Lab Module 4: Streaking for Isolation
INTRODUCTION
In the 1850s, Louis Pasteur developed the concept of the pure culture. This was an important
intellectual step forward because the ability to work with pure cultures allowed Pasteur (and
subsequent microbiologists) to study individual bacterial species. Pasteur’s method for developing
pure cultures—repeatedly diluting broth cultures until he thought he had individual species—was
very inefficient.
In the 1880s, Robert Koch developed the procedure of streaking for isolation. The purpose of this
procedure was to obtain isolated colonies on a solid medium. Remember that a colony is a
population of millions of cells that are identical and are descended from a single parent cell by
binary fission. An isolated colony is one that is not touching any other colony on an agar plate.
Koch picked cells from a single colony to inoculate another sterile medium to produce a pure
culture. This process presented a powerful new way to develop pure cultures. Streaking for
isolation is now a standard procedure in all microbiology labs.
The steps used in streaking for isolation:
The idea underlying the streaking process is that we want to spread cells out on an agar surface so
that individual cells can grow to form isolated colonies. We’ll do this in three sectors. Sector 1 will
have the most bacterial growth, and the other sectors should have successively less.
Step 1. Necessary equipment is shown in the following picture.
Stock culture = a culture that already contains cells. It is used a source of cells from which to
inoculate new sterile media.
CAUTION:
Lids on test tubes are loose. Always firmly grasp the glass test tube (not the lid)
when carrying the test tubes. REMEMBER, ALWAYS TRANSPORT TEST TUBES IN A TUBE RACK.
Bacticinerator
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Step 2. Label around the perimeter of the bottom of the agar plate (name, date, medium,
inoculum, incubation temperature). Next, draw lines on the bottom of the plate to visually
separate the plate into three sectors. Make sure you do all of this on the agar plate, not
the lid of the Petri dish. Note: Do not write too large because that makes it harder to see
the colonies.
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Step 3. Use aseptic technique (bacticinerator OR Bunsen burner) to transfer cells from the stock
culture to Sector 1 of the agar plate. REMEMBER TO COOL THE LOOP on a sterile portion
of the agar each time you flame it.
a.
Start from one end of the sector and move in a zigzag path to cover the sector
entirely. Do this gently to avoid puncturing the agar.
b. Be careful not to cross the sector lines. See the picture below.
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Step 4. Spread cells from sector 1 into sector 2.
a.
First, sterilize the loop. This will kill all the cells on the loop. This is important
because we want to spread out some of the cells from sector 1 into sector 2. We
do not want to add more cells to the plate.
b. Cool the loop on an uninoculated part of the agar. Now, move the loop back and
forth from sector 1 to sector 2 several (4-5) times. There are now cells on the loop.
Now, spread these cells out into sector 2 using the same zigzag path you used to
inoculate sector 1, without crossing any sector lines again. See the picture here:
Step 5. Repeat step 4 to spread cells from sector 2 to sector 3. See the picture here:
Step 6. Incubate at an appropriate temperature.
DAY ONE ACTIVITIES
Today, EACH PERSON will complete the following activities.
Materials needed: Test tube rack, Bunsen burner OR Bacticinerator, Inoculating loop, any
bacterial stock culture, two sterile NAYE agar plates
Activity 1: Streak for isolation.
1. Follow the steps above to streak two NAYE plates. You may streak with any of the bacterial
stock cultures available, but inoculate the two plates from different stock cultures.
Incubate your inoculations at 30º C.
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DAY TWO OBSERVATIONS
1. Observe your two streaked plates. On each plate, you should see several to many wellisolated colonies.
2. Compare the colonies. Detect and describe any differences in the characteristics of the
colonies on the two plates.
3. Does it appear that the stock cultures you used to streak your plates were pure?
QUESTIONS
1. There’s an old saying, “slow and steady wins the race.” Why is this not necessarily true when
streaking for isolation?
2. What is the goal when streaking for isolation?
3. What might happen if you unintentionally cross a sector line?
4. What happens if you forget to sterilize the loop in between sectors?
5. What happens if you transfer from the stock culture to inoculate each of the sectors?
6. Suppose you successfully streak a plate and obtain isolated colonies. How can you use this
plate to create a new pure culture?
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