an angle so that a large surface area for growth is available. When the agar solidifies in a vertical tube, it is called a deep. Petri dishes are shallow dishes with a lid that nests over the bottom to prevent contamination. When the dish is filled, it is called petri plates.

### Chemically Defined Medium

To support microbial growth, the media provide energy sources, as well as sources of carbon, nitrogen, sulfur, phosphorus, and any organic growth factors that the organism is unable to synthesize. A chemically defined media is where the exact chemical composition of the media is known. For a chemoheterotrophic microorganism, the chemically defined media contains organic growth factors that serve as a source of carbon and energy. For example, glucose is included in the media for growing the chemoheterotrophic E.coli.

Organisms that require many growth factors are described as fastidious. Organisms of this type such as Lactobacillus are sometimes used in tests that determine the concentration of a particular vitamin in a substance. To perform microbiological assay, a growth media is prepared that contains all of the growth requirements of the bacteria except the vitamin being assayed. Then the medium, test substance, and the bacteria are combined, and the growth of the bacteria are measured. The bacterial growth, which is reflected by the amount of lactic acid produced, will be proportional to the amount of the vitamin in the test substance. The more lactic acid, the more the Lactobacillus cells that are able to grow, so the more vitamin that is present.

### Complex Media

Chemically defined medium is usually reserved for laboratory experimental activities or for the growth of autotrophic bacteria. Most heterotrophic bacteria and fungi are grown on complex media made up of nutrients including extracts from yeasts, meat, or plants, or digests of proteins from these sources. The exact chemical composition varies slightly from batch to batch.

In complex media, the energy, carbon, nitrogen, and sulfur requirements of the growing microorganisms are provided by protein. Protein is a large, insoluble molecule that a minority of microorganisms can utilize directly; however, partial digestion of the proteins by acids or enzymes reduces protein to shorter chains of amino acids called peptones. These small soluble fragments can be digested by bacteria.

Vitamins and other organic growth factors are provided by meat extracts or yeast extracts. The soluble vitamins and minerals from the meats or yeasts are dissolved in the extracting water which is evaporated so that the factors are concentrated. If a complex media is in liquid form, it is called nutrient broth. When agar is added, it is called nutrient agar.

### Anaerobic Growth Media and Methods

The cultivation of anaerobic bacteria poses a special problem. Because anaerobes can be killed by exposure to oxygen, special media called reducing media must be used. These media contain ingredients, such as sodium thioglycolate, that chemically combine with dissolved oxygen and deplete the oxygen in the culture medium. To routinely grow and maintain pure cultures of obligate anaerobes, microbiologists use reducing media stored in ordinary, tightly capped test tubes. These media are heated shortly before use to remove absorbed oxygen.

When cultures must be grown in Petri plates to observe individual colonies, laboratories use systems that incubate microorganisms in sealed boxes and jars in which oxygen is chemically removed after the culture plates have been introduced and the container is sealed.

### Special Culture Techniques

Many bacteria have not been successfully grown on artificial laboratory media. Mycobacterium leprae, the leprosy bacillus, is grown in armadillos, which have a relatively low body temperature that matches the requirements of the microbe. With few exceptions, the obligate intracellular bacteria, such as rickettsia and chlamydia, do not grow on artificial media. Viruses can only reproduce in a living host cell, as well. Special techniques are employed to isolate these types of organisms.

### Selective and Differential Medium

It is frequently necessary to detect the presence of specific microorganisms associated with disease or poor sanitation. For this task, selective and differential media are used. Selective medium is designed to suppress the growth of unwanted bacteria and to encourage the growth of the desired microbe. Bismuth sulfite agar is a medium used to isolate the typhoid bacterium, gram negative Salmonella typhus, from feces. Bismuth sulfite inhibits gram positive bacteria and most gram negative intestinal bacteria. Sabouraud’s dextrose agar, has a pH of 5.6 and it is used to isolate fungi that outgrow bacteria at this pH.

Differential medium makes it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. Pure cultures of microorganisms have identifiable reactions with differential media in tubes or on plates. Blood agar is used to identify bacterial species that destroy red blood cells. These species such as streptococcus progenies show a clear ring around their colonies where they have lysed the surrounding blood cells.

Selective and differential characteristics are combined in a single media. In isolating the bacteria Staphylococcus aureus it is known that this organism has a tolerance for sodium chloride and that it ferments carbohydrate mannitol to form acid. A Mannitol salt agar contains 7.5% sodium chloride, which discourages the growth of competing organisms and the media selects for S. aureus. The media also contains a pH indicator that changes color if the mannitol in the media is fermented to acid. The mannitol fermenting colonies of S. aureus are differentiated from colonies of bacteria that do not ferment mannitol. Bacteria that grow at salt concentrations and ferment mannitol to acid are identified by the color change, and they are the colonies of S. aureus. The use of differential media can be used to identify toxin producing E. coli also.

