Prior to ~ 1995 all bacteria, fungi, spores and virus (excluding prion) were consider heat sensitive (destroyed) by autoclave treatment at 121C for 15 minutes measured from the time the sample reaches 121C. The extremophile, Geogemma barosii, can survive at temperatures above 121 °C. See http://en.wikipedia.org/wiki/Strain_121 I suspect your autoclave is not reaching the desired temperature. Also, keep in mind that the media you describe can precipitate on cooling. You could try a simple experiment where you autoclave, let's say 2 x 1 liter screw cap pyrex flasks containing 250 - 500 ml of your media. Then remove both flasks from autoclave (still hot) screw one cap tight (anaerobic culture) and the second flask leave cap loose (aerobic culture). Incubate as usual but tighten cap on the aerobic culture and shake gently (avoiding splashing the lid) then loosen cap repeating this approximately every 15 minutes for the first two hours to introduce sufficient air to establish an aerobic environment. Incubate as usual for 7 days or until you see evidence of growth. Do gram's stain if you get growth to insure it is a bacterium and not precipitate. If you confirm microbial growth it's likely your autoclave but could be a very unusual bacterium. If you get turbidity in both it is likely a precipitate.

Without knowing all your handling steps and raw materials one can only guess what might be the reason for the contamination. However, my first guess would be the autoclave itself. Presuming that there is a water reservoir I would suggest changing the water, cleaning the autoclave and running it for 30 min at 134°C or-if not possible- at 121°C for 60 min.

How long do you let the autoclave run at said temp and pressure? As long at > 15 mins. or so I think not likely autoclave resistant spores. Contamination is prob. coming in after autoclaving somehow like the air if you open your tubes/containers for too long while not in a clean hood.

If you're using culture tubes one tip that took me awhile to figure out is to aliquot the broth into each tube, then autoclave the rack of tubes so you don't have to pour or pipette after sterilization.

Hi, I think that if your glass bottle is the source of contamination because of the biofilm, you should clean with bleach. We always use distilled water in order to prepare media. You should ask manufacturer company in orde to learn that you autoclave works or not. As you know some water can go inside of your medium because of the steam pressure so you should clean the autoclave water. After autoclaving you should close tightly the lid of bottle in order to prevent air transition or contamination. Is the place that you pour your plates sterile or not may be this is the reason. Because much contamination comes from air when you pour your media in to plates.

Why dont you just sterilize your lab once and for all. Either you use UV, or fumigate. If you try this and it doesn't work, then, see a specialist. I believe you'll see an improvement. You may just be incapacitated for a while, as you might not be able to able to work while sterilizing. You shall overcome, its just one of those things. Cheers

Prior to ~ 1995 all bacteria, fungi, spores and virus (excluding prion) were consider heat sensitive (destroyed) by autoclave treatment at 121C for 15 minutes measured from the time the sample reaches 121C. The extremophile, Geogemma barosii, can survive at temperatures above 121 °C. See http://en.wikipedia.org/wiki/Strain_121 I suspect your autoclave is not reaching the desired temperature. Also, keep in mind that the media you describe can precipitate on cooling. You could try a simple experiment where you autoclave, let's say 2 x 1 liter screw cap pyrex flasks containing 250 - 500 ml of your media. Then remove both flasks from autoclave (still hot) screw one cap tight (anaerobic culture) and the second flask leave cap loose (aerobic culture). Incubate as usual but tighten cap on the aerobic culture and shake gently (avoiding splashing the lid) then loosen cap repeating this approximately every 15 minutes for the first two hours to introduce sufficient air to establish an aerobic environment. Incubate as usual for 7 days or until you see evidence of growth. Do gram's stain if you get growth to insure it is a bacterium and not precipitate. If you confirm microbial growth it's likely your autoclave but could be a very unusual bacterium. If you get turbidity in both it is likely a precipitate.

Hi,

The problem can be with the water reservoir in the autoclave, change this with a fresh water and always use distilled water for preparing the media. Dispense the prepared media in tubes and do sterilization. You can wash the tubes by soaking it in acid and rinse it with distilled water and use the tubes for preparing and sterilizing the media. Do not prepare test tubes and media separately as it might cause contamination while pouring it into the tubes. Make fresh cotton plug for plugging it in the test tubes and sterilize it as it can also be a source for contamination.

Hello, Thank you all for your suggestions, I did not think about changing the water of the autoclave reservoir as we have been refilling it ever since without completely removing it and replacing it. So that is one thing which i am going to perform now. Also i do not use screw cap bottles, instead i use conical flask of 5L capacity with cotton plugs with approx 2-3 L media with autoclave cycle of 121 degree and 30 -40 min. also the nutrient broth is getting contaminated with one specific film forming bacteria when left still at room temperature without opening or moving it anywhere, also i may need to use bleach or a strong detergent to clean my flasks also i will try using distilled water in media preparation.

@ Kolapo Fasina - I have performed fumigation sometime back and it did not help, its just one specific contamination so its something i am doing wrong which could be water in autoclave reservoir too.

Many thanks again @ Alexander Huwig, Ryan lynch, Ebru Tekin, Kolapo Fasina, Frank Gelder, Baskaran viswanathan for all your valuable input, any other suggestion is highly welcome.

Cheers

Hey, Ishan! I see you're using a conical flash with a cotton plug? I would try loosely covering the cotton plug..i.e. the mouth of the flask with foil to prevent any contamination. It can be autoclaved and handled after autoclaving with minimal risk of contamination. Also, the tape that you've used isn't a good measure of sterility...it would only tell you if the item has been exposed to heat/steam from the autoclave. I think it would be worthwhile for you to use a biological indicator such as ProSpore2 to ensure that your autoclave is indeed working!

Hi again, in the past I also used cotton plugs and it was not such a good idea to reuse them. In case that they are reused it may be possible that they get contaminated by spore-forming microorganisms. The spores can survive partly the autoclave process causing a contamination of the broth - very often from top of the broth by forming a flower at the edge of liquid and air. So both could idea, changing the water/cleaning the water reservoir and using only new cotton plugs with foil as suggested by Elie.

If you are using a tabletop autoclave, changing out the water would be a good idea. Check the drain as well. I have a plumbed autoclave so this isn't a problem, but I've seen this before.

As far as the types of vessels and lids, I've never used cotton plugs. I have had success with just covering the top with a piece of foil folded in two so that there is enough coverage over the lips that I can grip to open the foil on the folded edge without having to put my fingers too close to the opening. The point of having it folded is because a single layer tears too easily. I do this frequently with flasks, but have't tried this on tubes since I have reusable autoclavable plastic tube covers. I have a mixture of glassware so some of my flasks and tubes actually have screw cap lids and the rest I use foil. My foil capped media seems to keep just fine for months without contamination right alongside my media in screw-capped vessels.