Chapter IX: Appendix: 492 (7)

In plate No. 1, probably, the colonies will be so numerous and crowded, and therefore so small, as to render it useless. In plate No. 2 they will be more widely separated, but usually No. 3 is the plate reserved for careful examination, as in this the colonies are usually widely separated, few in number, and large in size.

_Agar plates_ are poured in a similar manner, but the agar must be melted in boiling water and then allowed to cool to 45 deg. C. or 42 deg. C. in a carefully regulated water-bath before being inoculated, and the entire process must be carried out very rapidly, otherwise the agar will have solidified before the operation is completed.

NOTE.--In pouring plates, since tube No. 1 (for the first
dilution) rarely gives a plate that is of any practical
value it is frequently replaced by a tube of bouillon or
sterile salt solution, and in such case plate No. 1 is not
poured.

~Surface Plates.~--

This method of pouring what may be termed "whole" plates (since colonies may appear both on the surface and in the depths of the medium) is essential to the accurate study of the formation of colonies under various conditions, but when the main object of the separation of the bacteria is to obtain subcultivations from a number of individual bacteria, "surface" plates must be prepared, since here colony formation is restricted to the surface of the medium. The method adopted varies slightly according to whether the medium employed is gelatine or agar, or one of the derivatives or variants of the latter.

(a) ~Gelatine Surface Plates.~--

1. Liquefy three tubes of nutrient gelatine.

2. Pour each tube into a separate Petri dish and allow it to solidify. Then turn each plate and its cover upside down.

3. When quite cold raise the bottom of plate 1, revert it and deposit a drop of the inoculum (whether a fluid culture or an emulsion from solid culture) upon the surface of the gelatine with a platinum loop--close to one side of the plate; replace the bottom half of the Petri dish in its cover.

4. Take a piece of thin glass rod, stout platinum wire or best of all a piece of aluminium wire (say 2 mm. diameter) about 28 cm. long. Bend the terminal 4 cm. at right angles to the remainder, making an L-shaped rod (Fig. 126). Sterilise the short arm and adjacent portion of the long arm, in the Bunsen flame, and allow it to cool.

5. Now raise the bottom of the Petri dish in the left hand, leaving the cover on the laboratory bench, and holding it vertically, smear the drop of inoculum all over the surface of the gelatine with the short arm of the spreader by a rotatory motion, (Fig. 127). Replace the dish in its cover.

6. Raise the bottom of plate 2 and rub the infected spreader all over the surface of the gelatine--then go on in like manner to the third plate in the series.

7. Sterilise the spreader.

8. Label and incubate the plates.

After incubation, plate No. 1 will probably yield an enormous number of colonies; plate 2 will show fewer colonies, since only those bacteria adhering to the rod after rubbing over plate 1 would be deposited on its surface, and by the time the rod reached plate 3 but very few organisms should remain upon it. So that the third plate as a rule will only show a very few scattered colonies, eminently suitable for detailed study.

(b) ~Agar Surface Plates.~--

1. Liquefy three tubes of nutrient agar--nutrose agar or the like.

2. Pour each tube into a separate Petri dish and allow it to solidify.

3. When quite solid invert each dish, raise the bottom half and rest it obliquely on its inverted cover (Fig. 128) and place it in this position in an incubator at 60 deg. C. for forty-five minutes (or in an incubator at 42 deg. C. for two hours). This evaporates the water of condensation and gives the medium a firm, dry surface.

4. On removing the plates from the incubator close each dish and place it--still upside down--on the laboratory bench.

5. Inoculate the plates in series of three, as described for gelatine surface plates 3-8.

Hanging-drop Cultivation.

~Apparatus Required.~--

Hanging-drop slides.
Cover-slips.
Section rack (Fig. 75).
Blotting paper.
Bell glass to cover slides.
Original culture.
Tubes of broth, or liquefied gelatine or agar.
Forceps.
Platinum loop.
Bunsen burner.
Grease pencil.
Sterile vaseline.
Lysol.

(a) ~Fluid Media.~--

1. Prepare first and second dilutions of the inoculum as directed for plate cultivations (_vide_ pages 228-229, sections 4 to 6), substituting tubes of nutrient broth for the liquefied gelatine.

2. Sterilise a hanging-drop slide by washing thoroughly in water and drying, then plunging it into a beaker of absolute alcohol, draining off the greater part of the spirit, grasping the slide in a pair of forceps, and burning off the remainder of the alcohol in the flame.

3. Place the hanging-drop slide on a piece of blotting paper moistened with 2 per cent. lysol solution and cover it with a small bell glass that has been rinsed out with the same solution and _not dried_.

4. Raise the bell glass slightly and smear sterile vaseline around the rim of the metal cell by means of a sterile spatula of stout platinum wire.

5. Remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the alcohol; again raise the bell glass and place the sterile cover-slip on the blotting paper by the side of the hanging-drop slide.

6. Remove a drop of the broth from the second dilution tube with a large platinum loop; raise the bell glass and deposit the drop on the centre of the cover-slip. Sterilise the loop.

7. Raise the bell glass sufficiently to allow of the cover-slip being grasped with forceps, inverted, and adjusted over the cell of the hanging-drop slide. Remove the bell glass altogether and press the cover-slip firmly on to the cell.

8. Either incubate and examine at definite intervals, or observe continuously with the microscope, using a warm stage if necessary (Fig. 53).

(b) ~Solid Media.~--Observing precisely similar technique, a few drops of liquefied gelatine or agar from the second dilution tube may be run over the surface of the sterile cover-slip and a hanging-drop plate cultivation thereby prepared.

This method is extremely useful in connection with the study of yeasts, when the circular cell on the hanging-drop slide should be replaced by a rectangular cell some 38 by 19 mm., and the gelatine spread over a cover-slip of similar size. After sealing down the preparation, the upper surface of the cover-slip may be ruled into squares by the aid of the grease pencil or a writing diamond and numbered to facilitate the subsequent identification of the colonies which are observed to develop from solitary germs.

~Hanging-block Culture~ (Hill).--

_Apparatus required_: As for hanging-drop cultivation with the addition of a scalpel.

Carry out the method as far as possible under cover of a bell glass, to avoid aerial contamination.

1. Liquefy a tube of nutrient agar (or gelatine) and pour into a Petri dish to the depth of about 4 mm. and allow to set.

