Microbiology Methods for Drinking Water Laboratories · 2018-04-04 · Microbiology Methods for Drinking Water Laboratories Erica Fox. Origins of Drinking Water ... •Sample Collection,

Microbiology Methods for

Drinking Water Laboratories

Erica Fox

Origins of Drinking Water

Bacteriological Testing1854 – Cholera epidemic in London

– Dr. John Snow determined source of Cholera to be public water supply

– Traced nearly all deaths to water from the Broad Street pump, which supplied water from a public well dug 3 feet from an old cesspit

– Cholera epidemic stopped after removal of pump handle

– Showed connection between fecal contamination of drinking water and disease

3

Origins of Drinking Water

Bacteriological Testing

1885 – “Bacterium coli

commune” described by

Theodor Escherich

– Found in higher numbers than

enteric pathogens in fecal

material

– Suggested that detection of B.

coli would indicate presence of

fecal matter

– Later renamed Escherichia coli,

this organism remains the main

indicator of fecal pollution

Regulations

• Safe Drinking Water Act (SDWA), 1974 –

passed by Congress to protect public health by

regulating public drinking water supplies

• National Primary Drinking Water Regulations –

standards set by Environmental Protection

Agency (EPA), as required by SDWA– Found in Code of Federal Regulations (CFR) Part 141

– Establish regulated contaminants and Maximum Contaminant Levels

(MCLs) if applicable, as well as approved methods for analysis

– Microbiological contaminants include Total coliforms (includes Fecal

coliforms and E. coli)

5

Regulations

• National Primary Drinking Water Regulations

– Revised Total Coliform Rule (RTCR) – Establishes MCL for E. coli and uses

E.coli and total coliforms to initiate “find and fix” approach to address fecal

contamination that could enter distribution system.

– Requires public water systems to perform assessments to identify sanitary

defects and take action to correct them

– Surface Water Treatment Rule (SWTR) – HPC are regulated under the SWTR

with a limit of 500 cfu/mL

• Virginia a “Primacy” state – enforces national regulations

– Virginia Waterworks Regulations: 12 VAC 5-590

• Virginia certifies drinking water laboratories through Division of Consolidated

Laboratory Services (DCLS)

– Labs are required to comply with EPA Manual for the Certification of Laboratories

Analyzing Drinking Water

Regulations

• To comply with regulations, certified laboratories must use an approved method. Each of the methods presented today is listed below with the applicable regulation and method reference:

• Surface Water Treatment Rule, CFR 141.74– SimPlate

®

- IDEXX SimPlate®

HPC Test Method for Heterotrophs in Water

• Revised Total Coliform Rule, CFR 141.21– ONPG-MUG - SM 9223 B

• Ground Water Rule, CFR 141.402– ONPG-MUG - SM 9223 B

• Long Term 2 Enhanced Surface Water Treatment Rule, CFR 141.704– Quanti-Tray

®

- SM 9223 B

7

Critical Elements for Microbiology(from EPA Lab Cert Manual, Chapter V)

• Personnel

• Laboratory Equipment and Supplies

• General Laboratory Practices

• Analytical Methodology

• Sample Collection, Handling and Preservation

• Quality Assurance

• Records and Data Reporting

• Action Response to Laboratory Results

Personnel

• Supervisor

– Bachelor’s degree in microbiology, biology or equivalent

– Minimum 2 weeks training at federal/state agency or academic

institution in drinking water microbiological analysis or 80 hours

on-the-job training in water microbiology at certified laboratory

• Analyst

– At least 3 months bench experience in water, milk, or food

microbiology

– Training acceptable to the state in drinking water microbiology

analysis and at least 30 days of on-the-job training in drinking

water microbiology under an experienced analyst

– Demonstrate acceptable results on unknown samples before

analyzing compliance samples

Laboratory Equipment and

Supplies• pH meter

– Accuracy with ± 0.1 units

– Use buffers once

– Standardize before each use with pH 4.0 and 7.0 or 7.0 and 10.0 buffers

– Record slope monthly

– Maintained according to manufacturer’s instructions

• Balance

– Readability of 0.1 g

– Sensitivity of 0.1 g for a load of 150 g and 1 mg for load of 10 g or less

– Readability and Sensitivity checks performed monthly

– Maintenance and calibration performed annually

• Thermometers

– Graduated in 0.5°C increments

• Exception: 1°C for Refrigerator

– Calibration checked annually, discard thermometer if differs more than 1 degree

