E. Coli Enumeration in Drinking Water
-
Upload
fredrick-perez -
Category
Documents
-
view
219 -
download
0
Transcript of E. Coli Enumeration in Drinking Water
-
8/11/2019 E. Coli Enumeration in Drinking Water
1/36
-
8/11/2019 E. Coli Enumeration in Drinking Water
2/36
E. coliHappens
Fecal Coliform Group
- group of total coliform bacteria
found in intestinal tracts of warm-
blooded animals.
E. co li
- a species of bacteria within the fecalcoliform group
- dominant bacteria found in waste ofhumans and warm-blooded animals.
- produce a positive total coliform
response- possess an enzyme called
(-glucuronidase) which releasesfluorogen that is detected using a365 nm UV lamp.
-
8/11/2019 E. Coli Enumeration in Drinking Water
3/36
E. colias an indicator organism
Determines the sanitary quality of water
Impossible to test for ALL pathogenic microorganisms,
so test for easily detectable indicator organisms.
Hundreds of E. colistrains Most are non pathogenic (some beneficial)
Some pathogenic strains
Although generally not pathogenic, their presence
indicates apathwayfor human pathogens (ex. Viruses,
bacteria) to enter the water source.
-
8/11/2019 E. Coli Enumeration in Drinking Water
4/36
-
8/11/2019 E. Coli Enumeration in Drinking Water
5/36
Analytical Methods
EPA approved methods for testing for E. coliunder theGround Water Ruleinclude:
Membrane Filtration (MF)
- EPA Method 1604
- Standard Methods 9222 G
Multiple Tube/ Multiple Well Fermentation (enumeration)
- Standard Methods 9221 F
- Standard Methods 9223- Enzyme Substrate Methods
- Quanti-Tray and Quanti-Tray 2000 (SM 9223 B)
Link: www.epa.gov/ogwdw/methods/pdfs/methods/methods_gwr.pdf
-
8/11/2019 E. Coli Enumeration in Drinking Water
6/36
Enzyme Substrate Methods
Enzyme based methodologies detect both total coliforms
and E. colisimultaneously.
Easy, rapid, accurate, economical
Widely accepted as standard for microbiological analysisof water.
Enzymes for Quanti-Tray method: Colilert, Colisure,Colilert-18
-
8/11/2019 E. Coli Enumeration in Drinking Water
7/36
Enzyme Substrate Methods
Colilert:
Ability to detect either presence/absence or to
enumerate organisms.
Detects a single, viable organism per sample
Enhancements for enzyme expression
Buffers, salts, micro-nutrients
Suppression of non-coliforms
Ex. Antibiotics
Suppresses up to 2 million heterotrophs per 100 ml
-
8/11/2019 E. Coli Enumeration in Drinking Water
8/36
Equipment Needed for
Method 9223 B
Autoclave
Sterilize TSB media for 15
minutes at 119 - 121
Sterilize blank water
< 500 ml = 30 mins
> 500 ml = 45 mins
-
8/11/2019 E. Coli Enumeration in Drinking Water
9/36
Equipment
Autoclave Supplies
Autoclave tape
Autoclave bags : run waste
cycle 45 mins at proper temp.
Autoclave biological indicator
(monthly QC)
Log book: record time in/out,
temp., cycle time
Autoclave tape
Autoclave bags
BT Sure Biological
Indicator
http://www.vwrsp.com/catjpg/y00/y0062.jpg -
8/11/2019 E. Coli Enumeration in Drinking Water
10/36
Equipment
Refrigerator
Storage of reagents at 0-5 CTSB media
Bacterial cultures
ex. Kwik Stiks
Refrigerator 0-5C
-
8/11/2019 E. Coli Enumeration in Drinking Water
11/36
Equipment
Oven
Sterilize measuring glassware
for 2 hrs. at 180 C
pipettes
graduated cylinders
Supplies:
aluminum foil
-
8/11/2019 E. Coli Enumeration in Drinking Water
12/36
Equipment
Incubator
Incubate Quanti-Trays forE. colianalysis at 35C +/-
0.5 C for indicated amount of
time
Incubator 35 C +/- 0.5
-
8/11/2019 E. Coli Enumeration in Drinking Water
13/36
Equipment
Testing Supplies
Quanti-Tray sealer and rubber
inserts
Quanti-Tray (57 wells)
range: 1-200 MPN/100 mls
OR
Quanti-Tray 2000 (97 wells)
range: 1-2419 MPN/100 mls
Quanti-Tray Sealer and
rubber inserts
Quanti-Tray / Quanti-Tray 2000
-
8/11/2019 E. Coli Enumeration in Drinking Water
14/36
Equipment
Testing Supplies (cont.)
