Since I'm stuck waiting for my agar plates, I'm forging ahead with culturing the freeze dried
P. chrysogenum that I ordered from Carolina Biological supply. The point of this exercise was to determine if an intrepid ZS'er could, under ZPAW conditions of sanitation and using ZPAW materials, use this type of preserved culture to grow a viable penicillin mold.
In order to approximate the (probable) inability of an average ZS'er to maintain strict aseptic technique in a world of shamblers, I decided early on that I would not lose my mind trying to keep things sterile and uncontaminated. Basic sanitation applied: Hands were washed frequently with soap and hot water (both of which we should have in the ZPAW), utensils and containers were likewise washed and dried with a clean dishtowel, cutting surfaces were wiped down with a soapy wet rag in between items, the utensils that came in direct contact with the freeze dried culture were flamed or dunked in boiling water between contacts, etc. In short I basically tried to keep things as clean as I do when I'm cooking, which is clean but far short of having everything I touch autoclaved for lab sterility.
First stop was selecting appropriate media and supplies. I tried to pick a variety of items for my media, at least some of which would likely be availble to an average homesteader. The finalists were:
1. Standard Idaho potato
2. Sweet potato
3. Unflavored gelatin (Knox brand)
4. Unflavored gelatin (Knox brand) + 1 TSP organic honey
5. Plain white rice (not instant)
6. 'Artisan' white bread from a local bakery (represents the homemade bread we'll be living on).
My media trays are disposable ziploc style tupperware.
I had to make a 'control' tupperware as well as an 'active' tupperware for each media, since we need to know exactly how aseptic my half-ass aseptic technique was. Also, a control media should tell us if there are naturally occurring molds in the items I slected that might cause the
P. chrysogenum to be either impure or inhibited altogether. Media prep was fairly basic: peel, wash, and quarter the potatoes, prepare the rice and gelatin according to package directions, and lop off a few slices of bread.
The full set of tupperware (control and innoculated) is shown here:
A close up is here:
From left to right, the picture shows the potato slices (Idaho and sweet), the bread, and the rice (still warm from cooking, so it's a bit foggy). The 'C' marker shows the un-inoculated control plate, the 'P' marker shows the plate that I inoculated with the freeze dried spores. The other information is the date and time of inoculation or preparation.
A close up of the gelatin plates is shown here:
One of the plates is plain gelatin, the other is the one with the honey added. You'll note that both carry the 'P' designation. I innoculated both plates, mostly because I ran out of gelatin, but also because I figured the gelatin was made with boiling water and poured while still hot, so it was likely about as bug free as it was going to get - - hence, no control needed (we'll see about that...).
Reconstituting the freeze dried P. chrysogenum was essentially idiot proof - - you just follow the directions in the package. The package contents are shown here:
This shows the directions (very easy to follow), the freeze dried spores (small glass container), and the rehydrating solution (large tube, which in this case was plain water). Squirt the water into the tube, wait 1/2 hour, and then suck out the sediment which has settled to the bottom of the tube. These are the spores you use to innoculate your media. The freeze dried media basically looks like a hunk of yellow chalk before you reconsititute it, after you add the water it separates into two layers, shown here:
The bottom layer (kinda hard to see) is the layer of spores.
So, slurp up some spore goo with a pipette and put 3 or four drops on all the media items I had labeled 'P'. Fairly straightforward. I'm keeping these all in my basement, which stays around 70 degrees F - not the ideal temperature (which is a bit higher at 25C according to the literature), but we likely won't have automatic thermostats and central air in the ZPAW either! As an additional control, I inoculated one of my lab-grade potato dextrose agar plates with the reconstituted spores. If we fail to get growth on the lab grade agar, it might give us a heads up that I did something wrong during the reconstituting process.
Anyway, I'll let these grow for a few days and check back, including pictures of any growth. If we get growth, I can use the 'homemade' penicillin and check for antibacterial properties.
