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Coliforms

Coliform Testing As An Andicator Of Sewage Contamination

Coliform testing is routinely used as an indicator of sewage contamination. Sewage contamination is a concern because it may contain potentially infectious organisms such as such as Escherichia coli, Salmonella, and Shigella. Exposure to sewage contamination increases the risk of gastrointestinal infections and other related illnesses. Sources of sewage contamination may include raw sewage overflows, severe flooding and leaking sewer lines or septic tanks.

To determine the potential health risks from sewage contamination swab samples could be collected and tested for sewage contamination indicators. Indicator organisms include Total Coliform, Fecal Coliform and Enterococcus.

Coliform

Total Coliform

Surface Sampling Method for Total Coliform, Fecal Coliform and Enterococcus.

Sampling Procedure

  1. Measure the area of the surface to be sampled (both in the suspected area and a control in an area where low or no contamination is expected) with a tape measure and record the area in square centimetres (a 100 cm2 is recommended);
  2. Collect swab samples by removing a sterile, rayon (non-cotton) swab from a sterile tube, moisten it by inserting it into a second tube which contains a sponge soaked with sterile 1.5 mL of phosphate-buffered saline (PBS) at pH 7.2;
  3. Swab the selected surface by moving the swab back and forth across the surface with several horizontal strokes, then several vertical strokes. Rotate the swab during sampling to ensure that the entire surface of the swab was used.
  4. After sampling, return the swab to the sterile tube (with the sponge);
  5. Before collecting another sample, label the sample appropriately;
  6. On the chain of custody clearly indicate what indicator organisms you want tested, i.e., total coliforms, fecal coliforms, E. Coli and/or Enterococcus. Submit the samples to Mold & Bacteria Laboratories for testing.

Results Interpretation

Total Coliform

Coliform are found in the intestines of warm-blooded animals including human beings and hence they end up in sewage. However, they are also present in the environment as part of the natural microflora. These naturally occurring coliforms can potentially cause false positive results when testing environmental samples. For environmental samples, positive tests for total coliforms must be interpreted with caution and always be confirmed by more specific testing.

Fecal Coliform

Fecal coliform include bacteria such as E. coli, Klebsiella, Enterobacter, and Citrobacter. The fecal coliforms test is more specific than the total coliform test in indicating potential sewage contamination. However, this test is also subject to giving false negatives since these bacteria with the exception of E. coli could also be associated with plants. Therefore, as with the total coliform test, the results of the fecal coliform assay must be interpreted with caution, and positive results should be confirmed.

Escherichia coli (E. coli)

Presence of E. coli has been demonstrated to be a very specific indication of fecal or sewage contamination. E. coli has also been isolated from soil suggesting that it may not be 100% reliable indicator of fecal or sewage contamination.

Enterococcus

Alternative indicator organisms are the enterococci. The enterococci, belong to the genus Enterococcus. They are less ubiquitous than the coliform in the environment and are always present in the feces of warm-blooded animals. Although research has indicated that enterococci might be a more stable indicator of sewage contamination than E. coli and fecal coliform, some species of Enterococcus are also associated with Plants.

Conclusion.

No single indicator organism on it’s own is 100% reliable to demonstrated sewage or fecal contamination. Some experts therefore recommend use of 3 or more indicators, i.e., total coliforms, fecal coliforms, E. coli and/or enterococci.

For additional information or if you have any questions please contact us by phone at 905-290-9101.

Microbial Contamination Of Wastewater

Microbial Contamination Of Wastewater And Associated Health Risks

Wastewater consists of water that carries wastes (dissolved or suspended solids) from homes, businesses, and industries and also stormwater discharges. This water is rich in chemical and biological pollutants. Therefore, wastewater effluents, if not properly treated, can cause a number of ecological, economical, and human health problems such as:

  • algal blooms from nutrient enrichment;
  • depletion of dissolved oxygen – sometimes resulting in fish death;
  • destruction of habitats from sedimentation;
  • health risks from drinking water contaminated with pathogens or toxic chemical substances;
  • loss of revenue from closures of shellfish growing areas; and
  • loss of tourism and recreational opportunities as a result of beach closures and restrictions of beneficial uses of water.

Decontamination of wastewater before it’s released into the environment is therefore very important. This article will focus on microbial contamination of wastewater and sludge and associated health risks.

