1. Common Findings in Microbiology
Jessica Hoch
TCEQ Laboratory Accreditation Assessor,
Senior Microbiologist
TCEQ Environmental Trade Fair and Conference
May 17, 2017

2. Overview References and Acronyms Presentation Format for Findings
Used throughout the presentation.
Presentation Format for Findings
7 Common Findings
Microbiological Method Modifications
Microbiological Nomenclature
Questions?!

3. References TNI SM EPA Volume 1 (2009) of the standards adopted by TNI
Standard Methods for the Examination of Water and Wastewater
22nd Edition
EPA
Title 40, Code of Federal Regulations, Part 141 (40 CFR 141)
40 CFR 136

4. Presentation Format Discussion for each finding will include:
Commonly Seen Deficiencies
References & Requirements
TNI SM
EPA
Important Points
Things to think about
Reasons why

5. Using Improper Procedures When Manipulating Sample Volumes.
Common Finding #1
Using Improper Procedures When Manipulating Sample Volumes.

6. Commonly Seen Deficiencies Manipulating Sample Volume
Not homogenizing / shaking a sample prior to:
Collecting an aliquot for a chlorine check.
Adjusting a volume for sample analysis.
Collecting an aliquot for dilution of a sample.
Collecting an aliquot for filtering a dilution of a sample.
Not having adequate detail in the SOP for homogenizing the sample.

7. References & Requirements Manipulating Sample Volume
CRITICAL Finding
Due to the nature of microorganisms tending to clump together and samples not being shaken, the assessor will judge this finding to be critical based on the potential for false negative results if improper procedures are observed.
V1M a
Not having adequate detail in the SOP to allow a similarly qualified analyst to reproduce the procedures used to generate the test result.

8. Important Points Manipulating Sample Volume
This finding requires client notification.
Data CRITICAL step.
All manipulations of a sample are critical steps due to the nature of microorganisms to clump together.
Microorganisms generally do not like to exist alone.
Total coliforms, including E. coli, are facultative anaerobes.
SOP needs to describe procedures at each point when samples should be shaken.

9. Using an Incorrect Volume for Presence/Absence Testing.
Common Finding #2
Using an Incorrect Volume for Presence/Absence Testing.

10. Commonly Seen Deficiencies Volume for P/A Testing
Analyzing 100 ± 2.5 mL sample volumes.
Using volume comparators for sample analysis.
Analyzing any volume varying from 100 mL.
Such as volumes greater than verified 100 mL line to below the neck of the bottle.

11. References & Requirements Volume for P/A Testing
40 CFR a.1
rTCR
Regardless of method used, sample volume is 100 mL.
SM 9223.B.2.b (2004)
Add enzyme medium to 100 mL sample.
Colilert®, Colilert®-18, and Colisure® Test Kit Instructions
Add contents of one pack to a 100 mL sample.

12. Important Points Volume for P/A Testing
This finding requires client notification.
Most stringent requirement applies.
40 CFR 141
All analyses are on 100 mL sample volume.
There is an allowable tolerance of 2.5% for the accuracy of the 100 mL line on sample analysis vessels.
This is related to the uncertainty of the line on the vessel.
It is not related to the sample volume analyzed.

13. Using Improper Procedures for Water Sterility Testing.
Common Finding #3
Using Improper Procedures for Water Sterility Testing.

14. Commonly Seen Deficiencies Water Sterility Testing
Using a selective media.
Such as Colilert®, Colilert®-18, Colisure®, etc.
Using single strength broth.
With procedures that create a significant dilution of the media in the water.
Placing water directly in the incubator.
Using a manufacturer’s Certificate of Analysis (CoA) which does not attest to having been analyzed with a non-selective growth media..
ISO Sterility Assurance Level (SAL).
Not testing for sterility prior to use.

15. References & Requirements Water Sterility Testing
TNI V1M b.iv
A sterility check shall be performed on each batch/lot with non-selective growth media (NSGM).
SM 9020 B.9.d (2005)
Check each new batch (or lot) before first use.
50 mL of water to 50 mL of a double-strength NSGM broth.
Alternatively, filter ≥100 mL and place filter on NSGM.

16. Important Points: Water Sterility Testing
Perform QC prior to use.
*Procedures need to create a single-strength broth.
Media is very specifically formulated with nutrient concentrations to encourage the growth of microorganisms present.
Too few nutrients make it difficult for any microorganisms to reproduce to a concentration that would result in turbidity, the visible growth in a broth.
Too many nutrients can overwhelm microorganisms and cause an inhibitory effect.
*Proposed 2017 TNI Standard requirement.

17. Not Recording Temperatures Every Day of Use.
Common Finding #4
Not Recording Temperatures Every Day of Use.

18. Commonly Seen Deficiencies Temperature Recording
Temperatures not recorded:
over weekends and holidays, when the equipment is “in use.”
for the freezer section of a refrigerator/freezer, when that section of the equipment is “in use.”
If anything pertaining to the analysis is kept in the piece of equipment, then that equipment is considered to be “in use.”
For example, storage of:
Media.
QC organisms.
Reagents.
QC Samples.
PT Samples.

