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/
1
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
Copyright © C. R. Cooper, Jr.
http://hoopmanscience.pbworks.com/w/page/47828206/Water%20Monitoring%3A%20%20Coliform
2
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)
Copyright © C. R. Cooper, Jr.
http://fr.nextews.com/fd561c0d/
3
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
Copyright © C. R. Cooper, Jr.
4
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
Copyright © C. R. Cooper, Jr.
5
Procedure (cont.)
• Both Groups: Filter the appropriate dilutions (see https://youtu.be/Ev5B6PxITh0, especially from 3:37 minutes to the end)
Copyright © C. R. Cooper, Jr.
6
Coliform-Enterococcus Isolation Fall 2019
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
Copyright © C. R. Cooper, Jr.
7
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)
Copyright © C. R. Cooper, Jr.
8
Procedure (cont.)
Copyright © C. R. Cooper, Jr.
mEI agar with Enterococcus colonies (left) and mFCagar with E. coli colonies (right)
9
Procedure (cont.)
• Both Groups: Observe both TSB tubes• Original sample = “++++”
(expected growth)• Growth in other tube =
“0” to “++++”
Copyright © C. R. Cooper, Jr.
10
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
Copyright © C. R. Cooper, Jr.
11
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
12
Coliform-Enterococcus Isolation Fall 2019
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
Copyright © C. R. Cooper, Jr.
13
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.
Copyright © C. R. Cooper, Jr.
14
Questions?
Copyright © C. R. Cooper, Jr.
https://www.huffingtonpost.com/barbara-jacoby/asking-questions-is-really-hard_b_7052722.html
15