10


Supplies• Incubator

– Must maintain a constant temperature of 35 ± 0.5 °C and provide a source of humidity

– Thermometers on top and bottom shelves of the use area

– Record temperature twice each day in use, separated by at least 4 hours

11


Supplies• Autoclave

– Maintain sterilization temperature during cycle

– Complete cycle within 45 minutes when 12-15 minute sterilization used

– Record date, contents, initials,sterilization time & temperature, total time in autoclave each use

– Maintenance performed annually

– Maximum registering thermometer used during each cycle

• Check automatic timer each quarter

with stop watch

• Spore strips or spore ampules should

be used monthly as bioindicators to

confirm sterilization.

12


Supplies• Conductivity Meter

– Suitable for checking reagent-grade water

– Readable in micromhos/cm or microsiemens/cm

– Calibrate monthly

• Refrigerator

– Maintain temperature of 1 – 5°C

– Record temperature once each day in use

• Inoculating equipment

– Sterile or presterilized disposable loops and sticks should be used

13


Supplies• Pipets

– Presterilized plastic or glass

– 10 mL or less must be accurate to within 2.5%

– Reseal packages between use periods

• Glassware and Plasticware– Borosilicate glass or clear, non-toxic plastic

– Tube closures should be screw caps with non-toxic liners, stainless steel, plastic or aluminum

• Sample Containers– Wide mouth plastic or glass bottles or sterile plastic bags with sodium

thiosulfate

14

General Laboratory Practices

• Sterilization procedures

– Autoclave at 121°C for recommended time

– Media should be removed immediately after end of cycle

• Sample Containers

– Randomly test 1 container from each batch or lot for sterility using 25 mL non-selective broth (such as Tryptic Soy Broth), incubate for 48 hours & check for growth

• Reagent grade water

– Quality should meet criteria for Conductivity (<2.0 micromhos/cm), Metals (Pb, Cd, Cr, Cu, Ni, Zn) less than 0.05 mg/L per contaminant and no greater than 0.1 mg/L collectively, Total Chlorine Residual (<0.1 mg/L), and HPC (<500 CFU/ml)

• Glassware washing

– Use distilled or deionized water in final rinse

– Inhibitory residue test should be performed prior to initial use of detergent or whenever different washing procedure or detergent is used; at the very least, every five years

– pH residue check

15

Analytical Methodology

• Media– Discard media by manufacturer’s expiration date

– Store prepared medium in dark at recommended temperature

– For lab-prepared media, record date prepared, type, lot #, volume, sterilization time & temperature, final pH, expiration date, and initials

– For commercially-prepared media, record date received, type, lot # and pH verification if specified by method, and expiration date

– Each batch of lab-prepared and each lot of commercially-prepared media should be checked before use for sterility and with positive & negative control cultures

– Run positive & negative controls each quarter for commercially-prepared media with shelf-lives longer than 90 days

• Examples of control cultures:– Total coliforms

• Positive: Escherichia coli Negative: Pseudomonas aeruginosa

– Fecal coliforms• Positive: Escherichia coli Negative: Enterobacter aerogenes

– E. coli• Positive: Escherichia coli Negative: Enterobacter aerogenes

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Analytical Methodology

• Analysis– For compliance samples, use only methods specified in Revised

Total Coliform Rule, Surface Water Treatment Rule and Groundwater Rule

– Laboratory must be certified for all methods used on compliance samples

• Minimum of 1 total coliform method and 1 E. coli method

• Recommend certification in second total coliform method if one method cannot be used due to interference in some drinking water samples

– Shake samples at least 25 times before analyzing

– Tolerance of dilution buffer should be ± 2 mL for volumes of 90 or 99 mL

– RTCR sample volume analyzed must be 100 mL

17

Sample Collection, Handling and

Preservation• Sample collectors should be trained in aseptic sampling

procedures

• Sampling– Locations must be representative of distribution system

– Sample taps must be free of attachments (aerator, strainer, hose)

– Use only cold water taps

– Flush tap until steady temperature

– Collect at least 100 mL, but allow 1 inch air space

• Sample Icing– RTCR: recommended to hold drinking water samples at < 10 °C

during transport

– SWTR: must hold at < 10 °C during transport, sample temperature should be recorded upon receipt at lab

