  In this chapter are collected a number of media which have been elaborated by various workers for special purposes, grouped together under headings which indicate their chief utility. In many instances the name of the originator of the medium is given, but without reference to his original instructions, since these are in many cases inadequate to the requirements of the isolated worker, who would probably fail to reproduce the medium in a form giving the results attributed to it by its author. Such modifications have therefore been introduced as make for uniformity between the different batches of media. A considerable number of coloured media, chiefly intended for work with intestinal bacteria, have been included; but beyond the fact that the author's modification of the Drigalski-Conradi medium has been included amongst the routine media of the laboratory, no comment has been made upon their relative values, since only by observation and practice can the skill necessary to utilise their full value be acquired. The instructions as to sterilisation are rarely given in full; the routine method of exposure in the steam steriliser at 100° C. (without pressure) for twenty minutes on each of three successive days for all fluid media, and thirty minutes on each of three successive days for all liquefiable or solid media must be carried out; and only when these general rules are to be departed from are further details given. Media for the Study of the Chemical Composition of Bacteria. Asparagin Medium (Uschinsky).— 1. Weigh out and mix
2. Dissolve the mixture in distilled water 1000 c.c. 3. Add glycerine, 40 c.c. 4. Tube, and sterilise as for nutrient bouillon. Asparagin Medium (Frankel and Voges).— 1. Weigh out and mix
and dissolve in
2. Tube, and sterilise as for nutrient bouillon. Note.—Either of the above asparagin media, after the addition of 10 per cent. gelatine or 1.5 per cent. agar, may be advantageously employed in the solid condition. Proteid Free Broth (Uschinsky).— 1. Weigh out and mix
2. Dissolve the mixture in distilled water 1000 c.c. 3. Add glycerine 30 c.c. 4. Tube and sterilise as for nutrient broth. Media for the Study of Biochemical Reaction. Inosite-free Media—Bouillon (Durham).— 1. Prepare meat extract, 1000 c.c. (vide page 148), from bullock's heart which has been "hung" for a couple of days. 2. Prepare nutrient bouillon (+10), 1000 c.c. (vide, page 161), from the meat extract, and store in 1-litre flask. 3. Inoculate the bouillon from a pure cultivation of the B. lactis aerogenes, and incubate at 37° C. for forty-eight hours. 4. Heat in the steamer at 100° C. for twenty minutes to destroy the bacilli and some of their products. 5. Estimate the reaction of the medium and if necessary restore to +10. 6. Inoculate the bouillon from a pure cultivation of the B. coli communis and incubate at 37° C. for forty-eight hours. 7. Heat in the steamer at 100° C. for twenty minutes. Now fill two fermentation tubes with the bouillon, tint with litmus solution, and sterilise; inoculate with B. lactis aerogenes. If no acid or gas is formed, the bouillon is in a sugar-free condition; but if acid or gas is present, again make the bouillon in the flask +10, reinoculate with one or other of the above-mentioned bacteria, and incubate; then test again. Repeat this till neither acid nor gas appears in the medium when used for the cultivation of either of the bacilli referred to above. 8. After the final heating, stand the flask in a cool place and allow the growth to sediment. Filter the supernatant broth through Swedish filter paper. If the filtrate is cloudy, filter through a porcelain filter candle. 9. Tube, and sterilise as for bouillon. Bouillon prepared in the above-described manner will prove to be absolutely sugar-free; and from it may be prepared nutrient sugar-free gelatine or agar, by dissolving in it the required percentage of gelatine or agar respectively and completing the medium according to directions given on pages 166 and 167. The most important application of inosite-free bouillon is its use in the preparation of sugar bouillons, whether glucose, maltose, lactose, or saccharose, of exact percentage composition. Sugar (Dextrose) Bouillon.— 1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6) or sugar-free bouillon (vide supra). 2. Weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and dissolve in the fluid. 3. Tube, and sterilise as for bouillon. Ordinary commercial glucose serves the purpose equally well, but is not recommended, as during the process of sterilisation it causes the medium to gradually deepen in colour. Note.—In certain cases a corresponding percentage of lactose, maltose, or saccharose is substituted for glucose. Sugar Gelatine.— 1. Prepare nutrient gelatine (vide page 164, sections 1 to 7). Measure out 1000 c.c. 2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the hot gelatine. 3. Filter through papier Chardin. 4. Tube, and sterilise as for nutrient gelatine. Sugar Agar.— 1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c. 2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the clear agar. 3. Tube, and sterilise as for nutrient agar. Note.—Other "sugar" media are prepared by substituting a corresponding percentage of lactose, maltose (or any other of the substances referred to under "Sugar Media," page 177) for the glucose. Iron Bouillon.— 1. Measure out nutrient bouillon, 1000 c.c. (vide page 141, sections 1 to 6). 2. Weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon. 3. Tube, and sterilise as for bouillon. Note.—The lactate of iron may be substituted for the tartrate. Lead Bouillon.— 1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6). 2. Weigh out lead acetate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon. 3. Tube, and sterilise as for bouillon. Nitrate Bouillon.— 1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6). 2. Weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and dissolve it in the bouillon. 3. Tube, and sterilise as for bouillon. Note.—The nitrate of sodium or ammonium may be substituted for that of potassium, or the salt may be added in the proportion of from 0.1 to 1 per cent. to meet special requirements. Iron Peptone Solution (Pakes).— 1. Weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap water, previously heated to about 60°C. 2. Wash the emulsion into a litre flask with 800 c.c. tap water. 3. Weigh out salt, 5 grammes, and sodium phosphate, 3 grammes, and dissolve in the mixture in the flask. 4. Heat the mixture in the steamer at 100° C. for thirty minutes, 5. Fill into tubes in quantities of 10 c.c. each. 6. Add to each tube 0.1 c.c. of a 2 per cent. neutral solution of ferric tartrate. (A yellowish-white precipitate forms.) 7. Sterilise as for nutrient bouillon. Lead Peptone Solution.— Prepare as for iron peptone solution but in step 6 substitute 0.1 c.c. of a 1 per cent. neutral aqueous solution of lead acetate. Nitrate Peptone Solution (Pakes).