Environmental Sampling and Experimental Design

1. Environmental Sampling and Experimental Design

2. Media Air Water Surfaces Food Organisms Viruses Bacteria Protozoans Fungi Algae By-products Sampling Dependent on:

3. Sampling Also Dependent on: • Detection Endpoint • Culture • Microscopy • Molecular Methods • Immunological Methods • Biochemical Methods • Logistics

4. Replication • Imperative for data interpretation • Should be multi-level • Experiment level • Assay level • 3-5 replicates typical at experiment level • Minimum of duplication required at assay level; triplicate better

5. Accuracy and Precision • Depends on methods and distribution of organisms • Precision between intra-assay replicates should be ~20%

6. Controls • Negative Controls • Sterility Controls • Experimental Controls • Positive Controls • Bacteriostasis/Fungistasis Controls • Seeded Controls • Recovery Controls

7. Sampling Considerations What we want: • Fast • Sensitive • Specific • Easy to Perform • Reliable (Accurate/Precise) • Compatible with Downstream Detection What do we have???

8. The Challenge of Environmental Sampling for Pathogens • Variation in microbe type and distribution • Low microbe numbers: need to concentrate them • Non-random distribution and physical state of microbes of interest: aggregated, particle-associated, embedded, etc. • Volume considerations • Environmental factors may inhibit or interfere with downstream detection • Separate them from interfering and excess other material

9. Detection of Pathogens in The Environment • Three main steps: • (1) recovery and concentration, • (2) purification and separation, and • (3) assay and characterization.

10. Standard Methods • Water and Wastewaster • Standard Methods for the Examination of Water and Wastewater, 20 ed., AWWA, APHA, WEF • EPA Methods • http://www.epa.gov/nerlcwww/index.html • http://www.epa.gov/waterscience/methods/ • ISO Methods • CEN/ISSS Methods • ASTM Methods

11. Standard Methods • Food • FDA Methods • http://www.cfsan.fda.gov/~ebam/bam-toc.html • http://www.cfsan.fda.gov/~comm/microbio.html • Codex Alimentarius standards • Standard Methods for the Examination of Dairy Products, 17th ed., APHA • NSSP • CEN/ISSS Methods • ISO Methods

12. Standard Methods • Disinfection and Disinfectant Efficacy Testing • EPA Methods • http://www.epa.gov/oppbead1/methods/atmpa2z.htm • http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/885_Microbial_Pesticide_Test_Guidelines/Series/ • ASTM Methods • ISO Methods • CEN/ISS methods

13. Standard Methods • Air • ISO standards • CEN/ISS standards • ACGIH standards • Surfaces • ISO standards • ASTM methods • CEN standards

14. Good Laboratory Practice

15. GLP and GMP • GLP- Good Laboratory Practices • GMP- Good Manufacturing Practices

16. Where does GLP/GMP come from? • OECD- Organization for Economic Cooperation and Development • International organization tasked with assuring data or products of member states • Who belongs? • 30 Member Countries • Relations with 70 other countries • NGOS, and Civil Society

17. AUSTRALIA: 7 June 1971AUSTRIA: 29 September 1961BELGIUM: 13 September 1961CANADA: 10 April 1961CZECH REPUBLIC: 21 December 1995DENMARK: 30 May 1961FINLAND: 28 January 1969FRANCE: 7 August 1961GERMANY: 27 September 1961GREECE: 27 September 1961HUNGARY: 7 May 1996ICELAND: 5 June 1961IRELAND: 17 August 1961ITALY: 29 March 1962JAPAN: 28 April 1964KOREA: 12 December 1996 LUXEMBOURG: 7 December 1961MEXICO: 18 May 1994NETHERLANDS: 13 November 1961NEW ZEALAND: 29 May 1973NORWAY: 4 July 1961POLAND: 22 November 1996PORTUGAL: 4 August 1961SLOVAK REPUBLIC: 14 December 2000SPAIN: 3 August 1961SWEDEN: 28 September 1961SWITZERLAND: 28 September 1961TURKEY: 2 August 1961UNITED KINGDOM: 2 May 1961UNITED STATES: 12 April 1961 Member Countries

18. How Enforced? • Left to authorities in individual countries • Who in the US? • FDA • EPA (TSCA and FIFRA)

19. What is Required? • Overall QA/QC Manual • QAPPs • Biosafety and Chemical Hygiene • Operational Controls • Routine Maintenance and Calibration • Training of Personnel • Audit Schedule

20. QA/QC Manual Personnel • Staff Organization • Training Schedules • Personnel Files

21. Facilities Physical Description Size Floorplan Benchspace Lighting Storage Security Workflow HVAC Water Environmental Monitoring Maintenance Schedule QA/QC Manual

22. QA/QC Manual Biosafety/Chemical Hygiene • Gowning and PPE • Emergencies/Spills • Decontamination • SOPs • Description of Safety Practices

23. Equipment • Calibration and Maintenance • Autoclaves • Balances • Spectrophotometers • Centrifuges • Temperature Dependent Devices • Thermometers • Etc. • Relative vs. Traceable Calibration

24. Operational • Purchasing • Inspection/Acceptance • Tracking • Sterility • Media and Reagent Prep

25. QA/QC • Staff • Master Schedule • Lab Notebooks • SOPs • Project Plans • Reporting of Study Results • Storage and Retention • Audits

26. Data Management • Detailed plan for collection, recording, and maintenance of data • Specifies what data collected with observations • Specifies how and where data kept • Laboratory notebooks • Equipment/procedure logs • LIMs system • Specifies Data Retention and Storage • Frequently 3-5 years on-site; upto 10 in off-site or archived storage

27. Keeping a Good Lab Notebook • Generally: • Be organized • Date (and sign) each new entry • Write everything down, no matter how trivial it may seem • Do not erase; rather strike through and initial corrections • Permanently affix additions to lab notebook • Have supervisor review and sign-off on data at least weekly. • When in Doubt write it out

28. Transferring Data from Lab Notebook to Electronic Database • Best to use LIMS with date stamping. • Location of original data should be recorded • Once input, data entry should be validated (ideally by another person; unrealistic) • Data analysis procedures should be annotated where possible