18

Sample Collection, Handling and

Preservation• Sample Holding/Travel Time (all times are from

collection to placing sample in incubator)– Total coliforms in drinking water: 30 hours

– Total coliforms & E.coli in source water: 8 hours

– E. coli under Ground Water Rule: 30 hours

• Sample Information Form– Record information after collection: system name;

sample identification and site location; sample type; date & time of collection; analysis requested; disinfectant residual; sampler name

19

Quality Assurance

• Laboratory should prepare & follow a

Quality Assurance (QA) plan

• Analyze a set of Proficiency Testing (PT)

samples once every 12 months for each

method for which laboratory is certified

• Participate in laboratory audit every three

years

20

Records and Data Reporting

• Laboratory should keep thorough and accurate

records

• QA Plan should describe procedures used for

record retention

• Laboratory should maintain easily accessible

records for 5 years (includes raw data,

calculations and QC data)

• Electronic data should be backed up and should

state how long you will retain records

21

Records and Data Reporting

• Data should be recorded in ink; changes should be lined through and dated & initialed

• Record date & time of sample receipt and name of person receiving sample, along with any comments on sample condition

• Record laboratory sample identification, date & time analysis begins, initials of analyst, method used, media lot number, items noted as QC and results

• Maintain preventive maintenance and repair records for all instruments and equipment for five years

22

Action Response to Laboratory

Results

• RTCR samples: laboratory must promptly notify appropriate authority of positive Total coliform (in excess of 5%) or an E. coli result

• E. coli positive results: water system must notify state by end of day when notified of result

Basic Aseptic Technique

• Disinfect bench tops prior to analysis

• Long hair should be pulled back

• Wash hands prior to analysis

• Take every precaution not to contaminate sterile bottles or media during analysis by talking, coughing, sneezing, etc

• Do not touch any surfaces that are sterile (interiors of bottles or bottle tops, tips of pipets, etc.)

• Keep sterile pipets, bottles, etc. closed until ready to use

• Use care when removing pre-sterilized pipets from bulk bags, to avoid contaminating inside of bag

24

Break

Total Coliforms/E. coli by Colilert®

and Quanti-Tray®

26

Coliforms

The term total coliforms refers to a broad group of bacteria that belong to the taxonomic family Enterobacteriaceae. This group includes the genera Enterobacter, Klebsiella, Citrobacter, and Escherichia. Total coliforms are present in the environment and are used as an indicator to determine the efficacy of treatment plant operation and the integrity of the distribution system. In the ONPG-MUG method, total coliforms are defined as any bacteria possessing the enzyme β-D-galactosidase.

27

Coliform GroupTotal Coliforms

Escherichia coli

Klebsiella

Citrobacter

Escherichia

Enterobacter

Klebsiella

Citrobacter

Escherichia

28

Escherichia coli

Escherichia coli (commonly abbreviated as

E. coli) is a member of the fecal coliform

group. E. coli is found in the digestive tract

of warm blooded animals and is considered

an indicator of fecal contamination. In the

ONPG-MUG method, E. coli is defined as

bacteria giving a positive total coliform

response and possessing the enzyme β-

glucuronidase.


and Quanti-Tray®

These methods allow for the simultaneous

detection of total coliform bacteria and E.

coli through the use of nutrient indicators

ortho-nitophenyl-β-D-galactopyranoside

(ONPG) and 4-methyl-umbelliferyl-β-D-

glucuronide (MUG). Coliform bacteria and

E. coli produce enzymes that can

metabolize ONPG and MUG respectively.

Method Summary

30


and Quanti-Tray®

• When ONPG is metabolized, a yellow color change

occurs, indicating the presence of total coliforms.

Method Summary

31


and Quanti-Tray®

• When MUG is metabolized, a fluorescent product is

produced, indicating the presence of E. coli.

Method Summary

32


and Quanti-Tray®

• Colilert®

is a Presence/Absence test.

• Quanti-Tray®

is an enumeration test.

Method Summary

33

Helpful Hints

• Make sure media is not discolored or caking

before opening snap pack.

• If a sample is yellow or fluorescing, but is less

intense than the comparator, it can be

reincubated, but do not exceed a total of 28

hours incubation.