— 1. Weigh out WittÉ's peptone, 10 grammes, and emulsify it with 200 c.c. ammonia-free distilled water previously heated to 60°C. 2. Wash the emulsion into a flask and make up to 1000 c.c., with ammonia-free distilled water. 3. Heat in the steamer at 100° C. for twenty minutes. 4. Weigh out sodium nitrate, 1 gramme, and dissolve in the contents of the flask. 5. Filter through Swedish filter paper. 6. Tube, and sterilise as for nutrient bouillon. Litmus Bouillon.— 1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6). 2. Add sufficient sterile litmus solution to tint the medium a dark lavender colour. (Media rendered +10 will usually react very faintly alkaline or occasionally neutral to litmus.) 3. Tube, and sterilise as for bouillon. Rosolic Acid Peptone Solution.— 1. Weigh out rosolic acid (corallin), 0.5 gramme, and dissolve it in 80 per cent. alcohol, 100 c.c. Keep this as a stock solution. 2. Measure out peptone water (Dunham), 100 c.c., and rosolic acid solution, 2 c.c., and mix. 3. Heat in the steamer at 100° C. for thirty minutes. 4. Filter through Swedish filter paper. 5. Tube, and sterilise as for nutrient bouillon. Capaldi-Proskauer Medium, No. I.— 1. Weigh out and mix
2. Dissolve in water 1000 c.c. in a 2-litre flask 3. Weigh out and mix
and add to contents of flask. 4. Measure out 25 c.c. of the solution and titrate it against decinormal sodic hydrate, using litmus as the indicator. Control the result and estimate the amount of sodic hydrate necessary to be added to render the remainder of the solution neutral to litmus. Add this quantity of sodic hydrate. 5. Filter. 6. Add litmus solution 47.5 c.c. (= 5 per cent.). 7. Tube, and sterilise as for nutrient bouillon. Capaldi-Proskauer Medium No. II.— 1. Weigh out and mix
2. Dissolve in water 1000 c.c. in a 2-litre flask. 3. Neutralise to litmus as in No. I (vide supra, Step 4). 4. Filter. 5. Add litmus solution 47.5 c.c. (= 5 per cent.). 6. Tube, and sterilise as for nutrient bouillon. Urine Media. Bouillon.— 1. Collect freshly passed urine in sterile flask. 2. Place the flask in the steamer at 100° C. for thirty minutes. 3. Filter through two thicknesses of Swedish filter paper. 4. Tube, and sterilise as for nutrient bouillon. (Leave the reaction unaltered.) Urine Gelatine.— 1. Collect freshly passed urine in sterile flask. 2. Take the specific gravity, and, if above 1010, dilute with sterile water until that gravity is reached. 3. Estimate (with control) at the boiling-point, and note the reaction of the urine. 4. Weigh out gelatine, 10 per cent., and add to the urine in the flask. 5. Heat in the steamer at 100° C. for one hour to dissolve the gelatine. 6. Estimate the reaction and add sufficient caustic soda solution to restore the reaction of the medium mass to the equivalent of the original urine. 7. Cool to 60° C. and clarify with egg as for nutrient gelatine (vide page 166). 8. Filter through papier Chardin. 9. Tube, and sterilise as for nutrient gelatine. Urine Gelatine (Heller).— 1. Collect freshly passed urine in sterile flask. 2. Filter through animal charcoal to remove part of the colouring matter. 3. Take the specific gravity, and if above 1010, dilute with sterile water till this gravity is reached. 4. Add WittÉ's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10 per cent. 5. Heat in the steamer at 100° C. for one hour, to dissolve the gelatine, etc. 6. Add normal caustic soda solution in successive small quantities, and test the reaction from time to time with litmus paper, until the fluid reacts faintly alkaline. 7. Cool to 60° C. and clarify with egg as for nutrient gelatine (vide page 166). 8. Filter through papier Chardin. 9. Tube, and sterilise as for nutrient gelatine. Urine Agar.— 1. Collect freshly passed urine in sterile flask. 2. Take the specific gravity and if above 1010, dilute with sterile water till this gravity is reached. 3. Weigh out 1.5 per cent. or 2 per cent. powdered agar, and add it to the urine. 4. Heat in the steamer at 100° C. for ninety minutes to dissolve the agar. 5. Cool to 60° C. and clarify with egg as for nutrient agar (vide page 168). 6. Filter through papier Chardin, using the hot-water funnel. 7. Tube, and sterilise as for nutrient agar. (Leave the reaction unaltered.) Serum Sugar Media (Hiss).— In these media the fermentation of carbohydrate substance by bacterial action is indicated by the coagulation of the serum proteids in addition to the production of an acid reaction. Serum Dextrose Water (Hiss).— 1. Measure out into a litre flask
2. Weigh out
and dissolve in the serum water. 3. Filter through Swedish filter paper. 4. Measure out and add to the medium
5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100° C. for twenty minutes on each of three successive days. LÆvulose, galactose, maltose, lactose, etc., can be substituted in similar amounts for dextrose and the medium completed as above. Omeliansky's Nutrient Fluid (For Cellulose Fermenters).— 1. Weigh out and mix
2. Dissolve in distilled water 4000 c.c. 3. Flask in quantities of 250 c.c. 4. Weigh out and add 5 grammes precipitated chalk to each flask. 5. Sterilise in the steamer at 100° C. for twenty minutes on each of three successive days. Media for the Study of Chromogenic Bacteria. Milk Rice (Eisenberg).— 1. Measure out nutrient bouillon, 70 c.c., and milk, 210 c.c., and mix thoroughly. 2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture. 3. Fill the paste into sterile capsules, spreading it out so as to form a layer about 0.5 cm. thick, over the bottom of each. 4. Heat over a water-bath at 100° C. until the mixture solidifies. 5. Replace the lids of the capsules. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days. (A solid medium of the colour of cafÉ au lait is thus produced.) Milk Rice (Soyka).— 1. Measure out nutrient bouillon, 50 c.c., and milk, 150 c.c., and mix thoroughly. 2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture. 3. Fill the paste into sterile capsules, to form a layer over the bottom of each. 4. Replace the lids of the capsules. 5. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days. (A pure white, opaque medium is thus formed.) Media for the Study of Phosphorescent and Photogenic Bacteria. Fish Bouillon.— 1. Weigh out herring, mackerel, or cod, 500 grammes, and place in a large porcelain beaker (or enamelled iron pot). 2. Weigh out sodium chloride, 26.5 grammes; potassium chloride, 0.75 gramme; magnesium chloride, 3.25 grammes; and dissolve in 500 c.c. distilled water. Add the solution to the fish in the beaker. 3. Place the beaker in a water-bath and proceed as in preparing meat extract—i. e., heat gently at 40° C. for twenty minutes, then rapidly raise the temperature to, and maintain at, the boiling-point for ten minutes. 4. Strain the mixture through butter muslin into a clean flask. 5. Weigh out peptone, 5 grammes, and emulsify with about 200 c.c. of the hot fish water; incorporate thoroughly with the remainder of the fish water in the flask. 