## Enrichment Culture

Bacteria in small numbers can be missed, especially if other bacteria are present in larger numbers. For this reason, it is necessary to use an enrichment culture. This technique is used especially for soil and fecal samples. The medium for an enrichment culture is liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe and not other microbes. It is a selective medium in this sense. It is designed to increase small numbers of the desired type of organism to detectable levels. The technique includes placing the sample into a liquid enrichment medium that favors the species that is desired to be isolated. The culture medium is allowed to incubate for a few days, and then a small amount of it is transferred into another flask of the same medium. After a series of transfers, the surviving population will consist of the bacteria that are capable of metabolizing the ingredients in the medium. The bacteria are given time to grow in the medium between transfers. This process is the enrichment stage. Any microbes in the original inoculum are rapidly diluted out with the successive transfers. When the last dilution is streaked onto the solid medium of the same composition, only the colonies of the desired organisms are capable of using the special ingredients to grow.

### Pure Cultures

Most samples include several different kinds of bacteria. If these samples are plated onto the surface of a solid medium, colonies will form that are exact copies of the original organism. A visible colony theoretically is derived from a single spore or vegetative cell or from a group of the same microorganisms attached to one another in clumps or chains. Microbial colonies often have a distinctive appearance that distinguishes one microbe from another species. The bacteria must be distributed widely enough on the plate so that the colonies are visibly separated from other microbial species.

Most investigations of microbes require a pure culture, or clones of bacteria. The isolation method used to achieve pure cultures is the streak plate method. A sterile inoculating loop is dipped into a mixed culture that contains more than one type of microbe, and it is streaked in a pattern over the surface of the nutrient medium. As the pattern is traced, bacteria are rubbed off the loop onto the medium. The last cells to be rubbed off the loop are far enough apart to grow into isolated colonies. These colonies can be picked up with an inoculating loop and transferred to a test tube for nutrient medium to form a pure culture containing only one type of bacteria.

Streak plate methods work well when the organism to isolate presents in a large number relative to the total population. When the microbe to be isolated is present in small numbers, its numbers must be increased by selective enrichment before it can be isolated with the streak plate method.

## Coliform Bacterial Analysis

The discharge of wastes from municipal sewers is one of the most important water quality issues. It is of particular significance to sources of drinking water. Municipal sewage contains human feces and water contaminated with these effluents may contain pathogenic (disease-causing) organisms and, consequently, may be hazardous to human health if used as drinking-water or in food preparation. Fecal contamination of water is routinely detected by microbiological analysis.

It is impractical to attempt the routine isolation of pathogens because they are present in relatively small numbers compared with other types of micro-organisms. Moreover, many types of pathogen exist, and each organism requires a unique microbiological isolation technique. The approach that has been adopted is to analyze samples for indicator organisms that inhabit the gut and are excreted in human feces. The presence of these indicator organisms in water is evidence that fecal contamination is present, and a risk exists that pathogens are present.

If indicator organisms are present in large numbers, the contamination is considered to be recent and/or severe. Bacteria in water are, in general, not present individually, but as clumps or in association with particulate matter. When enumerating bacteria in water, it is not the number of individual bacteria present which is counted, but the number of clumps of bacteria or the particles and their associated bacteria. Each clump or particle may have many bacteria associated with it.

### Total Coliforms

The term total coliforms refers to a large group of Gram-negative, rod-shaped bacteria that share several characteristics. The group includes thermos-tolerant coliforms and bacteria of fecal origin, as well as some bacteria that may be isolated from environmental sources. The presence of total coliforms may or may not indicate fecal contamination. In extreme cases, a high count for the total coliform group may be associated with a low, or zero, count for thermos-tolerant coliforms. A result of this nature would not necessarily indicate the presence of fecal contamination. It might be caused by the entry of soil or organic matter into the water or by conditions suitable for the growth of other types of coliform. In the laboratory total coliforms are grown in or on a medium containing lactose, at a temperature of 35 or 37 °C. They are provisionally identified by the production of acid and gas from the fermentation of lactose.

The term fecal coliform has been used in water microbiology to denote coliform organisms that grow at 44 or 44.5 C and ferment lactose to produce acid and gas. In practice, some organisms with these characteristics may not be of fecal origin and the term thermos-tolerant coliform is, therefore, more correct and is becoming more commonly used. Nevertheless, the presence of thermos-tolerant coliforms nearly always indicates fecal contamination.

Usually, more than 95 percent of thermos-tolerant coliforms isolated from water are the gut organism Escherichia coli, the presence of which is definitive proof of fecal contamination. As a result, it is often unnecessary to undertake further testing to confirm the specific presence of E. coli.

