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takeshikovacs
Stranger
Registered: 07/28/14
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Last seen: 9 years, 3 months
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Improper sterilization with pressure cooker?
#20428393 - 08/16/14 04:50 PM (9 years, 7 months ago) |
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I have attempted twice to culture a batch, both ending with contamination. I have a couple questions about my technique.
My first attempt yielded a monstrosity.
And my second attempt appears to not fare any better.
With the same contamination in all the jars, I can only conclude that with my following of PF-TEK, something is not going according to plan during the sterilization and inoculation process.
I use a Mirro 92180 (8 quart) to pressure cook the jars for 45 minutes - would leaving the jars for a longer time improve the sterilization?
During inoculation I place all items in a plastic tub with a hole cut in it and alcohol sterilize all my objects while wearing gloves. I flame sterilize the needles, yet the flame is not constant as from a bunsen burner or an alcohol lamp - would having a constant flame improve the sterilization process to avoid further contamination?
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HostileX420
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Re: Improper sterilization with pressure cooker? [Re: takeshikovacs]
#20428410 - 08/16/14 04:57 PM (9 years, 7 months ago) |
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Quote:
takeshikovacs said: I have attempted twice to culture a batch, both ending with contamination. I have a couple questions about my technique.
My first attempt yielded a monstrosity.
And my second attempt appears to not fare any better.
With the same contamination in all the jars, I can only conclude that with my following of PF-TEK, something is not going according to plan during the sterilization and inoculation process.
I use a Mirro 92180 (8 quart) to pressure cook the jars for 45 minutes - would leaving the jars for a longer time improve the sterilization?
During inoculation I place all items in a plastic tub with a hole cut in it and alcohol sterilize all my objects while wearing gloves. I flame sterilize the needles, yet the flame is not constant as from a bunsen burner or an alcohol lamp - would having a constant flame improve the sterilization process to avoid further contamination?
The recommended PC time is 90 min. 45 min is precisely the reason you are contaminating. If after you switch up your times and you still keep contaminating your syringes are probably the problem at that point. If after that it's time to look at your sterile technique.
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Edited by HostileX420 (08/16/14 05:03 PM)
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takeshikovacs
Stranger
Registered: 07/28/14
Posts: 8
Last seen: 9 years, 3 months
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Re: Improper sterilization with pressure cooker? [Re: HostileX420]
#20428438 - 08/16/14 05:06 PM (9 years, 7 months ago) |
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Thank you for the quick reply.
Will try again today! =)
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Mushiez
Registered: 04/28/14
Posts: 1,057
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Re: Improper sterilization with pressure cooker? [Re: takeshikovacs]
#20432370 - 08/17/14 11:44 AM (9 years, 7 months ago) |
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Keep trying
leave the foil off next time, it helps them breathe better and can prevent contams/stalling
good luck
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HostileX420
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Re: Improper sterilization with pressure cooker? [Re: Mushiez]
#20438967 - 08/18/14 06:58 PM (9 years, 7 months ago) |
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Quote:
Mushiez said: Keep trying
leave the foil off next time, it helps them breathe better and can prevent contams/stalling
good luck
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Workman
1999 Spore War Veteran
Registered: 03/01/01
Posts: 3,601
Loc: Oregon, USA
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Re: Improper sterilization with pressure cooker? [Re: HostileX420]
#20453542 - 08/21/14 11:13 AM (9 years, 7 months ago) |
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If it is the same contamination in all jars, then you can assume it is from a common source. Molds are easily killed with heat so it is unlikely that your sterilization is at fault. Pressure sterilization is meant for endospore forming bacteria which are extremely tough. If they didn't exist, simple pasteurization temperatures would work.
Look at the pattern of the growth. It forms a top to bottom line that matches the inoculation path of the needle. I think it is safe to say that you have contaminated inoculate and no amount of PF jar technique is going to work until you replace it with a clean one. Good luck!
