Quality Assurance/Quality Control Manual

Ohio Water Microbiology Laboratory

ABSTRACT

The U.S. Geological Survey (USGS), Ohio Water Microbiology Laboratory (the OWML) provides water-quality data on microorganisms of public health significance for a variety of projects within the USGS. Currently, the OWML analyzes samples for and provides training on bacterial indicators, coliphage, enteric viruses, and two protozoan pathogens—Cryptosporidium and Giardia.

Quality-assurance and quality-control (QA/QC) practices for the operation of OWML are described in this manual. The Laboratory Manager, Laboratory Coordinator, Chemical Hygiene Officer, and laboratory and field staff are responsible for implementing QA/QC procedures. This includes correctly following methods of analysis, media and reagent preparation and storage, and analytical quality-control procedures. A sample management and documentation system involves the use of service request forms and login ID’s for each sample. A laboratory information management system (LIMS) has been implemented to store sample login information and results. Laboratory equipment maintenance and calibration records are also stored in the LIMS.

INTRODUCTION

The USGS Ohio Water Center Ohio Water Microbiology Laboratory (the OWML) mainly provides analytical data for projects within the Ohio Center. With the growth of microbiological monitoring within the WRD, however, the OWML has begun analyzing samples for other Water Center programs by request. Samples are collected to determine the presence of microbiological organisms of public health significance in ground waters and surface waters for a variety of study objectives. For example, some local studies are done to judge compliance with standards for protection of public health in swimmable or drinkable waters. Other studies investigate the occurrence, distribution, and trends of pathogenic organisms and indicators in surface and ground waters and relate these to environmental and water-quality factors.

The OWML fulfills analytical requirements of the WRD by analyzing environmental samples for bacterial indicators, coliphage, enteric viruses, and two protozoan pathogens—Cryptosporidium and Giardia. OWML personnel provide assistance for project planning and training on three major groups of microorganisms of public health significance in the United States: bacteria, viruses, and protozoa. As funds become available for expansion, the OWML plans to add other analytical methods and types of microorganisms to its analytical list. The OWML is not involved in method development at the present time, but instead tests new methods developed by others for applicability to ambient monitoring programs.

The OWML is committed to providing quality microbiological analytical services to the USGS. The quality assurance/quality control (QA/QC) program is designed to ensure the production of scientifically sound, legally defensible data of known and documented quality. The effectiveness of this program relies on clearly defined objectives, well-documented procedures, and management support.

Purpose and Scope

The purpose of this manual is to identify and document practices and standard operating procedures for those activities of the OWML that affect quality of data. The manual provides OWML personnel and customers with general descriptions of quality practices and goals to aid in the interpretation of data. This manual is intended to be an unpublished, dynamic document that will be frequently updated as laboratory activities expand or change.

Organizational Structure

The Laboratory Manager (1) oversees the daily operations of the OWML, (2) directs technical personnel in the proper performance of laboratory procedures and the reporting of results, (3) ensures that appropriate methods are used, (4) plans activities leading to testing and modification of analytical procedures, and (5) designs and implements a comprehensive QA/QC program. The Laboratory Manager is responsible for initiating the QA/QC program, providing information and training to the staff, and periodically reviewing QA/QC activities.

The Laboratory Coordinator oversees the daily operations of the OWML, ensures that the equipment is properly maintained and calibrated, orders supplies and equipment, and oversees and performs analytical work. The Laboratory Coordinator implements the QA/QC program in the daily tasks of conducting analyses, performing quality control checks, and calculating and reporting results.

The Chemical Hygiene Officer oversees safety operations in the laboratory with assistance from the Laboratory Manager and Laboratory Coordinator.

The laboratory and field staffs are responsible for correctly implementing collection and analysis procedures and for identifying and working with supervisors to correct and avoid potential problems.

Table 1. Current laboratory personnel and qualifications.

