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Biological testing of water •

Escherichia coli, enterococci, Cryptosporidium, and Giardia.

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The studies support the use of E. coli and enterococci (instead of fecal coliform) as indicators of microbiological pollution. The studies also show that Cryptosporidium and Giardia can cause gastrointestinal illness when they are present in ambient waters.

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Cryptosporidium (crip-toe-spor-ID-ee-um) is a protozoan, a single-celled parasite that lives in the intestines of animals and people. This microscopic pathogen causes a disease called cryptosporidiosis (crip-toe-spor-id-ee-Osis). The dormant (inactive) form of Cryptosporidium, called an oocyst (O-o-sist), is excreted in the feces (stool) of infected humans and animals. The tough-walled oocysts survive under a wide range of environmental conditions The most common symptom of the disease is watery diarrhea. There may also be abdominal cramps, nausea, low-grade fever, dehydration, and weight loss.

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Drinking untreated surface water (such as streams, rivers, and lakes) or swallowing a small amount of water when swimming, even in a chlorinated pool, can cause cryptosporidiosis

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. Unwashed fresh fruits or vegetables may carry oocysts if manure was used or animals grazed where the crop was grown.

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Water treatment methods alone cannot solve the problem

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Physical removal of particles, including oocysts, fromwater by filtration is an important step in the municipal water treatment process. Typically, water pumped from rivers or lakes into a treatment plant is mixed with coagulants that help settle out particles suspended in the water. If sand filtration is used, even more particles are removed. Finally, the clarified water is disinfected and piped to customers. Filtration is the only conventional method now used in the United States for controlling Cryptosporidium.

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. Even well operated water treatment plants cannot ensure that drinking water will be completely free of Cryptosporidium oocysts.

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Unfortunately, there are no water quality indicators that can reliably predict the occurrence of cryptosporidiosis Boiling water is the best method of killing Cryptosporidium

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Only filters with an "absolute" (not "nominal") pore size of one micron (1 u) or smaller will remove all the oocysts(viruses, however, can pass through a 1 u filter). The pores of reverse osmosis (RO) membranes are too small for oocysts to pass through.

Max. contaminant level: Coliform Bacteria: Less than one total coliform organism per 100 ml of water. Non-coliform Bacteria: Concentrations less than 200 organisms per 100 ml of water considered acceptable as long as the total coliform count is • One of the standard methods of checking water quality is a test for coliform bacteria; other tests include nitrate, hardness and pesticides • •

37 percent of disease outbreaks were linked to microbial contamination from the overflow or seepage of sewage into ground water. Over the same time period, chemical contamination accounted for less than 10 percent of disease outbreaks. Bacteria of many kinds occur in both soil and surface water. Bacterial numbers in soil can approach 100 million per gram of soil. One group, not commonly found in soil or water, are the fecal coliforms, that reside in high numbers in the digestive tract of all warm-blooded animals. Because they prefer the animal system, fecal coliforms typically survive less than four days in soil or water. While ground water generally contains bacteria, it should not contain any fecal coliform.

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The presence of coliform bacteria signals the possibility of disease-causing bacteria or virus in the water. Certain fecal coliform bacteria are good indicators of pathogenic microbes since they can be identified and measured much easier than more harmful pathogens. Pathogens occur at very low levels, making testing difficult and very expensive.

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When water supplies contain coliform bacteria in levels greater than one per 100 ml of water, the water may also contain pathogens that cause acute intestinal infections.

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Water-related disease outbreaks tend to occur in the summer. Although rarely encountered today, typhoid, hepatitis and cholera, can spread through water supplies

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Obtain a coliform bacteria test if any of the following occur: a change in color, odor or taste of the water - flooding near the well

- repairs or changes are made to the plumbing or treatment system - a person or animal becomes sick with a suspected water- borne disease •

Poorly maintained water filter systems, such as activated carbon or other materials, can be a source of bacteria in the water system.

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Fecal/E. Coli - Escherichia Coli is a group of bacteria which are a subspecies of coliform bacteria. Most E. Coli bacteria are essential to the healthy life of all warm-blooded animals including humans, but certain strains can cause serious problems.

BART Biodetector With BART, you can monitor for Iron Related Bacteria (IRB), Sulfate Reducing Bacteria (SRB) and Heterotrophic Aerobic Bacteria (HAB) - the three most important agents involved in biofouling. Other BART systems are described below. These bacteria can cause corrosion, clogging, fouling of the water, and increased hygiene risks, so it is important to have an easy and accurate method of determining their presence and level of activity.

