Isolation of Pure Cultures Of Food Bacteria

Brevibacterium linens from Limberger Cheese

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.

Detailed Procedure for Isolation of Brevibacterium linens

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.

Suggested Equipment. Also see LINK for more details

• Pressure cooker to autoclave plates and stuff; 15 pounds pressure; 15 minutes
• 1 or 2 petri plates. Incubate at room temperature to 30C, maybe 37C. Flat bottles or any container such as a plastic jar lid covered with foil can serve as a petri dish.
• 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. 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.

Preparing the Agar Plates. Also see Preparing LINK for more details.

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 to Isolate the Bacterium

Materials needed

• 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%). I usually use a bunsen burner, but I frequently just dip my loop in 70% isopropanol, tap the handle on my finger to shake off the alcohol and use it. If alcohol remains on the loop it will kill the bacteria, but I touch the loop to a sterile surface such as the surface of the sterile agar before use. I have done this 250,000 times without a known contamination in my tissue culture lab.

Streaking a plate is simple and easy, but most beginners fail by pressing the loop to hard against the agar or rotating the loop.

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. Such care of your master culture shows you are a careful, dependable bacteriologist. 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.

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!

Trouble shooting

• Most beginners press the loop too tightly against the agar causing the loop to penetrate the agar. Never do that as it ruins the uniform distribution of the bacteria across the surface.
• After incubation of your first streaked plate you will see where you could have done better. Every microbiologist has his favorite way to streak a plate. Do whatever it takes to get nicely spaced pure colonies.
• In place of a loop, sterile sticks (autoclaved) or glass rod with rounded tip could be used, but the loop is easier.
• If your streaked plate is moldy, then dust got into the plate. If you must, you can put the plate in a paper bag, but handling the bag just pumps dust into the plate.
• Never take the lid off the plate, just raise one edge.
• Put a piece of tape on the bottom of the plate at one edge so you know the starting point, you can write on the tape to label the plate.
• When unwrapping the limburger cheese, just unwrap one side to get a slab of cheese for your medium. That leaves most of the block surface uncontaminated as a source for the inoculum to streak your plate.

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.

• Introduction to Bacteria
• Pure Culture Techniques for Microbiology
• Bacterial Media Preparation
• Introduction to Bacteria Genera
• Microbiological Safety

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

Revised 1997 Feb 1 (incomplete)

Isolating bacteria from dairy foods should be about as safe as microbiology gets. However, keep in mind that some very serious human pathogens have been found in milk. You must always treat any culture as potential pathogen until proven otherwise.

Isolating a pure culture from milk means we use some method to obtain a culture in which every cell is descentant of the same original cell. See our pageLINK which gives such methods. Pay particular attention to isolation by streaking. In this exercise we assume you have read those pages and are quite competent at streaking.

You now have to choose suitable medium, temperatrue, atmosphere,

Some good media for dairy bacteris. There are two approaches you cant find a list of suggest media used by dairy microbiologists. 2) you can read about the bacteria the candidate bacteria and pick a mmediums that fits. But if you don't have the ingredients needed for professional media, you take some of what the bacteria are growing on and autoclave it.

Media used by dairy microbiologitsts.

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This is a nice starting project because the desired organism has a distinctive color and produces a distinctive odor on certain medium. Therefore, when you isolate the organism you can be pretty sure you have Brevibacterium linens which is safe enough to eat. However, do not eat it because you are a beginner may have made some mistake.

of what you have. You can then be somewhat confident that it will be safe to work with.

Step one is to buy some Limberger cheese. We will use it both as medium and source of live organism cut Cut off say 5 mm slice at one end. and leave it in the wrapper and place in a clean jar. That leaves most of the cube of cheese for medium and a treat for the family. It tastes good on rye or other special bead, with sweet cream, with any type of jelly. Savor it, The flavor came from leftorvr products of the bacteria. Note the plural Many species of bacteria were present. Notice there is a salt flavor. Lots of salt was used. many kiinds of undrireabvel bact couldn't grow arroujnd all that salt.

try 300 g cheese per lliter = 30 gr per 100 ml

autoclave the medium, streak the plates, restreak the cultue. save on a slant or stab.