Health Risks Associated With Wastewater And Sewage Sludge

Microbial contamination of wastewater and sludge is monitored to determine the degree of pollution and potential exposure to harmful bacteria, fungi and enteric viruses. Many of the microorganisms found in sewage sludge are potentially pathogenic. These microorganisms include bacteria, fungi, parasites, and viruses.

The most common type of bacteria found in sewage sludge are of enteric origin (i.e., from the intestines) since sewage contains human waste. Also found are members of the genera Streptococcus, Clostridium, Mycobacterium, and Listeria. The presence and concentration of a given pathogenic microorganism depend, however, on the presence of healthy or sick carriers in a population. The sewage sludge can contain as high as 108 to 109 total coliforms, 106 to 107 of fecal coliforms, and 102 to 103 salmonellae bacteria per gram of dry matter. Generally, bacteria can survive in the soil for a few months. Some are partially or completely rendered inactive by extreme temperatures below the freezing point or by summer dry spells. Before using farmland to which sludge has been applied, a waiting period of a full year, or at least one summer is recommended.

Sludge may also contain a number of pathogenic fungi, such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. Since human infection by these fungi is through direct contact or inhalation spores and hyphal fragments, workers who handle sludge, and especially those who are immunocompromised, are at the greatest risk.

Parasites pose health risks primarily to workers at sites where sludge has been applied. The important parasites are protozoa (e.g., amoebae, Toxoplasma gondii, Giardia lamblia, and Cryptosporidium sp.) and helminthes (such as Ascaris sp., Trichuris sp., and Taenia sp). These parasites are often present in sludge in the form of cysts or eggs. The eggs and cysts are very resistant and can be destroyed only by heat treatment at temperatures of at least 70 °C. The number of egg and cysts in a kilogram of dry matter range from several hundred to several thousand. Such numbers are very high, given that only a few parasites are required to cause infection. If present in the soil after application of sludge on land, eggs or cysts can survive several years.

Viruses may be present in raw sludge at concentrations as high as 1000 virus particles per gram of dry matter. Most of the viruses are those that attack the digestive system, i.e., enteric viruses including the hepatitis A virus. In the soil, some viruses can survive for several months in the first 5-15 cm of soil thus posing a health risk to humans.

Monitoring Microbial Contamination Of Wastewater

filter membrane with colonies

Since it’s not practical to test for every potentially pathogenic organism in wastewater, the degree of pollution of wastewater is determined by use of indicator organisms. Two groups of organisms used as indicators of pollution are the total coliforms and fecal coliforms. The fecal coliform group, however, is considered the most significant by majority of laboratories.

The coliform group of organisms consist of primarily the genera Escherichia, Enterobacter, Citrobacter, and Klebsiella. The coliforms are widely distributed in nature, and many are in the gut of humans and warm-blooded animals.

A more rigorous test to assess the extent of fecal contamination may involve testing a suite of microbial indicators (e.g., total and fecal coliforms, enterococci, Escherichia coli, coliphage, Clostridium perfringens, and human enteric viruses).

Collection Of Samples

Sampling is a critical part of sanitary water testing. As often stated, the laboratory results are only as good as the sample collected. Therefore, it’s not only important that the sample accurately represents the mass of water being tested but also to prevent deterioration and contamination of the sample before analysis.

Generally there are two methods for sampling wastewater. The grab sampling and composite sampling. Grab sampling refers to a sample collected at one time. The disadvantage of a grab sample is that it reflects the condition of the water only at the point in time that the sample was collected. Composite sampling, on the other hand consists of a collection of numerous individual discrete samples (about 100 mL each) taken at regular intervals, usually of 1 hour over a period of 24 hours. The collected samples are poured into a larger bottle and kept refrigerated at around 4 oC over the sampling period. The analysis of this composite sample, collected over a period of time, represent the average condition of the wastewater during the collection period.

counting coliform colonies

Sample containers

Two types of sample containers may be used: a wide-mouth, 120 mL borosilicate glass bottle with glass stopper or screw-cap closure or an autoclavable, nontoxic polypropylene bottle. The containers must be sterilized before use.

Sample Treatment And Handling

If the sample to be tested contains residual chlorine, it has to be dechlorinated. This is achieved by adding appropriate amount of sodium thiosulphate dechlorination agent to the bottle before sample collection. This amount is typically 1.0 mL of 1% solution of sodium thiosulphate for a 120-mL bottle and is typically added before sterilization of the bottle.

