[35 Pa.B. 519]
[Continued from previous Web Page] § 252.403. Essential quality control requirements--toxicity testing.
(a) In addition to the requirements of § 252.401 (relating to basic requirements), an environmental laboratory that measures the toxicity or bioaccumulation of contaminants, including testing of effluents, receiving waters, sediments, elutriates, leachates and soils shall comply with this section.
(b) When the method selected by an environmental laboratory in accordance with § 252.307 (relating to methodology) contains more stringent requirements than the requirements of this section, the environmental laboratory shall follow the more stringent requirements contained in the method.
(c) An environmental laboratory that measures toxicity or bioaccumulation of contaminants shall comply with guidance issued by the Department regarding counting of neonates, algae cells and weighing of fish for selected endpoints.
(d) Negative control requirements are as follows:
(1) In addition to the negative controls specified by the method, permit or regulation, additional negative controls shall be included when sample adjustments (for example, pH adjustments or dechlorination) or solvent carriers are used in the test.
(2) The results of the negative controls shall be compared to the acceptance criteria published in the method. When there are no established acceptance criteria for the negative control in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(3) The test acceptability criteria for negative controls as specified in the method must be achieved for both the reference toxicant and the environmental sample toxicity test.
(e) The requirements for reference toxicants are as follows:
(1) The environmental laboratory shall demonstrate the ability to obtain consistent results with reference toxicants before performing toxicity tests on environmental samples.
(i) Intralaboratory precision shall be determined by performing a minimum of five acceptable reference toxicant tests for each method and species using different batches of organisms and negative controls (water, sediment or soil) before performing testing or analysis on environmental samples.
(ii) An environmental laboratory shall maintain control charts for the control performance and reference toxicant statistical endpoint (such as NOEC or ECp) and shall evaluate the intralaboratory variability with a specific reference toxicant for each method.
(iii) The results of the toxicant test shall be compared to the acceptance criteria published in the method. When there are no established acceptance criteria for the toxicant test in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(2) The following minimum frequency of reference toxicant testing shall be met:
(i) Each batch of test organisms obtained from an outside source, field collection or from laboratory spawning of field-collected species not amenable to routine laboratory culture shall be evaluated with a reference toxicant test of the same type as the environmental toxicity test within 7 days preceding the test or concurrently with the test.
(ii) Test organisms obtained from in-house laboratory cultures shall be tested with reference toxicant tests at least once each month for each method.
(iii) If a species produced by in-house laboratory cultures is used less than once per month, a reference toxicant test of the same type shall be performed with each environmental toxicity test.
(iv) When methods and species commonly used in the laboratory are only tested on a seasonal basis, reference toxicant tests shall be conducted each month the method is in use.
(3) Ongoing environmental laboratory performance shall be documented by maintaining laboratory quality control charts that meet the following requirements:
(i) For endpoints that are point estimates (ICp, ECp), control charts shall be constructed by plotting the cumulative geometric mean and the limits that consist of the upper and lower 95% confidence limits (+ 2 standard deviations).
(ii) For endpoints from hypothesis tests (NOEC, NOAEC), control charts shall be constructed by plotting the values directly and the control limits shall consist of one concentration interval above and below the concentration representing central tendency or the mode.
(iii) After 20 data points are collected for a method and species, the control charts shall be maintained by using only the most recent 20 data points.
(iv) Test results that fall outside of control chart limits at a frequency of 5% or less shall be retested and confirmed before reporting and all results shall be documented in the report of the testing and analysis.
(v) The endpoint shall be compared to the acceptance criteria published in the method.
(vi) When there are no established acceptance criteria for the endpoint in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(vii) If the reference toxicant fails to meet acceptance criteria, the results of environmental toxicity tests conducted during the affected period shall be examined for defects and the test repeated using a different batch of organisms or the results shall be reported with appropriate data qualifiers.
(4) Reference toxicant tests conducted for a method and species must use the same reference toxicant, test concentrations, dilution water and data analysis method as the environmental toxicity tests for which the precision is being evaluated unless otherwise specified in the method.
