Agar Preparation

Producing agar is truly a simple task. The most basic agar is just that, agar and water. About 20 grams of agar to one liter of water will gel just fine under nearly all home laboratory conditions. Regardless of what organisms you are working with however, you will likely want to supplement this basic mix with extra nutrients. One good supplement is yeast or yeast extract. Two grams of yeast, or one gram of yeast extract per liter of agar solution will greatly extend the range of microbes that can utilize the media, and mycological specimens will retain their original characteristics much longer with a more nutritive agar.

It should be noted that adding excessive nutrients hinders rather than helps. Too many nutrients, and the agar will support too many species, and will tend to result in higher contamination rates. You are better off finding a happy medium in between. No pun intended!

Malt Agar

One of the most basic formulas for agar, and one that works well, is as follows:

This is commonly refered to as MEA, ME agar, or malt extract agar. The malt extract can be found in any home brewing store, most health food stores, and most chemical catalogs carry at least a few basic microbiological supplies. The agar can also be found in health food stores and in chemistry supply catalogs. A few very well stocked home brew stores may carry agar supplies for maintaining strains of beer yeast. Common variants to this formula include adding 5 grams of peptone and/or 5 grams of ground grain. The grain can be of just about any variety that strikes your fancy, but it should be a food grade grain, and not an animal feed or seed grain. These are often treated with variable degrees of antibiotics in the case of animal food, and fungicides in the case of seed grain. Common grain additives include rolled oatmeal, ground rye grain, and ground corn. Peptone is a commercially prepared mix of meat protiens that have been enzymatically digested to render most or all of the protein into small fragments of just a few tens of amino acid bases each. You might also add straight amino acids, though this is somewhat more temperature sensative, I believe. Peptone is generally obtained only from reliable scientific supply houses. A crude extract of meat broth may suffice for many applications, particulary mycological endeavors. However for detailed research into bacteriology, peptone with well tested and defined parameters is likely to be a necessary condition for reproduceable results.

Potato Dextrose Agar

Alternating basic agar types is a good way to help maintain culture strength. One might wish to swap the above formula with a potato dextrose agar after ever generation of two. Potato dextrose agar, or PDA generally contains the following:

This PDA is also often supplemented with 2 grams of yeast, or one gram of yeast extract. As a general rule tap water should be sufficient quality to use for the amateur researcher. If the quality of the tap water is in doubt, bottled or distilled water may be used. Mineral water or spring water should prove equally useful. Agar should be balanced to the PH most appropriate for the organism being cultured. Most organisms do quite well with a near nuetral PH of approximately 7. A good PH probe is rather inexpensive and invaluable for this purpose. PH paper may also be used. In general, the formulae presented at this site will not require any buffering, and should be in a useful PH range as described, at least for the vast majority of micro-organisms. Only once one begins developing ones own formulas does this testing of PH become critical. It should be noted that the agar formulae listed here assume tap water of approximately PH 7 (neutral). In some areas well water can approach PH 8 or 9 depending on the season, and the amount of run off water present in the well. Likewise some municipalities and cities deliberately basify their water sources to PH 8 or 9. This is due to the presence of old houses with lead pipes. The lead salts that accumulate in the pipes tend to be less soluble in basic water than in acidic or neutral water. I have seen some internet reports of great success using pond water. The assumption is that pond water contains many micro-nutrients and organics that promote health and wellness to growing cultures. I would caution anyone wishing to try this to be absolutely certain that the pond water is free from sewer and farm run-off contaminates.

You can check out my list of selected media types taken from published material. Not all of these are intended for a wide range of organisms. Where the media was meant to have a restricted use, I've tried to document its intended use. I would encourage persons just starting out to stick to the two basic formulas I've supplied on this page until they have a little practice and know what to expect.

Sterilization

A word about sterilizing agar is in order. You might get away with sterilizing agar in a hot water bath at around 100C for an hour. I wouldn't count on it however. Even if you get away with it once or twice, you are likely to have problems with it sooner or later. Instead I would recommend that if you are serious about this at all, you should get a pressure cooker. Preferably one with variable pressure settings, at least for 10 and 15 pounds. A check of garage sales, flea markets and auction garage sales will often yield a very useable and affordable cooker. Pressure cookers are very easy to use and very safe to use when operated properly. However you must operate them according to the instructions or you risk causing yourself grievous bodily harm. Sterilization in the pressure cooker can be accomplished with 30 minutes at 15 psi. Resist the temptation to think that more is better. Pressure cooking for excessively long periods is as bad or worse than for too short a period of time. Excess sterilization can carmelize the sugars and/or agar in the media. This is harmful to most microbes, and will often skew the results of whatever you were working on. Using a narrow necked container to hold the agar facilitates aseptic pouring. After filling the container with the agar to be sterilized, but before pressure cooking it, fit a cotton plug into the opening of the container, and wrap the top with tinfoil. Allow the pressure cooker to return to normal atmospheric pressure before opening. It is very important to prevent sudden changes in temperature while cooking the agar, and while cooling it. Quick changes will cause the agar to boil over. Also do not needlessly play with the steam vent or weight. A sudden release of steam from the cooker during processing will cause decompression inside the cooker, resulting in a boil over, even after the unit is removed from the heat. When the agar is warm to the touch, but cooled enough to comfortably handle, you are ready to pour your plates or slants.

Autoclaving is very similiar to pressure cooking in that they both use steam under pressure to achieve higher temperatures. Autoclaves are more sophisticated than pressure cookers and tend to be more efficient and effective. Small models are priced within reach of the amateur, but certainly not for the beginner just starting out. For the sake of this web site, the terms autoclaving and pressure cooking will be used interchangeably. Please be advised that there are in reality important differences between autoclaves and pressure cookers, but this will not concern us here.

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