Lesson 10:

Direct Measurement of Microbial Growth

 

 

Objective

In this lesson we will learn the following:

 

 

Reading Assignment

In addition to the online lecture, read chapter 13 in Microbiological Examination of Water and Wastewater.

 

Lecture

There are different methods of counting microbial growth. For the purpose of this lesson we will focus on the Chromogenic/Fluorogenic (C/F) Substrate Analyses.

 

 

Chromogenic/Fluorogenic (C/F) Substrate Analyses

Presence-Absence and MPN Formats

Purpose

Enzymatic substrate systems were designed to detect Total Colilforms and E. coli in a single test procedure without the requirement for confirmation steps. Depending on the substrate used, test results can be available in as few as 18 hours. These test systems have been applied to the presence-absence (qualitative) and MPN (quantitative) formats, as well as to membrane filtration techniques discussed elsewhere in this lesson.

Enzymatic substrate systems have a number of advantages, as well as disadvantages, over conventional microbiological tests.

 

 

Advantages

 

 

Disadvantages

 

 

 

Test Protocol

These substrate systems are based on the ability of total coliforms to produce the enzyme ß-galactosidase and E. coli to produce the enzyme ß-glucuronidase. Each of these enzymes is tagged with either a chromogen or a fluorogen indicator compound, depending on the manufacturer design of the substrate system. As the target bacteria grow and produce the applicable enzyme, the indicator is cleaved, thus releasing a color or a fluorescence visible under longwave UV light.

At the current time, the EPA has approved the substrate systems listed on the attached table for testing distribution samples for compliance with the Total Coliform Rule (TCR). Some products, when used in an MPN format, can be used for determining Total Coliform levels as required with the Surface Water Treatment Rule (SWTR).

 

Chromogenic/Fluorogenic Test Systems

Method Incubation Total Coliform E. coli Applicable Program
Temperature Time (hrs) Indicator Reaction Indicator Reaction TCR SWTR
Colilert 18 35.0° + 0.5°C* 18-22 ONPG Yellow MUG Fluorescence Yes Yes
Colilert 24 35.0° + 0.5°C 24-28 ONPG Yellow MUG Fluorescence Yes Yes
Colisure 35.0° + 0.5°C 24-48 CPRG Magenta MUG Fluorescence Yes No
E*ColiteTM 35.0° + 0.5°C* 28-48 BCIG Blue-green MUG Fluorescence Yes No
Readycult 35.0° + 0.5°C 23-25 BCIG Blue-green MUG* Fluorescence* Yes No
ColitagTM 35.0° + 0.5°C 22-26 ONPG Yellow MUG Fluorescence Yes No

 

Manufacturers:

 

 


Colilert 18, Colilert 24, Colisure-Idexx Corporation
E*ColiteTM-Charm Sciences
Readycult-EM Sciences
ColitagTM-CPI International

 

Temperature:

 

*Colilert 18 must be incubated in a water bath at 35.0° + 0.5°C for the first 20 minutes.
*E*ColiteTM must be placed in a 35°C water bath for 10 minutes prior to incubation.

Time:

 

Colilert 18 and 24 may be incubated an additional 4 hours if the initial reading is indeterminate. Colisure may be incubated up to 48 hours.
E*ColiteTMis initially incubated fo r28 hours. If positive for Total Coliforms, the sample is reincubated an additional 20 hours (total 48) and examined for the presence of E. coli.

Indicators:

 

 

ONPG
CRPG
BCIG
MUG
*KOVAC's
o-nitrophenol-ß-D-galactopyranoside
chlorophenol red ß-D-galactopyranoside
5-bromo-4-chloro-3-indoxyl-ß-D-glucuronide
4-methylumbelliferyl-ß-D-glucuronide
used to determine the presence of indole (Optional confirmation test for E. coli)

 

 

 

Qualitative C/F Technique

Colilert/Colisure

Single-bottle test

 

Reagents

Colilert 18, Colilert 24 or Colisure

Individual reagent packets for 100 mL sample volume, or
Premeasured quantity dispensed into test containers (100 mL sample volume)

Note: If Colisure reagent has been stored under refrigeration, it must be warmed to room temperature prior to use.

 

Each new lot of chromogenic/fluorogenic reagent must be performance tested prior to use. (See "Media' section for instructions.)