(5) The test duration, dilution or control water, feeding, organism age, age range and density, test volumes, renewal frequency, water quality measurements, number of test concentrations, replicates and organisms per replicate must be the same as the environmental toxicity test. A dilution factor of greater than 0.5 shall be used for both acute and chronic tests.
(f) Sensitivity requirements are as follows:
(1) If the Dunnett's procedure or hypothesis test (NOEC, NOAEC) is used, the statistical minimum significant difference (SMSD) by species shall be calculated according to the formula specified by the method and reported with the test results. The SMSD must be estimated for nonnormal distribution or heterogeneous variances, or both.
(2) Confidence intervals for point estimates (LCp, ICp or ECp) shall be reported as a measure of the precision around the point estimate value.
(g) When required, the data shall be plotted in the form of a curve relating the dose of the chemical or concentration of sample to cumulative percentage of test organisms demonstrating a response, such as death.
(h) At least once every 30 days, an environmental laboratory shall verify and document that the reagent grade water meets the following criteria:
(1) Conductivity must be less than 0.1 µmhos/cm or resistance greater than 17 megohms at 25°C.
(2) pH must be between 5.5 to 7.5.
(3) Total residual chlorine must be nondetectable.
(i) Reagent water used for culturing and testing shall be analyzed for toxic metals and organics whenever the minimum acceptability criteria for control survival, growth or reproduction are not met and no other cause can be identified.
(j) An environmental laboratory shall demonstrate that any analyte at a measured concentration or the reported detection limit does not exceed one tenth the expected chronic value for the most sensitive species tested or cultured.
(k) Air used for aeration of test solutions, dilution waters and cultures must be free of oil and fumes.
(l) The requirements for test organisms are as follows:
(1) An environmental laboratory shall positively identify test organisms to species on an annual basis. The taxonomic reference (citation and pages) and the names of the taxonomic experts shall be documented. When organisms are obtained from an outside source, an environmental laboratory shall obtain the information from the supplier.
(i) Organisms used for a test must be from the same source. When available, certified seeds shall be used for soil tests.
(ii) Organisms used in tests or as brood stock to produce neonate test organisms must appear healthy, show no signs of stress or disease and exhibit survival of greater than 90% during the 24-hour period immediately preceding use in tests.
(iii) An environmental laboratory shall document the health and culturing conditions of all organisms used for testing. The documentation shall include culture conditions and observations of any stress, disease or mortality.
(iv) When organisms are obtained from an outside source, the laboratory shall obtain written documentation of the water quality parameters and biological observations for each lot of organisms received.
(v) An environmental laboratory shall record the water quality parameters and the biological observations when the organisms arrive at the environmental laboratory.
(vi) Supporting information such as hatch dates and times, times of brood releases and metrics (for example, chironomid head capsule width) shall be documented.
(vii) Organisms obtained from an outside source may not be from different batches.
(viii) The control population of Ceriodaphnia in chronic effluent or receiving water tests may not contain more than 10% males.
(ix) Test soils and sediments must be within the geochemical tolerance range of the test organism.
(2) The requirements for feeding of test organisms are as follows:
(i) For each new batch of laboratory-prepared food or lot of commercial food used by the environmental laboratory, the performance of organisms fed with the new food shall be compared with the performance of organisms fed with a food of known quality. The suitability of food used for culturing shall be determined using a measure that evaluates the effect of food quality on survival and growth or reproduction of each of the relevant test species.
(ii) Foods used only in chronic toxicity tests shall be evaluated using the reference toxicant employed in the environmental laboratory quality assurance program, and shall be compared with results of previous tests using a food of known quality.
(iii) In the case of algae, rotifers or other cultured foods, which are collected as a continuous batch, the quality of the food shall be assessed as described in subparagraphs (i) and (ii) each time new nutrient stocks are prepared, a new starter culture is employed or when a significant change in culture conditions occurs.
