Environmental Microbiology

The dedicated staff in the Environmental Microbiology Section have impacted the lives of everyone in West Virginia who has consumed water from a Public Water System or purchased and consumed Grade A dairy products.  The Bardane District Environmental Laboratory staffs three individuals to handle the drinking water testing for the nine counties in the Eastern panhandle.  This includes Berkeley, Jefferson, Grant, Hampshire, Hardy, Mineral, Morgan, Pendleton, and Tucker.

Water Testing

This Unit tests water from the majority of Public Water Systems in the state as a requirement under the Safe Drinking Water Act (SDWA) and the Total Coliform Rule (TCR).  In addition, this Unit also tests water from private, individual households, recreational areas such as swimming pools and bathing beaches, and bottled water companies.  Dairy plants and dairy farms also submit water for testing.

What are we looking for?

Water samples are first tested for Coliform Bacteria.  This group of bacteria, normally found in the environment, is used as an indicator, to indicate the possible presence of pathogens (disease-causing organisms).  Generally, when pathogens are present in drinking water, coliforms are present as well.  The reverse is not necessarily true - when coliforms are found in drinking water, pathogens may or may not be present.  However, treating the water to eliminate the coliforms would most likely also eliminate the pathogens.  The level of coliform bacteria allowed in drinking water is zero.  If coliforms are found in the water sample, further testing is performed to look for Fecal Coliforms (a subgroup of Total Coliforms) or E. coli (one member of the Fecal Coliform Group).  Since both Fecal Coliforms and E. coli are found in the intestinal tract and feces of warm-blooded animals, their presence in drinking water can indicate a sewage contamination problem.

Where do water samples come from?

Water samples are collected by Water Plant Operators from Public Water Systems, Engineers from the Office of Environmental Health Service (OEHS) - Environmental Engineering Division (EED), Sanitarians from OEHS - Public Health Sanitation Division (PHSD) and Local County Health Departments (LHD).  Samples are also received from other laboratories that are not certified to conduct drinking water tests.  On a limited basis and by prior arrangement, samples are received from private citizens.  Private citizens needing to have their well water tested should first contact their local county health departments.  This way a trained sanitarian can collect the sample and ensure that the well is properly protected.  In the event that the sample does have coliform bacteria present, the sanitarian can instruct the well owner on how to treat the well, remedy the problem and provide follow-up testing.

Samples must be collected in containers that are provided by the Office of Laboratory Services.  These containers are specially prepared, contain a dechlorinating agent and are tested for sterility.  They are supplied to clients upon submission of the Bottle Request Form.  This form may be mailed, faxed, submitted with samples, or hand-delivered.  Samples must be received and testing begun within 30 hours of collection for compliance with the SDWA.  The EM-1 Water Bacteriological Report Form must be completed in ink and submitted along with the sample.  Water collection containers are state property and must be returned to the Office of Laboratory Services for testing.  They cannot be sent to any other laboratory.

Samples can be mailed or sent by other delivery services such as UPS or FedEx or can be hand delivered.  If samples are hand-delivered to the South Charleston Laboratory after operating hours, they can be left in the refrigerator located in the building by the back parking lot gate.  Samples are received at the South Charleston Laboratory, 8:00 a.m. to 5:00 p.m. Monday through Friday (with the exception of State holidays), by U.S. Mail, and by special arrangements on Saturday.  The Bardane District Environmental Laboratory receives samples 8:00 a.m. to 4:00 p.m. Monday through Wednesday and 8:00 a.m. to 12:00 p.m. on Thursday.

How is water tested?

The Environmental Microbiology Section has at their disposal, several different methods to test water for coliform bacteria.  All of the test methods require 100 mL (approximately 3.4 oz) of sample.

Enzyme Substrate Method
The Enzyme Substrate Test is used primarily for all types of water.  A reagent packet is added to 100 mLs of sample and then the mixture is incubated for 24 hours.  A color change from clear to yellow confirms the presence of coliform bacteria.  At the same time, a blue fluorescence when subjected to UV light confirms the presence of E. coli.  A variation of this test is also available when there is a need to know the amount of coliform bacteria and E. coli present.  A slightly different formulation is available that will allow for confirmed results in as little as 18 hours.

Fermentation Method
The Fermentation Method is a backup method used on certain types of dairy plant samples.  One hundred milliliters of sample is added to a growth medium and incubated for up to 48 hours.  A change in color from purple to yellow indicates a presumptive positive test for coliform bacteria.  All presumptive positive samples are then subjected to a confirmation test and a fecal coliform test which can take an additional 48 hours to complete.  High numbers of non-coliform bacteria can interfere with this test causing invalid results.

