Exercise 6 - Pure Culture Isolation

Exercise 6.
Isolation of Bacteria into Pure Culture

Laboratory Techniques in Biological Sciences
Biology Unit-Philippine Science High School-Main Campus
In nature, microorganisms commonly occur as mixed culture. It is necessary to isolate
microorganisms into pure cultures before a species can be characterized. A pure culture
contains only a single species of organisms. Pure culture techniques require that:
1. All apparatus and media must be previously sterilized or freed
from microorganisms.
2. The desired bacterium must be separated from the naturally
occurring microbial populations into the sterile medium, and
3. Aseptic techniques which prevent the entry of containing microorganisms
must be followed.
Materials:
Nutrient agar slant or slope
Nutrient agar in flask
Mixed bacterial culture in broth
Petri dishes
9-ml water blanks
Wire loop
Procedure:
I. Methods of Isolation
A. Streak plate
1. Pour a melted nutrient agar into a dish and allow the agar to solidify.
2. Sterilize the wire loop and get a loopful of culture broth.
3. Streak very gently over the surface of the agar as in figure below. Do not cut into the
agar with the wire loop.
4. Invert the plate and incubate at 30 C for 48 hrs.

5. Describe the result.
B. Pour plate
1.
2.
3.
4.
5.
Liquefy nutrient agar by boiling them in water. Maintain it at 50 C until ready for
pouring.
Obtain three 9-ml sterile water blanks in screw-capped tubes and label each tube from
1 to 3.
By using a pipettor, transfer 1 ml from the stock culture to tube no. 1. Shake the tube 25
times vigorously.
Subsequently, transfer 1 ml from tube 1 to tube 2. Mix as in tube 1.
Repeat the process of transferring and shaking to tube 3.
Exercise 6. Isolation of Bacteria into Pure Culture

6.
Transfer 1 ml from each tube to sterile plates. Pour nutrient agar onto plates and mix
contents gently by following the figure below.
7. Allow the agar to solidify and incubate as directed in A 4.
8. Describe the result.
C. Spread plate
1.
2.
3.
4.
5.
6.
Pour 15-20 ml of melted nutrient agar into each of 3 sterile petri plates and allow the
agar to solidify.
Using a micropipettor, transfer 10 ul from each tube used in pour plate technique.
Sterilize an L-shaped glass rod by dipping in alcohol and flaming until alcohol has
burned off. Cool rod by touching in the uninoculated portion of agar.
Spread the inoculum evenly over the surface of the agar with an L-shaped rod.
Incubate as in A 4.
Describe the result.
Other methods that can be employed in isolation of bacteria are:

1. Enrichment culture
2. Single-cell isolation
3. Serial dilution
4. Selective media (Eosin Methylene Blue Agar or EMBA)
II. Study of Plate Culture
1. For each method of isolation, choose a plate with well-separated colonies.
2. Select two colonies of different types and mark the colonies A and B.
3. Examine each colony under a dissecting microscope. Do not open the plates while
examining the colonies.
4. Based on the description chart (next page), describe the colonies selected.
III. Obtaining Pure Culture
1. Sterilize the wire needle or loop.
2. Raise the cover of the Petri dish slightly and touch the hot wire loop or needle on
the agar without growth to cool the loop.
3. Obtain the marked colonies separately with the wire loop. Replace the dish cover.
4. Remove the plug and flame the mouth of the tube of agar slope.
5. Make a streak from the bottom to the top of the agar, flame the mouth of tube
and return the plug. Sterilize the wire loop.
1. Incubate the agar slope at 30C for 48 hrs.
2. Based on the chart (next page), study the growth on the agar slope and describe.
3. From this agar slope you can make transfers to other culture media.

This lab. exercise was modified from:

FERNANDEZ, W.L., I.F. DALMACIO, A.K. RAYMUNDO, AND A.F. ZAMORA. 1995. Laboratory Manual: General
Microbiology. 2nd ed. University of the Philippines Los Baos (UPLB). Microbiology Laboratory, IBS, CAS, UPLB. 115
pp.
LIST OF REFERENCES (or any microbiology book or microbiology manual)
PRESSCOTT, L.M., J.P. HARLEY and D.A. KLEIN. 1993. Microbiology. 2nd ed. Wm. C.
Brown. Melbourne, Australia. 912 pp.
RAYMUNDO, A.K., A.F. ZAMORA, and I.F. DALMACIO. 1991. Manual on Microbiological
Techniques. TLRC-UPLB. 112 PP.
STAINER, R.Y.,J.L. INGRAHAM, M.L. WHEELIS and G.R. PAINTER. 1986. The Microbial
World. 5th ed. Prentice-hall, N.J. Englewood Cliffs, N.J. 689 pp.
QUESTIONS TO ANSWER
1.Compare the pour plate and the streak plate methods for isolating bacteria.
2.In which plate do you find both the surface and the deep or buried colonies?
3.How do surface colonies differ from deep or buried colonies?
4.Which is more rapid in proving the purity of a culture on an agar or in a broth? Why?
5.Why are Petri dishes incubated in an inverted position?
6.What is a selective media?
7.What is Eosin Methylene Blue Agar?

I. A.
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Observations:
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Observations:
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I.B.
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Observations:
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Observations:
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I.C.
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Observations:
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Observations:
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Observations:
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II. A.
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Observations:
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Observations:
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II.B.
Figure ___. ____________________

Observations:
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Figure ___. ____________________

Observations:
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II.C.
Figure ___. ____________________

Observations:
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Observations:
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Observations:
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III. A.
Figure ___. ____________________

Observations:
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Observations:
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III.B.
Figure ___. ____________________

Observations:
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Figure ___. ____________________

Observations:
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III.C.
Figure ___. ____________________

Observations:
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Figure ___. ____________________

Observations:
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Figure ___. ____________________

Observations:
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Exercise 6.

Isolation of Bacteria into Pure Culture

4. Invert the plate and incubate at 30 C for 48 hrs.

Exercise 6. Isolation of Bacteria into Pure Culture

Other methods that can be employed in isolation of bacteria are:

Exercise 6. Isolation of Bacteria into Pure Culture

This lab. exercise was modified from:

Exercise 6. Isolation of Bacteria into Pure Culture

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Exercise 6. Isolation of Bacteria into Pure Culture

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Exercise 6. Isolation of Bacteria into Pure Culture

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Exercise 6. Isolation of Bacteria into Pure Culture

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Exercise 6. Isolation of Bacteria into Pure Culture

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Exercise 6. Isolation of Bacteria into Pure Culture

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