(iv) The environmental laboratory shall have written procedures for the statistical evaluation of food acceptability.
(v) Food used to culture organisms used in bioaccumulation tests shall be analyzed for the compounds to be measured in the bioaccumulation tests.
(m) Equipment requirements are as follows:
(1) If closed refrigerator-sized incubators are used, culturing and testing of organisms shall be separated to avoid loss of cultures due to cross-contamination.
(2) Temperature control equipment must be adequate to maintain the required test temperature. The average daily temperature of the test solutions shall be maintained within 1°C of the selected test temperature for the duration of the test. Temperature measurements shall be made at least once per 24-hour period. The test temperature for continuous-flow toxicity tests shall be monitored and recorded continuously.
(3) The test chambers used in a test must be identical.
(4) Materials used for test chambers and any material coming in contact with test samples, solutions, control water, sediment, soil or food must be nontoxic and cleaned according to the method. Materials may not add to nor reduce sample toxicity.
(5) Light intensity shall be maintained as specified in the method. Measurements shall be made and recorded at least once per 12 months.
(6) The photoperiod shall be maintained as specified in the method and be documented at least once every 90 days.
(7) For algal and plant tests, the light intensity shall be measured and recorded at the start of each test.
(n) The requirements for sample holding times and conditions are as follows:
(1) The sample holding time may not exceed 36 hours.
(2) The last use of the sample in renewal tests may not exceed 72 hours unless specifically approved by the Department.
(3) Samples shall be chilled to 4°C during or immediately after collection and held at that temperature until time of analysis.
(o) Chronic tests must have a minimum of four replicates per treatment.
(p) The requirements for testing conditions are as follows:
(1) Dissolved oxygen and pH in aquatic tests must be within acceptable ranges published in the method. When there are no established acceptance criteria in the method, the environmental laboratory shall establish internal criteria and document the method used to establish the acceptance limits.
(2) During aquatic chronic testing, dissolved oxygen and pH shall be measured daily in at least one replicate of each concentration.
(3) In static-renewal tests, dissolved oxygen shall be measured at both the beginning and end of each 24-hour exposure period.
(4) The pH shall be measured at the end of each exposure period after organism transfer.
(5) Minimal aeration may be provided to tests only if acceptable dissolved oxygen concentrations cannot be otherwise maintained or if specified by the method.
§ 252.404. Essential quality control requirement--microbiology.
(a) In addition to the requirements of § 252.401 (relating to basic requirements), environmental laboratories performing testing or analysis in the area of microbiology shall comply with this section.
(b) When the method selected by an environmental laboratory in accordance with § 252.307 (relating to methodology) contains more stringent requirements than the requirements of this section, the environmental laboratory shall follow the more stringent requirements contained in the method.
(c) The following pieces of equipment shall be maintained according to this subsection:
(1) Autoclave.
(i) An environmental laboratory shall use autoclaves that meet specified temperature tolerances of the method. Pressure cookers may not be used.
(ii) Prior to first use, an environmental laboratory shall evaluate and document the performance of an autoclave by establishing its functional properties and performance (for example, heat distribution characteristics with respect to typical uses).
(iii) A continuous temperature-recording device or a maximum-temperature-registering thermometer shall be used during each autoclave cycle.
(iv) An environmental laboratory shall verify the sterilization capability of each autoclave by utilizing appropriate biological indicators (for example, spore strips or ampoules) once a month. Records of biological indicator tests shall be maintained in a laboratory notebook and include the autoclave identification, date, incubation time and temperature, results and initials of the responsible individual.
(v) An environmental laboratory shall verify the mechanical timing device, if used, for each autoclave every 3 months. Records of mechanical timer verification shall be maintained in a laboratory notebook and include the autoclave identification, date, mechanical timing device time, actual time and initials of the responsible individual. Correction factors shall be documented and used.
(vi) Autoclaves shall be properly cleaned and maintained. A qualified person shall service autoclaves at least once per year. Servicing must include a pressure check and calibration of temperature devices. Records of annual service shall be maintained and the service date shall be recorded on the autoclave.