NAME	LABORATORY TITLE	USGS TITLE	Education/experience
Donna Francy	Microbiology/ Water Quality Specialist	Hydrologist GS-13	B.A. Biology, M.S. Environmental Science, Certified Clinical Microbiologist 15 years experience in water quality and environmental microbiology
Rebecca Bushon	Laboratory Manager	Hydrologist GS-12	B.S. Biology 10 years experience in microbiology
Don Stoeckel	Project Chief/ Special Projects	Hydrologist GS-12	B.S. Microbiology M.S. Environmental Science Ph.D. Soil Science 10 years experience in microbiology
Chris Kephart	Molecular Analyst	StuTrain(Hyd) GS-9	B.S. Microbiology Working on M.S. Environmental Science 7 years experience in microbiology
Amie Brady	Laboratory Coordinator	Hydrologist GS-9	B. S. Environmental Science B.S. Plant Biology M.S. Environmental Science 7 years experience in microbiology
Christina Likirdopulos	Specialized Lab Analyst/ Chemical Hygiene Officer	Hydrologist GS-9	B.A. Chemistry M.S.P.H. Environmental Sciences & Engineering 7 years experience in microbiology
Erin Bertke	General Lab Analyst	Biologist GS-7	B.S. Environmental Science 3 years experience in microbiology

GENERAL LABORATORY QUALITY ASSURANCE/QUALITY- CONTROL PRACTICES

An overview of analytical methods, training policies, safety, laboratory maintenance, sample management, and data documentation is given in this section.

Analytical methods

The methods used by the OWML can be categorized into four groups: compliance, official, provisional, and experimental. The United States Environmental Protection Agency (USEPA) and others in the research community are continuously developing new methods for detecting and quantifying microbiological pathogens and indicators in water; therefore, several types of methods for target organisms may be currently in use at the OWML.

Compliance methods are those published by USEPA in the Federal Register and are used to determine compliance with standards for protection of public health in swimmable or drinkable waters. Analytical methods for fecal-indicator bacteria are often in this group because they are straightforward, quantitative, and routinely used.

Official methods are those noncompliance methods published by water-analysis authorities such as American Public Health Association, the U.S. Environmental Protection Agency, or the USGS. Official methods should be well established, have known levels of bias and variability, and be relatively easy to apply in field operations or have holding times long enough to allow shipping to a central laboratory for analysis.

Provisional methods are published methods that are still being validated by the method developer, usually the USEPA. For these methods, the method developer establishes precision and accuracy and ensures the methods are adequately tested. Because methods for detection of protozoa are complex, qualitative to semiquantitative, expensive, and very time consuming, these methods are often provisional.

Experimental methods are unpublished methods that are currently being testing to establish QA/QC practices and determine applicability to ambient monitoring programs.

Training

The Laboratory Manager and Laboratory Coordinator are responsible for ensuring that laboratory employees receive proper training in analytical methods and laboratory procedures and for documenting any training received. In particular, laboratory employees will be trained in sterile technique before handling samples for microbiological analysis. A new employee will receive orientation and skills training. New or established employees may receive training on new methods given by the method developer. The Laboratory Coordinator will maintain training records for microbiological methods on file by employee; this includes on-the-job training as certification of proficiency in microbiology.

The Laboratory Coordinator, Chemical Hygiene Officer, and Water Center Safety Officer provide safety orientation to new employees and safety education to all employees. The employee orientation covers general safety issues, emergency procedures, standard-safety operations, the chemical-hygiene plan, hazardous-waste management, waste disposal, and location of safety equipment.

Safety

Detailed laboratory safety practices and responsibilities are described in the Chemical Hygiene Plan. Safety activities include safeguards to avoid electric shock; prevent fire; prevent accidental chemical spills; and minimize microbiological dangers, facility deficiencies, and equipment failures.

Laboratory personnel that are isolating microorganisms from natural sources must be made aware that pathogens may be present in environmental samples. Technicians are to wear disposable gloves and lab coats when handling samples that are likely to contain pathogens. Safety glasses are worn if there is a chance of projectiles, aerosols, or other foreign matter entering the eye. This includes when using positive-pressure air to blow out any remaining liquid during the ultrafiltration process for Cryptosporidium and Giardia. Laboratory personnel will receive immunizations for pathogens on a project-specific basis. Each project sending samples to the OWML is required to have a project safety plan--copies are available for OWML employees. Immunizations are offered to all OWML workers for Hepatitis A virus, Hepatitis B virus, and tetanus.

