Sterilizing and Storing your Media

Using Your Autoclave (Pressure Cooker)

By Harold Eddleman, Ph. D.

Incomplete but perhaps of some use to you.

Rapid advances in microbiology and food preservation came after pressure sterilization of media and foods began. In laboratories and canneries, huge steel vessel called autoclaves are used. The steel or glass cans of food on giant carts are rolled into the autoclaves. Then live steam is forced into the autoclave with the exhaust vents open so that the steam can drive all the air from the autoclave. Then the exhaust vents are closed and the steam pressure inside the autoclave is raised about 15 pounds or higher for many minutes. That temperature will kill all growing organisms, spores, and viruses.

Home Canning of non-acid Foods Requires an Autoclave

Canning meat, beans, peas at home by the cold pack method is very risky and people have died from eating such foods. Those foods can be home canned safely using large home pressure cookers. Refer to the manual that came with the cooker for safe canning.

General Information about Pressure Cookers & Canners

First, read and follow all safety instructions in the manual that came with your pressure cooker.

While I have used some very large industrial autoclaves for sterilizing huge volumes of medium, I have used home pressure cookers for 90% of my loads. I have never had any problems with using home cookers except for routine maintainence such as replacing valves and seals. Spare parts are available at most hardware stores. I have often purchased old non-working pressure cookers and installed new seals and parts for some savings of money. However, rubber seals for old off-brand cookers may not be available. You should be able to get parts for Presto and Mirro cookers.

Most homes use four quart cookers, but the six quart sizes are will hold larger culture tubes. Pipets are too long to fit into any of them. Pipets and gallon glass jugs will fit into some of the home pressure canners.

General Safety Considerations for using pressure cookers

If you have not read page B024, read it first.

There are some safety problems and an inexperienced child is likely to suffer moderate to disabling injuries. An experienced user of pressure cookers, including me, have made errors and had accidents.

Loading the Pressure Cooker

Some ideas to avoid accidents and produce quality sterile media.

Tyndallization

Heat the tubed medium in moist flowing steam for one hour. Repeat for a total of 3 days. The principle of Tyndallization depends upon spores germinating within the 49 hour period. On day 1, most of the vegetative bacteria, molds and mold spores are killed, but some bacterial spores will survive. The surviving spores may germinate overnight and be killed during the 2nd steaming. The third steaming is an added precaution. Tyndallization is used for media which can't withstand autoclaving. Students and field workers who have no autoclave (pressure cooker) can try Tyndallization. Tyndallization is not 100% dependable. If you use this method, incubate some of the uninoculated tubes of medium along with the experiments. If the uninoculated tubes rot, then you know the experiments were contaminated. If you want to try an experiment. Mix a little garden soil with milk and immediately autoclave half the tubes and tyndallize the other tubes. Incubate all the tubes for a week or so and compare the results.

Sterilization by Dry Heat

This method is used for equipment which can withstand high temperature dry heat but can't withstand autoclaving. This method is often used for pipets and glassware because it dries and sterilizes in one operation. The pipets must be wrapped in dust proof aluminum foil or placed in metal pipet cans. The can lids are removed during heating and replaced before dust can get to them.

Heat the pipets at xxC for x hours. Markings on disposeable pipets may burn off. This method may only be good for permanent reuseable pipets.

Chemical Sterilization

Students who have no method for sterilizing long pipets and glassware may consider placing the items in 10% Chlorox for 20 minutes or longer. They then need to remove the Chlorox (sodium hypochlorite) by rinsing with sterile water or rubbing alcohol (70% iso-propanol) without recontaminating the glassware or tools. Do not use Chlorox on metal devices as it corrodes metals rapidly.

There are gases which can be used by professionals to sterilize equipment. If do not already know the names of those gases and how to use them safely you are not qualified to use them. This site is intended for beginners and will not discuss those gases because beginners do not have the special equipment needed.

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.

Store your Sterile Items in a Dust-free Place.

The air of every workplace contains dust which will blow into or fall into your sterile medium. Many laboratories have dust free cabinets for storing their sterile medium and equipment. I use two milk cartons to make one "dust-free" box. I prefer to used cardboard milk cartons, but also use plastic milk jugs.