Are Drinking Water Laboratories certified?

To ensure uniformity and the validity of test results, Public Water Systems must have their water routinely checked for coliform bacteria and chemical analytes by a laboratory that is certified to perform such testing.  The Environmental Microbiology Section is certified by the United States Environmental Protection Agency (USEPA) to perform Coliform Testing on drinking water.  All commercial and private laboratories that test drinking water samples from public supplies in West Virginia must be certified by the State.  Several staff members of the Environmental Microbiology Section have been trained as Certification Officers (CO) by the USEPA at the Andrew W. Breidenbach Environmental Research Center in Cincinnati, OH.  These staff members oversee the Drinking Water Microbiology Certification Program for the state of West Virginia.  Certified Drinking Water Laboratories must pass an on-site inspection by the CO every three years.  This is based on the USEPA's Manual for the Certification of Drinking Water Laboratories.  Laboratories must successfully test Proficiency Test (PT) Samples annually for each method that they are certified for.  All laboratories certified by the State of West Virginia along with their testing capabilities can be found on the Certified Lab List.  Certification for chemical analysis is handled by the Environmental Chemistry Section.

Milk Testing

The Environmental Microbiology Section is the only fully certified Grade A Dairy Laboratory in West Virginia.  This certification is kept by following stringent guidelines set forth by the National Conference on Interstate Milk Shipments (NCIMS) and enforced by the Laboratory Proficiency and Evaluation Team (LPET) of the Food and Drug Administration.  The Environmental Microbiology Section is subject to an on-site inspection every three years by a Federal Laboratory Evaluation Officer and must pass Split Samples every year.  These Samples are prepared by the LPET and then "split" among every state's central milk laboratory.  Every single size of every single Grade A Milk Product produced in West Virginia is tested by the Environmental Microbiology Section.  Milk shipped into West Virginia from other states is also tested as is raw milk from the producers (farmers).

Where do milk samples come from?

Because of the complexity associated with the testing of milk samples, a testing schedule is set up with the OEHS-PHS and the local county health departments.  This schedule is unknown to the dairy industry.  The Interstate Milk Shipment (IMS) Program consists of the milk produced from dairy plants located within the state.  This milk is picked up in its original containers by state sanitarians with the OEHS-PHS and delivered to the laboratory for testing.  Ten weeks throughout the year are set aside for this testing.  Raw milk (unpasteurized and from the farm) is also picked up by a state sanitarian and delivered to the laboratory.  Another 10 weeks throughout the year is set aside to test raw milk.  The County Program consists of milk products that are shipped into the counties from other counties and states.  These samples are picked up by sanitarian from the local county health departments.  Instead of shipping the original container to the lab as required in the IMS Program, the sanitarian will collect a portion of the milk in a sterile vacuum tube and ship the tubes to the laboratory.  See Milk Case Packing Instructions. Testing must begin at the laboratory within 60 hours of collection and IMS and Raw Milk must be received at a temperature between 0.0° to 4.5°C.  If these conditions are not met, the milk samples are rejected.

What type of tests are conducted on milk?

Total Bacteria
The Standard Plated Count is a test performed to determine the total bacterial count of a milk sample.  This test is conducted on almost all milk samples (IMS, Raw and County Samples) except those that are made with bacterial cultures, such as buttermilk, dips, sour cream, and yogurt.  The test is performed by removing a very small portion of milk, 0.1 mL and 0.01 mL for pasteurized products and 0.01 mL and 0.001 mL for raw milk, then mixing them with a growth medium.  To put these volumes in perspective, 1 ounce equals 29.57 mL.  The bacteria are allowed to grow in an incubator for 48 hours and then counted.  Pasteurized milk is allowed to contain 20,000 colonies of bacteria, referred to as colony forming units (cfu) per mL of sample (cfu/mL).  Raw milk, prior to pasteurization, from each farm can contain up to 100,000 cfu/mL and once it is mixed together with other farms, it can have up to 300,000 cfu/mL.

Coliform Bacteria
All pasteurized milk and milk products (from both the IMS and County Programs), including the buttermilks, dips, sour creams and yogurts are tested for Coliform Bacteria.  For this test, 1.0 mL of sample is mixed with a growth media that is specific for coliform bacteria.  The coliform bacteria are then allowed to grow in an incubator for 24 hours and then counted.  Coliform, if present in a milk sample, is generally considered to be a contamination problem that occurs after pasteurization.  Milk is allowed to have 10 coliform colonies per mL.

