Microorganisms can be grown in either solid or liquid media. The chemical composition of the medium can be varied to suit the needs of the investigator. Liquid media-- in tubes or bottles-- is best used for growing large numbers of organisms, for measuring growth rates, and for performing certain biochemical tests. Solid media--slants and petri plates--are used to isolate single colonies (pure cultures), to obtain information about colony morphology and to perform certain biochemical tests. In addition, slants are used to store cultures of organisms for a long period of time.

In this lab you will be exposed to a variety of techniques that you will use throughout the term. These techniques are demonstrated on the video "Microbiology Techniques," that is kept in the learning center. You may wish to refer to it at various times throughout the term.

A. Use of the Touch-o-matic Bunsen burner

This burner is a special burner designed to reduce the use of open flames; it contains a pilot light that is used to ignite the burner.

1. Make certain that the circular indentation on the burner is opposite the gas intake valve. Open the gas valve all the way and wait a few seconds. With the striker, the pilot light. You must squeeze the striker toward you and across the flint at the same time.

2. When the pilot is lit, a continuous flame suitable for flaming can be obtained by depressing the platform. Before depressing the platform, reduce the gas supply by closing the gas valve about half way.

3. The platform can be locked in a depressed position by rotating it slightly in either direction. To release the platform, turn it so that the indentation is opposite the intake valve.

NOTE: In some cases the pilot light may not work. To light the burner if this is the case, turn gas on about half way, depress platform, and hold striker over pilot and strike as above.

Try to keep the flame at a height of  no more than two inches when in use.

B.  Flaming loops and transferring cultures

1. Place the loop or needle in the blue flame until the wire turns red. Position the wire so that at least half the length is in the flame.

2. Remove the cap of the tube with the fourth and fifth  fingers of the hand that is holding the loop. Do not let the cap touch anything while it is off.

3. Quickly flame the mouth of the test tube, always keeping the tube at a slight angle.

4. If going into liquid, dip the loop into the liquid. If going into solid, cool the loop or needle first by sticking it into the agar, and then touching the colony.

5. Remove the loop or needle, flame the top of the tube, and replace the cap.

6. Repeat step 3 and transfer the loop to a new tube. A colony on the surface of a petri plate or slant can be transferred in the same manner.

7. If you are transferring to a plate, remove the liquid of the plate with your free hand. DO NOT SET THE LID DOWN ON THE TABLE.

8. Rapidly, but gently, move the needle across the surface of the agar. DO NOT GOUGE THE AGAR.

Loops and needles should be sterilized before and after each use.

C. Pipeting

Sterile pipettes are provided in sterilized canisters. The canisters contain wither 10.0 ml or 1.0 ml pipettes. Ten ml pipettes have an orange and on them, and 1.0 ml; pipettes have a red or yellow band on them. Canisters should be stored and carried in a horizontal position unless they  are empty.

1. Place the canister flat on the table

2. Remove the top of the canister and place it near the canister. Tops can be left off until you are finished using the pipettes.

3. Grasp the end of the pipette and quickly remove it from the canister. DO NOT TOUCH THE BOTTOM PART OF THE PIPETTE!

4. Remove the top from the bottle or tube and flame the mouth of the container.

5.  Place pipette in the liquid and by sucking withdraw the desired amount of liquid. Press your index finger, NOT YOUR THUMB,  over the top of the pipette to prevent the liquid from coming out.

6. Reflame the mouth of the container and replace the top.

 7. Remove the top of the new container, and if it is glass, flame the mouth.

8. Pipette the desired amount of liquid by removing the index finger and blowing the contents of the pipette into the new tube.

9. When finished with the pipette, place it in the discard bucket, tip down.

NOTE: If you are transferring bacteria, do not suck up the contents of the culture. Use capillary action or the propipette. You should always use a pipette that is about ten times the volume of liquid to be transferred

D. Mixing tubes

Liquid cultures should always be mixed to insure uniform distribution of microorganisms. Grasp the tube firmly between thumb and middle finger; your index finger should be on top of the tube. With the index finger of the other hand, vigorously strike the bottom of the tube several times so that a noticeable whirlpool or swirl is produced.

E. Spreading

This procedure is used to produce a uniform distribution of liquid over a petri plate.

1. Using the piping procedure above, remove the desired amount of liquid from the tube or bottle.

2. With your free hand, remove the lid of the plate and pipette  the desired amount of liquid onto the plate. Discard the pipette and replace the lid of the plate.

3. Sterilize the triangular shaped spreader by dipping it in alcohol and igniting it by quickly passing it through the flame. BE SURE TO REPLACE THE LID TO THE ALCOHOL BEFORE FLAMING.

4. Remove the lid of the plate and place the spreader in the puddle of liquid and move the spreader across the surface of the plate many times. Rotate the plate as you are spreading. Be careful not to gouge the agar.

