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Microbiology Methods and Applications


Natural Bathing Beaches. Whirlpools, Hot Tubs and Spas (NJAC 8:26) ... Tubes are inoculated at 44.5 C for 22-26 hours in a water bath ... – PowerPoint PPT presentation

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Title: Microbiology Methods and Applications

MicrobiologyMethods and Applications
  • Office of Quality Assurance
  • debra.waller_at_dep.state.nj.us

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

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

  • 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

Public Recreational Testing
  • Swimming Pools
  • Natural Bathing Beaches
  • Whirlpools, Hot Tubs and Spas (NJAC 826)
  • Testing for Pseudomonas aeruginosa, fecal
    coliforms, fecal streptococci, enterococci,
    salmonella and E.Coli, etc.

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.

SM 9215B Heterotrophic Plate Count
  • Pour Plate Method
  • Most commonly used growth media is Standard Plate
    Count agar
  • pH of media 7.00.2 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 35C 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

  • 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 35C for 48 hours for drinking water

Multiple Tube Fermentation or MPN
  • Samples usually in a dilution series
  • Can also be run as a single 100ml sample
  • Incubation at 35C, 44.5 and 45C
  • 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

SM 9221B Total Coliforms
  • Presumptive Phase
  • Growth media is Lauryl Tryptose Broth (LTB)
  • pH 6.80.2 (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 35C, if negative incubate for an
    additional 24 hours.

SM 9221B
  • Confirmation Phase
  • Growth media is Brilliant Green Lactose Bile
    Broth (BGLBB)
  • final pH 7.20.2 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 35C

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
  • Media is then sterilized and final pH 6.80.2
  • A 100ml sample is then added to the sterilized
    media and incubated at 35C 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)

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
  • 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 6.90.2 after sterilization
  • A-1 6.90.1 after sterilization

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 0.2C.
  • 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.

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 35C for 3 hours then is transferred
    to and incubated in a 44.5C water bath for 19-23
  • Any amount of gas production is a positive test.

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 6.90.2 after sterilization
  • Tubes are inoculated at 44.5C 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

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 35C for
    22-24 hours.

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 7.20.1 after preparation.
    The media is heated to near boiling and contains
    95 alcohol. The media is not sterilized by

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.

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

SM 9222D Fecal Coliforms
  • Technique is the same as SM 9222B but with
    different media and incubation times and
  • Media is m-FC broth or agar
  • pH of media 7.40.2 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

SM 9222D
  • Sample is filtered and then transferred to the
    agar plate or plate containing a media saturated
  • The inverted pads are secured into plastic bags
    and submerged into a 44.5C 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.

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.

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

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.

SM 9230 C Membrane Filter
  • Fecal Streptococci
  • m Enterococcus agar for 48hrs at 35 0.5C
  • light and red colonies are counted with a
    fluorescent light and magnifying lens as fecal
  • Enterococci
  • mE agar for 48hrs at 41 0.5C
  • after 48hrs filter is transferred to EIA medium
    and incubated at 41 0.5C 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

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 0.5C.
  • Positive growth from the BHI agar is transferred
    to a tube of BHI broth and to two clean glass
  • The BHI broth is incubated at 35 0.5C for
  • 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.

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

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 350.5C. 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
    450.5C and BHI broth with 40 bile (oxgall)
    incubated at 350.5C.
  • Growth at 45C and in 40 bile verifies fecal

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

  • Thermometers
  • calibrated to NIST traceable thermometers
    (accurate to 0.2C)
  • 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

  • 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
  • hardwood applicators that are dry heat sterilized
    (no autoclaving)
  • Pipets
  • sterile glass or plastic pipets, cotton plugs are
  • Culture dishes
  • sterile plastic with loose or tight lids or glass
    with loose lids

  • 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
  • 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

  • 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

  • Media prepared according to manufacturers
    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
  • 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

  • Records are critical to support the legal
    defensibility and reliability of the data
  • 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,
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