2003 NELAC StandardsMicrobiology Checklist: How to “Pass” your Assessment By Denise K. Williams Biological Scientist III Florida Department of Health Environmental Laboratory Certification Program FSEA Microbiology Workshop Rev. 2; 5-22-07
Do Your Homework • Use the Microbiology Checklist! • Read Appendix D.3 to NELAC Chapter 5 • Make sure previous deficiencies are still corrected (if still applicable) • Review original methods and your current Standard Operating Procedures [SOP] • Do internal audits of each method • Maintain Demonstrations of Capability [DOC]
Work Area • D.3.8 (a) – Are the laboratory floors and work surfaces where Microbiology testing takes place non-absorbent and easy to clean and disinfect? • D.3.8 (a) – Are microbiology work surfaces adequately sealed? • D.3.8 (a) – Are laboratory storagespaces for Microbiology testing sufficient, clean, and free from accumulation of dust? • D.3.8 (a) – Does the laboratory prohibit plants, food, and drink from the Microbiology work area?
Incubator/Water Bath Records • D.3.8(b)(6)(i) - Does the laboratory document temperatures of incubators & water baths twice daily, at least 4 hours apart, on each day of use? • Are your times documented to demonstrate that checks were performed 4 hours apart? • Temperature Units? (oC?)
Incubator/Water Bath Records • Do the laboratory's analytical records on strip charts, tabular printouts, computer data files, analytical notebooks, & run logs include the following essential information? • 126.96.36.199.5.3(f) - Analyst or operator initials/ signature…(Initials?) • 188.8.131.52.5.3(c) - Instrumentation identification & instrument operating conditions/parameters (or reference to such data)…(Equipment ID?) • 184.108.40.206(b) - Manufacturer's name, type identification, & serial number or other unique identification… (Unique ID?)
Incubator/Water Bath Records • 220.127.116.11.1(d) - Is the support equipment acceptability for use according to the needs of the analysis or the application for which the equipment is being used? • Are your incubation temperatures in the proper range per method requirements? • (Typically 35oC+/- 0.5 or 44.5oC+/- 0.2)
Incubator/Water Bath Records • If temperatures were out of range, did you: • (1) Qualify the data? NELAC 18.104.22.168.1 • (2) Take corrective action (and document that action)? NELAC 22.214.171.124 • (3) Result of corrective action: when did temperature become acceptable?
Incubator/Water Bath Records • D.3.8(b)(6)(i) - Has the laboratory established the stability, uniformity of temperature distribution, & time to re-establish thermal equilibrium conditions (after test sample additions) in incubators & water baths? • Two parts: (1) Stability/Uniformity (2) Equilibrium • One time study (can you find your records?)
Refrigerator • 126.96.36.199.1(d) - Temperatures checked once per day? • Equipment ID? Analyst Initials? Temperature Units? • Standard Methods for the Examination of Water and Wastewater [SM], 20th Edition specifies: 1-4oC • Out of range? Need corrective action (Clean vents? Too close to wall?)
Thermometers • 188.8.131.52.1(d) - Is the following support equipment associated with microbiological testing checked with NIST traceable materials (where available)? • Includes Refrigerator(s) for sample storage and/or media storage; Water Baths; Incubators • Traceability: Do you have the NIST certificates??
Thermometers • D.3.8(b)(1) - Is each temperature measuring device (e.g., liquid-in-glass thermometers, thermocouples, platinum resistance thermometers) calibrated at least annually to national or international standards for temperature? • SM 20th Ed.: Requires semi-annual calibration for microbiology
Thermometers • D.3.8(b)(1) - Are the available temperature monitoring devices that are used in incubators, autoclaves, refrigerators, or other equipment where temperature accuracy has a direct effect on the Microbiological analysis of appropriate quality to achieve specifications in the test method (e.g., no separations in liquid column for liquid-in-glass thermometers)? • LOOK at your thermometers
Thermometers • D.3.8(b)(1) - Is the scale of graduations for each temperature measuring device appropriate for the required accuracy of measurement? • LOOK at your thermometers: • 35.0oC- need at least 0.5 units • 44.5oC- need at least 0.1 units
Thermometers • Recording temperatures is dependent on the graduations of the thermometer. • For example, if your thermometer is in 0.5 increments, temperature recordings must either end in “NN.0” or “NN.5”.
