Why doesn't our autoclave sterilize our broth?

April 23, 2012 3:18 PM

Why is weird stuff growing in my flasks of 1% tryptone broth after I've autoclaved them to mix and sterilize the broth?

We routinely make 1% tryptone broth solutions in glass flasks, then to completely mix the broth and sterilize it, we put it in the autoclave. We set the temperature to 255 F and run it for 25 minutes before allowing it to cool/vent for 10 minutes. Since the liquid is boiling, to keep it from blowing up the flask, we unscrew the lid a bit so that it is still on but loose enough to allow pressure to vent from the flask. Additionally, we use "autoclave tape" to make sure the proper temp/pressure has been met. When the autoclave has run its course, we open it up, screw the caps on and call it good. There are further steps in the process but that's not germain to my current issue...

Within a few days (as few as 2 and as many as 7) something starts to grow (bacterial? yeast?) in what should be sterile broth. We've tried two different autoclaves. We have not yet tried new tryptone powder (but are about to...), nor have we tried a new source of DI water (but are too about to...) Additionally, we've tried upping the time in the autoclave to 35 minutes with no time for venting. The problem continues.

Does this sound familiar to anyone? Has our tryptone or water been contaminated with something that can survive the autoclave?

posted by pwb503 to science & nature (14 answers total)

One possibility is that the contaminant is in the powdered media and when you mix the media into the water, it clumps and doesn't fully dissolve until late in the autoclave cycle. Any contaminants in the powder won't be exposed to moisture for the full cycle and might make it through. I would try dissolving the powdered media better before autoclaving which you can usually do with extra shaking and time.
posted by Durin's Bane at 3:41 PM on April 23, 2012 [1 favorite]

Sounds like it didn't sterilize. Is the autoclave temperature going all of the way to where it is supposed to and staying there? (The tape just says it went past 121 at all). You need to heat the entire solution to 121 or above for 20 minutes. Larger volumes take longer: for example, when my lab does 2L growths, they autoclave for 40 minutes.
posted by overhauser at 4:11 PM on April 23, 2012 [1 favorite]

Is it possible that you're just seeing precipitation/cloudiness that isn't microbiological contamination? Have you tried plating out the contaminated broth to see what (if anything) grows?
posted by pullayup at 4:12 PM on April 23, 2012 [1 favorite]

I'm confused by your description that the liquids are 'boiling'. Is the autoclave reaching the proper pressure? If it were, the liquids inside should not boil as they do at atmospheric pressure. One leave the bottles/flasks slightly open so that the hot steam can best penetrate into the bottle, not so that gasses can escape. Sterilization requires both the proper temp and pressure to kill.

Also, the tape that changes color is not an adequate control for sterilizing liquids -- you need some diaks that can be suspended in a control bottle. Ideally, you would also have a microbe control (spore strips), but the diak will be a huge improvement on the tape. What volume are you autoclaving? 25 min seems long enough but may not be depending on the volume and there's really no way to know without the proper controls
posted by Tandem Affinity at 4:26 PM on April 23, 2012

Some quick answers:

We are autoclaving 500 mL or so of broth. We shake and mix the broth well prior to putting it in the autoclave but yes, it could conceivably not be truly mixed before putting it in the autoclave.

The readout on the autoclave suggests the temperature is going all the way up and staying at 255 F (121-ish C) but beside trusting the autoclave to report temperature, we are counting on the tape. I should have mentioned this, but yes it definitely isn't just cloudiness; it smells terrible.

Thank you Tandem Affinity for the info on what's actually going on inside. I was wrong. I assumed it was boiling but now that I think about it, you are right. I really don't know what's going on in there as there are no windows... It most likely isn't boiling.
posted by pwb503 at 4:48 PM on April 23, 2012

If it's the water, you could try running it through a 0.2-0.45 um filter before mixing it in the broth, and see if that doesn't help. Alternatively, it could be the powder, or even the glassware. Have you tried using another lab's tryptone or glassware? If these attempts don't fix it, it very well could be the autoclave somehow. It seems like the contamination would be pretty impressive if that much crud could grow in that short time.
Other possibilities....do you keep the bottles near an air vent? Perhaps the vent could be blowing crap onto them? When you screw the lids on, are you wearing clean gloves? Do you have any enemies in nearby labs who think sneezing in your media is a great joke?
posted by nasayre at 4:57 PM on April 23, 2012

