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Offlinemonkeylove
Stranger
Registered: 03/26/02
Posts: 14
Last seen: 14 years, 9 months
CRASHED AND BURNED
    #604202 - 04/10/02 12:48 PM (15 years, 21 days ago)

My first foray into the field of mushroom cultivation has been a complete failure. I need to know where the key fuck-up was in my procedure. Here's what I did:

I mixed and packed the substrate as per the MMGG technique. The first time I packed the eight jars, I waited a few days until a safe place to sterilize became available. Big mistake. The jars got severely contaminated and I had to start over. I used the same jars the second time around. All I did to clean them was to wipe down the insides with a dry cloth. Should I have cleaned them more meticulously? Packed the jars once again using the same technique as I did the first time. Boiled them for one hour with the lids on the jars, holes in the lids, and aluminum foil over the tops, crumpled around the sides. Being the genius that I am, of course, I forgot to put a lid on the pot I was boiling the jars in. The next morning I realized the error in my ways, and decided to resterilize the jars without repacking them again. That's what I did. One more hour, this time with a lid. Did I boil too much of the moisture out of the jars? Also, there was no washcloth on the bottom of the pan. I took it out when too much water vapor was building up underneath it, causing the jars to move around and almost tip over. Could too much heat have transferred to the jars?

I allowed the jars to cool for about three and a half hours. Upon my pre-inoculation inspection, I took note of two of the jars having cracked. They weren't broken. They just had long cracks in them. I decided that it might not be too big of a deal and proceeded to innoculate them with a 10cc Puerto Rican syringe that had been sitting at room temperature since I got it in the mail from Sporeworks. That had been, oh, let's say, about three weeks.

I built an incubator out of a plastic cooler with a fish tank heater inside a cup on the inside. The heater was at its highest setting (92 deg. F), but the highest temperature I could get inside was about 76 deg. F. This pissed me off greatly, but there was nothing I could think to do to raise the temperature another 10 degrees or so. Five days passed, and I saw nothing. No mycelium growth, but no contamination, either. I then left on a trip for another five days, and when I got back, two of the jars had definitely begun to colonize, but there was a whole bunch of black mold or some shit that had grown in there with it. Another cake had some white stuff growing all through it, though it wasn't fuzzy white. It was all milky and disgusting looking. There were a few jars with no signs of growth whatsoever. I figured that if I haven't seen something by now (approx. 10 days), there just wasn't going to be any happenings.

My guess is this: The two biggest mistakes I made were a) I boiled the jars twice, thus drying them out too much, and b) The temperature was about 10 degrees too cool for efficient colonization, and that opened up too large of a window of time where the jars were vulnerable to contamination. What do you think? I'm so sad. I feel like crying. Crying like a... like a little girl...

Oh yeah, and is there any way I can get a new syringe via Prioriy Mail? I want to get back in the saddle before I allow myself to become too discouraged. Someone tell me it will all be OK...


--------------------
"Fireflies bring the stars down to us" -Jonathan Greene
"Gravity is a myth- the world sucks" -Chad M. Horn


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OfflineXAZIA
glitter goddess
Registered: 08/22/01
Posts: 678
Loc: oklahoma
Last seen: 14 years, 7 months
Re: CRASHED AND BURNED [Re: monkeylove]
    #604213 - 04/10/02 01:08 PM (15 years, 21 days ago)

it will be okay...if being okay is starting from scratch again. but really don't worry. mistake teach us alot. first of all you are right on two of your assumption. you probably dried out your substarte a little too much by sterilizing twice but this is not the cause of your diasater.
i believe you didn't let your jars cool long enough and killed your spores at innoculation. three hours i don't is long enough to cool. and if you noticed cracks in the jars you should through them out. its a futile point man. if the jar has a crack thats a breech of the sterility and anything you do after that is a mute point. if you need to get the temp up inside your incubator place it on top of a heating pad.

good luck!!


--------------------
"Emancipate yourself from mental slavery, none but ourselves can free our minds."

