CULTURES OF MICROORGANISMS. solutions intermittently. Such intermittent observations of. and decrease of the number of bacteria, of the consumption of

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A NEW APPARATUS FOR INTERMITTENT OBSERVA- TIONS OF PHYSIOLOGICAL CHANGES IN CULTURES OF MICROORGANISMS Botanical Department, Faculty of Science, Tokyo Imperial University Received for publication

1 A NEW APPARATUS FOR INTERMITTENT OBSERVA- TIONS OF PHYSIOLOGICAL CHANGES IN CULTURES OF MICROORGANISMS Botanical Department, Faculty of Science, Tokyo Imperial University Received for publication October 6, 1925 In the study of physiological changes in cultures of bacteria, yeasts or other microorganisms, it is of great importance to note the development of various phenomena, by investigating the solutions intermittently. Such intermittent observations of physical and chemical changes in the culture-media, of increase and decrease of the number of bacteria, of the consumption of nutritive substances, of the formation of new substances such as alkali and acids, of changes in hydrogen-ion concentration, etc., are very widely carried out in recent years especially in bacterial chemistry. Hitherto, two methods had been generally adopted for such investigations. In one method the culture medium is divided into equal volumes and each portion placed in a test-tube, Erlenmeyer flask, or Florence flask. An equal number of microorganisms, which have been cultivated under the same conditions, is inoculated into each portion. All vessels are incubated alike, the same physiological changes occurring in each of them, and the cultures are examined one after another, so that we may know the gradual changes that should take place in any one of them. In the other method a large quantity of the culture is provided in one vessel, and its changes are observed successively by removing a portion with a sterile pipette. In the first method we can observe the correct results under constant conditions, if the same number of organisms are inoculated into each vessel, and if every vessel is placed in the same 125

2 126 conditions, and the same changes occur in all vessels with the same velocity. But these conditions can not always be attained with ease, and it is often the case that various vessels do not show the same stage of change at a given time. Therefore this method can not be relied on except when the experiment needs no great accuracy or when the growth of the microorganisms is very vigorous and not affected by differences in various circumstances. In the second method the volume of the solution will become a FIG. 1 *0 I;u smaller and smaller, as the portions are taken from it one by one. For this reason the ratio of the surface of culture-medium to its volume does not remain constant all the time during the experiment, i.e., the air supply for the medium varies as the observation goes further. For example, suppose that we use for the second method a flask with round bottom, whose body has the shape of an exact sphere. Place the center of the sphere at the origin of co6rdinate axes (fig. 1, a). The distance from the center of sphere to the surface of the' liquid being p, and the I 1 : I : b

APPARATUS FOR OBSERVATIONS OF MICROORGANISMS 127 radius of the sphere being R, the quotient, u e varies very irregularly as p varies, that is, the volume of liquid decreases.

3 APPARATUS FOR OBSERVATIONS OF MICROORGANISMS 127 radius of the sphere being R, the quotient, u e varies very irregularly as p varies, that is, the volume of liquid decreases. If we use a large test-tube for the same purpose, imagine the tube as a cylinder, the bottom having the radius R and the length being h, (fig. 1, b), the quotient, surface, increases proportionally as the surface goes down. Now let us call the ratio of the surface to the volume of the liquid "the surface-coefficient" (or "the surface-volume quotient"). In both of the above examples this coefficient is not constant during the experiment, so when the life-phenomena of the observed micro6rganisms is appreciably affected by the airsupply the results will be atypical. The new vessel invented by the author is so constructed, that the quotient, sure is not altered by taking off the portions of the liquid in it. In figure 2, a curve represented by the formula y - f (x) is rotated around the X-axis, so that a body of revolution will be formed by it. This body is cut with a plane which is vertical to the X-axis, through a point, x, y. Then we have the equation kx y= Ae2 where e is the base of the natural logarithm, and A is a constant, k being the surface coefficient. This formula represents the curve to be obtained. Since this curve is represented by an exponential function it never intersects the X-axis. For this reason the body of revolution given by it does not satisfy the conditions except when calculated from - o. But since, in practice, it is impossible to extend the vessel to infinite length, we must either extend the curve sufficiently to approach the X-axis, or cut the. body of revolution at some place, which is allowable for the experiment, and substitute the remaining volume in some proper shape.

