|
Some of these posts are very old and might contain outdated information. You may wish to search for newer posts instead.
|
Themadhatter106
Psychedelic Teacher



Registered: 06/18/09
Posts: 100
Loc: USA
Last seen: 7 years, 8 months
|
100% bacterial contamination
#22352315 - 10/08/15 10:21 PM (8 years, 3 months ago) |
|
|
After taking a long break from growing I came back and decided I want to start again. So I made spore syringes of a 5 year old B+ print that I had using a glove box. I make syringes by steaming a half pint mason jar with the right amount of water in a pot along with the syringes I need wrapped with foil. I put everything in my glove box which was sprayed with alcohol and nuked with Lysol air sanitizer. I then use a flame sterilized inoculation loop to scrape spores off the foil into the mason jar which I opened. Then I mix the solution with a spore syringe needle and fill all of the syringes.
During everything I wear a surgery mask, latex gloves and tyvek arm sleeves sprayed down with alcohol periodically.
I then inoculated about 40 jars in the open air since my glove box was too small. No mushrooms spores germinated and eventually the jars got infected with trich and bacteria after about 2-3 weeks.
I thought that the spores were just too old to germinate and instead contaminates did so I dumped out all of the jars and made a new batch of jars. I borrowed a spore print from a free spore thread and made more syringes. Although, the print was quite light and I'm not sure but someone else might have also used it by the way it looked. The syringes turned out completely clear.
Anyway, I had inoculated all of the jars inside a new bigger glovebox and I kept most in my incubator from day 1. After about a week I noticed that most of the jars that I had kept outside the incubator had germinated but none of the ones in the incubator had. After 12 days I dumped all of the jars because I could smell that they had all bacteria contaminated even the not incubated ones that had some injection sites with germination smelled of bacteria. I suspect at the injection sites with no growth especially. So I dumped all of the jars and made new ones.
What do you guys think? I am having a very high contamination rate.
I don't know what is causing my bacteria problem but I suspect it was that the print was dirty. So I am trying one syringe worth of another print that I got from someone else and it looks less sketchy. Then I will also make sure the uninoculated jars don't grow anything otherwise I have other problems.
Does incubation actually speed germination or only speed bacteria growth?
-------------------- "Turn on, Tune in, Drop Out"- Dr.Timothy Leary "If I had a world of my own, everything would be nonsense. Nothing would be what it is, because everything would be what it isn't. And contrary wise, what is, it wouldn't be. And what it wouldn't be, it would. You see?" - Alice (Alice in Wonderland) Don't Grow mushrooms just because you want to trip. Grow them to stop drug dealers from going to jail and for the satisfaction of being self-sufficient.
|
Arush
Godfather


Registered: 05/06/13
Posts: 312
Loc: Canada
Last seen: 5 years, 9 months
|
|
High temperatures seem to favor contamination. Probably the print if you were as clean as you possibly could.
-------------------- http://www.shroomery.org/forums/showflat.php/Number/22409475 “Psychedelics are illegal not because a loving government is concerned that you may jump out of a third story window. Psychedelics are illegal because they dissolve opinion structures and culturally laid down models of behaviour and information processing. They open you up to the possibility that everything you know is wrong.” ― Terence McKenna
|
Themadhatter106
Psychedelic Teacher



Registered: 06/18/09
Posts: 100
Loc: USA
Last seen: 7 years, 8 months
|
Re: 100% bacterial contamination [Re: Arush]
#22355581 - 10/09/15 05:49 PM (8 years, 3 months ago) |
|
|
I think I will go without incubation this time around since from what I've read it seems to increase the risk of bacteria taking over jars.
-------------------- "Turn on, Tune in, Drop Out"- Dr.Timothy Leary "If I had a world of my own, everything would be nonsense. Nothing would be what it is, because everything would be what it isn't. And contrary wise, what is, it wouldn't be. And what it wouldn't be, it would. You see?" - Alice (Alice in Wonderland) Don't Grow mushrooms just because you want to trip. Grow them to stop drug dealers from going to jail and for the satisfaction of being self-sufficient.
|
Themadhatter106
Psychedelic Teacher



Registered: 06/18/09
Posts: 100
Loc: USA
Last seen: 7 years, 8 months
|
|
Well, the new print looked a lot better and more professional but I still got a large amount of contamination. I didn't use an incubator and did everything as sterile as I possibly could. Out of 40 jars only two germinated, all have bacteria and smell bad.
I guess the print was just dirty.
Since I don't have a pressure cooker I made BRF agar. In my glovebox I birthed one of the germinated cakes that had bacteria and cut out a sector of the germination. Dipped that in hydrogen peroxide and transferred to a brf agar jar. I've made several.