(vii) Required times for autoclaving items at 121°C are set forth in this subparagraph. The following items must be at temperature for the required amount of time. Except for membrane filters and pads and carbohydrate-containing media, indicated times are minimum times and may necessitate adjustment depending upon volumes, containers and loads. For autoclave runs that include membrane filters and pads and media, the total cycle time may not exceed 45 minutes. Autoclaved membrane filters and pads and media shall be removed immediately after completion of the autoclave cycle.
(A) Membrane filters & pads 10 minutes (B) Carbohydrate-containing media 12-15 minutes (C) Contaminated test materials 30 minutes (D) Membrane filtration units 15 minutes (E) Sample containers 15 minutes (F) Individual glassware 15 minutes (G) Dilution water 15 minutes (H) Rinse water 15-30 minutes (viii) Records of each autoclave run shall be maintained in a laboratory notebook and include the date, contents, sterilization time and temperature, total cycle time (recorded as time in and time out) and initials of the responsible individual.
(ix) If an autoclave cycle fails to meet any requirement, corrective action shall be documented. Media may not be reautoclaved.
(2) Hot air oven.
(i) Prior to first use, an environmental laboratory shall evaluate the performance of each hot air oven by establishing its functional properties and performance (for example, heat distribution characteristics with respect to typical uses).
(ii) An environmental laboratory shall maintain a thermometer, graduated in 10°C increments or less with the bulb placed in sand, in each hot air oven.
(iii) An environmental laboratory shall verify the sterilization capability of each hot air oven by utilizing appropriate biological indicators (for example, spore strips) once a month. Records of biological indicator tests shall be maintained in a laboratory notebook and include the hot air oven identification, date, incubation time and temperature, results and initials of the responsible individual.
(iv) An environmental laboratory shall sterilize items in a hot air oven maintaining a temperature of 170-180°C for a minimum of 2 hours. Only dry items may be sterilized in a hot air oven.
(v) Records of each hot air oven operation shall be maintained and include the date, contents, sterilization time and temperature, and initials of the responsible individual.
(3) Optical counting equipment.
(i) An environmental laboratory shall use appropriate optical counting equipment to view and enumerate colonies.
(ii) A dark field colony counter shall be used to count heterotrophic plate count colonies.
(iii) A 10X to 15X stereomicroscope with a fluorescent light source shall be used to count sheen colonies.
(4) Inoculating equipment.
(i) An environmental laboratory shall use appropriate sterile inoculating equipment.
(ii) Metal loops and needles must be made of nickel alloy or platinum.
(iii) Wooden applicator sticks must be sterilized using dry heat.
(iv) For oxidase tests, nickel alloy loops may not be used.
(5) Membrane filtration equipment.
(i) Membrane filtration funnels must be stainless steel, glass or autoclaveable plastic. Membrane filtration funnels may not be scratched or corroded and may not leak.
(ii) Membrane filtration units shall be autoclaved before the beginning of a filtration series. A filtration series ends when 30 minutes or longer elapses after a sample is filtered.
(iii) Forceps must be blunt and smooth-tipped without corrugations on the inner sides of tips.
(iv) Membrane filters must meet the following requirements:
(A) Membrane filters must be made of cellulose ester, white, grid marked, 47 mm diameter and 0.45-µm pore size unless otherwise specified by the method.
(B) Membrane filters must be either purchased presterilized or autoclaved for ten minutes at 121°C before use. Membrane filters may not be brittle or distorted.
(C) Membrane filters must be approved (based upon manufacturer data from tests for toxicity, recovery, retention and absence of growth-promoting substances) for the specified analysis for which they are to be used.
(v) Records of membrane filters shall be maintained and include the type, lot number, date received and date opened. The manufacturer's specification/certification sheet shall be retained for each lot of membrane filters.