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification
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takeshikovacs
Stranger
Registered: 07/28/14
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Last seen: 9 years, 3 months
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Re: Improper sterilization with pressure cooker? [Re: Workman]
#20486920 - 08/28/14 12:23 AM (9 years, 6 months ago) |
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Hey everyone! Thank you for all your replies.
Quote:
Mushiez said: Keep trying
leave the foil off next time, it helps them breathe better and can prevent contams/stalling
good luck
By taking all the foil off, do you mean for me to do it after the jars are placed in the incubation chamber?
Quote:
Workman said: If it is the same contamination in all jars, then you can assume it is from a common source. Molds are easily killed with heat so it is unlikely that your sterilization is at fault. Pressure sterilization is meant for endospore forming bacteria which are extremely tough. If they didn't exist, simple pasteurization temperatures would work.
Look at the pattern of the growth. It forms a top to bottom line that matches the inoculation path of the needle. I think it is safe to say that you have contaminated inoculate and no amount of PF jar technique is going to work until you replace it with a clean one. Good luck!
On my third try, I tried a different strain (from a selection of 5) and got similar yellow-looking results as before with one semi-success that was quickly surrounded by yellow neon. The idea that the online source was faulty is disconcerting, and seems to be consistent.
Could the needles be dirty? I always flame sterilize the entire needle several times but surely foreign bacteria surviving the flaming process is highly unlikely.
I plan to try the remaining syringes (un-used and un-tipped) in replicate after PCing for 90 minutes, and I guess that would give a clearer idea of the contamination.
On a further note, I have not used these syringes for at least 6 months after obtaining them and have kept them bundled in a plastic bag in a cabinet ever since. Could contamination have happened to the syringe during storage?
Edit: Upon further reading, would it be prudent to try to "save" some mycelium through culturing on agar plates? (Though I wouldn't even know where to obtain said agar plates legally on such short notice)
Edited by takeshikovacs (08/28/14 03:08 AM)
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Mushiez
Registered: 04/28/14
Posts: 1,057
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Re: Improper sterilization with pressure cooker? [Re: takeshikovacs]
#20487492 - 08/28/14 04:59 AM (9 years, 6 months ago) |
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Quote:
takeshikovacs said: Hey everyone! Thank you for all your replies.
Quote:
Mushiez said: Keep trying
leave the foil off next time, it helps them breathe better and can prevent contams/stalling
good luck
By taking all the foil off, do you mean for me to do it after the jars are placed in the incubation chamber?
Yes leave the foil off when it is removed for inoculation. Foil constricts air exchange, and even worse, causes moisture build up, such that any contaminants that land on the foil during the inoc period even if just a few will multiply under the moist conditions when it is placed back on the jars. The vermiculite barrier is sufficient and does its best job when it is dry
Better to start from anew. Contam jars are bad for tissue samples for two reasons, 1) exposes grow area to contams that could hinder future projects, 2) hard to achieve clean culture.
No more incubating. Place the jars to grow happy on the top shelf of a bookshelf (contams lower to the ground).
The optimal temp for mycelium growth is 75F room temperature. Any more than that and mycelium has a very hard time outrunning the much faster division rates of molds, and especially bacteria. Incubating causes big problems namely condensation, will creates wet spots in the substrate and where there is standing water, there will be bacteria in due time
RR said if you are comfortable in a shirt where your jars are sitting, the temperature is good. Mushroom mycelium has the same temperature preferences as humans
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Workman
1999 Spore War Veteran
Registered: 03/01/01
Posts: 3,601
Loc: Oregon, USA
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Re: Improper sterilization with pressure cooker? [Re: takeshikovacs]
#20489599 - 08/28/14 03:31 PM (9 years, 6 months ago) |
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Quote:
takeshikovacs said: On my third try, I tried a different strain (from a selection of 5) and got similar yellow-looking results as before with one semi-success that was quickly surrounded by yellow neon. The idea that the online source was faulty is disconcerting, and seems to be consistent.
Could the needles be dirty? I always flame sterilize the entire needle several times but surely foreign bacteria surviving the flaming process is highly unlikely.
