Microbiology Methods and Applications

1. MicrobiologyMethods and Applications NJDEP Office of Quality Assurance debra.waller@dep.state.nj.us

2. BSDW Potable Water Supplies • Total Coliform Rule (PWS systems) • Surface / Source Water Testing • Private Well Testing Act • Additional water certifications include: • Cryptosporidium • Giardia Cysts

3. NJPDES Wastewater • Monitoring in Support of Permit Requirements • Fecal Coliform • Enterococci / Fecal Streptococci • Concurrent Monitoring

4. NJPDES SQAR • Monitoring in compliance with Federal 40CFR Part 503 testing (Appendix F) • Verification for Class Alternative Options for Pathogen Reduction • Coliform Bacteria • Salmonella • Ascaris Ova (Helminth Ova) • Enteric Viruses

5. Public Recreational Testing • Swimming Pools • Natural Bathing Beaches • Whirlpools, Hot Tubs and Spas (NJAC 8:26) • Testing for Pseudomonas aeruginosa, fecal coliforms, fecal streptococci, enterococci, salmonella and E.Coli, etc.

6. Certified Laboratories • Microbiological testing of distilled water* used in support of microbiological testing • Heterotrophic Plate Count • Inhibitory Residue testing • Suitability testing • Use Test • *Certification is not required for this testing if used for lab distilled water monitoring only.

7. SM 9215B Heterotrophic Plate Count • Pour Plate Method • Most commonly used growth media is Standard Plate Count agar • pH of media = 7.0±0.2 after autoclaving • All plates (dilutions) are run in duplicate • Sample volumes of 0.1 - 2.0mls • Blank plates of sterilized media included • Humidity is maintained in the incubator • Incubated at 35°C and read at 24 and 48 hours • Results are based on a calculation utilizing the number of colonies on the plate • Media is tested before use or quality certificates are maintained

8. Simplate Simplate is the only alternative method for heterotrophic plate count bacteria Does not require duplicate analysis Media is prepared Expands counting range from 30-300 colonies to 738 colonies May negate the need for dilutions Run at 35°C for 48 hours for drinking water testing

9. Multiple Tube Fermentation or MPN • Samples usually in a dilution series • Can also be run as a single 100ml sample • Incubation at 35°C, 44.5° and 45°C • Requires a preliminary step to grow bacteria population and the a confirming step to identify sub-groups or individual species • Calculation based on number of + tubes

10. SM 9221B Total Coliforms • Presumptive Phase • Growth media is Lauryl Tryptose Broth (LTB) • pH= 6.8±0.2 (after sterilization) • fermentation tube (gas production) • bromcresol purple indicator (yellow color, acid reaction, suggested for single 100ml sample volumes to reduce false positives from bubbles in larger test tubes) • 24 hours at 35°C, if negative incubate for an additional 24 hours.

11. SM 9221B • Confirmation Phase • Growth media is Brilliant Green Lactose Bile Broth (BGLBB) • final pH = 7.2±0.2 after sterilization • All tubes showing growth, gas or acid reaction in LTB must be transferred to BGLBB to confirm Total Coliform growth. • Incubation at 24 and / or 48 hours at 35°C

12. SM 9221D Coliforms • Presence-Absence (P-A) Coliform Test • 3x strength for single 100ml samples • P-A broth is dissolved in water and then 50ml portions are transferred into 250 milk dilution bottles. • Media is then sterilized and final pH = 6.8±0.2 • A 100ml sample is then added to the sterilized media and incubated at 35°C and checked at 24 and 48 hours. • Yellow color with or without gas is a positive presumptive test • Positives must be confirmed in BGLBB (SM 9221B)

13. SM 9221E Fecal Coliforms • All potable water tests with positive presumptive LTB tests that have been confirmed or awaiting confirmation in BGLBB as total coliforms, must be tested for fecal coliform or E.Coli. Source water can be directly tested for fecal coliform by this procedure. • Two types of growth media • EC Medium with inverted vial (drinking water and source water) or • A-1 Medium with inverted vial (source water only) • pH of media • EC 6.9±0.2 after sterilization • A-1 6.9±0.1 after sterilization

14. SM 9221E • EC medium can be utilized in two ways • As a confirmation for fecal coliform from TC + tubes. Inoculated from positive BGLBB tubes and incubated for 24-26hrs at 44.5 ± 0.2°C. • Or as a direct test for fecal coliform. Requires three sample volumes, 10, 1 and 0.1mls, five or ten EC tubes for each sample volume. • Any amount of gas production in the EC tubes is considered to be a positive reaction.

15. SM 9221E • A-1 Medium can be used to enumerate fecal coliform in source water samples • Requires same dilutions as EC Medium • Can be directly inoculated with sample • Incubated at 35°C for 3 hours then is transferred to and incubated in a 44.5°C water bath for 19-23 hours. • Any amount of gas production is a positive test.

