LAB4_PURE CULTURES: STREAK PLATE

When a single bacterium (bacterial cell) is placed on a suitable solid nutritional medium, it will
continually replicate to form a visible mass called a colony. For rapidly growing bacteria such as E.
coli, a visible colony appears after overnight incubation at 37oC. Each individual colony consists of a
population of organisms descended from a single bacterial cell and is referred to as a pure culture.
Most work in microbiology relies on using pure cultures. As mentioned previously we must use
sterile technique to keep a culture pure, but how do we originally obtain pure cultures? The easiest
way is to use a streak plate. This technique increasingly dilutes bacteria on the surface of an agar
plate so that individual bacteria are eventually deposited far enough apart to grow as isolated
colonies. Not only do isolated colonies provide a source of a pure culture, but morphological
characteristics such as color and shape of an individual colony can be used to distinguish different
isolates and begin to identify them.
In this procedure you will use the streak plate technique to obtain isolated colonies of bacteria from two
different broths, each containing a mixture of bacteria.
NOTE: We may perform the Pour Plate Method as well depending on time/media availability.

Reference: Brown, A.E. Benson’s Microbiological Applications , Laboratory Manual in General

Microbiology, Short Version, 13th Edition, McGraw Hill, 2015. LAB EXERCISE 9, Pure Culture
Technique.

PERIOD 1
Materials per student:
2 TSA plates

Materials per table of 4:

2 TSB broth cultures labeled “Mixture A” and “Mixture B”. Each mixture contains a suspension
of 3 different types of bacteria. In general, you will find 1 set of cultures for each table of four
students in the refrigerator on the green racks. The stocks cultures and green racks should always
be returned to the same spot in the refrigerator before you leave. Also, your table will need 6 TSA
plates.

Procedure:

1. Swirl broth culture “A” slightly to mix it (careful not to spill anything!). Using sterile technique,
withdraw a loopful of the culture and streak it over 1/3 of the surface of an agar plate as
illustrated (step A in picture below). Then sterilize and cool the loop. This step deposits the
inoculum relatively evenly over the first quarter of the plate. It's best to simply slide the loop over
the agar since pressing down on the agar will tend to gouge the surface (you do not want to create
cracks in the agar). The closer together the streak lines are, the more thoroughly the inoculum
will be diluted. Note that this time we are using a broth culture but the same technique is used
with a solid culture or a clinical specimen such as a throat swab. From a solid culture start with a
barely visible amount on the loop.

2. Use the cool sterile loop to streak from the first into the second section several times and then,
without going back into the first section, continue streaking until another third of the plate has
been used (step B). Sterilize and cool the loop. This step uses the first section as an inoculum and
spreads that out over part of the plate, thus diluting the original inoculum. Again, it is best to have
 the streak lines very close together. If the loop sizzles when it touched the agar then you didn't
allow time for it to cool sufficiently. A hot loop will kill the culture, but more importantly, will
create aerosols that may contain bacteria that can then be inhaled.

3. Use the cool sterile loop to streak from the second section several times into the third section
and then, without going back into any previous sections, continue streaking until another third
of the plate has been used (step C). Sterilize the loop. This step dilutes the inoculum even
further, thus hopefully depositing individual bacteria on the agar surface.

4. Repeat the procedure with broth culture “B” and the second TSA plate.

5. Label the plates on the bottom of the plates. You can write directly on the plastic, and
EVERYTHING you label in lab should have: 1) your name or table/team designation, 2) the date,
3) lab section, 4) type of media, 5) incubation temperature, and 6) name of the sample

A. Streak one loopful here.

Sterilize loop.

A B. Use sterile loop to streak through section A into section B

several times (3 to 5). Continue streaking in section B without
touching section A. Sterilize loop.
B
C

C. Use sterile loop to streak through section B into section C several times (3 to 5).
Continue streaking in section C without touching section A. Sterilize loop. (Optional: If you
have space, you can follow the same procedure to set up a section D also) Incubate plate.

Plate should look something like this after incubation. Dark lines
indicate confluent growth and dots indicate isolated colonies.

Optional procedure: Pour plate method

In addition to the streak plate method, your instructor may also ask you to obtain isolate your
organisms using the pour plate method. Please consult p.78 in your Benson’s Lab Manual about
information regarding the pour plate method
 PERIOD 2: SUBCULTURE of ISOLATED COLONIES

1. Observe the plates for isolated colonies, and make colored drawings of what you see in your lab
notebook. The goal of a streak plate is to obtain isolated colonies; it doesn't matter which section
has the isolated colonies. How many different colony types do you see? Describe their
appearance. For each different colony type that you draw, you should write down 3 or more
colony characteristics for that colony (reference figure = Benson Figure 35.4). Show instructor
to receive a signature for “Streak Plate” if you have isolated colonies (if you don’t, your
instructor will tell you what you need to set up to get checked off next period). Record all
observations and make colored drawings of the plate appearance.

2. As a table of 4 subculture all 6 different colony types (A1, A2, A3, B1, B2, B3) onto new TSA
plates. Choose one isolated colony of each type (you can either use a colony from your plate
from period 1, or use a colony from demonstration plates provided by your instructor): use
your inoculating needle to obtain a small portion of the colony that you will streak onto
quadrant 1 of a sterile TSA plate. Then use your inoculating loop to streak the remaining
section for isolation, as in Steps 2 and 3 from the Period 1 Procedure above). Use masking tape
to put your plates together and incubate at 37oC by placing in the 37oC white tubs). Your table
of 4 should have 6 new streak plates, one for each of the different types of colonies.

You will use these new plates to perform your simple stains and your Gram Stains (LAB 5) as
well as for Aerobic/anaerobic growth (LAB 6), and selective/differential media (LAB 7).
Please DO NOT DISCARD THESE CULTURES, and also subculture for the next few weeks as
instructed – some experiments will not work when your cultures are too old.
 Name_________________________________________________________

Section ______________________________________________________

4_ PURE CULTURE ISOLATION Post Lab Report (10pts)

Answer the questions that follow (Note that this lab exercise and the Benson lab manual can help
you answer some of these questions):

1. How many different bacteria were in Mixture A? Include a drawing (you can simply copy
drawings from your lab notebook) of the each of the colony types you observed, and use at
least 3 descriptive terms to describe the visible colony characteristics of EACH of the 3
colony types (color of colony is one characteristic, and you can use Benson Figure 35.4 to
learn some additional appropriate terms to describe each colony type) – include a separate
sheet or write on the back of this sheet.
2 How many different bacteria were in Mixture B? Include a drawing of the each of the colony
types you observed, and use at least 3 descriptive terms to describe the visible
characteristics of EACH of the 3 colony types (color of colony is one characteristic, and you
can use Benson Figure 35.4 to help find some additional appropriate terms to describe each
colony type) – include a separate sheet or write on the back of this sheet.

3. Why is it necessary to flame the loop between quadrants on the quadrant streak plate?

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

1:

2:

5. Define “subculture”. Why is it necessary to subculture the colony to be sure it is a pure

culture?

6. Describe at least two different ways you could end up with a contaminant if you do not
properly use aseptic technique when inoculating a plate:

7. Let’s say you perform a streak isolation with 4 total sections on a TSA plate (from a bacterial
sample in a TSB tube). How many total times did you sterilize your inoculating loop
(include the times before and after, as you would when performing proper aseptic
technique)?