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Counting Vibrios


Roger Kelso ( While I have some experience doing plate counts of various bacteria, I’m curious about how other folks do them.


Giovanni Chasin ( Roger, for seawater and brackishwater Vibrios, we prepare TCBS agar plates with the same salinity as the samples (or very close to them).  After proper sampling, inoculation and incubation, we do a direct count of the bacterial colonies, grouped by morphology (size, color and type of border) expressed in colony forming units (CFUs/ml), taking into account the volume of inoculum and any dilution that was required.  For samples from the hemolymph and hepatopancreas, the procedure is similar, except samples from hepatopancreas are expressed at CFU/g.  Normally, an electronic device is not required for counting well-defined colonies, however, with multiple very tiny colonies and a lot of samples to run, the task is not too easy, and an automatic colony counter is recommended.  I used to have problems counting or quantifying colonies occurring as large non-uniform spots.


If you know how to quantify large, non-uniform, spot colonies, please advise.


To differentiate among Vibrios (alginolyticus, vulnificus and parahaemolyticus), I use ChromAgar.  Nowadays, however, I am just monitoring V. parahaemolyticus.


Daniel Gruenberg ( Roger, I have an app on my iPhone called “CFU Scope”.  Just plate 0.1 ml with a sterile insulin syringe onto TCBS plates and then wait 12 hours and count with the app and multiply the result by ten to get CFUs per ml.


Roger Kelso ( Daniel, does V. parahaemolyticus normally produce blue-green colonies on TCBS agar?


Giovanni, I have been using heterotrophic plate count as a procedure for estimating the number of live heterotrophic bacteria in our water and the efficiency of our disinfection/filtration systems.  Your suggestion of TCBS agar is very helpful for my screening process.  I will look into the ChromAgar.


I use a counting program named “OpenCFU” to speedup/standardize our counting procedures.  I will look into the iPhone app.


Your question about the large non-uniform spot colonies is interesting.  Sometimes they are caused by a contaminate.  Can you send me a photo of your plate?


Giovanni Chasin ( Roger, you must use ChromAgar Vibrio to screen V. cholera, V. vulnificus, V. alginolyticus and V. parahaemolyticus.  In TCBS agar parahaemolyticus always occurs as a green colony, but with some minor differences in pigmentation.  Depending on the brand of agar used when plating parahaemolyticus, TCBS agar leads to confusion with vulnificus.  ChromAgar Vibrio resolves this misinterpretation.  I will look into my digital file for an image of a large irregular sacarose positive spot-like colony to send you.


Nelson Gerundo ( When doing green and yellow diversity tank assessment, always rely on fast, easy to install and use advanced digital counting software.  It’s very accurate and not prone to human errors.  Digital counting software also has higher visual sharpness in recognizing spots on the head of the plate.


Sources: 1. The Shrimp List (a mailing list for shrimp farmers).  Subject: Vibrio Plate Counts.  April 11–12, 2017.  2. Bob Rosenberry, Shrimp News International, April 19, 2017.

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