Microorganisms • D.3.7(a) - Does the laboratory use reference cultures of microorganisms for positive & negative controls obtained from a recognized national collection, organization, or manufacturer recognized by the NELAP Accrediting Authority? • Typically ATCC • Do you have your certificates? • Do you use these organisms for your media QC? • Are your reference cultures not expired? • Traceability of use?
Microorganisms • D.3.7(a) - Note: Microorganisms can be single-use preparations or cultures maintained by documented procedures that demonstrate continued purity & viability of the organism. • Documented: Do you have a written procedure? • Do you have a subculture record (traceability)? • [184.108.40.206.1 - Does the laboratory have standard operating procedures that accurately reflect all phases of current laboratory activities?]
Microorganisms • D.3.7(a) - Note: Microorganisms can be single-use preparations or cultures maintained by documented procedures that demonstrate continued purity & viability of the organism. • Purity: How do you know there is only a single type organism? • Gram stain? Streak for isolated colony? API? • Viability: How do you know the organism is still alive after storage under your specified conditions? • How do you know your negative control culture is viable?
Microorganisms • D.3.7(a)(1) and (2): • Are reference cultures of microorganisms revived (if freeze-dried) or transferred from slants & subcultured only once to provide reference stocks? • Are reference stocks preserved by a technique that maintains the desired characteristics of the strain? • Are the working stocks of microorganisms for routine work prepared from the reference stocks? • Are reference stocks that have been thawed not re-frozen & re-used? • Are microorganism working stocks not sequentially subcultured more than 5 times? • Are working stocks of microorganisms not subcultured to replace reference stocks?
Autoclave • D.3.8(b)(2)(i) - Has the laboratory evaluated the functional properties & performance (e.g., heat distribution characteristics) for each autoclave with respect to typical uses? • One time study- can you locate your records? • Can be done with biological indicators or maximum registering thermometer.
Autoclave • D.3.8(b)(2)(i) - Is the autoclave capable of meeting specified temperature tolerances? • Check manufacturers specifications. • [SM 20th Ed. media: 121-124oC]
Autoclave • D.3.8(b)(2)(ii) - Does the laboratory demonstrate sterilization temperature by using a continuous temperature recording device or maximum registering thermometer with each cycle? • D.3.8(b)(2)(iv) - Does the laboratory perform autoclave maintenance annually (either internally or by service contract) which includes a pressure check & calibration of the temperature device? [SM requires semi-annual calibration of temperature devices.]
Autoclave • D.3.8(b)(2)(iv) - Does the laboratory perform autoclave maintenance annually (either internally or by service contract) which includes a pressure check & calibration of the temperature device? • Pressure check: Is there a leak around the seal/gasket of the autoclave? [Is the pressure 15-20 psi during the sterilization portion of the autoclave cycle? Check manufacturer’s specifications.]
Autoclave • D.3.8(b)(2)(iii) - Does the laboratory record the  date,  contents,  maximum temperature reached,  pressure,  time in sterilization mode,  total run time (may be documented as time in & time out), and  analyst’s initials for every cycle of autoclave operations? • Equipment Identification?
Autoclave • D.3.8(b)(2)(v) - Does the laboratory check the autoclave mechanical timing device quarterly against a stopwatch and document the actual elapsed time? • Includes “automatic” autoclaves with continuous recorders. • D.3.8(b)(2)(ii) - Does the laboratory use temperature sensitive tape with the contents of each autoclave run to indicate that the autoclave contents have been processed? -Tape only indicates that materials have been inside an autoclave, not that autoclave conditions have been met. • D.3.8(b)(2)(i) -Are pressure cookers not used for sterilization of growth media?