Using Carboard Milk Cartons

Cut the top off one carton by cutting all the way around the top of the straight sides at the "roof line". Pinch each corner and cut about 1/2 inch. That gives a 1/2 inch "V notch" at each coner so the top of each side can slope in a little. Now make a lid from the second carton by cutting around the box about 60 mm above the bottom. Turn this lid upside down and let it telescope over the box made from the first carton. Wash the carton immediately after emptying the milk.

Using Plastic Milk Jugs

Make a box from 2 plastic milk jugs about the same as above. You might want to make the lid first and then keep trimming away on the other jug until you obtain a dust-free box that fits your needs.

I also make dust-free boxes from pint and quart jugs and boxes for holding test tubes and cultures. These dust-free boxes are very important for keeping your cultures contamination free. Pint milk cartons are very good for storing 13 x 100 tubes.

A strong draft or wind will blow dust into petri dishes and contaminate them. Therefore, when the agar is solid, you may want to store the dishes in dust-free containers such as gallon milk cartons. During storage, the excess water on the lids may evaporate. Cardboard milk cartons work best, but you may also use plastic milk jugs.

Remember Dust Falls

If you remember that dust falls or is blown by wind, you will have fewer contamination problems. Thus, a loose foil cover or lid will protect a container from contamination. Loose covers such as those on petri dishes are effective because dust only falls, unless it is blown by a wind. Remember to shut off electric fans when you are working with loose fitting covers. Get your cultures into your dust-free boxes soon as possible.

Wrap Plates and Tubes

I often use a strip of any clear plastic wrap about 1 inch wide to seal bottles and petri dishes. Just wind the plastic strip tightly around the neck of the bottle so dust can't get into the culture. I have sealed about 20,000 cultures in this way with good results. I often cut a roll of plastic wrap into 1.2 inch long pieces. Like cutting a log into firewood.

Other Useful Items to Autoclave


Revision #2     1999 Jan 23
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
Please send an e-mail containing your ideas.

For safety, have someone familiar with pressure cookers help you with this half of Expt 2. We still have a problem. The ingredients and the glass or jars will already contain bacteria. Lets try the most important step in Pure Culture Methods. That is autoclaving the medium. Pour the remaining gelatin-broth-syrup medium in a few glasses covered with aluminum foil and place them in a pressure cooker. Be sure to use the rack; do not set containers directly on the bottom of the pressure cooker because they will crack from the heat. Be sure to put about 1/2 inch or inch of water in the cooker. Place on fire, and when steam is coming out nicely, turn flame down and continue cooking for 15 minutes at 15 pounds pressure. Fiveteen pounds of steam pressure for 15 minutes will kill all bacteria, viruses, spores, and other living things. Let it cool, it is best to let it cool to cold gelled medium before you remove the lid. The cooker is a good sterile place for your sterile glasses of medium. Warning: if your containers had screw lids, they must be loose during autoclaving so steam can get in. You can now use the sterile containers of gelatin as in Expt 1.

More points about autoclaving media. A pressure cooker having 15 pounds pressure is a dangerous device. It is very hot. If the lid is not on properly, it could fly off hard enough to kill someone. It is easy to let all the water boil away and damage the pan and containers by overheating. I find I use 8 oz (one glass) of water. I never use less than 2 glasses of water in a 4 or 6 quart cooker. A beginner should use 3 or 4 glasses of water. Four glasses of water may cause your containers to float, upset, and be made useless. Use blocks of metal, rocks, or canner rings, etc to lift the rack up off bottom so containers do not float or upset. Stay in the room and watch. If you forget to turn fire off after 15 minutes, the water will all boil away and the pressure cooker may be damaged. Here is a way to avoid laboratory accidents. Place an object in your hand labelled, for exmaple, pressure cooker, 2:15 done, and never lay that reminder down before 2:15 when the flane is to be turned off. Use a timer bell to aid you if you have one.

When the cooker is cold and you open it, measure the left over water. If it is less than 1.5 glasses, be sure to use 1 extra glass of water next time. Occasionally, containers will break. If you remove hot containers and sit them on cold surfaces they are likely to break and scald someone. I always let cooker get cold before I open it. If it is hot. The hot steam inside containers condenses to a liquid sucking in one volume of non-sterile air. If you let cooker get cold, the dust in the room air collects on the surfaces it strikes and no non-sterile air gets inside the containers. Always leave some of your containers unused as sterility controls. They should not rot. I have such containers still free of contamination after many years.