Bob Marley

Remember, EGOISM is the beginning, the source, and the root of EVIL!

http://www.fanaticus.com/pf-tek.htm
http://www.mycotopia.net/teks/hongus.html


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OfflineAnnoA
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Re: CRASHED AND BURNED [Re: monkeylove]
    #604215 - 04/10/02 01:10 PM (15 years, 21 days ago)

Don?t worry, first time failure is nothing unusual to happen, you will do better next time whith the knowledge you got the first time.

Deffinitely wash out contaminated jars very good using a diluted bleach solution next time.
If you want a method how to get a more reliable substrate moisture, take a look here:
http://www.fungifun.org/pf/pf_en.htm

For an icubator setup that will use more available heat from your heater, look here:
http://www.fungifun.org/pf/incubator.htm


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OfflineSeussA
Error: divide byzero

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Re: CRASHED AND BURNED [Re: monkeylove]
    #604219 - 04/10/02 01:12 PM (15 years, 21 days ago)

You have a series of mistakes, one on top of the other.  This isn't meant to be a flame, please don't take it that way.  Here are a few of the bigger things that need fixing:

I waited a few days until a safe place to sterilize

Never wait to sterilize the jars.  You just give the contaminates time to build up their army.

I used the same jars the second time around

Some people will reuse contaminated jars.  I don't recommend it.

All I did to clean them was to wipe down the insides with a dry cloth.

Use a 10% bleach solution and soak them for a few hours next time.

I forgot to put a lid on the pot I was boiling the jars in.

Couple that with the repeat cooking and the substrate was probably much too dry.

there was no washcloth on the bottom of the pan

If there is no spacer between your jars and the pan, then the jars will overheat and dry out the substrate or crack.

Somebody else can take over from here.  :smile: 


--------------------
Just another spore in the wind.


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OfflineDrAbeLincoln
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Registered: 08/31/01
Posts: 248
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Re: CRASHED AND BURNED [Re: monkeylove]
    #604220 - 04/10/02 01:13 PM (15 years, 21 days ago)

Well you should have really washed the jars that were contaminated to begin with...wiping with a dry cloth isnt doing a whole lot....boiling them twice wasnt really a good idea either..you could have very well dryed them out especially if they were sitting directly on the bottom of a pan and boiled two times....I dont think its the incubator thats your problem...I've had jars colonize at room temp before...just takes forever and being that your jars were at 76 degress F they could take up to 14 days to show signs of germination...be patient....my suggestion is to CLEAN everything....boil your jars again like you did for an hour...just this time put something on the bottom of the pan like a washcloth so all the heat isnt directly transferred into the cake and your jars dont crack....and if you want a syringe quick...order from the Little Guy...he uses priority mail and has kick ass delivery time....dont give up...just have patience


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OfflineParticleMan
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Registered: 02/05/02
Posts: 240
Last seen: 14 years, 11 months
Re: CRASHED AND BURNED [Re: monkeylove]
    #604223 - 04/10/02 01:16 PM (15 years, 21 days ago)

i would say your biggest mistake was using the jars that were contaminated, i have a pressure cooker and i am stil a little hesitant about using jars that had contams in them. i at least pc them twice before putting in substrate and doing a final PC. The temp difference would not make a difference in the 10 days pretaining to letting in contams, its going to take more than 10 days anyway.

All in all i would say first dont cut corners.
dont use something you already think might be messed up
get a pressure cooker


--------------------
___________________________________________________________
"the weekend has landed all that exists now is clubs,drugs, pubs, and parties" - Human Traffic


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OfflineLSD_4me
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Registered: 02/09/02
Posts: 416
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Re: CRASHED AND BURNED [Re: ParticleMan]
    #604228 - 04/10/02 01:20 PM (15 years, 21 days ago)

you guys throw away jars just becasue there was a contam in them? if your careful this isnt necessary at all... its kinda crazy if you ask me... as long as you clean them good you wont have problems, the jars are made of glass remember... not some other porus material, if your really paranoid wash them in a bleech solution