6. Heat in the steamer at 100° C. for twenty minutes to complete the solution of the peptone. 7. Filter through Swedish filter paper. 8. When the fish bouillon is cold, if it is to be used as fluid medium, make up to 1000 c.c. by the addition of distilled water. If, however, it is to be used as the basis for agar or gelatine media store it in the "Double Strength" condition. 9. Tube and sterilise as for nutrient bouillon. As an alternative method "Marvis" fish food (16 grammes) may be substituted for the 500 grammes of fresh fish. Fish Gelatine.— 1. Measure out double strength fish bouillon, 500 c.c., into a "tared" 2-litre flask. 2. Add sheet gelatine, 100 grammes, cut into small pieces. 3. Bubble live steam through the mixture for fifteen minutes to dissolve the gelatine. 4. Weigh the flask and its contents; adjust the weight to the calculated figure for one litre of medium (1135.5 grammes) by the addition of distilled water at 100° C. (vide page 166). 5. Cool to below 60°C., and clarify with egg. 6. Filter through papier Chardin. 7. Tube, and sterilise as for nutrient gelatine. Shake well after the final sterilisation, to aerate the medium. Fish Gelatine-Agar.— 1. Weigh out powdered agar, 5 grammes, and emulsify it with 200 c.c. double strength fish bouillon. 2. Wash the emulsion into a "tared" 2-litre flask with 300 c.c. fish bouillon. 3. Weigh out sheet gelatine, 70 grammes, cut it into small pieces and add it to the contents of the flask. 4. Bubble live steam through the mixture to dissolve the gelatine and agar. 5. Weigh the flask and contents. Adjust the weight to the calculated figure for one litre of medium (1110.5 grammes) by the addition of distilled water at 100° C. (vide page 166). 6. Cool to below 60° C. and clarify with egg. 7. Filter through papier Chardin. 8. Tube, and sterilise as for nutrient gelatine. Shake well after the final sterilisation, to aerate the medium. Media for the Study of Yeasts and Moulds. Pasteur's Solution.— (Reaction alkaline). 1. Weigh out and mix the ash from 10 grammes of yeast; ammonium tartrate, 10 grammes; cane sugar, 100 grammes. 2. Dissolve the mixture in distilled water, 1000 c.c. 3. Tube or flask, and sterilise as for nutrient bouillon. Yeast Water (Pasteur).— 1. Weigh out pressed yeast, 75 grammes; place in a 2-litre flask and add 1000 c.c. distilled water. 2. Heat in the steamer at 100° C. for thirty minutes. 3. Filter through papier Chardin. 4. Tube or flask, and sterilise as for nutrient bouillon. Cohn's Solution.— 1. Weigh out and mix
and dissolve in
2. Tube, or flask and sterilise as for nutrient bouillon. Naegeli's Solution.— 1. Weigh out and mix
and dissolve in
2. Tube or flask; sterilise as for nutrient bouillon. Plaster-of-Paris Discs.— 1. Take large corks, 2.5 cm. diameter, and roll a piece of stiff note-paper round each, so that about a centimetre projects as a ridge above the upper surface of the cork, and secure in position with a pin (Fig. 112). 2. Mix plaster-of-Paris into a stiff paste with distilled water, and fill each of the cork moulds with the paste. 3. When the plaster has set, remove the paper from the corks, and raise the plaster discs. 4. Place the plaster discs on a piece of asbestos board and sterilise by exposing in the hot-air oven to 150° C. for half an hour. Fig. 112.—Cork and paper mould for plaster-of-Paris disc. Fig. 112.—Cork and paper mould for plaster-of-Paris disc. 5. Remove the sterile discs from the oven by means of sterile forceps, place each inside a sterile capsule, and moisten with a little sterile water. 6. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days. Gypsum Blocks (Engel and Hansen).— These are in the form of truncated cones and for their preparation small tin moulds are required, each having a diameter of 5.5 cm. at the base and 4 cm. at the truncated apex. The height (or depth) of a mould is 4.5 to 5 cm. 1. Mix powdered calcined gypsum into a stiff paste with distilled water. 2. Fill the paste into the moulds and allow it to set and dry by exposure to air. 3. Remove the block from the mould and transfer it to a double glass dish of adequate size (7 cm. diameter × 7 cm. high). 4. Sterilise block in its dish for one hour in the hot-air oven at 115°C. 5. Carefully open the dish and add sterile distilled water to moisten the block and form a layer in the bottom of the dish 1 cm. deep. Wine Must.—(Wine must is obtained from Sicily, in hermetically sealed tins, in a highly concentrated form—as a thick syrup—but not sterilised.) 1. Weigh out "wine must," 200 grammes, place in a 2-litre flask and add distilled water, 800 c.c. 2. Weigh out ammonium tartrate, 5 grammes, and add to the dilute must. 3. Place the flask in a water-bath regulated to 60° C. for one hour and incorporate the mixture thoroughly by frequent shaking. 4. Filter through papier Chardin. 5. Tube, and sterilise as for nutrient bouillon. Wheat Bouillon (Gasperini).— 1. Weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5 gramme; potassium nitrate, 1 gramme; glucose, 15 grammes. 2. Dissolve the mixture in 1000 c.c. of water heated to 100°C. 3. Filter through papier Chardin. 4. Tube, and sterilise as for nutrient bouillon. Bread Paste.— 1. Grate stale bread finely on a bread-grater. 2. Distribute the crumbs in sterile Erlenmeyer flasks, sufficient to form a layer about one centimetre thick over the bottom of each. 3. Add as much distilled water as the crumbs will soak up, but not enough to cover the bread. 4. Plug the flasks and sterilise in the steamer at 100° C. for thirty minutes on each of four consecutive days. Media for the Study of Parasitic Moulds. French Proof Agar (Sabouraud).— 1. Weigh out Chassaing's peptone, 10 grammes, and emulsify it with 200 c.c. distilled water previously heated to 60°C. 2. Weigh out powdered agar, 13 grammes, and emulsify with 200 c.c. cold distilled water. 3. Mix the two emulsions and wash into a tared 2-litre flask with 600 c.c. distilled water. 4. Bubble live steam through the mixture for twenty minutes, to dissolve the agar. 5. Cool to 60° C. and clarify with egg as for nutrient agar (vide page 168). 6. Filter through Papier Chardin, using the hot-water funnel. 7. Weigh out French maltose, 40 grammes, and dissolve in the agar. 8. Tube, and sterilise as for nutrient agar. English Proof Agar (Blaxall).—Substitute WittÉ's peptone for that of Chassaing, and proceed as for French proof agar. French Mannite Agar, Sabouraud.—(For cultivation of Favus.) Proceed exactly as in preparing French Proof agar vide supra substituting Mannite (38 grammes) for maltose. Media for the Study of Milk Bacteria. Gelatine Agar.—This medium is prepared by adding to nutrient gelatine sufficient agar to ensure the solidity of the medium when incubated at temperatures above 22° C. If it is intended 1. Measure out 400 c.c. double strength meat extract into a "tared" 2-litre flask, and add to it gelatine, 100 grammes. 2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold distilled water and add to the contents of the flask. 