Isolation of Pure Culture

Objectives:
Upon completion of the laboratory activity, the students should be able to:
 Demonstrate two standard methods of obtaining a pure culture.
 Stress importance of pure cultures in microbiology.
 Give one practical application.
 Correlate staining with cultural methods.

Introduction:
A pure culture theoretically contains a single bacterial species. There are a number of
procedures available for the isolation of pure cultures from mixed populations. A pure culture
may be isolated by the use of special media with specific chemical or physical agents that allow
the enrichment or selection of one organism over another. The differential and selective
procedures will be utilized later in this course. Simpler methods for isolation of a pure culture
include: (i) spread plating on solid agar medium with a glass spreader and (ii) streak plating with
a loop. The purpose of spread plating and streak plating is to isolate individual bacterial cells
(colony-forming units) on a nutrient medium.

Both procedures (spread plating and streak plating) require understanding of the aseptic
technique. Asepsis can be defined as the absence of infectious microorganisms. However, the
term is usually applied to any technique designed to keep unwanted microorganisms from
contaminating sterile materials.
Materials:
 Nutrient agar in sterile Petri dishes  Sterile cotton swabs
 Nutrient agar in sterile agar slants  Water bath
 Nutrient agar in sterile tubes  Glass slide
containing about 10 mL
 Distilled water
 Sterile Petri dishes
 E. coli & S. aureus, mixed broth
 Sterile culture tubes culture

Procedures:
A. Streak plate method
1. Isolating bacterial colonies
a. Use a mixed broth culture of E. coli & S. aureus
b. Dip the flame sterilized platinum loop into the mixed broth culture. Shake
off excess fluid.
c. Streak surface of the nutrient agar in Petri dish and label.
d. Invert the dish and incubate at 37˚C for 24 hours.

B. Pour Plate
1. Mark the bottom of three sterile Petri dishes. Mark one plate 1:10, 1:100, and
1:1000.
2. Place the three test tubes of melted nutrient agar (about 9 mL each in a beaker
containing hot water.
3. Aseptically transfer 1 mL of turbid broth culture to a tube of melted agar. Mix
well. Using different sterile pipette transfer 1 mL to the second tube. Now
aseptically pour the contents of the first tube in the Petri plate. This is your 1:10
dilution.
4. Get another pipette, transfer 1 mL from the second tube to the third tube. Mix.
Pour contents of the second tube into 1:100 Petri plate. Likewise, pour the
contents of the third tube to the 1:1000 Petri plate.
5. Let the agar solidify, incubate all the plates at 37˚C for 24 hours in an inverted
position.
6. After incubation, count the colonies in the Petri plates.
 Fishing the colonies
a. Select a discrete example of each different kind of colony present in the
series. Use the plate in which bacterial growth is best isolated.
b. Fish the respective example to an agar slant. Label.
c. Observe growth after 24 hours incubate at 37˚C.
d. Make a bacterial smear from each slant, stain by Gram’s method, and
study microscopically.
e. Correlate findings on the slant with those on the Petri dishes.

B. Practical application of pure culture techniques: Throat culture

Note: The instructor will show you how a sterile cotton swab is used to take a throat culture.
The swab after it has been rubbed over the back part of the throat of a given subject is then
passed over the surface of a suitable culture medium

1. Taking the culture

a. pass a sterile cotton swab over the back of the throat.
b. Pass the swab, moistened by throat secretions, over the surface nutrient
agar in Petri dish.
2. Plating the culture
a. Streak the inoculum over the surface of the agar with a flame sterilized
platinum loop according to the method described previously.
b. Invert the dish and inoculate at 37˚C for 24 hours

Safety first
The water, which is used in washing glassware and other materials
not contaminated with chemicals, should be disposed of into the sink.

Food sample must securely wrap in paper/plastic bags and disposed

in design bin.

Disposable gloves must be collected in a container with appropriate

lid cover. The same is done with used disposable masks.
 Report Sheet

Name: JOSHETTE CHEN ANTONIO Date submitted:

Course/Year/Section/Set: BSN 1-L SET B Score:

Documentation of Results:

Streak plate – part A

Appearance Margin Elevation Color
1. The colonies are Entire Flat Milky
intact
2.

3.

4.

Streak plate – part B

Appearance Margin Elevation Color
1.

2.

3.

4.

Throat culture – part C

Appearance Margin Elevation Color
1. The colonies are Entire Flat White
little circles and
shiny in
appearance
2.

3.

4.
Study Questions:

1. Why is aseptic technique so important in the isolation of pure cultures?

Aseptic technique is so important in working in the laboratory, such as

performing the isolation of pure cultures in order to keep unwanted microorganisms from
contaminating sterile materials and the culture itself.

2. Tell how you know from your results that pure cultures were obtained by the
streak plate or the pour plate methods.

From the results given, I would be able to know if the pure culture was obtained
by the streak plate or the pour plate method in a way that the result of the streak plate
method was that the colonies were not spread or intact. They were also clearly visible. While
in the pour plate, there are little circles that are spread, and the 1: 1000 plate has no colonies
formed.

3. Predict what would happen if the plates were incubated for more than a week.

Incubating the plates inappropriately will lead to poor results. If the plates were
incubated for more than a week, the bacteria would die because the already dead bacteria
could release a toxic substance that could affect the other live bacteria. As a result, no
colonies would grow.

4. Give the advantage of the streak plate and the pour plate method.

The advantage of the streak plate method is that it allows for the isolation of
colonies and the obtaining of discrete colonies from a mixed culture, while the pour
plate method counts the number of colonies and or uses it to count the colony-forming
bacteria present in a liquid specimen.