I plan to try the remaining syringes (un-used and un-tipped) in replicate after PCing for 90 minutes, and I guess that would give a clearer idea of the contamination.
On a further note, I have not used these syringes for at least 6 months after obtaining them and have kept them bundled in a plastic bag in a cabinet ever since. Could contamination have happened to the syringe during storage?
Edit: Upon further reading, would it be prudent to try to "save" some mycelium through culturing on agar plates? (Though I wouldn't even know where to obtain said agar plates legally on such short notice)
Flaming eliminates the needle being dirty as a source. Storing slightly contaminated syringes that may have worked when fresh can make them worse as the contaminate grows in the syringe. It is possible they were contaminated during storage if they were stored in damp dirty conditions (like inside a moist plastic bag that grew a little mold). It would be difficult to isolate out good mycelium, even on agar. But you did get some growth, so it is still possible. You can quickly make some agar "plates" using small jars with lids and food grade grocery store agar (often in health food stores and asian markets). You need a nutrient for your agar such as light malt powder from a brewing supply store or some finely ground grains, boiled potato water, instant potatoes or even the juice from soaked dog food (cubensis isn't picky).
You haven't mentioned the source of the spores (which is good, because they are for microscopy purposes only). But an insider from Spores101(nonsponsor) revealed that they intentionally mislabel whatever spores they have available to what the customer orders. This means that all the strains are likely the same and if the bulk spore solution they loaded the syringes with is contaminated, all syringes are contaminated. But this may not apply to you, depending on your source.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification
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takeshikovacs
Stranger
Registered: 07/28/14
Posts: 8
Last seen: 9 years, 3 months
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Re: Improper sterilization with pressure cooker? [Re: Workman]
#20524888 - 09/05/14 03:51 PM (9 years, 6 months ago) |
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I did everything again except this time I took the lids off after inoculation and left them in the top bookshelf.
Here were the results of the 6 jars.
Strain A: Jar 1, Jar 2 Strain B: Jar 1, Jar 2 Strain c: Jar 1, Jar 2
All jars appeared to have some promising growth, but had contaminants (especially in Strain A!) found throughout the substrate.
Should my next step be to acquire new spore syringes?
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tchyted
miestro
Registered: 09/03/01
Posts: 526
Loc: WA near seattle
Last seen: 9 years, 6 months
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Re: Improper sterilization with pressure cooker? [Re: takeshikovacs]
#20526909 - 09/06/14 02:24 AM (9 years, 6 months ago) |
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i have experienced this type of contam. you have very little if any mushroom spore in your original syringe. yes they get bad over time. get a supply of fresh ones.
you are using whole rice. look up the PF tek and start out with brown rice flour, not whole grain, that is for more advanced growers.
you also have some humidity problems, try using a smaller sized jar, it takes less time for the mycelium to colonize and does not give the contams so much of a chance.
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Kanji
Registered: 01/04/09
Posts: 86
Loc: Near the edge
Last seen: 5 years, 11 months
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Re: Improper sterilization with pressure cooker? [Re: Workman]
#20531411 - 09/07/14 12:11 AM (9 years, 6 months ago) |
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Quote:
takeshikovacs said: On a further note, I have not used these syringes for at least 6 months after obtaining them and have kept them bundled in a plastic bag in a cabinet ever since. Could contamination have happened to the syringe during storage?
I thought spore syringes were only good for a month at most. I make mine and let them set for a week or two in a closed jar with the needle up and covered. Inoc a few LC and use the first one that colonizes fully.
I know spores on a properly stored print can last for much longer than 6 months.
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Kanji
Registered: 01/04/09
Posts: 86
Loc: Near the edge
Last seen: 5 years, 11 months
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Re: Improper sterilization with pressure cooker? [Re: Workman]
#20531420 - 09/07/14 12:15 AM (9 years, 6 months ago) |
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Quote:
Workman said: But an insider from Spores101(nonsponsor) revealed that they intentionally mislabel whatever spores they have available to what the customer orders.
