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alabamamushie
Tmoney


Registered: 01/04/13
Posts: 522
Loc: Alabama
Last seen: 3 years, 4 months
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Agar
#19153410 - 11/18/13 09:59 AM (10 years, 2 months ago) |
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so i have An AA+ sporeprint that i tried to inoculate Agar Jars with This was my Procedures
9 Grams Agar powder approx 4 grams insta-potato mix 1 cup water Simmered to right before boiling loaded jars PC'Ed at 15 psi for 30 mins
let depressurize/cool over night woke up Washed my SAB with warm water and soap as to Franks G2G Tek loaded my jars after wiping with 91% Isopropyl outside of the box ofcourse close lid and put caps on armholes took a shower while stuff settled and put on clean clothes a facemask my gloves
i then preceeded to do a quick alcohol glove wipe got inside my SAB removed tinfoil and loosened ring on my jar unfolded tin foil corner of sporeprint, i then flamed my loop outside of my SAB with my Vector torch while loop was red hot for a good sec i wiped gloves again took my red hot loop cooled on the receiving Agar Jar then quickly and as swiftly as possible touched the print and swiped across my agar
Okay now heres the real part of my question
2 days after my innoculations which i did 4 jars 2 different times
My jars had some serious Bacterial contamination and Green mold Contamination
so i was wondering if the spore print was just that contaminated or was it my sterile techniques and each 8 jars had the same light brown bacterial contam
my only clue was the print is just way to contaminated or my procedures i will be trying with a syringe and new print
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dutchfunkle
subowski


Registered: 02/10/11
Posts: 132
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contaminated print, you should be letting spores germinate on an agar filled petri dish, not grain;and the spores need hydrated if youre gonna go directly to grain
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Aero
Orea


Registered: 11/01/13
Posts: 2,253
Last seen: 1 year, 2 months
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try a syringe or lc, which u know for sure that 100% clean. that will clear things up
-------------------- SPREAD THE SPORES
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Re: Agar [Re: Aero]
#19153831 - 11/18/13 12:09 PM (10 years, 2 months ago) |
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Quote:
Aero said: try a syringe or lc, which u know for sure that 100% clean. that will clear things up 
 How is op supposed to know if the syringe is clean without testing them first? LC syringes must be tested to be determined if clean and ms syringes are made from prints which are rarely sterile. Agar is usually how I test my syringes to begin with. OP if you want to test your procedure, I recommend doing a dry run with your plates. Go through all the motions of inoculation, without actually putting the spores to agar. This will let you know if the contam vector was your inoculate or your technique.
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Aero
Orea


Registered: 11/01/13
Posts: 2,253
Last seen: 1 year, 2 months
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Quote:
Pastywhyte said:
Quote:
Aero said: try a syringe or lc, which u know for sure that 100% clean. that will clear things up 
 How is op supposed to know if the syringe is clean without testing them first? LC syringes must be tested to be determined if clean and ms syringes are made from prints which are rarely sterile. Agar is usually how I test my syringes to begin with. OP if you want to test your procedure, I recommend doing a dry run with your plates. Go through all the motions of inoculation, without actually putting the spores to agar. This will let you know if the contam vector was your inoculate or your technique.
easy cowboy...
i said try a syringe that he knows for sure its clean, it means the one he used before...
-------------------- SPREAD THE SPORES
Edited by Aero (11/18/13 12:15 PM)
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Re: Agar [Re: Aero]
#19153853 - 11/18/13 12:16 PM (10 years, 2 months ago) |
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Still not guaranteed. He could have contamed the syringe inadvertently on his last use of it, it could have one mold spore hiding in the last cc. MS syringes are almost never 100% clean, even ones from sponsors, as anyone who has grown a few time could tell you. If OP is not sure of his technique then the dry run I suggested will be a lot more definitive in determining the true source of his contams.
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Aero
Orea


