Presentation on theme: "Microbiology Methods and Applications"— Presentation transcript:
1Microbiology Methods and Applications NJDEPOffice of Quality Assurance
2BSDW Potable Water Supplies Total Coliform Rule (PWS systems)Surface / Source Water TestingPrivate Well Testing ActAdditional water certifications include:CryptosporidiumGiardia Cysts
3NJPDES Wastewater Monitoring in Support of Permit Requirements Fecal ColiformEnterococci / Fecal StreptococciConcurrent Monitoring
4NJPDES SQARMonitoring in compliance with Federal 40CFR Part 503 testing (Appendix F)Verification for Class Alternative Options for Pathogen ReductionColiform BacteriaSalmonellaAscaris Ova (Helminth Ova)Enteric Viruses
5Public Recreational Testing Swimming PoolsNatural Bathing BeachesWhirlpools, Hot Tubs and Spas (NJAC 8:26)Testing for Pseudomonas aeruginosa, fecal coliforms, fecal streptococci, enterococci, salmonella and E.Coli, etc.
6Certified Laboratories Microbiological testing of distilled water* used in support of microbiological testingHeterotrophic Plate CountInhibitory Residue testingSuitability testingUse Test*Certification is not required for this testing if used for lab distilled water monitoring only.
7SM 9215B Heterotrophic Plate Count Pour Plate MethodMost commonly used growth media is Standard Plate Count agarpH of media = 7.0±0.2 after autoclavingAll plates (dilutions) are run in duplicateSample volumes of mlsBlank plates of sterilized media includedHumidity is maintained in the incubatorIncubated at 35°C and read at 24 and 48 hoursResults are based on a calculation utilizing the number of colonies on the plateMedia is tested before use or quality certificates are maintained
8SimplateSimplate is the only alternative method for heterotrophic plate count bacteriaDoes not require duplicate analysisMedia is preparedExpands counting range from colonies to 738 coloniesMay negate the need for dilutionsRun at 35°C for 48 hours for drinking water testing
9Multiple Tube Fermentation or MPN Samples usually in a dilution seriesCan also be run as a single 100ml sampleIncubation at 35°C, 44.5° and 45°CRequires a preliminary step to grow bacteria population and the a confirming step to identify sub-groups or individual speciesCalculation based on number of + tubes
10SM 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.
11SM 9221B Confirmation Phase Growth media is Brilliant Green Lactose Bile Broth (BGLBB)final pH = 7.2±0.2 after sterilizationAll 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
12SM 9221D Coliforms Presence-Absence (P-A) Coliform Test 3x strength for single 100ml samplesP-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±0.2A 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 testPositives must be confirmed in BGLBB (SM 9221B)
13SM 9221E Fecal ColiformsAll 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 mediaEC Medium with inverted vial (drinking water and source water) orA-1 Medium with inverted vial (source water only)pH of mediaEC 6.9±0.2 after sterilizationA-1 6.9±0.1 after sterilization
14SM 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.
15SM 9221EA-1 Medium can be used to enumerate fecal coliform in source water samplesRequires same dilutions as EC MediumCan be directly inoculated with sampleIncubated at 35°C for 3 hours then is transferred to and incubated in a 44.5°C water bath for hours.Any amount of gas production is a positive test.
16SM 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 samplesGrowth 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 sterilizationTubes are inoculated at 44.5°C for hours in a water bathInverted fermentation tube is obmittedIncubated tubes are checked for fluorescence, and if they do, + resultFalse positives can be eliminated by including a tube inoculated with a known + and - culture for reference purposes with each batch tested
17SM 9222B Total Coliforms Membrane Filter Method Samples are filtered through a membrane designed to retain bacteriaFilter 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 hours.
18SM 9222BMedia 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.
19SM 9222BAll 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.
20SM 9222B Counting range is 20-80 colonies per membrane Colony counts are either for total counts with the assumption of TC + resultsTotal colonies / 100mlsor are verified coliform counts based on the ratio of verified / total colonies verification as a percentagePercentage verified coliforms / 100mls
21SM 9222D Fecal ColiformsTechnique is the same as SM 9222B but with different media and incubation times and temperatures.Media is m-FC broth or agarpH 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 colonies on each membrane
22SM 9222DSample is filtered and then transferred to the agar plate or plate containing a media saturated padThe inverted pads are secured into plastic bags and submerged into a 44.5°C water bath within 30 minutes of filtration, for hours.Can also use approved solid heat sink incubatorsBlue colonies detected indicate a positive fecal coliform test.