The presence of fecal streptococci is evidence of fecal contamination. Fecal streptococci tend to persist longer in the environment than thermos-tolerant or total coliforms and are highly resistant to drying. It is, therefore, possible to isolate fecal streptococci from water that contains few or no thermos-tolerant coliforms as, for example, when the source of contamination is distant in time or space from the sampling point. Fecal streptococci grow in or on a medium containing sodium azide, at a temperature of 37-44 °C. They are usually detected by the reduction of a dye (a tetrazolium-containing compound) or the hydrolysis of aesculin. Routine methods may give false positives and additional confirmatory tests may be required.

### Heterotrophic Plate Count

The heterotrophic plate count includes all of the microorganisms that are capable of growing in or on a nutrient-rich solid agar medium. Two incubation temperatures and times are used. They are 37 °C for 24 hours to encourage the growth of bacteria of mammalian origin, and 22 °C for 72 hours to enumerate bacteria that are derived principally from environmental sources. The primary value of colony counts lies in comparing the results of repeated samples from the same source. If levels increase substantially from normal values, a cause for concern may exist.

### Selecting a Bacteriological Analytical Technique

Two techniques are commonly used to detect the presence of coliforms in water. The first technique is called the multiple fermentation tube or most probable number technique. In this method measured portions of a water sample are placed in test-tubes containing a culture medium. The tubes are then incubated for a standard time at a standard temperature. In the second technique, a measured volume of sample is passed through a fine filter that retains bacteria. The filter is then placed on culture medium and incubated. This is called the membrane filter technique.

### Multiple Fermentation Tube Technique

The technique has been used for the analysis of drinking water for many years with satisfactory results. It is the only procedure that can be used if water samples are very turbid or if semi-solids such as sediments or sludge are to be analyzed. The procedure followed is fundamental to bacteriological analyses and the test is used in many countries. It is customary to report the results of the multiple fermentation tube test for coliforms as a most probable number (MPN) index. This is an index of the number of coliform bacteria that, more probably than any other number, would give the results shown by the test. It is not a count of the actual number of indicator bacteria present in the sample.

Separate analyses are usually conducted on five portions of three serial dilutions of a water sample. The individual portions are used to inoculate tubes of culture medium that are incubated at a standard temperature for a standard period of time. The presence of coliforms is indicated by turbidity in the culture medium, by a pH change and/or by the presence of gas. The MPN (Most Probable Number) index is determined by comparing the pattern of positive results (the number of tubes showing growth at each dilution) with statistical tables. The tabulated value is reported as MPN per 100 ml of sample. A number of variants exist for the multiple fermentation tube technique. The most common procedure is to process five aliquots of water from three consecutive 10-fold dilutions; for example, five aliquots of the sample (an aliquot is a portion of a larger whole), five of a 1/10 dilution of the sample, and five of a 1/100 dilution. Aliquots may be 1-ml volumes, each added to 10 ml of single strength culture medium, or 10-ml volumes, each added to 10 ml of double-strength medium.

Each part of the test requires a different type of medium. For example, when enumerating coliforms, lauryl tryptose (lactose) broth is used in the first (isolation or presumptive) part of the test. In the second (confirmation) part, brilliant green lactose bile (BGLB) broth is used to confirm total coliforms and E. coli medium to confirm fecal coliforms.

### Membrane Filter Technique

The membrane filter technique can be used to test relatively large numbers of samples and yields results more rapidly than the multiple fermentation tube technique. It was originally designed for use in the laboratory but portable equipment is now available that permits the use of the technique in the field.

The membrane filter method gives a direct count of total coliforms and fecal coliforms present in a given sample of water. A measured volume of water is filtered, under vacuum, through a cellulose acetate membrane of uniform pore diameter, usually 0.45 µm. Bacteria are retained on the surface of the membrane which is placed on a suitable selective medium in a sterile container and incubated at an appropriate temperature. If coliforms and/or fecal coliforms are present in the water sample, characteristic colonies form that can be counted directly.

The technique is unsuitable for natural waters containing very high levels of suspended material, sludge, and sediments. Each circumstance could block the filter before an adequate volume of water passes through. When small quantities of a sample (sewage effluent or grossly polluted surface water) are to be tested, it is necessary to dilute a portion of the sample in a sterile diluent to ensure that a sufficient volume to filter is present across the entire surface of the membrane.

If doubt concerning the probable bacterial density exists, it is advisable to test two or more volumes in order to ensure that the number of colonies on the membrane will be in the optimum range for counting (20-80 colonies per membrane). If a suitable volume of sample cannot be filtered through a single membrane, the sample may be filtered through two or more membranes and the numbers of colonies on the membranes added to give the total count for the sample.