(vi) An environmental laboratory using an ultraviolet sanitation lamp to sanitize filtration funnels between successive filtrations shall test the ultraviolet sanitation lamp every 3 months for effectiveness with an appropriate UV light meter or by plate count agar spread plates. Records of ultraviolet lamp tests shall be maintained and bulbs shall be replaced if output is less than 70% of original for light tests or if count reduction is less than 99% for a plate containing 200 to 300 organisms.
(6) Culture dishes.
(i) Culture dishes must be presterilized plastic or sterilizable glass and of appropriate size for the method.
(ii) Stainless steel canisters, aluminum canisters or a wrap of heavy aluminum foil or char-resistant paper, shall be used for autoclave sterilization of glass culture dishes.
(iii) Loose-lid culture dishes shall be incubated in a tight fitting container containing a moistened paper towel.
(iv) Opened packs of disposable culture dishes shall be resealed between use periods.
(7) Culture tubes and closures. Culture tubes and containers must be of sufficient size to contain medium and sample without being more than three quarters full. Tube closures must be stainless steel, aluminum, plastic or a screw cap with a nontoxic liner.
(8) Pipettes.
(i) Pipettes must have legible markings and may not be chipped or etched.
(ii) Stainless steel canisters, aluminum canisters or a wrap of heavy aluminum foil or char-resistant paper shall be used for autoclave sterilization of pipettes.
(iii) Opened packs of disposable sterile pipettes shall be resealed between use periods.
(9) Sample containers.
(i) Sample containers must be sterile plastic bags or wide-mouth plastic or noncorrosive glass bottles with nonleaking ground glass stoppers or caps with nontoxic liners that can withstand repeated sterilization. Sample containers must be capable of holding sufficient volume of sample for all required tests while maintaining adequate air space for mixing.
(ii) Glass stoppers must be covered with aluminum foil or char-resistant paper for sterilization.
(iii) Glass and plastic bottles that have not been presterilized shall be sterilized by autoclaving. Glass bottles may be sterilized by dry heat. Empty containers shall be moistened with several drops of water prior to autoclaving.
(10) Plastic and glassware washing procedure.
(i) Prior to the initial use of a lot of detergent or washing procedure, an environmental laboratory shall perform an inhibitory residue test utilizing the method described in the currently approved editions of Standard Methods for the Examination of Water and Wastewater (available from American Public Health Association, 1015 Fifteenth Street NW, Washington, D.C. 20005). Records of inhibitory residue tests shall be maintained and include the detergent identification, date, calculations, results and initials of responsible individual.
(ii) Washed plastic and glassware shall be tested at least once each month for possible acid or alkaline residue by testing at least one piece of plastic and glassware with a suitable pH indicator such as 0.04% bromothymol blue. Records of pH tests shall be maintained.
(11) Ultraviolet lamp. An environmental laboratory shall use a 365-nm, 6-watt ultraviolet lamp in a darkened room to view sample fluorescence.
(12) Quanti-TrayTM Sealer.
(i) An environmental laboratory shall perform a sealer check on each Quanti-Tray Sealer once a month by adding a dye to a water sample and performing the sealing procedure.
(ii) Records of the sealer check shall be maintained and include the sealer identification, date, results and initials of responsible individual. If dye is observed outside the wells, the Quanti-Tray Sealer may not be used.
(d) The requirements for reagent water areas as follows:
(1) An environmental laboratory shall use reagent water in the preparation of media, solutions and buffers.
(2) An environmental laboratory shall demonstrate that reagent water meets the following criteria on a monthly basis or whenever maintenance is performed on the water treatment system or at startup after a period of nonuse longer than 1 month:
(i) Total chlorine residual must be less than 0.1 mg/L.
(ii) Conductivity must be less than 2.0 µmhos/cm or resistance greater than 0.5 megohms at 25°C.
(iii) Heterotrophic plate count must be less than 500 CFU/mL.
(3) An environmental laboratory shall demonstrate that reagent water meets the following criteria every 12 months:
(i) The individual concentration of lead, cadmium, chromium, copper, nickel and zinc must be less than 0.05 mg/L.
