By Donna S. Francy, Rebecca N.
Bushon, Erin E. Bertke, Amie M. G. Brady, Christopher M. Kephart, Christina A.
Likirdopulos, and Donald M. Stoeckel
Updated
November 2006
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
Micropipettors
Vacuum pump
Thermometers, incubators,
water baths, refrigerators, and freezers
Microscope and centrifuge
METHODS OF ANALYSIS, MEDIA AND
REAGENT PREPARATION, AND ANALYTICAL QUALITY-CONTROL PROCEDURES
Fecal-indicator bacteria
Enteric viruses
Coliphage
Cryptosporidium and Giardia
APPENDIXES
D.
Analysis of E. coli and total coliforms using
Colilert Quantitray
E.
Presence/absence
analysis of E. coli and total coliforms
using Colilert, large volume samples
F.
mTEC agar and
urea-phenol solution preparation
I.
Analysis of Clostridium perfringens in
environmental water samples
J.
Media
quality-control procedures
K.
Media and buffer
quality-control log sheet
L.
Expendable supplies
request form
N.
RT-PCR elution
protocol
N1. Inhibitor
removal protocol
N2. RT-PCR analysis
O.
Coliphage detection
by USEPA Method 1602: Single-Agar Layer (SAL)
O1. NWIS coding
for coliphage results
P.
QC for Method 1602:
Single-Agar Layer (SAL)
Q.
Coliphage detection
by USEPA Method 1601: Two-Step Enrichment
R.
QC for Method 1601:
Two-Step Enrichment
V. Master Cycler QC
instructions and form
TABLES
1. Current laboratory
personnel and qualifications
2. Acceptance criteria for laboratory water
quality-assurance checks
3. Acceptance criteria for laboratory
thermometers
4. Acceptance criteria for laboratory
refrigerators, freezers, incubators, and water baths
5. Methods for fecal-indicator bacteria analysis
used by the OWML
6. Information on media, buffered-dilution
water, and reagents prepared and stored in the OWML
The
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.
The
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.
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.
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 |
An overview of analytical methods, training policies,
safety, laboratory maintenance, sample management, and data documentation is
given in this section.
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
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.
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
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.
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).
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.,
·
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.
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 124oC
(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.
The OWML has three types of laboratory water:
(1) Type III deionized water (“deionized water”)
produced from City of
(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 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.
The
·
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 (
The biosafety
cabinets, laminar-flow hood, and
·
The working surfaces of the laminar-flow
hood, the biosafety cabinets, and the
·
The
biosafety cabinets and
·
Biannually, nonselective agar plates are
exposed to airflow in the laminar-flow hood, the biosafety cabinets, and
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.
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 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.