2. With a sharp scalpel cut out a block some 8 mm. square, from the entire thickness of the agar layer.

3. Raise the agar block on the blade of the scalpel and transfer it, under side down, to the centre of a sterile slide.

4. Spread a drop of fluid cultivation (or an emulsion of growth from a solid medium) over the upper surface of the agar block as if making a cover-slip film.

5. Place the slide and block covered by the bell glass in the incubator at 37 deg. C. for ten minutes to dry slightly.

6. Take a clean dry sterile cover-slip in a pair of forceps, and with the help of a second pair of forceps lower it carefully on the inoculated surface of the agar (avoiding air bubbles), so as to leave a clear margin of cover-slip overlapping the agar block.

7. Invert the preparation and with the blade of the scalpel remove the slide from the agar block.

8. With a platinum loop run a drop or two of melted agar around the edges of the block. This solidifies at once and seals the block to the cover-slip.

9. Prepare a sterile hanging-drop slide, and smear hard vaseline or melted white wax on the rim of the metal cell.

10. Invert the cover-slip with the block attached on to the hanging-drop slide, and seal the cover-slip firmly in place.

11. Observe as for hanging-drop cultivations.

ANAEROBIC CULTIVATIONS.

Numerous methods have been devised for the cultivation of anaerobic bacteria, the majority requiring the employment of special apparatus. The principle upon which any method is based and upon which it depends for its success falls under one or another of the following headings:

(a) ~Exclusion of air~ from the cultivation.

(b) ~Exhaustion of air~ from the vessel containing the cultivation by means of an air pump--i. e., cultivation _in vacuo_.

(c) ~Absorption of oxygen~ from the air in contact with the cultivation by means of pyrogallic acid rendered alkaline with caustic soda--i. e., cultivation in an atmosphere of nitrogen.

(d) ~Displacement of air~ by an indifferent gas, such as hydrogen or coal gas--i. e., cultivation in an atmosphere of hydrogen.

(e) A combination of two or more of the above methods.

A selection of the simplest and most generally useful methods is given here.

Whenever possible, the nutrient media that are employed in any of the processes should contain some easily oxidisable substance, such as sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 per cent.), which will absorb all the available oxygen held in solution by the medium. The further addition of glucose, 2 per cent., favors the growth of anaerobic bacteria (_vide_, pages 189-190).

Further, it is advisable to seal all joints between india-rubber stoppers and tubulures or the mouths of the tubes with melted paraffin; glass stoppers and taps should be lubricated with resin ointment or a mixture of beeswax 1 part, olive oil 4 parts.

(A) ~Method I~ (Hesse's Method).--

1. Make a stab culture in gelatine or agar, choosing for the purpose a straight tube containing a deep column of medium, and thrusting the inoculating needle to the bottom of the tube.

2. Pour a layer of sterilised oil (olive oil, vaseline, or petroleum), 1 or 2 cm. deep, upon the surface of the medium.

3. Incubate.

~Method II.~--This method is only available when dealing with pure cultivations.

1. Liquefy a tube of gelatine (or agar) by heat, pour it into a Petri dish, and allow it to solidify.

2. Inoculate the surface of the medium in one spot only.

3. Remove a cover-slip from the pot of absolute alcohol with sterile forceps; burn off the alcohol in the gas flame.

4. Lower the now sterile cover-slip carefully on to the inoculated surface of the medium, carefully excluding air bubbles, and press it down firmly with the points of the forceps. (A sterile disc of mica may be substituted for the cover-slip.)

5. Incubate.

~Method III~ (Roux's Physical Method).--

1. Prepare tube cultures of fluid media (or solid media rendered fluid by heat) in the usual way.

2. Aspirate some of the inoculated media into capillary pipettes.

3. Seal both ends of each pipette in the blowpipe flame.

4. Incubate.

~Method IV~ (Roux's Biological Method).--

1. Plant a deep stab, as in method I.

2. Pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous aerobe--e. g., B. aquatilis sulcatus or B. prodigiosus--upon the surface of the medium; or an equal depth of liquefied gelatine, which is then inoculated with the aerobic organism.

3. Incubate.

The growth of the aerobe will use up all the oxygen that reaches it and will not allow any to pass through to the medium below, which will consequently remain in an anaerobic condition.

(B) ~Method V.~--

1. Prepare tube or flask cultivations in the usual way.

2. Replace the cotton-wool plug by an india-rubber stopper perforated with one hole and fitted with a length of glass tubing which has a constriction about 3 cm. above the stopper and is then bent at right angles (Fig. 129). The stopper and glass tubing are sterilised by being boiled in a beaker of water for five minutes.

3. Connect the tube leading from the culture vessel with a water or air pump, interposing a Wulff's bottle fitted as a wash-bottle and containing sulphuric acid.

4. Exhaust the air from the culture vessel.

5. Before disconnecting the apparatus, seal the glass tube from the culture vessel at the constriction, using the blowpipe flame.

6. Incubate.

(C) ~Method VI~ (Buchner's Method).

~Apparatus and Solutions Required.~--

Buchner's tube (a stout glass test-tube 23 cm. long and 4
cm. in diameter, fitted with india-rubber stopper, Fig.
130).

Pyrogallic acid in compressed tablets each containing 1
gram.

Dekanormal solution of caustic soda.

METHOD.--

1. Prepare the tube cultivation in the usual way.

2. Moisten the india-rubber stopper of the Buchner's tube with water and see that it fits the mouth of the tube accurately.

3. Remove the stopper from the caustic soda bottle.

4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube (roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity of the receiving vessel).

5. Add about 1 c.c. of the soda solution.

6. Place the inoculated tube inside the Buchner's tube. The pyrogallic tablet acts as a buffer and prevents damage to either the inoculated tube or the Buchner's tube even should it be slipped in hurriedly.

7. Fit the india-rubber stopper tightly into the mouth of the Buchner's tube.

The pyrogallic acid tablet dissolves slowly in the soda solution and its oxidation proceeds very slowly at first so that ample time is available when this method is adopted.

8. Restopper the caustic soda bottle.

9. Place Buchner's tube in a wire support, and incubate.

~Method VII~ (Wright's Method).--

1. Prepare tube cultivation in the usual way.

2. Cut off that portion of the cotton-wool plug projecting above the mouth of the tube with scissors, then push the plug into the tube for a distance of 2 or 3 cm.

3. By means of a pipette drop about 1 c.c. of pyrogallic acid 10 per cent. aqueous solution on to the plug. It will immediately be absorbed by the cotton-wool.

