When I used to do it, I just used a loop to pick up the colonies I wanted to, then put them on a prepared slant.
Denny is correct. One starts by cleaning one's work area very well followed wiping it down with 70% alcohol. After that is done, one needs to wipe any dust off of one's alcohol lamp with an alcohol soaked paper towel and place it in the center of one's work area. The next step is to wipe down one's nichrome inoculation loop with alcohol and place it near the alcohol lamp before gloving up. Allow all of the alcohol to flash off all of the surfaces that were wiped down with alcohol before lighting the alcohol lamp.
This technique relies on rising air to create an aseptic work area. The air above the alcohol lamp is rising within a radius of around 6" from the flame; therefore, we need to do all of our transfers within this 12" diameter circle. The plate is placed within this circle with the colonies one wants to transfer pointing toward one's dominant hand (one will be holding the loop in one’s dominant hand). The blank slants are placed in a clean holder that has also been wiped down with alcohol that is located near one's non-dominant hand. Believe or not, I use a small diameter ceramic mug to hold the slants I plan to inoculate. A mug works as good as any lab rack I have used for this task and it is much smaller.
With everything setup, take the loop and place its tip in the hottest part of the flame, which is the tip of the inner cone with respect to the outer cone of the flame. The loop will start to glow. Move the loop back and forth until all of the nichrome wire portion has been passed through the flame and about an inch of the loop wire near the end is glowing. Now, one needs to carefully lift up the side of the plate cover with one’s non-dominant hand high enough to be able to scrape off a colony with the loop. Tne needs to be cooled before scraping the colony, so pick an area of the agar solidified media that has nothing growing on it and stick the tip of the nichrome wire there for a couple of seconds. We then scrap the colony off of the plate that we wto use to inoculate a slant and carefully remove the loop from the plate without touching anything. Now, while holding tip of the loop within the circle where the air is rising, one needs to grab a slant with one's non-dominant hand and carefully remove the screw cap with the pinky one's dominant hand while holding the slant with one’s non-dominant hand and carefully cradle the cap in the pinky of one's dominant hand (this step takes a little practice). Now, we pass the opening of the culture tube quickly through the flame a couple of times before moving it away from the flame and inserting the nichrome loop into the tube all the way to bottom of the slanted media. The loop is placed on the surface of the media and squiggly line is drawn on top of the media while slowly removing the loop. Now, we quickly pass the mouth of the culture tube through the flame a couple of times followed doing the same thing with the open end of the screw cap before screwing the cap back onto the culture loosely to allow CO2 gas to escape during incubation and then place the slant in another holder that will be placed in a draft-free area to incubate until the surface of the slant is covered with a film of yeast. If one wants to create another slant from a different colony (technically a different isolate), repeat this process before placing the slants in a draft-free area for incubation. After the surface of the slants have been covered with a film of yeast, one needs to tighten the screw caps and seal the slants with parafilm before placing them in a refrigerator in a sealed container that has been kept very sanitary.
Here is a photo of my old bank:
One can see that the inoculated slants are sealed with parafilm in this photo:
Topic: RVA Manchester consistent low attentuation (Read 2182 times)