Easy to use

The BART Biodetector requires no microscope, no laboratory, and no incubator! The test is done at room temperature in your office or treatment room, on a desk, shelf, or in a cupboard, and is viewed daily. Different microorganisms like to grow at different heights in a column of water to which nutrients have been added. BART biodetectors contain nutrients in the base of a column and a ball. The ball restricts the amount of oxygen entering the water column, so that aerobic organisms grow around the ball and anaerobic organisms grow deep down in the water column. By changing the nutrients in the base of the column, different organisms are encouraged to grow. BART determines presence and activity levels.

Easy to analyze The time taken for a color change (reaction) to occur gives a measure of the population size and activity. A color change occurs in the BART tube as a result of the oxygen gradient diffusing from the

bottom upward. The change of color indicates a presence of bacteria within that sample. Interpretation is provided with the kit.

The Test Full instructions for the use of BART biodetectors are included with your purchase. Each individual test consists of: • •

Test vial with media and BART ball Outer tube for spill containment, odor control, disinfection, and disposal

To Order Each kit number below includes nine (9) BARTs, except the 5-0031 which contains seven (7) BARTs and reaction caps. Each BART test is color-coded for quick and easy recognition.

Test Factor Iron Related Bacteria - IRB-BART Sulfate Reducing Bacteria - SRB-BART* Slime Forming Bacteria - SLYM-BART Heterotrophic Aerobic Bacteria - HAB-BART Micro-Algae - ALGE-BART Fluorescent Pseudomonas - FLOR-BART*

BART Color Order Code Red Black Lime green Blue Dark green Yellow

5-0024 5-0025 5-0026 5-0027 5-0028 5-0029

Denitrifying Bacteria - DN-BART

Gray

5-0030

Nitrifying Bacteria - N-BART

White

5-0031

Combo

5-0032

Three each of IRB-, SRB-, and SLYM-BART

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Coliform bacteria are not pathogenic (disease causing) organisms, and are only mildly infectious. For this reason these bacteria are relatively safe to work with in the laboratory. If large numbers of coliforms are found in water, there is a high probability that other pathogenic bacteria or organisms, such as Giardia and Cryptosporidium, may be present

Approved tests for total coliform bacteria include the membrane filter, multiple tube fermentation, MPN and MMO-MUG ("Colilert") methods. The membrane filter method uses a fine porosity filter which can retain bacteria. The filter is placed in a petri (culture) dish on a pad with growth enrichment media (mEndo)

and is incubated for 24 hrs at 35 degrees C. Individual bacteria cells which collect on the filter grow into dome-shaped colonies. The coliform bacteria have a gold-green sheen, and are counted directly from the dish. Since some other bacteria may develop a similar color, a confirmation test using more specific media is required. The confirmation procedure requires an additional 24 to 48 hrs to complete the test for suspected positive total coliform tests.

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The MPN (most probable number) method uses a test tube full of media with a smaller inverted test tube inside which captures carbon dioxide gas released from the growth of coliform bacteria. A series of dilutions and replicates are set up, and those producing gas in 24 hrs at 35 degrees C are counted. A statistical analysis is used to determine the most probable number of bacteria cells present.

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Our laboratory is certified for the membrane filter technique. The sample should be collected in a specially prepared, sterile whirl pack bag for the test to be valid. The bags contain a small amount of sodium thiosulfate to remove any chlorine present, and have been sterilized. Sample collection should be done very carefully and directly into the bottle from the tap to avoid contamination of the bottle from hands or a transfer vessel such as a cup. The sample should be kept cool and delivered to the lab within 24 hrs for analysis. Total coliform bacteria testing is a relatively inexpensive when compared to the cost for the determination of the concentration or presence of viruses, Giardia, or Cryptosporidium

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Cryptosporidium is a protozoan organism which causes the parasitic infection, cryptosporidiosis. It exists in either the free-swimming (trophozoite) form or the oocyst (dormant) form. Cryptosporidium parvum is now recognized as a human pathogen which can cause severe diarrheal illness.

