med 18

 

7. Use 40X objective; if needed, use the 100X oil

immersion by inserting the funnel stop into it.

Demerits

1. Focusing and/or centering of condenser is difficult as

is the alignment of the lamp.

2. Difficulties may arise under the following circumstances:

• Smear traces on the slide or coverslip

• If the specimen is dense

• A bubble is present in the immersion oil

• Insufficient oil contact below or above the slide.

Importance

1. This method is of particular importance for the

examination of Treponema group of organisms.

2. It can also be of use for microfilariae, for the sheath

of the pathogenic forms can be clearly seen which

otherwise needs to be stained.

3. For examining the rapid movement of Vibrio cholerae.

4. In addition, this method can be used for:

• Leptospira

• Borrelia, and

• Spirillum species.

• The ideal objective for dark ground illumination

is the 50X fluorite as this lens gives a clear, sharp

and a well-illuminated image.

Fluorescence Microscopy

This method entails the illumination of particles/microorganisms (previously stained with a fluorescent dye) with

ultraviolet (UV) light into visible light (yellow or orange),

by lengthening their wavelength. This procedure is made

use of for visualizing, besides other things, mycobacteria

glowing against a black background.

All other wavelengths emitted by the lamp except the

ultraviolet (UV) are to be filtered off (by using appropriate

optical filters) and no harmful rays of UV light should reach

the observer’s eye (by using an immersion dark ground

condenser as described for previous method). Again,

another filter is used to remove all unwanted fluorescent

light by placing a secondary or a barrier filter above the

eyepiece (Fig. 1.12).

Equipment Needed

1. A fluorescent lamp (mercury vapor or quartz iodine, the

latter is better, being cheaper, lighter and easier to use).

2. A blue (primary or exciting) filter, generally a BG 12.

3. A yellow (secondary or barrier) filter.

4. An immersion dark ground condenser.

5. A nonfluorescent immersion oil, e.g. liquid paraffin.