It's turned fuzzy after one day, but only time will tell how the mycelium grows.
-------------------- "Turn on, Tune in, Drop Out"- Dr.Timothy Leary "If I had a world of my own, everything would be nonsense. Nothing would be what it is, because everything would be what it isn't. And contrary wise, what is, it wouldn't be. And what it wouldn't be, it would. You see?" - Alice (Alice in Wonderland) Don't Grow mushrooms just because you want to trip. Grow them to stop drug dealers from going to jail and for the satisfaction of being self-sufficient.
Edited by Themadhatter106 (10/20/15 09:03 PM)
|
Mr.GuessWork
Stranger

Registered: 03/30/13
Posts: 4,563
|
|
Agar is your best bet to get a clean culture out of that print. Do a streak plate with the spores and isolate from there.
|
Themadhatter106
Psychedelic Teacher



Registered: 06/18/09
Posts: 100
Loc: USA
Last seen: 7 years, 8 months
|
Re: 100% bacterial contamination [Re: Mr.GuessWork]
#22432330 - 10/25/15 03:10 PM (8 years, 3 months ago) |
|
|
Well with the jars that I transferred to the bit of the PF cake that I dropped in colonized completely. In the brf agar jars I made there is a big white fluffy mass of mycelium where I did the transfer. It definitely grew there. However, it wont spread to the brf. Its just a big mass in one spot. Even after 1 week, 2 of the jars show no spreading and in one of the jars there is a tiny bit of growth on the brf but not much at all.
I think either the moisture content is wrong or I didn't sterilize long enough, although I followed the tek and what others have done (5g BRF, 16ml of water). Instead of pressure cooking I steamed for 45 mins thinking that the time should be less than a PF jar because its not as dense. I opened up one of the jars I didn't use and it smelled a little funky. Can't say for sure its bacteria but its possible. So I made a new batch of jars steaming for 90 mins and with varying water content in case that was the problem.
I will try transferring some mycelium to the new jars and see if I get some growth.
-------------------- "Turn on, Tune in, Drop Out"- Dr.Timothy Leary "If I had a world of my own, everything would be nonsense. Nothing would be what it is, because everything would be what it isn't. And contrary wise, what is, it wouldn't be. And what it wouldn't be, it would. You see?" - Alice (Alice in Wonderland) Don't Grow mushrooms just because you want to trip. Grow them to stop drug dealers from going to jail and for the satisfaction of being self-sufficient.
Edited by Themadhatter106 (10/25/15 03:11 PM)
|
TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
|
Re: 100% bacterial contamination [Re: Mr.GuessWork]
#22432353 - 10/25/15 03:16 PM (8 years, 3 months ago) |
|
|
Quote:
Mr.GuessWork said: Agar is your best bet to get a clean culture out of that print. Do a streak plate with the spores and isolate from there.
Agar is your best friend.
|
BL4Z3D247
Stranger


Registered: 09/25/15
Posts: 41
Last seen: 7 years, 8 months
|
|
Steam sterilize your jars for at least 90 minutes from the time you dial down the stove. 45 minutes is not enough time at all. I bet that was your issue. Also I put microspore tape and a sheet of tin foil over the top so no extra moisture can get it. Worked for me and I really had no idea what I was doing when I did my first 5 PF jars. Good luck on this next try. Have patience and if you can try to keep the ambient temps at 70-75 during the germination/colonization stage. Oh and use a soft white bulb(2600k) during the colonization period too. I used indirect sunlight and they took forever to colonize. I used a 2600k soft white bulb for my last jars and the finished in half the time as my first ones.
Edited by BL4Z3D247 (10/26/15 08:44 AM)
|
TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
|
Re: 100% bacterial contamination [Re: BL4Z3D247]
#22436532 - 10/26/15 03:42 PM (8 years, 3 months ago) |
|
|
Using a light for colonization is pointless. There are many factors which could have contributed to the last batch of jars taking half the time. It's possible the heat from the bulb was a factor as well. Could've been genetics, moisture content, temp, GE, etc. Why are you using 2600k specifically, and not say 6500k?
|
BL4Z3D247
Stranger