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OfflineGaNjAShRooM
===SPUN===

Registered: 02/22/02
Posts: 2,954
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Re: CRASHED AND BURNED [Re: monkeylove]
    #604247 - 04/10/02 01:53 PM (15 years, 21 days ago)

all i can say is this-dude if you are going to culitvate,you have to pay attention-u made simple mistakes-mistakes that if you read a little,would not have happened-


--------------------
Cultivation Laws Of America Suck


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OfflineParticleMan
enthusiast
Registered: 02/05/02
Posts: 240
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Re: CRASHED AND BURNED [Re: LSD_4me]
    #604265 - 04/10/02 02:25 PM (15 years, 21 days ago)

LSD 4me,
I know it may be kind of stupid to throw away jars, but 12 of them cost like $8, i would rather spend a dollar than use a contam'ed jar and wait 15-20 days to find out it messed it all up. I would rather be overly cautious than anything else.


--------------------
___________________________________________________________
"the weekend has landed all that exists now is clubs,drugs, pubs, and parties" - Human Traffic


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OfflineAnnoA
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Re: CRASHED AND BURNED [Re: ParticleMan]
    #604319 - 04/10/02 02:59 PM (15 years, 21 days ago)

Throwing jars away is really not called for. If you can wash your dishes, then you can wash the contaminated jars, as simple as that.


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Offlineseattlekid
Stranger

Registered: 02/26/02
Posts: 87
Last seen: 11 years, 4 days
Re: CRASHED AND BURNED [Re: Anno]
    #604351 - 04/10/02 03:29 PM (15 years, 21 days ago)

I think i would have to agree with what everyone else said in this post but i just wanted to add a small side note about the temp. Living is seattle my room is rarely above 60-65 during the winter thus jars are rarly colonized above that temp. And I havn't really seen any problems with contamination or non-growth due to the low temp so far. If anything the jars just take alittle longer...i think a 1/2 pint jar using PF tek would take around 3 weeks to a month to completely colonize. Also if you?re just boiling jars now it might be worth it to you to upgrade to a pressure cooker...it defiantly is worth its money over the long run...you'll have lots issue with contamination which sounds like the root of your problem. Try ebay, you can find really good ones super cheap...i think i got mine for like 50 bucks and its a presto with a capacity of 7 1 quart jars or 24 1/2 pint jars.


--------------------
Life moves pretty fast, if you don't stop and look around once in a while you could miss it.


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OfflineLSD_4me
addict
Registered: 02/09/02
Posts: 416
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Re: CRASHED AND BURNED [Re: seattlekid]
    #604484 - 04/10/02 05:36 PM (15 years, 21 days ago)

boiling works, but a pressure cooker is the best investment you will make in this hobby if your even 1% serious about it....


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Offlineshianggu
member
Registered: 03/24/02
Posts: 171
Loc: Asia
Last seen: 12 years, 10 months
Re: CRASHED AND BURNED [Re: monkeylove]
    #604618 - 04/10/02 08:14 PM (15 years, 21 days ago)

As a newbie I really feel your pain. Luckily you got some great advice from others here.


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Offlineitalic
seer
Registered: 02/13/02
Posts: 15
Loc: Midwest, USA
Last seen: 14 years, 15 days
Re: CRASHED AND BURNED [Re: monkeylove]
    #604647 - 04/10/02 08:47 PM (15 years, 21 days ago)

The first line of your post bothers me.

"I mixed and packed the substrate as per the MMGG technique."

I hope that you did not mean pack literally, as this would lead to imminent failure as well as the other things pointed out in this post. Loose as a goose is the key.

italic.