Well cubes are cubes. I can't tell one strain from another, aside from unique varieties like albino or penis envy.
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blindingleaf
blue collar underworld
Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: Improper sterilization with pressure cooker? [Re: Workman]
#20532784 - 09/07/14 10:55 AM (9 years, 6 months ago) |
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Quote:
Workman said:
Quote:
takeshikovacs said: On my third try, I tried a different strain (from a selection of 5) and got similar yellow-looking results as before with one semi-success that was quickly surrounded by yellow neon. The idea that the online source was faulty is disconcerting, and seems to be consistent.
Could the needles be dirty? I always flame sterilize the entire needle several times but surely foreign bacteria surviving the flaming process is highly unlikely.
I plan to try the remaining syringes (un-used and un-tipped) in replicate after PCing for 90 minutes, and I guess that would give a clearer idea of the contamination.
On a further note, I have not used these syringes for at least 6 months after obtaining them and have kept them bundled in a plastic bag in a cabinet ever since. Could contamination have happened to the syringe during storage?
Edit: Upon further reading, would it be prudent to try to "save" some mycelium through culturing on agar plates? (Though I wouldn't even know where to obtain said agar plates legally on such short notice)
Flaming eliminates the needle being dirty as a source. Storing slightly contaminated syringes that may have worked when fresh can make them worse as the contaminate grows in the syringe. It is possible they were contaminated during storage if they were stored in damp dirty conditions (like inside a moist plastic bag that grew a little mold). It would be difficult to isolate out good mycelium, even on agar. But you did get some growth, so it is still possible. You can quickly make some agar "plates" using small jars with lids and food grade grocery store agar (often in health food stores and asian markets). You need a nutrient for your agar such as light malt powder from a brewing supply store or some finely ground grains, boiled potato water, instant potatoes or even the juice from soaked dog food (cubensis isn't picky).
You haven't mentioned the source of the spores (which is good, because they are for microscopy purposes only). But an insider from Spores101(nonsponsor) revealed that they intentionally mislabel whatever spores they have available to what the customer orders. This means that all the strains are likely the same and if the bulk spore solution they loaded the syringes with is contaminated, all syringes are contaminated. But this may not apply to you, depending on your source.
workman, if u are reading this, i had a question about ur post. say u have an LC or a spore syringe that is clean. it was used a few times, all good clean growth, then u decide u r going to store it in the fridge. i have never read anything about cleaning the cap before storing, and always wondered if it was possible for bacteria or mold to form in the condensate in the cap, and go up the syringe? is that ever a concern with you? i was mainly thinking of bacteria starting from a tiny drop of nutrient LC that got squirted out accidentally, staying in the unsterile cap, forming bacteria, than traveling up the needle into the syringe. is that possible, or does the pressure from the plunger prevent that, so a simple flaming is all that is required?
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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HostileX420
Webcast Hunter!!!!!
Registered: 04/11/13
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Re: Improper sterilization with pressure cooker? [Re: blindingleaf]
#20535943 - 09/07/14 10:48 PM (9 years, 6 months ago) |
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What on earth is going on over their?
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Edited by HostileX420 (09/07/14 10:49 PM)
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takeshikovacs
Stranger
Registered: 07/28/14
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Last seen: 9 years, 3 months
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Re: Improper sterilization with pressure cooker? [Re: tchyted]
#20536240 - 09/08/14 12:15 AM (9 years, 6 months ago) |
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Quote:
tchyted said: i have experienced this type of contam. you have very little if any mushroom spore in your original syringe. yes they get bad over time. get a supply of fresh ones.
you are using whole rice. look up the PF tek and start out with brown rice flour, not whole grain, that is for more advanced growers.
you also have some humidity problems, try using a smaller sized jar, it takes less time for the mycelium to colonize and does not give the contams so much of a chance.
You are right about the whole rice; for some reason I thought grinding the whole rice would suffice as brown rice flour.