4. With another pipette run in an equal quantity of the caustic soda solution.

5. Quickly close the mouth of the tube with a tightly fitting india-rubber stopper.

6. Incubate.

~Method VIII~ (McLeod's Method).--

~Apparatus and Solutions Required.~--

McLeod's plate base (a hollow glazed earthenware disc 9 cm.
in diameter and 2 cm. deep: the upper surface is pierced by
a central hole, 2 cm. in diameter, giving access to the
interior, the lower part of which is divided into two by a
low partition. A shallow groove encircles the upper surface
near to the edge).

Plasticine.
Pyrogallic acid (1 gramme) compressed tablets.
Sodic hydroxide (0.4 gramme) compressed tablets.
Wash bottle of distilled water.
Surface plates of one or other agar medium (in petri dishes
of 8 cm. diameter).
Surface plate spreader.

METHOD.--

1. Roll out a long cylinder of plasticine and fit it into the groove on the upper surface of the earthenware base.

2. Place a tablet of pyrogallic acid in one division of the interior of the plate base, and two tablets of sodic hydroxide in the other.

3. Prepare surface plate culture of the organism to be cultivated.

4. Run a few cubic centimetres of distilled water into that division of the plate base containing the sodic hydroxide.

5. Invert the bottom half of the surface plate over the plate base and press its edges firmly down into the plasticine filling the groove.

6. Label and incubate.

(D) ~Method IX.~--

~Apparatus Required.~--

Small Ruffer's or Woodhead's flask (Fig. 33).
Sterile india-rubber stopper.
India-rubber tubing.
Glass tubing.
Metal screw clips.
Cylinder of compressed hydrogen; or hydrogen gas apparatus

METHOD.--

1. Sterilise a glass vessel, shaped as in a Ruffer's or Woodhead's flask, in the hot-air oven. (The tubulure and the side tubes are plugged with cotton-wool.) After sterilisation, fix a short piece of rubber tubing occluded by a metal clip to each side tube.

2. Inoculate a large quantity (e. g., 200 c.c.) of the medium. Where solid media are employed they must first be liquefied by heat.

3. Remove the cotton-wool plug from the tubulure and pour the inoculated medium into the glass vessel.

4. Close the tubulure by means of an india-rubber stopper previously sterilised by boiling in a beaker of water.

5. Connect up the india-rubber tubing on one of the side tubes with a cylinder of compressed hydrogen (or the delivery tube of a Kipp's Fig. 132 or other hydrogen apparatus, Fig. 133), interposing a short piece of glass tubing; and in like manner connect a long piece of rubber tubing which should be led into a basin of water, to the opposite side tube.

6. Open both metal clips and pass hydrogen through the vessel until the atmospheric air is replaced by hydrogen. This is determined by collecting some of the gas which bubbles through the water in the basin in a test-tube and testing it by means of a lighted taper.

7. Close the metal clip on the tube through which the gas is entering; close the clip on the exit tube.

8. Disconnect the gas apparatus.

9. Incubate.

~Method X~ (Botkin's Method).--

~Apparatus Required.~--

Large glass dish 20 cm. diameter and 8 cm. deep. Flat leaden
cross slightly shorter than the internal diameter of the glass dish.
Bell glass about 15 cm. diameter and 20 to 25 cm. high.
Metal frame for plate cultivations.
_Or_, glass battery jar for tube cultivations.
Cylinder of compressed hydrogen.
Rubber tubing.
Two pieces of ~U~-shaped glass tubing (each arm 8 cm. in length).
Half a litre of glycerine (or metallic mercury).

METHOD.--

1. Place the leaden cross inside the glass dish, resting on the bottom.

2. Prepare the cultivations in the usual way.

3. Place the tube cultivations in a glass battery jar (or the plate cultivations on a metal frame), resting on the centre of the leaden cross.

4. Cover the cultivations with the bell jar.

5. Adjust the U-shaped pieces of glass tubing in a vertical position on opposite sides of the bell jar, one arm of each inside the jar, the other outside. These tubes are best held in position by embedding the U-shaped bends in two lumps of plasterine stuck on the bottom of the glass dish. Fix a short length of rubber tubing clamped with a metal clip to each of the outside arms (Fig. 134).

6. Fill the glass dish with glycerine or metallic mercury to a depth of about 5 cm.

7. Connect up one U-shaped tube with the hydrogen cylinder (or gas apparatus) by means of rubber tubing. Replace the atmospheric air by hydrogen, as in method IX.

8. Clamp the tubes and disconnect the gas apparatus.

9. Incubate.

~Method XI~ (Novy's Method).--

~Apparatus Required.~--

Jar for plate cultivations (Fig. 135).
_Or_, jar for tube cultivations (Fig. 136).
Lubricant for stopper of jar.
Rubber tubing.
Cylinder of compressed hydrogen.

METHOD.--

1. Prepare cultivations in the usual way.

2. Place these inside the jar.

3. Lubricate the stopper and insert it in the mouth of the jar, with the handle in a line with the two side tubes.

4. Connect up the delivery tube a with the hydrogen gas supply by means of rubber tubing.

5. Attach a piece of rubber tubing to the exit tube b and collect samples of the issuing gas (over water) and test from time to time.

6. When the air is completely displaced by hydrogen, turn the handle of the stopper at right angles to the line of entry and exit tubes; this seals the orifice of both tubes.

7. Disconnect the gas apparatus and incubate.

(E) ~Method XII~ (Bulloch's Method).--

~Apparatus Required.~--

Bulloch's jar.
Pot of resin ointment.
Small glass dish 14 cm. diameter by 5 cm. deep.
Vessel for tube cultures or metal rack for plate cultures.
Pyrogallic acid tablets.
Cylinder of compressed hydrogen.
Geryk or other air pump.
Rubber pressure tubing.
10 c.c. pipette.
Glass tubing.
Dry granulated caustic soda or compressed tablets each, containing
0.4 grammes sodic hydroxide.
Small beaker of water.

METHOD.--

1. Prepare the cultivations in the usual way.

2. Place the glass dish in the centre of the glass slab, and stand the cultivations inside this.

3. Place a sufficient number of pyrogallic acid tablets at one side of the glass dish (i. e., 1 tablet for each 100 cubic centimeters air capacity of the bell jar). Place a small heap of dry granulated soda (or half a dozen tablets of sodic hydroxide) by the side of the pyro tablets.

