3. Sterilise the loop.

4. Hold the slide firmly on the bench with the thumb and forefinger of the left hand applied to the end nearest the drop of fluid.

5. Take another clean 3 by 1 slide in the right hand and lower its short end obliquely (at an angle of about 60) transversely on to the mixed ink and culture on the first slide, and allow the fluid to spread across the slide and fill the angle of incidence.

6. Maintaining the original angle, draw the second slide firmly and evenly along the first toward the end farthest from the left hand (Fig.

69, b).

7. Throw the second slide into a pot of disinfectant; allow the first slide to dry in the air.

[Ill.u.s.tration: FIG. 69.--Spreading negative film.]

8. Place a drop of immersion oil on the centre of the film, lower the 1/12-inch objective into the oil and examine microscopically without the intervention of a cover-slip.

(The film of ink may be covered with a long cover-gla.s.s and xylol balsam as a permanent preparation.)

(

1. Smear a layer of sterile vaseline on the upper surface of the ring cell of a hanging-drop slide by means of the gla.s.s rod provided with the vaseline bottle, and place the slide on a piece of filter paper.

2. "Flame" a cover-slip and place it on the filter paper by the side of the hanging-drop slide.

3. Place a drop of water on the centre of the cover-slip by means of the platinum loop.

4. Obtain a small quant.i.ty of the material it is desired to examine, in the manner detailed above (pages 74-76, steps 2 to 11 must be followed in their entirety and with the strictest exact.i.tude whenever tube contents are being handled), and mix it with the drop of water on the cover-slip.

5. Raise the cover-slip in the points of the forceps and rapidly invert it on to the ring cell of the hanging-drop slide, so that the drop of fluid occupies the centre of the ring. (Carefully avoid contact between the drop of fluid and either the ring cell or the layer of vaseline.

Should this happen, the now _infected_ hanging-drop slide and its cover-slip must be dropped into the pot of lysol and a new preparation made.)

6. Press the cover-slip firmly down into the vaseline on to the top of the ring cell. (This spreads out the vaseline into a thin layer, and besides ensuring the adhesion of the cover-slip, seals the cells and so r.e.t.a.r.ds evaporation.)

7. Examine microscopically.

The examination of a "fresh" specimen or a "hanging-drop" preparation is directed to the determination of the following data:

1. The nature of the bacteria present--e. g., cocci, bacilli, etc.

2. The purity of the cultivation; this can only be determined when gross morphological differences exist between the organisms present.

3. The presence or absence of spores; when present, spores show their typical refrangibility exceedingly well by this method.

4. The presence or absence of mobility. In a hanging-drop specimen some form of movement can practically always be observed, and its character must be carefully determined by noting the relative positions of adjacent micro-organisms.

(a) Brownian or molecular movement. Minute particles of solid matter (including bacteria), when suspended in a fluid, will always show a vibratory movement affecting the entire field, but never altering the relative positions of the bacteria. (Cocci exhibit this movement, but with the exception of the Micrococcus agilis, the cocci are non-motile.)

(b) Streaming movement. This is due to currents set up in the hanging drop as a result of jarring of the specimen or of evaporation, or to the fact that the cover-slip is not perfectly level, and although the relative positions of the bacteria may vary, still the flowing movement of large numbers of organisms in some one direction will usually be sufficient to demonstrate the nature of this motion.

(c) Locomotive movement, or ~true motility~, is determined by observing some one particular bacillus changing its position in the field independently of, and in a direction contrary to, other organisms present.

When the examination is completed and the specimen finished with, the "fresh specimen"--i. e., the slide with the cover-slip attached--must be dropped into the lysol pot. In the hanging-drop specimen, however, the cover-slip only is infected, and this may be raised from the ring cell by means of forceps and dropped into the disinfectant.

_Permanent Staining of the Hanging-drop Specimen._--Occasionally it is necessary to fix and stain a hanging-drop preparation. This may be done as follows:

1. Remove the cover-slip from the cell by the aid of the forceps.

2. If the drop is small, fix it by dropping it face downward, whilst still wet, on to the surface of some Gulland's solution or corrosive sublimate solution (_vide_ page 82) in a watch-gla.s.s. If the drop is large, place it face upward on the rubber mat, cover it with an inverted watch-gla.s.s, and allow it to dry. Then fix it in the alcohol and ether solution (_vide_, page 82).

3. Dip the cover-gla.s.s into a beaker containing hot water in order to remove some of the vaseline adhering to it.

4. Wash successively in alcohol, xylol, ether, and alcohol, to remove the last traces of grease.

5. Wash in water.

6. Stain, wash, dry, and mount as for an ordinary cover-slip film preparation (_vide_ pages 83-85).

~2. Killed, Stained.~--In this method three distinct processes are necessary:

"Preparing" and "fixing" the film.

Staining.

Mounting.

_Preparing the Film._--

1. Flame a cover-slip and place it on a piece of filter paper.

2. Place a drop of water on the centre of the cover-slip by means of platinum loop.

3. Obtain a small quant.i.ty of the material to be examined upon a sterilised platinum loop (see pages 74-76, steps 2 to 11) and mix it with the drops of water on the cover-slip.

4. Spread the drop of emulsion evenly over the cover-slip in the form of a square film to within 1 mm. of each edge of the cover-slip.

5. Allow it to dry completely in the air.

_Fixing._--Fix by pa.s.sing the cover-slip, held in the fingers, three or four times through the flame of a Bunsen burner.

In some instances (e. g., when the films after staining are intended for micrometric observations) it is almost essential to fix by exposure to a uniform temperature of 115 C., for twenty minutes. This is best done in a carefully regulated hot-air oven.

Fixation may also be effected by immersing in some fixative fluid, such as one of the following:

1. Absolute alcohol, for five to fifteen minutes.

{ equal parts, for five to thirty 2. Absolute alcohol, { minutes (e. g., for blood or Ether, { milk).

3. Osmic acid, 1 per cent. aqueous solution, for thirty seconds.

4. Corrosive sublimate, saturated aqueous solution, for five minutes.

5. Corrosive sublimate (Lang), for five minutes. This solution is prepared by dissolving: