§ 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) A continuous temperature-recording device or a maximum-temperature-registering thermometer shall be used during each autoclave cycle.
(iii) 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.
(iv) 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.
(v) Autoclaves shall be properly cleaned and maintained. Copies of service contracts or internal maintenance protocols and maintenance records shall be kept.
(vi) 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 and 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 (vii) 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.
(viii) If an autoclave cycle fails to meet any requirement, corrective action shall be documented. Media may not be reautoclaved.
(2) Hot air oven.
(i) 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.
(ii) 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.
(iii) 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.
(iv) 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) 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.
(4) Membrane filtration equipment.
(i) Membrane filtration funnels must be stainless steel, glass, porcelain or autoclaveable or presterilized plastic. Membrane filtration funnels may not be scratched or corroded and may not leak.
(ii) Membrane filtration units shall be sterilized 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 10 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) 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.
(5) 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.
(6) 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.
(7) Pipettes.
(i) Pipettes must have legible markings and may not be chipped or etched and must be accurate to within 2.5% tolerance.
(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.
(8) 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.
(9) 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 the American Public Health Association, 800 I Street, NW, Washington, D.C. 20001). 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 and include the date, results and identification of the responsible individual.
(10) Ultraviolet lamp. An environmental laboratory shall use a 365-nm, 6-watt ultraviolet lamp in a darkened room to view sample fluorescence.
(11) 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 are 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 (d)(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 the American Public Health Association, 800 I Street, NW, Washington, D.C. 20001).
(4) The metals analyses may only be performed by an environmental laboratory accredited under this chapter for those fields of accreditation.
(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 section 1080 of the currently approved editions of Standard Methods for the Examination of Water and Wastewater (available from the American Public Health Association, 800 I Street, NW, Washington, D.C. 20001), for Type I (high-quality) or Type II (medium-quality) reagent water.
(7) The heterotrophic plate count and bacteriological water quality test ratio analyses described in paragraphs (2) and (3) shall be performed by an environmental laboratory accredited under this chapter for the appropriate field of accreditation.
(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 the American Public Health Association, 800 I Street, NW, Washington, D.C. 20001).
(2) Stock buffers shall be autoclaved or filter-sterilized. Stock buffers shall be refrigerated and 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 the American Public Health Association, 800 I Street, NW, Washington, D.C. 20001).
(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 prepares media from dehydrated stock shall follow method specifications.
(3) Media may not be reautoclaved.
(4) After preparation, 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) Fermentation media stored in a refrigerator shall be brought to room temperature before use. Media that shows growth or false positive results 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 preprepared, 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 and time of the start and end of incubation, results and initials of the responsible individuals. If sterility blank indicates contamination, the media may not be used.
(i) For chromogenic/fluorogenic media, add single-strength media to sterile reagent water and incubate at the appropriate temperature and time.
(ii) For all other media, incubate uninoculated, single-strength at the appropriate temperature and time.
(2) For each reusable membrane filtration unit used during a filtration series, the laboratory shall prepare at least one sterility blank at the beginning and at the end of the series. A series is considered ended when more than 30 minutes elapses between filtrations. The laboratory shall insert a sterility blank after every ten sample aliquots filtered through each membrane filtration unit or sanitize filtration units by UV light after each sample filtration in addition to the regular rinsing procedure. Records of sterility blank results shall be maintained in the same manner as the associated sample and include the date and time of the start and end of the incubation, results and initials of the responsible individuals. If sterility blanks indicate contamination, the laboratory must treat each affected sample according to program requirements.
(3) For presterilized single use filtration funnel units, a sterility check shall be performed on one funnel unit per lot.
(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 and time of the start and end of incubation, results and initials of responsible individuals. 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 preprepared, ready-to-use dilution water with an appropriate nonselective 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 and time of the start and end of incubation, results and initials of the responsible individuals. 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 and time of the start and end of incubation, results and initials of the responsible individuals. If the membrane filter sterility check indicates contamination, the affected membrane filters may not be used.
(7) Sterility checks on Quanti-TrayTM sample trays shall be performed on at least one sample tray for each lot of purchased presterilized sample trays with an appropriate nonselective growth media. Results shall be maintained and include sample tray identification, date and time of the start and end of incubation, results and initials of the responsible individuals. If the sample tray sterility check indicates contamination, the affected lot of sample trays may not be used.
(h) The requirements for positive and negative culture control checks are as follows:
(1) Each preprepared, ready-to-use lot of medium and each batch of medium prepared in the laboratory shall be tested by the laboratory 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 and time of the start and end of incubation, media lot or batch number, type of media, positive culture control organism identification, results and initials of the responsible individuals. If positive culture control checks do not meet expected results, the affected media may not be used.
(2) Each preprepared, ready-to-use lot of selective medium and each batch of selective medium prepared in the laboratory shall be tested by the laboratory 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 and time of the start and end of incubation, media lot or batch number, type of media, negative culture control organism identification, results and initials of the responsible individuals. 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 after the manufacturer’s expiration date.
(5) Culture controls may be single use or cultures maintained by the laboratory using a documented procedure that maintains the purity and viability of the organisms.
(6) For cultures maintained by the laboratory, the following criteria must be met:
(i) Reference control cultures may be revived and subcultured once to provide reference stocks.
(ii) Reference stocks shall be preserved using a method which maintains the characteristics of the organism strains. If reference stocks are thawed, they may not be refrozen and reused.
(iii) Working stocks shall be prepared from reference stocks for routine laboratory work.
(iv) If the laboratory sequentially cultures working stocks, the laboratory shall prepare a second working stock. Sequential culturing may not be performed from a working stock that has been used for routine laboratory work.
(v) Working stocks may not be used for more than 30 days.
(vi) Working stocks may not be sequentially cultured more than five times and may not be subcultured to replace reference stocks.
(vii) Secondary working stocks shall be used to prepare sequential working stocks.
(7) Positive and negative controls must be processed under the same conditions and using the same equipment as routine environmental samples, including all steps of the preparation and analytical procedure.
(i) 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%.
(j) Quality control checks, including sterility checks and positive and negative controls, shall be conducted after the laboratory receives the material or supply and before or during first use. These checks shall be performed by an environmental laboratory accredited under this chapter and utilizing the same supplies, reagents and media to be used during laboratory analysis of environmental samples. Certificates of analysis from a manufacturer may not be used to demonstrate compliance with the requirements of this subsection.
(k) Records of all equipment, reference materials, reagents, media and supplies shall be maintained in accordance with § 252.306 (relating to equipment, supplies and reference materials).
Authority The provisions of this § 252.404 amended under 27 Pa.C.S. § § 4103(a), 4104 and 4105; and section 1920-A of The Administrative Code of 1929 (71 P.S. § 510-20).
Source The provisions of this § 252.404 amended April 9, 2010, effective April 10, 2010, 40 Pa.B. 1898; amended July 28, 2017, effective July 29, 2017, 47 Pa.B. 4085. Immediately preceding text appears at serial pages (348826) to (348834).
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