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SanitizersvenusflyCbee25 5 12-25-01  02:24 am
How to make a Clean Room - 1
Pure Culture - 1

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Nan (Nanook)
Posted on Sunday, October 14, 2001 - 01:45 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Sanitizing and Sterile Technique


People tend towards sloppiness with their Tek until a batch of contams sweeps over their jars/cakes/beds like dominoes in a row. Sure you can be lazy and sloppy. But one day your number will come up. It happens and a cleanup is required.

In a clean room all horizontal surfaces should be wiped. EVERYTHING should be wiped, but you can get away with just horizontal surfaces... Most of the time... Bins, counters, sinks: How do you “sanitize” them?

Sanitary or "clean" practices should be used from the time a jar is removed from the PC, through Birthing, until the cake fails.

Rags: You need lots of really clean rags. Pillowcases too with some Straw Teks. Collect or buy secondhand, but get a decent bag of clean cotton rags together and wash them in very hot water with unscented detergent and liquid bleach. Dry in a clean dryer, transfer them with clean (gloved?) hands into a clean plastic bag. Rags for Glovebox use can be wrapped in foil or bagged up in an Oven Bag and sterilized in the pc for 45 min after washing.

The cheapest and simplest sanitizing solution is one part Clorox added to nine parts water. This sanitizer needs a wetting agent when it is used on plastic: ½ teasp of liquid dish soap to one litre of Clorox solution; wash plastic down with a soaked clean rag. Buy new plastic containers for Straw Tek. Never, ever, use anything scratchy to wash and sanitize plastic. Scratches in plastic instantly fill with contams that are impossible to remove. The surface must be new, and kept in smooth, as new condition, in order to sanitize properly.

Mix up a litre of 10% Clorox with wetting agent (dish soap) and pour it into the plastic bin. I mix it a little strong with Clorox (15%) if the bottoms are really cruddy. Wear rubber gloves, and scrub it out with a clean soft rag… Rinse. If the bin was really cruddy, toss the rag into the “used” bag to be rewashed, grab a fresh rag: don't carry contams with you.

Mix up a fresh batch of 10% Clorox sol., plain this time, no wetting agent or soap. Wipe a chair back down with it, using a clean rag (of course). As the chair back is drying, rinse and wipe the bin with the fresh Clorox solution and clean rag. Get the insides of the bins first, then the outsides: Wipe down lids too. Place wet bins upside down on the clean chair back to drip dry. When you are ready to spawn, wipe down the inside of the bin with a clean rag soaked in rubbing alcohol, then wipe a chair back and hang upside down to dry: spawn after the alcohol evaporates. Sanitize bins used for PF cakes the same way.

Rubber gloves… Keep a couple pairs of cheap grocery store or Playtex gloves on hand and wear them. They will protect your hands from the strong sanitizer, and they are clean. Wear gloves when you spawn. Rinse gloved hands with alcohol, or spray with Lysol, rub them together and wipe on an alcohol soaked rag. Beware of fire risk, always keep water on hand when working with alcohol and flame of any type. I keep the two separated.

Pour one cup bleach into the dishwasher during the soap cycle. All dishes coming out of a properly functioning dishwasher run at 180* with Clorox are sanitized. Water bottles & carboys should be sanitized by filling with 10% Clorox, soak for 20 minutes, drain and rinse. Bottles should contain no visible filth before sanitizing.

Also, go down to the sporting goods section at Wally Mart and buy a box of gun cleaning patches for 12 guage shotguns… Makes great alcohol wipes for needles: toss a few in a small bowl and splash in just enough rubbing alcohol to wet them. Cover with a saucer.

Counters and work surfaces should be sanitized where you are grinding cakes; grinders should be wiped with alcohol and allowed to dry. Jar Incubators should be sanitized periodically. And the things you touch: doorknobs, light switches, faucets, cabinet and fridge handles... You pick up contams at every one of these spots. If they are not clean, don't touch them during clean glove work.