Membrane filtration and colony count techniques assume that each bacterium, clump of bacteria, or particle with bacteria attached, will give rise to a single visible colony. Each clump or particle is, therefore, a colony-forming unit (cfu), and the results are expressed as colony-forming units per unit volume. In the case of thermos-tolerant coliform bacteria, the result should be reported as thermos-tolerant coliforms [No.] cfu per 100 ml.

For the examination of raw or partly treated waters, presumptive results may be adequate but, in certain circumstances, it is important to carry out confirmatory tests on pure subcultures. To confirm the membrane results for total coliforms, each colony (a representative number of colonies) is subcultured to tubes of lactose peptone water and incubated at 35 or 37 °C for 48 hours. Gas production within this period confirms the presence of total coliforms. To confirm thermos-tolerant coliforms and E. coli on membranes, whether incubated at 35, 37 or 44 °C, each colony (a representative number of colonies) is subcultured to a tube of lactose peptone water and a tube of tryptone water. Tubes are incubated at 44 °C for 24 hours. Growth with the production of gas in the lactose peptone water confirms the presence of thermos-tolerant coliforms. Confirmation of E. coli requires the addition of 0.2-0.3 ml of Kovac’s reagent to each tryptone water culture. Production of a red color indicates the synthesis of indole from tryptophan and confirms the presence of E. coli. The use of lauryl tryptose mannitol broth with tryptophan allows gas production and indole synthesis to be demonstrated in a single tube.

## Review Questions

1. Describe a biofilm.
2. Bacteriologically, what does the term coliforms refer to?
3. Describe the Multiple Fermentation Tube Technique for culturing coliform bacteria.
4. Describe a chemically defined medium.

## Chapter Quiz

1. A nutrient material prepared for the growth of microorganisms in a laboratory is called a(n) ___________.
1. Enrichment medium
2. Culture medium
3. Selective media
4. Complex media
2. ___________ is designed to suppress the growth of unwanted bacteria and to encourage the growth of the desired microbe.
1. Enrichment medium
2. Culture medium
3. Selective media
4. Complex media
3. Bacteria in small numbers can be missed, especially if other bacteria are present in larger numbers. For this reason, it is necessary to use a(n) ___________.
1. Enrichment medium
2. Culture medium
3. Selective media
4. Complex media
4. Most investigations of microbes require a pure culture, or clones of bacteria. The isolation method used to achieve pure cultures is the ___________.
1. Selective selection
2. Streak plate method
3. Inoculation method
4. Defined medium method
5. The term ___________ refer(s) to a large group of Gram-negative, rod-shaped bacteria that share several characteristics. The group includes thermos-tolerant bacteria of fecal origin.
1. Virus
2. Protozoan
3. Parasite
4. Total coliforms
6. This technique for identifying coliforms is the only procedure that can be used if water samples are very turbid or if semi-solids such as sediments or sludge are to be analyzed. The procedure followed is fundamental to bacteriological analyses and the test is used in many countries. It is customary to report the results as a most probable number (MPN) index.
1. Multiple Fermentation Tube Technique
2. Membrane Filter Technique
3. Heterotrophic Plate Count
4. Sabouraud
7. ___________ assume that each bacterium, clump of bacteria, or particle with bacteria attached, will give rise to a single visible colony. Each clump or particle is, therefore, a colony-forming unit (cfu), and the results are expressed as colony-forming units per unit volume. In the case of thermos-tolerant coliform bacteria, the result should be reported as thermos-tolerant coliforms [No.] cfu per 100 ml.
1. Multiple Fermentation Tube Technique
2. Membrane Filter Technique
3. Heterotrophic Plate Count
4. Sabouraud
8. Bismuth sulfite inhibits gram positive bacteria and most gram negative intestinal bacteria. ___________ agar has a pH of 5.6 and it is used to isolate fungi that outgrow bacteria.
1. Multiple Fermentation Tube Technique
2. Membrane Filter Technique
3. Heterotrophic Plate Count
4. Sabouraud
9. ___________ count includes microorganisms that are capable of growing in or on a nutrient-rich solid agar medium. Two incubation temperatures and times are used. They are 37 °C for 24 hours to encourage the growth of bacteria of mammalian origin, and 22 °C for 72 hours to enumerate bacteria that are derived principally from environmental sources.
1. Multiple Fermentation Tube Technique
2. Membrane Filter Technique
3. Heterotrophic Plate Count
4. Sabouraud
10. More than 95-percent of thermos-tolerant coliforms isolated from water are the gut organism, ___________, the presence of which is definitive proof of fecal contamination.
1. Streptococcus
2. E. coli
3. Staphylococcus
4. Bacteroides

This page titled 1.8: Total Coliform Rule is shared under a not declared license and was authored, remixed, and/or curated by John Rowe (ZTC Textbooks) .