16. SM 9221E + MUG E.Coli • MUG (4-methylumbelliferyl-ß-D-glucuronide) • Substance that is cleaved off of an E.coli cell containing the specific enzyme (ß-glucuronidase) • Used as a confirmation test for total coliform positive samples • Growth media is EC Medium with 50µg/ml MUG added before sterilization. Medium is tested for fluorescence prior to use. • pH of media = 6.9±0.2 after sterilization • Tubes are inoculated at 44.5°C for 22-26 hours in a water bath • Inverted fermentation tube is obmitted • Incubated tubes are checked for fluorescence, and if they do, + result • False positives can be eliminated by including a tube inoculated with a known + and - culture for reference purposes with each batch tested

17. SM 9222B Total Coliforms • Membrane Filter Method • Samples are filtered through a membrane designed to retain bacteria • Filter is antiseptically transferred from the filtration apparatus and placed on either a pad saturated with media or agar plate. • Plates are inverted and incubated at 35°C for 22-24 hours.

18. SM 9222B • Media is M-Endo (broth or agar) or LES Endo agar. M-Endo used more often than LES Endo agar. • pH of M-Endo medium is 7.2±0.1 after preparation. The media is heated to near boiling and contains 95% alcohol. The media is not sterilized by autoclaving.

19. SM 9222B • All bacteria that produce a red colony with a metallic (golden) sheen, are considered to be members of the coliform group. However, some non-coliform bacteria (ie. Proteus mirabilis) can produce sheen colonies. • The MF test requires confirmation with LTB and BGLBB. Only colonies that ferment lactose (found in BGLBB) can be confirmed as coliforms.

20. SM 9222B • Counting range is 20-80 colonies per membrane • Colony counts are either for total counts with the assumption of TC + results • Total colonies / 100mls • or are verified coliform counts based on the ratio of verified / total colonies verification as a percentage • Percentage verified coliforms / 100mls

21. SM 9222D Fecal Coliforms • Technique is the same as SM 9222B but with different media and incubation times and temperatures. • Media is m-FC broth or agar • pH of media = 7.4±0.2 after preparation. • Media is brought just to the boiling point and is not autoclaved. • Dilutions are used to bring counting range to 20-60 colonies on each membrane

22. SM 9222D • Sample is filtered and then transferred to the agar plate or plate containing a media saturated pad • The inverted pads are secured into plastic bags and submerged into a 44.5°C water bath within 30 minutes of filtration, for 22-26 hours. • Can also use approved solid heat sink incubators • Blue colonies detected indicate a positive fecal coliform test.

23. SM 9223B + UV T. Coliform / E.Coli • Chromogenic Substrate ColiformTest • Similar to SM 9221E + UV in that MUG is the det ecting substance for E. Coli bacteria. • Colilert • Looking for the enzyme ß-Ð-galactosidase as an indicator of total coliforms • ONPG is the chromogenic substrate for this procedure and once broken down will produce a color change as a positive result.

24. SM 9223B + UV • Colilert: • 18 hour test (sampled is warmed to 35°C water bath, media is added and the test is moved to a 35°C incubator for 18 hours) or • 24 hour test • Clear to Yellow • Colisure (enzyme is CPRG) • Yellow to Red • E*Colite (enzyme is X-Gal) • Yellow to Blue Green

25. SM 9223B + UV • Final color is verified against a color comparator purchased from the media manufacturer. • If E. Coli is present then the samples will fluorescence under a 365nm, 6 watt long wavelength UV lamp. • If results are questionable after 24 hours of incubation then the incubation period can be extended for an additional 4 hours.

26. SM 9230 C Membrane Filter • Fecal Streptococci • m Enterococcus agar for 48hrs at 35 ± 0.5°C • light and red colonies are counted with a fluorescent light and magnifying lens as fecal streptococci • Enterococci • mE agar for 48hrs at 41 ± 0.5°C • after 48hrs filter is transferred to EIA medium and incubated at 41 ± 0.5°C for 20 minutes • pink to red enterococci colonies with black or reddish brown precipitate on the underside of the filter are counted with with a fluorescent light and magnifying lens

27. SM 9230C • Both tests are verified to determine whether or not the bacteria is definitively fecal streptococci or enterococci. • Growth is first streaked onto the surface of a brain heart infusion (BHI) plate and incubated for 24 to 48 hrs at 35 ± 0.5°C. • Positive growth from the BHI agar is transferred to a tube of BHI broth and to two clean glass slides. • The BHI broth is incubated at 35 ± 0.5°C for 24hrs. • To one of the prepared slides 3% hydrogen peroxide is added. If bubbles appear then the a positive catalyst test then the organism is not a member of the fecal streptococci group. • If the peroxide test is negative a gram stain of the second slide is done.