19. References & Requirements Temperature Recording
TNI V1M d
On each day the equipment is used it shall be checked and documented.
SM 9020 B.4.i and B.4.j (2005)
Check and record temperature of refrigerator and freezer daily when in use.
TNI V1M
The laboratory shall retain records of original observations and derived data, with sufficient information to establish an audit trail.

20. Important Points Temperature Recording
Options for recording temperatures when laboratory is not staffed include:
Minimum/Maximum (min/max) recording thermometers.
Must have procedures for proper use.
Continuous recording data loggers.
Must ensure the original observations are not overwritten by the software.

21. Not Bracketing the Range of Use for Thermometer Calibrations.
Common Finding #5
Not Bracketing the Range of Use for Thermometer Calibrations.

22. Commonly Seen Deficiencies Thermometer Calibration
Single point calibration.
Thermometer calibrations that do not bracket the range of use.
Used in incubators, water baths, refrigerators, freezers, autoclaves, sample receipt, dry-block incubator, etc.

23. References & Requirements Thermometer Calibration
TNI V1M b
All support equipment shall be calibrated annually, bracketing the range of use.
SM 9020 B.4.a (2005)
Annually check the accuracy of working thermometers and determine the necessary calibration correction factor.
Record calibration-corrected temperature measurements.
TNI V1M b.ii
The laboratory shall maintain records of established correction factors.

24. Important Points Thermometer Calibration
BRACKET ALL THERMOMETER CALIBRATIONS.
Bracket the range of temperatures measured.
Ensure calibration certificates bracket range.
When bracketed calibrations give rise to differing correction factors (CF), the laboratory needs to have procedures on how to determine the CF.
Keep the bracketing reasonably narrow to avoid this issue.
Consider performing a third temperature calibration at the point of use.

25. Using Improper Procedures for Air Quality Control Checks.
Common Finding #6
Using Improper Procedures for Air Quality Control Checks.

26. Commonly Seen Deficiency Air Quality Checks
Using Simplate for monthly air quality control checks.
Must use a settling plate in which everything that lands on the plate is given an opportunity to grow.
Not performing the checks monthly.
Performed quarterly.
Not done at all.
Not performing the air quality checks in areas where testing occurs.

27. References & Requirements Air Quality Checks
SM 9020 B.3.e (2004)
At least monthly, monitor air quality in areas where aseptic work is conducted.
Use air density settling plates.
Results should be ≤15 colonies / plate / 15 minutes, on a standard Petri plate.

28. Important Points Air Quality Checks
This check is a passive sampling process, wherein particles can settle on the agar surface.
Simplate procedures include pouring off excess media into the plate’s absorbent cotton well.
These procedures create an opportunity for the “settled” microorganisms to be poured off.
Simplate result detection relies on microorganisms to be captured within the wells on the Simplate®.
Using Simplate® could skew air quality control results.

29. Using Incorrect Incubation Procedures for Colilert-18®.
Common Finding #7
Using Incorrect Incubation Procedures for Colilert-18®.

30. Commonly Seen Deficiencies Colilert-18® Incubation
Incubating longer than 10 minutes when using the °C warm-up option.
Not recording the time in and out of the 44.5 °C waterbath warm-up step.
Total incubation time starts with the warm-up step.

31. References & Requirements Colilert-18® Incubation
SM 9223 B.2.c (2004)
Incubate as specified in manufacturer’s instructions.
Colilert-18 Package Insert.
TNI V1M f
Lab shall keep records necessary for historical reconstruction of the data.

32. Important Points Colilert-18® Incubation
Warm-up step is initiated after the addition of media.
The warm-up step is the beginning of the incubation period.
44.5 °C for 7-10 minutes.
Records of incubation are necessary to reconstruct the history of the sample pre-warming steps.
Total coliforms are temperature sensitive.
Do not expose to high temperature for long periods.
Waterbath water levels should reach the level of the top of the sample.

33. Modification of Microbiological Methods

34. Method Modifications
Method modifications are not allowed for microbiological and biological methods.
This includes both drinking water and non-potable matrices.
EPA Alternative Test Procedures FAQ
questions-and-answers
40 CFR definition of a method-defined analyte
An analyte defined solely by the method used to determine the analyte.
Incubation time, temperature, duration, media used, sample handling, etc.

35. Proper Microbiological Nomenclature

36. Microbiology Nomenclature
Laboratory management should ensure the use of proper microbiological nomenclature in quality documentation.
Genus names are always capitalized.
Species names are always lower case.
Both genus and species should always be:
Italicized (Genus species) OR
Underlined (Genus species)

37. Microbiology Nomenclature
When referring to a microorganism in a document for the first time:
Both genus and species should be included in full.
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa.
Any following references to a microorganism in a document may be made in the form of:
An abbreviated genus with full species name.
E. coli
K. pneumoniae
P. aeruginosa.

38. Questions?!

39. TCEQ Laboratory Accreditation Program
Jessica Hoch, Senior Microbiologist
Ken Lancaster, Program Manager
General Accreditation Questions