Alkaline Phosphatase
All milk with the IMS Program is subject to the Alkaline Phosphatase Test.  Alkaline Phosphatase (ALP) is an enzyme that naturally occurs in raw milk.  This enzyme is deactivated during the pasteurization process.  ALP can be found in pasteurized milk if it has been contaminated with raw milk.  This is referred to as Residual Phosphatase.  ALP can be reactivated in pasteurized milk that is not properly handled after pasteurization and allowed to warm up for an extended period of time.  This is referred to as Reactivated Phosphatase.  Certain types of microorganisms can produce a type of ALP, this is referred to as Microbial Phosphatase.  The Environmental Microbiology Section has an instrument that is capable of quantifying the amount of ALP in milk samples.  The procedure consists of adding a milk sample to a reagent and then using an instrument to measure the production of a special compound.  This is then converted to the amount of ALP activity and reported in milli units per liter (mU/L).  Activity above 350 mU/L is considered positive of ALP.  The laboratory has special procedures that can then be used to determine the type of ALP present (Residual, Reactivated or Microbial).  Residual ALP above 350 mU/L will result in an immediate product recall.  As little as 0.006% raw milk can be detected with this instrument.

Somatic Cells
Somatic Cell Counts are conducted only on raw milk.  Somatic cells are also referred to as leucocytes or white blood cells and their presence in high numbers can indicate an infection in the cow's udder, such as mastitis.  The somatic cell count can be determined electronically by using an electronic somatic cell counter or by preparing and staining microscope slides and then counting the somatic cells under 1,000X magnification.  Somatic Cell Counts above 750,000/mL are considered high.

Drug Residues
Antibiotic Drug Residues are one of the hottest subjects in the dairy industry to date.  The push to eliminate drug residues in the nation's milk took a radical turn in December 1989 with a Wall Street Journal Article announcing milk contaminated with antibiotic drug residues.  Exposing the population to low levels of antibiotics increases the risk of creating bacteria that are resistant to these antibiotics.  Prior to this date, random milk samples were checked in the laboratory for drug residues.  After this date, the NCIMS and FDA set up a protocol that required all raw milk be checked for drug residues before it is received by the dairy plant.  Also included was a protocol that provided oversight by the states to ensure that dairy plant personnel were adequately trained to perform antibiotic drug residue testing.
Several staff members of the Environmental Microbiology Section has been trained by the FDA-LPET as a Laboratory Evaluation Officer (LEO).  The LEO has the responsibility of training the Dairy Industry Supervisors/Back-up Supervisors by conducting hands-on training sessions once every three years, providing oversight by performing bi-annual on-site inspections of Dairy's drug testing facilities/analysts and annually, preparing milk samples spiked with drug concentrates for every analyst in the state to test. 

Even with all this training and oversight, the dairies are only approved to screen the milk.  Any milk that is found to contain drug residues must be confirmed in a certified facility by certified analysts.  The analysts of the Environmental Microbiology Section are the only certified analysts in West Virginia that can confirm drug residues in milk.  This puts someone on call 7 days a week for instances that occur at off hours.  Tanker loads of milk that are found to contain drug residues are no longer available for human consumption and must be disposed of. 

Both raw and pasteurized milk (with the exception of a few pasteurized products) received at the laboratory are subjected to antibiotic drug residue testing.  Milk samples are monitored for Penicillin, Ampicillin, Amoxicillin, Cloxacillin, Ceftiofur, and Cephapirin.  Tests that are used must be able to detect at least 4 of the 6 drugs.  The Environmental Microbiology Section has at its disposal, 4 different methods that can detect anywhere from 4 to all 6 of the drugs and even identify the drug. 

Pasteurized Milk Containers
Even the empty containers that are used for milk products are tested.  The entire inner surface of these containers is rinsed with a special reagent.  This reagent is then checked for residual bacteria and residual coliform bacteria.

Are milk laboratories certified?

The Environmental Microbiology Section is the only fully certified dairy laboratory in the state of West Virginia.  A listing of the certified dairy labs throughout the country can be found in the IMS Listing.  Any facility wishing to become certified to test Grade A Milk and Milk Products must first contact the State's Laboratory Evaluation Officer (LEO).  This individual must attend FDA training every three years and successfully conduct a joint on-site evaluation with a Federal LEO.  

Forms and Instructions

Water Sample Submission Form (not available electronically; call for copy)
Milk Sample Submission Form (not available electronically; call for copy)

Resource Links

EPA (general information on drinking water)

Contact Information

South Charleston Location

Phone: 304-558-3530
Water Testing - extension 2711
Milk Testing - extension 2701
Laboratory Certification - extension 2710

Kearneysville (Bardane) Location

Phone: 304-725-5832