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Pyrotrons
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I don't know what the stuff from Hobby Lobby is, but even in a 10% concentration it melts before human body temperature.
confused, you said I need to sterilize my agar first. Does this mean I shouldn't touch all my plates to test if they are solid? I mean, the bacteria
haven't formed yet, so it should be ok right?
"Thus ethyl alcohol is inferior to xylene, although usable for certain applications." - Gerald Hurst
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confused
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dont touch them, if you want you can shake the petri dish slightly to see if it's still liquid
the bacteria havn't grown yet, but some bacteria can form endospores which would then grow when the agar sets
its better to leave it alone to set, if you have a fridge that isn't used for food, that would work as well
you should dissolve the agar first, then sterilize and pour
also you should incubate the plates upside down to minimize condensation on the plate cover, which can cause contamination
[Edited on 27-12-2013 by confused]
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Tsjerk
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You can also sterilize the agar before melting in then melt after sterilization in a microwave, doesn't really matter. If the agar you have melts at
such low temperatures it could be the "low melting" variant, in my lab it is used to immobilize zebrafish by pouring it over their tail, then it can't
get hotter then 30oC.
Edit: When sterilizing before melting, do add the water while sterilizing! You can't sterilize dry agar.
[Edited on 27-12-2013 by Tsjerk]
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Pyrotrons
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I fully understand the sterilization concept, thanks.
I just will be pissed if my third try...buying commercial Agar...results in it melting at less than body temp.
Another update on my condition while I'm at it:
1. Small white spot of what the Doctor and I assume to be Streptococcus...is still present on my left tonsil. I have long since finished the
Azithromycin
2. Thumbnails are still white, and I am still a little "high" all the time, indicative of continuing low blood Oxygen.
3. Going to the Doc again soon.
[Edited on 27-12-2013 by Pyrotrons]
"Thus ethyl alcohol is inferior to xylene, although usable for certain applications." - Gerald Hurst
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phlogiston
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With respect to agar concentration, I have always used 20 g/l agar which is more than enough.
1. Dissolve your nutrients (sugar/amino acids/etc) in water at the right concentration in a bottle with a cap
2. add the agar
3. Leave the cap on, but unscrew slightly and sterilize by boiling. Ideally, 20 minutes in a pressure cooker (120 deg C), but it is very likely that
boilin bit longer at atmospheric pressure is good enough. While you are at it, sterilise the petri dishes as well.
4. Allow to cool down slowly till it is still a little too hot to hold for very long with bare hands and not yet solid.
5. If desired, add antibiotics at this point.
6. Mix well
7. Pour a layer in your petri dishes. For 100mm dishes, use about 20 ml per dish. Put the lid on to maintain sterility as much as possible.
8. Leave to solidify for some time. A few hours at least. Then leave longer (e.g overnight) to dry a bit (when the plates are very moist, the layer of
water can 'redistribute' colonies/bacteria over the surface, remixing the colonies you were trying to keep separate
9. place a few of your plates overnight in your incubator (upside down) to see if you managed to keep them sterile. If you can't see anything after 24
hours: good job! If you see things growing, you need to work on better sterility.
10. Keep the rest of the plates in a sterile bag in the fridge until you want to use them.
PS. Don't boil in bleach. Boiling decomposes the hypochlorite in bleach. Just add room temperature bleach and wait an hour or so. It is very effective
and cheap. Alternatively, immerse in 1 M NaOH or heat everything in superheated steam.
[Edited on 27-12-2013 by phlogiston]
[Edited on 27-12-2013 by phlogiston]
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"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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Tsjerk
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If you don't have a pressure cooker and have trouble with getting your stuff sterile it could be due to spore forming bacteria, a trick against these
is to sterilize two times at atmospheric pressure with 24-48 hours in between. The spores surviving the first cook will "come to live" in the period
in between and die the second time.
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Pyrotrons
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While this thread is still at the top of the list, I'd like to address something that was said previously... that Streptococcus was one of the few
strains that was still resistant to penicillin.
This really doesn't appear to be true. Not only are the white patches on my left tonsil resistant to Azithromycin (I say that because the colony
never changed size while I was on the course of the Abx.), doing a Google search for: "streptococcus still resistant to penicillin?" seems to show
that they aren't. At least with Streptococcus Pneumoniae.
I still plan to test the Strep. on my tonsil for resistance to a slew of Abx Amoxicillin, Azithromycin, Ciprofloxacin. I will need to yank the lone
white patch off of my tonsil, and store it somehow. This scares me. Anyhow, someone suggested storing them at 4 degrees C, so I will do that. I'm
going back to the doctor today, I assume he will give me the "next step up" for Abx. Cipro I wonder? I hate the idea... but what can I do...
Quick side bitch: Why does this forum have a ton of smiley's available...but no symbol buttons...such as the small circle that represents the word
"degrees", or Pi, or any math symbols?
"Thus ethyl alcohol is inferior to xylene, although usable for certain applications." - Gerald Hurst
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