Total Coliforms/E. coli by Colilert® and

Quanti-Tray®

34


Quanti-Tray®

Interferences

• Some non-coliform bacteria produce small amounts of

the enzyme β-D-galactosidase. These bacteria are

suppressed, and will not produce a positive response

unless 104

CFU/mL are present.

• A combination of hydrogen sulfide and high levels of

either manganese or iron may cause a greenish-black to

black color change to occur after incubation.

• Some water samples may have background color. If

there is background color, compare the inoculated

sample to a control containing only sample.

35

• Some samples may fluoresce without having the yellow

color. According to the manufacturer, an ONPG(-)/ MUG(+)

test (colorless with fluorescence) is considered a

nonspecific reaction as it does not meet the test definition

of a total coliform or E. coli. Therefore this test would be

interpreted as negative for both total coliforms and E. coli.

Per the method, only samples that test positive for total

coliforms should be checked under UV light for E. coli.


Quanti-Tray®

Interferences

36


and Quanti-Tray®

• Colilert®

media QC requirements– Media Fluorescence Check

• Each lot of media must be checked for fluorescence prior to use.

• Media dissolved in sterile reagent grade water should not fluoresce.

– Positive/Negative Control Check

• Each lot of media must be checked for selectivity and proper performance prior to use, and every 3 months while in use. Media dissolved in sterile reagent grade water and inoculated with the following cultures should exhibit the appropriate reactions after a 24 hour incubation at 35 ± 0.5 °C.

Culture Reaction

E. coli Positive yellow color, fluorescence

K. pneumoniae or

E. aerogenesPositive yellow color, no fluorescence

P. aeruginosa No color, no fluorescence

37


and Quanti-Tray®

• Vessel QC requirements– Vessel sterility check

• Each lot of vessels must be checked for sterility prior to use.

• Add 25mL of single strength Tryptic Soy Broth to a randomly selected vessel and incubate for 48 hours at 35 ± 0.5 °C.

• If no growth is present, the vessel lot may be put into service.

– Vessel volume check• Each lot of vessels must be checked for volume accuracy prior to

use.

• Fill a Class A 100mL graduated cylinder with reagent grade water and transfer to a randomly selected vessel.

• The level of water should be at the 100mL mark on the vessel for the lot to be placed in service.

– Vessel fluorescence check• A randomly selected vessel should be checked for fluorescence

using the UV light box prior to use.

• Vessel should not exhibit any fluorescence.

38

Total Coliforms/E. coli by

Colilert®

and Quanti-Tray®

• Quanti-Tray®

QC requirements

– Quanti-Tray®

sterility check

• Each lot must be checked for sterility prior to use.

• Add 25mL of single strength tryptic soy broth to a randomly

selected Quanti-Tray®

, seal, and incubate for 48 hours at 35 ±

0.5 °C.

• If no growth is present, the Quanti-Tray®

lot may be put into

service.

– Quanti-Tray®

Sealer QC check

• The sealer is checked each month for proper sealing.

• Add 100mL of a solution of water and a dark dye to a Quanti-

Tray®

, and run through the sealer. Check for leaks between

the wells and on edges of the tray.

Total Coliforms/E. coli by

Colilert®

40


• Materials:

– Sterile sample vessels

– Colilert®

media

– Sterile Reagent Grade

Water

– Sterile pipettes

– Vessel rack

– Incubator, 35 ± 0.5 ºC

– UV light

41


• Shake sample thoroughly

(at least 25 times)

• Use sterile pipet to draw

volume of sample down to

100 mL.

• Tap Colilert®

media snap

pack on counter to settle

contents. Snap pack

open, and empty media

into sample vessel.

42


• Replace cap and shake sample vigorously. Media does not have to be completely dissolved in the sample prior to incubation.

• Place samples in a rack, and incubate at 35 ± 0.5 °C for 24-28 hours.

43


• After incubating for 24

– 28 hours, the

bottles are read using

a Colilert®

Presence/Absence

comparator for

reference.

44


• Samples that show a

yellow color equal to

or greater than that of

the Comparator are

total coliform positive.

• Samples with no color

are total coliform

negative.

45


• Total coliform positive (yellow) samples and the Comparator are checked with UV light.