Safety equipment is tested at regular intervals. Safety showers and eyewash stations are tested annually and recorded in the LIMS. Fire extinguishers are inspected annually. The Chemical Hygiene Officer maintains a list of chemicals and arranges for a contract for disposal of hazardous waste.

Laboratory materials and equipment

The Laboratory Manager sets policies for preventive maintenance and calibration of laboratory materials and equipment. Two QA/QC logbooks are kept in the laboratory bookshelf with records of quality-assurance checks of materials and equipment up through September 30, 2006. The logbooks are for (1) equipment -autoclaves, balances, pipettors, hoods, and thermometers; and (2) laboratory water. Examples of equipment log sheets are in Appendix AA. Results of quality-assurance checks of materials and equipment starting in FY 04 are stored in the LIMS. Quality-control checks that are required LIMS entries are listed in italics below.

· The Laboratory Manager or Laboratory Coordinator must review QA/QC quarterly reports from LIMS to ensure procedures are followed and problems are properly addressed.

For some pieces of equipment, the use of daily logbooks to record operating times and other types of frequent entries are required. A daily logbook is kept with the autoclaves and the water-quality meters (pH, specific conductance, and turbidity).

General sterility and cleanliness

The sterility and cleanliness of the laboratory is necessary to ensure the integrity of samples and analytical procedures.

· Traffic through the laboratory is restricted to those doing work in the laboratory, especially when analytical work is being done.

· The countertops are wiped down with surface disinfectants, such as Conflikt (Decon Labs, Inc., King of Prussia, PA) or 70 percent ethanol, before and after use.

· Antimicrobial soap is available at various laboratory sinks to facilitate hand washing before and after laboratory work.

Clean and sterile glassware that is free of detergent residue is crucial to ensure valid results in microbiology.

· Dirty dishes are placed on a moveable laboratory cart after use and are not to be stored on countertops. Dishes are washed in a dishwasher or by hand with hot water and laboratory-grade phosphate-free detergent. Dishes are rinsed with tap water and then deionized water.

Autoclaves

Sterilization is the process that eliminates living organisms from substances or objects. The OWML is equipped with three autoclaves for sterilization of glassware, reagents, media, and disposables—two medium-sized autoclaves (Market Forge) that are operated in the side laboratory and one large autoclave (Consolidated) that is operated in the warehouse.

· Dishes that need to be sterilized are wrapped in aluminum foil or kraft paper and placed in the autoclave for moist heat sterilization. Clean and sterile dishes are stored in closed cupboards until use.

· The autoclaves are operated at 15 lb/in² steam pressure, producing an inside temperature of 121 to 124^oC (American Public Health Association, 1998, Section 9020B). Do not overload the autoclave. Autoclave time depends on the type and amount of equipment as follows:

· Glassware and up to 250 mL of liquid—15 minutes

· 500 to 2,000 mL liquid—30 minutes

· Greater than 2,000 mL to 6,000 mL liquid—15 minutes per 1,000 mL

· Greater than 6,000 mL liquid—90 minutes

· Carbohydrate-containing media—15 minutes (no more than 250 mL volumes)

· Contaminated materials and discarded cultures—30 minutes, allow autoclave chamber pressure to decrease, then run for a 60 minute cycle

· Operating temperature and pressure are checked once a week. Heat-sterilizing tape is used with each run to identify supplies that have been properly sterilized and checks the performance of the autoclave. The performance is also checked monthly by using spore indicators and recorded in the LIMS.

· If the autoclave does not reach the specified temperature or fails the spore indicator test, service the autoclave and re-sterilize all glassware and reagents that were insufficiently sterilized.

For the two medium-sized autoclaves, general maintenance is as follows:

· The autoclaves are operated using deionized water.

· At the end of the day, autoclaves are drained. Twice a month, autoclaves are cleaned with mild soap, rinsed with water, and drained. The condensate holding tank is drained daily or as needed. The cleaning date is recorded in the LIMS.

· Twice a year, have a contractor inspect and calibrate the autoclaves and perform preventive maintenance. Preventive maintenance dates are recorded in the LIMS.

· Twice a year, clean the chambers with 10% muriatic acid and flush well with water. Cleaning dates are recorded in the LIMS.

