SIR 1001

ACADEMIC SESSION 2, 2021/2022

LAB REPORT 1
Introduction to microscope, smear preparation and simple stains
techniques

NAME : QAISARA ARISSA MOHAMMED NAJIB

STUDENT ID : U2103586
 PRACTICAL 1: Introduction to microscope, smear preparation and simple
stains techniques

1.1 Introduction

Microbiology is a branch of biology that deals with microorganisms and their effect on
living organisms. Microbes are very small organisms which can only be viewed with the aid of
microscope. The type of microscope used in most courses is a bright field, binocular, compound
microscope. It is a bright field because it projects bright light through the image on the slide it is
binocular because you can use both eyes to view the object, and it is compound because it uses
a series of lenses to achieve magnifications of up to 1000 times.

In this experiment, four different bacterial cultures were used for smear preparation and

simple staining, which includes Escherichia coli, Staphylococcus aureus, Pseudomonas

aeruginosa and Bacillus megaterium.

Escherichia coli is a gram-negative facultative, anaerobic, and rod-shaped bacterium.

Escherichia coli are usually motile by flagella and produce gas from fermentable carbohydrates.
E. coli is also the most common member of the normal flora of the human intestinal tract.
Because E. coli occur naturally in the gut, this bacterium causes the infection is by the faucal–
oral route. This is also why this bacterium is common in food poisoning and why we test for E.
coli.

Staphylococcus aureus is a gram-positive, round-shaped bacterium and is a usual member

of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It
is often positive for catalase and nitrate reduction and is a facultative anaerobe that can grow
without the need for oxygen. Although S. aureus usually acts as a commensal of the human
microbiota it can also become an opportunistic pathogen, which can cause abscesses and food
poisoning.
 Pseudomonas aeruginosa is a gram-negative, aerobic rod bacterium of the
Pseudomonadaceae family. P. aeruginosa is commonly found in soil and water as well as in
plants and humans. Importantly, P. aeruginosa has become an emerging opportunistic
pathogen in the clinics. Recent epidemiological studies demonstrate its nosocomial pathogen
status, particularly those strains with increased antibiotic resistance.

Bacillus megaterium is a rod-like, Gram-positive, mainly aerobic spore forming bacterium

found in widely diverse habitats. With a cell length of up to 4 µm and a diameter of 1.5 µm, B.
megaterium is amongst the biggest known bacteria. The cells often occur in pairs and chains,
where the cells are joined together by polysaccharides on the cell walls.
1.2 Objective

1. Demonstrate the appropriate procedures used while using the compound light
microscope correctly.
2. Prepare a proper bacterial smear.
3. Properly stain and observe microbes using a simple dye.
4. Differentiate between different cell shapes.

1.3 Materials and Procedure

Materials:

• Light compound microscope

• Bacterial cultures (Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa)
• Glass slides
• Distilled water
• Inoculation loop
• Bunsen burner
• Staining tray and kit
• Bibulous paper
• Lens tissue
• Cover slip
• Immersion oil
Procedure:

1. A clean glass slide was labelled E. coli using a marker.

2. Thin bacterial smears on clean glass slides were prepared.

3. The smears were heated fix carefully without overheating the slides.

4. The specimen was cooled and is ready for the simple stain.

5. The heat fixed slide was placed on the staining tray.

6. Smear was covered completely with crystal violet or methylene blue and was allowed to sit
for 30 sec (crystal violet) or 1-2 minutes (methylene blue).

7. Slide was tilted slightly and rinsed off excess stain gently with distilled water using a wash
bottle.

8. The slide was then gently blotted with bibulous paper. Excess water was removed from the
slide by touching one corner of the slide to the blotting paper, the slide was then placed
between clean sheets of paper in the blotting pad and was blotted dry.

9. Slide was then examined under the microscope and observation was recorded.

10. Steps 1-9 was repeated by replacing E. coli with S. aureus, P. aeruginosa and B.

Megaterium.
1.4 Results

Image 1: Escherichia coli with 100x magnification.

Image 2: Pseudomonas aeruginosa with 100x magnification.

Image 3: Staphylococcus aureus with 100x magnification.

Image 4: Bacillus megaterium with 100x magnification.

1.5 Discussion

From the observation above, E. coli are rod-shaped bacteria with rounded ends. S. aureus is
spherical in shape, and they appear in cluster, resembling a bunch of grapes. P. aeruginosa can
be seen as rod-shaped and flagellated bacterium. B. megaterium is rod-shaped and it is a spore
forming bacterium.

1.6 Precautions

1. All the bacterial smears must be heat-fixed.

2. Avoid cross contamination during preparation of the smear to ensure an accurate result.
3. Wash the stained slide gently with tap water by holding it parallel to water stream to avoid
loss of cells.
4. Use clean, grease free glass slides.
1.7 Conclusion

These results are accurate figuring out they are stained correctly. Finding their correct shape
and charge are exact as the actual results. Simple staining is necessary because the naked eye is
unable to view these microbes, viewing under a microscope is helpful. Although they are from
distinct genus, E. coli, S. aureus, Pseudomonas, and B. megaterium all have rod-shaped
morphology. However, all four specimens belong to the same kingdom, that of bacteria.

1.8 References

1. Tortora, G. J., Funke, B. R., & Case, C. L. (2013). Microbiology: An

Introduction (11th ed.). Boston: Pearson.
2. Mandal, Ananya. “Staphylococcus Aureus Microbiology.” News, 5 June
2019, www.news-medical.net/health/Staphylococcus-Aureus-
Microbiology.aspx.
3. Anderson, D. G., Salm, S., Allen, D., & Nester, E. W. (2016). Nester’s
microbiology: a human perspective. New York, NY: McGraw-Hill
Education.
4. Maxwell, R. (2017). Lab 2: Biological Stains. GSU iCollege. Retrieved on
September 17,2017 from http://gsu.edu/lab2.pdf.
5. Wikipedia contributors. (2022, March 17). Escherichia coli. Wikipedia.
https://en.wikipedia.org/wiki/Escherichia_coli