This is a good bacterium for beginning students to isolate at home because you can buy a package of limburger cheese and use it both as a source of the bacterium and food for the bacterium. Since you were going to eat the cheese, any bacterium you isolate from is likely to be safe to humans. There seem to be two main organisms in Limburger Cheese. Brevibacterium linens colonies are yellowish orange brown and have a strong, rotten odor with a trace of ammonia(?). Those traits are distinctive and help you find the colonies of B. linens. The other main organism is white in color and I won't discuss it now as I have little information about it. You may save a pure culture of the white bacterium and feel fairly safe working with it because it came from food.
DISCLAIMER: Remember that pathogenic (disease-producing) bacteria may contaminate food or be picked up during faulty isolation. Therefore, there is always a risk that you will isolate a pathogenic organism. Therefore, do not eat the bacteria you isolate and observe all other microbiological safety precautions. See the laboratory safety page on this site.
This is a very useful experiment, as you will learn to isolate a bacterium by streaking a sample from cheese onto agar. The principle is to touch your inoculation loop to a yellowish region of the limburger cheese and then rub the loop onto a petri plate of agar which is able to support good growth of Brevibacterium linens. Then incubate the inoculated plate at a temperature suitable for growth of the organism. After a day or more, you will see bacteria colonies on the plate. There will be a confluent mass of bacteria where the loop first touched the plate. Farther along when most of the bacteria have been rubbed off the loop you will see ridges of white and brown-yellow bacteria. Finally, you hope to find isolated white and yellow-brown colonies. The orange yellow brown colonies should be Brevibacterium linens.
There is a risk that your yellow-brown colony is not pure Brevibacterium linens; it might contain some of the other bacteria occurring in the cheese. Therefore, you must repeat the streaking on a fresh sterile plate of the suitable medium. This time you should get a plate containing nothing but uniform yel-brown colonies of Brevibacterim linens. If not, restreak one of the yel-brown colonies.
Since this is your first attempt to isolate a clone (colony) of a bacterium, you may not understand all of the above. We will now repeat from the beginning in greater detail.
I am going to assume you are working at home and have one or more glass petri plates. If you do not have a glass petri plate you can use plastic plates or a flat glass bottle. Standard plates are 100 mm diameter and 15 mm deep; smaller plates are just as good. You can't autoclave ordinary plastic plates. You need agar to streak the bacteria on. Once you get a pure culture you can store it on agar slants or in liquid. Usually, agar cultures of bacteria live longer because fresh nutrients diffuse through the agar slowly and feed the bacteria for weeks or years.
THIS WILL BE MOVED TO THE STERILIZATION PAGE. Any size presssure cooker is fine, if it is large enough to hold your equipment. I like 6 quart pressure cookers. I often let things cool in the pressure cooker overnight or longer because the dust can't get to the sterile supplies inside the cooker. The big secret to keeping things sterile is to keeping the dust out. Dust carries bacteria and molds. Dust mostly falls (settles) or is moved sideways by the wind. If you keep sterile things covered the dust cant fall onto them. Do not aim an electric fan at your sterile equipment. Do not talk (splash saliva) or cough at your sterile equipment. Slow human breath is fairly sterile, but can blow dust from surfaces into your sterile equipment.
Glass petri dishes are best because you can put the food agar in them and then autoclave, let it cool a long time and the plates are ready to use. If you don't have glass petri plates, you can use flat glass bottles stoppered with fairly tightly rolled cotton.
It is easier to melt small quantities of medium in a Erlenmeyer flask or jar in a microwave oven than on a stove in a pan.
How to use plastic petri dishes. Be sure to keep such dishes in original plastic sleeve until used. Be careful to keep dust out of them. Melt your agar medium and pour into test tubes or bottles and autoclave. Allow to cool somewhat (to 60C approx). Before the medium gels, pour it into the plastic petri plates. There is no danger that the hot agar will melt the plastic plates. The plates can stand boiling water (100C), but autoclave temperature (125C) deforms them.
Beginners are likely to have contaminated plates by careless methods. Never take the lid off a petri plate, just lift the lid on one side to pour the agar in. Never work in a draft as it will carry dust into the open or closed dish. As an extra precaution you can incubate or store your poured plates in clean cardboard, metal, or plastic boxes. On your first attempt, you are only making 100 mL of medium and I wouldn't worry excessively. Learn whether convenient methods are adequate. An expert can work in pretty dusty conditions and have very few contaminated plates.
Screw cap tubes are nice, but most people use cotton plugs. Dust which accumulates over a period of weeks may get inside the tube when the plug is pulled out to transfer the culture. If this becomes a problem, you can use paper or foil over the cotton plug to exclude dust.
I use TGY medium and supplement it with some limburger cheese. One needs 25 ml of medium for a standard 100 mm dia petri plate. The exact amount of medium in the plate is not critical. I usually use 20 to 30 ml. TGY (Tryptone-Glucose-Yeast extract) agar medium, sometimes called Plate Count Agar, is the best agar there is for growing bacteria because it has everything common bacteria need. Tryptone supplies amino acids and peptides. Glucose is a carbon source and energy source which most bacteria can use. Yeast extract contains most known vitamins, some sugars, nucleic acid building blocks, and amino acids and peptides. Minerals are present in milk and yeast which are the foods used to manufacture tryptone and yeast extract. seeLINK for details about ingredients for microbiological media.
Combine the TGY medium, cool tapwater, and some limburger cheese in an Erlenmeyer or other utensil and melt. The cheese will melt easily, but the agar will not melt until a few seconds or minutes of slight boiling. Use 22g of TGY powder per liter and 10 to 25 grams of limburger cheese per liter. Cottage cheese or dry milk would probably work just as well. You probably do not need the TGY. If you don't have a balance to weigh things, recall that 1ml = 1 cubic centimeter = 1 gram of water. Most media are heavier or lighter than water, but you can ignore the difference.
I would only mix 100 ml as that will make 3 petri plates and a few slants (tubes). Use 100 mL water, 2.2 grams of TGY (can omit this if you use 1 gram of agar), and 2 grams of limburger cheese.
Streaking a plate is simple and easy, but most beginners fail by pressing the loop to hard against the agar or rotating the loop.
Now be proud of yourself, few people have the patience to isolate a pure culture. Do you know any student or adult who has done so. If you know such an adult, isn't she a leading citizen? We welcome you to our profession!
You should read these pages before you try the above experiment because these include safety and experimental information not repeated above. Those in blue are completed and linked from this page.
Pages on WWW Related to This Page
You may click on these
Microbiological Safety Precautions
See our safety page.
If you isolate your own bacteria for experiments, avoid environments where pathogens are common. But if you isolate bacteria from your own mouth, perhaps they are not causing you harm. It should be safer to isolate bacteria from plant leaves, and perhaps soil. However, Staphylococcus aureus from plants can cause skin infections, and Clostridium tetani (lockjaw = tetnaus) Clostridium perfrigens?? (gas gangrene) are from soil and manure and are serious pathogens.
Indiana Biolab provide supplies for isolation of Brevibacterium linens if you need any. We require the order include a signed statement that you have read and understand our safety pages. If the order is from a student, parents must include a permission statement. Teachers need only order on school letterhead. Include your e-mail address if you have one. We prefer you send an e-mail first as we usually just gather the items you need.
Revised 1998 Jan 22