Variances seen in Bacterial Analysis for Water and Waste Water Sampling

1. Variances seen in Bacterial Analysis for Water and Waste Water Sampling Gretchen Hathaway Whatman Sales Representative July 19, 2007

2. Topics • M-Endo (LTB, BGB, & ECB) • M-FC & M-FC with rosolic acid • MI Broth and Agar • ColiCheck & ColiCheck with MUG

3. M-Endo

4. M-Endo • M-Endo Broth is used for the enumeration of coliforms by membrane filtration. • Ideal for drinking water and waste water contaminant testing, M-Endo Coliform Broth is used to differentiate lactose from non-lactose fermenting intestinal organisms.

5. M-Endo: Description • M-Endo is a red colored media, which needs to be stored in the dark to prevent discoloration of the media. • Gram-positive bacteria are inhibited on this media by the desoxycholate and lauryl sulfate. The addition of ethanol increases the antibacterial nature of the formulation. • Lactose fermenting organisms form aldehydes, which react with Schiffs reagent (basic fuchsin and sodium sulfite) to give red colored zones around the colonies. • Coliform colonies are therefore red with a characteristic metallic sheen.

6. M-Endo: Interpretation • Production of both acid and aldehyde by lactose fermenters, such as Escherichia coli, produce deep red colonies that color the surrounding medium and have a green metallic sheen. • Non-lactose fermenters form colorless, translucent colonies

7. M-Endo Verification • The chart to the side demonstrates the need of verification steps due to the type of growth. • Verification is done in durham tubes with Lauryl Tryptose Broth, Brilliant Green Broth (to confirm Total coliform) and EC Broth (to confirm Fecal coliform).

8. ColiCheck and ColiCheck with MUG

9. ColiCheck and ColiCheck with MUG • ColiCheck is used for the presumptive identification of coliforms in water samples by a presence/absence technique. • Two product types are available, ColiCheck that identifies coliforms and ColiCheck with MUG that checks for the presence/absence of Escherichia coli.

10. ColiCheck and ColiCheck with MUGDescription • Bromocresol purple is a pH indicator which demonstrates a color change from purple to yellow in the presence of acid. • Lactose fermenting organisms produce acid which initiates the color change. • The presence of coliforms is detected with greater sensitivity by use of a relatively large sample volume (100ml) in a single bottle. • The addition of MUG (4-methyl-umbellrifryl-β- glucuronide) which is a fluorogenic enzyme allows the media to selectively identify E. coli. • MUG is hydrolyzed by the E. coli specific enzymeβ-glucuronidase to release 4-Methylumbelliferone which fluoresces under UV light (366nm).

11. ColiCheck and ColiCheck with MUG Interpretation • Yellow color in the sample after 24 hours incubation at 35° C indicates the presence of coliform organisms. • A red sample after 24 hour incubation at 35° C should be incubated for further 24 hours. • A red sample at 48 hours incubation at 35° C indicates no coliform organisms present in the sample. • ColiCheck w/ MUG: Florescence under UV Light is specific for E. coli.

12. M-FC broth

13. M-FC • M-FC media (membrane Fecal Coliform media) is used for the detection of fecal coliforms as an index of water pollution.

14. M-FC: Description • Allows the development of fecal coliforms at elevated temperatures (44.5° C).

15. M-FC: Interpretation • Bile salts included in the medium inhibit the growth of gram-positive bacteria. • Fecal coliforms ferment lactose at elevated temperatures and produce blue colonies. • Other organisms form grey to cream colonies.

16. M-FC with Rosolic Acid

17. M-FC with Rosolic Acid • M-FC Broth with Rosolic Acid is used in waste water analysis. M-FC (Membrane Fecal Coliform) media is designed for the detection of fecal coliforms as an index of water pollution.

18. M-FC with Rosolic Acid: Description • M-FC with Rosolic Acid acts and functions in the same way as m-FC Broth. • Rosolic acid inhibits bacterial growth in general, except for fecal coliforms.

19. M-FC with Rosolic Acid: Interpretation • Bile salts inhibit non-enteric bacteria. • Aniline blue indicates the ability of fecal coliforms to ferment lactose to acid that causes a pH change in the medium. • Other organisms form grey to cream colonies.

20. MI Broth and Agar

21. MI Broth and Agar • Used for the simultaneous detection of total coliforms and Escherichia coli in water according to the Surface Water Treatment Rule (USEPA) and the Total Coliform Rule (USEPA), which applies to drinking water.

22. MI Broth and Agar: Description • MI Broth detects the presence of coliform bacteria by the production of β-galactosidase, which cleaves the substrate MUGal to produce 4- Methylumbelliferone, which fluoresces on exposure to UV light (366nm). • Non-coliforms do not produce this enzyme and therefore do not fluoresce on the medium. • Escherichia coli is detected by the compound IBDG. • The β-glucuronidase produced by E. coli cleaves the substrate to produce a blue indigo color in the colonies. • As E. coli is also a total coliform, and also produces β-galactosidase it will also fluoresce. • The antibiotic cefsulodin is added to inhibit the growth of gram-positive bacteria and some non-coliform gram negative bacteria that can cause false positive reactions. • Fewer False Positives are seen with MI Broth.

23. MI Broth and Agar: Interpretation • Fluorescent blue colonies are Escherichia coli. • Colonies that demonstrate blue/white fluorescence where the colonies are clear, cream or pale yellow in color are other coliform organisms. • Clear colonies that do not fluoresce are non-coliforms. • Short incubation period… Positive control for E. coli ATCC 25922 and Enterobacter aerogenes ATCC 13048 can be achieved in 18-24 hours at 35°C. • Negative control… Complete inhibition of Pseudomonas aeruginosa ATCC 10145 occurs at 35°C after 24 hours.

24. Other Products offered by Whatman • Monitors/ Analytical Filter Funnels • Membrane Systems and Membrane Butler • Dilution Bottles • Petri dishes with pads • Filter papers • Glass Fiber for Total Suspended and Volatile Solids, & TCLP • pH papers • Syringe filters

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