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Offlineegolesss
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Registered: 10/26/00
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Re: CRASHED AND BURNED [Re: monkeylove]
    #604675 - 04/10/02 09:17 PM (15 years, 21 days ago)

.First the jar controversy, Contams die at 165 degree's, you will boil around 212 degree's killing all contams so throwing away jars is a total waste... My friend doesnt's do anything but wash them out and even that is really not needed as long as they are sterilized "correct" during canning. The only benifit of a P.C. is pounds per square inch fully sterilizing faster and thorough in a shorter time which is fine for 1/2 pints or even pints of rice,careful not to dry them out. You are using the MMG?If so like someone mentioned you don't pack at all the mmg uses stright rice right? If so you may want to trry adding some vermiculite to your mix to keep from drying out and airate the mix a bit. Your jars cracked because you need to place a spacer under the jars or a washcloth. Remember to flame your needle between each jar AND MORE spore liquid doesn't mean better results it means more water. Also wait longer than 3 hrs to innoculate.. Top off the jars with a verm layer before puttin the lid on it will form a barrier over substrate from contams...........You said this "It was all milky and disgusting looking. There were a few jars with no signs of growth whatsoever" That sounds like they were too wet,so does the black mold its' easy to do with MMG tek. A good rule is this...THE GRAIN SHOULD BE SEPERATE WHEN YOU STIR IT, ONLY FORMING SMALL CLUMP OR BALLS, IF YOU SQUEEZE IT TOGETHER IT SHOULD JUST BARELY HOLD TOGETHER


--------------------
Going crazy will drive you mad, but once you get there the rest is easy....All spores are not created equal!!!!!!!!!!! Sporeworks, Hawkseye, PF, they are completely viable with very strong genetics.




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OfflineLSD_4me
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Registered: 02/09/02
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Re: CRASHED AND BURNED [Re: egolesss]
    #604793 - 04/10/02 11:57 PM (15 years, 21 days ago)

actually some contams can survive 212... some types of bacteria can live in much hotter temps than that..... but most that you will encounter will die with boiling but with PCing you are a lot safer.


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Offlinefidget
Fungus Hunter

Registered: 03/18/02
Posts: 245
Loc: Oregon
Last seen: 11 years, 4 months
Re: CRASHED AND BURNED [Re: LSD_4me]
    #604814 - 04/11/02 12:20 AM (15 years, 21 days ago)

My first time was a huge bomb and i quit for 3 years. Now im having fun and doing it right again. Dont give up.


--------------------
- fidget

"With ordinary consciousness you can't even begin to know what's happening."



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Offlineegolesss
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Re: CRASHED AND BURNED [Re: LSD_4me]
    #604820 - 04/11/02 12:24 AM (15 years, 21 days ago)

What are the names of some of these contams? I am curious, because, the only one food related I know of that takes more than 165 is botulism and that dies at 170 max...........Here is a reference chart

TERM DEFINITION EXAMPLE
Sterilization Complete destruction 121oC/15 min; 170oC/2h
Disinfection Application of chemicals to objects Chlorination of water; kill pathogens
Antisepsis Application of chemicals to living tissue Treatment of wounds
Bacteriostasis Halts growth but not killed Refrigeration, dyes in food
Asepsis Absence of pathogens; aseptic techniques Air filtration, uv light, gloves, gowns
Sanitization Public health; mechanical / chemical cleansing Palatability of food

2. Factors influencing the effectiveness of control methods
A number of factors affect the usefulness (efficacy) of control methods & all factors should be considered to devise control measures
Size of microbial population:
Death is exponential ie more microbes = more time to kill the population
Plot the log of the no. of surving microbes vs time = straight line, the slope = killing rate
Initial microbe concentrations one can predict the kill time

Exposure time of the agent:
Increasing exposure time increases kill rates; kill time used is usually well past the required minimal contact time
large volumes / vessels require more time for complete destruction Container Size* Liquid Volume (ml) Sterilization time (min)
Test tubes / Erlenmeyer flask 10 - 900 15
Erlenmeyer flask 1000 - 2000 20
Bottle 6750 70
* A container is not usually filled past 75% of its capacity.

