Because we are dealing with fungi cleanliness is of the utmost importance. The three elements of lab design well touch on in this post are establishing positive pressure within the lab, the importance of multiple doors between the cleanroom and outside world, and inoculation procedure.
Establishing Positive pressure
Step one is ensuring your lab is air-tight except for where you want your air to dump out. In most cases, your laminar flow hood will be enough to establish positive pressure in your lab. Larger scale-spaces will require external air handlers and equipment with external HEPA filters and UV lightboxes to properly clean outside air brought in. It is essential to maintain positive pressure, if positive pressure were lost or reversed the potential for microbial contaminants entering the space is high.
Creating an air lock
Creating a little space between your cleanroom and the outside/other rooms is essential. In this space non-organic storage areas with lab coats and a place to put shoes is important. This space can be achieved in a number of different ways. A popular DIY method is by using sliding glass doors for smaller scale rooms or swinging doors for larger rooms. Having an airlock area will help prevent microbial contaminants as well as give you a space to store your cleanroom attire.
Inoculation procedure
There is a lot that goes into the inoculation procedure but the three things we will touch on today are mass inoculation of grain spawn, inoculation of petri dishes from spores, and cloning mushrooms. When inoculating a large number of bags/jars of hydrated grain for spawn, it is important to have your team assembled in an assembly line fashion. You’ll need someone holding the bags, someone dropping the inoculation medium into the bags, and someone sealing and storing the inoculated bags. To prevent contamination team members should wear lab coats, masks and avoid talking during the procedure. The actual act of inoculating the grain should take place within 6-8 inches of the laminar flow hood to ensure the transfer is done in a clean environment. I have found when inoculating petri dishes from spores using a loop is an ideal tool. The first step in inoculating Petri dishes from spores is to sterilize your loop over an alcohol lamp. After you have allowed it to cool collect a good deal of spores from the print and spread them evenly in the middle of the petri dish, in an area about the size of a quarter. As the spores hatch and grow out it will allow you to isolate favorable phenotypes. When cloning mushrooms it is important that the mushroom you plan to clone is healthy and not at the end of its life cycle. Taking a tissue sample from the meat of the cap is appropriate for most mushroom species. The size of the tissue sample should be no larger than 1-2 centimeters. keep in mind this all takes place within 6-8 inches of your laminar flow hood. Something to be aware of when cloning mushrooms is there is a limit to the number of times you can clone. Cloning from 1-2 generations is usually best. Once you start getting into third and fourth-generation clones wasting diseases start to appear.
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