Use a drop of sterile water to deposit spores onto the plate, then streak using the T or quadrant method to thin cells on for pure culture isolation away from contaminants.
At first glance it may appear perfectly reasonable to grow mushrooms straight from spore; plants grow from seeds planted in soil, after all, so why not reduce the complexity of mushroom cultivation by doing essentially the same with spores? Let’s look at what agar is first before we move on to the why and how.
Robert Koch initially utilized potato slices to culture bacteria during his investigations into the germ theory of disease, Koch postulated that:
1. The microorganism must be abundant in all organisms suffering from the disease but should not be found in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.
To prove these postulates, Koch would require media to both grow and then isolate these organisms for identification. Gelatin was widely available but failed to produce a growth medium that could reliably culture bacteria due to the protein structure of gelatin. Bacteria would often utilize those proteins as an energy source, gelatin failed to remain stiffened at higher incubation temperatures and would often melt when bacteria produced protease enzymes that break down proteins.
Agar, on the other hand, is made up of carbohydrates extracted from seaweed. These carbohydrates have a much higher melting temperature than animal proteins which bacteria do not readily catabolize for energy. Koch would eventually graduate from potato slices to agar-stiffened liquid media poured into jars; Julius Richard Petri would further develop Koch’s culture dishes into the Petri dishes we recognize today.
How is this germane?
When streaking microbes for identification there may be a wide variety of bacterial species present including yeasts and molds; sound familiar?
To I.D. the species of microorganisms responsible for disease, there must be a method for isolating a pure culture, the same is true for the cultivation of mushrooms; this is achieved by streaking to agar.
Agar-filled Petri dishes provide a sterile environment to germinate and grow all present microorganisms allowing you to isolate a pure culture of your target organism. Mushroom fruit bodies grown in an open environment like tubs or tents will be exposed to environmental contaminants like molds and bacteria.
The removal of caps for printing, swabbing, etc, risks further contamination via handling. Spores are to be considered septic/contaminated by default. When spore syringes, spore prints, and swabs are viewed from this perspective it becomes obvious why agar is necessary: to produce a pure mushroom culture free of contaminant organisms. Failing to produce a pure culture before inoculation will result in a very high risk of failure.
7 окт 2024
