Variances seen in Variances seen in Bacterial Analysis Bacterial Analysis

for Water and for Water and Waste Water Waste Water

SamplingSamplingGretchen HathawayGretchen HathawayWhatman Sales RepresentativeWhatman Sales Representative

July 19, 2007July 19, 2007

TopicsTopics

M-Endo (LTB, BGB, & ECB)M-Endo (LTB, BGB, & ECB) M-FC & M-FC with rosolic acidM-FC & M-FC with rosolic acid MI Broth and AgarMI Broth and Agar ColiCheck & ColiCheck with MUGColiCheck & ColiCheck with MUG

M-EndoM-Endo

M-EndoM-Endo M-Endo Broth is used for the enumeration M-Endo Broth is used for the enumeration

of coliforms by membrane filtration. of coliforms by membrane filtration. Ideal for drinking water and waste water Ideal for drinking water and waste water

contaminant testing, M-Endo Coliform Broth contaminant testing, M-Endo Coliform Broth is used to differentiate lactose from non-is used to differentiate lactose from non-lactose fermenting intestinal organisms.lactose fermenting intestinal organisms.

M-EndoM-Endo : Description: Description M-Endo is a red colored media, M-Endo is a red colored media,

which needs to be stored in the dark which needs to be stored in the dark to prevent discoloration of the to prevent discoloration of the media. media.

Gram-positive bacteria are inhibited Gram-positive bacteria are inhibited on this media by the desoxycholate on this media by the desoxycholate and lauryl sulfate. The addition of and lauryl sulfate. The addition of ethanol increases the antibacterial ethanol increases the antibacterial nature of the formulation.nature of the formulation.

Lactose fermenting organisms form Lactose fermenting organisms form aldehydes, which react with Schiffs aldehydes, which react with Schiffs reagent (basic fuchsin and sodium reagent (basic fuchsin and sodium sulfite) to give red colored zones sulfite) to give red colored zones around the colonies. around the colonies.

Coliform colonies are therefore red Coliform colonies are therefore red with a characteristic metallic sheen.with a characteristic metallic sheen.

M-Endo: InterpretationM-Endo: Interpretation

Production of both acid Production of both acid and aldehyde by lactose and aldehyde by lactose fermenters, such as fermenters, such as Escherichia coliEscherichia coli, produce , produce deep red colonies that deep red colonies that color the surrounding color the surrounding medium and have a green medium and have a green metallic sheen. metallic sheen.

Non-lactose fermenters Non-lactose fermenters form colorless, translucent form colorless, translucent coloniescolonies

M-Endo VerificationM-Endo Verification The chart to the side The chart to the side

demonstrates the demonstrates the need of verification need of verification steps due to the type steps due to the type of growth.of growth.

Verification is done in Verification is done in durham tubes with durham tubes with Lauryl Tryptose Lauryl Tryptose Broth, Brilliant Green Broth, Brilliant Green Broth (to confirm Broth (to confirm Total coliform) and Total coliform) and EC Broth (to confirm EC Broth (to confirm Fecal coliform).Fecal coliform).

ColiCheck and ColiCheck and ColiCheck with ColiCheck with

MUGMUG

ColiCheck and ColiCheck ColiCheck and ColiCheck with MUGwith MUG

ColiCheckColiCheck is used for the is used for the presumptive identification of presumptive identification of coliforms in water samples by a coliforms in water samples by a presence/absence technique.presence/absence technique.

Two product types are available, Two product types are available, ColiCheck that identifies coliforms ColiCheck that identifies coliforms and ColiCheck with MUG that and ColiCheck with MUG that checks for the presence/absence of checks for the presence/absence of Escherichia coli.Escherichia coli.

ColiCheck and ColiCheck ColiCheck and ColiCheck with MUGwith MUG

DescriptionDescription Bromocresol purple is a pH indicator which Bromocresol purple is a pH indicator which

demonstrates a color change from purple to demonstrates a color change from purple to yellow in the presence of acid.yellow in the presence of acid.

Lactose fermenting organisms produce acid Lactose fermenting organisms produce acid which initiates the color change. which initiates the color change.

The presence of coliforms is detected with The presence of coliforms is detected with greater sensitivity by use of a relatively large greater sensitivity by use of a relatively large sample volume (100ml) in a single bottle. sample volume (100ml) in a single bottle.

The addition of MUG (4-methyl-umbellrifryl-β- The addition of MUG (4-methyl-umbellrifryl-β- glucuronide) which is a fluorogenic enzyme glucuronide) which is a fluorogenic enzyme allows the media to selectively identify allows the media to selectively identify E. coliE. coli..

