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.