4. Smear the flange of the bell jar with resin ointment and apply the jar firmly to the glass slab, covering the cultivations--so arranged that the long tube passes with its lower end into the glass dish at a point directly opposite to the pyrogallic acid tablets. Lubricate the two stop-cocks with resin ointment (Fig. 137).

5. Connect up the short tube a with the gas-supply by means of rubber pressure tubing and open both stop-cocks.

6. Connect a long, straight piece of glass tubing to the long tube b by means of a piece of rubber tubing interposing a screw clamp: and collect samples of the issuing gas from time to time and test.

7. When the air is displaced, shut off the stop-cock of the entry tube, then that of the exit tube b. Screw down the clamp and remove the glass tube from the rubber connection and connect up the short tube a to the air pump by means of pressure tubing.

8. Open the stop-cock of tube a and with two or three strokes of the air pump, aspirate a small quantity of gas, so creating a slight vacuum. Then shut off the stop-cock and disconnect the air pump.

9. Fill the 10 c.c. bulb pipette with water; insert its point into the rubber tubing on the long tube b as far as the screw clamp. Open the screw clamp and run in water until stopped by the internal pressure. Shut off stop-cock. (The water dissolves the soda and pyrogallic acid converting the latter into alkaline pyro. and so bringing its latent capacity for oxygen into action).

10. Reverse the tubes from the tubulures so that they meet, out of harm's way, over the top of the bell glass; again see that all joints are tight and transfer the apparatus to the incubator.

This last method is the most satisfactory for anaerobic cultivations, as by its means complete anaerobiosis can be obtained with the least expenditure of time and trouble.

FOOTNOTES:

[8] See also method of opening and closing culture tubes, pages 74-76.

[9] If compressed tablets of pyrogallic acid cannot be obtained make up a stock "acid pyro" solution

Pyrogallic acid, 10 grammes
Hydrochloric acid, 1.5 c.c.
Distilled water, 100 c.c.

and at step 4, run in 10 c.c. of the solution.

XV. METHODS OF ISOLATION.

The work in the preceding sections, arranged to demonstrate the chief biological characters of bacteria in general, is intended to be carried out by means of cultivations of various organisms previously isolated and identified and supplied to the student in a state of purity. A cultivation which comprises the progeny of a single cell is termed a "pure culture"; one which contains representatives of two or more species of bacteria is spoken of as an "impure," or "mixed" "cultivation," and it now becomes necessary to indicate the chief methods by which one or more organisms may be isolated in a state of purity from a mixture; whether that mixture exists as an impure laboratory cultivation, or is contained in pus and other morbid exudations, infected tissues, or water or food-stuffs.

Before the introduction of solid media the only method of obtaining pure cultivations was by "dilution"--by no means a reliable method. "Dilution" consisted in estimating approximately the number of bacteria present in a given volume of fluid (by means of a graduated-celled slide resembling a haematocytometer, Fig. 138), and diluting the fluid by the addition of sterile water or bouillon until a given volume (usually 1 c.c.) of the dilution contained but one organism. By planting this volume of the fluid into several tubes or flasks of nutrient media, it occasionally happened that the resulting growth was the product of one individual microbe. A method so uncertain is now fortunately replaced by many others, more reliable and convenient, and in those methods selected for description here, the segregation and isolation of the required bacteria may be effected--

A. ~By Mechanical Separation.~

1. By surface plate cultivation:

(a) Gelatine.
(b) Agar.
(c) Serum agar.
(d) Blood agar.
(e) Hanging-drop or block.

[2. By Esmarch's roll cultivation:

This archaic method (see page 226) is no longer employed for the isolation of bacteria.]

3. By serial cultivation.

B. ~By Biological Differentiation.~

4. By differential media.

(a) Selective.
(b) Deterrent.

5. By differential incubation.

6. By differential sterilisation.

7. By differential atmosphere cultivation.

8. By animal inoculation.

The selection of the method to be employed in any specific instance will depend upon a variety of circumstances, and often a combination of two or more will ensure a quicker and more reliable result than a rigid adherence to any one method. Experience is the only reliable guide, but as a general rule the use of either the first or the third method will be found most convenient, affording as each of them does an opportunity for the simultaneous isolation of several or all of the varieties of bacteria present in a mixture.

~1. Surface Plate Cultivations.~--

(a) _Gelatine_ (_vide_ page 164).

(b) _Agar_ (_vide_ page 167).

(c) _Alkaline serum agar_ (_vide_ page 211).

These plates are prepared in a manner precisely similar to that adopted for nutrient gelatine and agar surface plates (_vide_ pages 231-233).

(d) _Serum Agar._--

1. Melt three tubes of nutrient agar, label them 1, 2, and 3, and place them, with three tubes of sterile fluid serum, also labelled 1a, 2a, and 3a, in a water-bath regulated at 45 deg. C.; allow sufficient time to elapse for the temperature of the contents of each tube to reach that of the water-bath.

2. Take serum tube No. 1a and agar tube No. 1. Flame the plugs and remove them from the tubes (retaining the plug of the agar tube in the hand); flame the mouths of the tubes, pour the serum into the tube of liquefied agar and replace the plug of the agar tube.

3. Mix thoroughly and pour plate No. 1 _secundum artem_.

4. Treat the remaining tube of agar and serum in a similar fashion, and pour plates Nos. 2 and 3.

5. Dry the serum agar plates in the incubator running at 60 deg. C. for one hour (see page 232).

6. Inoculate the plates in series as described for gelatine surface plates (page 231).

(e) _Blood Agar, Human._--

1. Melt a tube of sterile agar and pour it into a sterile plate; let it set.

2. Collect a few drops of human blood, under all aseptic conditions, in a sterile capillary teat pipette.

3. Raise the cover of the Petri dish very slightly, insert the extremity of the capillary pipette, and deposit the blood on the centre of the agar surface. Close the dish.

4. Charge a platinum loop with a small quantity of the inoculum. Raise the cover of the plate, introduce the loop, mix its contents with the drop of blood, remove the loop, close the dish and sterilise the loop.

5. Finally smear the mixture over the surface of the agar with a sterilised L-shaped rod.

6. Label and incubate.

(If considered necessary, two, three, or more similar plates may be inoculated in series.)

(f) _Blood Agar, Animal._--

When preparing citrated blood agar (page 171) it is always advisable to pour several blood agar tubes into plates, which can be stored in the ice chest ready for use at any moment for surface plate cultures.

