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Vibrio test; Axillary to Sylvio's Utah Zuchter thread
Topic Started: Feb 11 2009, 08:43:20 AM (251 Views)
Post #1 Feb 11 2009, 08:43:20 AM	Clint
For background on this read Sylvio's Utah Zuchter thread. In short we want to see if vibrio spp are present in two common sources of Brine Shrimp cysts. The two sources I am checking are the San Francisco and Great Salt Lake varieties sold by Brine Shrimp Direct. I plan to plate regularly hatched and decapsulated cysts from both cyst varieties. I mixed salt water at ~1.023 sg let it sit for about a week then split the water into canning bottles. I pressure cooked the bottles of water for about 1 hour at 15 psi. After letting the water cool, I floated regular cysts of both varieties in separate bottles. later I decapsulated samples of both varieties useing the method spelled out in Wilkerson's clownfish book. I modified it slightly because I was trying to do this using sterile technique. I did not use a dechlorinator; I rinsed the cysts using sterile salt water and then added a few of the cysts to about 300 ml of sterile salt water. I will plate a control of my sterile salt water to verify sterile technique. I am working with some time constraints windows for use of the lab to plate and grow the samples. I hope the cysts hatch in time to allow me to plate them today. I may run into some trouble with hatching because I did not have a sterile way to aerate the decapsulated cysts. Additionally I had to move the floating bottles of regular cysts after about 13 hours and the cysts settled to the bottom of the bottle. I hope enough will hatch to do what I need. If I don't get enough to hatch I will have to find and use a shaker table to improve hatch.	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215
Post #2 Feb 11 2009, 10:43:50 AM	jpiotrowski
I have expired dryslide (oxidase testing). If you get a culture we can use it to test oxidase +/- (Vibrio is oxidase positive).	breeding stock Posts: 85 Group: Members Member #48 Joined: September 16, 2008
Post #3 Feb 11 2009, 10:48:47 AM	Suzy
I find this very exciting! So, if you are not able to hatch using a sterile method, how can we be sure that the bacteria is not introduced while hatching? That is assuming it is there in the end. I still am not convinced that Vibrio spp are pathogenic. It seems to me to be an opportunist bacteria like Strep is in the terrestrial world. Thanks for doing this, guys!	Seahorse Whisperer Posts: 3,047 Group: Admin Member #1 Joined: June 28, 2008
"i was informed of some dolphin related testing going on up there" Too Funny!
Post #4 Feb 11 2009, 01:35:20 PM	Clint
Even if there are non pathologic manifestations you could still have very problematic manifestations. Even many of the Streps and Staphs of the world that are very common, and as you know, they can be really bad actors as well. Most vibrios are not thought to be a problem, but Sylvio has two species that are supposed to be the MRSA of the fish world.	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215
Post #5 Feb 12 2009, 12:13:12 AM	Amie
Hey Clint, I'm really excited that you are working on this. I can't wait to hear about the results. It seems like if you hatched the brineshrimp in a dish without aeration it would cut down on your exposure to pathogens in the air. And couldn't you put the disc in a container, before adding the bs cysts, that you could close - that would cut down the exposure even further. You are more than welcome to use the 2 discs that I have if you need them. I could make a clear acrylic box for them to sit in that just slightly bigger than they are....or just a 'top' to set over them, that might be even better.	stormy, stormy nights Posts: 2,362 Group: Admin Member #4 Joined: June 29, 2008
http://www.spawar.navy.mil/sandiego/technology/mammals/interns.html Tell them Adam sent you.
Post #6 Feb 12 2009, 08:25:12 AM	Josh W
To cut down on pathogen exposure in the air, you could always run a pre-filter, I have some 0.2 micron filters that might fit nicely in an air line. The one problem is they will most likely create some back pressure.	breeding stock Posts: 236 Group: Members Member #33 Joined: September 3, 2008
Post #7 Feb 12 2009, 10:47:58 AM	Amie
That might be good, but I've been thinking that you shouldn't worry about it for now. Just run it the way you are doing now and see what the outcome is. If it's filled with vibrio, then narrow it down from there. You've eliminated the possibility that's it's coming from the water or the cysts themselves, so if it's there, it will be coming from the air or the embryos. Edited by Amie, Feb 12 2009, 10:49:04 AM.	stormy, stormy nights Posts: 2,362 Group: Admin Member #4 Joined: June 29, 2008
http://www.spawar.navy.mil/sandiego/technology/mammals/interns.html Tell them Adam sent you.
Post #8 Feb 12 2009, 11:49:28 AM	jpiotrowski
Right, simply run it the way you normally do to see if you can even detect vibrio. Are you going to use bile salt plates? I tried growing some salt water on TSA and the result wasn't pretty, nor did it smell good. I am not sure what I grew.	breeding stock Posts: 85 Group: Members Member #48 Joined: September 16, 2008
Post #9 Feb 12 2009, 04:22:21 PM	Clint
I am using TCBS which I'm told is selective for Vibrio. I did get a few shrimp to hatch and if the bugs are there that should be enough to infect the water I would think. At 18 hrs I cannot detect any growth whatsoever. I'll check again later.	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215
Post #10 Feb 12 2009, 04:24:56 PM	Clint
Maybe I should have started with blood agar then transferred to selective media but that seemed like an extra step considering that I am only interested in Vibrio in the first place.	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215
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