Problem with slants. What causes this?

[Charlie]

Lately it seems like 30% of my slants blow up from the bottom and jam the agar against the cap. Here's a pic.



This is a fairly new agarose* slant that was innoculated with WY-2112 about 5 days ago. I've been using the same agarose for 2 years and this has only started happening recently. It's really pissing me off! What do you suppose causes it?

Charlie

*IIRC agarose is a more pure form of agar, and it melts at a slightly lower temperature.

[spiderwrangler]

Dude, that's fucking racist. If you have problems with Asians, you need to go to sensitivity classes with JP.

How are they being stored? Could it just be gravity? If they are being blown up, then there may be something in the bottom producing gas. Dunno man.

[Charlie]

How are they being stored? Could it just be gravity? If they are being blown up, then there may be something in the bottom producing gas. Dunno man.

I store blank slants in the fridge, and no problem there. This happens during the growth phase (7 days at RT, upright in a culture tube rack, burped daily). I'm guessing that there's a channel where the agar is separated from the tube and some yeast is getting down in the bottom of the tube. Then the channel miraculously seals off and the evolved CO2 blows the slant up. But I don't really know. Like I said, it's never happened before. Then one or two would do it, and now it happens to about 30% of my new slants.

I wonder if it's bad caps. Repeated autoclaving has caused them to take a set so they don't seal as well as they used to, but if it is then I don't understand the mechanism.

Charlie

[ajdelange]

It's clear that the yeast have penetrated well into the agar (how deep do you stab?) such that some of the CO2 propagates to the bottom and some to the top. You release the pressure at the top and that makes the pressure at the bottom higher than at the top. This pushes the piston up. I havent fiddled with slants in quite a while but seem to remember some plugs being blown apart by internal CO2 pressure when the caps were removed.

[spiderwrangler]

I havent fiddled with slants in quite a while

Sorry AJ...

[ajdelange]

I havent fiddled with slants in quite a while

Sorry AJ...

It's jsut I haven't been to Bangkok in several years.

[Charlie]

how deep do you stab?

I don't. I innoculate the slants by squiggling the charged loop along the surface. I could understand the problem if I was stabbing them, but I don't do stabs.

Absent other ideas I'm going to soak the next round of tubes with 6N NaOH for several days to expose some -OH groups and get them really squeaky prior to making the slants and see if that helps. Maybe I'm getting some slippery organic buildup on the inside of the tubes.

Charlie

[CRBrewHound]

Not sure how you are doing your agar, but it looks like the problem is how you are forming your slants. The way I do mine is I rest them on a pencil and the width of the tube is never covered at any point. Not sure if that is clear.

Check out the picture at the bottom of the following site:

http://braukaiser.com/wiki/index.php/Ma ... and_Slants

See how you can see the bottom of the tube, this prevents a gas build up underneath the agar. Which appears to be what is pushing your agar towards the top of the slant. I do mine this way and have yet to see the problem you are encountering.