3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes. 4. Weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100 c.c. double strength meat extract previously heated to 60°C., and add to the contents of the flask. 5. Replace in the steamer for fifteen minutes. Then adjust the weight to the calculated figure for one litre (in this instance 1120 grammes) by the addition of distilled water at 100°C. 6. Estimate the reaction; control the result. Then add sufficient caustic soda solution to render the reaction +10. 7. Replace in the steamer at 100° C. for twenty minutes. 8. Cool to 60° C. Clarify with egg as for nutrient agar. 9. Filter through papier Chardin, using the hot-water funnel. 10. Tube, and sterilise as for nutrient agar. Agar Gelatine (Guarniari).— 1. Measure out double strength meat extract, 400 c.c., into a "tared" 2-litre flask, and add to it gelatine, 50 grammes. 2. Weigh out powdered agar, 3 grammes; emulsify with cold distilled water, 50 c.c., and add to the contents of the flask. 3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes. 4. Weigh out WittÉ's peptone, 25 grammes; salt, 5 grammes, and emulsify with 100 c.c. double strength meat extract previously heated to 60°C., and add to the contents of the flask. 5. Replace in the steamer for fifteen minutes. 6. Weigh the flask and make up the medium mass to the calculated figure for one litre (1083 grammes) by the addition of distilled water at 100°C. 7. Neutralise carefully to litmus paper by the successive additions of small quantities of normal soda solution. 8. Replace in the steamer at 100° C. for twenty minutes. 9. Cool to 60° C. Clarify with egg as for nutrient agar. 10. Filter through papier Chardin, using the hot-water funnel. 11. Tube, and sterilise as for nutrient agar. Whey Gelatine.— 1. Curdle fresh milk by warming to 60°C., and adding rennet; filter off the whey into a sterile "tared" flask. 2. Estimate and note the reaction of the whey. 3. Weigh out gelatine, 10 per cent., and add it to the whey in the flask. 4. Bubble live steam through the mixture fifteen minutes to dissolve the gelatine; and weigh. 5. Estimate the reaction of the medium mass; then add sufficient caustic soda solution to restore the reaction of the medium mass (i. e., total weight minus weight of flask) to the equivalent of the original whey. 6. Cool to 60° C. and clarify with egg as for nutrient gelatine (vide page 166). 7. Filter through papier Chardin. 8. Tube, and sterilise as for nutrient gelatine. Whey Agar.— 1. Curdle fresh milk by warming to 60°C., and adding rennet; filter off the whey into a sterile flask. 2. Weigh out agar, 1.5 or 2 per cent., and add it to the whey in the flask. 3. Bubble live steam through the mixture for twenty minutes, to dissolve the agar. 4. Cool to 60°C.; clarify with egg as for nutrient agar (vide page 168). 5. Filter through papier Chardin, using the hot-water funnel. 6. Tube, and sterilise as for nutrient agar. Litmus Whey.— 1. Curdle fresh milk by warming to 60° C. and adding rennet. 2. Filter off the whey through butter muslin into a sterile flask. 3. Neutralise to litmus by the cautious addition of citric acid solution 4 per cent. (Do not neutralise with mineral acid.) 4. Heat in the steamer at 100° C. for one hour to coagulate all the proteid. (If the whey is cloudy when removed from the steamer allow it to stand for forty-eight hours in the ice chest and then decant off the clear fluid—or filter through a Berkefeld filter candle.) 5. Filter into a sterile flask. 6. Tint the whey with litmus solution to a deep purple red. 7. Tube, and sterilise as for milk. Litmus Whey (Petruschky).— 1. Measure out into a flask
2. Add
and boil. 3. Filter off coagulated casein. 4. Neutralise to litmus by the addition of n/1 caustic soda solution and boil. Whey now cloudy and acid again. 5. Again neutralise to litmus by addition of n/10 caustic soda solution. 6. Filter. 7. Tint the whey with neutral litmus solution to a deep purple colour. 8. Tube and sterilise as for milk. Litmus Whey Gelatine.— 1. Measure out milk 1000 c.c. into a tared 2-litre flask. 2. Add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and boil for five minutes. 3. Filter off the casein, and make the whey faintly alkaline to litmus. 4. Weigh out
and emulsify in a few cubic centimeters of the whey and return to the flask. 5. Weigh out
add it to the whey in the flask and incorporate the mixture by bubbling through live steam. 6. Clear with egg and filter. 7. Make the weight of the medium mass to the calculated figure for one litre (1060 grammes) by the addition of distilled water. 8. Weigh out
and dissolve in the fluid whey gelatine. 9. Add sterile litmus solution to the required tint. 10. Tube and sterilise for twenty minutes in steamer at 100°C. on each of five successive days. This medium will remain semi-fluid at the room temperature, and may be used for cultures in the cool or hot incubator. Litmus Whey Agar is prepared in a similar manner to Whey Gelatine, with the substitution of 15 grammes of agar for the gelatine. Malt Extract Solution (Herschell).— 1. Measure into a flask distilled water 1000 c.c. 2. Weigh out
and add to distilled water in flask. 3. Boil for five minutes, allow to stand, and decant off clear fluid from sediment. 4. Tube and sterilise as for nutrient bouillon. Media for the Study of Earth Bacteria, Nitrogen Fixers. Earthy Salts Agar (Lipman and Brown).—(For the enumeration of soil organisms.) 1. Measure out
Emulsify in 200 c.c. distilled water. 2. Wash the agar emulsion into a tared 2-litre flask with 400 c.c. distilled water. 3. Weigh out
Emulsify in 50 c.c. distilled water and add to the contents of the flask. 4. Bubble live steam through the mixture for twenty minutes to dissolve the agar. 5. Weigh out and mix
and add to the contents of the flask. 6. Adjust the weight of the medium mass to the calculated figure for one litre (1025 grammes) by the addition of distilled water at 100°C. 7. Titrate the medium mass and adjust the reaction to +5. 8. Cool to 60° C. Clarify with egg and filter. 9. Tube in quantities of 10 c.c. and sterilise as for nutrient agar. Beyrinck's Solution. I.—(For the cultivation of nitrogen fixing organisms.) 1. Weigh out and mix 1 gramme potassium hydrogen phosphate, 0.2 gramme magnesium sulphate, and 0.02 gramme sodium chloride. 2. Dissolve in water 1000 c.c., in a 2-litre flask. 3. Add 1 c.c. of a one per thousand aqueous solution of ferrous sulphate. 4. Add 1 c.c. of a one per thousand solution manganese sulphate. 5. Weigh out 20 grammes dextrose and add to the contents of the flask (dextrose up to 40 grammes may be used for the different organisms). 6. Steam for twenty minutes, filter. 7. Tube, and sterilise as for nutrient bouillon. Beyrinck's Solution. II.—(For growth of Azobacter.) Proceed as in preparing solution No. I, substituting mannite for dextrose in step 5. Winogradsky's Solution (for Nitric Organisms).— 1. Weigh out and mix.
