INOCULATING 4 PLATES FROM MASTER SLANT FOR MASTER SPAWN JARS.
I execute all standard aseptic technique in front of a flowhood.
I lay out my utensils on a bleach alcohol soaked papertowel and fold it over to cover them up.
I grab my PDA plates. I like to use ziploc bags instead of micropore tape, parafilm, and etc. It is easy for me to use and increases efficiency. I bought a 1000 for 7 bucks i think from Amazon.
I spray the bag down with alcohol then remove it and place in a container. Most of the time i toss them and use new bags but this time i did not. If im reusing the bags i make sure to open them with ooening facing away from the air flow. If im using new bags i load my container with the quantity i need and spray with alcohol before putting the lid on.
I wipe every plate down with alcohol.
I grab my container i use to store my master slants, a deli container filled with pollyfill.
I select the slant i want to use and wipe it down with alcohol and remove the micropore tape while leaving the pollyfill in. I bore out my caps and pack with pollyfill before covering with micropore tape.
I place it in the holder that has been cleaned and steralized.
Its cramped but we are ready to go. Everything you see has been cleaned. Once i get everything laid out i spray everything with alcohol again and let it sit on there.
This was hard to do and take pics but i pulled it off. My only concern is it required me to leave the lids off longer then usual.
I lay out 4 plates that i want to inoculate. I work front to back so that if anything where to drop off of me and make it down to the working surface the plate has already been inoculated.
When ready to go i grab my loop and flame steralize it. For photography purposes i set it so i didnt have to hold it. I would never recommend doing this as it is a blow torch and it gets knocked over it could be really bad.
I then touch the agar in the plate that is going to receive the transfer. This cools it down and helps the myc to stick to the loop.
I then go to the slant and smear the surface.
Take the loop with the myc now on it and go back to the plate and smear the center.
I do this for all 4 plates making sure i flame steralize the inoculation loop between plates.
I put a fresh piece of micropore tape on my slant and it will go into incubation to recover and colonize the agar surface again.
I lable my plates with any information i need. If isolating or making alot of transfers i use a spread sheet i made that tracks plates and transfers utilizing diagrams and space to input information such as temp, nutrient agar ingredients, colonization times, and etc. But for this instance just strain and date is fine. I wrote iso on the tag but it is not a true monoculture but rather a very agressive, nice fruiting specimen.
I would say after a few more transfers it would be a true mono.
I will incubate these at room temp under my flowhood. Its a mess right now and requires some organization.
At the end of my session i wipe down my flowhood and replace the dust cover.
Edited by Microbe77, 22 November 2014 - 04:57 PM.