• Samples that show fluorescence equal to or greater than that of the Comparator are E. colipositive.

• Samples with no fluorescence are E. colinegative.


Questions?

Total Coliform/E. coli by

Quanti-Tray®

48

Total Coliform/E. coli by Quanti-

Tray®

This method is used for the detection of total

coliform bacteria and E. coli in source water and

ground water. This method is approved for the

analysis of samples for Long Term 2 Enhanced

Surface Water Treatment Rule (LT2) and

Ground Water Rule compliance.

49

Total Coliform/E. coli by Quanti-Tray®

• Quanti-Tray®

trays are available in two different

sizes

• The 51-well Quanti-Tray®

contains only large wells,

and is appropriate for samples containing less than

200.5 CFU (colony forming units) per mL.

• The 97-well Quanti-Tray®

/2000 contains 49 large and

48 small wells. This tray is appropriate for samples

containing less than 2419.6 CFU per mL.

• Samples may be diluted as needed

50


• Materials:– Package of sterile Quanti-

Tray®

trays

– Sterile sample bottles

– Colilert®

media

– Sterile dilution water (90 and 99 mL)


– Quanti-Tray®

sealer and inserts


– UV light

– MPN table

51


• Preheat Quanti-Tray®

sealer.

• Shake sample thoroughly (at least 25 times)

• Use sterile pipet to draw volume of sample down to 100 mL. Sample may be diluted using sterile deionized water if necessary.

• Tap Colilert®

media snap pack on counter to settle contents. Snap pack open, and empty media into sample bottle.

Procedure

52


• Replace cap and mix sample until media dissolves. Media needs to be completely dissolved in the sample prior to pouring into the Quanti-Tray

®

.

• Open Quanti-Tray®

by squeezing in at the sides while pulling the foil tab away. Pour sample into tray.

Procedure

53


• Fit Quanti-Tray®

in

rubber insert and

slide into the

preheated sealer.

Procedure

54


• The Quanti-Tray®

will

go through the sealer

and the sealed

Quanti-Tray®

will

come out the back.

• Incubate sealed trays

at 35 ± 0.5 °C for 24-

28 hours.

Procedure

55


• Wells with a yellow color

equal to or greater than

that of the Quanti-Tray®

Comparator are total

coliform positive.

• Count the number of

positive wells, and refer

to the provided Quanti-

Tray®

MPN table to find

the total coliform MPN.

Reading Samples

56


• Trays with total coliform positive (yellow) wells and the Comparator are checked for fluorescence with a UV light.

• Wells that show fluorescence equal to or greater than that of the Comparator are E. coli positive.

• Count the number of positive wells, and refer to the provided MPN table to find the E. coli MPN.

• The number of positive wells is converted to a Most Probable Number result in the Quanti-Tray

®

or Quanti-Tray®

/2000 MPN Table. The MPN is multiplied by the appropriate dilution factor, if needed.

Reading Samples

57


MPN table for Quanti-Tray®

/2000

58


MPN table for Quanti-Tray®

Total Coliform/E. coli by Quanti-

Tray®

Questions?

HPC by SimPlate®

This method is used for the quantification of

heterotrophic bacteria in drinking and raw

water and can be used for raw, in process and

finished water samples. This method is

approved for the analysis of samples for

Surface Water Treatment Rule compliance.

Summary

HPC by SimPlate®

62

Heterotrophs

• Population of microorganisms that

commonly occur in drinking water.

• Include bacteria, yeasts and molds.

• Dependent upon organic carbon source

for growth.

• Growth in distribution system often found

near dead ends or where loss of

disinfectant residual occurs.

63

Why HPC?

• Used to verify adequate disinfection in distribution system in lieu of monitoring for disinfectant residual, or when residual is low.

• Monitoring of distribution system for biofilm growth.

• Monitoring for nitrification in distribution system when chloramines are used.

• Evaluating effectiveness of treatment process.

64

Consideration of SimPlate®

for HPC as

replacement for HPC Pour Plate

method

• Approved by EPA in late 2002 for compliance

monitoring of drinking water samples under

the Surface Water Treatment Rule, as an

alternative to the HPC Pour Plate Method.

• SimPlate® for HPC has the potential to

reduce staff time, while getting similar results.

65

How does it work?

