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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 storage spaces 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?
Refrigerator
5.5.5.2.1(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
Thermometers
5.5.5.2.1(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 singleuse 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)?
[5.5.4.1.1 - 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 singleuse 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 refrozen & 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 manufacturers specifications.]
Autoclave
D.3.8(b)(2)(iii) - Does the laboratory record the [1] date, [2] contents, [3] maximum temperature reached, [4] pressure, [5] time in sterilization mode, [6] total run time (may be documented as time in & time out), and [7] analysts 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 sporeforming organism, typically Bacillus stearothermophilus -Have you ever processed a non-autoclaved control? (See NELAC 5.4.6.2) -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 manufacturers 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.
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 laboratorys 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.
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 laboratorys procedures for sample receipt but checks are typically required. 5.5.8.3.1(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- pH
5.5.8.3.1(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 [nondisposable] 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 tipsDELETE] once per lot?
Environmental Conditions
NELAC 5.5.3.2 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.
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.
Media- pH
Does the pH measured meet method (or sometimes, manufacturer) specifications? Have you checked the pH of Colilert and other similar media? (Suspend in reagent water and test pH).
D.3.1(c) - Does the laboratory test each lot of prepared, ready-to-use medium & each batch of medium prepared in the laboratory with at least one or more known negative culture controls (non-target organisms) as appropriate to the method? [N/A for general purpose (non-selective) media such as TSB, HPC, PCA, etc.]
Media- General
D.3.6 - Does the laboratory ensure that the quality of reagents & media is appropriate for the test concerned? D.3.6(a) - Does the laboratory only use culture media from commercial dehydrated powders or purchased ready-to-use?
Media- SM Requirements
SM 20th Ed. 9020 B, 4 (i):
Store opened bottles in a desiccator.
Media Records
D.3.6(d) - Does the laboratory have records on media preparation in the laboratory that includes the (1) date of preparation, (2) preparer's initials, (3) type & (4) amount of media prepared, (5) manufacturer, & (6) lot number, (7) final pH of the media, & (8) expiration date [of the prepared media]? D.3.6(d) - Does the laboratorys documentation on media purchased pre-prepared, ready-to-use include (1) manufacturer, (2) lot number, (3) type & (4) amount of media received, (5) date of receipt, (6) pH of the media [even Colilert!], and (7) expiration date?
Media-Misc.
See checklist and method requirements. D.3.6(d) - Are the media, solutions, & reagents prepared, used, & stored according to a documented procedure that follows the manufacturer's instructions or the test method?
HPC/PCA- Sterile agar medium melted not more than once; Melted agar used within 3 hours, agar tempered at 44-46oC before pouring. Media preparation- Is media autoclaved or brought just to boiling point? Colilert, Colilert-18, etc.: Protect from light m-Endo Preparation: Ethanol used is NOT denatured
Media- Storage
Storage of prepared media (SM9020B, 3h4; EPA-600/878-017, Part IV-A, 7.9; & EPA 9131, 8.3.7): Membrane Filter broth 4C 96 hours Membrane Filter agar plates 4C 2 weeks Media (loose-fitting closures) 4C 2 weeks HPC plates (sealed in plastic bags) 4C 2 weeks Broth media (with screw caps) [4C] 3 months HPC agar (screw-cap container) 4C 3 months Refrigerated fermentation tube media incubated overnight prior to use; media indicating growth not used OR Fermentation tube media stored at 25oC used within 1 week, evaporative losses < 1 mL
Dilution Water
Laboratory Prepared:
Water quality records Preparation record [see 5.5.6.4(d)] Sterility check (double strength TSB) [see NELAC D.3.1(a)(5)] pH check [see D.3.6(d)]
Support Equipment
5.5.5.2.1(d) - Is the following support equipment associated with microbiological testing checked with NIST traceable materials (where available):
pH meter [retain buffer certificate?], Balance(s), Conductivity meter, Chlorine meter, Refrigerator(s), Water Baths, Incubators
D.3.8(b)(5) - Are conductivity meters, oxygen meters, pH meters, hygrometers, & other support equipment calibrated according to the method-specified requirements?
Water Quality
D.3.6(c) - Is the laboratory reagent water used in the preparation of media solutions & buffers free from bactericidal & inhibitory substances? D.3.6(c) - Is the laboratory reagent water tested monthly, when maintenance is performed on the water treatment system, or at start-up when the period of disuse exceeds one month, for [1] chlorine residual, [2] specific conductance, & [3] HPC? D.3.6(c) - Does the laboratory test its Microbiology reagent water annually for [1] toxic metals & [2] Bacteriological Water Quality? Note: The Bacteriological Water Quality Test [Ratio] is not required for laboratories that have documentation to show that their water source is Type I or Type II reagent water. (See SM 1080 C)
Water Quality
D.3.6(c) - Does the laboratory maintain records on all water quality checks (for 5 years) & meet the following criteria for acceptance (SM9020B, 4d & EPA-600/8-78017, Part IV-A, 5.2): **Pay attention to the required test intervals and the actual data!
Water Quality
pH 5.5-7.5 (measured each use) Residual Chlorine < 1.0 mg/L (monthly) Conductivity < 2.0 umho/cm at 25oC (with each use) Heterotrophic Plate Count < 1000 CFU/ mL (monthly) [Bacteriological ratio 0.8 3.0 (annually, EPA-600/8-78017 only)*] Cd, Cr, Cu, Ni, Pb, Zn each < 0.05 mg/L, collectively < 0.1 mg/L (annually) NH3, Organic Nitrogen < 0.1 mg/L (monthly check) TOC < 1 mg/L (monthly) Student's t < 2.78 for Use Test (quarterly & for new water source)
Reagent water lots stored at the laboratory >1 month must be tested at the required frequencies. Laboratories may obtain the same or different lots of water from the manufacturer on a monthly basis, along with the relevant, current certificates, to avoid having to perform additional tests other than pH and conductivity.
Demonstration of Capability
C.1 Does the laboratory document in its Quality Manual other adequate approaches to Demonstration of Capability if this procedure is not required by the mandated test method or regulation and if the laboratory elects not to perform this procedure?
If your method or regulation does not describe how your lab will perform the DOC, then the lab must define this procedure and place it into the QM. If there is no proficiency test available, how do you demonstrate capability?
Demonstration of Capability
NELAC 5.5.4.2.2 (d) Does the laboratory retain all associated supporting data necessary to reproduce the analytical results summarized in the appropriate certification statement? A logical place to link or have a copy of these records are in a personnel DOC file. NELAC 5.5.4.2.2 (e) and Appendix C.1 The laboratory does not complete a demonstration of capability each time there is a change in instrument type, personnel, or test method.
Method Requirements
Review actual method and its Quality Control, as well as any additional Quality Controls that may be a NELAC requirement. Review checklists for some method requirements. A checklist is a tool and guide only.
Membrane Filtrations
Do dilutions yield the appropriate number of colonies? Do you verify typical AND atypical colonies, if required by the method?
Microbiological Sludge
If your lab is analyzing microbiology sludge samplesthe lab must have certification in the Solid and Chemical Materials matrix.
DEP QA Rule 62-160.120 (16)(c), the Solid and Chemical Materials (SCM) matrix includes sludges; biosolids are solids.
DEP-SOP-001/01, Table FS 1000-9: preserve Cool 4oC; Holding time 24 hrs
The End