(g) Hanging-drop or block culture, (_vide_ page 233).

~3. Serial Cultivations.~--These are usually made upon agar or blood-serum, although gelatine may also be used.

The method is as follows:

1. Take at least four "slanted" tubes of media and number them consecutively.

2. Flame all the plugs and see that each can be readily removed.

3. Charge the platinum loop with a small quantity of the inoculum, observing the usual routine, and plant tube No. 1, smearing thoroughly all over the surface. If any water of condensation has collected at the bottom of the tube, use this as a diluent before smearing the contents of the loop over the surface of the medium.

4. Without sterilising or recharging the loop, inoculate tube No. 2, by making three parallel streaks from end to end of the slanted surface.

5. Plant the remainder of the tubes in the series as "smears" like tube No. 1.

6. Label with distinctive name or number, and date; incubate.

The growth that ensues in the first two or three tubes of the series will probably be so crowded as to be useless. Toward the end of the series, however, discrete colonies will be found, each of which can be transferred to a fresh tube of nutrient medium without risk of contamination from the neighbouring colonies.

~"Working" up Plates.~--

Having succeeded in obtaining a plate (or tube cultivation) in which the colonies are well grown and sufficiently separated from each other, the process of "working up," "pricking out," or "fishing" the colonies in order to obtain subcultures in a state of purity from each of the different bacteria present must now be proceeded with.

Occasionally it happens that this is quite a simple matter. For example, the original mixed cultivation when examined microscopically was found to contain a Gram positive micrococcus, a Gram positive straight bacillus and a Gram negative short bacillus. The third gelatine plate prepared from this mixture, on inspection after four day's incubation, showed twenty-five colonies--seven moist yellow colonies, each sinking into a shallow pit of liquefied gelatine, fourteen flat irridescent filmy colonies, and four raised white slimy colonies. A film preparation (stained Gram) from each variety examined microscopically showed that the yellow liquefying colony was composed of Gram positive micrococci; the flat colony of Gram positive bacilli and the white colony of gram negative bacilli. One of each of these varieties of colonies would be transferred by means of the sterilised loop to a fresh gelatine culture tube, and after incubation the growth in each subculture would correspond culturally and microscopically with that of the plate colony from which it was derived,--the object aimed at would therefore be achieved.

Usually, however, the colonies cannot be thus readily differentiated, and unless they are "worked up" in an orderly and systematic manner much labour will be vainly expended and valuable time wasted. The following method minimises the difficulties involved.

(A) Inspection.

a. Without opening the plate carefully study the various colonies with the naked eye, with the assistance of a watchmaker's lens or by inverting the plate on the stage of the microscope and viewing with the 1-inch objective through the bottom of the plate and the layer of medium.

b. If gross differences can be detected mark a small circle on the bottom of the plate around the site of each of the selected colonies, with the grease pencil.

c. If no obvious differences can be made out choose nine colonies haphazard and indicate their positions by pencil marks on the bottom of the plate.

(B) Fishing Colonies.--

a. Take a sterile Petri dish and invert it upon the laboratory bench. Rule two parallel lines on the bottom of the dish with a grease pencil, and two more parallel lines at right angles to the first pair--so dividing the area of the dish into nine portions. Number the top right-hand portion 1, and the central bottom portion 8 (Fig. 139). Revert the dish. The numbers 1 and 8 can be readily recognised through the glass and by their positions enable any of the other divisions to be localised by number. This is the stock dish.

b. Slightly raise the cover of the dish, and with a sterile teat-pipette deposit a small drop of sterile water in the centre of each of the nine divisions.

c. With the sterilised platinum spatula raise one of the marked colonies from the "plate 3" and transfer it to the first division in the ruled plate and emulsify it in the drop of water awaiting it. Repeat this process with the remaining colonies, emulsifying a separate colony in each drop of water.

(C) Preliminary Differentiation of Bacteria.--

a. Prepare a cover-slip film preparation from each drop of emulsion in the "stock dish" and number to correspond to the division from which it was taken. Stain by Gram's method.

b. Examine microscopically, using the oil immersion lens and note the numbers of those cover-slips which morphologically and by Gram results appear to be composed of different species of bacteria.

(D) Preparing Isolation Subcultures.--

a. Inoculate an agar slope and a broth tube from the emulsion in the stock dish corresponding to each of these specially selected numbers.

b. Ascertain whether the cover-slips from the nine emulsions in the stock dish include all the varieties represented in the cover-slip film preparation made from the original mixture before plating.

c. If some varieties are missing prepare a second stock dish from other colonies on plate 3, and repeat the process until each morphological form or tinctorial variety has been secured in subculture.

_d._ Place the stock dishes in the ice chest to await the results of incubation. (If any of the subcultures fail, further material can be obtained from the corresponding emulsion; or if it has dried, by moistening it with a further drop of sterile distilled water.)

_e._ Incubate all the subcultures and identify the organisms picked out.

4. Differential Media.--

(a) _Selective._--Some varieties of media are specially suitable for certain species of bacteria and enable them to overgrow and finally choke out other varieties; e. g., wort is the most suitable medium-base for the growth of torulae and yeasts and should be employed when pouring plates for the isolation of these organisms. To obtain a pure cultivation of yeast from a mixture containing bacteria as well, it is often sufficient to inoculate wort from the mixture and incubate at 37 deg. C. for twenty-four hours. Plant a fresh tube of wort from the resulting growth and incubate. Repeat the process once more, and from the growth in this third tube plant a streak on wort gelatine, and incubate at 20 deg. C. The resulting growth will almost certainly be a pure culture of the yeast.

(b) _Deterrent._--The converse of the above also obtains. Certain media possess the power of inhibiting the growth of a greater or less number of species. For instance, media containing carbolic acid to the amount of 1 per cent. will inhibit the growth of practically everything but the Bacillus coli communis.

~5. Differential Incubation.~--

In isolating certain bacteria, advantage is taken of the fact that different species vary in their optimum temperature. A mixture containing the Bacillus typhosus and the Bacillus aquatilis sulcatus, for example, may be planted on two slanted agar tubes, the one incubated at 40 deg. C., and the other at 12 deg. C. After twenty-four hours' incubation the first will show a pure cultivation of the Bacillus typhosus, whilst the second will be an almost pure culture of the Bacillus aquatilis.