4 128 When, in figure 2, we cut the body at the Y-axis, the total volume of the part which exists on its left hand may be substi- FIc.. 2 I I. FIG. 3

5 APPARATUS FOR OBSERVATIONS OF MICROORGANISMS 129 tuted with a spherical body having the radius R, which can easily be calculated from the following formula. The bottle which is 12 kr2-3 k2r2r2 - k2r4-9 r2 = 0 constructed according to such a principle Downloaded from FIG. 4 (1) Surface-coefficient: k = 5, can be used between 100 cc cc. (2) Surface-coefficient: k = 2, can be used between 100 cc.-1.7 cc. (3) Surface-coefficient: k = 1, can be used between 100 cc.- 1 cc. (4) Surface-coefficient: k = 2, can be used between 100 cc.-1.8 cc. (5) Surface-coefficient: k =, can be used between 100 cc.-5.5 cc. (6) Surface-coefficient: k = - can be used between 100 cc.-11 cc. (7) Surface-coefficient: k = can be used between 100 cc.-50 cc. 'The maximum capacity of each being 100 cc. on September 3, 2018 by guest

6 130 has the form shown in figure 3. The range in which vume = k is satisfied is only the part that is surrounded by the curve kx y = Ae2 namely, the part which is included between the lines I and II in figure 3. In practice it is unusual that all of the solution must FIG. 5 be tested. Generally some portion of it will remain at the end of the experiment. If we take this fact into consideration and make R proportionately great, the shape of the bottle will be remarkably simple. Figure 4 shows some examples of the bottles, which have different values for k, but the same V. Any type that is appropriate for the experiment can be selected. By the use

7 APPARATUS FOR OBSERVATIONS OF MICROORGANISMS 131

8 132 of the set it is possible to investigate the maximum, optimum and minimum "surface-coefficient" for the growth of microorganisms. The bottle which is constructed for the purpose described has two tubes attached to the spherical part, one horizontal and the other vertical, both with glass stopcocks. To the horizontal tube is attached a short rubber tubing with a pinchcock and a small glass out-let (fig. 5); the solution for test should be taken out by this tubing. The vertical one should not be used during the experiment, but serves for washing out the precipitates or the residuum of the solution at the end of the experiment. The upper opening is closed with the usual cotton plug. The wall of the bottle is graduated to indicate the volume of the contents. The bottles are held by a holder as shown in figure 5. Figure 6 shows a set of the bottles, the maximum V of each being 250 cc., while k is respectively, 2, 1, 1, -, -o and?w. The whole set can be placed in the usual thermostat. In the use of this apparatus, it must not be forgotten that before a portion of the solution is taken out, the bottle must be shaken thoroughly in order to make the solution homogeneous, because it is important to take out numbers of the micro6rganisms which are representative of the numbers present in the solution as a whole. By the use of this bottle the irregular variation of the air-supply, which had been, hitherto, carelessly overlooked, will be corrected, and at least in this respect our experiments will be rectified. The author hopes that this bottle may be of importance not only in the physiological study of bacteria and other microorganisms, but also in other branches of physiological or physical chemistry, for example, in the study of the gas-liquid or liquidliquid junction. The experimental study using this bottle in bacteriology is now being carried on by the author and will be reported later.

Basic Equipments and Instruments used in Chemistry laboratory: Balance: It is an instrument for measuring mass.

Basic Equipments and Instruments used in Chemistry laboratory: Balance: It is an instrument for measuring mass. Pipettes: They are used to transfer of known volumes of liquids from one container to another.