I'm forgetting right now what, but was also have one thing that just never really get's sterile when you autoclave it. Our solution has been to make only as much as you'll use. It's not a very satisfying solution though (so, sorry to not be that helpful, but wanted to post to at least say you're not the only one).
posted by lab.beetle at 5:00 PM on April 23, 2012

I agree that their could be contamination in the autoclave(s). There are some spores that pretty hardy. Does the same autoclave get used for autoclaving biohazard and media? This can lead to problems like you're having.

Also, how long do you wait to close the caps? Don't wait too long -- when you can comfortably touch the bottle with a glove on, it's cool enough. Also, you're probably nervous about the caps now that you're having these problems, but don't let it keep you from leaving them open the usual amount -- it's important to make sure they're open, or you're not getting the full steam effect. (You can make a hood for the caps with aluminum foil if you want to keep things from falling on the opening after the autoclave cycle.)

Also as a crude check of whether the autoclave is reaching the right pressure, how is the volume of the liquids in the flasks before and after? If you are seeing a big reduction in volume, this could mean that things are actually boiling over (that the pressure is not being maintained). You'd probably have a lot of spillage on the bottom of the autoclave, too.

If you're worried that it's your particular solution (i.e., the tryptone, the water), make up some broth using a different media and different solvent (say some prepackaged water or PBS), autoclave it together with your tryptone media, and see if the same thing happens.
posted by Tandem Affinity at 5:08 PM on April 23, 2012

35 minutes with no time for venting

That sounds like you're saying that you took stuff out of the autoclave without giving it time for the pressure to go back down. Is that right? AFAICR, that's okay for solid material, but not a super good idea for liquids.
posted by Made of Star Stuff at 5:26 PM on April 23, 2012 [1 favorite]

At least in my lab sterile in the autoclave is never sterile forever--even in the cleanest labs most of the time bacteria will find a way to make it into your broth whether you like it or not. For example, the condensation that's going to form on the inside of your cap as it cools in the autoclave is a possible haven for bacteria. That's why you flame the flask and cap when removing media for cultures, sterilize the area where you're opening the bottle, all that.

In my lab we generally immediately add whatever antibiotic we're going to be using in our cultures to the media as soon as it's cool enough that it won't break the antibiotic down. We store it like that. This helps with the contamination. Do you do this?
posted by schroedinger at 6:08 PM on April 23, 2012 [1 favorite]

Another biologist I know once figured out - after a lot of grief with a similar situation - that he was selecting for some obligate thermophiles! Crazy! Maybe that's what you have.
posted by Cygnet at 6:12 PM on April 23, 2012

Additional things I might try:

• looking at the contaminant under the light microscope (to get a better idea of what you're dealing with)
• autoclaving a bottle of media and then not opening it again for a week (to determine whether problem is the lab environment/handling or the autoclaving)
• re-cleaning the glassware with a strong detergent (e.g. Decon)
• filter-sterilizing the media instead

posted by en forme de poire at 7:49 PM on April 23, 2012

Problems I've seen (or at least we surmised - the problem with this stuff is that you can never "see" the vector of contamination, only the result, and then guess at where it got in later -

1. depressurizing the autoclave too fast causing a brief, rapid boil-up in the flask, depositing a film of solution up the neck/around the rim/in the upper surface of the flask. Contamination was following the nutrient path up and under the lid.

2. Too much airflow in the autoclave room/not being slow and gentle enough opening and unloading it could mean airborne contaminants are sneaking in under the cap during the opening.

3. Are the screw caps you're using definitely meant to be autoclaved? The procedure could be warping them just enough that they're not really giving an airtight closure any more. One person I worked with swore by an erlenmeyer with a one-hole stopper with the hole stuffed with cotton wool and then used a square of aluminum foil to lightly wrap the stoppered top of the flask, mainly to prevent the tops from popping out during depressurization.
posted by nanojath at 9:44 AM on April 24, 2012

Thanks for all the suggestions. This will help a lot!
posted by pwb503 at 9:54 AM on April 24, 2012