The Elements of Bacteriological Technique Part 106

Pota.s.sium acetate 160 grammes.

Sodium fluoride 80 grammes.

Chloral hydrate 80 grammes.

Cane sugar (Tate's cubes) 3,500 grammes.

Saturated thymol water 8,000 c.c.

6. After twenty-four hours in this solution, or as soon as the tissue sinks, transfer to a museum jar, fill with fresh mounting solution, and seal.

_6a._ Or transfer to museum jar and fill with liquefied gelatine, to which has been added 1 per cent. formalin. Cover the jar and allow the gelatine to set. When solid, seal the cover of the jar in place.

7. To seal the museum preparation first warm the gla.s.s plate which forms the cover. This is most conveniently done by placing the cleaned and polished cover-plate upon a piece of asbestos millboard over a bunsen flame turned low.

8. Smear an even layer of hot cement over the f.l.a.n.g.e of the jar. The cement is prepared as follows:

Weigh out and mix in an iron ladle

Gutta percha (pure) 4 parts.

Asphaltum 5 parts.

and melt together over a bunsen flame, stirring with an iron rod until solution is complete.

9. Invert the gla.s.s plate over the jar and press down firmly into the cement. Place a piece of asbestos board on the top and on that rest a suitable weight until the cement is cold and has thoroughly set.

10. Trim off any projecting pieces of cement with an old knife, burr over the joint between jar and cover-plate with a hot smooth piece of metal (e. g., the searing iron).

11. Paint a narrow band of j.a.pan black to finish off, round the joint, overlapping on to the cover-plate.

_II. Tube Cultivations of Bacteria._--When showing typical appearances these may be preserved, if not permanently, at least for many years, as museum specimens, by the following method:

1. Take a large gla.s.s jar 25 cm. high by 18 cm. diameter, with a firm base and a broad f.l.a.n.g.e, carefully ground, around the mouth. The jar must be fitted with a disc of plate gla.s.s ground on one side, to serve as a lid.

2. Smear a thick layer of resin ointment (B.P.) on the f.l.a.n.g.e around the mouth of the jar.

3. Cover the bottom of the jar with a layer of cotton-wool and saturate it with formalin.

4. Remove the cotton-wool plug from the culture tubes and place them, mouth upward, inside the jar. (If water of condensation is present in any of the culture tubes, it should be removed by means of a capillary pipette before placing the tubes in the formalin chamber.)

5. Adjust the gla.s.s disc, ground side downward, over the mouth of the jar and secure it by pressing it firmly down into the ointment, with a rotary movement.

6. Remove the tubes from the formalin chamber after the lapse of a week, and dry the exterior of each.

[Ill.u.s.tration: FIG. 202.--Bulloch's tubes.]

7. Seal the open mouth of each tube in the blowpipe flame and label.

If the cultivations are intended for museum purposes when they are first planted, it is more convenient to employ Bulloch's tubes. These are slightly longer than the ordinary tubes, and are provided with a constriction some 2 cm. below the mouth (Fig. 202)--a feature which renders sealing in the blowpipe flame an easy matter.

XX. THE STUDY OF THE PATHOGENIC BACTERIA.

The student, who has conscientiously worked out the methods, etc., previously dealt with, is in a position to make accurate observations and to write precise descriptions of the results of such observations.

He is, therefore, now entrusted with pure cultivations of the various pathogenic bacteria, in order that he may study the life-history of each and record the results of his own observations--to be subsequently corrected or amplified by the demonstrator. In this way he is rendered independent of text-book descriptions, the statements in which he is otherwise too liable to take for granted, without personally attempting to verify their accuracy.

During the course of this work attention must also be directed, as occasion arises, to such other bacteria, pathogenic or saprophytic, as are allied to the particular organisms under observation, or so resemble them as to become possible sources of error, by working them through on parallel lines--in other words the various bacteria should be studied in "groups." In the following pages the grouping in use in the author's elementary cla.s.ses for medical and dental students and for candidates for the Public Health service is adopted, since a fairly long experience has completely vindicated the value and utility of this arrangement, and by its means a fund of information is obtained with regard to the resemblances and differences, morphological and cultural, of a large number of bacteria. The fact that some bacteria appear in more than one of these groups, so far from being a disadvantage, is a positive gain to the student, since with repet.i.tion alone will the necessary familiarity with the cultural characters of important bacteria be acquired. The study of the various groups will of course vary in detail with individual demonstrators, and with the student's requirements--the general line it should take is indicated briefly in connection with the first group only (pages 410-411). This section should be carefully worked through before the student proceeds to the study of bacterioscopical a.n.a.lysis.

It is customary to commence the study of the pathogenic bacteria with the Organisms of Suppuration. This is a large group, for all the pathogenic bacteria possess the power, under certain conditions, of initiating purely pyogenic processes in place of or in addition to their specific lesions, (e. g., Bacillus tuberculosis, Streptococcus lanceolatus, Bacillus typhosus, etc.). There are, however, a certain few organisms which commonly express their pathogenicity in the formation of pus. These are usually grouped together under the t.i.tle of "pyogenic bacteria," as distinct from those which only occasionally exercise a pyogenic role.

The organisms included in this group are:

1. Staphylococcus pyogenes albus.

2. Staphylococcus pyogenes aureus.

3. Staphylococcus pyogenes citreus.

4. Streptococcus pyogenes longus.

5. Micrococcus tetragenus.

6. Bacillus pyocyaneus.

7. Bacillus pneumoniae.

and in certain special tissues

8. Micrococcus gonorrhoeae.

9. Micrococcus intracellularis meningitidis (Meningococcus).

10. Micrococcus catarrhalis.

11. Bacillus aegypticus (Koch-Weeks Bacillus).

The group may with advantage be subdivided as indicated in the following pages:

I. _Pyogenic cocci._

Staphylococcus pyogenes albus.

Staphylococcus pyogenes aureus.

Staphylococcus pyogenes citreus.

to contrast with Micrococcus candicans.

Micrococcus agilis.

1. Prepare subcultivations from each:

Bouillon, } Agar streak, } Blood serum, } Litmus milk. } and incubate at 37C.

Agar streak, } Gelatine stab, } Potato. } and incubate at 20C.

Compare the naked-eye appearances of the cultures from day to day. Note M. agilis refuses to grow at 37C.

2. Make hanging-drop preparations from the bouillon and agar cultivations after twenty-four hours' incubation. Examine microscopically and compare. Note the locomotive activity of M. agilis and the Brownian movement of the remaining micrococci.

3. Prepare cover-slip films from the agar cultures, after twenty-four hours' incubation. Stain for flagella by the modified Pitfield's method.

Note M. agilis is the only micrococcus showing flagella.

4. Make microscopical preparations of each from all the various media after twenty-four and forty-eight hours and three days' incubation.