5. Replace the lid of the plate and resterilize the spreader as above. Allow the plate to stand about 5 minutes.

6. Tape the plate, invert it, and place in the appropriate incubator.

NOTE: All petri plates should be stored in an inverted position to prevent condensation from accumulating on the surface of the plate.

F. Making dilutions

1. Obtain culture and 4 sterile tubes. Label tubes 10-2,  10-4, 10-5 and 10-6.

2. Add 9.9 ml broth or sterile water to first two tubes. Add 9.0 ml liquid to the last two tubes. Use the same 10 ml pipette to fill all four tubes.

3. Mix original culture. Remove 0.1 ml of culture to 10-4. Discard pipette.

4. Mix 10-2 tube. Remove 0.1 ml from this tube and add to 10-4 tube. Discard pipette.

5. Mix 10-4, remove 1.0 ml, add to 10-5 tube and discard pipette.

6. Mix 10-5 tube, remove 1.0 ml to 10-6 tube and discard pipette.

Be sure you understand this procedure. In future labs, the procedure will only say prepare a 10-6 dilution.

7. Spread 0.1 ml samples from the last three dilutions. If you begin with the most dilute culture, you can use the same pipette for all the dilutions.

8. Incubate plates for the desired amount of time and return to count colonies.

BACTERIAL QUANTITATION

Many procedures require that the investigator know the number of organisms/ml culture. Bacteria can be counted microscopically with the aid of a hemocytometer, or with an electrical machine., but these methods do not distinguish between dead and live bacteria or between bacteria and particles of dirt, etc. In contrast, the quantitative plating method gives the concentration of live bacteria. One makes a series of dilutions and plates known volumes from some of the dilutions. After incubation, one counts the number of colonies and determines the concentration of bacteria in the original sample.

Normally, then number of bacteria in the original culture is not known. Therefore, it is necessary to plate samples from a number of different dilutions. the dilution that is ultimately used to determine the concentration is the dilution that gives between 30 and 300 colonies on the plate. Dilutions that give less than 30 colonies are subject to wide variation due to random sampling. Dilutions that give more than 300 colonies are not accurate because some of the colonies will touch each other. Greater accuracy is achieved by plating 2-4 samples for each dilution, and then averaging the number of colonies. The original concentration is calculated as follows:
 

Concorig  = number of colonies  X  ______1______
                volume plated               dilution factor

Example: 40 colonies from 10-5 dilution with 0.1 ml plated

Conc = (40/0.1) X  ___1___  =  ____40____  =  40 X 106  or 4 X 107/ml
                                  10-5                 (10-1) (10-5))
 

Bacteria are typically grown on some kind of all purpose medium such as tryptic soy agar (TSA). Other media allow one to measure certain biochemical properties of the bacteria. MacConkeys agar is a medium that allows one to distinguish between lac+ and lac- bacteria. The medium contains both lactose and glucose, so that all bacteria will grow. Those organisms that can use lactose will appear as hot pink colonies; those that cannot use lactose will appear as beige colonies.

In this lab, you will determine the concentration of bacteria in an original sample and also determine the ratio of lac+ to lac-
 
 

Materials per pair

3 TSA plates
3 MacConkeys plates
4 sterile tubes
1 bottle sterile water
pipettes
bacterial culture

Procedure

1. Record the number on the culture on your table.

2. Prepare 10-2, 10-4, 10-5 and 10-6 dilutions of the culture. Use sterile water to make the dilutions. See unit 2. You can use the same 10 ml pipette to fill all four tubes. When transferring bacteria, REMEMBER TO USE A NEW PIPETTE FOR EACH STEP OF THE TRANSFER. YOU SHOULD ONLY GO INTO THE ORIGINAL TUBE ONE TIME!
 

                                        0.1 ml          0.1 ml              1.0 ml              1.0 ml
 
 

                                            10-2             10-4                 10-5                 10-6
 

                 original             9.9 ml          9.9 ml             9.0 ml             9.0 ml
 

3. Plate duplicate 0.1 ml samples from the last three dilutions. Use the same 1.0 ml pipette for plating, and begin with the most dilute tube. Each dilution should be plated on one plate of TSA and one plate of MAC.

4. Incubate the plates overnight at 40o C.

5. Count the colonies on each of the six plates. Count each colony and continue counting until you reach 500. If there are more than 500 colonies, record the result as too many to count (TMC). Calculate bacteria/ ml in the original culture. Calculate the ratio of lac+ to lac-
 
 

RESULTS

    TSA

dilution            # colonies                bact/ml
10* -4
10*-5
10*-6
 
 

MacConkey

dilution                 # lac+             #lac-                         ratio +/-
10-4
10-5
10-6