6. Differential Sterilisation.--

(a) _Non-sporing Bacteria._--Similarly, advantage may be taken of the varying thermal death-points of bacteria. From a mixture of two organisms whose thermal death-points differ by, say, 4 deg. C.--e. g., Bacillus pyocyaneus, thermal death-point 55 deg. C., and Bacillus mesentericus vulgatus, thermal death-point 60 deg. C.--a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to 58 deg. C. and keeping it at that point for ten minutes. The mixture is then planted on to fresh media and incubated, when the resulting growth will be found to consist entirely of the B. mesentericus.

(b) _Sporing Bacteria._--This method finds its chief practical application in the differentiation of a spore-bearing organism from one which does not form spores. In this case the mixture is heated in a water-bath at 80 deg. C. for fifteen to twenty minutes. At the end of this time the non-sporing bacteria are dead, and cultivations made from the mixture will yield a growth resulting from the germination of the spores only.

Differential sterilisation at 80 deg. C. is most conveniently carried out in a water-bath of special construction, designed by Balfour Stewart (Fig. 140). It consists of a double-walled copper vessel mounted on legs, and provided with a tubulure communicating with the space between the walls. This space is nearly filled with benzole (boiling-point 80 deg. C.; pure benzole, free from thiophene must be employed for the purpose, otherwise the boiling-point gradually and perceptibly rises in the course of time), and to the tubulure is fitted a long glass tube, some 2 metres long and about 0.75 cm. diameter, serving as a condensing tube (a tube half this length if provided with a condensing bulb at the centre will be equally efficient). The interior of the vessel is partly filled with water and covered with a lid which is perforated for a thermometer. This latter dips into the water and records its temperature. A very small Bunsen flame under the apparatus suffices to keep the benzole boiling and the water within at a constant temperature of 80 deg. C. The bath is thus always ready for use.

METHOD.--To use the apparatus.

1. Place some of the mixture itself, if fluid, containing the spores, or an emulsion of the same if derived from solid material, in a test-tube.

2. Immerse the test-tube in the water contained in the benzole bath, taking care that the upper level of the liquid in the tube is at least 2 cm. beneath the surface of the water in the copper vessel.

3. The temperature of the water, of course, falls a few degrees after opening the bath and introducing a tube of colder liquid, but after a few minutes the temperature will have again reached 80 deg. C.

4. When the thermometer again records 80 deg. C., note the time, and fifteen minutes later remove the tube containing the mixture from the bath.

5. Make cultures upon suitable media; incubate.

7. Differential Atmosphere Cultivation.--

(a) By adapting the atmospheric conditions to the particular organism it is desired to isolate, it is comparatively easy to separate a strict aerobe from a strict anaerobe, and _vice versa_. In the first case, however, it is important that the cultivations should be made upon solid media, for if carried out in fluid media the aerobes multiplying in the upper layers of fluid render the depths completely anaerobic, and under these conditions the growth of the anaerobes will continue unchecked.

(b) When it is desired to separate a facultative anaerobe from a strict anaerobe, it is generally sufficient to plant the mixture upon the sloped surface agar, incubate aerobically at 37 deg. C., and examine carefully at frequent intervals. At the first sign of growth, subcultivations must be prepared and treated in a similar manner. As a result of these rapid subcultures, the facultative anaerobe will be secured in pure culture at about the third or fourth generation.

(c) If, on the other hand, the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes, pour plates of glucose formate agar (or gelatine) in the usual manner, place them in a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick off the colonies of the required organism when the growth appears, and transfer to tubes of the various media.

Incubate under suitable conditions as to temperature and atmosphere.

~8. Animal Inoculation.~--

Finally, when dealing with pathogenic organisms, it is often advisable to inoculate some of the impure culture (or even some of the original _materies morbi_) into an animal specially chosen on account of its susceptibility to the particular pathogenic organism it is desired to inoculate. Indeed, with some of the more sensitive and strictly parasitic bacteria this method of animal inoculation is practically the only method that will yield a satisfactory result.

XVI. METHODS OF IDENTIFICATION AND STUDY.

In order to identify an organism after isolation, tube, plate, and other cultivations must be prepared, incubated under suitable conditions as to temperature and environment, and examined from time to time (a) ~macroscopically~, (b) by ~microscopical methods~, (c) by ~chemical methods~, (d) by ~physical methods~, (e) by ~inoculation methods~, and the results of these examinations duly recorded.

It must be stated definitely that no micro-organism can be identified by any _one_ character or property, whether microscopical, biological or chemical, but that on the contrary its entire life history must be carefully studied and then its identity established from a consideration of the sum total of these observations.

In order to give to the recorded results their maximum value it is essential that they should be exact and systematic, therefore some such scheme as the following should be adhered to; and especially is this necessary in describing an organism not previously isolated and studied.

SCHEME OF STUDY.

Designation:

Originally isolated by (_observer's name_) in (_date_), from (_source of organism_).

~1. Cultural Characters.~--(_Vide_ Macroscopical Examination
of Cultivation, page 261.)

Gelatine plates, }
Gelatine streak, } at 20 deg. C.
Gelatine stab, }
Gelatine shake, }

Agar plates, }
Agar streak or smear, }
Agar stab, }
Inspissated blood-serum, } at 20 deg. C. and 37 deg. C.
Bouillon, }
Litmus milk, }
Potato, }

Special media for the purpose of demonstrating
characteristic appearances.

~2. Morphology~.--(_Vide_ Microscopical Examination of
Cultivations, page 272.)

Vegetative forms:
Shape.
Size.
Motility.
Flagella (if present).
Capsule (if present).
Involution forms.
Pleomorphism (if observed).
Sporing forms (if observed). Of which class?
Staining reactions.

~3. Chemical Products of Growth.~--(_Vide_ Chemical
Examination of Cultivations, page 276.)

Chromogenesis.
Photogenesis.
Enzyme formation.
Fermentation of carbohydrates:
Acid formation.
Alkali formation.
Indol formation.
Phenol formation.
Reducing and oxidising substances.
Gas formation.

~4. Biology.~--(_Vide_ Physical Examination of Cultures, page
295.)

Atmosphere.
Temperature.

Reaction of nutrient media.
Resistance to lethal agents:
Physical:
Desiccation.
Light.
Colours.
Chemical germicides.
Vitality.

~5. Pathogenicity:~

Susceptible animals, subsequently arranged in order of susceptibility.
Immune animals.
Experimental inoculation, symptoms of disease.
Post-mortem appearances.
Virulence:
Length of time maintained.
Optimum medium?
Minimal lethal dose.
Exaltation and attenuation of virulence?
Toxin formation.