and dissolve in
2. Fill into flasks, in quantities of 20 c.c. and add to each a small quantity of freshly washed magnesium carbonate. 3. Sterilise in the steamer at 100° C. for twenty minutes on each of three consecutive days. 4. Add to each flask containing 20 c.c. solution, 2 c.c. of a sterile 2 per cent. solution of ammonium sulphate. 5. Incubate at 37° C. for forty-eight hours and eliminate any contaminated culture flasks. Store the remainder for future use. Winogradsky's Solution (for Nitrous Organisms).— 1. Weigh out and mix
and dissolve in
2. Add 5 to 10 grammes basic magnesium carbonate, previously sterilised by boiling. 3. Fill into flasks and sterilise, etc., as for previous solution. Silicate Jelly (Winogradsky).— 1. Weigh out and mix
and dissolve in
Label—Solution A. 2. Weigh out and mix and dissolve in
Label—Solution B. 3. Weigh out
and dissolve in
4. Pour the silicic acid solution into a large porcelain basin. 5. Mix equal quantities of the solutions A and B; then add successive small quantities of the mixed salts to the silicic acid solution, stirring continuously with a glass rod, until a jelly of sufficiently firm consistence has been formed. 6. Spread a layer of this jelly over the bottom of each of several large capsules or "plates." 7. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days. Media for the Study of Water Bacteria. Naehrstoff Agar (Hesse and Niedner).—(For enumeration of water organisms.) 1. Weigh out: agar, 12.5 grammes and emulsify in 250 c.c. distilled water. 2. Wash the agar emulsion into a tared 2-litre flask with a further 250 c.c. distilled water. 3. Dissolve by bubbling live steam through the mixture. 4. Emulsify Naehrstoff-Heyden (albumose) 7.5 grammes in 200 c.c. cold distilled water and add to melted agar. 5. Adjust weight of medium mass to the calculated figure for one litre (1020 grammes) by addition of distilled water at 100° C. 6. Clarify with white of egg and filter. 7. Tube in quantities of 10 c.c. and sterilise in the steamer at 100° C. for twenty minutes on each of three successive days. Bile Salt Broth—Double Strength.— 1. Weigh out WittÉ's peptone, 40 grammes, and emulsify with 300 c.c. distilled water previously warmed to 60° C. 2. Wash the peptone emulsion into a litre flask with 600 c.c. distilled water. 3. Weigh out sodium taurocholate, 10 grammes, and glucose, 10 grammes; dissolve in 100 c.c. distilled water and add to the peptone emulsion in the flask. 4. Heat in the steamer at 100° C. for twenty minutes. 5. Filter through Swedish filter paper into a sterile flask. 6. Add sterile neutral litmus solution sufficient to colour the medium to a deep purple. 7. Fill into small Erlenmeyer flasks in quantities of 25 c.c. 8. Sterilise as for nutrient bouillon. Media for the Study of Plant Bacteria.
Hay Infusion.— 1. Weigh out dried hay, 10 grammes, chop it up into fine particles and place in a flask. 2. Add 1000 c.c. distilled water, heated to 70° C.; close the flask with a solid rubber stopper. 3. Macerate in a water-bath at 60° C. for three hours. 4. Replace the stopper by a cotton-wool plug, and heat in the steamer at 100° C. for one hour. 5. Filter through Swedish filter paper. 6. Tube, and sterilise as for nutrient bouillon. Haricot Bouillon.—(For cultivation of bacteria from tubercles of Legumes.) 1. Measure out 1000 c.c. distilled water into a 2-litre flask. 2. Weigh out 250 grammes haricot beans and add to the water in the flask. 3. Weigh out 10 grammes sodium chloride and add to the contents of the flask. 4. Add 1 c.c. of a 1 per cent. solution of sodium bicarbonate. 5. Place in the steamer at 100° C. for thirty minutes. 6. Filter. 7. Weigh out 20 grammes saccharose and add to the filtrate. 8. Tube, and sterilise as for nutrient bouillon. Haricot Agar.— 1. Measure out 400 c.c. distilled water into a "tared" 2-litre flask. 2. Weigh out 15 grammes agar and mix into a thick paste with 100 c.c. cold distilled water, and add to the flask. 3. Dissolve the agar by bubbling live steam through the mixture as in making nutrient agar. 4. Weigh out 250 grammes haricot beans, place in the flask with the agar mixture. 5. Add 1 c.c. of 1 per cent. aqueous solution sodium bicarbonate. 6. Weigh out 10 grammes sodium chloride and add to the contents of the flask. 7. Place in the steamer at 100° C. for thirty minutes. 8. Adjust the weight of the medium mass to 1030 grammes (the figure per litre obtained experimentally) by the addition of distilled water at 100° C. 9. Cool to 60°C., clarify with egg and filter. 10. Weigh out 20 grammes saccharose and add to the contents of the flask. 11. Tube, and sterilise as for nutrient agar. Wood Ash Agar.— 1. Measure 400 c.c. distilled water into a tared 2-litre flask. 2. Weigh out 10 grammes agar and make into a thick paste with 100 c.c. cold distilled water. 3. Add this agar paste to the distilled water in the flask. 4. Dissolve the agar by passing live steam through it, as in preparing nutrient agar. 5. Weigh out 5 grammes clean wood ash and place in a second flask containing 200 c.c. distilled water with some sterile glass beads: shake thoroughly in a mechanical shaker for ten minutes. 