I use plates to isolate colonies (4 quadrant streaking as mentioned earlier) and to make sure that the source of the culture is not contaminated.
My method is to start by innoculating 2 to 4 plates from the same source, based on how "rare" the source is. Then, after incubating for several days, I select the strongest/cleanest colonies in each plate to innoculate 2 or 3 slants per plate.
Assuming that the source was not contaminated and my sterile technique was, well, sterile, I could have up to 12 possible slants. In reality, I usually only bother making 4-6 slants.
When I am down to two fresh slants, I innoculate another couple of slants when I open the 2nd to last slant. This way, I always have a known clean culture in my "yeast bank"
I use medical-grade agar (expensive, but I can't find Asian market agar locally) and DME:
- 40g extra light, hopped DME to 450ml water (around 1040 SG after boiling)
- boil for 5-10 min
- 1 tbsp agar
- boil for 5 min
- put into a glass, auto-clave safe medium bottle
- place into pressure cooker for 30min @ 15psi
- pour into plates and test tubes
- microwave extra agar/dme medium to boiling for 1 min and refrigerate for later use
This last step is just to raise temp to > 200degF before cooling.
I microwave the agar/dme medium or place back into the pressure cooker to sterilize before pouring more slants/plates.
I use hopped DME for the anti-bacterial properties of hops. If I'm out of hopped DME, I just place a medium sized hop pellet into the 400ml of water and make sure to boil for at least 10 min.
I'm not sure if my technique has improved or if it's the hops, but since I started hopping my agar media, the number of bacterial contaminations has dropped significantly!
My next improvement will be to place the hopped DME into the pressure cooker for 30min @ 15psi (without the agar) and then chill overnight before boiling and adding agar. This should precipitate the hot/cold breaks and result in much clearer agar medium.