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Exercise III

Aseptic Transfer Techniques II: Streak Plate and Spread Plate Methods

The appearance of colonial growth on agar media can be very distinctive for individual

species. Observation of the noticeable gross features of cultures, that is, of their colonial

morphology, is very important. The colony density, consistency, surface texture, shape

and size of colonies all should be observed, for these features are probable clues as to the

identity of the organism, although final identification cannot be made by morphology

alone.

When a bacterium grows on a solid surface, the number of cells increases

geometrically until a visible mass of cells, called a colony appears. The purpose of the

streak plate and spread plate methods is to isolate individual bacterial cells on a nutrient

medium so that during subsequent incubation, a cell grows and divides sufficiently to

form a single colony. That colony is considered a pure culture.

The streak plate technique uses the spreading of a loopful of bacteria over the surface

of the media until an isolated cell is obtained on the surface. When the plate is incubated,

each cell gives rise to a colony, a visible mass of cells resulting from repeated divisions

of a single cell and its daughter cells. Colonies can then be transferred to other media.

Such transfer is called sub-culturing and must be carried out under sterile conditions

using aseptic technique to prevent possible contamination. A variety of streaking

methods may be used.

The spread plate method uses a glass rod to spread a broth culture of bacteria over the

surface of the plate. If the inoculum is too concentrated, individual isolated colonies will

not appear.

Materials Needed

culture of Micrococcus luteus

culture of Escherichia coli

broth culture of Micrococcus luteus

broth culture of Escherichia coli

4 sterile nutrient agar plates

inoculating loop

Bunsen burner

striker

test tube rack

wax pencil

dish of alcohol

glass spreader

2 pipettes

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goggles

gloves

spray bottle of disinfectant

paper towels

Procedure 1: Streak Plate Method

1. Obtain a nutrient agar plate of Micrococcus luteus.

2. Using a wax pencil, label the bottom of a nutrient agar plate with the initials of

the organism and the initials of someone in the group.

3. Sterilize the inoculating loop by holding it at a downward angle into the center of

the flame of the Bunsen burner.

4. Flame the whole wire until it turns red by moving it through the flame from the

loop to the top.

5. Holding the loop, let it cool for 20 seconds.

6. Raise the lid of the nutrient agar plate of Micrococcus luteus at an angle like a

clam shell.

7. Place the loop on the culture and gently (do not apply a lot of pressure) move it

back and forth in a zigzag pattern. Do not dig or gouge into the media.

8. Close the lid of the culture plate.

9. Open the lid of the sterile nutrient agar plate at an angle and gently streak the loop

of bacteria back and forth in a zigzag pattern across the surface of the media.

10. Turn the plate and repeat the process.

11. Close the lid of the inoculated plate.

12. Sterilize the loop in the flame.

13. Tape the edges of the plate and turn it upside down.

14. Repeat the procedure with Escherichia coli.

Procedure 2: Spread Plate Method

1. Obtain a broth culture of Micrococcus luteus.

2. Using a wax pencil, label the bottom of a sterile nutrient agar plate with the

initials of the organism and the initials of someone in the group.

3. Mix the broth culture by rolling the tube between your hands.

4. Dip the pipette into the broth culture and obtain .3ml of the mixture.

5. Open the lid of the sterile nutrient agar plate at an angle, like a clam shell.

6. Squeeze the bulb of the pipette and place the inoculum in the center of the plate.

7. Close the lid of the plate.

8. Dispose of the pipette in a biohazard bag.

9. Obtain a glass spreader and immerse the short end in the alcohol dish.

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10. Flame the glass rod in the Bunsen burner to evaporate the alcohol and sterilize the

spreader.

11. Let the spreader cool 20 seconds.

12. Open the lid of the inoculated dish at an angle and evenly spread the inoculum

over the surface of the media.

13. Rotate the plate and spread again.

14. Close the lid and let the inoculum soak into the agar for 5 minutes.

15. Immerse the glass spreader in the alcohol and flame to sterilize.

16. Once the inoculum has soaked into the agar, tape the lid shut and turn the plate

upside down.

17. Repeat the procedure using Escherichia coli.

Clean-up Procedure

1. Tape all four plates together and label with the initials of someone in the group.

2. Place the plates in the incubator upside down to prevent condensation that may

form on the lid from dropping down onto the surface of the media.

3. Make sure all used pipettes are placed in a biohazard bag

4. Return the Bunsen burner, striker, wax pencil, alcohol dish, glass spreader,

inoculating loop, test tube rack, and original cultures to the appropriate place.

5. Spray and wipe the table top with disinfectant.

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