• SimPlate® media contains multiple

enzyme-substrates, each targeted to a

different bacterial enzyme.

• When bacteria metabolize the enzyme

substrates provided by the media, they

produce a fluorescence that is visible

under UV light.

66

SimPlate®

for HPC Analysis

• Materials:– Sleeve of sterile plates

– Bottle of dehydrated SimPlate

®

media

– Sterile reagent grade water


– Sterile phosphate buffered dilution water


– UV light

67

SimPlate®

for HPC Analysis

• Shake sample thoroughly (at

least 25 times)

• Use sterile pipet to transfer 1 mL

total volume of sample into

middle of SimPlate®

, then touch

tip of pipet off on dry portion of

plate. Sample can be diluted

using sterile phosphate buffered

dilution water as needed.

68

SimPlate®

for HPC Analysis

• 9 mL of SimPlate®

media

is aseptically transferred

from the media bottle to

the center of the base

plate.

• Sample and media mix

and begin to spread out

over the surface of the

plate.

69

SimPlate®

for HPC Analysis

• The plate is gently

swirled, dispersing the

sample and media to

the wells.

• Gently tapping the

plate on the counter

helps bring any

remaining air bubbles

to the surface.

70

SimPlate®

for HPC Analysis

• Air bubbles will

frequently remain on

the surface of the

wells after tapping the

plate.

• After waiting a minute

or two for the bubbles

to pop, the excess

media is poured off

the base plate.

71

SimPlate®

for HPC Analysis

• After incubating for

45 – 72 hours, the

plates are read under

a UV light.

• A blank plate is

shown next to a raw

plate, and both are

upside down to

facilitate viewing of

fluorescing wells.

72

SimPlate®

for HPC Analysis

• The number of

positive wells is

converted to a Most

Probable Number

result in the SimPlate®

for HPC MPN Table.

The MPN is multiplied

by the appropriate

dilution factor, if

needed.

73

SimPlate®

for HPC AnalysisLessons learned

• Up to 10 samples and one blank can be run out of one bottle of prepared SimPlate

®media.

• Prepared media that has been refrigerated must be pre-warmed before next use.

• Sterile phosphate buffered dilution water may be used to dilute raw water samples prior to analysis.

• Although air bubbles in the wells do not interfere with the test, we have found that it can make interpretation of fluorescence more difficult. To help prevent this interference, we have found that the majority of bubbles popped if the plates are allowed to sit a minute or two after tapping, prior to pouring off the sample.

74

SimPlate®

for HPC AnalysisLessons learned

• Swirling the plates should be done GENTLY to avoid spilling excess media.

• Gently tapping the plate on the counter can help pop bubbles up out of the wells.

• Use care with the plates while they are inverted. Bumping, dropping, or setting the plates down roughly can result in the sample being knocked out of the wells.

• Any amount of fluorescence greater than the blank is considered positive when counting wells.

• Keeping the lid on or reading plates upside down can help prevent background fluorescence from interfering with reading.

75

SimPlate® for HPC Analysis

• Analysis QC:

– A blank is analyzed with each set of samples. If more than one bottle of media is needed for a set, a blank should be run for each bottle, and the samples that correspond to that bottle/blank should be noted.

• Media QC:

– All lots of media must be checked for proper performance prior to being placed in service. If the lot is in service for more than a quarter, the media check must be repeated.

• Analyze a plate using raw water or inoculated sterile water as the sample. Incubate for 48 ± 3 hours at 35 ± 0.5 °C. The sample must produce growth for the media to be put in service.

• Check for sterility by incubating an uninoculated plate (blank) for 48 ± 3 hours at 35 ± 0.5 °C. No growth should be observed.

• Sterile Reagent Grade Water QC:

– All batches of sterile water must be confirmed sterile prior to being placed in service.

• Add 50mL of sterile water to 50mL of Double Strength Tryptic Soy Broth and incubate for 48 hours. Check for turbidity indicating bacterial growth.

Quality Control

HPC by SimPlate®

Questions?

Thank you for your attention!

Questions?

For more information, please contact:

Erica Fox,

[email protected]

mailto:[email protected]

Documents

Microbiology Methods for Drinking Water Laboratories · 2018-04-04 · Microbiology Methods for Drinking Water Laboratories Erica Fox. Origins of Drinking Water ... •Sample Collection,