Registered: 11/01/13
Posts: 2,253
Last seen: 1 year, 2 months
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Quote:
Pastywhyte said: Still not guaranteed. He could have contamed the syringe inadvertently on his last use of it, it could have one mold spore hiding in the last cc. MS syringes are almost never 100% clean, even ones from sponsors, as anyone who has grown a few time could tell you. If OP is not sure of his technique then the dry run I suggested will be a lot more definitive in determining the true source of his contams.
i agree with you, but as far as u have a syringe which u previously used with success, im fairly sure that it will be just as fine on the plate. another thing is to use antibacterial agar if u dont have a flowhood handy iv used my MS syringes with 99% of success, i made the print, i made the syringe, they were all working fine..
-------------------- SPREAD THE SPORES
Edited by Aero (11/18/13 12:26 PM)
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Re: Agar [Re: Aero]
#19153891 - 11/18/13 12:31 PM (10 years, 2 months ago) |
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If op is trying to determine if it was his technique or his inoculate that was the source of his contam its easier to eliminate one of the variables from the equation. I don't care if he has used the syringe before, it could still be contamed. That's why when doing pf tek it is recommended to use as little solution as possible for each hole. Because we expect that there could be contams lurking in the syringe. Agar is contam heaven and things will grow on it that might not do as well on brf. If op can do the dry run without the plate contaminating then he can suspect the print. If he gets more contams with the syringe, there is still some doubt as to what the vector truly was.
I'm done arguing, op do what feels best to you and good luck, agar is well worth the learning curve
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alabamamushie
Tmoney


Registered: 01/04/13
Posts: 522
Loc: Alabama
Last seen: 3 years, 4 months
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Quote:
Pastywhyte said: If op is trying to determine if it was his technique or his inoculate that was the source of his contam its easier to eliminate one of the variables from the equation. I don't care if he has used the syringe before, it could still be contamed. That's why when doing pf tek it is recommended to use as little solution as possible for each hole. Because we expect that there could be contams lurking in the syringe. Agar is contam heaven and things will grow on it that might not do as well on brf. If op can do the dry run without the plate contaminating then he can suspect the print. If he gets more contams with the syringe, there is still some doubt as to what the vector truly was.
I'm done arguing, op do what feels best to you and good luck, agar is well worth the learning curve 
alright awesome ima do a dry run do all the steps accept for actually putting spores in being my first of agar work and SAB work
i didn't expect to be 8/8 but i expected one to atleast grow mycelium but they all contaminated to hell and back and i had this hunch it was the spore print or it was me only two things it could ever be
your answer was what i was looking for and you can always count on a Syringe/print to always atleast have 1 source of contamination IMO
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alabamamushie
Tmoney


Registered: 01/04/13
Posts: 522
Loc: Alabama
Last seen: 3 years, 4 months
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So finally got new prints a few weeks ago and put spores to agar 9 days ago inside my SAB made to Transcendinglifes Tek
1 got a green mold contam only in one spot and its slow i awoke today the 9th morning, Same with Green Mold now has Little Circles of mycelium!!!!
YESSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS The Door Has been Opened Now the Trek from Spore To Pin porn Continues for this mycologist
well be doing some transfers later then once i get a clean culture ill make a master jar also G2G about 5 jars
i will be relying on doing a Grow So i can Buy some more Much Needed Supplies Stay Tuned
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cronicr



Registered: 08/07/11
Posts: 61,436
Loc: Van Isle
Last seen: 2 years, 8 days
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Quote:
alabamamushie said:
1 got a green mold contam only in one spot and its slow i awoke today the 9th morning, Same with Green Mold now has Little Circles of mycelium!!!!
YESSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS The Door Has been Opened Now the Trek from Spore To Pin porn Continues for this mycologist
thats awesome, all excited about some mold lol, glad things are working for ya though
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  It doesn't matter what i think of you...all that matters is clean spawn I'm tired do me a favor
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