MACROSCOPICAL EXAMINATION OF CULTIVATIONS.

In describing the naked-eye and low-power appearances of the bacterial growth the descriptive terms introduced by Chester (and included in the following scheme) should be employed.

SOLID MEDIA.

~Plate Cultures.~--

_Gelatine._--Note the presence or absence of liquefaction of the surrounding medium. If liquefaction is present, note shape and character (_vide_ page 269, "stab" cultures).

_Agar._--No liquefaction takes place in this medium. The liquid found on the surface of the agar (or at the bottom of the tube in agar tube cultures) is merely water which has been expressed during the rapid solidification of the medium and has subsequently condensed.

_Gelatine and Agar._--Examine the colonies at intervals of twenty-four hours.

(a) With the naked eye.

(b) With a hand lens or watchmaker's glass.

(c) Under a low power (1 inch) of the microscope, or by means of a small dissecting microscope.

Distinguish superficial from deep colonies and note the characters of the individual colonies.

(A) ~Size.~--The diameter in millimetres, at the various ages.

(B) ~Shape.~--

Punctiform: Dimensions too slight for defining form by naked eye; minute, raised, hemispherical.

Round: Of a more or less circular outline.

Elliptical: Of a more or less oval outline.

Irregular: Outlines not conforming to any recognised shape.

Fusiform: Spindle-shaped, tapering at each end.

Cochleate: Spiral or twisted like a snail shell (Fig. 141, a).

Amoeboid: Very irregular, streaming (Fig. 141, b).

Mycelioid: A filamentous colony, with the radiate character of a mould (Fig. 141, c).

Filamentous: An irregular mass of loosely woven filaments (Fig. 142, a).

Floccose: Of a dense woolly structure.

Rhizoid: Of an irregular, branched, root-like character (Fig. 142, b).

Conglomerate: An aggregate of colonies of similar size and form (Fig. 142, c).

Toruloid: An aggregate of colonies, like the budding of the yeast plant (Fig. 142, d).

Rosulate: Shaped like a rosette.

(C) ~Surface Elevation.~--

1. _General Character of Surface as a Whole_:

Flat: Thin, leafy, spreading over the surface (Fig. 143, a).

Effused: Spread over the surface as a thin, veily layer, more delicate than the preceding.

Raised: Growth thick, with abrupt terraced edges (Fig. 143, b).

Convex: Surface the segment of a circle, but very flatly convex (Fig. 143, c).

Pulvinate: Surface the segment of a circle, but decidedly convex (Fig. 143, d).

Capitate: Surface hemispherical (Fig. 143, e).

Umbilicate: Having a central pit or depression (Fig. 143, f).

Conical: Cone with rounded apex (Fig. 143, g).

Umbonate: Having a central convex nipple-like elevation (Fig. 143, h).

2. _Detailed Characters of Surface_:

Smooth: Surface even, without any of the following distinctive characters.

Alveolate: Marked by depressions separated by thin walls so as to resemble a honeycomb (Fig. 144).

Punctate: Dotted with punctures like pin-pricks.

Bullate: Like a blistered surface, rising in convex prominences, rather coarse.

Vesicular: More or less covered with minute vesicles due to gas formation; more minute than bullate.

Verrucose: Wart-like, bearing wart-like prominences.

Squamose: Scaly, covered with scales.

Echinate: Beset with pointed prominences.

Papillate: Beset with nipple or mamma-like processes.

Rugose: Short irregular folds, due to shrinkage of surface growth.

Corrugated: In long folds, due to shrinkage.

Contoured: An irregular but smoothly undulating surface, resembling the surface of a relief map.

Rimose: Abounding in chinks, clefts, or cracks.

(D) ~Internal Structure of Colony~ (_Microscopical_).--

Refraction Weak: Outline and surface of relief not strongly defined.

Refraction Strong: Outline and surface of relief strongly defined; dense, not filamentous colonies.

1. _General_:

Amorphous: Without any definite structure, such as is specified below.

Hyaline: Clear and colourless.

Homogeneous: Structure uniform throughout all parts of the colony.

Homochromous: Colour uniform throughout.

2. _Granulations or Blotchings_:

Finely granular.

Coarsely granular.

Grumose: Coarser than the preceding, with a clotted appearance, and particles in clustered grains (Fig. 145, a).

Moruloid: Having the character of a mulberry, segmented, by which the colony is divided in more or less regular segments (Fig. 145, b).

Clouded: Having a pale ground, with ill-defined patches of a deeper tint (Fig. 145, c).

3. _Colony Marking or Striping_:

Reticulate: In the form of a network, like the veins of a leaf (Fig. 146, a).

Areolate: Divided into rather irregular, or angular, spaces by more or less definite boundaries.

Gyrose: Marked by wavy lines, indefinitely placed (Fig. 146, b).

Marmorated: Showing faint, irregular stripes, or traversed by vein-like markings, as in marble (Fig. 146, c).

Rivulose: Marked by lines like the rivers of a map.

Rimose: Showing chinks, cracks, or clefts.

4. _Filamentous Colonies:_

Filamentous: As already defined.

Floccose: Composed of filaments, densely placed.

Curled: Filaments in parallel strands, like locks or ringlets (Fig. 147).

(E) ~Edges of Colonies.~--

Entire: Without toothing or division (Fig. 148, a).

Undulate: Wavy (Fig. 148, b).

Repand: Like the border of an open umbrella (Fig. 148, c).

Erose: As if gnawed, irregularly toothed (Fig. 148, d).

Lobate.

Lobulate: Minutely lobate (Fig. 149, e).

Auriculate: With ear-like lobes (Fig. 149, f).

Lacerate: Irregularly cleft, as if torn (Fig. 149, g).

Fimbriate: Fringed (Fig. 149, h).

Ciliate: Hair-like extensions, radiately placed (Fig. 149, j).

Tufted.

Filamentous: As already defined.

Curled: As already defined.

(F) ~Optical Characters~ (after Shuttleworth).--

1. _General Characters_:

Transparent: Transmitting light.

Vitreous: Transparent and colourless.

Oleaginous: Transparent and yellow; olive to linseed-oil coloured.

Resinous: Transparent and brown, varnish or resin-coloured.

Translucent: Faintly transparent.

Porcelaneous: Translucent and white.

Opalescent: Translucent; greyish-white by reflected light.

