Working With Agar
Working With Agar
Working With Agar
What is agar?
Agar is a polysaccharide found in the cell walls of some red algae from which it's extracted by boiling. This raw agar is then purified. When agar is dissolved in boiling water and left to cool it will form a gel (very much like gelatin). It can be bought in many different grades from food-grade to purified tissue culture grade. It's main uses are in the field of microbiology, tissue culture and food preparation (as a vegetarian substitute for gelatin).
This medium will support the growth of most saprophytic fungi. Other often used formulations are PDA (Potato Dextrose Agar) and DFA (Dog Food Agar).
When making your own recipes keep in mind that more is not always better. When media are too rich in nutrients mycelium will not grow or grow very poorly secreting yellowish metabolites.
Pouring dishes
When the flasks come out of the pressure cooker or autoclave the temperature of the agar will be close to 100C. The medium will be liquid until the temperature drops below 40C. The agar is left to cool until it has almost reached this temperaure. As a rule of thumb: If you can hold the bottle in your bare hand for 10 seconds without real discomfort the temperature is right. Do not let it cool too much or it will solidify in the bottle. Better to pour a little too hot than having to reliquify the agar. Since the lids of the dishes will be removed for a few seconds it's very important to prepare a clean workplace. A laminar flowhood is best but an improvised transfer hood will usually give adequate results. Whichever is used the table top is cleaned with alcohol or lysol (CAREFUL! THESE ARE FLAMMABLE!) and the dishes are taken out of the tube in which they were packaged and put in stacks of 10 dishes. When working in less then totally sterile environments it's important to work as fast as possible, limiting the direct contact of agar with outside air. The flask is swirled to mix the ingredients and the aluminum foil and polyifll are removed. The lid of the bottom dish is lifted (in fact lifting the lid and the 9 dishes on top). Swiftly a layer of nutient medium is poured in the dish (to 0,3-0,5 cm depth) and the lid is replaced. Quickly lift the second lid (and the 8 remaining dishes) and work your way up in this fashion. By letting the agar cool before pouring and by stacking the dishes condensation on the lids is minimized. When a mug or jar with near boiling water is put on the top dish of a stack condensation will be even less. When the agar in the has solidified the dishes are ready to use.
The nutrient medium is prepared by bringing the appropiate amount of water to a near boil in a pot on the stovetop. The ingredients are added and the mixture is stirred to let them dissolve. Agar should first be mixed with a little bit of cold water before adding or it will form lumps. Careful, adding agar to boiling water will cause the medium to foam and boil over. Boiling agar can cause serious burns! Let the agar boil for a few minutes, then turn off the heat. Tubes are filled with 5-6 ml of medium and loosely capped. A small funnel and a big syringe are very useful here. The tubes are sterilized for 25 minutes at 121C. When they come out they are left to cool on a sloped surface to increase to surface area of the agar. When the agar has solidified the slants are ready to use.
If contaminants are encountered one can choose to discard the whole dish or one can transfer the mycelium away from the contamination to a new dish. When bacterial contamination is present this can take place in a flowhood. If sporulating molds are present isolation should take place in a still-air environment to prevent the release of mold spores. The germinating spores will form a mycelium that by itself is not capable of fruiting called a 'monokaryon'. When the mycelia of two spores fuse they will form a new type of mycelium that IS capable of fruiting, a 'dikaryon'. This process will happen by itself and one dish with germinated spores will often contain dozens of these combinations. A dish with germinated spores will often look a little bit messy, with monokaryons and dikaryons competing with each other for nutrient sources. Here one has two options: use this
mixture of substrains (a 'multispore') or select a pure substrain. Each substrain has it's own properties but generally resembles it's parents. Generally speaking flushes from pure substrains are more uniform and better yeilding than a mixture of substrains. To select pure substrains small agar squares from a multispore germination are transferred to fresh media. Growth from these squares will still be pretty messy but often some sectors will develop that show healthy growth. These healthy sectors should be transferred to new dishes again. This process is repeated until the growth in a dish is uniform and non-sectoring. This is a pure substrain.
One can also use the multispore cultures to directly inoculate spawn. Since mushrooms are composed of mycelial threads (of one substrain, not of a mixture) one can always isolate pure strains from mushrooms as soon as they emerge from this multispore culture.
Often within a few days the fragment will spring to life. Becoming fuzzy and showing new growth on the agar surface. If contaminants are visible one can try to isolate the mycelium away from the contaminants.
Even though a pure (sub)strain should be growing on the agar sectoring may be visible, often healthy zones mixed with cottony or fuzzy ones. The reason for this is not known. Some strains have a tendency to show this phenomenon more than others. The healthy mycelium is then transferred to a new dish until a healthy pure culture is established.
Storing cultures
To store cultures slants are inoculated with small agar squares. The cap is put on loosely and the tube neck is taped with polyethylene clingfilm. Once the agar is fully colonized the slants are put in the fridge where they can be kept for at least a year. It's wise to check the viability of cultures each year by inoculating some dishes with the culture.
Incubating cultures
Cultures should be incubated at the right temperature. If the temperature is too high mycelia will often start to sweat, sickening the culture. If the temperature is too low mycelia will grow slowly. In general too low is better than too high. It's also very important not to let the temperature fluctuate too much as this causes condensation to form on the lids. The temperature should be kept as constant as possible. Dishes are incubated upside down so even if condensation forms it will not disturb mycelial growth.