The way in which culture media shall be used depends on the purpose in view. By far the larger part of bacteriological work is done with cultures in “bacteriological culture tubes.” Various laboratories have their own special types but all are more or less after the “Board of Health” form. They differ from ordinary chemical test-tubes in that they are usually longer, have no “lip” and have much thicker walls to prevent breakage and consequent loss of the culture as well as danger from pathogenic organisms. The author finds two sets of tubes most serviceable for student use—one size 15 cm. long by 19 mm. outside diameter (No. 9, Fig. 119), the other 15 cm. long by 13 mm. (Nos. 1 to 7, Fig. 119). Culture tubes are conveniently used in “wire baskets” circular or square in section and of a size to correspond with the length and number of tubes used. These baskets are light, do not break, and if made of good galvanized wire netting do not readily rust (Figs. 117 and 118).
Liquid media such as broth, milk, litmus milk, indol and nitrate broths are used in the above-mentioned tubes when small quantities only are to be worked with. The tubes are filled approximately one-third full, then plugged with non-absorbent cotton and sterilized. Cotton plugs are used so much in bacteriological work because they permit a free circulation of air and gases and at the same time act as filters to keep out the bacteria of the air.
Sugar broths or other media in which gas may be produced are used in fermentation tubes (Smith tubes) of the type shown in Fig. 120 so that the gas may be collected in the closed arm of the tube, measured (Fig. 121) and tested if desired.
One method of using gelatin and also agar is as “puncture” or “stab” cultures. The tubes (the narrower tubes are to be preferred for most “stab” cultures) are filled one-third full of the medium while it is still fluid, plugged, sterilized and allowed to cool in the vertical position. The medium is then “inoculated” with a straight platinum needle by plunging this into the center of the surface down to the bottom of the tube (Fig. 119, Nos. 4 and 5).
Agar and blood serum are frequently used in the form of “slope” or “slant” cultures. That is, the medium solidifies with the tubes lying on their sides which gives a long, sloping surface on which the bacteria are inoculated (Fig. 119, Nos. 6 and 7).
Potato tubes are likewise used for “slant” or “slope” cultures (Fig. 119, No. 8). Potatoes as “plate cultures” have been referred to. Agar and gelatin are very largely used in the form of “plate cultures” also. For this purpose Petri dishes are first sterilized, then the melted agar or gelatin poured into them and allowed to “set” while the plates are kept horizontal. The melted media may be “inoculated” before they are poured, or a portion of the material to be “plated” may be placed in the dish, then the melted medium poured in and distributed over the dish by tilting in various directions, or the medium after solidifying may be inoculated by “strokes” or “streaks” over its surface, according to the purpose in view in using the plate. The larger sized tubes should be used for making plates in order to have sufficient medium in the plate (No. 9, Fig. 119).
For using large quantities of medium, Florence flasks, Ehrlenmeyer flasks, special toxin flasks (Fig. 122) or various other devices (Vaughan and Novy’s “mass cultures,” Figs. 123 and 124) have been employed.
For growing anaërobic organisms it is evident that some method for removing and excluding the oxygen of the air must be used. A very great variety of appliances have been devised for these purposes. Some are based on the principle of the vacuum, exhausting the air with an air pump; some on replacing the air with a stream of hydrogen; others on absorbing the oxygen by chemical means, as with an alkaline solution of pyrogallic acid, or even by growing a vigorous aërobe in the same culture or in the same container with the anaërobe, the aërobe exhausting the oxygen so that the anaërobe then develops, or finally by excluding the air through the use of deep culture tubes well filled with the medium, or in the closed arm of fermentation tubes. For many purposes a combination of two or more of the above methods gives good results.
In any event the culture medium should have been freshly sterilized just before use, or should be boiled in order to drive out the dissolved oxygen. For most, anaërobes the presence in the medium of about 1 per cent. of a carbohydrate, as dextrose, is advisable.
A description of all the various devices is unnecessary in this work, but the following have answered most of the purposes of general work in the author’s laboratories.
A. “Vignal tubes” of the style shown (Fig. 125) are made from glass tubes of about 6 to 8 mm. outside diameter, sealed at the small end, plugged with cotton above the constriction and sterilized. The medium, agar or gelatin, which has been previously inoculated with the anaërobic culture, is then drawn up into the tube, after breaking off the tip, as far as the constriction. The tube is then sealed in the flame at the small end and also at the constriction. Since it is full of the medium and sealed, access of air is prevented. This forms an excellent means for “isolation” (Chapter XVIII); the tube needs merely to be cut with a file at the point where colonies appear, then these may be readily transferred.
B. “Fermentation tubes” form a simple means for growing liquid cultures of anaërobes, the growth occurring in the closed arm only, while with facultative anaërobes, growth occurs both in the closed arm and in the open bulb. A little “paraffin oil” (a clear, heavy petroleum derivative) may be poured on the fluid in the open bulb as a very efficient seal, though it is not usually necessary.
C. “Deep culture tubes.”—The medium, agar, gelatin or a liquid is poured into tubes until they are approximately one-half full, a little paraffin oil is poured on the surface (not essential always), then the tubes are plugged and sterilized. Inoculation is made to the bottom and anaërobes grow well (Fig. 126).
D. For slope or plate, or any type of surface cultures the Novy jar (Fig. 127) is the most practical device. It is good practice to combine the vacuum method, the hydrogen replacement method and the oxygen absorption method in using these jars. In operation a solution of 20 per cent. NaOH is poured on the bottom of the jar to a depth of 1 or 2 cm., the cultures are placed on glass supports above the alkali and a short wide tube of strong pyrogallol is set in on the bottom in such a way that it may be easily upset and mixed with the alkali when it is desired to do so. The cover is now clamped in position with all joints well vaselined. Then the outlet tube is connected with a suction pump and the air drawn out. Meanwhile the inlet tube has been connected with a hydrogen generator, and after the jar is exhausted hydrogen is allowed to flow in, and this process is repeated until one is satisfied that the air is replaced. The suction exhausts the air from the tubes or plates so that much less time is required to replace the air with hydrogen. Finally the stop-cock is closed, and the pyrogallol solution is gently shaken down and mixed with the alkali so that any remaining oxygen will be absorbed.
It must be remembered that facultative anaërobes as well as anaërobes will grow under any of the above conditions, so that cultures of organisms so obtained must be further tested aërobically in order to determine to which group the organisms belong.
Reference has been made above to the “inoculation” of culture media, which means introducing into the medium used the desired material in the proper way. For small quantities this is most conveniently done with platinum “needles,” that is, pieces of platinum wire inserted into the ends of glass rods. The “straight” needle is a piece of heavy platinum wire of about 0.022 inch in diameter (Fig. 128). It is used most frequently to inoculate all forms of solid media. The platinum loop is of lighter wire, 0.018 inch. The loop in the end is conveniently made by twisting the wire around the lead of an ordinary lead-pencil. The “loop needle” (Fig. 129) is most used in transferring liquid media. On account of the high price of platinum, the author has substituted “nichrome” wire for student use. This is stiffer, not so easily made into loops and breaks out of the rods more easily. The latter defect is remedied to some extent, by imbedding the wire only slightly for about one-fourth of an inch on the side of the end portion of the rod. The low cost, less than one-twentieth of platinum, justifies its use.
Sterile graduated pipettes varying in capacity from 1 cc graduated in hundredths, upward, permit the transfer of definite amounts of liquids. Large quantities are conveniently transferred by means of Pasteur flasks (Fig. 130). The details of inoculation are best derived from laboratory practice.