Registered: 09/25/15
Posts: 41
Last seen: 7 years, 8 months
|
|
Quote:
RogerRabbit said: The purpose of the plastic bag is to shrink along with the substrate, preventing the gap around the edges that leads to side pinning, which seems to upset some folks. Personally, I see no problem in having five sides fruiting as opposed to only the top, but different strokes. . .
There is no reason whatsoever to have a mushroom substrate in total darkness. Normal ambient day/night light is good from day one, and with many species, it actually speeds up colonization. Avoid direct sunlight until after you've increased fresh air exchange at full colonization in order to stimulate pinning.
Light will help to get a much nicer pinset, but not unless you also increase fresh air exchange and keep the humidity in the correct range. Light is even more important once the fruits are growing, because it's needed to develop large, meaty mushrooms. Mushrooms grown without sufficient light are small, light weight, and have thin, small caps. RR
Is this not relevant anymore? I know I read another quote from RR stating the strength bulb to use for colonization(2600k) and fruiting(6500k).
All I know is my 1st 5 jars took well over a month to colonize in indirect sunlight. My other 12 were kept near a light with a soft white bulb(2600k) and they finished in just under 3 weeks. Maybe a fluke? I don't know. This is still pretty new to me and I'm still learning.
Edited by BL4Z3D247 (10/26/15 09:01 PM)
|
TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
|
Re: 100% bacterial contamination [Re: BL4Z3D247]
#22437812 - 10/26/15 09:25 PM (8 years, 3 months ago) |
|
|
The only light bulb I intentionally use for any part of the process is fruiting. Every other step gets ambient/indirect light, and I have no issues with my colonization speeds. At no point does any of my mycelium experience prolonged darkness past normal night hours, aside from cold storage of my cultures.
If you're noticing improvement, go for it. You might be onto something, but without side by side testing with an isolate, it's hard to say for sure. I don't want to piss on your party, and giving your mycelium light while they colonize will certainly cause no harm. 
I'm curious how much of this theory is conjecture or if it has actually been tested.
|
BL4Z3D247
Stranger


Registered: 09/25/15
Posts: 41
Last seen: 7 years, 8 months
|
|
You are correct, ambient light for colonization. I don't put the jars directly in the light, sorry for not being more clear.
Anyway, you're probably right, it could've been a variation of things with the preparation of the substrate and temps. My first 5 jars were in a slightly cooler room but I'm talking only a degree or two cooler.
|
TheEaglesGift
The Nagual


Registered: 04/10/11
Posts: 10,554
Loc: Ixtlan, Mexico
|
Re: 100% bacterial contamination [Re: BL4Z3D247]
#22438078 - 10/26/15 10:19 PM (8 years, 3 months ago) |
|
|
When dealing with spores, each time they mate they're configuring a different set of traits. Something to always keep in mind when looking at the differences from grow to grow.
OP, sorry for any derailment I may have caused.
|
Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
|
|
Might wanna try those spores on agar to see if contams are coming from them. Or maybe its the sterilising going bad. Anyway whatever the problem you can always fix it on agar. Its what I would try next if I had that much contams.
|
micro
bunbun has a gungun



Registered: 05/09/03
Posts: 7,532
Loc: Brick City
|
|
ampicillin plates
bacteria problem solved
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
|
Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
|
Re: 100% bacterial contamination [Re: micro]
#22454190 - 10/30/15 04:05 PM (8 years, 3 months ago) |
|
|
Quote:
micro said: ampicillin plates
bacteria problem solved
I found it funny when the first contam I got on my antibiotic agar was bacteria. Then again I didn't know what the hell I was doing so it wasn't really a suprise
|
micro
bunbun has a gungun



Registered: 05/09/03
Posts: 7,532
Loc: Brick City
|
|
Did you put the antibiotic in after sterilizing and cooling it a bit?
What did you use and how much?
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
|
Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
|
Re: 100% bacterial contamination [Re: micro]
#22455570 - 10/30/15 09:12 PM (8 years, 2 months ago) |
|
|
It was premade. Doesn't say how much they put in it. Here's the recipe.
24 g agar-agar 20 g malt extract 2 g dry yeast 1 g peptone Aminoglycoside antibiotics
|
micro
bunbun has a gungun



Registered: 05/09/03
Posts: 7,532
Loc: Brick City
|
|
Wrong antibiotic to use; it's not effective against a large range of bacteria. I couldn't find information on stability in heat at a glance, but gentamycin is one of the only antibiotics that can be autoclaved so chances are that kind of heat would destroy it anyway. Also, dry yeast that is hopefully dead o.O
Also, you may want to add simple sugars.
Pre-made powdered stuff is fine, and is easier to work with IMO, but you'll probably want to get potato dextrose agar or the like and after sterilizing it let it cool down a bit first and add ampicillin or another broad-spectrum antibiotic. I wouldn't recommend gentamycin as it can cause rather serious health complications.
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
|
Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
|
Re: 100% bacterial contamination [Re: micro]
#22455817 - 10/30/15 10:08 PM (8 years, 2 months ago) |
|
|
I meant premixed not premade sorry.
Yea I finally found sea veg agar in a health shop after weeks of getting wierd looks. No luck with dextrose or malt extract though. I did find liquid malt but I really wanna stick to the teks when it comes to agar.
Maybe ill see how much they are online
|
|