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TREATMENT: Water treatment for Cryptosporidium relies on properly designed and operated filtration systems. Chlorine disinfection of the organism is ineffective, as it has been shown that even one oocyst can withstand pure bleach (50,000 ppm chlorine) for 24 hours and still cause an infection. Filter systems usually consist of several filters. A "roughing filter" containing a 5µ - 10µ (micron) cartridge filter is installed to remove any large diameter sediments, such as iron sediments, sand, salt , etc. Downline from the roughing filter, a "polishing filter" containing a <1µ absolute cartridge filter is installed to remove small particles including Cryptosporidium, from the water. Most reputable water system vendors are

currently recommending a filter porosity of <1µ to submicron or membrane filters to remove Cryptosporidium cysts and trophozoites from drinking water. The filters are available at most water supply dealers. Testing for Groundwater Sources and Potential for Surface Water Influence

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TESTING: Testing procedures are available for detecting Cryptosporidium oocysts in both raw and treated drinking water. The testing procedure involves filtering a large volume of water through a 1 micron, yarn-wound, polypropylene filter. The filter is then treated to remove any oocysts which may be present and the sample is concentrated. A monoclonal antibody to Cryptosporidium is added to the sample to bind to oocyst wall antigens. The reaction can be visualized by the addition of fluorescein isothiocyanate (FITC) - conjugated anti-immunoglobulin and scanning with an epifluorescence microscope.

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Giardia:

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Giardia (say "gee-ar-dee-ah") cysts are elliptically shaped and range in size from 6 to 10 microns. The cyst stage could be thought of as the equivalent of a "seed" or "spore",

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Treatment Options In adults, giardiasis is commonly treated with a drug called metronidazole (otherwise known as Flagyl). Usually the drug is taken 3 times a day for 5 to 10 days. For children less than 5 years old, the treatment is typically with a drug known as furazolidone (or Furoxone). Typically the drugs have side effects similar to the disease.

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Detection of Giardia in Drinking Water The procedure used for Giardia is known as the immunofluorescent antibody method. The procedure requires that a minimum of 100 to 1000 gallons of water is filtered through a 1 micron filter. The filter is then cleaned and the material collected and centrifuged. The centrifuged pellet is then floated using a Percoll Sucrose density gradient technique. The floated material is then cleaned and centrifuge and the pellet is then stained with a fluorescent antibody.

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Slides of the pellet are prepared and the slides are examined for the cysts. If a cyst is suspected, the slide is then examined using Hoffman Modulation to confirm the proper shape, size, and at a minimum two internal structures.

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Presumptive test: lauryl so4 lactose broth

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Confirmative test: Eosin methylene blue agar(EMB) agar

Materials Needed: coliform test Sterile collection container (sterile bottle or test tube) with water sample Sterile pipets or sterile transfer pipets (dropper pipets) Sterile petri dish Coliscan Easygel (Micrology Laboratories) Incubator set at 37° C

Cryptosporidium TESTING: Testing procedures are available for detecting Cryptosporidium oocysts in both raw and treated drinking water. The testing procedure involves filtering a large volume of water through a 1 micron, yarn-wound, polypropylene filter. The filter is then treated to remove any oocysts which may be present and the sample is concentrated. A monoclonal antibody to Cryptosporidium is added to the sample to bind to oocyst wall antigens. The reaction can be visualized by the addition of fluorescein isothiocyanate (FITC) - conjugated antiimmunoglobulin and scanning with an epifluorescence microscope

Material provided

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Each test kit contains: 1 x 12 Microtiterstrips with 8 wells each; coated with monoclonal antibodies (mouse) against Cryptosporidium. Vial 1 Universal Stool Diluent (100 ml); buffered NaCl solution for sample dilution; ready to use

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Vial 2 Washing Buffer (100 ml; 10x conc., brown lid) pH 7.2, contains 0.01 % Thimerosal Vial 3 Positive Control (3 ml); .Cryptosporidium antigen from stool, heat inactivated ready to use. Contains 0,1 % Thimerosal. Vial 4 Enzyme Conjugate (12 ml); HRP- conjugated mAb(mouse) against Cryptosporidium ; dyed blue, ready to use. Vial A Substrate (6 ml); urea peroxide, ready to use Vial B Chromogen (6 ml); tetramethylbenzidine (TMB), ready to use Vial C Stop Solution (6 ml); 1 M sulfuric acid Instructions for use

Material needed but not provided

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Distilled or deionized water Test tubes Transfer-Pipets Vortex mixer Micropipet for volumes of 100 µl and 1 ml Microplate washer or multichannel pipet (250 µl) Microplate reader (450 nm, optional reference wavelength ³ 600 nm) Absorbent paper