Advanced Agar Techniques

This is far from an exhaustive list of what can be done with agar, but it should help get your imagination flowing a bit. Working with agar is as much art as it is science. The best lab techs can clean up cultures that Ph.D's might decalre unviable due to contamination. Truly miracles can be worked by those with the gift. For many years small tricks of the trade were guarded secrets passed only from teacher to pupil and never written down. Perhaps some of the best tricks still are.

Antibiotic Media

Antibiotic media can be useful for a number of things. Genetic researches use it to select for genetically engineered bacteria. When they splice in the gene code they want, they include the code for an antibiotic resistance. When they have finished splicing, they culture the bacteria on antibiotic media. Not all the splices will take, but the ones which do take will survive on the selective media. I seriously doubt that anyone reading this page will be in the business of "building" bacteria. However antibiotics in the media can still be a useful tool for the part time enthusiast. It is particularly useful in mycological applications, as most fungi are considerably more tolerant to antibiotics than are most bacteria. A fungi perfecti culture is started by innoculating a plate from one of several sources, a fresh tissue sample, a spore print, or perhaps a swap of a dirty room corner. A judicious use of antibiotic media in making a plate from a spore print can improve the chances of getting a clean mycelial culture. Selective media may also help in circumstances where there is reason to believe a tissue sample may not be completely sterile, such as is often the case with home labs.

Cautions and Safeguards in Using Antibiotic Media

Caution must be employed with respect to antibiotics. Today we have come to percieve antibiotics as a sort of magic bullet that can cure anything. Partly because of this, many disease organisms are gaining resistance to antibiotics. With repeated exposure bacteria that are suseptible to antibiotics die, but those with some resistance may survive. These survivors are constantly changing, and will develop further resistance to the antibiotics. Eventually they will become tolerant to the point where the specific antibiotic they have been exposed to will no long affect them. This unfortunately is exactly the case with some of the most deadly opportunistic infections known to man. There are strains of staphococcus which are resistant to all but a few antibiotics, and there is a wide spread belief that there are currently bacterial diseases which simply cannot be treated with any antibiotic we now have. The point of this is that in using antibiotics as a tool, we accept responsibility to limit the amount of damage done by the use of said medicines. If you are use antibiotic media, do so only very sparingly, and switch back to normal media as soon as you have accomplished your purpose. If you are mixing your own antibiotic media, I recommend you make sure you are using a full dose of antibiotics in it. Partial doses will allow bacteria to acclimate themselves to the antibiotics even faster. If you have purchased prepared, dehydrated antibiotic media, carefully follow the directions for mixing it. If you are not deliberately culturing antibiotic resistant bacteria, and bacteria crops up on your antibiotic media, destroy colonies by autoclaving. Better yet, carbonize the media if at all possible. If you are using a plastic plate, incinerate the whole thing, as the plate cannot be reused anyway. Do this for your own saftey, as well as for the sake of your experiments. Only a few antibiotics will survive the autoclaving necessary to sterilize the media, and you cannot risk having your lab contaminated with these bugs. Beyond that, you do not wish to have such creatures escaping into your home! If this is now already the law where you live, it should be.

Actual Methods of Preparing Selective Media

Perhaps the simplest and most effective means of preparing antibiotic plates for the isolation of various resistant organisms is to pour the plate half full of anti-biotic media. Then allow it to cool and harden while resting on a pencil or other object so that the anti-biotic media hardens with a definite slant. When it cools, lay it level, and finish pouring the plate with regular agar. You can then innoculate the section of the plate with the thickest top layer. This section will have the lowest antibiotic concentration. As the culture grows across the plate, the contaminants will succumb to the antibiotics.

Alternatively, an entire plate can be poured with antibiotic media, but if the culture you are trying to clean does not have complete resistance to that antibiotic, growth will be slow or non-existant. This also has the disadvantage of using much more antibiotic media, which is usually a good bit more expensive.

A more complicated, but quite effective means involves using sterile technique to gouge a trough across the agar of a regular plate. The idea is to section the plate into two halves. Then fill this trough with warm liquid anti-biotic media, perhaps from an eye dropper, and allow to cool. The plate can be innoculated on one side, and the cleaned culture cllected from the far side of the plate. The strip or band of anti-biotic media need not be more than 1/4 to 1/2 inch wide.

Aerial Hyphae Seperation

Some fungi have hyphae that extend quite a distance above the agar surface. If a culture of this needs to be cleaned up from lower crawling contaminants such as bacteria or non-aerial hyphal-type fungi it may be possible to isolate them based on growth habit alone. A glass petri dish is lined with moist paper towels and sterilized. A chunk of agar containing the culture to be cleaned is placed on the paper towel and incubated. The paper towel promotes humidity which in turn promotes greater hieghts of hyphal growth. After good, tall hyphae are seen, a clean chunk of agar is brought close to the original culture chunk until the aerial hyphae just touch the new agar. Incubate a bit longer, and the aerial culture should make the bridge to the new agar, leaving the contaminants behind. Watch out however, for fungal cultures that are capable of colonizing the paper towel and crossing by that bridge! In this case you will need to isolate the agar from the paper towel by some method.

Substrate Selections

Speaking of colonizing the paper towel, there is a wide open playing field when it comes to cleaning up cultures by means of substrate material. Crystal violet is harmful to some bacteria but not others. Cellulose can be digested by some organisms and not others. Nitrogen fixing bacteria can be isolated by providing a carbon rich, nitrogen free enviroment. This will unfortuneatly involve setting the agar aside, as it contains nitrogen, and working with a liquid culture for a time. The formulae page lists a number of selective media which can be useful for various bacteria or fungal species. I'll post specific methods here as they become available to me, and as I find time to type them up. I welcome input from anyone who has experience or ideas to try out.

Humorous Advanced Techniques

A friendly sort of compatriot has suggested that agar could be poured into animal shaped jello molds and allowed to harden. These then could be exposed to the air for an extended period of time. They would no doubt quickly pick up bread mold spores, and similiar, hairy looking colonies of common house molds. A sort of an instant agar-chia pet, if you will. I sort of like the idea myself.

Updated May 18, 2010; August 20, 1999 by Glider Onair

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