Ideally, the sample should be processed within 1 hour of collection or refrigerated below 10 °C and then processed within 6 hours. When collecting samples, sufficient space (approximately 2.5 cm) in the bottle should be left to allow mixing of the sample by shaking. Contaminating the mouth of the bottle with hands or other non-sterile objects should be avoided.

Testing For Total Coliforms And Fecal Coliforms In Wastewater

The two accepted methods used to test for total coliforms and fecal coliforms are the membrane filter procedure and the most probable number (MPN) method. The former is the most commonly used today because it’s easy to use, accurate, inexpensive, and results are obtained within 24 hours.

On M-Endo medium coliform colonies appear golden-green sheen at 10x magnification under a fluorescent light source. The coliform count of the sample size tested is expressed as the number of colony forming units (CFU) per 100 mL of water. Although each colony of organisms recovered on the membrane filter typically represents one organism, occasionally, more than one organism will merge to form a larger than normal colony.

To be considered valid, the number of coliform colonies on the filter surface should not exceed 80 CFUs, and the total number of all colonies should not exceed 200 CFUs. If these limits are exceeded, the sample should be diluted appropriately to give between 20 to 80 coliform colonies and less than 200 of total number of all colonies.

Fecal Coliforms ferment lactose at elevated temperatures as well as at 35 °C. The incubation temperature required is 44.5 plus or minus 0.2 °C. When grown on M-FC medium, they appear as blue colonies. No more than 60 fecal coliforms, or more than 200 of total colonies should be present on the membrane for a valid count.

coliform colonies on filter membrane

Results Interpretation

The recovery of fecal coliform organisms in wastewater is an indication of possible presence of enteric pathogens.

Jackson Kung’u is the Principal Microbiologist, Mold & Bacteria Consulting Laboratories (MBL) Inc.

Testing For Sewage Contamination

Sewage contamination in building is the result of septic system backups or sewage pipe damages.  Sewage backflow in a building can damage the integrity of its structure and render it inhabitable as well. Sewage contains a range of pathogenic microorganisms like E.coli, Salmonella, Vibrio, mycobacteria, moulds, viruses and protozoa like Cryptosporidium and Giardia. Recurrent sewage leaks or spills in particular areas such as the basement of residential or commercial buildings, allow all these micro-organisms to proliferate. Exposure to these pathogens poses a serious health risk.

What Should One Do After Sewage Back-up?
Once a building gets contaminated with sewage, the most important steps to be taken are immediate removal of sewage water, disinfection of the contaminated surfaces, and prompt drying of the surfaces. Sewage remediation may require special equipment such as heavy duty vacuum cleaners. Hiring the services of remediation or restoration professionals is therefore recommended.

Documentation of contaminated materials is important. It’s difficult to clean contaminated porous materials. Therefore, any material that is porous (such as carpet, gypsum wallboard, insulation, upholstered furniture, clothing) and directly got contaminated by sewage is supposed to be discarded since the pathogenic micro-organisms have already penetrated the material.  Semi-porous material like wood furniture or pressed wood products may be thoroughly cleaned, disinfected and dried. Non-porous surfaces like metal, glass and ceramic tile floorings can be cleaned and disinfected.

coliformDetermining the effectiveness of sewage cleanup
Opinions differ as to whether microbial sampling to document effectiveness of sewage remediation is necessary. Those who think it’s not necessary argue that the primary objective of sewage remediation is to remove the sewage water and disinfecting and drying of contaminated interior surfaces. However those who support sampling recommend screening for E. coli and fecal coliforms.  E. coli (Escherichia coli in full) are found in the intestines of humans and animals and hence in fecal matter which happens to be a major component of sewage. Direct or indirect contact with E. coli contaminated water, food, air or surfaces could lead to harmful health effects. E. coli is only used as an indicator since not all strains of E. coli are pathogenic. However, E. coli strain O157:H7 is a toxin producing strain and potentially dangerous.

Even though sewage may contain many other pathogens, E. coli is the easiest to detect and identify. Hence the reason it’s used as a marker of sewage contamination.