Nacreous: Translucent, greyish-white, with pearly lustre.

Sebaceous: Translucent, yellowish or greyish-white.

Butyrous: Translucent and yellow.

Ceraceous: Translucent and wax-coloured.

Opaque.

Cretaceous: Opaque and white, chalky.

Dull: Without lustre.

Glistening: Shining.

Fluorescent.

Iridescent.

2. _Chromogenicity_:

Colour of pigment.

Pigment restricted to colonies.

Pigment restricted to medium surrounding colonies.

Pigment present in colonies and in medium.

~Streak or Smear Cultures.~--

_Gelatine and Agar._--Note general points as indicated under plate cultivations.

_Inspissated Blood-serum._--Note the presence or absence of liquefaction of the medium. (The presence of condensation water at the bottom of the tube must not be confounded with liquefaction of the medium.)

_All Oblique Tube Cultures._--

1. Colonies Discrete: Size, shape, etc., as for plate cultivations (_vide_ page 261).

2. Colonies Confluent: Surface elevation and character of edge, as for plate cultivations (_vide_ page 263).

Chromogenicity: As for plate cultures.

~Gelatine Stab Cultures.~--

(A) _Surface Growth._--As for individual colonies in plate cultures (_vide_ page 261).

(B) _Line of Puncture._--

Filiform: Uniform growth, without special characters (Fig. 150, a).

Nodose: Consisting of closely aggregated colonies.

Beaded: Consisting of loosely placed or disjointed colonies (Fig. 150, b).

Papillate: Beset with papillate extensions.

Echinate: Beset with acicular extensions (Fig. 150, c).

Villous: Beset with short, undivided, hair-like extensions (Fig. 150, d).

Plumose: A delicate feathery growth.

Arborescent: Branched or tree-like, beset with branched hair-like extensions (Fig. 150, e).

(C) _Area of Liquefaction_ (if present).--

Crateriform: A saucer-shaped liquefaction of the gelatine (Fig. 151, f).

Saccate: Shape of an elongated sack, tubular cylindrical (Fig. 151, g).

Infundibuliform: Shape of a funnel, conical (Fig. 151, h).

Napiform: Shape of a turnip (Fig. 151, j).

Fusiform: Outline of a parsnip, narrow at either end, broadest below the surface (Fig. 151, k).

Stratiform: Liquefaction extending to the walls of the tube and downward horizontally (Fig. 151, l).

(D) _Character of the Liquefied Gelatine._--

1. Pellicle on surface.

2. Uniformly turbid.

3. Granular.

4. Mainly clear, but containing flocculi.

5. Deposit at apex of liquefied portion.

(E) _Production of Gas Bubbles._

~Shake Cultures.~--

1. Presence or absence of liquefaction.

2. Production of gas bubbles.

3. Bulk of growth at the surface--aerobic.

4. Bulk of growth in depths--anaerobic.

~Fluid Media.~

~1. Surface of the Liquid.~--

Presence or absence of froth due to gas bubbles.

Presence or absence of pellicle formation.

Character of pellicle.

~2. Body of the Liquid.~--

Uniformly turbid.

Flocculi in suspension.

Granules in suspension.

Clear, with precipitate at bottom of tube.

Colouration of fluid, presence or absence of.

~3. Precipitate.~--

Character.

Amount.

Colour.

~Carbohydrate Media.~--

Growth.

Reaction.

Gas formation.

Coagulation or not of serum albumen (when serum water media are employed).

~Litmus Milk Cultivations.~--

{Unaltered.
1. Reaction: {Acid.
{Alkaline.
2. Odour.

3. Formation of gas.

{Unaltered.
4. Consistency: {Peptonised (character of solution).
{Coagulated.

{hard: solid.
5. Clot: Character {soft: floculent.
{ragged and broken up by gas
{bubbles.

(a) Coagulum undissolved.

(b) Coagulum finally peptonised, completely: incompletely.

Resulting solution, clear: turbid.

{Abundant.
{Scanty.
6. Whey: {Clear.
{Turbid.
{Coagulated by boiling, or not.

~BY MICROSCOPICAL METHODS.~

As a council of perfection preparations must be made from pure cultivations 4, 6, 8, 12, 18, and 24 hours; and subsequently at intervals of, say, twenty-four hours, during the entire period they are under observation, and examined--

(A) ~Living.--1.~ In ~hanging drop~, to determine _motility_ or _non-motility_.

In this connection it must be remembered that under certain conditions as to environment (e. g., when examined in an unsuitable medium, atmosphere, temperature, etc.) motile bacilli may fail to exhibit activity. No organism, therefore, should be recorded as non-motile from one observation only; a series of observations at different ages and under varying conditions should form the basis of an opinion as to the absence of true locomotion.

_Size._--In the case of non-motile or sluggishly motile organisms, endeavour to measure several individuals in each hanging drop by means of the eyepiece micrometer or the eikonometer (_vide_ page 63), and average the results.

If the organism is one which forms spores, observe--

(a) _Spore Formation._--Prepare hanging-drop cultivations (_vide_ page 78) from vegetative forms of the organism, adding a trace of magenta solution (0.5 per cent.) or other intra vitam stain (see page 77) to the drop, on the point of the platinum needle, to facilitate the observation of the phenomenon by rendering the bacilli more distinct.

Place the preparation on the stage of the microscope; if necessary, using a warm stage.

Arrange illumination, etc., and select a solitary bacillus for observation, by the help of the 1/6-inch lens.

Substitute the 1/12-inch oil-immersion lens for the sixth, and observe the formation of the spore; if possible, measure any alteration in size which may occur by means of the Ramsden micrometer.

(b) _Spore Germination._--Prepare hanging-drop cultivations from old cultivations in which no living vegetative forms are present, and observe the process of germination in a similar manner.

The comfort of the microscopist is largely enhanced in those cases where the period of observation is at all lengthy, by use of some form of eye screen before the unemployed eye, such as is figured on page 58 (Fig. 49).

If it is impossible to carry out the method suggested above, proceed as follows:

(a) _Spore Formation._--Plant the organism in broth and incubate under optimum conditions.

At regular intervals, say every thirty minutes, remove a loopful of the cultivation and prepare a cover-slip film preparation.

Fix, while still wet, in the corrosive sublimate fixing solution.

Stain with aniline gentian violet, and partially decolourise with 2 per cent. acetic acid.

Mount and number consecutively; then examine.