How to use agar

Agar is a great tool for many mycological purposes and offers unique and essential benefits.  If you are wondering what I’m referring to when I say agar, I am talking about nutrient rich gelatin used to cultivate a colony or “culture” of microbes usually fungus or bacteria.  The essential benefit that inoculating with agar offers over various popular methods such as inoculating with liquid culture or spores, is a visual confirmation that your culture is clean.  This is one of few circumstances you can be sure that you will be inoculating with a 100 percent clean culture.  If you get a contamination it is much easier to solve your issue as you know you can completely rule out you culture as the culprit. The other advantage is if your culture on agar gets contaminated can you take a sector from a clean spot as far away from the contamination as possible and transfer it to a fresh clean plate. So long as there is a small amount of clean mycelium on the sector transferred to the clean plate your fresh plate will colonize and remain clean.  Sometimes it can take a couple transfers, but it will be worth it to know you have a clean mycelium, which will make all your next steps much easier and more successful. 

  1.  Take agar plate to a clean environment, the more sterile the better.  The nature of agar is to be very nutrient rich, so anything not properly cleaned can result in unwanted contaminations.  Because of this you can never be too sterile, a still air box should be  used the very least when working with agar, although laminar flow hoods will  give you the best success rates by keeping the air sterile. 
  2.  Thoroughly clean your work area with 70 percent iso alcohol or something better.  Once you have your clean environment set up once again stressing you can never be too sterile and EVERYTHING including your hands should be wiped. 
  3.  If you have a bag of sterile agar plates quickly check the bag to see if the rim of the plates are individually sealed.  If they not, you should go through and seal them all individually (even if you do not plan on using them at that time) with parafilm, or parafilm substitute (cling wrap also works). This will keep any unwanted contaminations  out.
  4.  With the plates you’re working with set aside, also set aside what you are wishing to inoculate with and remember from now on to make movements as direct and calm as possible. Avoid sudden movements, you should be keeping the air as still as possible.
  5.  Inoculate: this step will vary depending on what you are using but these general rules will apply to all methods:
  6.  Open agar plate as quickly as possible, usually by getting the inoculation sample ready in one hand and other hand on the lid to quickly crack it open and shut it.  Do not open the agar dish fully unless necessary, even if you need to drop spores in the middle of the dish, the less the dish opens the less the nutrient rich medium is exposed to contaminations.  If you do need to open it fully work as quickly as possible to get it back closed.   After finishing inoculations don’t  waste time and seal all plates immediately.
  7.  If using gourmet spore prints it is important, I stress how little you need, the less the better.  Only 2 spores need to meet to germinate, if you can see ANY spores at all, its thousands upon thousands in a condensed area.  So take a small sample (barely visible to your eye) and drop it into the middle of the plate.
  8.  You can also use an inoculation loop by heating it up to red hot, sticking into the agar to cool down. After hearing a sizzle  take it out  and you can notice agar residue covering  the loop.  
  9.  Dip the residue covered loop into the spore print and roll around until the  residue is covered in  desired spores. 
  10.  Take the loop covered in spores and drag across agar in an S pattern for many inoculation points. 

 Inoculating with gourmet spore

1.        Method #1: is to quickly open agar plate, plant the entire swab directly into the middle of the plate so it stays on its own. Then take small scissors flame sterilize the blades quickly and cut as close to the base of the swab as possible, ensuring you cut out the wooden stick part and leave just a cotton ball on the agar. Seal the plate when you are finished!

2.     Method #2: you can aggressively swab or spin the cotton tip in the agar and sea.

3.    Method #3: you can use a scalpel or small tweezers that have been flamed sterilized and allowed to cool to rip off a small piece of the swab and place it onto the agar. Seal and you are finished! 

 Inoculating with agar wedge

1.  As with all inoculation steps make sure you have your entire work area clean including the air.

2.  Decide which sector to take from and flame sterilize scalpel. Crack the lid on colonized agar plate and swiftly cut a triangle in the agar ensuring the triangle has only healthy mycelium somewhere on it.   

3.  Carefully stab the precut triangle and lift it out of the agar. So it is completely free from the original plate.

4.  Quickly move the wedge from the cracked plate, to the fresh plate making sure to only crack the lid on the clean agar plate as little as possible.

5.  Place wedge in middle of clean agar plate, colonized side down for best results.

  1. Close plate and seal both plates. Put into colonization temperatures  and let  growJ.