MUG is hydrolyzed by the MUG is hydrolyzed by the E. coliE. coli specific enzyme specific enzyme β-β- glucuronidase to release 4-glucuronidase to release 4-Methylumbelliferone which fluoresces under UV Methylumbelliferone which fluoresces under UV light (366nm).light (366nm).

ColiCheck and ColiCheck ColiCheck and ColiCheck with MUG Interpretationwith MUG Interpretation

Yellow color in the sample Yellow color in the sample after 24 hours incubation after 24 hours incubation at 35° C indicates the at 35° C indicates the presence of coliform presence of coliform organisms. organisms.

A red sample after 24 hour A red sample after 24 hour incubation at 35° C should incubation at 35° C should be incubated for further be incubated for further 24 hours. 24 hours.

A red sample at 48 hours A red sample at 48 hours incubation at 35° C incubation at 35° C indicates no coliform indicates no coliform organisms present in the organisms present in the sample.sample.

ColiCheck w/ MUG: ColiCheck w/ MUG: Florescence under UV Florescence under UV Light is specific for Light is specific for E. coliE. coli..

M-FC brothM-FC broth

M-FCM-FC

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

M-FC: DescriptionM-FC: Description

Allows the development of fecal coliforms at elevated temperatures (44.5° C).

M-FC: InterpretationM-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.

M-FC with M-FC with Rosolic AcidRosolic Acid

M-FC with Rosolic AcidM-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.

M-FC with Rosolic Acid: M-FC with Rosolic Acid: DescriptionDescription

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.

M-FC with Rosolic Acid: M-FC with Rosolic Acid: InterpretationInterpretation

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.

MI Broth and MI Broth and AgarAgar

MI Broth and AgarMI 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.

MI Broth and Agar: MI Broth and Agar: DescriptionDescription

MI Broth detects the presence of MI Broth detects the presence of coliform bacteria by the production coliform bacteria by the production of β-of β-galactosidasegalactosidase, which cleaves the , which cleaves the substrate MUGal to produce 4- substrate MUGal to produce 4- Methylumbelliferone, which Methylumbelliferone, which fluoresces on exposure to UV light fluoresces on exposure to UV light (366nm). (366nm).

Non-coliforms do not produce this Non-coliforms do not produce this enzyme and therefore do not enzyme and therefore do not fluoresce on the medium.fluoresce on the medium.

Escherichia coli Escherichia coli is detected by the is detected by the compound IBDG. compound IBDG.

The β-The β-glucuronidase glucuronidase produced by produced by E. E. coli coli cleaves the substrate to produce cleaves the substrate to produce a blue indigo color in the colonies.a blue indigo color in the colonies.

As As E. coli E. coli is also a total coliform, and is also a total coliform, and also produces β-galactosidase it will also produces β-galactosidase it will also fluoresce.also fluoresce.

The antibiotic cefsulodin is added to The antibiotic cefsulodin is added to inhibit the growth of gram-positive inhibit the growth of gram-positive bacteria and some non-coliform bacteria and some non-coliform gram negative bacteria that can gram negative bacteria that can cause false positive reactions. cause false positive reactions.

Fewer False Positives are Fewer False Positives are seen with MI Broth.seen with MI Broth.

MI Broth and Agar: MI Broth and Agar: InterpretationInterpretation

Fluorescent blue colonies are Fluorescent blue colonies are Escherichia coliEscherichia coli..

Colonies that demonstrate Colonies that demonstrate blue/white fluorescence blue/white fluorescence where the colonies are clear, where the colonies are clear, cream or pale yellow in color cream or pale yellow in color are other coliform are other coliform organisms. organisms.

Clear colonies that do not Clear colonies that do not fluoresce are non-coliforms.fluoresce are non-coliforms.

Short incubation period… Short incubation period… Positive control for Positive control for E. coli E. coli ATCC 25922 and ATCC 25922 and Enterobacter aerogenes Enterobacter aerogenes ATCC 13048 can be achieved ATCC 13048 can be achieved in 18-24 hours at 35°C.in 18-24 hours at 35°C.

Negative control… Complete Negative control… Complete inhibition of inhibition of Pseudomonas Pseudomonas aeruginosa aeruginosa ATCC 10145 ATCC 10145 occurs at 35°C after 24 occurs at 35°C after 24 hours.hours.

Other Products offered by Other Products offered by Whatman Whatman

Monitors/ Analytical Filter FunnelsMonitors/ Analytical Filter Funnels Membrane Systems and Membrane ButlerMembrane Systems and Membrane Butler Dilution BottlesDilution Bottles Petri dishes with padsPetri dishes with pads Filter papersFilter papers Glass Fiber for Total Suspended and Volatile Glass Fiber for Total Suspended and Volatile

Solids, & TCLPSolids, & TCLP pH paperspH papers Syringe filtersSyringe filters

The EndThe End