Lab Exercise 5: Pure culture techniques

OB JE C TIV E S

1. Perform a streak-plate to separate the cells of a mixed culture so that discrete

colonies can be isolated.
2. Perform a pour-plate (loop) dilution to separate cells of a mixed culture and
compare growth characteristics beneath and on the surface of the agar.

INT R O DU C TI O N

As you learned in Lab 4, microbes exist everywhere,

and very rarely do they occur as a single species.
Robert Koch, known as the father of medical
microbiology, was one of the first to recognize that
isolating a microbe (in his case, bacterium) away from
other microbes was crucial for his own argument that
microbes cause disease, as well as understanding
characteristics of the microbe itself. His studies on
Bacillus anthracis contributed to many of the laboratory
techniques we still use today, including the method for
isolating pure c ul tures of bacteria.

The most commonly used method in the laboratory for

isolating microbes is the s trea k pla te, and to a lesser
extent, the po ur pla te. Both methods rely on dilution of bacterial cells in a sample
to the point at which a single cell can divide giving rise to a single pure colo ny . The
pure colony is essentially a clone of cells that all are identical copies of the original
cell and can be used for further study.

Lab E xercis es

I. Streak plate m ethod

Ta ble s upplies Indivi dual suppli es

1 mixed culture of Serratia marcescens, 1 environmental body broth (from
Escherichia coli, and Chromobacterium Lab Exercise 4)
violaceum

2 nutrient agar plates

Inoculating loop

Pro tocol :
1. Prepare lab bench by removing extraneous items and cleaning surface with table
disinfectant.
2. Label the bottom surface of your sterile agar plates.
3. Using the T-method (illustrated below) or quadrant method, outline the sections
on the bottom of the agar plate.
4. Obtain mix culture and shake gently to suspend organisms.
5. Flame the loop until it is red-hot and let cool.
6. Remove cap of mix culture and flame the mouth of the tube (do NOT place cap
down on the table).
7. Insert the loop into the tube and remove a loopful of broth with bacterial cultures.
8. Lightly flame the mouth of the culture tube again.
9. Return the cap to the tube and set in your tube rack.
10. Streak the plate, following either the T-method or quadrant streak shown below.
Do not gouge into the medium with the loop and keep the lid over the plate as
much as possible.
11. Flame the loop before setting it down.
12. Repeat the above protocol (# 4-11) using your body broth from Lab 4 and a new
agar plate.

Step 1:Do the initial inoculation then FLAME the loop, let cool about 15 seconds.

Step 2: Use the sterile cooled loop and draw over the agar surface in the first section,
flame and cool.
Step 3: Use the sterile cooled loop and draw of the agar surface in the second
section. Note that you do NO T go back to the broth and you ove rla p with the first
section a few times. Flame the loop and allow to cool.

Step 4. Use the sterile cooled loop and draw of the agar surface in the third section.
Again do NOT go back to the broth and overlap with the second section only. Now
flame loop. Now place the plate in incubator.

13. Incubate the plates inverted at 30 C for 2 days. (NOTE: we will have to set up a
separate 30 C incubator for this experiment. Follow instructions on where to put
these plates).
See animation at:
http://www.sumanasinc.com/webcontent/animations/content/streakplate.html

II. P our plate m ethod

Ta ble s upplies Tea m s upplies

Mixed culture of S. marcescens, 3 nutrient agar pours
E. coli, and C. violaceum
3 empty Petri dishes
Inoculating loop
1. Label the bottom of three sterile Petri plates I, II, and III.
2. Remove 3 tubes of nutrient agar from the 50 C water bath in the back of the
room. (N O TE: you must work quickly at this point or the nutrient agar will solidify
in the tube).
3. Using aseptic technique, take one loopful from the mixed culture and add to the
nutrient agar in tube I.
4. Mix the tube by rolling vigorously between
the palms of both hands. DO NOT shake
the tubes as the caps are not secure and
medium will splash out.
5. Immediately, and using aseptic technique,
take a loopful of the agar from tube I and
add it to tube II.
6. As soon as this is done, you may pour the
contents of tube I into the plate labeled
plate I, being sure to pour it into the
bottom of the plate.
7. Repeat steps 4-6, transferring a loopful of
tube II to tube III, and then pouring the contents into plates II and III.
8 . Allow media to solidify and then incubate the plates inverted at 30 C for 2 days.
(NOTE: we will have to set up a separate 30 C incubator for this experiment.
Follow instructions on where to put these plates).

DA TA A ND O B SE RVA TI O NS
1. Eval ua tio n of strea k pla te s: Show within the circle the distribution of the
colonies on your streak plate.

Did you get clear isolation of purple Chromobacterium violaceum, red Serratia
marcescens, and white E. coli?

If not, you maybe need to practice the technique again by subculturing the colonies
(your instructor might want you to do this regardless). To subculture, use your wire
loop to pick an isolated colony that you will use as inoculum for another streak plate.
2. Eval ua tio n of po ur pla te s: Show the distribution of colonies on the pour plate
II or III. Indicate which colonies are growing on the medium and which are growing
within it.

DI SC USSI O N

1. Compare your results from the streak-plate and pour-plate methods. Which
method achieved the best separation of species?

2. Which specie(s) are able to grow both on the surface and within the medium? In
which location are the colonies larger? Why?

3. In regards to bacterial growth on solid media, define the term colony.

4. Explain how dilution is a common approach employed by any pure culture

technique.

5. What advantage does the streak-plate method have over the pour-plate method?

6. Before inoculating and pouring molten nutrient agar into a plate, why must the
agar first be cooled to 50 C?

7. Provide two reasons why plates should be inverted during incubation