 

 

Materials and Equipment

Sterile, clear glass or plastic bottles with screw caps; graduated at 100 mL

Sterile 100 mL measuring device (graduated cylinder or pipets) - Optional

Incubator - capable of maintaining a temperature of 35.0° + 0.5°C.

Waterbath - capable of maintaining a temperature of 35.0° + 0.5°C (if using Colilert 18)

Longwave UV light, 366 nm, 6-watt

Color comparator - Colilert products only

 

 

Procedure

  1. Obtain one reagent packet or sterile bottle with premeasured reagent for each sample to be tested. Label each bottle with the sample identification.
  2. Shake the sample vigorously 25 times.
  3. Aseptically pour the water sample into the bottle until the liquid reaches the 100 mL mark. If test samples are received in the bottles to be used for testing, aseptically pour off any excess sample. If the bottles are not graduated, each sample must be measured with a sterile graduated cylinder or a sterile pipet.
  4. If using reagent packets, carefully open the packet, avoiding inhalation of any dust material, and aseptically add the reagent to the 100 mL sample. Do not tap the reagent packet against the side of the test bottle.
  5. Cap the bottle and shake to dissolve the test reagent.
  6. Repeat steps 2-5 for the remaining samples.
  7. Indubate the test bottles at 35.0° + 0.5°C for the required time period. If Colilert 18 is used, the first 20 minutes of incubation must be in a waterbath at 35.0° + 0.5°C. If the initial reading is indeterminate, reincubate the samples up to the time listed below.
  Incubation Times (hours)
  Minimum Maximum
Colilert 18 18 22
Colilert 24 24 28
Colisure 24 48

  1. After incubation, carefully remove the bottles from the incubator and examine for a color change indicative of the presence of Total Coliform.

    Reagent Total Coliform
    Positive Reaction Negative Reaction
    Colilert 18 or 24 Yellow Clear, no color
    Colisure Magenta Yellow


  2. Examine any Total Coliform positive sample for the presence of E. coli by holding the longwave UV light 4-6 inches from the sample bottle in a dark location. For both test substrates the reaction is as follows:

    Reagent E. coli
    Positive Reaction Negative Reaction
    Colilert or Colisure Fluorescence No fluorescence


  3. If the color still cannot be determined after the maximum incubation time, invalidate the sample. Collect another sample and retest using an alternate Total Coliform method.

 

 

 

 

Qualitative C/F Technique

E*ColiteTM

Single-bottle test

 

Reagent

E*ColiteTM

Available in a complete packet which includes sample bag, reagent and bacteriocide.

 

Each new lot of chromogenic/fluorogenic regent must be performance tested prior to use. (See "Media" section for instructions.)

 

 

Materials and Equipment

Sterile E*ColiteTM sample bags

Sterile 100 mL measuring device (Class A graduated cylinder)

Incubator - capable of maintaining a temperature of 35.0° + 0.5°C

Waterbath - capable of maintaining a temperature of 35.0° + 0.5°C

Longwave UV light, 366 nm, 6-watt

 

 

Procedure

  1. If the sample was not collected in an E*ColiteTM bag, obtain one for each sample to be tested. Label each with the sample identificaiton.
  2. Shake the sample vigorously 25 times.
  3. Tear the perforated strip and open by pulling the white tabs. Aseptically pour 100 mL of sample into the bag and flatten to remove any trapped air. Twirl the bag around the wire strip two to three times and bend the wires to seal. Shake the sample bag to dissolve the sodium thiosulfate tablet.
  4. Hold the twirled end of the sample bag flat on a table and push the water sample against Divider 1 while firmly holding the bag to keep it from unsealing. Push the water sample into Divider 1 only.
  5. Shake to dissolve the test reagent.
  6. Repeat 2-5 for the remaining samples.
  7. Place the sealed bag in a 35.0° + 0.5°C water bath for 10 minutes.
  8. Incubate the bags at 35.0° + 0.5°C for 28 hours.
  9. After incubation, carefully remove the bags from the incubator, and examine for a color change indicative of the presence of Total Coliforms.

    Reagent Total Coliform
    Positive Reaction Negative Reaction
    E*ColiteTM Blue-green Yellow


  10. When a positive Total Coliform response is observed, reincubate the sample an additional 20 hours, for a total of 48 hours. Examine for the presence of E. coli by holding the longwave UV light 4-6 inches from the sample bag in a dark location. For this substrate, the reaction is as follows:

    Reagent E. coli
    Positive Reaction Negative Reaction
    E*ColiteTM Fluorescence No fluorescence

 

 

 

 

Qualitative C/F Technique

Readycult

Single-bottle test

 

 

Reagents

Readycult Coliform 100

Individual reagent packets for 100 mL sample volume

Each new lot of chromogenic/fluorogenic reagent must be performance tested prior to use (See "Media" section for instructions.)

