Agaric

“ Luxuriant cultures of the desired organism are grown upon inclined agar; the growth is then removed with salt solution and a glass rod, and thoroughly emulsified by shaking in a test-tube, in order to get the germs well distributed and the clumps broken up. The number of germs in a given quantity of the emulsion is then determined by comparing the number of germs and red blood-corpuscles in a mixture of one part emulsion, one part freshly drawn blood, and three parts of normal salt solution. ”

“ In the process of division the cells gradually separate at the suture, and a new half-cell is formed upon each old half, which grows until it attains the size and form of the original half of the parent-cell. The conjugation is effected by two cells approximating so that their sutures are near together, the cells then{74} open at the sutures, and the effused contents become united to form a spore or sporange, from which one or more individuals are formed. These spores are often elegantly spinous on the surface. ”

“ Though filtration through paper is possible as with gelatin, if the agar solution is thoroughly boiled and filtered boiling hot, it is more satisfactory for beginners to use absorbent cotton wet with boiling water and to pour the hot agar through the same filter if not clear the first time. The solidified agar medium is never perfectly clear, but always more or less opalescent. The agar medium may be sterilized in the autoclave for fifteen minutes at 15 pounds pressure as the high temperature does not injure the agar. ”

“ In this method the bacteria are distributed in a gelatine or agar medium liquefied by heat, and the medium is then poured out on sterile glass plates or in shallow glass dishes, and allowed to solidify. Each bacterium capable of growth gives rise to a colony visible to the naked eye, and if the colonies are sufficiently apart, an inoculation can be made from any one to a tube of culture-medium and a pure culture obtained. ”

“ Pick off coliform or typhiform colonies; make streak or smear subcultivations upon nutrient agar; incubate aerobically for twenty-four hours at 37° C.
15. Examine the growth in each tube carefully both macroscopically and microscopically. If the growth is impure, replate on nutrose agar, pick off colonies and subcultivate again. When the growth in a tube is pure, add 5 c.c. sterile normal saline solution or sterile broth, and emulsify the entire surface growth with it. ”

“ The cells of the Bacteria are constructed like other plant-cells in so far as their diminutive size has allowed us to observe them. The cell-wall only exceptionally shows the reactions of cellulose (in Sarcina, Leuconostoc; also in a Vinegar-bacterium, Bacterium xylinum) ; a mucilaginous external layer is always present. The body of the cell mostly appears to be an uniform or finely granulated protoplasm. Very few species (e.g. Bacillus virens) contain chlorophyll; others are coloured red (purple sulphur Bacteria) ; the majority are colourless. ”

“ Prepare a sloped agar tube cultivation and incubate under optimum conditions as to spore formations. [Pg 305]
2. Pipette 5 c.c. sterile normal saline into the culture tube and emulsify the entire surface growth in it. Examine microscopically to determine the presence of spores in large numbers.
3. Spread thin even films on twelve sterile cover-slips and place each cover-slip in a separate sterile capsule.
4. Expose each capsule in turn for ten minutes to a different fixed temperature, varying 5°C, between 100° C. and 160°C.
5. Complete the examination as for vegetative forms. ”

“ Cranmer the elder, through use of an undescribed (in the notebook) growth medium of which one constituent was agar-agar, and the use of radium emanations, had succeeded in bringing about apparently unrestricted growth in the paramœcium protozoan, certain of the vegetable growths (among which were bacteria) , and in the amorphous cell of protoplasm known as the amœba—the last a single cell containing only neucleolus, neucleus, and a space known as the contractile vacuole which somehow aided in throwing off particles impossible [30] to assimilate directly. ”

“ Bacteria are minute fungi, each consisting of a single cell enclosed in a cell membrane of cellulose which can be demonstrated by iodine, the latter causing the protoplasm to retract from the cell wall. There is no nucleus or central core. Some of the bacteria are colorless, others pigmented, yellow, blue or red. The cells vary in shape and in size in different species as well as in their mode of growth, and [Pg 7] are named in accordance with these peculiarities. The round or oval cells are called cocci; the rod-shaped organisms are termed bacilli. ”

“ It will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. ”

“ The protein used as antigen to stimulate its formation, or some part of the protein molecule (haptophore group) , which acts as stimulus to the cell is spoken of as a “precipitinogen,” both terms after the analogy of “agglutinin” and “agglutinogen.” In fact the specific precipitation and agglutination are strictly analogous phenomena. Precipitins act on proteins in (colloidal) solution and cause them to settle out, agglutinins act on substances within cells which cells are in suspension in a fluid and cause the cells to settle out. ”

“ To show that Bacteria are not given off from a Moist Surface.—A culture of harmless, brilliant yellow bacteria was smeared on the inside of a large glass tube. Through this tube a strong current of air was directed on to agar in a Petri dish. If bacteria were given off into the air from the moist surface, they would be blown upon the agar, where they would develop into colonies. The Petri dish used in this experiment developed no yellow colonies. ”

“ For any particular experiment prepare a smear culture on agar and incubate at 37° C. for 24 hours anaerobically. Then emulsify the whole of the surface growth in 10 c.c. of sterile water. Transfer the emulsion to a sterile test-tube with some sterile glass beads and shake thoroughly to ensure homogenous emulsion. Transfer to a centrifuge tube and centrifugalise the emulsion to throw down any masses of bacteria which may have escaped the disintegrating action of the beads. Pipette off the supernatant emulsion for use in the test. ”