To ensure that sewage decontamination has been effectively done, a swab test is performed on surfaces. A suspect area is identified and marked. A swab is uniformly rolled over the entire marked area (e.g. 10cm x10cm) and enclosed back into its container. Such swabs are sent to the lab at the earliest for analytical purposes. The lab would then process the swabs for detection of E. coli and other coliforms. Along with determining the presence or absence of E. coli, it is highly recommended to quantify them. Quantification gives us an idea of the bacterial load of the particular suspect area. This helps also determine the effectiveness of the disinfecting agent.

If the lab results are positive for E. coli and other coliforms, further cleaning would be required.

E.coli

Here we go again…talking about E.coli. Just a few days ago, we heard about the outbreak in North Bay, Ontario where the number of confirmed and suspected cases of E. coli O157:H7 poisoning has risen to 159. The source of the bacterium has been linked to a fast food restaurant this time.

So what is this E.coli and why is it so important? You can’t see it, smell it or taste it. But it can leave you fighting for your life, especially if your immune system is weak or compromised. E. coli or Escherichia coli (named after Escherich who first discovered it) are a type of bacteria commonly found in the intestines of animals and humans. There are hundreds of strains of the bacterium, but E. coli O157:H7 is possibly the most dangerous as it produces a powerful toxin that can cause severe illness.

E. coli O157:H7 was first recognized in the United States in 1982, when an outbreak of severe, bloody diarrhoea was traced to contaminated hamburgers. It was then called the “hamburger disease.” E. coli O157:H7 can contaminate ground beef during the butchering process. If it is present in the intestines of the slaughtered animal, it can get into the meat as it is ground into hamburger.

Canada’s worst-ever E. coli outbreak occurred in  Walkerton, Ontario in May 2000, after the bacteria got into the town’s water supply. More than 2,300 people were affected. The source of the contamination was manure spread on a farmer’s field near one of the town’s wells. Health authorities across the country normally deal with a few thousand cases of E. coli illness every year.

Sources of E. coli
E. coli comes from human and animal wastes. During precipitation, E. coli may be washed into creeks, rivers, streams, lakes, or groundwater. When these are used as sources of drinking water — and the water is not treated or inadequately treated — E. coli may end up in drinking water. This is what we call the sewage or fecal contamination of water. It indicates that the water is not fit for human consumption.

Although the bacteria are mainly found in meats, it is also present in unpasteurized milk and fruit juices, ham, turkey, chicken, roast beef, sandwich meats, raw vegetables, fruits and cheese. Once someone has eaten contaminated food, the infection can be passed from person-to-person, and by hand-to-mouth contact. The bacteria are most often spread from person-to-person.

Symptoms
These are characterized by severe abdominal cramping that can appear within hours of eating any contaminated food but could also take up to 10 days to show up. Some people may also be afflicted with bloody diarrhoea or non-bloody diarrhoea, nausea and fever. Some people may show no symptoms at all, but can still carry the bacteria and pass it on to people who will become sick.

Precautionary Measures
Since most cases of E. coli infections are contagious (passed from person to person), good personal hygiene is critical to protecting yourself.

  • Wash your hands thoroughly and frequently.
  • Don’t handle food if you are suffering from diarrhoea.
  • Wash raw fruits and vegetables thoroughly before cooking or cutting them.
  • Sanitize food preparation surfaces and utensils.
  • Anyone known to be infected with E. coli, should not share dishes, cutlery or glasses with anyone else. Their towels, face cloths and bedding should be washed separately in hot water and bleach.
  • Call your family doctor for any unusual symptoms that you notice in your health or that of your family.

Some of the proper food handling techniques to minimize exposure to E.coli include:

  • Refrigerate or freeze meat as soon as possible after buying it and then thaw frozen meat in the refrigerator, not on the counter.
  • Use a digital food thermometer when cooking ground beef, which should be cooked to an internal temperature of at least 71 C (160 F).
  • Serve cooked meat immediately or keep it hot (60 C or 140 F).
  • Clean and sanitize countertops and utensils after contact with raw meat.
  • Don’t store raw and cooked food together.
  • If you marinate meat, don’t use the liquid as a dip or to pour over cooked meat.
  • Drink only pasteurized milk or juice fruit juices. 
  • Drink water from a supply known to be safe. If you have a private water supply (well) it should be tested several times a year.

If you need further information on E.coli or performing a water quality test, please visit our web-site www.moldbacteria.com or call us at 905-290-9101.

Article by: Sneha Panchal, M.Sc.