 

KOVAC's Indole reagent (Optional)

 

 

Materials and Equipment

Sterile, clear glass or plastic bottles with screw caps; 120 mL or 150 mL capacity; graduated at 100 mL

Sterile 100 mL measuring device (graduated cylinder or pipets) - Optional

Incubator - capable of maintaining a temperature of 35.0° + 0.5°C

Longwave UV light, 366 nm, 6-watt

 

 

Procedure

  1. Obtain one reagent packet for each sample to be tested.
  2. Shake the sample vigorously 25 times.
  3. If samples are received in opaque bottles, aseptically pour the water sample into a properly labeled sterile bottle until the liquid reaches the 100 mL mark. If test samples are received in the bottles to be used for testing, aseptically pour off any excess sample. If the bottles are not graduated, each sample must be measured with a sterile graduated cylinder or a sterile pipet.
  4. Carefully open the reagent packet, avoiding inhalation of any dust material, and aseptically add the reagent to the 100 mL sample. Do not tap the reagent packet against the side of the test bottle.
  5. Cap the bottle and shake to dissolve the test reagent.
  6. Repeat steps 2-5 for the remaining samples.
  7. Incubate the test bottles at 35.0° + 0.5°C for a 24 + 1 hour time period.
  8. After incubation, carefully remove the bottles from the incubator and examine for a color change indicative of the presence of Total Coliform.

    Total Coliform
    Positive Reaction Negative Reaction
    Blue-green Yellow


  9. Examine any Total Coliform positive sample for the presence of E. coli by holding the longwave UV light 4-6 inches from the sample bottle in a dark location and examining for the presence of fluorescence.

    E. coli
    Positive Reaction Negative Reaction
    Fluorescence No fluorescence


  10. OPTIONAL: If confirmation of E. coli is desired, to any bottle with positive fluorescence add 2.5 mL KOVAC's reagent directly to the broth. A red ring will immediately form to confirm the presence of E. coli.

 

 

 

Qualitative C/F Technique

ColitagTM

Single-bottle test

 

 

Reagents

ColitagTM

Individual reagent packets for 100 mL sample volume

 

Each new lot of chromogenic/fluorogenic reagent must be performance tested prior to use. (See "Media" section for instructions.)

 

 

Materials and Equipment

Sterile, clear glass or plastic bottles with screw caps; graduated at 100 mL

Sterile 100 mL measuring device (graduated cylinder or pipets) - Optional

Incubator - capable of maintaining a temperature of 35.0° + 0.5°C

Longwave UV light, 366 nm, 6-watt

 

 

Procedure

  1. Obtain one reagent packet for each sample to be tested. Label each bottle with the sample identification.
  2. Shake the sample vigorously 25 times.
  3. Aseptically pour the water sample into the bottle until the liquid reaches the 100 mL mark. If test samples are received in the bottles to be used for testing, shake and aseptically pour off any excess sample. If the bottles are not graduated, each sample must be measured with a sterile graduated cylinder or a sterile pipet.
  4. Carefully open the packet, avoiding inhalation of any dust material, and aseptically add the reagent to the 100 mL sample. Do not tap the reagent packet against the side of the test bottle.
  5. Cap the bottle and shake to dissolve the test reagent.
  6. Repeat steps 2-5 for the remaining samples.
  7. Incubate the test bottles at 35.0° + 0.5°C for 24 + 2 hours.
  8. After incubation, carefully remove the bottles from the incubator and examine for a color change indicative of the presence of Total Coliform.

    Reagent Total Coliform
    Positive Reaction Negative Reaction
    ColitagTM Yellow/Orange Clear, no color


  9. Examine any Total Coliform positive sample for the presence of E. coli by holding the longwave UV light 4-6 inches from the sample bottle in a dark location.

    Reagent E. coli
    Positive Reaction Negative Reaction
    ColitagTM Fluorescence No fluorescence


  10. If the color cannot be determined after the maximum incubation time, invalidate the sample. Collect another sample and retest using an alternate Total Coliform method.

