Isolation of Pure Cultures Of Food Bacteria

Brevibacterium linens from Limberger Cheese

This is a good bacterium to isolate 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. 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.

Detailed Procedure for Isolation of Brevibacterium linens

Requirements: Petri dishes/plates, autoclave, 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.

Suggested Equipment:
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Autoclave: To autoclave plates and stuff; 15 pounds pressure; 15 minutes 1 or 2 petri dish. Incubate at room temperature to 30C, maybe 37C.

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2 test tubes filled with medium and plugged with cotton for storage of your pure cultures. Wire loop for streaking bacteria onto the petri plate; any steel wire will work. Nichrome or stainless steel is better, but copper may have some toxic effect. Rich people use platinum. Microwave ovens are great for melting media without scorching. If bumping occurs (steam explosion), add a few 3 mm sized pieces of crushed limestone for nucleation of small bubbles.

THIS WILL BE MOVED TO THE STERILIZATION PAGE. Autoclaving: 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. It is easier to melt small quantities of medium in a Erlenmeyer flask or jar in a microwave. 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. 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. 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.

Preparing the Agar Plates. Also see Preparing LINK for more details.
TGY medium and supplement it with some limburger cheese. TGY ** Tryptone Glucose Yeast
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Tryptone: 5.0g, Agar:10.0g, Yeast extract:5.0g, Glucose: 1.0g K2HPO4: 1.0g Spring water: 1000 mL

This is called Plate Count Agar by many people. It supports more species of bacteria than any other medium. If in doubt try TGY. Bacterial pigments which are pale on some media are usually much brighter on TGY in air at room temperature. Add 1 gram of powdered CaCO3 to counteract the acid generated by many bacteria from the glucose. With the carbonate, many stock cultures last years instead of months. If you don't have calcium carbonate powder try ground agriculural limestone or any limestone or blackboard chalk. To avoid settling of CaCO3, stir while filling tubes. The low level of glucose helps reduce acid production and cultures live longer. 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. 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. Combine the TGY medium, cool tapwater, and some limburger cheese in an Erlenmeyer flask or beaker 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 to Isolate the Bacterium

Materials needed
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Sample of limburger cheese which you have just unwrapped partially. We will touch loop to the rind of the cheese in an orangish area. An inoculation loop this is a piece of wire with a 1 mm loop bent on one end. Petri plate of suitable medium or medium slanted in a flat bottle. Some method for sterilizing the loop: bunsen burner or kitchen stove flame, alcohol lamp, or a tube of alcohol or chlorine bleach. Isopropyl alcohol (100% or 70%).

Streaking a plate is simple and easy, 1. Barely touch the edge of the loop (not the flat of the loop) against an orange-yellow region of the rind of the limburger cheese (freshly opened). 2. Now touch that exact edge of the loop to the agar of the petri plate and move loop back and forth across the top 1/3 of the petri plate without hitting the same path twice. 3. Lower cover of plate and resterilize the loop by heating it red hot.

4. Rotate the dish 1/5 or 1/4 revolution and draw the loop across the recently streaked area so that edge of loop picks up a little bacteria and streak as before. 5. Repeat Steps 3 and 4 two times and that uses up the entire plate surface. 6. Incubate the plate at a suitable temperature for Brevibacterium linens. Room temperature to 30C should do nicely. 7. After the colonies have grown, select an isolated yellow-brown colony and restreak as before. 8. When this second plate is grown, all the colonies should be identical yellow-brown and the plate should smell horrible just like limburger cheese complete with a trace of ammonia from breakdown of amino acids in the casein. 9. Select a typical colony of Brevibacterium linens and use a sterile loop to inoculate a TGY slant and incubate at suitable temperature. Label the culture with species and ORIG or MASTER. When he tube is grown store it in the refrigerator if you have one. Make a working culture by using a sterile loop to inoculate a second tube of TGY. Label this with species and WORK to mean working culture, never use the master culture for anything except to make a working culture when you must. You can wrap the top of your master with foil to reduce drying and risk of contamination. Your refrigerator should be dry enough that molds do not grow on all the surfaces.