Pre-sterilized clear samplebottles with dechlorination
chemicals
Squeeze bottles for blankwater
- used for dilutions
Enzymes
- ex. Colilert
Long wave UV lamp
Sterile sample bottle
Squeeze bottles
Long wave UV lamp
365-366 nm
-
8/11/2019 E. Coli Enumeration in Drinking Water
15/36
Equipment
Measuring Items
Sterile graduated cylinders
Sterile pipettes
Balance for weighing dry
media
- if preparing TSB
Sterile graduated cylinders
Sterile pipettes
Balance
http://www.vwrsp.com/catjpg/041/041487.jpg -
8/11/2019 E. Coli Enumeration in Drinking Water
16/36
Equipment
Misc. Items
Pipette washer
Conductivity meter
-monthly QC of blank water
pH meter
-for checking pH of TSB
Pipette washer
pH meter
Conductivity
meter
-
8/11/2019 E. Coli Enumeration in Drinking Water
17/36
Reagents and Standards
Colilert Snap packs for sample size 100 ML
Sample turnsYELLOWwhen totalcoliform bacteria are present andfluoresces BLUEto indicate the presenceof E. coli
Distilled Water/RODI Water Do NOTuse phosphate buffered rinse
water with this method
pH Buffers For calibration of pH meter used for
monthly QC of RODI water
DPD reagent packets For determination of residual chlorine
(monthly QC on RODI water)
-
8/11/2019 E. Coli Enumeration in Drinking Water
18/36
-
8/11/2019 E. Coli Enumeration in Drinking Water
19/36
Sample Collection
Samples to berepresentative of the waterbeing tested Use aseptic technique for
collection
Keep sample containerclosed until collection begins Avoid contact with inside of
bottle and/or cap
Collect directly into sterilecontainer containing de-chlorinate agent Do not rinse the bottle
Leave air space to allow formixing
-
8/11/2019 E. Coli Enumeration in Drinking Water
20/36
Sample Collection
If not analyzed
immediately:Recommended but not required
to hold samples at
-
8/11/2019 E. Coli Enumeration in Drinking Water
21/36
Procedure
Turn on Quanti-Tray Sealer
Warm up time approx. 10 mins.
Vigorously shake water sample bottle 25 times within 7 seconds.
Interval between shaking and measuring the test portion should
not exceed 3 minutes.
-
8/11/2019 E. Coli Enumeration in Drinking Water
22/36
Procedure
Aseptically remove lid and adjust sample volume to the
calibrated 100 ml line on sample container.
Test requires the use of 100 ml of sample
-
8/11/2019 E. Coli Enumeration in Drinking Water
23/36
Procedure
Aseptically add 1 packet of Colilert reagent to the 100
ml test bottle**If sample flashes blue: excessive chlorine and invalid for analysis
Re-cap the bottle and shake until reagent is mostly
dissolved.
-
8/11/2019 E. Coli Enumeration in Drinking Water
24/36
Procedure
Use one hand to hold open the Quanti-Tray or Quanti-
Tray/2000
Well side is facing the palm of the hand.
Squeeze upper part of tray so it bends toward the palm.
Gently pull foil tab to open the tray.
Avoid touching inside of tray or foil tab.
Pour 100 ml sample into the tray.
-
8/11/2019 E. Coli Enumeration in Drinking Water
25/36
Procedure
Tap small wells 2-3 times to release air bubbles.
Label back of tray with sample ID and dilution used.
