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This is lab report describing the observation under microscope on bacteria staining with different techniques, include gram staining and endospore staining....
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Universiti Tunku Abdul Rahman (Kampar Campus)
Faculty of Science, Engineering, and Technology
Bachelor of Science (Hons) Biotechnology
Year 2 Semester 1
UESB 2142 Laboratory 2A
(III) Microbiology
Lecturer: Dr. Teh Yok Lan
Student’s Name: Cheah Hong Leong
Student’s ID: 08AIB03788
Experiment No. 2
Title: The Staining of Microorganisms
Date: 8 June 2009
Title: The Staining of Microorganisms
Objectives:
- To learn the technique in preparation of smear for staining.
- To learn the four technique of staining of microorganisms: Basic staining, acidic
staining, differential staining, and endospore staining.
Results:
1. Staining Techniques: Basic Staining
Bacteria: Escherichia coli
Magnification: 10 x 100
Observation: The bacteria cells were appeared purple in color and in rod or
bacillus shape. Most of the bacteria cells appeared in clusters.
2. Staining Techniques: Acidic Staining
Bacteria: Escherichia coli
Magnification: 10 x 100
Observation: At lower magnification, only star-like dots can be viewed against the
dark background. At higher magnification (10 x 100), the bacteria were hardly
observed against the dark background.
3. Staining Techniques: Differential Staining (Gram staining)
Bacteria: Escherichia coli and Micrococcus luteus
Magnification: 10 x 100
Observation: Two kinds of bacteria can be observed. One kind was the pink color
and rod shape bacteria, while another one was the purple color and spherical
shape.
4. Staining Techniques: Endospore Staining
Bacteria: Bacillus cereus
Magnification: 10 x 100
Observation: Bacillus cereus were observed in clustered appeared in two colors:
red and green. Some of the bacteria appeared red in color while some of the
bacteria appeared wholly green in color. There was no bacterial cell that observed
to has green body enclosed in a red colored cell.
Discussion:
Escherichia coli are the bacteria have rod or bacillus shape.
The slide specimen of Escherichia coli, stained with negative stain of nigrosine were
hardly be observed under the light microscope, especially at high magnification. This
might due to the nigrosine stain was put too much on the slide, thus prevent the light
penetration through the slide. In order to view a clearer specimen, only little nigrosine
stain (less than one drop) should be applied on the slide surface, and the stain should be
spread over the whole slide without leaving any part of the slide surface thick with the
nigrosine stain.
From the result of the basic staining, the bacteria that appeared pinkish in color and have
rod shape were identified as the Escherichia coli while another kind of bacteria that were
appeared purple in color and have spherical shape were identified as the Micrococcus
luteus. The pinkish color of Escherichia coli indicates that it is Gram-negative bacteria
that have less peptidoglycan but structurally more complex with additional
lipopolysaccharides membrane layer. The purple color of Micrococcus luteus indicates
that it is Gram-positive bacteria that have cell wall with large amount of peptidoglycan
that traps the crystal violet dye in cytoplasm that cannot be removes by alcohol, thus the
crystal violet masks the added Safranin counter stain.
The red color cells that were observed were actually the bacterial cells of Bacillus cereus
while the green cells were actually the endospores of Bacillus cereus. The expected
observation that the red-stained Bacillus cereus cells would have a green-stained
endospores inside the cells, were not obtained. The possible explanation for this
observation was the endospore formation had been completed and the cells lyses, leaving
the endospores that were stained with Malachite green.
Conclusion:
In staining the microorganisms for observing under the microscope, techniques and
prevention steps of smear preparation, staining, as well as techniques of using microscope
are important in order to obtain a clear observation of the microorganisms.
References:
Campbell, N. A., Reece, J. B. (2005). Biology, 7th ed., San Francisco, CA: Pearson
Benjamin Cummings.
Madigan, M. T., Martinko, J. M., Dunlap, P. V., & Clark, D. P. (2009). Brock Biology of
Microorganisms, 12th ed., San Francisco, CA: Pearson Benjamin Cummings.
Whitman, K. A., Macnair, N. G. (2004). Finfish and Shellfish bacteriology Manual.
Techniques and Procedures, 1st ed., Danvers, MA: Blackwell Publishing Company.