23SM 9223B + UV T. Coliform / E.Coli Chromogenic Substrate ColiformTestSimilar to SM 9221E + UV in that MUG is the det ecting substance for E. Coli bacteria.ColilertLooking for the enzyme ß-Ð-galactosidase as an indicator of total coliformsONPG is the chromogenic substrate for this procedure and once broken down will produce a color change as a positive result.
24SM 9223B + UV Colilert: Colisure (enzyme is CPRG) 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) or24 hour testClear to YellowColisure (enzyme is CPRG)Yellow to RedE*Colite (enzyme is X-Gal)Yellow to Blue Green
25SM 9223B + UVFinal 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.
26SM 9230 C Membrane Filter Fecal Streptococci Enterococci m Enterococcus agar for 48hrs at 35 ± 0.5°Clight and red colonies are counted with a fluorescent light and magnifying lens as fecal streptococciEnterococcimE agar for 48hrs at 41 ± 0.5°Cafter 48hrs filter is transferred to EIA medium and incubated at 41 ± 0.5°C for 20 minutespink 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
27SM 9230CBoth 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.
28SM 9230CGrowth from the BHI tube is then transferred to each of the following media:Bile esculin agar (BEA) incubated at 35 ± 0.5°C for 48hrsBHI broth incubated at 45 ± 0.5°C for 48hrsBHI broth with 6.5% NaCl incubated at 35 ± 0.5°C for 48hrsGrowth 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.
29Fecal Streptococci EPA Page 136 Reference: Microbiological Methods for Monitoring the Environment (EPA-600/ )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.
30Essential QA / QC QA Manual Details the QC procedures that the lab will follow and the frequency for eachQC requirements are conducted annually, monthly, daily or each batchPertains to equipment, instrumentation and supplies including growth media
31Equipment Thermometers calibrated to NIST traceable thermometers (accurate to 0.2°C)glass, annually and metal, quarterlycalibrated over entire operating rangepH metercalibrated to 2 certified buffers, usually 4.00 and 7.00Incubators and Water Bathstwice daily temperature checks (4 hours apart)Refrigeratorsdaily temperature checksAutoclave (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 thermometerBalanceannual calibration, monthly or daily checks with two Class 1weights
32Equipment Hot air ovens (for sterilizing glassware) temperature checks Optical, counting and lighting equipment10 to 15x magnification source and fluorescent light sourcemechanical hand tallycolony counter (HPC)Inoculation equipmentpresterilized plastic, one use or reusable metal loopshardwood applicators that are dry heat sterilized (no autoclaving)Pipetssterile glass or plastic pipets, cotton plugs are recommendedCulture dishessterile plastic with loose or tight lids or glass with loose lids
33Equipment Culture tubes and closures Membrane filtration equipment glass, sufficient size to accommodate media and sample w/o being more than 3/4 fullglass fermentation tubes for gas producing proceduresMembrane filtration equipmentstainless steel, glass or autoclavable plasticautoclaved or UV sterilized before useUV sterilizer must be checked quarterly with a light meter and spread plate irradiation testfiltration unit resterilized after 30 minutessterile beginning and ending blanks to check sterility of filtration unitsSample Containerssterile and checked with non-selective brothMaintenance records and annual service protocols met
34Equipment Membrane filters and pads usually 47 mm, 0.45µm pore size, cellulose ester, white and grid markedautoclaved or pre-sterilized before userecords are maintained as to date received and lot numberparallel testing of membrane filter lots no longer requiredLaboratory pure watertested 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 waterautoclaved (or filter sterilized), stock buffer solution made from lab pure water, labeled, dated, stored properly and free of turbidityeach batch of lab prepared or lot of purchased sterile water is checked with non-selective broth
35MediaMedia prepared according to manufacturer’s directions and method, labeled as date received and date first openedLactose broth may not be usedDehydrated media must be tightly closed and stored properlyMF broth in screw cap containers may be stored for three months, if not screw caps then used in 96 hoursPoured MF agar plates may be stored for 2 weeksPrepared media must be kept in the refrigerator unless the method allows for a different storage temperaturePre-purchased sterile media must be used before the expiration dateEach 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
36RecordsRecords are critical to support the legal defensibility and reliability of the data generatedWhen in doubt write it downYou can never have too many details in support of the hard work done at the benchRetain records for 5 years or 10 if an epidemiological concernKeep all supporting records, certificates, logs, etc.