Standard Methods 22 Edition 9000 Methods...Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005) • Samples should be transported to the laboratory at

Standard Methods 22nd Edition 9000 MethodsBy Legend Technical Services

Microbiology Quality ControlJenny CahoonQA/QC CoordinatorLegend Technical Services

9020-Quality Assurance/Quality Control• Most of the QC updates are already outlined in

the EPA Drinking Water Manual.• “The items listed in 9020 are not mandatory, but

represent practices that should be followed.”▫ Certain items that are removed may not

necessarily mean you have to stop doing them.• Always check with your other licensing agencies

as they may have more stringent requirements.

9020-Quality Assurance/Quality ControlStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Liquid bearing thermometers need to be calibrated semi-annually (9020B.3.a).

• Water baths, ex. 44.5°C, use a submersible thermometer to 0.2°C or less (9020B.3.a).

• No timer check for autoclave (9020B.3.h).

• Liquid bearing thermometers need to be calibrated annually (9020B.4.a).▫ Note: Preferably semi

annually.

• Water baths, ex. 44.5°C, use a total immersion thermometer to 0.1°C (9020B.4.a).

• Autoclave timer must be checked quarterly with a stopwatch (9020B.4.h).

9020-Quality Assurance/Quality ControlStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Maintain refrigerator temperature from 1-4°C (9020B.3.i).

• Monthly plate count for Biological Safety Cabinet (9020B.3.m).

• Maintain refrigerator temperature from 2-8°C (9020B.4.i).▫ EPA Drinking Water

Manual states that the temperature needs to be 1-5°C

• This was removed.

9020-Quality Assurance/Quality Control

TABLE 9020:I. KEY QUALITY CONTROL PRACTICES  

Item Action Frequency Further Information in Section 9020B, ¶  

Reagent water Monitor quality   See Table 9020:IIBench surface Monitor for contamination Weekly 2eAir in workplace Monitor bacterial density Monthly 2eThermometers Check accuracy Semiannually 3aBalances and weights Check accuracy Monthly 3bBalances Service and recalibrate Annually 3bpH meter Standardize Each use 3c

Check against another meter Monthly 3cMedia-dispensing apparatus Check volume accuracy Each use 3fHot-air oven Check performance Monthly 3gAutoclave Check performance Monthly 3h, 4i2)Refrigerator Check temperature Daily 3iFreezer Check temperature Daily 3j

Defrost Semiannually 3jMembrane filtration equipment Check for leaks and surface scratches Each use 3kUV lamps Test with UV meter Quarterly 3l Biohazard hood Monitor air and UV lamps Monthly 3m

Inspect for airflow Quarterly 3mIncubator Check temperature Twice daily 3n and oMicroscope Clean optics and stage Each use 3pGlassware Inspect for cleanliness, chips, and etching Each use 4a

Check pH Each batch 4a1)Conduct inhibitory residue test Annually 4a2)

Dilution water bottles Check pH and volume Each use 4cMedia Check pH and appearance Each use 4i1)Plate counts Perform duplicate analyses Weekly 8a4)  Repeat counts Monthly 8a2)

TABLE 9020:I. KEY QUALITY CONTROL PRACTICES     

Item Action Frequency  

Reagent water Monitor quality See Table 9020:II

 Further

Information in Section 9020B,

¶

Air in workplace Monitor bacterial density Monthly 3e Temperature devices: 4a

Working units Check accuracy Annually Reference units Recertify Every 5 years

Balances Check zero Each use 4b Check accuracy Monthly/each use preferably Service and recalibrate Annually

Weights: 4b Working Check with reference weights Annually Reference Recertify Every 5 years

pH meter Standardize Determine slope

Each use 4c Monthly

Media-dispensing apparatus Check volume dispense accuracy Each use 4f Hot-air sterilizing oven Check performance Monthly 4g Autoclave Check temperature with max-registering thermometer Weekly 4h

Check performance with bioindicator Monthly Timer:

Autoclave Check timing with stopwatch Quarterly 4h Stopwatch Check against National Time Signal Annually

Refrigerator Check temperature Daily 4i Freezer Check temperature Daily 4j

Defrost Annually Membrane filtration equipment Check for leaks and surface scratches Each use 4k

Check sterility 100-mL volume check

Pre- and post-test Initially

UV lamps (shortwave) Test with UV meter or perform plate count check Quarterly 4l Biosafety cabinet Inspect for airflow Each use 4m

Have certified Annually Incubator Check temperature Twice daily 4n and o Microscope Clean optics and stage, check alignment Each use 4p Conductivity meter Calibrate Monthly 4q Micropipettors Check dispense accuracy and precision Quarterly or more frequently if 4s

heavily used Calibrate Annually

Glassware Inspect for cleanliness, chips, and etching Each use 5a Check pH with bromthymol blue Each wash batch Conduct inhibitory residue test Initial use and new washing procedure

(also may be annual) Check for autofluorescence if used for testing Each batch or lot

Dilution water bottles Check sterility, pH, and volume Each batch or lot 5c and 9050C.1a Sample bottles Check sterility Each batch or lot 5d

Check dechlorination agent efficacy Each batch or lotCheck 100 mL line Each lotCheck for autofluorescence if also used for testing Each lot

Multi-well sealer Check performance Monthly 5eMembrane filters Check sterility and properties Each new lot 5iMedia Check sterility, pH and appearance Each batch or lot 5j

Check recovery of new vs. old media Before first use

Plate counts Check performance with + and - culture controlsPerform duplicate analyses

Each batch or lotMonthly 9a

Repeat counts Monthly

9020-Quality Assurance/Quality Control

Chemical tests:

TABLE 9020:II. QUALITY OF REAGENT WATER USED IN MICROBIOLOGY TESTING  Test Monitoring Frequency Maximum Acceptable Limit

Conductivity pH

Continuously or with each use With each use

>0.5 megohms resistance or <2 1-mhos/cm at 25°C5.5–7.5

Total organic carbon Heavy metals, single (Cd, Cr, Cu, Ni, Pb, and Zn) Heavy metals, total Ammonia/organic nitrogen Total chlorine residual

Monthly Annually* Annually* Monthly Monthly or with each use

<1.0 mg/L <0.05 mg/L <0.10 mg/L <0.10 mg/L <0.01 mg/L

Bacteriological tests: Heterotrophic plate count (See Section 9215)Use test (see 4e)

MonthlyQuarterly and for a new source

< 1000 CFU/mLStudent’s t < 2.78

*Or more frequently if there is a problem.

Chemical tests:

TABLE 9020:II. QUALITY OF REAGENT WATER USED IN MICROBIOLOGY TESTING  Test Monitoring Frequency Maximum Acceptable Limit

Conductivity Total organic carbon Heavy metals, single (Cd, Cr, Cu, Ni, Pb, and Zn) Heavy metals, total Total chlorine residual

Monthly* Monthly Annually† Annually† Monthly or with each use

<2 f.Lmhos/cm (f.Lmsiemens/cm) at 25°C<1.0 mg/L <0.05 mg/L <0.10 mg/L <0.1 mg/L

Bacteriological tests: Heterotrophic plate count (See Section 9215)Use test [(see 9020B.5f2)]

MonthlyFor a new source

<500 CFU/mLStudent’s t < 2.78

Water quality test [see 9020B.5f1)]‡ Annually 0.8–3.0 ratio

* Monthly, if meter is in-line or has a resistivity indicator light; otherwise with each new batch of reagent water. † Or more frequently if there is a problem. ‡ This bacteriological quality test is not needed for Type II water or better, as defined in Standard Methods (18th and 19th Editions), Section 1080C, or medium-quality water or better, as defined in Standard Methods (20th, 21st, and Online Editions), Section 1080C.

9050 Preparation of Culture MediaStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Does not include the final pH and no expiration date was included.

• More clarification on the working solution for Buffered Water (9050.C.1.a).