“ The substance within the introduced cell which acts as a stimulus (antigen) to the body cells is called an “agglutinogen.” That “agglutinogen” is present in the cell has been shown by injecting animals experimentally with extracts of cells (bacterial and other cells) and the blood serum of the animal injected showed the presence of agglutinin for the given cell. It will be noticed that the receptors which become the free agglutinins have at least two functions, hence at least two chemical groups. ”

“ Blood Agar, Human.—
1. Melt a tube of sterile agar and pour it into a sterile plate; let it set. [Pg 251]
2. Collect a few drops of human blood, under all aseptic conditions, in a sterile capillary teat pipette.
3. Raise the cover of the Petri dish very slightly, insert the extremity of the capillary pipette, and deposit the blood on the centre of the agar surface. Close the dish.
4. Charge a platinum loop with a small quantity of the inoculum. Raise the cover of the plate, introduce the loop, mix its contents with the drop of blood, remove the loop, close the dish and sterilise the loop. ”

“ In agar plates incubated at 37° C. numerous colonies may be found in twenty to thirty hours. In the gelatin plates, after two to three days' incubation at 20° to 22° C., numerous typical colonies can be got. (A more modern method which is very efficacious is Dieudonne's blood-alkali agar. Equal parts of defibrinated ox-blood and normal caustic soda solution are mixed with 70 c.c. of neutral peptone agar. On this medium few organisms other than the cholera vibrio develop.) ”

“ 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. ”

“ By diluting the blood serum from the animal to be tested the agglutinins for the related organisms (so-called “coagglutinins” or “partial agglutinins”) will become so much diminished as to show no action, while the agglutinin for the specific organism is still present in an amount sufficient to cause its clumping. Agglutinins are specific for their particular agglutinogens, but since a given blood serum may contain many agglutinins, the serum’s specificity for a given bacterium can be determined only by diluting it until this bacterium alone is agglutinated. ”

“ Recently Francis has shown that the organism will grow scantily on serum or blood agar. However, by adding a piece of fresh sterile spleen to such media he has obtained a more satisfactory culture medium, although for routine work the egg yolk medium is preferred. The organism refuses to grow on ordinary media such as nutrient broth or agar. ”

“ Two other signs of degeneration are the appearance of granules in the body of the cell protoplasm known as metachromatic granules, owing to their different staining propensities, and the polar bodies which are seen in some species of bacteria. Surrounding the mass of mycoprotein, we find in most organisms a capsule or membrane composed, in part at least, of cellulose. This sheath plays a protective part in several ways. During the adult stage of life it protects the mycoprotein, and holds it together. ”

“ 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. ”

“ The behavior toward oxygen is another character upon which considerable reliance may be placed in the identification of strains recently isolated from the animal body. This is tested by transplanting the colonies from the serum-agar plates to two series of agar streak sub-cultures, of which one series is incubated in the atmospheric air and the other in the closed jar together with cultures of B. subtilis. Unless the growth under the latter condition is much better than the growth outside the jar, the culture may be discarded as one not belonging to this species. ”

“ The cells of which all plants and animals consist increase in numbers by the division of [Pg 10] each cell into two cells through the formation of a division wall across the cell. The new cells divide and the plant or animal continues to grow. The same cell division occurs in the bacteria but since the bacteria are single celled, division of the cells means an increase in numbers rather than growth as in the higher forms of life. ”

“ Others consider the bacterium as formed of protoplasm containing granules capable of being colored, which are a part of the protoplasm itself, and not a nuclear substance. On the latter view, bacteria would consist of cell plasm enclosed in a membrane and destitute of a nucleus. Whatever be the nature of the granule-containing material, each bacterium is regarded as a cell, the minutest and simplest living particle capable of an independent existence that has yet been discovered. ”

“ Hanging-block Culture (Hill) .—
Apparatus required: As for hanging-drop cultivation with the addition of a scalpel.
Carry out the method as far as possible under cover of a bell glass, to avoid aerial contamination.
1. Liquefy a tube of nutrient agar (or gelatine) and pour into a Petri dish to the depth of about 4 mm. and allow to set.
2. With a sharp scalpel cut out a block some 8 mm. square, from the entire thickness of the agar layer.
3. Raise the agar block on the blade of the scalpel and transfer it, under side down, to the centre of a sterile slide. ”

“ Plate Cultures.—
Gelatine.—Note the presence or absence of liquefaction of the surrounding medium. If liquefaction is present, note shape and character (vide page 269, "stab" cultures) .
Agar.—No liquefaction takes place in this medium. The liquid found on the surface of the agar (or at the bottom of the tube in agar tube cultures) is merely [Pg 262] water which has been expressed during the rapid solidification of the medium and has subsequently condensed.
Gelatine and Agar.—Examine the colonies at intervals of twenty-four hours. ”

“ Another method which is frequently applicable with material from human or animal sources is to (3) rub the material over the surface of a slope tube or of medium solidified in a Petri dish with a sterile heavy platinum needle, glass rod, or cotton swab. If the bacteria are not too numerous, pure cultures may frequently be obtained. ”