Microbiology Guide: Media Overview

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Document ID TE8827

Published Date

Published Date 05/21/2019
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Microbiology Guide: Media Overview
Summary
Microbiology Guide: Media and Equipment Selection; Media Overview.
Answer
From: Microbiology Guide: Media and Equipment Selection
  • Membrane Filtration (MF) Technique  
    • The Membrane Filtration (MF) method is a fast, simple way to estimate bacterial populations in water that is low in turbidity. This method is especially useful when evaluating large sample volumes or performing many tests daily. In the initial step, an appropriate sample volume passes through a membrane filter with a pore size small enough (0.45 micron) to retain the bacteria present. The filter is placed on an absorbent pad (in a petri dish) saturated with a culture medium that is selective for the growth of a specific organism. The petri dish containing the filter and pad is incubated, inverted, for 24 hours at the appropriate temperature. After incubation, the colonies that have grown are identified and counted using a low-power microscope.
    • MF media can be received as a Hach’s PourRite™ Ampule or Prepoured Agar Plates.
      • Hach PourRite™ Ampules contain prepared selective media. This eliminates the measuring, mixing, and autoclaving needed when preparing dehydrated media. The ampules are designed with a large, unrestrictive opening that allows media to pour out easily. Simply break off the top of the ampule and pour the medium onto an absorbent pad in an MF petri dish. Each ampule contains enough medium for one test. Medium packaged in PourRite™ Ampules has a shelf-life of one year. Ampules are shipped with a Certificate of Analysis and have an expiration date printed on the label.
      • Hach’s Prepared Agar Plates contain prepared media, which eliminates the measuring, mixing, and autoclaving associated with preparing dehydrated medium. The plates are ready to use without the need for further preparation and contain enough medium for one test. The shelf life of the prepared agar plates varies from 3 months to 1 year. Plates are shipped with a Certificate of Analysis and have an expiration date printed on the label.
 
  • Most Probable Number or Multiple Tube Fermentation Technique (MPN)
    • Method uses a specified number of test tubes to statistically predict the number of organisms present (based on the expected population of organisms in the sample). The MPN method is ideal for wastewater samples and nonpotable samples, because the analyst can accommodate highly turbid samples by diluting prior to analysis. No filtering is necessary.
    • The MPN method is performed by using screw-capped tubes that contain sterile broth medium. Some tubes contain inverted inner vials (durham tubes) for gas collection. Simply add sample to a tube of prepared medium and incubate. If coliforms are present, gas is produced and is trapped in the inner vial. Cloudiness, turbidity and/or fluorescence also can indicate the presence of coliforms.
 
  • Presence / Absence Technique (PA)
    • PA Broth is ideal for screening drinking water samples for total coliforms. The method is a simple modification of the multiple-tube method. It uses Presence–Absence Broth containing bromcresol purple (BCP). BCP detects acidity formed during lactose fermentation by the bacteria.
      • Simply combine 100 mL of sample and PA Broth, incubate for 24–48 hours, and check for a color change. A yellow color indicates the presence of total coliforms.
    • PA Broth with MUG allows simultaneous detection of total coliform bacteria and Escherichia coli (E. coli). In addition to the PA Broth, this medium contains MUG (4-methylumbelliferyl-b-D-glucuronide) reagent. MUG reagent produces a fluorogenic product when hydrolyzed by b-D-glucuronidase, an enzyme produced by E. coli. MUG detects non-gas producing (anaerogenic) strains of E. coli and works well when competitive organisms are present.
      • Simply combine 100 mL of sample and P–A Broth with MUG, incubate for 24–48 hours and check for a color change and fluorescence. A yellow color indicates the presence of total coliforms. To detect E. coli examine the sample under a long-wave ultraviolet (UV) light (4–6 watts). Fluorescence indicates the presence of E. coli.
    • PA Broth and PA Broth with MUG come packaged in disposable bottles and in glass ampules. To reduce the cost of medium, both bottles and ampules contain 20 mL of 6X strength medium. The medium is sterilized by membrane filtration to prevent degradation. Disposable bottles and ampules are shipped with a Certificate of Analysis and have an expiration or manufacture date printed on the label.
    • The Modified Colitag™ Presence/Absence (PA) method detects as few as 1 colony-forming unit (CFU) of E. coli and other coliform bacteria in 100 mL of water. This media is the fastest PA media with methods that can be performed in 16 or 22 hours. Results are valid for 32 hours.
      • The Modified Colitag™ method comes packaged in snap packs. Store the snap packs at room temperature away from light. The Modified Colitag™ method shelf life and warranty period is 22 months from the manufacture date that is on the snap packs. The product expiration date is on the box and on the individual blisters.
 
  • Biological Activity Reaction Test (BART™) is a water testing system for nuisance bacteria and can involve several different tests. These tests detect the activity (aggressivity) of these nuisance bacteria by the time lag (TL, measured in the number of days from the start of the test to when the first reaction is observed). The longer the TL before the observation of activity, the less aggressive the bacteria are in that particular sample. BART™ testers can be incubated at room temperature (an incubator is not required).
 
  • Dip Slides & Paddle Testers: Self-contained, ready-to-use, and provides semi quantitative results for screening purposes. Can be used for liquid or solid surface testing. The paddle tester is double sided with dual agar.  One side gives a non-selective total count of bacteria.  The other side is selective for a specific group of bacteria.
 
  • PathoScreen™: PathoScreen™ will detect the presence of hydrogen sulfide - producing bacteria (including Salmonella, Citrobacter, Proteus, Edwardsiella, and some species of Klebsiella). Indigenous E. Coli will not interfere with this test. The test uses an incubation temperature at 25 - 35 °C. Therefore, an incubator is not required. The test is well-suited for monitoring drinking water systems in developing tropical countries, in remote field locations, and in disaster or emergency situations. PA and MPN type analyses can be run using PathoScreen™.
 
  • HPC Peel Plate®: The Heterotrophic Plate Count (HPC) Peel Plate® method is a simplified method for estimating heterotrophic bacteria populations in water. Easy and convenient to use, the HPC method does not require an incubator as incubation can be done at ambient temperature.
 
  • LuminUltra: LuminUltra methods measure the Adenosine Triphosphate (ATP) concentration produced by living organisms in a sample (including heterotrophic bacteria, SRBs, yeast, algae, molds, nitrifiers, and protozoa), and thus, provide an estimate of the total biological activity of the sample.  These methods use a photomoter to measure ATP and do not require incubation. They are rapid methods that can be done in a little over 5 minutes.

Testing Technique Overview


Additional information regarding microbiological methods can be found on the Hach website in the LIT7015 document.

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