The Project Gutenberg eBook of The Elements of Bacteriological Technique, by John William Henry Eyre

3. Moisten the cotton-wool pads at the bottom of the potato tubes with a 25 per cent. solution of glycerine.

5. Sterilise in the steamer at 100° C. for twenty minutes on each of five consecutive days.

1. Take a number of sterile test-tubes 16 × 3 or 4 cm., plugged with cotton wool, and into each insert a 2 cm. length of stout glass tubing (about 1 cm. diameter); fill in glycerine (6 per cent.) bouillon to the upper level of the piece of glass tubing. Sterilise in the steamer at 100° C. for twenty minutes on each of three successive days.

3. Under strictly aseptic precautions remove the lungs, liver and other solid organs and transfer them to a sterile double glass dish.

4. With the help of sterile scissors and forceps divide the organs into roughly rectangular blocks 3 × 1.5 × 1 cm.

5. Pour into the dish a sufficient quantity of sterile glycerine solution (6 per cent. in normal saline), cover, and allow to stand for one hour.

6. Introduce a block of tissue into each tube so that it rests upon the upper end of the piece of glass tubing. (The surface of the tissue will now be kept moist by capillary attraction and condensation).

Tissues obtained at postmortems can also be used after preliminary sterilisation by boiling or autoclaving.

1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by aspiration directly into sterile flasks, under strictly aseptic precautions.

2. Mix the serum with twice its bulk of sterile nutrient bouillon (vide page 163).

3. If considered necessary (on account of the presence of blood, crystals, etc.), filter the serum bouillon through porcelain filter candle.

4. Tube, and sterilise in the water bath at 56° C. for half an hour on each of five consecutive days.

5. Incubate at 37° C. for forty-eight hours and eliminate contaminated tubes. Store the remainder for future use.

5. After the third sterilisation cool the tubes to 42°C., and add to each 3 c.c. of sterile hydrocele fluid, ascitic fluid, or pleuritic effusion (previously sterilised, if necessary, by the fractional method); allow the tubes to solidify in a sloping position.

6. When solid, incubate at 37° C. for forty-eight hours, and eliminate any contaminated tubes. Store the remainder for future use.

5. After the last sterilisation cool to 42°C., then add 5 c.c. sterile blood-serum from human placenta (sterilised, if necessary, by the fractional method) to each tube; slope the tubes.

6. When solid, incubate at 37° C. for forty-eight hours, and eliminate any contaminated tubes. Store the remainder for future use.

1. Collect ascitic, pleuritic, or hydrocele fluid in sterile flasks and allow to stand in the ice-chest for twelve hours to sediment.

2. Decant 1000 c.c. of the clear fluid into a measuring cylinder and transfer to sterile litre flask.

3. Add 0.5 c.c. dekanormal NaOH solution for every 100 c.c. serum (i. e., 5.0 c.c.), and mix thoroughly.

5. Weigh out 15 grammes agar, emulsify in a separate vessel with 200 c.c. of the alkaline fluid previously cooled to about 20°C., and then add to the remainder of the fluid in the flask.

6. Bubble live steam through the mixture for twenty minutes to dissolve the agar.

8. Weigh out glucose 10 grammes (= 1 per cent.), and dissolve it in the clear agar.

1. Prepare nutrient agar (vide, page 167) using, however, 1.5 per cent. peptone (that is 15 grammes per litre instead of 10 grammes).

5. Tube in quantities of 6 c.c.; and sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days.

6. After the third sterilisation cool to 42° C. and add to each tube 3 c.c. of sterile hydrocele fluid, ascitic fluid or pleuritic effusion (previously sterilised, if necessary, by the fractional method); allow the tubes to solidify in a sloping position.

7. When solid, incubate at 37° C. for forty-eight hours, and eliminate any contaminated tubes. Store the remainder for future use.

1. Break several fresh eggs (hens', ducks', or turkeys' eggs), and collect the "whites" in a graduated cylinder, taking care to avoid admixture with the yolks.

2. Add 40 per cent. distilled water, and incorporate the mixture thoroughly by the aid of an egg-whisk.

3. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid (or add the amount of dekanormal caustic soda solution calculated to yield the required percentage of soda in the total bulk of the fluid—i. e., 0.375 c.c. of dekanormal NaOH solution per 100 c.c. of the mixture).