My next attempt will have to wait until I get all the necessary materials.
Thanks for all your help guys
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HostileX420
Webcast Hunter!!!!!
Registered: 04/11/13
Posts: 1,124
Loc: An American Shroomery
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Re: Improper sterilization with pressure cooker? [Re: takeshikovacs]
#20545926 - 09/10/14 12:42 AM (9 years, 6 months ago) |
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Quote:
takeshikovacs said:
Quote:
tchyted said: i have experienced this type of contam. you have very little if any mushroom spore in your original syringe. yes they get bad over time. get a supply of fresh ones.
you are using whole rice. look up the PF tek and start out with brown rice flour, not whole grain, that is for more advanced growers.
you also have some humidity problems, try using a smaller sized jar, it takes less time for the mycelium to colonize and does not give the contams so much of a chance.
You are right about the whole rice; for some reason I thought grinding the whole rice would suffice as brown rice flour.
My next attempt will have to wait until I get all the necessary materials.
Thanks for all your help guys
You can! It must be ground into a powder not chunks! Like the dust in a bag of verm not like course verm. Catch my drift?
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tchyted
miestro
Registered: 09/03/01
Posts: 526
Loc: WA near seattle
Last seen: 9 years, 6 months
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Re: Improper sterilization with pressure cooker? [Re: HostileX420]
#20584028 - 09/18/14 02:36 PM (9 years, 6 months ago) |
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in PF cakes, you can indeed use blender-ground rice. i'm sure there are old threads here. original PF tec used brown rice flour because:
1. the flour milling process does a good job of eliminating contams that naturally get on the surface of grains.
2. the small globs and flour painted verm present a quick-nutrition easily colonized substrate without deep thicknesses of starch that invite other contams to set up shop.
3. in his experiments, it worked best. what can i say, success speaks for itself.
try this experiment: make up a set of cake jars and a set of whatever you have done allready. leave them sit, do nothing to them. do they get contamned all by themselves?
Edited by tchyted (09/18/14 02:39 PM)
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Workman
1999 Spore War Veteran
Registered: 03/01/01
Posts: 3,601
Loc: Oregon, USA
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Re: Improper sterilization with pressure cooker? [Re: blindingleaf]
#20584996 - 09/18/14 06:13 PM (9 years, 6 months ago) |
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Quote:
blindingleaf said:
workman, if u are reading this, i had a question about ur post. say u have an LC or a spore syringe that is clean. it was used a few times, all good clean growth, then u decide u r going to store it in the fridge. i have never read anything about cleaning the cap before storing, and always wondered if it was possible for bacteria or mold to form in the condensate in the cap, and go up the syringe? is that ever a concern with you? i was mainly thinking of bacteria starting from a tiny drop of nutrient LC that got squirted out accidentally, staying in the unsterile cap, forming bacteria, than traveling up the needle into the syringe. is that possible, or does the pressure from the plunger prevent that, so a simple flaming is all that is required?
I don't really use syringes, so my experience is limited. But given enough time, it seems reasonable to expect contaminates to work their way into a syringe. It is always a good idea to flame the needle.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification
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tchyted
miestro
Registered: 09/03/01
Posts: 526
Loc: WA near seattle
Last seen: 9 years, 6 months
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Re: Improper sterilization with pressure cooker? [Re: Workman]
#20597314 - 09/21/14 02:07 PM (9 years, 6 months ago) |
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blindingleaf, i believe the possibility of contamnation in a fridge stems from the fact that they are generally loaded with lots of contams. the temperature inside a fridge is nothing like constant, so thermal expansion and contraction with each on/off cycle will tend to draw contams into the syringe. taking steps to minimize temp changes is your best bet to avoid contams. then put that into the fridge to keep it cool.
i put mine in a foil-bubble envelope inside a 2-qt mason jar with the lid tightly screwed on. it tends to get sealed if i do it in warmish conditions and then put the jar in the fridge. (all these items meticulusly cleaned)
Edited by tchyted (09/21/14 02:12 PM)
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