6. Heat in steamer at 100°C., for thirty minutes. 7. After removal from the steamer dry the outside of the flask thoroughly, place it over a Bunsen flame and boil for one minute. 8. Filter directly into the flask containing the melted agar mixture. 9. Weigh out 4 grammes maltose. Add to the contents of the flask. 10. Adjust the weight of the medium mass to the calculated figure for one litre (1019 grammes) by the addition of distilled water at 100°C. 11. Replace the flask in the steamer for twenty minutes, cool to 60°C., and clarify with egg and filter. 12. Tube, and sterilise as for nutrient agar. Media for the Study of Special Bacilli. B. Acnes. Oleic Acid Agar (Fleming).— 1. Measure out into a sterile stout glass bottle which already contains about 10 sterile glass beads
2. Weigh out
and add it to the ascitic fluid in the bottle. 3. Emulsify evenly by shaking (either by hand or in a shaking machine) for ten minutes. 4. Liquefy and measure out into a flask
then cool to 55°C. 5. Mix the oleic acid emulsion with the agar. 6. Add 10 c.c. sterile neutral red, 1 per cent. aqueous solution. 7. Tube in quantities of 10 c.c., slant, and allow to set. 8. Incubate for forty-eight hours at 37° C. and reject any contaminated tubes. Store the sterile tubes for future use. Coli-typhoid Group. Parietti's Bouillon.— 1. Measure out pure hydrochloric acid, 4 c.c., and add to it carbolic acid solution (5 per cent.), 100 c.c. Allow the solution to stand at least a few days before use. 2. This solution is added in quantities of 0.1, 0.2. and 0.3 c.c. (delivered by means of a sterile graduated pipette) to tubes each containing 10 c.c. of previously sterilised nutrient bouillon (vide page 163). 3. Incubate at 37° C. for forty-eight hours to eliminate contaminated tubes. Store the remainder for future use. Carbolised Bouillon.— 1. Prepare nutrient bouillon (vide page 163, sections 1 to 6). Measure out 1000 c.c. 2. Weigh out carbolic acid, 1 gramme (2.5 or 5 grammes may be needed for special purposes), and dissolve it in the medium. 3. Tube, and sterilise as for bouillon. Carbolised Gelatine.— 1. Prepare nutrient gelatine (vide page 164, sections 1 to 7). Measure out 1000 c.c. 2. Weigh out carbolic acid, 5 grammes (= 0.5 per cent.), and dissolve it in the gelatine. 3. Filter if necessary through papier Chardin. 4. Tube, and sterilise as for nutrient gelatine. One or 2.5 grammes of carbolic acid (= 0.1 per cent. or 0.25 per cent.) are occasionally used in place of the 5 grammes to meet special requirements. Carbolised Agar.— 1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c. 2. Weigh out 1 gramme pure phenol and dissolve in the medium. 3. Filter if necessary through papier Chardin. 4. Tube, and sterilise as for nutrient agar. Litmus Gelatine.— 1. Prepare nutrient gelatine (vide page 164, sections 1 to 8). 2. Add sterile litmus solution, sufficient to tint the medium a deep lavender colour. 3. Tube, and sterilise as for nutrient gelatine. Lactose Litmus Bouillon (Lakmus Molke).— 1. Weigh out peptone, 4 grammes, and emulsify it with 200 c.c. meat extract (vide page 148), previously heated to 60°C. 2. Weigh out salt, 2 grammes, and lactose, 20 grammes, and mix with the emulsion. 3. Wash the mixture into a sterile litre flask with 200 c.c. meat extract and add 600 c.c. distilled water. 4. Heat in the steamer at 100° C. for thirty minutes, to completely dissolve the peptone, etc. 5. Neutralise carefully to litmus paper by the successive additions of small quantities of decinormal soda solution. 6. Replace in the steamer for twenty minutes to precipitate phosphates, etc. 7. Filter through two thicknesses of Swedish filter paper. 8. Add sterile litmus solution, sufficient to colour the medium a deep purple. 9. Tube, and sterilise as for bouillon. Lactose Litmus Gelatine (Wurtz).— 1. Prepare nutrient gelatine (vide page 164, sections 1 to 4). 2. Render the reaction of the medium mass -5. 3. Replace in the steamer at 100° C. for twenty minutes. 4. Clarify with egg as for gelatine. 5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the medium. 6. Filter through papier Chardin. 7. Add sufficient sterile litmus solution to colour the medium pale lavender. 8. Tube, and sterilise as for nutrient gelatine. Lactose Litmus Agar (Wurtz).— 1. Prepare nutrient agar (vide page 167, sections 1 to 4). 2. Render the reaction of the medium mass -5. 3. Replace in the steamer at 100° C. for twenty minutes. 4. Cool to 60° C. and clarify with egg as for nutrient agar. 5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the medium. 6. Filter through papier Chardin, using the hot-water funnel. 7. Add sterile litmus solution, sufficient to colour the medium a pale lavender. 8. Tube, and sterilise as for nutrient agar. Glycerine Potato Bouillon.— 1. Take 1 kilo of potatoes, wash thoroughly in water, peel, and grate finely on a bread-grater. 2. Weigh the potato gratings, place them in a 2-litre flask, 3. Strain the mixture through butter muslin and filter through Swedish filter paper into a graduated cylinder. Note the amount of the filtrate. 4. Place the filtrate in a flask, add an equal quantity of distilled water, and heat in the steam steriliser for sixty minutes. 