Universiti Tunku Abdul Rahman (kampar Campus) Faculty Of Science, Engineering,

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.

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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.