4. Strain the mixture through butter muslin and filter through a porcelain filter candle into a sterile filter flask.

6. Incubate at 37° C. for forty-eight hours and eliminate any contaminated tubes; store the remainder for future use.

1. Place unbroken hens' eggs in dekanormal caustic soda solution for ten days. (After this time the white becomes firm like gelatine.)

4. Place the egg slices in a large beaker and sterilise in the steamer at 100° C. for one hour.

5. Transfer each slice of egg by means of a pair of sterilised forceps to a Petri dish or large capsule.

6. Sterilise in the steamer at 100° C. for twenty minutes on each of three consecutive days.

2. Measure out distilled water 200 c.c. into a half-litre flask and warm to 37° C. in the incubator.

3. Add the water to the flask containing the albumin and beads and dissolve by shaking.

4. Add n/10-NaOH, 40 c.c. Allow the mixture to stand for thirty minutes with frequent shaking.

9. Incubate for forty-eight hours at 37°C.—discard any contaminated flasks and store the remainder for future use.

2. Liquefy and measure out ordinary nutrient agar 600 c.c. and add to the egg albumin solution (in place of the nutrient broth).

3. Heat over a bunsen flame, shaking constantly until the fluid boils, and the nutrose is dissolved.

Note.—The finished medium in this case measures 900 c.c. only since inconvenient fractions would be introduced in making up to one litre exactly.

1. Melt up several tubes of nutrient agar (vide page 167) and allow them to solidify in the oblique position.

2. Place the tubes, in the horizontal position, in the "hot" incubator for forty-eight hours, to evaporate off some of the condensation water.

3. Kill a small rabbit with chloroform and nail it out on a board (as for a necropsy). Moisten the hair thoroughly with 2 per cent. solution of lysol.

8. Sear the surface of the left ventricle with a red-hot iron and remove fluid blood from the heart by means of sterile pipettes (e. g., those shown in Fig. 13, c).

9. Deliver a small quantity of the blood on the slanted surface of the agar in each of the tubes, and allow it to run over the entire surface of the medium.

11. Return the "blood agar" to the hot incubator for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use.

2. Add 10 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes.

Tissue Medium (Noguchi).—For spirochætes (cultivations must be grown anaerobically).

2. Kill a small rabbit with chloroform vapour. Open the abdomen with all aseptic precautions, remove kidneys and testicles and transfer to a sterile glass dish. Cut up the organs with sterile scissors into small pieces—say 4 millimetre cubes. The four organs should yield from 25 to 30 pieces of tissue.

3. Drop a small piece of sterile tissue into the bottom of each sterilised tube.

4. Take a flask containing about 400 c.c. nutrient agar (+10 reaction), liquefy the medium by heat and cool in a water bath to 50°C.

5. Add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum may be employed, but is not so good) to the liquid agar and mix carefully to avoid formation of air bubbles.

6. Fill about 20 c.c. of the ascitic agar into each of the sterilised tubes which already contains a piece of sterile rabbit's tissue, stand all the tubes upright in racks or a jar, and allow agar to set.

7. After solidification pour sterile paraffin oil on the surface of the medium in each tube to the depth of 3 centimetres.

8. Incubate tubes at 37° C. for several days and discard any which prove to be contaminated.

XIII. INCUBATORS.

An incubator (Fig. 113) consists essentially of a chamber for the reception of cultivations, etc., surrounded by a water jacket, the walls of which are of metal, usually copper, and outside all an asbestos or felt jacket, or wooden casing. The water in the jacket is heated by gas or electricity and maintained at some constant temperature by a thermo-regulator. The cellular incubator (Fig. 114) which was made for me [7] some years ago is of the greatest practical utility. Here the central cavity is subdivided by five double-walled partitions (in which water circulates in connection with the water tanks at the top and base of the incubator) and again by iron shelves to form twenty-four pigeon holes. Into each of these slides an iron drawer 35 cm. long × 12 cm. wide × 22 cm. high forming a self-contained incubator. The drawer is fitted with a wooden form to which is fixed a handle and a numbered label. The thermo-regulating apparatus is the well-known Hearson capsule.

