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expt 8-revised

The document outlines Experiment No. 7 for an Introduction to Food Microbiology course, focusing on the isolation of microorganisms using streak-plate and spread-plate techniques. It details the objectives, materials, procedures for both techniques, and includes sections for data collection and key questions regarding the methods. The experiment aims to help students understand and perform these techniques to isolate pure cultures from mixed populations.

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Jerna Mae Pabilona
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14 views

expt 8-revised

The document outlines Experiment No. 7 for an Introduction to Food Microbiology course, focusing on the isolation of microorganisms using streak-plate and spread-plate techniques. It details the objectives, materials, procedures for both techniques, and includes sections for data collection and key questions regarding the methods. The experiment aims to help students understand and perform these techniques to isolate pure cultures from mixed populations.

Uploaded by

Jerna Mae Pabilona
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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FT 15: Introduction to Food Microbiology

Second Semester, AY 2013-2014

Experiment No. 7
ISOLATION OF MICROORGANISMS:
STREAK-PLATE AND SPREAD-PLATE TECHNIQUES

In natural habitats, microorganisms commonly occur as mixed culture. It is necessary to isolate


microorganisms into pure cultures before a species can be characterized. The spread-plate
technique is an easy, direct way of achieving this result. After incubation, the general form of the
colony and the shape of the edge or margin can be determined by looking down at the top of the
colony. After a well-isolated colony has been identified, it can then be picked up and streaked onto a
fresh medium to obtain a pure culture.

Isolated, pure colonies can also be obtained by the streak-plate technique. At some point on the
streaks, individual cells will be removed from the loop as it glides along the agar, and will give rise to
separate colonies. The key principle of this method is that by streaking, a dilution gradient is
established on the surface of the plate as cells are deposited on the agar surface. Because of this
gradient, confluent growth occurs on part of the plate where the cells are not sufficiently separated,
and individual, well-isolated colonies develop in other regions of the plate where few enough cells
are deposited to form separate colonies that can be seen with the naked eye.

Objectives:
1. To understand the purpose of the spread-plate and the streak-plate techniques
2. To perform above stated techniques

Materials and Equipment:


Broth culture of E. coli or mixed bacterial culture alcohol lamp
Wire loop
95% ethyl alcohol
L-shaped glass rod
500 ml beaker
12-15 petri dishes (depending on the number of group members)
4-5 1 ml pipettes (depending on the number of group members)

Procedures:
I. Glassware Preparation and Sterilization
Wash, rinse and air-dry all glassware to be used in the experiment. Sterilize using the autoclave.

II. Plating Techniques

A. Spread-Plate Technique
1. Pour 15-20 ml of melted PCA into each of the 2 sterile petri dishes and allow the agar to
solidify.
2. Pipette 0.1 ml of the bacterial culture onto the center of the PCA plate.
3. Dip the L-shaped glass rod into a beaker of ethanol and then tap the rod on the side of
the beaker to remove any excess ethanol.
4. Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol, and
allow it to cool by touching in the uninoculated portion of agar surface.
5. Spread the bacterial sample evenly over the agar surface with the sterilized spreader,
making sure the entire surface of the plate has been covered without touching the edge
of the plate.
6. Immerse the spreader in ethanol, tap on the side of the beaker to remove any excess
ethanol, and reflame.
7. Repeat the procedure to inoculate the 2nd plate.
8. Invert the plates and incubate for 24 to 48 hours at 30°C.

B. Streak-Plate Technique
1. Pour 15-20 ml of melted PCA into a sterile petri dish and allow the agar to solidify.
2. Sterilize the wire loop and get a loopful of the culture broth.
3. Streak very gently over the surface of the agar as follows:
a. Carefully lift the top of the petri dish just enough to insert the wire loop. The top
should cover the agar surface as completely as possible at all times in order to avoid
contamination. Insert a loopful of sample and spread it over a small area at one edge
of the plate and move the wire loop across the surface without cutting the agar.
b. Remove the wire loop and kill any remaining bacteria by flaming them. Then insert
the loop under the lid and cool it at the uninoculated surface of the agar.
c. Rotate the plate while carefully keeping in mind where the initial streaks ended and
cross over the streaks in area 1. Streak out any bacteria picked up as shown in area
2.
d. Remove the loop, flame it, cool in the agar as before, and repeat the streaking
process.

2
3

4. Invert the plates and incubate for 24 to 48 hours at 30°C.

Data and Results:


a. Examine each of the agar plates for colony distribution and amount of growth. Look for
discrete surface colonies as well as subsurface colonies and record your results. Refer to
next page for bacterial colony distribution. Include as well the color of the colonies.
b. Draw your streaking pattern. Did you obtain isolated colonies? If not, what went wrong?

Key Questions:
1. State the advantages and disadvantages of spread plate technique in the isolation of the
microorganisms.
2. What is the purpose of the ethanol in the spread-plate technique?
3. In the streak-plate technique, how are microorganisms diluted and spread out to form
individual colonies?
4. Which area of a streak plate will contain the greatest amount of growth? The least amount
of growth? Explain your answers.
5. How can a streak plate become contaminated?

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