To get highly clean, practically sterile environments: inside Gloveboxes primarily. Treat three times: rag in soaped Clorox sol., rag in plain Clorox sol., (allow to dry) and spray with Lysol. Straw bins should be finished with a wipe of rubbing alcohol on a rag instead of lysol. Wash rubber gloves in a few mils of alcohol periodically while wearing them, especially if you are using a pressurized box or hood where you have freedom of movement in and out of your sterile work area. Don't answer the phone and expect to have clean hands afterwards.
Sanitizing Perlite: It is reported that Perlite may be reused by washing in tap water, draining, rinsing with a solution of 9 parts clean water and one part bottled Hydrogen Peroxide, H2O2. It may also be boiled or baked after rinsing.

Syringes: The best way to sterilize syringes is to fill them with water, wrap them in foil, and pressure cook for 20 minutes... But I have successfully reused "clean" PF syringes without Pressure Sterilization.: Take a well rinsed syringe and draw it up full of water with no air gaps. Put the syringe and needle (separatly if need be) in a large saucepan, cover with Clean Water and bring to a boil. Cover tightly, reduce heat to a simmer, and cook for 30 minutes. Turn off the heat and allow to cool. Remove the syringe(s} with clean gloved hands and attach the needles. Turn the heat back on and bring the water to a boil. Suck boiling water up with the syringe (be careful it will be hot) several times, squirting the spent water out into the sink. Ready to suck inoculum and shoot jars.

Needles: needles should be wiped with rubbing alcohol or flammed over an Alcohol Flame for several seconds if they contact any non-sterile surface. I let the tip get red and squirt out some liquid after flamming. Needles should be wiped clean with an alcohol patch whenever they gum up during inoculation. Do not inoculate jars with a hot needle.

I Flame Needles before each use and have never had a contam in a syringe or needle prepared either way.

For sterile water to make up spore syringes from prints fill a clean half pint jar 3/4 full of Clean Water, put a dome lid on seal side down with band on loose. Foil Cover. 20 minutes in a PC at 15 lbs. Band lid may be tightened for storage.

Dextrose\Honey jars should be sprayed with lysol and placed in the glovebox to dry before removing the Foil Covers. Foil packs of sterilized instruments (exacto, sizzors, tweezers, dome lids, for Spore Printing, Innoculating, and Cloning) should also be sprayed with Lysol and allowed to dry in the glovebox before being unwrapped by gloved hands... This is the basics of sterile technique.

How far you advance in mycology is in part a function of how clean your work is. Take pride in it.

STERILIZATION
All utensils used in the cultivation of mycelia must be sterilized by heat before use. Glassware must be boiled in water for 30 minutes. Metalware used repeatedly must be held in a flame until glowing and then allowed a moment to cool before making contact with any cultures or specimens. When the inoculation loop has been used to transfer a fragment of mycelium it must be flame sterilized again before touching the next fragment. All medium containers must be sterilized after the medium has been poured. This process is known as autoclaving. Containers no more than half full with medium are placed in a canning type pressure cooker. The lids of these must be loose enough to allow escape of internal pressures. Otherwise the containers may crack. Seal the lid of the pressure cooker. Keep the stopcock valve open. Using high heat bring the cooker to boiling so that thick steam comes through the vent. Close the stopcock and let the pressure rise to 15-20 pounds. (250 Degrees F.) for 30 minutes. This should be enough to destroy any foreign spores or lifeforms. Any higher temperature or longer period would cause the Dextrose or Maltose sugars to Carmelize. This would inhibit growth and psilocybin production of the mycelium. When the autoclave period is up turn off the heat and let the cooker cool to room temperature. Do not release the stopcock until everything has thoroughly cooled or the sudden change in pressure will cause the containers to boil over. Discard any containers that have cracked during sterilization. Keep all containers of medium at incubated for three days to see if any foreign molds develop. If they do occur discard the medium in the contanminated jars and thoroughly clean and sterilize such jars before using again.

Clean Room : Iodophore : Straw Tek Tips : Simple Cubie Tek : Pressure Cooker Safety : Shroom Glossary