Autoclave • D.3.8(b)(2)(ii) - Does the laboratory use appropriate biological indicators once per month [if in use] to determine effective sterilization? -Biological indicator: contains thermophilic spore-forming organism, typically Bacillusstearothermophilus -Have you ever processed a non-autoclaved control? (See NELAC 220.127.116.11) -Do you keep records? Optimally, these records would include the incubator ID, incubation period, traceability to lot of spores used, retention of spore certificates, analyst initials? -Do you incubate at the proper temperature? Typically, 55-60oC (see manufacturer’s instructions).
UV Sterilization • D.3.8(b)(4) - If used for sanitation, are UV instruments tested quarterly for effectiveness with an appropriate UV light meter or by plate count agar spread plates? • Note: UV bulbs must be replaced if output is less than 70% of the original for light tests or if count reduction is less than 99% for a plate containing 200-300 organisms.
Ovens for Sterilization • D.3.8(b)(6)(ii) - Are ovens used for sterilization checked for sterilization effectiveness monthly with appropriate biological indicators? • D.3.8(b)(6)(ii) - Does the laboratory maintain records of each sterilization cycle for the oven that include date, cycle time, temperature, contents, & analyst’s initials? • Equipment ID?
Washing • D.3.8(b)(7)(i) - Does the laboratory have a documented [written] procedure for washing labware? • D.3.8(b)(7)(i) - Does the laboratory use detergents [Alconox, Liquinox] designed for laboratory use for washing labware? • D.3.8(b)(7)(ii) - Is the laboratory’s glassware used for Microbiological analysis made of borosilicate or other non-corrosive material, free of chips & cracks, and have readable measurement marks?
Washing- IRT • IRT- Inhibitory Residue Test • D.3.8(b)(7)(iii) - Is labware that is washed & reused tested for possible presence of residues which may inhibit or promote growth of microorganisms by performing the Inhibitory Residue Test annually? • D.3.8(b)(7)(iii) - Does the laboratory perform the Inhibitory Residue Test each time it changes the lot of detergent or washing procedures? • Either current (annual) certificate or annual test is OK. • Procedure is in SM 20th Ed, 9020 B, 4(a)(2).
Washing- pH test • D.3.8(b)(7)(iv) - Does the laboratory test washed labware at least once daily, each day of washing, for possible acid or alkaline residues by testing at least one piece of labware with a suitable pH indicator such as bromothymol blue? • Note: Records of these tests must be maintained.
Sample Containers- Sterility • D.3.1(a)(4) - Does the laboratory perform sample container sterility checks on at least one container for each lot of purchased, pre-sterilized containers, or on one container per sterilized batch for containers prepared & sterilized in the laboratory, with nonselective growth media [such as TSB]. -Includes sample bottles, sample bags, Quantitray. -Did you record the lot number?
Sample Containers- Volume Non-disposable: • D.3.8(b)(3)(ii) - Does the laboratory calibrate volumetric equipment such as filter funnels, bottles, non-Class A glassware, & other marked containers once per lot prior to first use? Disposable: • D.3.8(b)(3)(iii) - Does the laboratory check the volume of disposable volumetric equipment such as sample bottles, disposable pipettes, & [micropipette tips-DELETE] once per lot? • Did you record the batch number (sterilization date) or lot number?
Sample Containers-Chlorine • Requirement depends on laboratory’s procedures for sample receipt but checks are typically required. • 18.104.22.168.1(a)(2) -Has the laboratory checked samples for proper preservation (e.g. pH, absence of free chlorine, temperature) prior to or during sample preparation or analysis? • [Note: These checks are not required for chlorinated water systems as long as: The laboratory must have records showing that Chlorine was measured in the field & the actual concentration is documented; AND ] • The laboratory must check one sample container from each commercial lot or prepared batch (for adequate Na2S2O3), to prove that 5 mg/L Chlorine in Drinking Water & 15 mg/L Chlorine in Non-Potable Water can be neutralized.
