Light Microscopy

 

Light Microscopy for Observing Bacterial Morphology Using Permanent Slides

 

Objective:

To observe and identify the morphological characteristics of bacteria (such as shape and arrangement) using a compound light microscope and permanent stained slides.

 Principle:

Light microscopy uses visible light to magnify images of small specimens. In bacterial studies, staining techniques (e.g., Gram staining) enhance contrast, allowing for better observation of morphology—such as coccus, bacillus, spirillum, and their arrangements (e.g., diplo-, strepto-, staphylo-).

 Materials Required:

·         Compound light microscope

·         Permanent bacterial slides

·         Immersion oil (for 100× objective)

·         Lens cleaning tissue

·         Microscope slide box

·         Blotting paper or tissue

 Procedure:

1.      Place the microscope on a flat, stable surface.

2.      Clean all lenses gently with lens tissue before use.

3.      Turn on the light source and adjust the mirror or condenser if using a manual light source.

4.      Select a permanent slide of a stained bacterial specimen.

5.      Place the slide on the stage and secure it with stage clips.

6.      Start with the low power objective (10×) to locate the specimen. Use coarse focus to bring it into view.

7.      Switch to high power (40×) for a closer look. Adjust fine focus to sharpen the image.

8.      For detailed morphology, switch to the oil immersion lens (100×)

9.      Add a drop of immersion oil directly onto the cover slip, carefully rotate the 100 × objectives into place, and Use only fine focus to view the specimen clearly.

10.  Note the shape of the bacteria: cocci (spherical), bacilli (rod-shaped), spirilla (spiral). Observe the arrangement: pairs, chains, clusters, etc. Note the color/stain uptake to infer possible Gram characteristics.

Observation Table Example:

Slide No.	Bacterial Type	Shape	Arrangement	Staining Color	Likely Group
1	Bacillus subtilis	Rod	Chains	Purple	Gram-positive
2	Staphylococcus spp.	Cocci	Clusters	Purple	Gram-positive
3	E. coli	Rod	Single/paired	Pink	Gram-negative
4	Spirillum spp.	Spiral	Single	Pink	Gram-negative

Result:

Different bacterial species were observed with distinct shapes and arrangements. Gram-positive bacteria appeared purple, and Gram-negative bacteria appeared pink, due to their cell wall composition.

Conclusion:

The light microscope is a valuable tool to study bacterial morphology. Observing permanent stained slides allows clear identification of structural differences between bacterial species.

Precautions:

·         Always begin with the lowest objective lens.

·         Use only fine focus at high magnification (especially 100×).

·         Clean oil immersion lens immediately after use to avoid residue buildup.

·         Do not use immersion oil on 40× or lower lenses.

·         Handle permanent slides with care to avoid scratches or breakage.