 

 

 

Quantitative Technique

Colilert/Colisure

10-tube test

 

 

Reagents

Colilert 18, Colilert 24 or Colisure

Individual reagent packets for 100 mL sample volume

Premeasured quantity dispensed into test containers (100 mL sample volume)

For the MPN test only, Colilert 24 is available in test tubes for use with 10 mL sample volumes

 

Note: If Colisure reagent has been stored under refrigeration it must be warmed to room temperature prior to use.

 

Each new lot of chromogenic/fluorogenic reagent must be performance tested prior to use. (See "Media" section for instructions.)

 

 

Materials and Equipment

Sterile, clear glass or plastic bottles with screw caps; graduated at 100 mL

Sterile pipets, to deliver (TD), 10 mL capacity

Sterile test tubes, minimum 10 mL capacity, 10 per sample

Sterile 100 mL measuring device (Class A graduated cylinder or pipets) - Optional

Incubator - capable of maintaining a temperature of 35.0° + 0.5°C

Waterbath - capable of maintaining a temperature fo 35.0° + 0.5°C if using Colilert 18

Longwave UV light, 366 nm, 6-watt

Color comparator

 

 

Procedure

  1. Obatin one reagent packet or sterile bottle with premeasured reagent for each sample to be tested. Label each bottle with the sample identification.
  2. Shake the sample vigorously 25 times.
  3. Aseptically pour the water sample into the bottle until the liquid reaches the 100 mL mark. If test samples are received in teh bottles to be used for testing, aseptically pour off any excess sample. If the bottles are not graduated, each sample must be measured with a sterile graduated cylinder or a sterile pipet.
  4. Alternatively, if test tubes with premeasured Colilert 24 are to be used with 10 mL volumes, aseptically transfer 10 mL of sample to each of the 10 tubes. Replace the cap and shake each tube to dissolve.
  5. If using reagent packets, carefully open the packet, avoiding inhalation of any dust material, and aseptically add the reagent to the 100 mL sample. Do not tap the reagent packet against the side of the test bottle.
  6. Cap the bottle and shake to dissolve the test reagent.
  7. Aseptically transfer 10 mL of the prepared sample to 10 sterile test tubes.
  8. Repeat steps 2-7 for the remaining samples.
  9. Incubate the tubes at 35.0° + 0.5°C for the required time period. If Colilert 18 is used, the first 20 minutes of incubation must be in a waterbath at 35.0° + 0.5°C.

      Incubation Times (hours)
    Minimum Maximum
    Colilert 18 18 22
    Colilert 24 24 28
    Colisure 24 48
         


  10. After incubation, carefully remove the tubes from the incubator and examine for a color change indicative of the presence of Total Coliforms. Record the number of positive tubes on the datasheet.

    Reagent Total Coliform
    Positive Reaction Negative Reaction
    Colilert 18 or 24 Yellow Clear, no color
    Colisure Magenta Yellow


  11. Examine any Total Coliform positive test tubes for the presence of E. coli by holding the longwave UV light 4-6 inches from the tube(s) in a dark location. For both test substrates, the reaction is as follows. Record the number of positive tubes on the data sheet.

    Reagent E. coli
    Positive Reaction Negative Reaction
    Colilert or Colisure Fluorescence No fluorescence


  12. If the color still cannot be determined after the maximum incubation time, invalidate the sample. Collect another sample and test using an alternate Total Coliform method.

 

 

 

Reporting of MPN Results

Refer to the following table to determine the Most Probable Number (MPN) of Total Coliform and E. coli bacteria in the water sample.

 

 

 

Quantitative Technique

Colilert/Colisure

Quanti-Tray Tests

 

Note: Colisure cannot be used for the determination of Total Coliform and/or E. coli counts as required by the Surface Water Treatment Rule.

 

 

Reagent

Colilert 18, Colilert 24 or Colisure

Individual reagent packets for 100 mL sample volume

Premeasured quantity dispensed into test containers (100 mL sample volume)

 

Note: If Colisure reagent has been stored under refrigeration it must be warmed to room temperature prior to use.

 

Each new lot of chromogenic/fluorogenic reagent must be performance tested prior to use. (See "Media" section for instructions.)