Sample Receipt- Chlorine • 22.214.171.124.1(a)(2) - Has the laboratory checked samples for proper preservation (e.g., pH, absence of free chlorine, temperature) prior to or during sample preparation or analysis? Note: These checks are not required for chlorinated water systems as long as : Sufficient Na2S2O3 was added to each sample container to dechlorinate at least 5 mg/L Chlorine in Drinking Water samples & at least 15 mg/L Chlorine in Non-Potable Water samples. -How do you demonstrate this?
Sample Receipt- Chlorine • (1) The laboratory must have records showing that Chlorine was measured in the field & the actual concentration is documented; AND • (2) The laboratory must check one sample container from each commercial lot or prepared batch (for adequate Na2S2O3), to prove that 5 mg/L Chlorine in Drinking Water & 15 mg/L Chlorine in Non-Potable Water can be neutralized.
Sample Receipt- Chlorine • If no field chlorine level is documented for the sample, the laboratory must check the sample for chlorine. • The laboratory may check each sample for chlorine and not check each lot/batch of sample containers for adequate Na2S2O3. • Even sources “known” not to contain chlorine must have chlorine checks.
Sample Receipt- Temperature • 126.96.36.199.1(a)(2) - Has the laboratory checked samples for proper preservation (e.g. pH, absence of free chlorine, temperature) prior to or during sample preparation or analysis? • DEP-SOP-001/01, Table FS 1000-4: “However, even if ice is present when the samples arrive, it is necessary to immediately measure the temperature of the samples…” (document actual temperature) • Lab must also document if sample was received on ice or not on ice.
Sample Receipt- Temperature • DEP-SOP-001/01, Table FS 1000-8 : • Drinking Water (Total coliforms, fecal coliforms, E. coli, HPC): < 10oC • DEP-SOP-001/01, Table FS 1000-4: • Non-Potable Water (Total coliforms, fecal coliforms): 4oC • NEW: 40 CFR Part 136, Table II: </= 10oC
Sample Receipt- Temperature • How do I take sample temperature? • Temperature of ice water bath in cooler must not be used for sample temperature. • Can take temperature of each sample, representative sample, or “dummy” sample per cooler. • Lab needs to have a procedure! • If using non-invasive temperature measuring device (IR gun), have you calibrated it?
Sample Receipt- Temperature Acceptability • 188.8.131.52.1(a)(1) - For samples that require thermal preservation, does the laboratory consider acceptable only those samples where the arrival temperature is within 2oC of the required temperature or method-specified range OR is within 0-6oC (where the specified temperature is 4oC). • Note: For samples hand-delivered to the laboratory on the same day [calendar date] that they are collected, samples are considered acceptable if there is evidence that the chilling process has begun (e.g., arrival on ice).
Sample Receipt- Holding Times • DEP-SOP-001/01, Table FS 1000-8: • Drinking Water (Total coliforms, fecal coliforms, E. coli): 30 hrs; (HPC): 8 hrs • DEP-SOP-001/01, Table FS 1000-4: • Non-Potable Water (Total and fecal coliforms): 6 hrs • NEW: 40 CFR Part 136, Table II: 6 hrs transport with 2 hours to begin analysis after receipt • Hold time: Time between collection and analysis • Is the sample time and date documented? • Is the analysis time and date documented? • Do you reject the sample or qualify the data if holding times are exceeded (procedure should be in corrective action/contingency plan)?
Sample Receipt- pH • 184.108.40.206.1(a)(2) - Has the laboratory checked samples for proper preservation (e.g., pH, absence of free chlorine, temperature) prior to or during sample preparation or analysis? • pH not typically required for microbiology samples (virus method?)
