Roger's Agar method
Posted 01 February 2008 - 05:16 PM
I’ve posted a few methods of using agar in the past, and wrote a strain isolation tek last year, but have never explained my process for getting the agar mixed up, cooked, and into the petri dishes in the first place. Lots of new members have been showing interest in agar lately, so I thought it was time for an easy, successful way to do it. It’s a simple process actually, and the simpler the better. Unless you’re a long-term experienced mycologist and amateur biologist, I would not recommend mixing up your own agar. It’s too easy to buy it ready mixed, so all you do is add water. You can even get it, as shown below, with antibiotics already mixed in, to make it resistant to bacteria while you’re germinating your spores, or more importantly while you’re cloning mushroom or mycelial tissue.
The recipe on the above mix calls for 49 grams of agar per liter of water. I measured out a tablespoon of the mix and it weighed 7 grams, therefore 7 tablespoons per liter. The container I use for agar is awesome and highly recommended. It is a 750 ml liqueur bottle. I fill it about 3/4 full, so I figure it’s just over half a liter. I use 4 tablespoons of the pre mixed agar, and it works great.
Prepare Your Bottle
Simply find any long necked bottle with a screw on lid that will fit inside your pressure cooker. The longer the neck, the easier it is to pour while keeping your hands away from the petri dish and agar. Drill a 1/4” hole in the center of the lid. This will allow steam and pressure to vent from inside the bottle, while the filter protects the agar inside from contamination. I simply take one of the larger synthetic filter disks that are made to fit mason jars and cut it with scissors to the size of the liqueur bottle lid. One could also stuff polyfill into the hole. I don’t trust post office tyvek for agar work.
Fill and Mix the Agar
Four tablespoons of agar are added to the bottom of the bottle. Filtered, bottled water is then added, bringing the level to the bottom of the neck. Put your thumb over the opening and shake like hell. Continue to shake until it’s all dissolved. You may need to find a coat hanger or something else to stir with if you end up with a chunk or two stuck to the bottom. Don’t worry about contaminating the agar at this point, as the PC will take care of anything it picks up off your thumb.
After it’s been well shaken, place the lid with filter inserted onto the bottle, and cover with foil. Place, as shown below in the center of the PC with a larger volume of water than you would normally use. It’s hard to tell from the below picture, but the PC has about 4 inches of cold water in it.
The reason for using more water in the PC for agar is to allow for slower temperature fluctuations. A major problem many people have when using agar is boilover. They open the pc to find half their agar floating on the surface of the water inside. This will happen anytime the pressure inside the bottle of agar is greater than outside the bottle. If you have plenty of water in the PC, the pressure/temperature will drop very slowly, and your agar won’t boil over. If you have too little water in the PC, the jar of agar will still be very hot, while the air in the rest of the PC will be a few degrees cooler, and the agar will boil over. This is key. It should also go without saying, never lift the weight or do anything else to the pc to get it to cool down sooner. For agar, you want the slowest possible cool down you can achieve, so don’t be in a hurry.
The Pressure Cooker
Note that it was said above to always begin with cold water. You want to make sure that the water in the PC and the agar heat up together. Use a bit of Vaseline on the sealing surface of the lid and secure it. It is not necessary to tighten the wing nuts very tight. That only makes it hard to get the lid off later, and wears out the mated surfaces quicker. Simply snug the nuts, then give each about 1/8 to 1/4 turn extra. Make sure the lid is even. LEAVE THE WEIGHT OFF, and turn the stove to HIGH. It may take 20 minutes or so for steam to begin escaping the vent hole. Leave the weight off and allow steam to vent for at least ten minutes with the stove still on high. I leave the weight off until I can read 5 LBS pressure on the gauge, then the weight goes on. If your stove isn’t hot enough to allow you to get to five pounds as shown below, put the weight on only after it has blown steam vigorously for ten minutes. This is a very important step.
Once the weight has been placed on the PC, allow it to build up to 15 Pounds per square inch. At this point, the weight will begin to rattle. I try to be standing by, and when the pressure reaches 14lbs, I reduce the heat. I want the weight to rattle now and then, but not continuous. Once the PC has reached 15lbs, BEGIN to count your time. You want 45 minutes at 15psi. Be responsible and always use a timer, no matter how much of a clock watcher you are. Many PC's have been ruined because the owner fell asleep during a batch and woke up to a dry and warped unit.