(ii) The total concentration of lead, cadmium, chromium, copper, nickel and zinc must be less than 0.1 mg/L.
(iii) Except as provided in subsection (c)(6), the bacteriological water quality test ratio must be between 0.8 and 3.0. The bacteriological water quality test shall be performed according to the currently approved editions of Standard Methods for the Examination of Water and Wastewater (available from American Public Health Association, 1015 Fifteenth Street NW, Washington, D.C. 20005).
(4) The monthly and annual reagent water analyses may only be performed by an environmental laboratory accredited under this chapter for the field of accreditation that includes the analyte.
(5) Results of the monthly and annual reagent water analysis shall be maintained and include the date, type of test, results and initials of responsible individual. Reagent water that does not meet the required criteria may not be used.
(6) The bacteriological water quality test need not be performed if the environmental laboratory can supply documentation to show that their laboratory pure water or reagent water meets the criteria, as specified in the currently approved editions of Standard Methods for the Examination of Water and Wastewater (available from American Public Health Association, 1015 Fifteenth Street NW, Washington, D.C. 20005), for Type I or Type II reagent water.
(e) The requirements for dilution/rinse water are as follows:
(1) Stock buffer solution or peptone water shall be prepared as specified in the currently approved editions of Standard Methods for the Examination of Water and Wastewater (available from American Public Health Association, 1015 Fifteenth Street NW, Washington, D.C. 20005).
(2) Stock buffers shall be autoclaved or filter-sterilized. Stock buffer containers shall be labeled and dated. Stock buffers shall be refrigerated. Stored stock buffers must be free from turbidity.
(3) Dilution/rinse water solutions shall be prepared as specified in the currently approved editions of Standard Methods for the Examination of Water and Wastewater (available from American Public Health Association, 1015 Fifteenth Street NW, Washington, D.C. 20005).
(4) Records of stock buffers and dilution/rinse water preparation shall be maintained and include the date prepared, lot number or laboratory identification of solutions used, amounts measured, final pH and initials of the responsible individual.
(f) The requirements for media are as follows:
(1) An environmental laboratory shall use dehydrated or commercially manufactured prepared media. Dehydrated media shall be stored in a cool, dry location. Caked or discolored dehydrated media shall be discarded.
(2) An environmental laboratory that uses commercially prepared media shall maintain records on each lot received that includes the date received, type of media, lot number and pH verification. Media may not be used after the manufacturer's expiration date.
(3) An environmental laboratory that prepares media from dehydrated stock shall follow method specifications and maintain records of each batch that includes the date of preparation, type of media, lot number, amounts measured, sterilization time and temperature, final pH and initials of the responsible individual.
(4) Media may not be reautoclaved.
(5) After sterilization, prepared media shall be stored and maintained as follows:
(i) Stored away from sources of direct light.
(ii) Prepared plates shall be stored in sealed plastic bags or containers.
(iii) Each bag, container or rack of broth or agar media shall be labeled with the date prepared or expiration date.
(iv) Liquid media stored in a refrigerator shall be incubated overnight at room temperature before use. Media that shows growth or bubbles may not be used.
(v) Prepared liquid media shall be discarded if evaporation exceeds 10% of the original volume.
(vi) Poured agar plates and broth in tubes, bottles or flasks with loose-fitting closures shall be discarded if not used within 2 weeks of sterilization unless otherwise specified by the method.
(vii) Broth in tightly closed screw-cap tubes, bottles or flasks shall be discarded if not used within 3 months of sterilization unless otherwise specified by the method.
(g) An environmental laboratory shall demonstrate that the filtration equipment and filters, sample containers, media and reagents have not been contaminated through improper handling or preparation, inadequate sterilization or environmental exposure as follows:
(1) A sterility blank shall be analyzed for each lot of pre-prepared, ready-to-use medium and for each batch of medium prepared in the laboratory prior to first use of the medium. Records shall be maintained and include media identification, date, results and initials of responsible individual. If sterility blank indicates contamination, the media may not be used.