28. SM 9230C • Growth from the BHI tube is then transferred to each of the following media: • Bile esculin agar (BEA) incubated at 35 ± 0.5°C for 48hrs • BHI broth incubated at 45 ± 0.5°C for 48hrs • BHI broth with 6.5% NaCl incubated at 35 ± 0.5°C for 48hrs • Growth on the catalase-negative, gram-positive cocci on BEA and at 45°C verifies fecal streptococci. • Growth at 45°C and in 6.5% NaCl verifies enterococci. • Batches of media must be tested with an appropriate positive and negative culture (S. faecalis) before use.

29. Fecal StreptococciEPA Page 136 • Reference: Microbiological Methods for Monitoring the Environment (EPA-600/8-78-017) • Similar requirements to SM9230C but detecting media is KF Streptococcus Agar incubated for 48hrs at 35±0.5°C. Pink and red colonies are counted as streptococci. • Positive growth is subjected to verification by growth in BHI agar(plate or slant)and BHI broth. Transfer growth to two slides. Subect one slide to the catalase test with hydrogen peroxide. If negative, inoculate growth from BHI broth to another fresh tube of BHI broth, incubated at 45±0.5°C and BHI broth with 40% bile (oxgall) incubated at 35±0.5°C. • Growth at 45°C and in 40% bile verifies fecal streptococci.

30. Essential QA / QC • QA Manual • Details the QC procedures that the lab will follow and the frequency for each • QC requirements are conducted annually, monthly, daily or each batch • Pertains to equipment, instrumentation and supplies including growth media

31. Equipment • Thermometers • calibrated to NIST traceable thermometers (accurate to 0.2°C) • glass, annually and metal, quarterly • calibrated over entire operating range • pH meter • calibrated to 2 certified buffers, usually 4.00 and 7.00 • Incubators and Water Baths • twice daily temperature checks (4 hours apart) • Refrigerators • daily temperature checks • Autoclave (for sterilizing growth media and dilution/buffer water) • temperature checks and checks on efficiency include spore strips or ampules, heat sensitive tape or a maximum registering thermometer • Balance • annual calibration, monthly or daily checks with two Class 1weights

32. Equipment • Hot air ovens (for sterilizing glassware) • temperature checks • Optical, counting and lighting equipment • 10 to 15x magnification source and fluorescent light source • mechanical hand tally • colony counter (HPC) • Inoculation equipment • presterilized plastic, one use or reusable metal loops • hardwood applicators that are dry heat sterilized (no autoclaving) • Pipets • sterile glass or plastic pipets, cotton plugs are recommended • Culture dishes • sterile plastic with loose or tight lids or glass with loose lids

33. Equipment • Culture tubes and closures • glass, sufficient size to accommodate media and sample w/o being more than 3/4 full • glass fermentation tubes for gas producing procedures • Membrane filtration equipment • stainless steel, glass or autoclavable plastic • autoclaved or UV sterilized before use • UV sterilizer must be checked quarterly with a light meter and spread plate irradiation test • filtration unit resterilized after 30 minutes • sterile beginning and ending blanks to check sterility of filtration units • Sample Containers • sterile and checked with non-selective broth • Maintenance records and annual service protocols met

34. Equipment • Membrane filters and pads • usually 47 mm, 0.45µm pore size, cellulose ester, white and grid marked • autoclaved or pre-sterilized before use • records are maintained as to date received and lot number • parallel testing of membrane filter lots no longer required • Laboratory pure water • tested monthly for heterotrophic plate count, chlorine residual and conductivity and annually for Pb, Cd, Cr, Cu, Ni and Zn and Bacteriological Water Quality (Suitability) • Dilution/buffer sterile water • autoclaved (or filter sterilized), stock buffer solution made from lab pure water, labeled, dated, stored properly and free of turbidity • each batch of lab prepared or lot of purchased sterile water is checked with non-selective broth

35. Media • Media prepared according to manufacturer’s directions and method, labeled as date received and date first opened • Lactose broth may not be used • Dehydrated media must be tightly closed and stored properly • MF broth in screw cap containers may be stored for three months, if not screw caps then used in 96 hours • Poured MF agar plates may be stored for 2 weeks • Prepared media must be kept in the refrigerator unless the method allows for a different storage temperature • Pre-purchased sterile media must be used before the expiration date • Each batch of lab prepared media and lot of pre-sterilized purchased media must be checked with a known positive and known negative culture before use. • All records of receipt and details of preparation must be maintained

36. Records • Records are critical to support the legal defensibility and reliability of the data generated • When in doubt write it down • You can never have too many details in support of the hard work done at the bench • Retain records for 5 years or 10 if an epidemiological concern • Keep all supporting records, certificates, logs, etc.