Agar

“ So that the third plate as a rule will only show a very few scattered colonies, eminently suitable for detailed study.
(b) Agar Surface Plates.—
1. Liquefy three tubes of nutrient agar—nutrose agar or the like.
2. Pour each tube into a separate Petri dish and allow it to solidify.
3. When quite solid invert each dish, raise the bottom half and rest it obliquely on its inverted cover (Fig. 128) and place it in this position in an incubator at 60° C. for forty-five minutes (or in an incubator at 42° C. for [Pg 233] two hours) . ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ Nutrient Agar-agar is a substance prepared from seaweed which grows on the coasts of Japan and India, and is supplied in long crinkled strips. It boils at 90° C., and remains solid up to a temperature of about 45° C. It is therefore substituted for gelatine in the preparation of a jelly for the cultivation of those bacteria which will grow best in the incubator at the temperature of the blood, and also at ordinary temperature for bacteria which lignify gelatine. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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 Council found that the amount of agar-agar in Agar-lac and the identity of the “lactic ferment” are not declared; that the name “Agar-lac” is blown in the glass and that the method of its exploitation will lead laymen to use it to their detriment; that the claims that it “facilitates assimilation of proteids” and that it is of value as an aid to “gastro-intestinal digestion” give a false value to the mixture and that the claims emphasize the action of agar-agar when from the composition it is evident that the phenolphthalein action will predominate ”

“ The material to be plated out is diluted by transferring to three or more tubes of melted gelatin or agar as in the first method and then all the tubes are poured into Petri dishes and grown under suitable conditions. By proper mixing in the tubes the bacteria are well scattered through the medium which holds the individual organisms separate when it solidifies. ”

“ Experience is the only reliable guide, but as a general rule the use of [Pg 250] either the first or the third method will be found most convenient, affording as each of them does an opportunity for the simultaneous isolation of several or all of the varieties of bacteria present in a mixture.
1. Surface Plate Cultivations.—
(a) Gelatine (vide page 164) .
(b) Agar (vide page 167) .
(c) Alkaline serum agar (vide page 211) .
These plates are prepared in a manner precisely similar to that adopted for nutrient gelatine and agar surface plates (vide pages 231-233) .
(d) Serum Agar.—
1. ”

“ Plant tubes of gelatine or agar, liquefied by heat, from each of the higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and incubate. When growth is visible enumerate the number of organisms present in each, average up and calculate the number of bacteria present in one loopful of the inoculum. ”

“ 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. ”

“ 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. ”

“ 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.) ”

“ My plates were prepared by being wrapped in separate paper covers, sterilized in dry heat and transferred to a box still wrapped in their paper covers. The agar-agar was in separate tubes stoppered as usual with cotton plugs. Each morning before starting in quest of bacteria the required number of fresh plates were prepared. ”

“ 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. ”

“ 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. ”

“ Agar reveals a growth in about twenty-four hours at 37° C., which is the favourite temperature. A greyish, moist, irregular growth appears, but it is invariably attached to the track of the inoculating needle. On gelatine the growth is much the same, but its irregular edge is, if anything, more apparent. There is no liquefaction and no gas formation. On plates of gelatine the colonies appear large and spreading, with jagged edges. ”

“ Actinomyces may be cultivated, like other parasitic diseases, outside the body. Gelatine, blood serum, agar, glycerine agar, and potato have been used for this purpose. After a few days on glycerine agar at the temperature of the blood little white shining colonies appear, which in319crease and coalesce. In about ten days' time the culture often turns a bright yellow, though it may remain white or even take on a brown or olive tint. The entire mass of growth is raised dry and crinkled, and composed almost exclusively of threads. ”

“ As a result of these rapid subcultures, the facultative anaerobe will be secured in pure culture at about the third or fourth generation.
(c) If, on the other hand, the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes, pour plates of glucose formate agar (or gelatine) in the usual manner, place them in a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick off the colonies of the required organism when the growth appears, and transfer to tubes of the various media. ”

“ Our process of inoculating land with tubercle bacteria is to be carried out as follows: The active bacteria to promote the growth of the Leguminosæ are delivered to the farmer in glass tubes or other suitable packages, which contain pure colonies thereof in agar-gelatin having suitable additions for propagating such bacteria, for instance sugar, asparagin and an aqueous extract of the green substance of the Leguminosæ. In some cases the bacteria can also be prepared for transport in fluid cultures. ”

“ Soak agar-agar as for Orange Jelly. Cook fruit with 1/2 pint water until well done. Strain through muslin. Warm the agar-agar until dissolved in 1 gill of water. Put the fruit juice, sugar, and agar-agar into a saucepan. If liquid measures less than 1-1/2 pints, add enough water to make up quantity. Bring to the boil, pour through a hot strainer into wet mould. Turn out when cold and serve.
22. MINCEMEAT.
1/2 lb. raisins, 1/2 lb. sultanas, 1/2 lb. currants, 1/2 lb. castor sugar, 1/4 lb. nutter, 1/2 a nutmeg, grated rind of 2 lemons, 1-1/2 lb. apples. ”

“ Subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths. ”

“ The incubation process consists in placing the inoculated plates in a place where bacteria can grow. Leave the plates upside down at room temperature for 48 hours and in a place with no direct light. Then, analyze the colonies present in the agar and compare color, texture, form, etc.
After two days, you can store the plates in a refrigerator (4 a 6oC) for a couple of days.
Activity No3
“Growing microorganisms that live in our body”
Materials:
• Cotton swabs
• 2 Petri dishes
• Permanent marker
Using a swab, take a sample from inside the nose of a classmate. ”