When the 45 minutes expires, simply turn off the stove. Do not move the PC from where it is on the stove. Don’t do anything to speed up cooling, as was noted above. Simply leave it on the stove burner. It will gradually loose pressure as it cools. When the PC has returned to zero, and you can no longer hear any boiling sounds from inside, it is safe to open. At this time, place your bottle of very hot agar into your already waiting glovebox or preferably, in front of your flowhood to cool. Don’t allow your nasty potholders from the kitchen to ever touch your pc. Use ten or twenty fresh paper towels as hot pads, as they’re very clean. The filter in the lid will prevent the agar from contaminating as it cools. You want to wait until you can comfortably hold the bottle with latex gloves on. If you wait longer than that, the agar will be too thick to pour, if you pour much sooner than that, not only do you burn your hands, the petri dishes will be full of condensation because of the temperature difference between inside and outside.
The Work Station
The area in front of your flow hood must be completely clean. During the 45 minutes your agar was cooking, you should have shut off all fans and HEPA filters, and bleach bombed the entire room. Do this with a very fine mist. Mix up ten percent bleach with ninety percent water and put it in a good mister. Aim the mister up into the room, and spray every cubic foot of space in the room. Begin at the ceiling and wash down. Damp the carpet and any bedding in the room. I use only white sheets on the bed for this reason. The carpet in the room is now white as well. J By misting like this, contaminates are attracted to the much larger droplets of water, to which they attach and fall harmlessly to the floor, then are nuked by the bleach. Do this two or three times, fifteen minutes apart. After the last bleach bombing, wait ten minutes, then start the flow hood.
While the agar cools, go take a good shower. Wash your hair and brush your teeth twice. Use mouthwash. Put on a clean cotton shirt or work naked. Before putting on the gloves, hands and forearms should be thoroughly washed with 70% alcohol. The sleeve that holds the petri dishes should be very thoroughly washed with alcohol as well. The inside of the sleeve and the petri dishes are sterile, but the outside is not.
When everything is clean, put on your gloves, and then wash them with alcohol. The gloves usually are not shipped sterile. The four petri dishes you see up against the flowhood are also washed with alcohol at this time. They are only to be used as a ‘stand’ to get the dishes to be filled up into the airflow.
Carefully remove the foil from the agar bottle. You will notice from the picture below, six petri dishes stacked on top of the four that were previously shown. You always want to stack petri dishes vertically when you pour them. It’s very important not to ever pass your hands or any other part of your body between the flow hood and a petri dish, whether the petri dish is open or closed. The fan will blow contaminants off your body and into the agar. By stacking the dishes right up against the face of the flow hood, it allows us to handle the dishes from the back, so contaminants will be blown off our hands and arms and safely away from the work.
Filling the Petri Dishes
Note the hand position in the picture below. This is the proper way to lift a stack of petri dishes. There is no part of the hand that is between the flow hood and the work. Any contaminants that will blow off the hands or arms, will blow harmlessly back and away.
Lift the stack and pour the first dish. Be sure to always hold the bottle behind the work, with only the neck extending out and over the lip of the petri dish. Be sure the hand holding the petri dishes does not ever pass between the flowhood and the agar. Make sure no part of your fingers curl around under the lid of the dish you're holding.
Continue as shown below to pour each additional dish. Simply lift your grip up one dish, and then lift the stack, again pouring the bottom dish. Repeat until you fill the top dish.
Carefully set the six dishes you just poured off your stand and place the plastic cover back over them to keep them from cooling off too fast. This will help control the amount of condensation. Be sure to keep the dishes within the reaches of the flow hood or glove box if that’s what you’re using.
Repeat with the next stack of five or six dishes. Continue until all of the dishes have been filled. This amount of agar will be enough to fill 25 petri dishes.
When you’ve finished filling the entire sleeve of dishes, be sure to stack them all vertically then place the plastic sleeve back over them all. Stacking them vertically keeps the top and bottom surfaces of touching dishes the same temperature, so condensation only forms on the sides, and the top dish of each stack. This amount of condensation is easily absorbed back into the agar over the first week.
It’s best to leave the flowhood running until the petri dishes have cooled, at which time they should be wrapped with parafilm unless you’re going to inoculate them right away. Be sure to wrap all dishes in parafilm as soon as you inoculate. Be sure to pour out any leftover agar right away, and wash the bottle very well. If it becomes solid in your bottle, you’ll be looking for a new one.
Posted 01 February 2008 - 07:30 PM
Posted 01 February 2008 - 09:56 PM
Posted 10 March 2008 - 07:44 PM
Good tip about pouring the stack from the bottom plate up...
Posted 18 March 2008 - 11:35 AM
Posted 18 March 2008 - 12:00 PM