Standard Methods 22nd Edition

9000 Methods
By Legend Technical Services
Microbiology Quality Control
Jenny Cahoon
QA/QC Coordinator
Legend 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 Control
Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Liquid bearing thermometers • Liquid bearing thermometers

need to be calibrated annually
need to be calibrated semi- (9020B.4.a).
annually (9020B.3.a). ▫ Note: Preferably semi
annually.

• Water baths, ex. 44.5°C, use a • Water baths, ex. 44.5°C, use a
submersible thermometer to total immersion thermometer to
0.2°C or less (9020B.3.a). 0.1°C (9020B.4.a).

• Autoclave timer must be

• No timer check for autoclave checked quarterly with a
(9020B.3.h). stopwatch (9020B.4.h).
9020-Quality Assurance/Quality Control
Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Maintain refrigerator • Maintain refrigerator

temperature from 1-4°C temperature from 2-8°C
(9020B.3.i). (9020B.4.i).
▫ EPA Drinking Water
Manual states that the
temperature needs to be 1-
5°C

• Monthly plate count for

Biological Safety Cabinet • This was removed.
(9020B.3.m).
 9020-Quality Assurance/Quality
Control
TABLE 9020:I. KEY QUALITY CONTROL PRACTICES TABLE 9020:I. KEY QUALITY CONTROL PRACTICES
Further
Item Action Frequency Further Information in Section 9020B, ¶ Information in
Section 9020B,
Item Action Frequency ¶
Reagent water Monitor quality See Table 9020:II Reagent water Monitor quality See Table 9020:II
Bench surface Monitor for contamination Weekly 2e Air in workplace
Temperature devices:
Monitor bacterial density Monthly 3e
4a
Air in workplace Monitor bacterial density Monthly 2e Working units Check accuracy Annually
Reference units Recertify Every 5 years
Thermometers Check accuracy Semiannually 3a Balances Check zero Each use 4b
Balances and weights Check accuracy Monthly 3b Check accuracy
Service and recalibrate
Monthly/each use preferably
Annually
Balances Service and recalibrate Annually 3b Weights:
Working Check with reference weights Annually
4b

pH meter Standardize Each use 3c Reference Recertify Every 5 years

pH meter Standardize Each use 4c
Check against another meter Monthly 3c Determine slope Monthly
Media-dispensing apparatus Check volume dispense accuracy Each use 4f
Media-dispensing apparatus Check volume accuracy Each use 3f Hot-air sterilizing oven Check performance Monthly 4g
Hot-air oven Check performance Monthly 3g Autoclave Check temperature with max-registering thermometer
Check performance with bioindicator
Weekly
Monthly
4h

Autoclave Check performance Monthly 3h, 4i2) Timer:

Autoclave Check timing with stopwatch Quarterly 4h
Refrigerator Check temperature Daily 3i Stopwatch Check against National Time Signal Annually
Refrigerator Check temperature Daily 4i
Freezer Check temperature Daily 3j Freezer Check temperature Daily 4j
Defrost Semiannually 3j Membrane filtration equipment
Defrost
Check for leaks and surface scratches
Annually
Each use 4k
Membrane filtration equipment Check for leaks and surface scratches Each use 3k Check sterility
100-mL volume check
Pre- and post-test
Initially
UV lamps Test with UV meter Quarterly 3l UV lamps (shortwave) Test with UV meter or perform plate count check Quarterly 4l
Biosafety cabinet Inspect for airflow Each use 4m
Biohazard hood Monitor air and UV lamps Monthly 3m Have certified Annually
Incubator Check temperature Twice daily 4n and o
Inspect for airflow Quarterly 3m Microscope Clean optics and stage, check alignment Each use 4p
Incubator Check temperature Twice daily 3n and o Conductivity meter
Micropipettors
Calibrate
Check dispense accuracy and precision
Monthly
Quarterly or more frequently if
4q
4s
Microscope Clean optics and stage Each use 3p heavily used
Calibrate Annually
Glassware Inspect for cleanliness, chips, and etching Each use 4a Glassware Inspect for cleanliness, chips, and etching Each use 5a
Check pH with bromthymol blue Each wash batch
Check pH Each batch 4a1) Conduct inhibitory residue test Initial use and new washing procedure
Conduct inhibitory residue test Annually 4a2) Check for autofluorescence if used for testing
(also may be annual)
Each batch or lot
Dilution water bottles Check pH and volume Each use 4c Dilution water bottles
Sample bottles
Check sterility, pH, and volume
Check sterility
Each batch or lot
Each batch or lot
5c and 9050C.1a
5d
Media Check pH and appearance Each use 4i1) Check dechlorination agent efficacy Each batch or lot
Check 100 mL line Each lot
Plate counts Perform duplicate analyses Weekly 8a4) Check for autofluorescence if also used for testing Each lot
Multi-well sealer Check performance Monthly 5e
Repeat counts Monthly 8a2) Membrane filters Check sterility and properties Each new lot 5i
Media Check sterility, pH and appearance Each batch or lot 5j
Check recovery of new vs. old media Before first use
Check performance with + and - culture controls Each batch or lot
Plate counts Perform duplicate analyses Monthly 9a
Repeat counts Monthly
 9020-Quality Assurance/Quality
Control
TABLE 9020:II. QUALITY OF REAGENT WATER USED IN MICROBIOLOGY TESTING
Test Monitoring Frequency Maximum Acceptable Limit