5. Add glycerine, 4 per cent., mix thoroughly, and again filter. 6. Tube and sterilise as for nutrient bouillon. Potato Gelatine (Elsner).— 1. Take 1 kilo of potatoes, wash thoroughly in water, peel, and finally grate finely on a bread-grater. 2. Weigh the potato gratings, place them in a 2-litre flask, and add distilled water in the proportion of 1 c.c. for every gramme weight of potato. Allow the flask to stand in the ice-chest for twelve hours. 3. Strain the mixture through butter muslin, and filter through Swedish filter paper into a graduated cylinder. 4. Add 15 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes. 5. Estimate the reaction; adjust the reaction of the medium mass to +25. 6. Cool the medium to below 60°C.; clarify with egg as for nutrient gelatine (vide page 166). 7. Add 1 per cent. potassium iodide (powdered) to the medium. 8. Filter through papier Chardin. 9. Tube and sterilise as for nutrient gelatine. Aesculin Agar.—(B. coli and allied organisms give black colonies surrounded by black halo.) 1. Measure out 400 c.c. distilled water into a tared 2-litre flask. 2. Weigh out
and make into a thick paste with 150 c.c. distilled water. 3. Add this paste to the distilled water in the flask. 4. Dissolve the ingredients by bubbling live steam through the mixture. 5. Weigh out
and dissolve in a second flask containing 100 c.c. distilled water. 6. Mix the contents of the two flasks—adjust the weight to 7. Clarify with egg and filter. 8. Tube and sterilise as for nutrient agar. Bile Salt Agar (MacConkey).— 1. Weigh out powdered agar, 15 grammes (= 1.5. per cent.), and emulsify with 200 c.c. cold tap water. 2. Weigh out peptone, 20 grammes (= 2 per cent.), and emulsify with 200 c.c. tap water previously warmed to 60°C. 3. Mix the peptone and agar emulsions thoroughly. 4. Weigh out sodium taurocholate, 5 grammes (= 0.5 per cent.), dissolve it in 300 c.c. tap water, and use the solution to wash the agar-peptone emulsion into a tared 2-litre flask. 5. Bubble live steam through the mixture for twenty minutes. 6. Adjust the weight of the medium mass to the calculated figure for one litre (1040 grammes). 7. Cool to 60° C. and clarify with egg as for nutrient agar (vide page 168). 8. Filter through papier Chardin, using the hot-water funnel. 9. Weigh out lactose, 10 grammes (= 1 per cent.), and dissolve it in the agar. If desired, add 5 c.c. of a 1 per cent. (= 0.5 per cent.) aqueous solution of neutral red. 10. Tube, and sterilise as for nutrient agar. Litmus Nutrose Agar (Drigalski-Conradi).— This medium should be prepared in precisely the same manner as the Nutrose agar described on page 172 substituting meat extract for serum water, and increasing the percentage of agar added per litre to 3 per cent. Fuchsin Agar (Braun).— 1. Liquefy and measure out into a sterile flask:
2. Weigh out: lactose 10 grammes and dissolve in the fluid agar. 3. Adjust the reaction to -5 and filter. 4. Measure out and mix thoroughly with agar:
The fuchsin solution is prepared by mixing:
Allow to stand twenty-four hours, then centrifugalise thoroughly and decant the supernatant fluid into a well-stoppered bottle. 5. Measure out and add to the nutrient agar, sodium sulphite, 10 per cent. aqueous solution, freshly prepared 25 c.c. 6. Tube and sterilise as for nutrient agar. 7. Store in a dark cupboard. Fuchsin Sulphite Agar (Endo).— 1. Liquefy and measure out into a sterile flask:
2. Weigh out
and dissolve in the fluid agar. 3. Adjust the reaction to +3 and filter. 4. Measure out and mix thoroughly with the fluid agar.
5. Measure out and add to the medium
6. Tube and sterilise as for nutrient agar. Brilliant Green Agar (Conradi).— 1. Liquefy and measure out into a sterile flask
2. Adjust reaction to +30 by the addition of normal phosphoric acid; and filter. 3. Measure out and mix thoroughly with the fluid medium
4. Measure out and add to the medium
5. Tube and sterilise as for nutrient agar. Brilliant Green Bile Salt Agar (Fawcus).— 1. Weigh out agar 20 grammes and emulsify in 100 c.c. cold distilled water. 2. Wash the emulsion into a "tared" 2-litre flask with 500 c.c. distilled water. 3. Dissolve the agar by bubbling live steam through the flask. 4. Cool, clarify with egg and filter. 5. Weigh out
and add to the medium in the flask. 6. Weigh out
and add to the medium in the flask. 7. Adjust reaction to +15 and filter if necessary. 8. Measure out
and mix thoroughly with the fluid agar. 9. Measure out and add to the medium
10. Tube and sterilise as for nutrient agar. China Green Agar (Werbitski).— 1. Liquefy and measure out into a sterile flask
2. Adjust the reaction accurately to +13 and filter. 3. Measure out and mix thoroughly with the fluid agar
4. Tube and sterilise as for nutrient agar. Malachite Green Agar (Loeffler).— 1. Liquefy and measure out into a sterile flask
2. Weigh out
and dissolve in nutrient agar. 3. Adjust the reaction to +3, and filter. 4. Measure out and mix thoroughly in the fluid agar
4a. To the filtered agar add
5. Tube and sterilise as for nutrient agar. Double Sugar Agar (Russell).— 1. Liquefy and measure out into a sterile flask