Two incubators at least are required in the laboratory, for the cultivation of bacteria the one regulated to maintain a temperature of 37°C., and known as the "hot" incubator; the other, 20° C. to 22°C., and known as the "cool" or "cold" incubator.

Two other incubators, regulated to 42° C. and 60°C. respectively, whilst not absolutely, necessary very soon justify their purchase.

Thermo-regulators.—The thermo-regulator is the most essential portion of the incubator, as upon its efficient working depends the maintenance of a constant temperature in the cultivation chamber. It is also used in the fitting up of water and paraffin baths, and for many other purposes.

Of the many forms and varieties of thermo-regulator (other than electrical), two only are of sufficiently general use to need mention. In one of these the flow of gas to the gas-jet is controlled by the expansion or contraction of mercury within a glass bulb; in the other, by alterations in the position of the walls of a metallic capsule containing a fluid, the boiling-point of which corresponds to the temperature at which the incubator is intended to act. They are:

(a) Reichert's (Fig. 115), consists of a bulb containing mercury which is to be suspended in the medium, whether air or water, the temperature of which it is desired to regulate. Gas enters at A, and passes out to the jet by B. As the temperature rises the mercury expands and cuts off the main gas supply. As the temperature falls the mercury contracts and reopens the narrow tube C. By means of a thumbscrew D (which mechanically raises or lowers the column of mercury irrespective of the temperature) and the aid of a thermometer the apparatus can be set to keep the incubator at any desired temperature. With this form a special gas burner is required, with separate supply of gas to a pilot jet at the side.

(b) Hearson's capsule regulator consists of a metal capsule hermetically sealed and filled with a liquid which boils at the required temperature, this is adjusted in the interior of the incubator. Soldered to the upper side of the capsule is a thick piece of metal having a central cup to receive the lower end of a rigid rod, through which the movements of the walls of the capsule are transmitted to the gas valve fixed outside the incubator.

The gas valve or governor is shown in figure 116. A is the inlet for gas, C the outlet to burner heating the water jacket, B D a lever pivoted to standards at G, and acted upon by the capsule, through the rigid rod which enters the socket below the screw P.

The construction of the valve is such that, whenever the short arm of the lever B D presses on the disc below the end B, the main supply of gas is entirely cut off. At such times, however, a very small portion of gas passes from A to C, through an aperture inside the valve, the size of which aperture can be adjusted by the screw needle S, hence the gas flame below the incubator is never extinguished.

The expansion of the metal walls of the capsule, which takes place upon the boiling of its contents, provides the motive force, transmitted through the rigid rod to raise the long arm of the lever B D, and as this expansion only takes place at a predetermined temperature, the lever will only be acted upon when the critical temperature is reached, no sensible effect being produced at even 1° C. below that at which the capsule is destined to act.

W is a weight sliding on the lever rod D; by increasing the distance between the weight and the fulcrum of the lower increased pressure is brought to bear upon the walls of the capsule with the result that the boiling-point of the liquid in the capsule is slightly raised, and a range of about two degrees can thus be obtained with any particular capsule.

FOOTNOTES:

[7] Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.

XIV. METHODS OF CULTIVATION.

Cultivations of micro-organisms are usually prepared in the laboratory in one of three ways:

These may be incubated either aerobically (i. e., in the presence of oxygen) or anaerobically (i. e., in the absence of oxygen, or in the presence of an indifferent gas, such as hydrogen, nitrogen, or carbon dioxide).

With regard to the temperature at which the cultivations are grown, it may be stated as a general rule that all media rendered solid by the addition of gelatine are incubated at 20°C., or at any rate at a temperature not exceeding 22° C. (that is, in the "cold" incubator); whilst fluid media and all other solid media are incubated at 37° C. (that is, in the "hot" incubator). Exceptions to this rule are numerous. For instance, in studying the growth of the psychrophylic bacteria, the yeasts and the moulds, the cold incubator is employed for all media.

Tube cultivations are usually packed in the incubator in small tin cylinders, such as those in which American cigarettes are sold, or in square tin boxes. Beakers or tumblers may be used for the same purpose, but being fragile are not so convenient. Metal test-tube racks, long enough to just fit into the interior of the incubator and each accommodating two rows of tubes, are also exceedingly useful.

AEROBIC.

(a) Inoculating a tube of sterile nutrient medium with a portion of the material to be examined.