Chemical tests:
Conductivity Continuously or with each use >0.5 megohms resistance or <2 1-mhos/cm at 25°C
pH With each use 5.5–7.5
Total organic carbon Monthly <1.0 mg/L
Heavy metals, single (Cd, Cr, Cu, Ni, Pb, and Zn) Annually* <0.05 mg/L
Heavy metals, total Annually* <0.10 mg/L
Ammonia/organic nitrogen Monthly <0.10 mg/L
Total chlorine residual Monthly or with each use <0.01 mg/L
Bacteriological tests:
Heterotrophic plate count (See Section 9215) Monthly < 1000 CFU/mL
Use test (see 4e) Quarterly and for a new source Student’s t < 2.78
*Or more frequently if there is a problem.

TABLE 9020:II. QUALITY OF REAGENT WATER USED IN MICROBIOLOGY TESTING

Test Monitoring Frequency Maximum Acceptable Limit

Chemical tests:
Conductivity Monthly* <2 f.Lmhos/cm (f.Lmsiemens/cm) at 25°C
Total organic carbon Monthly <1.0 mg/L
Heavy metals, single (Cd, Cr, Cu, Ni, Pb, and Zn) Annually† <0.05 mg/L
Heavy metals, total Annually† <0.10 mg/L
Total chlorine residual Monthly or with each use <0.1 mg/L
Bacteriological tests:
Heterotrophic plate count (See Section 9215) Monthly <500 CFU/mL
Use test [(see 9020B.5f2)] For a new source Student’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 Media
Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2005)

• Does not include the final pH • More clarification on the

and no expiration date was working solution for Buffered
included. 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-Samples
Standard Method 20th Edition (1997)
• Sample volume should be no
less than 100 mL (9060.A.4).
Standard Method 22nd Edition (2005)
• 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-Samples
Standard Methods 20th Edition (1997)
• 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.
Standard Methods 22nd Edition (2005)
• 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 Methods
Heather Hobeck
Microbiology Department
Manager
Legend Technical Services
Overview
• Good News
• Focus on Most Commonly Performed Methods
• Most Changes More Organizational and
Cosmetic than Substantive
9223 Enzyme Substrate Coliform Test
Standard 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 Group
Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• Wording: Fecal Coliforms • Wording: Thermotolerant

Coliforms
9222B Standard Total Coliform MF
Procedure
Standard Methods 20th Edition (1997)
• No specific warning
• No admonition regarding data
usefulness
Standard Methods 22nd Edition (2006)
• 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 • Calculations section updated

most results units as to read “CFU/100mL”.
“coliforms/100mL”. • More examples for different
• Fewer examples scenarios that might be
encountered
• Desired count per filter = 20 to • Desired count per filter is still
80 CFU 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 • The same scenario would be

portions were filtered and the reported as estimated 30
counts were 15, 5 and <1, cfu/100mL.
respectively, the count would
be reported as 25
coliforms/100mL.
9222D Thermotolerant (Fecal) Coliform
MF Procedure
Standard Methods 20th Edition (1997) Standard Methods 22nd Edition (2006)

• No mention of Klebsiella • 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

• Desired count per filter = 20 to
60 cfu
60 cfu. Calculation section
references TCMF section, so
the same change in strategy
discussed earlier applies
9215 Heterotrophic Plate Count
Standard Methods 20th Edition (1994)
• Applications section lists 3
methods and 4 media
• Methods: Pour Plate, Spread
Plate, Membrane Filtration
• Media: PCA/ SMA, mHPC
agar, R2A, NWRI (HPCA)
• Spread Plate Method: use of
plastic pipets was expressly
forbidden
Standard Methods 22nd Edition (2004)
• 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 Group
Standard Methods 20th Edition(1994) Standard Methods 22nd Edition (2006)

• Reference to “Fecal Coliforms” • Updated term to

• MPN Tables present 95%
confidence values as
Approximate
“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?