Effect of concentration, temperature & pH:
Increasing concentration is similar to increasing exposure time
Temperature, pH & concentration at which the agent works best
Stability of the agent at various pHs & temepratures
Legionella pnuemophila (Legionnaires disease) - the effect of the microbe suspended in tap water & exposed to different temperatures, pH & chlorine concentrations
Protective features of the microbes (refer to microbial structures section)
Endospore formers such as C. tetani
Cysts -- Protozoa
Porins -- Pseudomonas
Cell wall -- Mycobacterium tuberculosis (opportunistic pathogen)
Active growth vs resting cells - antibiotics (penicillin - transpeptidase)
Interactions & protective features afforded by the environment:
sewage is rich in organics - polio virus protected
Disinfecting in hospitals - selection of ressistance
Lipids & fats in dairy industry - protect spoilage microbes
Remember that lab conditions used in testing are different to field conditions

Methods for controlling microbial growth
Many methods to choose from:
Physical
Heat (Dry, Moist, Pasteurization)
Filtration
Low Temperatures (fridge, freezer)
Desication & osmotic pressure
Radiation (ionizing and nonionizing)
Chemical
Phenol & phenolics
Halogens
Alcohol
Heavy metals & their compounds
Surface active agents ( anionic & cationic detergents)
organic acids
gaseous sterilants
oxidising agents
chemotherapeutic agents target
cell wall
protein synthesis
nucleic acids
cell membrane
essential metabolites (antimetabolites)

The method of choice depends on the situation & practicality of the approac
The material to be treated eg heat labile or heat stable???
Laboratory conditons differ from field conditions
Factors affecting growth eg growing or resting cells, ressistance factors

Physical Methods of controlling microbial growth
Method Mechanism of Action Comments Preferred Use
1. Moist Heat(A)
a. Boiling Denaturation Kills vegetative cells but not spores Equipment, dishes
b. Autoclaving Denaturation Sterilization (autoclaves, pressure cookers, retorts) Media, linens, equipment, dressings
c. UHT Denaturation Sterilization; 141oC / 2secs Milk falls in a thin film thro a chamber of superheated steam
2. Dry Heat(A)
a. Flaming Burning to ashes Sterilization Inoculating loops
b. Incineration Burning to ashes Sterilization animal cacasses, dressings, wipes
c. Hot-air
sterilization Oxidation 170oC / 2hrs Glassware, needles, glass syringes
3 Pasteurization(A)
a. Low temp
long time
(LTLT) Denaturation 63oC/30mins Milk: batch process in tanks
b. High temp,
short time
(HTST) Denaturation 72oC / 15secs Milk: Flash method thro continous winding pipe
4. Filtration(B) Separation Liquid thro screen Heat labile material
5. Low Temp
a. Fridge Growth slows Bacteriostatic Drug, Food
b. Deep Freezing Growth slows Preservation -70oC Drug, food & culture
6. Desiccation(C)
a. Lyophilization Growth arrested Long term preservation of microbes Food, Drug & culture
b. Osmotic Pressure Plasmolysis Loss of water Food preservation
8. Radiation(D)
a. Ionizing DNA destruction Not commonly used Sterilizing medical & dental supplies
b. Nonionizing DNA py-py dimers (eg UV) Not very penetrating radiation UV (germicidal) lamp

(A) Moist Heat is used extensively by the food (canning & milk) industry
Remember: microbe properties are important & so are the environmental conditions
Improve shelf life (sporeformers, anaerobes - hermetic sealing, thermodurics are a problem why?)
Kill pathogens to break the route of transmission
Non-spore formers in milk (M. bovis - TB, Coxiella burnetii - Q fever, Brucella - brucellosis); S. typhi & carriers eg the milk industry
C. botulinum in canning industry
Thermal Death Point (TDP): lowest temp required to kill all microbes in liquid suspension in 10 mins.
Thermal Death Time (TDT): Minimal time required to kill all microbes keeping temperature (or bacteriocidal agent) ) constant.
Decimal Reduction Time (DRT or D value): Used for heat ressistant bacteria; Time required to kill 90% of the population at a given temp. (Log growth and log death concept)
B. stearothermophilus & / or C. sporogenes is used to determine acceptable D values
C. botulinum endspore D value = 0.21 min at 121oC (heating food at 121oC for 2.52 mins means 10-12 chances of endospore survival ie 1 in trillion chance
Accidic canned foods (tomatoe, pineapples, orange juices) require lower temps. Eg 100oC for 10mins. Why?
Milk industry uses lower temps. Eg pasteurization preferred. Why?

