The Elements of Bacteriological Technique Part 36

~Lemco Broth.~--

1. Measure out 250 c.c. distilled water into a litre flask.

2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a piece of clean filter paper and add to the water in the flask. Shake the flask well to make an even emulsion of the meat extract.

3. Weigh out Witte's peptone (10 grammes), salt (5 grammes).

Mix into smooth paste with 100 c.c. distilled water previously heated to 60C.

4. Add the peptone salt emulsion to the meat extract emulsion in the flask and add 650 c.c. distilled water. Heat in the steamer for forty-five minutes.

5. Standardise the medium and complete as for nutrient bouillon.

~Nutrient Gelatine.~--

1. Weigh a 2-litre flask on a trip balance (Fig. 106) and note the weight, or counterpoise carefully.

[Ill.u.s.tration: FIG. 106.--Trip balance.]

An extremely useful counterpoise is a small sheet-bra.s.s cylinder about 38 mm. high and 38 mm. in diameter, with a funnel-shaped top and provided with a side tube by which its contents, fine "dust" shot, may be emptied out (Fig. 107).

[Ill.u.s.tration: FIG. 107.--Counterpoise; weight when empty, 35 grammes; when full of dust shot, 200 grammes.]

2. Measure out double strength meat extract, 500 c.c., into the "tared"

flask.

3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make into a thick paste with 150 c.c. distilled water; then add the emulsion to the meat extract in the flask; also add 100 grammes sheet gelatine cut into small pieces; place the flask in the water-bath and raise to the boil.

[Ill.u.s.tration: FIG. 108.--Arrangement of steam can and water-bath for the preparation of media.]

4. Arrange a 5-litre tin can (with copper bottom, such as is used in the preparation of distilled water) by the side of the water bath, fill the can with boiling water and place a lighted Bunsen burner under it. Fit a long safety tube to the neck of the can and also a delivery tube, bent twice at right angles; adjust the tube to reach to the bottom of the interior of the flask containing the gelatine, etc. (Fig. 108).

5. Keep the water in the steam can vigourously boiling, and so steam at 100C, bubbling through the medium ma.s.s, for ten minutes, by which time complete solution of the gelatine is effected. A certain amount of steam will condense as water in the medium flask during this process--hence the necessity for the use of double strength meat extract--but if the water bath is kept boiling this condensation will not exceed 100 c.c.

6. Weigh the flask and its contents; then (1115[4] grammes + weight of the flask) minus (weight of the flask and its contents) equals the weight of water required to make up the bulk to 1 litre. The addition of the requisite quant.i.ty of water is carried out as follows:

In one pan of the trip balance place the counterpoise of the tared flask (or its equivalent in weights) together with the weights making up the _calculated medium weight_. In the opposite pan place the flask containing the medium ma.s.s. Now add boiling distilled water from a wash bottle until the two pans are exactly balanced.

7. t.i.trate and estimate the reaction of the medium ma.s.s; control the result. Calculate the amount of soda solution required to make the reaction of the medium ma.s.s +10 (i. e., calculate for 1000 c.c., less the quant.i.ty used for the t.i.trations).

8. Add the necessary amount of soda solution and heat in the steamer at 100 C. for twenty minutes, to precipitate the phosphates, etc.

9. Allow the medium ma.s.s to cool to 60 C. Well whip the whites of two eggs, add to the contents of the flask and replace in the steamer at 100 C. for about half an hour (until the egg-alb.u.men has coagulated and formed large, firm ma.s.ses floating on and in clear gelatine).

10. Filter through papier Chardin into a sterile flask.

11. Tube in quant.i.ties of 10 c.c.

12. Sterilise in the steamer at 100 C. for twenty minutes on each of three consecutive days--i. e., by the discontinuous method.

~Nutrient Agar-agar.~--

1. Weigh a 2-litre flask and note the weight--or counterpoise exactly.

2. Measure out double strength meat extract, 500 c.c., into the "tared"

flask.

3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and 20 grammes of powdered agar, and make into a thick paste with 150 c.c.

distilled water, and add to the meat extract in the flask; place the flask in a water-bath.

4. Arrange the steam can and water-bath as already directed (for the preparation of gelatine) and figured.

5. Bubble live steam (at 100 C.) through the medium ma.s.s, for twenty-five minutes, by which time complete solution of the agar is effected.

6. Now weigh the flask and its contents; then (1035[5] grammes + weight of flask) minus (weight of flask and its contents) equals the weight of water required to make up the bulk of the medium to 1 litre. Add the requisite amount (see preparation of gelatine, page 166, step 6).

7. t.i.trate, and estimate the reaction of the medium ma.s.s; control the result. Calculate the amount of soda solution required to make the reaction of the medium ma.s.s + 10 (i. e., calculated for 1000 c.c., less the quant.i.ty used for the t.i.trations).

8. Add the necessary amount of soda solution and replace in the steamer for twenty minutes (to complete the precipitation of the phosphates, etc.).

9. Allow the medium ma.s.s to cool to 60 C. Well whip the whites of two eggs, add to the contents of the flask, and replace in the steamer at 100 C. for about _one hour_ (until the egg-alb.u.men has coagulated and formed large, firm ma.s.ses floating on and in clear agar.)

10. Filter through papier Chardin, by the aid of a hot-water funnel, if necessary (Fig. 101), into a sterile flask.

11. Tube in quant.i.ties of 10 c.c. or 15 c.c.

12. Sterilise in the steamer at 100 C. for thirty minutes on each of three consecutive days--i. e., by the discontinuous method.

~Blood-serum (Insp.i.s.sated).~--

1. Sterilise cylindrical gla.s.s jar (Fig. 109) and its cover by dry heat, or by washing first with ether and then with alcohol and drying.

2. Collect blood at the slaughter house from ox or sheep in the sterile cylinder.

3. Allow the vessel to stand for fifteen minutes for the blood to coagulate. (This must be done before leaving the slaughterhouse, otherwise the serum will be stained with haemoglobin.)

4. Separate the clot from the sides of the vessel by means of a sterile gla.s.s rod (the yield of serum is much smaller when this is not done), and place the cylinder in the ice-chest for twenty-four hours.

5. Remove the serum with sterile pipettes, or syphon it off, and fill into sterile tubes (5 c.c. in each) or flasks.

6. Heat tubes containing serum to 56 C. in a water-bath for half an hour on each of two successive days.

7. On the third day, heat the tubes, in a sloping position, in a serum insp.i.s.sator to about 72 C. (A coagulum is formed at this temperature which is fairly transparent; above 72 C., a thick turbid coagulum is formed.)

[Ill.u.s.tration: FIG. 109.--Blood-serum jar with wicker basket for transport.]

The serum insp.i.s.sator (Fig. 110) in its simplest form is a double-walled rectangular copper box, closed in by a loose gla.s.s lid, and cased in felt or asbestos--the s.p.a.ce between the walls is filled with water. The insp.i.s.sator is supported on adjustable legs so that the serum may be solidified at any desired "slant," and is heated from below by a Bunsen burner controlled by a thermo-regulator. The more elaborate forms resemble the hot-air oven (Fig. 26) in shape and are provided with adjustable shelves so that any desired obliquity of the serum slope can be obtained.