Procedure Procedure (cont.)

Coliform-Enterococcus Isolation Fall 2019

Dr. Cooper 1

BIOL 3702L:Membrane

Filter Detection of Coliforms and

Enterococci

Copyright © C. R. Cooper, Jr.

http://www.aviano.af.mil/News/Article-Display/Article/724709/mdss-airmen-ensure-success-through-support/

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Today’s Exercise

• Purpose of today’s exercise:• Determine fecal coliform counts and

enterococci counts from an environmental source by• Using previously acquired skills• Employing the filter membrane technique

• Draw conclusions regarding the water quality of the sample based upon the results derived


http://hoopmanscience.pbworks.com/w/page/47828206/Water%20Monitoring%3A%20%20Coliform

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Procedure

•Water is collected for use by both lab groups• Group A will use

mFC agar (to count coliforms)• Group B will use

mEI agar (to count enterococci)


http://fr.nextews.com/fd561c0d/

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Procedure (cont.)

• Both Groups: Use the electric pipette to place 1 ml of sample in a tube of TSB; incubate at 37°C for 18-24 hours• Video: https://youtu.be/4VTTE_oWs58• See portions: 0-50 seconds and 3:37 to end


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Procedure (cont.)

• Both Groups:• Place filter in filtration unit and properly secure

top of unit• Place approximately 50 ml of Butterfield’s

buffer in top unit• To one unit, add 10 ml of sample to buffer• To other unit, add 1 ml of sample to buffer


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Procedure (cont.)

• Both Groups: Filter the appropriate dilutions (see https://youtu.be/Ev5B6PxITh0, especially from 3:37 minutes to the end)


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https://youtu.be/4VTTE_oWs58

https://youtu.be/Ev5B6PxITh0


Dr. Cooper 2

Procedure (cont.)

• Remove filters and place on media• Group A – mFC agar• Group B – mEI agar

• Incubate plates• mFC agar at 44.5°C for 22-26 hours (Group A)• mEI agar at 41°C for 22-26 hours (Group B)

• Both Groups: Transfer 1 ml of eluate from each filter unit (the liquid that is in the bottom chamber) to a TSB tube and incubate at 37°C for 24 hours


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Procedure (cont.)

• Remove plates from incubators and count the number of colonies as follows:• mFC plates (Group A) presumptive E. coli

colonies (i.e., blue in color) • mEI plates (Group B) - presumptive

Enterococcus colonies (i.e., small colonies with blue halo surrounding them)


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Procedure (cont.)


mEI agar with Enterococcus colonies (left) and mFCagar with E. coli colonies (right)

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Procedure (cont.)

• Both Groups: Observe both TSB tubes• Original sample = “++++”

(expected growth)• Growth in other tube =

“0” to “++++”


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Procedure (cont.)

• Group A: From one mFC agar plate, streak a single presumptive coliform on EMB agar; incubate at 37°C for 18-24 hours• Group B: From one mEI agar plate, streak a

presumptive Enterococcus isolate on BHI (Brain Heart Infusion) agar; incubate at 37°C for 18-24 hours


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Procedure (cont.)• Groups A: • Select green-metallic

colony from EMB agar plate and transfer to TSA slant; incubate at 37°C for 18-24 hours• Confirm identification

as E. coli using EC Broth one or more of the IMViC tests• Save isolate for next

laboratory exerciseCopyright © C. R. Cooper, Jr.

http://www.socimage.net/user/_microbiology_/2208343490

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Dr. Cooper 3

Procedure (cont.)

• Groups B: • Select a single colony

from BHI agar plate and transfer to BHI slant; incubate at 37°C for 18-24 hours• Confirm using BHI-NaCl

broth and Esc/PYR test• Save isolate for next

laboratory exercise


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Lab Report Expectations

• SHARE the RAW data between groups• HOWEVER, all calculations and answers to

the questions on the data sheets must be completed WITHOUT COLLABORATION!• YOUR ANSWERS MUST BE CLEAR, CONCISE,

AND GRAMMATICALLY SOUND SENTENCES!•Where appropriate, cite resources.


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Questions?


https://www.huffingtonpost.com/barbara-jacoby/asking-questions-is-really-hard_b_7052722.html

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Documents

Procedure Procedure (cont.)