The details of the first of these processes must be varied somewhat according to whether the tubes of nutrient media are inoculated or "planted" from—

The method of preparing tube cultivations from pre-existing cultivations is as follows:

1. Flame the cotton-wool plug of the tube containing the cultivation and also that of the tube of sterile bouillon.

2. Hold the two tubes, side by side, between the left thumb and the first and third fingers, allowing the sealed ends to rest on the dorsum of the hand, and separating the mouths of the tubes (which are pointed to the right) by the tip of the second finger. Keep the tubes as nearly horizontal as is possible without allowing the fluid in the bouillon tube to reach the cotton-wool plug (Fig. 117).

4. Grasp the plug of the tube containing the cultivation between the little finger and palm of the hand and remove it from the tube.

5. Grasp the plug of the bouillon tube between the fourth finger and the ball of the thumb and remove it from the tube.

6. Pass the platinum loop into the tube containing the culture—do not allow the loop to touch the sides of the tube, or the handle to touch the medium—and remove a small portion of the growth; withdraw the loop from the tube, keeping the infected side of the loop downward.

7. Pass the loop into the bouillon tube almost down to the level of the fluid, reverse the loop so that the infected side faces upward, emulsify the portion of the growth in the moisture adhering to the side of the tube which is uppermost. Withdraw the loop.

10. Label the bouillon tube with (a) the name of the organism and (b) the date of inoculation.

1. Oblique tube or slanted tube (Fig. 118), in which the medium has been allowed to solidify whilst the tube was retained in an inclined position, so forming an extensive surface of medium extending from the bottom of the tube almost to its mouth.

2. Straight tube (Fig. 119), in which the medium forms a cylindrical mass in the lower portion of the tube and presents an upper surface which is at right angles to the long axis of the tube.

This is employed for "stab" or "stick" cultivations (Stichcultur), or by inoculating the medium whilst fluid, and allowing to solidify in this position, for "shake" cultivations.

1. Flame the plugs, sterilise the platinum loop (or spatula). Open the tubes and charge the loop as in previous inoculation.

2. Pass the infected loop to the bottom of the tube to be inoculated and draw it, as lightly as possible, along the centre of the surface of the medium, terminating the "streak" over the thin layer of medium near the mouth of the tube.

Smear Culture.—Proceed generally as in streak culture, but rub the infected loop all over the surface of the medium, instead of restricting the inoculation to a narrow line.

Note.—Gelatine and agar oblique tubes should be freshly "slanted" before use.

1. Flame the plugs, open the tubes, sterilise the platinum needle and charge it with the inoculum as in the previous cultivations.

2. Pass the platinum needle into the tube to be inoculated until it touches the centre of the surface of the medium. Now thrust it deeply into the substance of the medium, keeping the needle as nearly as possible in the axis of the cylinder of medium. Then withdraw the needle.

Note.—When gelatine is stored for some time the upper surface of the cylinder becomes concave owing to evaporation. Tubes showing this appearance should be liquefied and again allowed to set before use for stab culture, otherwise when the needle enters the medium, the surface tension will cause the gelatine cylinder to split.

1. Liquefy a tube of nutrient gelatine (or agar, or other similar medium), by heating in a water-bath (Fig. 121).

2. Inoculate the liquefied medium and label it, etc., precisely as if dealing with a tube of bouillon.

3. Place the newly planted tube in the upright position (e. g., in a test-tube rack) and allow it to solidify.

Esmarch's Roll Cultivation.—

1. Liquefy three tubes of gelatine by heat.

2. Prepare three dilutions of the inoculum (as described for plate cultivations, page 228, steps 4 to 7).

3. Roll the tubes, held almost horizontally, in a groove made in a block of ice, until the gelatine has set in a thin film on the inner surface of tube (Fig. 120); or under the cold-water tap.

Fig. 120. Esmarch's roll culture on block of ice. Fig. 120. Esmarch's roll culture on block of ice.

In order that the medium may adhere firmly to the glass, the agar used for roll cultivation should have 1 per cent. gelatine or 1 per cent. gum arabic added to it before sterilisation.

Roll cultivations, which served a most important purpose in the days before the introduction of Petri dishes for plate cultivations, are now obsolete in modern laboratories and are merely mentioned for the benefit of students, since examiners who are interested in the academic and historical aspects of bacteriology sometimes expect candidates to be acquainted with the method of preparing them.