 

 

 

Materials and Equipment

Sterile, clear glass or plastic bottles with screw caps; graduated at 10 mL

Sterile 100 mL measuring device (Class A graduated cylinder or pipet) - Optional

Quanti-Tray (QT) plastic container, sterile

200 MPN test format
2000 MPN test format

Incubator - capable of maintaining a temperature of 35.0° + 0.5°C

Waterbath - capable of maintaining a temperature of 35.0° + 0.5°C if using Colilert 18

Longwave UV light, 366 nm, 6-watt

Color comparator - for use in the Quanti-tray test

Heat sealer, with applicable templates

Quanti-Tray MPN tables - available from Idexx

Soft-tipped marker

 

 

 

Procedure

  1. Obtain one reagent packet or sterile bottle with premeasured reagent for each sample to be tested. Label each bottle with the sample identification. Also obtain one Quanti-Tray packet for each sample and label with the sample identification using a soft-tipped marker.
  2. Shake the sample vigorously 25 times.
  3. Aseptically pour the water sample into the bottle until the liquid reaches the 100 mL mark. If test samples are received in the bottles to be used for testing, aseptically pour off any excess sample. If the bottles are not graduated, each sample must be measured with a sterle graduated cylinder or a sterile pipet.
  4. If using reagent packets, carefully open the packet, avoiding inhalation of any dust material, and aseptically add the reagent to the 100 mL sample. Do not tap the reagent packet against the side of the test bottle.
  5. Cap the bottle and shake to dissolve the test reagent.
  6. Aseptically transfer the entire contents of the prepared sample to the desired Quanti-tray container. Avoid contamination of the sample. Gently tap the back of the QT container to release any trapped air bubbles.
  7. Heal seal the QT following manufacturer instructions.
  8. Repeat steps 2-7 for the remaining samples.
  9. Incubate the Quanti-Tray packets at 35.0° + 0.5°C for the required time period. If Colilert 18 is used, the first 20 minutes of incubation must be in a water at 35.0° + 0.5°C. Position the QT units throughout the incubator in a manner which wil not restrict air flow.

      Incubation Times (hours)
    Minimum Maximum
    Colilert 18 18 22
    Colilert 24 24 28
    Colisure 24 48
         


  10. After incubation, carefully remove the trays from the incubator, and examine for a color change indicative of the presence of Total Coliforms. Record the number of wells which are positive on the datasheet. When using Quanti-Tray 2000, individually record the number of large and number of small wells that are positive.

    Reagent Total Coliform
    Positive Reaction Negative Reaction
    Colilert 18 or 24 Yellow Clear, no color
    Colisure Magenta Yellow


  11. Examine any Total Coliform positive trays for the presence of E. coli by holding the longwave UV light 4-6 inches from the sample in a dark location. For all test substrates, the reaction is as follows. Record the number of positive wells on the data sheet. When using Quanti-Tray 2000, individually record the number of large and number of small wells that are positive.

    Reagent E. coli
    Positive Reaction Negative Reaction
    Colilert or Colisure Fluorescence No fluorescence


  12. If the color still cannot be determined after the maximum incubation time, invalidate the sample. Collect another sample and test using an alternate Total Coliform method.

 

 

 

Reporting of MPN Results

Refer to the Idexx Quanti-tray tables (included) to determine the Most Probable Number (MPN) of Total Coliform and E. coli bacteria in the water sample.

 

 

 

Chromogenic/Fluorogenic Quality Control

The following list quality control tests required for the C/F test procedures. This list is not intended to outline all QC tests, but to highlight those most critical. Other QC checks, such as monitoring of refrigerator temperatures and autoclave checks and records, are discussed in the Quality Control section of this course.

 

 

General

QC Check Frequency

Bottle Sterility Check Each new lot
Thermometer Calibrations Annually
Incubator temperatures Twice daily
Water bath temperatures Twice daily
Evaluation of test tubes for autofluorescence Each new lot
Proper function of UV light Weekly

 

 

Reagents

Receipt date and lot number

Use within expiration date

Each lot checked for autofluorescence before use

Sterility check of each new lot prior to use

Upon receipt and prior to use, perform control checks on each lot of reagent. Acceptable QC reactions must be obtained prior to use.

 

 

 

 

 

 

 

Assignment

Please answer the following questions and send via mail, fax or email (as an attachment) to your instructor.

  1. List the advantages of enzymatic substrate systems.
  2. List the disadvantages of enzymatic substrate systems.

 

 

 

Quiz

Answer the questions in the Lesson 10 quiz .  When you have gotten all the answers correct, print the page and either mail or fax it to the instructor. You may also take the quiz online and directly submit it into the database for a grade.