Isolation Techniques: Streak Plates
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1. Explain what is meant by a streak plate.
2. Explain what is meant by discrete colonies.
3. Demonstrate an understanding of the streak plate technique used for isolation of
discrete bacterial colonies.
As previously stated, in our surroundings we find that microorganisms are everywhere.
Not only do microorganisms live everywhere but they also live in mixed populations.
When samples of microorganisms are collected from the natural environment or a
controlled environment such as a cleanroom, to determine what microorganisms
specifically make up this mixed population, we must cultivate and isolate them from the
mix. Among the bacteria, there can be hundreds and thousands of different types or
species living together. To study them requires a pure culture.
A pure culture contains only one type or species of organism and allows for them to be
identified by using certain differential and biochemical tests. As you are now aware,
microorganisms such as a single bacterium are too small to see with the unaided eye and
it would be impossible to perform any kind of test procedures on this single microscopic
organism. So, along with isolation the microorganism(s) must also be cultured. Culturing
allows for optimal growth conditions (e.g. nutrients, temperature, moisture, pH) to be
provided so that the organism will thrive and reproduce.
Bacteria reproduce by asexual means in which a single bacterium cell "splits" into two
"new" independent bacteria cells that are identical replicas or clones of the original cell.
These two bacteria cells can then each "split" into two more bacterial cells for a total
count now of four bacteria cells. As this process continues, hundreds of thousands and
millions of bacteria cells are created that had originated from a single bacterium. The
compilation of all these clones from a single bacterium, constitute a colony. If a colony is
separated and isolated from another colony each individual colony represents a discrete
colony in which the origin of that colony was from a single discrete bacterium. When a
few members of a single colony are selected and recultured they result in what is called a
pure culture. In particular, bacteria can be cultured and isolated using a streak plate
technique from which a colony or colonies can be selected and recultured to attain pure
cultures.
In this laboratory exercise you will be using the streak plate technique by transferring
bacteria of a mass population growing on a slant agar to an agar plate in which the
resulting streak pattern will yield discrete colonies of bacteria. A colony can then be
selected as representing a pure culture that can then be used in future microbiological test
procedures.
25
Materials (per student)
1 Prepared Pure Culture Slant of Proteus vulgaris or Serratia marcescens
1 Prepared Pure Culture Slant of Micrococcus lutens or Enterococcus faecalis
2 TSA or Nutrient Agar Plates
1 Inoculating/Transfer Loop
China Marker or Sharpie
Bunsen Burner
Procedure
Streak Plate Technique
Acquire a prepared pure culture slant of P. vulgaris or S. marcescens and a prepared pure
culture slant of M. lutens or E. faecalis. Using aseptic technique (this will be
demonstrated by your instructor), transfer an inoculum from each of the prepared cultures
individually to an uninoculated agar plate as follows:
1. Setup and light a Bunsen burner.
2. Label the bottom perimeter of an uninoculated agar plate with the name of the
organism, your initials and the date. Note: Do not write on the lid of the plate.
3. Place the labeled plate on the bench lid-side up.
4. Place the base of the prepared pure culture slant tube in the palm of your hand and
secure the tube with your thumb.
5. Pick up the inoculating loop by the handle with your free hand, holding it between
your thumb and index finger. Flame the loop until the entire wire is glowing red. Allow
the wire to cool for 10 seconds before obtaining live culture! The loop is now sterilized,
so do not set the loop down on the bench or touch the wire to any object. If the wire
accidentally comes in contact with the bench surface or any other object; you must
reflame the entire wire. (Also you must reflame between inoculations to prevent crosscontamination of the pure cultures).
6. With the sterile loop in hand and using this same hand, place the cap of the tube
between the bent knuckles of your middle finger and ring finger. Grip and hold the cap
firmly between the knuckles.
7. Uncap the tube by pulling the cap from the tube with your clasped knuckles but do not
set the cap down, allow it to remain between your knuckles during the transfer.
8. Immediately flame the open end of the tube by passing the open end back-and-forth
once through the flame with a fluid motion and moderate speed.
9. Insert the wire loop into the open end of the tube containing the prepared pure culture
(the culture should appear as a wavy or zigzag smear along the length of the slant area
within the tube).
10. Gently dab the wire loop to the smear area of the live culture and acquire a very small
amount on the tip of the wire loop. Note: If you apply too much pressure with the wire
loop to the agar surface it will dig into the agar.
11. Withdraw the wire loop from the tube. Be careful not to contaminate the inoculum as
you proceed with steps 12 and 13.
26
12. Flame the open end of the tube as performed in step 8 and replace the cap on the tube.
Place the tube back on the bench or into a holding rack freeing up this hand.
13. Acquire the labeled agar plate, lid-side up, and using your free hand tilt the lid as if
opening a door to a 45 degree angle exposing the agar surface. In this way the lid and the
agar medium are kept to a minimal exposure of extraneous contamination.
Note: Refer to Figure 1 as you proceed with steps 14, 15, 16, 17, and 18.
Figure 1. Streaking Sequence for Colony Isolation. (http://www.addgene.org).
14. Refer to Figure 1. At position 1, gently touch the tip of the loop to the agar surface
and with a zigzag motion; gently drag the loop on the surface as indicated by pattern 1
and stop. Withdraw the loop from the plate and replace the lid.
15. Flame the loop and let it cool for a few seconds.
Note: It is convenient to rotate the plate so that the next region to be streaked is
oriented in the position of the previously streaked region!
16. Tilt the lid open again and streak from the end of region 1 across the edge of the plate
forming region 2 and stop. Withdraw the loop and replace the lid.
17. Flame the loop and let it cool for a few seconds.
18. Tilt the lid open again and streak from the end of region 2 across a third of the plate
forming region 3 and stop. Withdraw the loop and replace the lid. (Note: You may be
instructed to streak 4 quadrants rather than 3, apply the same procedure accept divide the
plate so that there would be 4 regions streaked rather than 3).
19. Flame the loop before setting it down.
20. Repeat the above procedure to streak plate the other prepared pure culture slant.
21. Incubate the two streak plates in an inverted position at 37°C for 24 to 48 hours.
22. Observe the plates for a fading zigzag growth pattern and isolated colonies, draw and
record your results.
27
Note: Do not discard your streak plates after observing. These cultures will be used
in the lab exercise: Gram Stain
Note: There is another option to cooling the loop wire after flaming, immediately
after flaming touch the loop to the agar surface of the tube or plate that you are
using for that particular step. Then proceed as if you had allowed it to air cool.
Results and Observations
For the streak plates record and draw your results below:
Streak Plate
Growth (+) or (-)
Drawing of Cultured Pattern
P. vulgaris or S. marcescens
M. lutens or E. faecalis
Laboratory Review
1. Explain what the expected out come is by performing the streak plate technique:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. Explain how discrete colonies are isolated by performing the streak plate technique:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
28
3. In your own words, explain what is meant by a bacteria colony:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. Explain why it is important to have a pure culture of one type of microorganism rather
than a mixed population of various types of microorganisms:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
29