Place tray with sample into rubber insert so that wells
sit within the cutouts
Place rubber insert on the input shelf of sealer. Slide rubber insert with tray
into the sealer
-
8/11/2019 E. Coli Enumeration in Drinking Water
26/36
Procedure
Once sealed, incubate the tray/trays for 24 hrs at
35 +/- 0.5 C
After 24 hrs, if fluorescence is questionable, incubate
for an additional 4 hrs.
-
8/11/2019 E. Coli Enumeration in Drinking Water
27/36
Counting and Calculations
Quanti-Tray (51 wells) and Quanti-Tray/2000 (97 wells)Counting Ranges:
Quanti-Tray: max. of 200 MPN/ 100 mls sample
Quanti-Tray 2000: max. of 2,419 MPN/ 100 mls sample
Count both small and large yellow wells.
Use color comparator to confirm positive result.
Document these as total coliform positive.
Use the UV lamp to check for fluorescence.
If no wells fluoresce, negative for E. coli
If wells do fluoresce, positive for E. coli
Count small and large fluorescing wells
Refer to table for MPN
-
8/11/2019 E. Coli Enumeration in Drinking Water
28/36
Counting and Calculations
-
8/11/2019 E. Coli Enumeration in Drinking Water
29/36
Counting and Calculations
-
8/11/2019 E. Coli Enumeration in Drinking Water
30/36
Quality Control Summary
Daily Monthly Quarterly Annually
Negative Control Autoclave Biological
Indicator Check
Autoclave Timer Working
Thermometers
Verification
Positive Control Balance Check
Class S1 weights
Autoclave
Thermometer
MRT1/3 years by
OEPA auditor
Incubator Temps
2/Day4Hrs apart
Reagent Water
pH, Cl & Cond.
Reagent Water
Metals
(Cd,Cr,Cu,Pb,Ni,Zn)
Refrigerator Temp
1/Day
Balance Service
Outside Vendor
-
8/11/2019 E. Coli Enumeration in Drinking Water
31/36
Quality Control
Monthly QC
Autoclave Biological Indicator Checks
Verifies autoclave is sterilizing properly
Reagent Water Analysis ( Blank Water)
Residual ChlorineNot Detected
Conductivity - < 2 mhos/cm
pH5.50 to 7.50 s.u.
Balance Calibration Check
(used for weighing medias)
BT Sure Biological
Indicator
BT Sure
Incubator
-
8/11/2019 E. Coli Enumeration in Drinking Water
32/36
Quarterly QC
Autoclave Timer Calibration
Autoclave External Thermometer
Calibration
Annual QC
Thermometer Calibrations
**Exception:
MRT = every 3 years at EPA
recertification for drinking
water analysis/ and when
new MRT received.
Reagent Water Contamination
Analysis
Cd, Cr, Cu, Pb, Ni, Zn
Balance Service Check
Outside Contractor
Quality Control
-
8/11/2019 E. Coli Enumeration in Drinking Water
33/36
Quality Control Summary
Media Bottles
TSB
Positive & Negative
Verification
Each new lot
PRIOR TO USE
Sterility Verification
Each new lot
PRIOR TO USE
Colilert
Each new lot
PRIOR TO USE
3 Control Bacteria
-
8/11/2019 E. Coli Enumeration in Drinking Water
34/36
QC per each new lot pr ior to use
Sample bottle sterility checks: each new lot
Use TSB media (Tryptic Soy Broth) Test 1% of each box received for growth
Quality Control
-
8/11/2019 E. Coli Enumeration in Drinking Water
35/36
Quality Control
Test each new batch (lot number) of mediaprepared/ received
TSB media check
1 positive control (using E. coli) ,1 negative control (no inoculation)
-
8/11/2019 E. Coli Enumeration in Drinking Water
36/36
Quality Control
Colilert check
( Each new lot received)
Inoculation with 3 control bacteria:
One control bacteria mustbe E.coli
total coliform (+), E. coli (+)
One control bacteria can be
Pseudomonas aeruginosa( or other
non-coliform)
total coliform (-), E. coli (-)
One control bacteria can be Klebsiella
pneumoniae( or other coliform)
total coliform (+), E.coli (-)