(B) Filtration is used for sterilizing heat-labile liquids and to sterilize air (gas) for creating aseptic environment (physical removal of microbes)
(i) Liquids:

Filters for removing pathogens come in different types:
Cellulose acetate
Celulose nitrate
Polycarbonate
Teflon
Filters vary in pore size (0.22um, 0.45um , 0.8um) but 0.2um are usually used but Mycoplasma and viruses may pass thro. Why?
(ii) Air:
To create an aseptic environment for microbiological procedures in semi contained areas. Eg laminar flow hood
Directs sterile air from the outside into the confined space via a 0.3um HEPA filter (High-efficiency Particulate Air) & stops contaminants coming into the space
Cotton wool used to "plug" test tubes, flasks, pipettes for growing ro working with cultures, surgical masks act in a similar way
(C) Cells require water for metabolic activities. Desiccation is a process by which water is removed and therefore growth is affected

Desciccation prevents microbial reproduction but is dependent on
Cell structure. Waxy material of cell wall protects M. tuberculosis, fungal spores (plant pathogens) disemmination
Environment. Nasal secretions can protect influenza virus to survive
Lyophilization (Freeze-Drying): Preserving microbial cultures
American Type Culture Collection (ATCC)
Australian Culture of Microorganisms (ACM)
Drying food prevents spoilage:
Aw < 0.9 inhibits bacteria
Aw < 0.65 inhibits fungi & most microbes
Examples: prunes, dates, figs, rasins - natural, Evaporated milk - removal of 60% water from milk, powdered milk - removal of 85% water
(D) Electromagnetic radiation causes cellular damage

Chemical methods of controlling microbial growth
Microbiology laboratory standards, design and safety

--------------------------------------------------------------------------------


--------------------
Going crazy will drive you mad, but once you get there the rest is easy....All spores are not created equal!!!!!!!!!!! Sporeworks, Hawkseye, PF, they are completely viable with very strong genetics.




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Offlinedelysid_1
journeyman
Registered: 01/17/02
Posts: 59
Last seen: 14 years, 8 months
Re: CRASHED AND BURNED [Re: egolesss]
    #604859 - 04/11/02 01:15 AM (15 years, 21 days ago)

Swid is waiting for his first batch to fruit. He inoculated 36 jars and one myco bag and not one loss due to contamination. He also did not wash out any of the jars before use. If you must wash them out first just use 91% isopropyl alcohol- it will kill everything. Even if it does not kill everything it still should not matter so long as you PC or steam the jars for an hour. There is now way you could get contams if you sterilize for an hour just no way. Also although I am a newbie I would still like to make a suggestion. Buy a pressure cooker damn it! It is so cheap. I can't imagine that people will invest all this time and money into buying spores, building terrariums and all that crap and will not spring $100 for an all american 915 PC.
Even without the PC swids friend grows and has never lost a jar to contams before birthing- and he just steams! I would try again and just make sure to sterilize for at least an hour.


--------------------
"Catholic church molestation scandel- the lord works in mysterius ways indeed........"

-delysid_1


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OfflineLSD_4me
addict
Registered: 02/09/02
Posts: 416
Last seen: 14 years, 11 months
Re: CRASHED AND BURNED [Re: delysid_1]
    #604867 - 04/11/02 01:27 AM (15 years, 21 days ago)

you can get a nice one on ebay if you look, hawkings 12 litre's go for like around 40 bucks and they sell online for 75 non auction....


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