ENVIRONMENTAL MICROBIOLOGY

WATER TESTING

The section tests water from the majority of the Public Water Supplies in the state as a requirement under the Safe Drinking Water Act (SDWA) and the Total Coliform Rule (TCR). In addition to testing water from Public Water Systems, water from Private Individual Household Wells, Recreational Areas such as Swimming Pools and Bathing Beaches, Bottled Water Companies, Dairy Plants and Dairy Farms are also routinely tested.

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 in 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 sub group 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 OHES - Public Health Sanitation Division (PHSD) and Local County Health Departments.   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.  And, 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.  Any unused containers must be returned to the Office of Laboratory Services for re-processing.

Samples can be mailed or sent by other delivery services such as UPS, FedEx, Airborne Express.  Samples can even be hand delivered.  If samples are hand delivered to the South Charleston Laboratory after operating hours, they can be left in the outside refrigerator located in the front of the building on the loading dock.  Samples are received at the South Charleston Laboratory, 8:00 a.m. to 4:30 p.m. Monday thru Friday and by U.S. Mail and special arrangements on Saturday.   The Bardane District Environmental Laboratory receives samples 8:00 a.m. to 4:00 p.m. Monday thru Wednesday and 8:00 a.m. to 12:00 p.m. (noon) 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.

Fermentation Method
The Fermentation Method is the choice for routine samples from Public Water Systems.  100 mLs 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.

Enzyme Substrate Method
The second method that is routinely used is the Enzyme Substrate Test.  This test is used primarily for all other types of water and for public water supplies when a rapid turn-around-time is required.  A reagent packed 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. 

Membrane Filtration
Membrane Filtration is the third method that can be performed.  Although it is not routinely used at the OLS, it does remain popular at other certified drinking water laboratories throughout the state.  The sample is run through a filter, trapping the coliform bacteria.  The filter is then placed on a growth medium and incubated for 24 hours.  Suspect coliform bacteria that grow on the filter are then subjected to a verification test.

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.  Two staff members of the Environmental Microbiology Section have been trained trained as Certification Officers (CO) by the USEPA at the Andrew W. Breidenbach Environmental Research Center in Cincinnati, OH.  These two 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 that is based on the USEPA's Manual for the Certification of Drinking Water Laboratories and 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 Quality Assurance Team (LQAT) 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 LQAT 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 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 48 hours of collection and IMS and Raw Milk must be received at a temperature between 0.0° to 4.4°C (32.0° to 40.0°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 buttermilks, dips, sour creams and yogurts.  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 protocol that required all raw milk be checked for drug residues before it is received by the dairy plant.  Also included was protocol that provided oversight by the states to ensure that dairy plant personnel were adequately trained to perform antibiotic drug residue testing.

One staff member of the Environmental Microbiology Section has been trained by the FDA-LQAT 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 5 analysts of the Environmental Microbiology Section and the two analysts at the Bardane District Environmental Laboratory 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.

RESOURCE LINKS

EPA - General Information on Drinking Water

EPA - Manual for the Certification of Laboratories Analyzing Drinking Water

EPA- Office of Ground Water and Drinking Water

EPA - Laboratories Certified for Cryptosporidium (Approval Pending)

EPA - Safe Drinking Water Act

EPA - Total Coliform Rule Highlights

FDA - Centers for Food Safety and Applied Nutrition

FDA - IMS Listing of Approved Dairy Laboratories

PUBLICATIONS

Laboratories Certified in West Virginia to Perform Drinking Water Analysis

FORMS AND INSTRUCTIONS

Water Bottle Requesition Form

Water Sample Collection Instructions

EM-1 Water Bacteriological Report Form

Proficiency Test Report From for Certified Drinking Water Laboratories

Laboratory Information Form for Laboratory Certification Renewals

Milk Sample Collection Instructions for County Health Departments

Milk Case Packing Instructions for County Health Departments

Milk Case Packing Instructions for Raw Milk

Pasteurized Milk Collection Form (PM-02)

Raw Milk Collection Form (RM-02)

Appendix N Drug Residue Screening Report

FDA Forms (2400 Series) for the On-site Evaluations of Dairy Laboratories

CONTACT INFORMATION

Environmental Microbiology Section - South Charleston Laboratory

Drinking Water Laboratory
304-558-3530 x2711

Milk Laboratory
304-558-3530 x2701

Laboratory Certification (Drinking Water and Dairy)
304-558-3530 x2705

Thomas L. Ong
Microbiologist Supervisor
Laboratory Certification Officer
Laboratory Evaluation Officer
tom.l.ong@wv.gov
304-558-3530 x2710

Tracy Goodson
Microbiologist III
Laboratory Certification Officer
tracy.g.goodson@wv.gov
304-558-3530 x2701

Michael Flesher
Microbiologist III
michael.a.flesher@wv.gov
304-558-3530 x2701

Carole Moore
Microbiologist I
304-558-3530 x2711

Bardane District Environmental Laboratory

Brenda K. Hall
Microbiologist III
brendakhall@wvdhhr.org
304-725-5832

Monica A. Whyte
Microbiologist II
monicawhyte@wvdhhr.org
304-725-5832