7. Clear with acetic acid and alcohol.

8. Stain and mount as an ordinary cover-slip film preparation, being careful to perform all washing operations with extreme gentleness.

~Microscopical Examination of the Unstained Specimens.~--

1. Place the body tube of the microscope in the vertical position.

2. Arrange the hanging-drop slide on the microscope stage so that the drop of fluid is in the optical axis of the instrument, and secure it in that position by means of the spring clips.

3. Use the 1/6-inch objective, rack down the body tube until the front lens of the objective is almost in contact with the cover-slip--that is, well within its focal distance. This is best done whilst bending down the head to one side of the microscope, so that the eyes are on a level with the stage.

4. Apply the eye to the ocular and adjust the plane mirror to the position which secures the best illumination.

5. Rack the condenser down slightly and cut down the aperture of the iris diaphragm so that the light, although even, is dim.

6. Rack up the body tube by means of the coa.r.s.e adjustment until the bacteria come into view; then focus exactly by means of the fine adjustment.

Some difficulty is often experienced at first in finding the hanging drop, and if the first attempt is unsuccessful, the student must not on any account, whilst still applying his eye to the ocular, rack the body tube down (for by so doing there is every likelihood of the front lens of the objective being forced through the cover-gla.s.s, and not only spoiling the specimen, but also contaminating the objective); but, on the contrary, withdraw his eye, rack the tube up, and commence again from step 2.

~Dark Ground Illumination.~--

1. Set up the microscope stand in the vertical position and insert the highest eyepiece available.

2. Remove the nosepiece from the microscope tube and fit the 2/3 inch objective in place.

3. Remove the substage condenser and replace it by the dark ground condenser.

4. Fit up the source of illumination some 30-50 cm. distant from the microscope. (This should be the Liliput Arc Lamp (Leitz), Nernst Lamp or incandescent gas lamp; if either of the two latter are employed, a bull's eye condenser to produce parallel rays must be interposed between light and microscope); and adjust illuminant and microscope so that the substage plane mirror is completely filled with light.

5. Focus the two concentric rings engraved upon the upper surface of the condenser and centre them accurately by means of the centring screws.

6. Prepare a "fresh" specimen (see pages 74-76) of the material it is desired to observe, using selected, new, 3 by 1 gla.s.s slips of less than 1 mm. thickness, and No. 1 cover-gla.s.ses (0.17 mm. thick), which should be cleaned with a piece of soft washleather and not with the emery paper, as scratches on the gla.s.s produce haziness in the preparation.

7. Deposit a large drop of immersion oil (or pure water) on the upper surface of the condenser and rack it down a few millimetres.

8. Adjust the fresh preparation on the microscope stage and fasten it in position with the stage clips.

9. Rack up the condenser until the immersion fluid makes contact with the under surface of the slide; avoid the formation of air bubbles.

10. Adjust the substage mirror so that the light is reflected upward. A bright spot will be seen on the fresh preparation near the centre of the field.

11. Replace the 2/3-inch objective by the 1/12-inch oil immersion lens which has been fitted with the special stop to reduce its N. A.; place a drop of immersion oil upon the centre of the cover-gla.s.ses of the fresh preparation and lower the microscope tube until the front lens of the objective has entered the oil drop.

12. Focus the bright spot referred to in step 10. If it no longer occupies the centre of the field, alter the angle of the substage mirror until it does.

13. Now focus the lens accurately on the film, cautiously vary the height of the dark ground condenser until the best position is found.

The intensely illuminated bacteria will stand out in vivid contrast to the dark background.

[Ill.u.s.tration: FIG. 70.--Immersion oil bottle.]

~Microscopical Examination of the Stained Specimen.~--(The body tube of the microscope may be vertical or inclined to an angle.)

1. Secure the slide on the stage of the microscope by means of the spring clips.

2. Place a drop of cedarwood oil on the centre of the cover-slip.

The immersion oil is pure cedarwood oil, and is kept in a small bottle of stout gla.s.s (Fig. 70), the cavity of which is shaped like an inverted cone, and is provided with a safety funnel (so that the oil does not escape if the bottle is accidentally overturned) and a dust cap of boxwood fitted with a wooden rod with which the drop of oil is applied to the cover-gla.s.s or lens.

3. Use the 1/12-inch oil immersion lens of the microscope. Rack down the body tube till the front lens of the objective is in contact with the oil and nearly touching the cover-slip.

4. Rack up the condenser until it is in contact with the under surface of the slide.

5. Apply the eye to the ocular and arrange the plane mirror so as to obtain the greatest possible amount of light.

6. Rack up the body tube until the stained film comes into view.

7. Focus the condenser accurately on the film.

8. Focus the film accurately by means of the fine adjustment.

VI. STAINING METHODS.

In the following pages are collected the various "stock" stains in everyday use in the bacteriological laboratory, together with a selection of the most convenient and generally useful staining methods for demonstrating particular structures or differentiating groups of bacteria. The stains employed should either be those prepared by Gruebler, of Leipzig, or Merck, of Darmstadt. The methods printed in ordinary type are those which a long experience has shown to be the most reliable, and to give the best results--those relegated to small type comprise such as are not so generally useful, but give excellent results in the hands of the experienced worker.

BACTERIA STAINS.

~Methylene-blue.~--

1. _Saturated Aqueous Solution._

Weigh out

Methylene-blue 1.5 grammes

Place in a stoppered bottle having a capacity of from 150 to 200 c.c.

and add

Distilled water 100.0 c.c.

Allow the water to remain in contact with the dye for two weeks, shaking the contents of the bottle vigourously for a few moments every day.

Filter.

2. _Saturated Alcoholic Solution._