which pass through the rest of the plate.

Supposing that the specimen is suspension free fluid,

the only light that reaches the eye is that which goes from

the annulus through the phase plate. Whereas presence

of organisms would diffract and scatter the light. The light

passing through the fluid gets out of phase with the light that

has the organisms stand out in contrast to their background.

Equipment Needed

An annulus, a phase plate and a telescope that is needed

for adjusting the rings of both annulus and the phase plate.

Method

1. Focus the specimen with the right objective after

illuminating the microscope.

2. Place the matching annulus at its position.

3. Remove the eyepiece and put the telescope in its place,

adjust it till the two rings, one bright and one dark are

in focus.

4. Adjust condenser screws till the bright annulus ring

fits exactly into the darker ring of the phase plate.

5. Remove the telescope, replace the eyepiece, focus and

examine the specimen.

Importance

This method is made use of for examining live organisms,

for examaple,

a. Cholera vibrios

b. Amebae

c. Trypanosomes

d. Trichomonas, and

e. Other flagellates.

It can also be used for platelet counting and for examining routine urine specimens.

Demerits

a. A halo is seen around each particle, it gives a false

appearance of its structure.

b. In addition, some resolution power is lost but this is

more than compensated for by the increased contrast

that is produced.

Dark Ground Illumination

This method too, is used for visualizing organisms

suspended in fluid, both the structure and the motility

FIG. 1.10: Working of an oil immersion objective

Laboratory 21

of the organisms can be seen. In this method, the light

enters the special condenser which has a central blackedout area so that light cannot pass directly through it to

enter the objective. Instead the light is reflected to pass

through the outer rim of the condenser at a wide angle

which illuminates the microorganisms by a ring of light

surrounding them (Fig. 1.11).

In this method, the light that is seen comes only from

the microorganisms themselves and not from the light

source. Hence, the organisms are brightly illuminated

against a dark background. Though useful, this method is

rather cumbersome.

Equipment Needed

1. An oil immersion dark ground condenser with the

centering screws.

2. A funnel stop for insertion in 100X objective to reduce

its NA and exclude light coming directly from the

source.

3. A brightly illuminated microscope lamp.

4. Scratchless slides not more than 1 mm thick.

Method

1. Fit the dark ground condenser and raise it to stage level.

2. Place the coverslipped specimen on the thin polished

glass slide. Both, the coverslip and the slide should be

absolutely clean.

3. Place a drop of immersion oil between the condenser

and the slide.

4. Adjust light source and the mirror properly.

5. Focus 10X objective and observe.

6. Focus condenser up or low, so that the ring ultimately

becomes just a spot of light. Focus this spot right in the

center.