2. Add 100 c.c. litmus solution to the fluid agar. 3. Weigh out and dissolve in the fluid agar.
4. Render the reaction of the medium neutral to litmus paper by the cautious addition of normal caustic soda. 5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100° C. for twenty minutes on each of three successive days. 6. Store for use in a cool dark place. B. DiphtheriÆ. Glycerine Blood-serum.— 1. Prepare blood-serum as described, page 168, sections 1 to 4. 2. Add 5 per cent. pure glycerine. 3. Complete as described above for ordinary blood-serum, sections 5 to 7. Note.—Different percentages of glycerine—from 4 per cent. to 8 per cent.—are used for special purposes. Five per cent. is that usually employed. Blood-serum (Loeffler).— 1. Prepare nutrient bouillon (vide page 163), using meat extract made from veal instead of beef. 2. Add 1 per cent. glucose to the bouillon, and allow it to dissolve completely. 3. Now add 300 c.c. clear blood-serum (vide page 168, sections 1 to 4) to every 100 c.c. of this bouillon. 4. Fill into sterile tubes and complete as for ordinary blood-serum. Blood-serum (Lorrain Smith).— 1. Collect blood-serum (vide page 168, sections 1 to 4), as free from hÆmoglobin as possible. 2. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid (or add 0.375 c.c. of dekanormal soda solution for every 100 c.c. of serum). 3. Tube, and stiffen at 100° C. in the serum inspissator. 4. Incubate at 37° C. for forty-eight hours to eliminate any contaminated tubes. Store the remainder for future use. Blood Serum (Councilman and Mallory).— 1. Collect blood serum in slaughterhouse, coagulate, remove serum and tube (vide page 168). Great care must be taken to avoid the inclusion of air bubbles—indeed if only a few tubes are filled at one time, it is a good plan to stand them upright in the receiver of an air pump and to exhaust as completely as possible before transferring to the serum inspissator. 2. Heat the tubes in a slanting position in hot-air steriliser at 90° C. till firmly coagulated, say half an hour. 3. Sterilise in steam steriliser at 100° C. for 20 minutes on each of three successive days. Resulting medium not translucent, but opaque and firm. B. Tuberculosis. Egg Medium (Lubenau).— This modification of Dorset's egg medium (quod vide page 174) is preferred by some for the growth of the tubercle bacillus of the human type. It consists in the addition of one part of 6 per cent. glycerine in normal saline solution, to the egg mixture between steps 4 and 5. Glycerine Bouillon.— 1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6). 2. Measure out glycerine, 60 c.c. (= 6 per cent.), and add to the bouillon. 3. Tube, and sterilise as for bouillon. Glycerine Agar.— 1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c. 2. Measure out pure glycerine, 60 c.c. (= 6 per cent.), and add to the agar. 3. Tube, and sterilise as for nutrient agar. Glycerine Blood-serum.— 1. Prepare blood-serum as described, page 168, sections 1 to 4. 2. Add 5 per cent. pure glycerine. 3. Complete as described above for ordinary blood-serum, sections 5 to 7. Note.—Different percentages of glycerine—from 4 per cent. to 8 per cent.—are used for special purposes. Five per cent. is that usually employed. Glycerinated Potato.— 1. Prepare ordinary potato wedges (vide page 174, sections 1 to 4). 2. Soak the wedges in 25 per cent. solution of glycerine for fifteen minutes. 3. Moisten the cotton-wool pads at the bottom of the potato tubes with a 25 per cent. solution of glycerine. 4. Insert a wedge of potato in each tube and replug the tubes. 5. Sterilise in the steamer at 100° C. for twenty minutes on each of five consecutive days. Animal Tissue Media (Frugoni).— 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. 2. Kill a small rabbit by means of chloroform vapour. 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). 7. Sterilise in the autoclave at 120° C. for thirty minutes. 8. Cap the tubes and store them in the ice chest for future use. Tissues obtained at postmortems can also be used after preliminary sterilisation by boiling or autoclaving. Media for the Study of Special Cocci. Diplococcus GonorrhoeÆ. Ascitic Bouillon (Serum Bouillon).— 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. Serum Agar (Heiman).— 1. Prepare nutrient agar (vide page 167), to following formula:
2. Make reaction of medium + 10. 3. Filter; tube in quantities of 6 c.c. 4. Sterilise as for nutrient agar. 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. Serum Agar (Wertheimer).— 1. Prepare nutrient agar (vide page 167), to the following formula:
2. Make reaction of medium +10. 3. Filter; tube in quantities of 5 c.c. 4. Sterilise as for nutrient agar. 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. Serum Agar (Kanthack and Stevens).— 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. 4. Heat in the steamer for twenty minutes. 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. 7. Filter through papier Chardin, using a hot-water funnel. 8. Weigh out glucose 10 grammes (= 1 per cent.), and dissolve it in the clear agar. 8a. If desired, add glycerine, 5 per cent., to the clear agar. 9. Tube, and sterilise as for nutrient agar. Serum Agar (Libman).— 1. Prepare nutrient agar (vide, page 167) using, however, 1.5 per cent. peptone (that is 15 grammes per litre instead of 10 grammes). 2. Adjust the reaction to 0 (i. e., neutral to phenolphthalein). 3. Filter and transfer 1000 c.c. liquefied medium to a sterile flask. 4. Weigh out dextrose 20 grammes and dissolve in the fluid agar. 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. Egg-albumen, Inspissated.— 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). 3a. Glucose to the extent of 1 to 2 per cent. may now be added, if desired. 4. Strain the mixture through butter muslin and filter through a porcelain filter candle into a sterile filter flask. 5. Tube, and stiffen at 100° C. in the serum inspissator. 6. Incubate at 37° C. for forty-eight hours and eliminate any contaminated tubes; store the remainder for future use. Egg-albumen (Tarchanoff and Kolesnikoff).— 1. Place unbroken hens' eggs in dekanormal caustic soda solution for ten days. (After this time the white becomes firm like gelatine.) 2. Carefully remove the shell and cut the egg into fine slices. 3. Wash for two hours in running water. 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. Egg Albumin Broth (Lipschuetz).— 1. Weigh out and place in a 2-litre flask with a number of sterile glass beads. 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. 5. Filter through Swedish filter paper. 6. Sterilise by boiling two or three times at intervals of two hours. 7. Add ordinary nutrient bouillon 600 c.c. 8. Fill into small Erlenmeyer flasks in quantities of 50 c.c. 9. Incubate for forty-eight hours at 37°C.—discard any contaminated flasks and store the remainder for future use. Egg Albumin Agar.— 1. Prepare egg albumin solution as above 1-6. 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. Complete as above 8-9. Diplococcus Meningitidis Intracellularis. Ascitic Fluid Agar (Wassermann) Synonym N-as-gar (Mervyn Gordon). 1. Liquefy and measure out into a sterile flask:
2. Measure out into a half litre flask
and add to it
3. Heat over a bunsen flame, shaking constantly until the fluid boils, and the nutrose is dissolved. 4. Add the nutrose ascitic solution to the fluid agar. 5. Heat in the steamer for thirty minutes, then filter. 6. Tube and sterilise as for nutrient agar. 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. Diplococcus PneumoniÆ. Blood Agar (Washbourn).— 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. 4. Sterilise several pairs of forceps, scissors, etc., by boiling. 5. Reflect the skin over the thorax with sterile instruments. 6. Open the thoracic cavity by the aid of a fresh set of sterile instruments. 7. Open the pericardium with another set of sterile instruments. 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. 10. Place the tubes in the slanting position and allow the blood to coagulate. 11. Return the "blood agar" to the hot incubator for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use. Media for the Study of Mouth Bacteria Generally. Potato Gelatine (Goadby).— 1. Prepare glycerine potato broth (see page 203, sections 1 to 5). 2. Add 10 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes. 3. Estimate the reaction; adjust the reaction of the medium to +5. 4. Cool the medium to below 60°C., clarify with egg as for nutrient gelatine. 5. Filter through papier Chardin. 6. Tube, and sterilise as for nutrient gelatine. Media for the Study of Protozoa. Tissue Medium (Noguchi).—For spirochÆtes (cultivations must be grown anaerobically). 1. Plug and sterilise test-tubes 20 × 2 cm. 2. Kill a small rabbit with chloroform vapour. Open the abdomen 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. 9. Store such tubes as are sterile for future use. |
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