Volumetric Equipment • D.3.8(b)(3)(i) - Does the laboratory calibrate volumetric equipment with movable parts, such as automatic dispensers, dispensers/diluters, & mechanical hand pipettes quarterly? • D.3.8(b)(3)(ii) - Does the laboratory calibrate [non-disposable] volumetric equipment such as filter funnels, bottles, non-Class A glassware, & other marked containers once per lot prior to first use? • D.3.8(b)(3)(iii) - Does the laboratory check the volume of disposable volumetric equipment such as sample bottles, disposable pipettes, [& micropipette tips- DELETE] once per lot?
Environmental Conditions • NELAC 220.127.116.11 – The laboratory does not provide for effective monitoring, control, and recording of appropriate environmental conditions (such as biological sterility, dust, electromagnetic interference, humidity, mains voltage, temperature, and sound and vibration levels). • Note: SM 9020 B, 2(e) requires monthly air monitoring to not exceed 15 colonies/plate/15 minutes.
Colony counts- Reproducibility • D.3.2 - If the test method specifies colony counts (e.g., membrane filtration, HPC), the laboratory does not verify the ability of individual analysts to count colonies at least once per month by having two or more analysts count colonies from the same plate. • Note: Counts must be within 10% between multiple analysts to be acceptable. • Note: An analyst in a 1-person laboratory may do repetitive counting on the same plate, with no more than 5% difference between the counts.
Sterility Checks- Misc. • D.3.1(a) - Does the laboratory demonstrate that filtration equipment & filters, sample containers, media, & reagents have not been contaminated through improper handling or preparation, inadequate sterilization, or environmental exposure? [Petri dishes? Blender? Pipettes? Quantitray?] • D.3.1(a)(4) - Does the laboratory perform sample container sterility checks on at least one container for each lot of purchased, pre-sterilized containers, or on one container per sterilized batch for containers prepared & sterilized in the laboratory, with nonselective growth media? • Non-selective growth media: typically TSB
Sterility Checks- Misc. • D.3.1(a)(5) - Does the laboratory perform a sterility blank on each batch of dilution water prepared in the laboratory, & on each batch of prepared, ready-to-use dilution water, with nonselective growth media? (may use double strength TSB) • D.3.1(a)(6) - Does the laboratory check at least one filter from each new lot of membrane filters for sterility with non-selective growth media?
Sterility Checks- Membrane Filtration Method • D.3.1 (a)(2) – Is one beginning and one ending sterility check conducted for each laboratory sterilized unit used in a filtration series?; Is a sterility check conducted once per lot for pre-sterilized single-use funnels? Note: The filtration series may include single or multiple filtration units that have been sterilized prior to beginning the series. • D.3.1 (a)(2)– Is the membrane filtration series ended when more than 30 minutes elapses between successive filtrations? • D.3.1 (a)(2) – Is a sterility blank analyzed every 10 samples (unless filtration units are sanitized by UV light after each filtration)? • Note: During a filtration series filter funnels must be rinsed with three 20-30 ml portions of sterile rinse water after each sample filtration.
Sterility Checks- Media • D.3.1(a)(1) - Is a sterility blank analyzed for each lot of pre-prepared, ready-to-use medium & for each batch of medium prepared in the laboratory? Note: This blank must be analyzed prior to first use of the medium. • Don’t forget to check TSB itself. • Don’t dilute other media with TSB. • D.3.1(a)(3) - For pour-plate technique does the laboratory make a sterility blank of the medium by pouring at least one uninoculated plate for each lot of prepared, ready-to-use media & for each batch of medium prepared in the laboratory? • Don’t forget HPC, PCA, etc.
Media QC TESTS: • (1) Sterility • (2) pH • (3) + Control • (4) – Control (if applicable) • Buy ready to use: test per lot • Lab must check even if certificate was supplied. • Make in lab: test per batch