Dispense your Media into Containers

After preparing your medium and before sterilizing it, you need to pour your medium into bottles, test tubes, or other container according to the form you plan to use. The choice of slants, stabs, deeps, petri dishes, bottles is determined by what you have available, organism, etc. You learn about these as you begin working with various organisms. Look in catalogs to see pictures of the glassware available.

Test tubes vs Culture tubes

Chemists use test tubes which flare out at the mouth. That lip makes it easier to pour contents from the tube. However, there is the disadvantage that the lips tend to chip and collect dust.

Culture tubes have no lip. Plastic caps are availabe which telescope over the mouth of culture tubes protecting them from dust. Plastic caps can't be used with test tubes. Microbiologists can use test tubes, but most prefer culture tubes. Culture tubes are often cheaper.

Screw cap tubes are more expensive, but the caps can be screwed on tightly to prevent drying and and to reduce contamination. Rubber lined caps have higher quality threads and when screwed on tightly form a tighter seal. To save money, most microbiologists use non-rubber lined screw caps for routine work and rubber-lined caps for stock cultures. Screw cap is abbreviated as S/C.

Cotton Plugs vs Caps

In the old days, bacteriologists rolled tight cotton plugs and pushed them into the mouth of test tubes to keep contamination out. The snug cotton plugs kept out dust which carries molds and other contamination. Now we have Kim Kaps and other plastic and metal caps which can be pushed on over the end of culture tubes. These caps cover the outside of the tube and help reduce contamination and they are easy to pull off the tubes when desired or by accident. Never use these loose caps for important stock cultures as contamination is likely to occur. Cotton plugs or screw caps are better for stock cultures.

Stabs

To make stabs pour agar medium into the tube about 3 or 4 cm deep. Autoclave (sterilize) and then cool these tubes vertically so the top of the agar is horizontal. To inoculate these tubes, you will get bacteria on a needle and carefully insert it in the center of tube all the way to the bottom. If you do not plan to study the shape of the growth along the stab, then you do not need to be so careful making the inoculating stab.

Deeps

Same as stabs, but deeper to insure that bottom is fully anaerobic. For deep stabs, fill the tube about half full. Deeps are primarily used for culturing anaerobic bacteria because anaerobes do not grow in the presence of air. When you inoculate a deep with an anaerobe, you usually find they grow only in the bottom of the tube.

Slants

Slants are the most widely used type of culture. Fill the tube about 3 cm deep, and then cap or plug with cotton and autoclave. Cool the tubes at a slanted angle. They are made by sterilizing the medium and then when cooled somewhat but not solid, remove from autoclave and lay the tube on its side so that you have the desired slant. TSI and certain other diagnostic medium require a generous butt and need to be slanted so there is still medium at bottom of the tube, ie. surface of slant lacks 1 or 2 cm of reaching the bottom of the tub. Laying tubes on a stick about 1 cm thick will give a suitable slant. As the tubes cool, dust may be sucked into the tube. Work in a dust-free room if available.

Petri Dishes

There are several ways to handle these. The standard dishes are 15 mm deep and 100 mm diameter. They are available in glass and several kinds of plastic. Most people use 20 to 25 mL of medium in each petri dish. They are all called culture dishes. Most platic dishes are make of polystyrene and can't be autoclaved because they deform in the heat. Some plastic dishes can be autoclaved, but they are more expensive.

Most people autoclave glass dishes and medium separately. When the medium is cooled to about 55o C, they lift the lid of the dish enough to pour the the medium to the desired depth and lower the lid in place.

If your dishes are autoclaveable, you may dispense the agar into the dish and then autoclave. In this case it is best to cool the dishes in the autoclave (pressure cooker) to reduce the amount of water which will condense on the lid of the dish.

A Useful Rule

Don't waste too much time wishing for equipment you don't have. Spend your time thinking about things you can do with what you have or how to make a substitute.


Revision #2 1998 Dec 1
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
Please help by sending me an e-message containing your ideas for improvement.

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