For the large autoclave, general maintenance is as follows:

· Once a month, clean chamber with water and liquinox. Cleaning dates are recorded in the LIMS.

· Twice a year, have a contractor perform preventive maintenance and inspection, clean and service the generator, clean the door gasket and head ring, apply graphite to the door gasket, oil the door hinge pins, and lubricate the door hub. Preventive maintenance dates are recorded in the LIMS.

· Twice a year, clean the chamber with 10% muriatic acid and flush well with water. Cleaning dates are recorded in the LIMS.

Laboratory water

The OWML has three types of laboratory water:

(1) Type III deionized water (“deionized water”) produced from City of Columbus tap water for general laboratory use. The deionized water unit and tap are stored in the warehouse. The system is described in Francy and others (1998). The vendor changes the cation and anion columns, moves forward the standby mixed-bed column, installs a new standby tank, and changes the carbon filter when the red service light illuminates. Maintenance checks are recorded in the LIMS.

(2) Reagent-grade water produced using a Millipore MilliQ system (“MilliQ water”). Deionized water is used as source water for the MilliQ system. Reagent water is used for cultivation media and additives (mTEC, MI, mEI, antibiotic stocks, and others) as well as for preparation of reagents for sensitive procedures (elutions, PCR, hybridization, and others). The MilliQ cartridges are changed by OWML laboratory personnel when the service light blinks and the display message reads “EXCH. CARTRIDGES.” Indicate the date of cartridge change in the LIMS.

(3) Deionized water is stored in a laboratory carboy (“stored water”) and used for rinsing of dishware and other supplies.

A variety of quality-control checks are routinely done on the three types of water and may differ depending on the type of water. Acceptance criteria are listed in table 2. For deionized water, two levels of acceptance criteria are listed—(1) a warning level wherein the system is inspected and constituents are retested and (2) a shut-down level. For MilliQ water, only a shut-down level is listed in table 2. For stored water, if criteria are not met, the container is cleaned out, refilled, and retested.

· Quarterly checks of specific conductance and turbidity are done on all three types of water and recorded in the LIMS. Instructions for performing this check are in the back of the equipment QA/QC logbook.

· Quarterly checks of bacterial growth are done on the MilliQ water and recorded in the LIMS. Instructions for performing this check are in the back of the equipment QA/QC logbook.

· A blank of deionized water is submitted to the National Water Quality Laboratory (NWQL) annually and analyzed for low level nutrients (Schedule 1217), and total-organic carbon (Labcode 114), and the results are recorded in the LIMS. We no longer analyze a blank for trace elements and low-level major ions because the need for these low-level analyses is project specific.

· The stored deionized water carboy is to be emptied completely and cleaned with Liquinox and water every other week. Record cleanings in the LIMS.

Table 2. Acceptance criteria for laboratory water quality-assurance checks

[Adopted from USEPA (1978), APHA (1998), and ASTM (1999); NA is not applicable; constituents highlighted in gray are no longer required tests]

	DEIONIZED		MILLIQ	STORED
ACTION	warning	shut down	shut down	clean and refill
Specific conductance (ms/cm)	3	5	2	3
Turbidity	1	5	1	1
Heterotrophic plate count (colonies/mL)	NA	NA	<1	NA
Total organic carbon (mg/L)	0.2	10	NA	NA
Sodium (mg/L)	0.1	1	NA	NA
Nutrients individual (mg/L)	0.1	1	NA	NA
Heavy metals, individual (Cd, Cr, Cu, Ni, Pb, Zn) (mg/L)	1	10	NA	NA
Other trace elements (mg/L)	3	50	NA	NA

Analytical balances

Analytical balances are used for accurate weighing of reagents and media. They are checked and calibrated annually by the manufacturer’s service technician, and the results are recorded in the LIMS. Balances must rest on a firm, level surface. Balance trays are wiped off after each use with water or a surface disinfectant, such as Conflikt or 70 percent ethanol.

Hoods

The Ohio Water Center has four types of hoods— (1) two biosafety cabinets, (2) a laminar-flow hood, (3) a hazardous-waste fume hood, and (4) a PCR workstation. To ensure proper use of hoods 1, 2, and 3, “quick check” criteria for properly running each hood are posted on the hoods.