The Preparation of Plate Cultures.

If a small number of bacteria are suspended in liquefied gelatine, agar, or other similar medium, and the infected medium spread out in an even layer over a flat surface and allowed to solidify, each individual micro-organism becomes fixed to a certain spot and its further development is restricted to the vicinity of this spot. After a variable interval the growth of this organism becomes visible to the naked eye as a "colony." This is the principle upon which the method of plate cultivation is based and its practice enables the bacteriologist to study the particular manner of development affected by each species of microbe when growing (a) unrestricted upon the surface of the medium, (b) in the depths of the medium. The method itself is as follows:

Apparatus Required.—

1. Tripod levelling stand.

2. Large shallow glass dish, with a square sheet of plate glass to cover it.

3. Spirit level.

4. Case of sterile Petri dishes.

5. Tubes of sterile nutrient media, gelatine (or agar) previously liquefied by heating in the water-bath and cooled to 42°C., otherwise the heat of the medium would destroy many, if not all, of the bacteria introduced.

6. Tube of cultivation to be planted from.

7. Platinum loop.

8. Bunsen burner.

9. Grease pencil.

Fig. 121.—Handy form of water-bath for melting tubes of agar and gelatine previous to slanting them; or to making shake cultures or pouring plates. Fig. 121.—Handy form of water-bath for melting tubes of agar and gelatine previous to slanting them; or to making shake cultures or pouring plates.

1. Place the glass dish on the levelling tripod (Figs. 122, 123); if gelatine plates are to be poured fill the dish with ice water—gelatine solidifies so slowly that it is necessary to hasten the process; if agar is to be used fill with water at 50°C.—agar sets almost immediately at the room temperature and by slightly retarding the process lumpiness is avoided; cover the dish with the square sheet of glass.

2. Place the spirit level on the sheet of glass and by means of the levelling screws adjust the surface of the glass to the horizontal.

This leveling is an important matter since the development of a colony is to some extent proportionate to the supply of medium available for its nutrition. Thus in a "smear" on sloped tube culture, the colonies at the upper part of the medium are stunted and small but increase in size and luxuriance of growth the nearer they approach to the bottom of the tube, where there is the greatest depth of medium.

3. Place three sterile Petri dishes in a row on the surface of the glass plate and number them 1, 2, and 3, from left to right.

4. Number the previously liquefied tubes of nutrient media 1, 2, and 3. Flame the plugs and see that each plug can be readily removed from the mouth of its tube.

5. Add one loopful of the inoculum to tube No. 1, treating the liquefied medium as bouillon. After replugging, grasp the tube near its mouth by the thumb and first finger of the right hand, and with an even circular movement of the whole arm, diffuse the inoculum throughout the medium; avoid jerky movements, as these cause bubbles of air to form in the medium.

The knack of mixing evenly without producing air bubbles, is not always easily acquired, by this method. An alternative plan is to hold the inoculated tube vertically upright between the opposed palms and to rotate it between them by rapid backward and forward movements of the two hands (Fig. 124).

6. Sterilise the platinum loop, and add two loopfuls of diluted inoculum to tube No. 2, and mix as before.

7. In a similar manner transfer three loopfuls of liquefied medium from tube No. 2 to tube No. 3, and mix thoroughly.

8. Flame the plug of tube No. 1, remove it, then flame the lips of the tube; slightly raise the cover of Petri dish No. 1, introduce the mouth of the tube; then, elevating the bottom of the tube, pour the liquefied medium into the Petri dish, to form a thin layer. Remove the mouth of the tube and close the "plate." If the medium has failed to flow evenly over the bottom of the plate, raise the plate from the levelling platform and by tilting in different directions rectify the fault.

10. Label the plates with the distinctive name or number of the inoculum, also the date; the number of the dilution having been previously indicated (step 3).

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° C. or 42° 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.

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.

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.

Agar	2.0 per cent.
Peptone	1.5 per cent.
Salt	0.5 per cent.
Meat extract	quantum sufficit.

Agar	2.0 per cent.
Peptone	2.0 per cent.
Salt	0.5 per cent.
Meat extract	quantum sufficit.

Ascitic fluid	90 c.c.
Nutrose	6 grammes