(2) For each membrane filtration unit used during a filtration series, the laboratory shall prepare at least one sterility blank at the beginning, after every ten samples, and at the end of the series and record the results. If the membrane filtration unit sterility blank indicates contamination, the data from affected samples shall be invalidated and an immediate resampling requested. When a filtration series is interrupted for more than 30 minutes, the filtration funnels shall be resterilized.
(3) For pour plate technique, sterility blanks of the medium shall be made by pouring at least one uninoculated plate for each lot of pre-prepared, ready-to-use media and for each batch of medium prepared in the laboratory. Results shall be recorded. If the sterility check indicates contamination, the data from affected samples shall be invalidated.
(4) Sterility checks on sample containers shall be performed on at least one container for each lot of purchased, presterilized containers with an appropriate nonselective growth media. For containers prepared and sterilized in the laboratory, a sterility check shall be performed on one container per sterilized batch with an appropriate nonselective growth media. Results shall be maintained and include sample container identification, date, results and initials of responsible individual. If sample container sterility check indicates contamination, the affected sample container may not be used.
(5) A sterility blank shall be performed on each batch of dilution/rinse water prepared in the laboratory and on each batch of pre-prepared, ready-to-use dilution water with an appropriate non-selective growth media. The concentration of media shall be single strength after addition of dilution water. Results shall be maintained and include dilution/rinse water identification, date, results, and initials of the responsible individual. If dilution/rinse water sterility check indicates contamination, the affected dilution water may not be used.
(6) At least one filter from each new lot of membrane filters shall be checked for sterility with an appropriate nonselective growth media. Results shall be maintained and include membrane filter identification, date, results and initials of the responsible individual. If the membrane filter sterility check indicates contamination, the affected membrane filters may not be used.
(h) The requirements for positive and negative culture control checks are as follows:
(1) Each pre-prepared, ready-to-use lot of medium and each batch of medium prepared in the laboratory shall be tested with at least one pure culture of a known positive reaction prior to first use of the medium. Records shall be maintained and include the date, media lot or batch number, type of media, positive culture control organism identification, results and initials of responsible individual. If positive culture control checks do not meet expected results, the affected media may not be used.
(2) Each pre-prepared, ready-to-use lot of selective medium and each batch of selective medium prepared in the laboratory shall be tested with at least one pure culture of a known negative reaction prior to first use of the medium. Records shall be maintained and include the date, media lot or batch number, type of media, negative culture control organism identification, results and initials of the responsible individual. If negative culture control checks do not meet expected results, the affected media may not be used.
(3) An environmental laboratory shall use stock positive and negative culture controls that are known and traceable to a recognized National collection. Documentation of traceability shall be maintained.
(4) Stock positive and negative culture controls shall be discarded upon the manufacturer's expiration date unless it is shown through appropriate biochemical and purity tests that the stock culture control has not been contaminated or altered.
(i) The requirements for test variability/reproducibility are as follows:
(1) For test methods that specify colony counts, duplicate counts shall be performed monthly on one positive sample for each month that the test is performed. If the laboratory has two or more analysts, each analyst shall count typical colonies on the same plate. Counts may not differ by more than 10%. In an environmental laboratory with only one analyst, the analyst shall count the same plate twice. Counts may not differ by more than 5%.
(2) If the protocol for a method does not require a positive culture control during sample analysis, the environmental laboratory shall analyze a positive culture control organism through the entire method on a monthly basis.
(3) If the method determines organism density, a control sample shall be prepared from stock culture to contain 20 to 80 viable organisms per the usual volume analyzed. The positive control shall then be processed through all steps of the method and the density of the positive control determined and recorded.
(4) If the environmental laboratory is using a method for detecting as opposed to counting organisms, a control sample may be inoculated by transferring a portion of the sample from a positive stock culture to 100-mL of reagent or dilution water.
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