Biological testing of water

• Escherichia coli, enterococci, Cryptosporidium, and Giardia.

• 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.

• 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.

• 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

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

• Water treatment methods alone cannot solve the problem

• 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.

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

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

• 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.

• 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.

• Water-related disease outbreaks tend to occur in the summer. Although rarely

encountered today, typhoid, hepatitis and cholera, can spread through water
supplies

• 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.

• 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 BART Color Order Code

Iron Related Bacteria - IRB-BART Red 5-0024
Sulfate Reducing Bacteria - SRB-BART* Black 5-0025
Slime Forming Bacteria - SLYM-BART Lime green 5-0026
Heterotrophic Aerobic Bacteria - HAB-BART Blue 5-0027
Micro-Algae - ALGE-BART Dark green 5-0028
Fluorescent Pseudomonas - FLOR-BART* Yellow 5-0029
Denitrifying Bacteria - DN-BART Gray 5-0030
Nitrifying Bacteria - N-BART White 5-0031
Three each of IRB-, SRB-, and SLYM-BART Combo 5-0032

• 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.

• 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.

• 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

• 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.

• 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

• 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.

• Giardia:

• 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",

• 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.

• 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.
 • 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.

• Presumptive test: lauryl so4 lactose broth

• 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 anti-
immunoglobulin and scanning with an epifluorescence microscope

Material provided

• 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
 • 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

• 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