• Includes the final pH of 7.2 ±0.1

• Expiration date of 6 months.• Refrigerate after opening.• Includes a note that pH values

will change with time.

9060-SamplesStandard Method 20th Edition (1997) Standard Method 22nd Edition (2005)

• Sample volume should be no less than 100 mL (9060.A.4).

• For potable water samples collect a minimum of 100 ± 2.5 mL. Larger volumes may be needed for bacterial pathogen, protozoan and viral analysis (9060A.4).▫ Ex. Giardia,

Cryptosporidium and Enteric Virus

9060-SamplesStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Samples should be transported to the laboratory at <10°C (9060B.1).▫ Includes: Drinking water

and nonpotable water samples for compliance, and other water types for noncompliance purposes.

• Samples should be transported to the laboratory at <8°C.▫ Includes: Drinking water

and nonpotable water samples for compliance, and other water types for noncompliance purposes.

Questions?

Microbiology MethodsHeather HobeckMicrobiology Department ManagerLegend Technical Services

Overview• Good News• Focus on Most Commonly Performed Methods• Most Changes More Organizational and

Cosmetic than Substantive

9223 Enzyme Substrate Coliform TestStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2004)

• No method changes • No method changes• Remember that QA changes

may apply

9222 Membrane Filter Technique for Members of the Coliform GroupStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• Wording: Fecal Coliforms • Wording: ThermotolerantColiforms

9222B Standard Total Coliform MF ProcedureStandard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• No specific warning

• No admonition regarding data usefulness

• Addition of a warning regarding hazards associated with Basic Fuchsin

• Addition of verbiage stating that, “quantitation information only provides a gross estimation of the actual coliform population at collection time due to non-uniform distribution within the matrix”.

9222B Standard Total Coliform MF Procedure (continued)Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• Calculations section presented most results units as “coliforms/100mL”.

• Fewer examples

• Desired count per filter = 20 to 80 CFU

• Calculations section updated to read “CFU/100mL”.

• More examples for different scenarios that might be encountered

• Desired count per filter is still 20 to 80 CFU/filter, but one example changed how to deal with varying counts on a sample split over multiple filters

9222B Standard Total Coliform MF Procedure (continued)Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• If 50-, 25-, and 10-mL portions were filtered and the counts were 15, 5 and <1, respectively, the count would be reported as 25 coliforms/100mL.

• The same scenario would be reported as estimated 30 cfu/100mL.

9222D Thermotolerant (Fecal) Coliform MF Procedure Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• No mention of Klebsiella

• Desired count per filter = 20 to 60 cfu

• Addition of a discussion of thermotolerant Klebsiella and limitations of the data’s usefulness in evaluating thermal waters and other types.

• Desired count per filter = 20 to 60 cfu. Calculation section references TCMF section, so the same change in strategy discussed earlier applies

9215 Heterotrophic Plate CountStandard Methods 20th Edition (1994) Standard Methods 22nd Edition (2004)

• Applications section lists 3 methods and 4 media

• Methods: Pour Plate, Spread Plate, Membrane Filtration

• Media: PCA/ SMA, mHPCagar, R2A, NWRI (HPCA)

• Spread Plate Method: use of plastic pipets was expressly forbidden

• Applications section lists 4 methods and 5 media

• Methods: same as in the 20th

Edition, with the addition of an Enzyme Substrate Method

• Media: same as in the 20th

Edition, with the addition of an Enzyme Substrate Medium

• Removed prohibition against using plastic pipets

9221 Multiple-Tube Fermentation Technique for Members of the Coliform GroupStandard Methods 20th Edition(1994) Standard Methods 22nd Edition (2006)

• Reference to “Fecal Coliforms”

• MPN Tables present 95% confidence values as Approximate

• Updated term to “Thermotolerant Coliforms”

• MPN Tables present 95% confidence values as Exact

• Addition of methods for special cases (Proposed)

MPN Tables, 20th Edition vs. 22nd Edition (examples)

Questions?