· The operation of all hoods are checked and certified by a qualified inspector annually and recorded in the LIMS.

The biosafety and laminar flow hoods have magnehelic pressure gauges (MAG) that are used to monitor operation of the hoods. When using these hoods, check to make sure the pressure gauge is reading at a level approximately equal to the annually recorded MAG level on the calibration sticker. A significant increase in pressure indicates that the filters are dirty whereas a significant decrease in pressure indicates an electrical problem.

The biosafety cabinets, laminar-flow hood, and PCR workstation (Hoods 1, 2, and 4) must be free from contamination by live organisms.

· The working surfaces of the laminar-flow hood, the biosafety cabinets, and the PCR workstation (Hoods 1, 2, and 4) are wiped down with a surface disinfectant, such as Conflikt or 70 percent ethanol before and after general use. When working with pathogenic organisms, the hoods are wiped down with Dispatch Hospital Cleaner Disinfectant towels with bleach (Caltech Industries Inc., Midland, MI) followed with 70 percent ethanol. For the biosafety cabinets, be sure to lift up the work surface and clean under this area periodically.

· The biosafety cabinets and PCR workstation (Hoods 1 and 4) have ultraviolet bulbs for germicidal purposes. The ultraviolet lights in the biosafety cabinets and PCR workstation are cleaned quarterly by wiping the bulbs with a soft cloth. Cleaning dates are recorded in the LIMS. A bulb that is dull in the center needs to be replaced. Record the bulb change in the LIMS.

· Biannually, nonselective agar plates are exposed to airflow in the laminar-flow hood, the biosafety cabinets, and PCR workstation for 1 hour (Hoods 1, 2, and 4). The plates are incubated at 35^oC for 24 hours and examined for contamination. The results are record in the LIMS.

The hazardous-waste fume hood (Hood 3) must be checked to ensure that it is operating properly.

· Check the operation of the hazardous-waste fume hood (Hood 3) quarterly by use of fume cartridges and record results in the LIMS.

Specific conductance, pH, and turbidity meters

With each use of the specific conductance, pH, or turbidity meter, calibrate the instrument according to the manufacturer’s instructions (kept with the meter). Use a calibrated solution that is within the range of the water sample to be measured. Label specific conductance and pH buffer solutions with the date opened and discard working solution weekly. Each piece of equipment has a daily logbook; record all calibrations in the appropriate logbook.

Micropipettors

Micropipettors are used for the accurate delivery of small volumes.

· Pipettors are sent to the manufacturer annually for cleaning, preventative maintenance, calibration, and adjustment, if necessary. Preventive maintenance dates are recorded in the LIMS. Preventative maintenance includes a new seal and piston cleaning annually, and a new shaft and reconditioned piston every 3 years.

Vacuum pump

The vacuum pump is mainly used for membrane filtration. The oil is changed in the pump every 2 years. Record the oil change in the LIMS.

Thermometers, incubators, water baths, refrigerators, and freezers

Thermometers are kept in three areas and are inventoried according to storage and use: (1) extra thermometers for general laboratory use, including the National Institute of Standards and Technology (NIST) thermometer (2) daily-use water-bath, incubator, and refrigerator thermometers, (3) digital thermometers, and (4) back-lab thermometers.

· The NIST thermometer is calibrated and certified annually by an outside service technician. Certification dates are recorded in the LIMS.

Quality Assurance/Quality Control Manual

Purpose and scope

Organizational structure

Sample management and documentation

Actinomycetes

A. OWML Service request form

G. Modified mTEC agar preparation

H. mEI agar preparation

S. Filtration and elution of samples by USEPA Method 1623 – ultrafiltration method

T. Detection of Actinomycetes in water

Quality Assurance/Quality Control Manual

Ohio Water Microbiology Laboratory

ABSTRACT

INTRODUCTION

Purpose and Scope

Organizational Structure

GENERAL LABORATORY QUALITY ASSURANCE/QUALITY- CONTROL PRACTICES

Analytical methods

Training

Safety

Laboratory materials and equipment

General sterility and cleanliness

Autoclaves

Laboratory water

Analytical balances

Hoods

Specific conductance, pH, and turbidity meters

Thermometers, incubators, water baths, refrigerators, and freezers