In cultivating microorganisms, it is possible to calculate the cell density of the microbes in the culture liquid by measuring the optical density or amount of light scattered by the liquid, so that it is of great importance to be able to make accurate measurements of these properties of the culture liquids.
However, in culturing aerobic microorganisms, air bubbles are produced in great quantities in the agitation tank as air agitation is conducted therein, so that in measuring the optical density of a test liquid by a colorimeter or spectrophotometer, or the turbidity of the liquid by a nephelometer, the air bubbles often enter into the measuring chamber to affect the light transmission or scattering rate because of irregular reflection caused by the air bubbles, resulting in inaccurate measurement.
Optical cells of different types are known in the art, but the cells which are used in turbulent environments have met with problems from the presence of small bubbles in the medium. The type of cell used to measure optical density of solutions can be of the type in which the cell is placed in the solution to be checked so as to detect the optical density or scattering of the solution with ease from the photoelectromotive force indicated by a voltmeter connected to a photocell by a cable. In this type of colorimeter, light is projected from a light source through a color filter and a lens to a specimen chamber designed to allow free ingress and egress of the solution to be examined so as to pass light from the light source through the solution and to a photocell or phototransistor, the light source and photocell or phototransistor being respectively connected by a cable to a power source and a voltmeter which automatically records the absorbance.
Turbidity or absorbance of the culture liquid can be determined by measuring the optical density of the liquid, and as it is known that the optical density is proportional to the number of microbes in the culture liquid, it is possible to know the number of microbes, i.e., the cell density of microbes in the culture liquid, by measuring the optical density of the liquid. In view of this, immersion types of colorimeters have been developed which are equipped with defoaming means wherein the degree of growth of the microorganisms in culture solution is determined by continuous measurement of the optical density of the liquid by the colorimeter.
Generally, the optical density (OD) can be expressed by the following formula: ##EQU1##
According to some immersion types of colorimeters, the device is introduced into the test liquid so as to detect the density or turbidity with ease from the photoelectromotive force indicated by a voltmeter connected to the device by a cable. Therefore, when it is desired to know, for example, the density of a culture liquid during cultivation of microorganisms, one may simply insert this throw-in type colorimeter into the fermentor. It may also be placed directly into a river, lake, water storage, or other such liquid environment when it is desired to know the turbidity of the water therein.
One existing type of experimental sensor consists of a light source and a facing sensor, immersed together in the liquid to be measured. Frequent measurements must be taken, with little time lag between measurements. The liquid flows freely through the space between the light and the sensor, and bubbles entering this space interfere with the measurement, giving false readings.
Another method of dealing with the problem of false readings from bubbles in the liquid medium is shown in Shibata et al., U.S. Pat. No. 4,075,062. In this patent, Shibata et al. disclose a throw-in type colorimeter equipped with a defoaming device wherein a downward flow is created in a cylinder by means of the liquid level column difference in the inlet of a cylinder to let bubbles in the solution escape upwardly therefrom. The device is also equipped with an upward solution flow passage to provide a communicating passage between the outer and inner cylinders to form a down flow in the outer cylinder and drive the remaining bubbles out of the solution in the outer cylinder by the twice reversed flow while removing the bubbles residing in the measuring chamber by the agitated flow.
The problems encountered with the Shibata et al. device are that it will not work if the flow is in the opposite direction in the culture flask. The colorimeter's defoaming device must always be in the same direction. The design of this device is rather complicated, hence requiring a fairly large device to obtain measurements. The device, which requires a screen, is difficult to clean after being removed from a culture. This device also provides a slow reading, requiring about one to three minutes.
Boe et al., in U.S. Pat. No. 3,560,099, disclose a colorimeter flow cell including a baffle to remove gas bubbles from the solution being measured. This flow cell is divided into two chambers by a wall extending parallel to a light beam which is to pass through the cell. Liquid enters the first chamber and flows to the second chamber through upper and lower apertures in the wall. Gas is separated from the liquid in the first chamber and passes to an outlet from the second chamber without interfering with the light beam, as the windows in the cell are at a lower level than the upper apertures.
The Boe et al. colorimeter is not a drop-in type of colorimeter, and it is a relatively complicated device. This device is also difficult to clean, and it does not work by right angle light scattering.
Neeley et al., U.S. Pat. No. 4,260,257, disclose a flow cell comprising an elongated tubular body member, a debubbler unit, and a tubular fluid outlet tower. One end of the debubbler unit functions as the fluid inlet. The tubular body member has an open ended bore therethrough constituting a sight passageway to lie along a portion of the length of the light path of a colorimeter.
The Neeley et al. flow cell is not a drop-in type of measuring device and does not make use of a large cross-section to reduce flow velocity to separate the bubbles in the liquid. This device is not intended to reduce the effects of fine bubbles, and does not include right angle light scattering. It is doubtful if this device will work, as any bubbles removed by the outlet tower must first have passed through the measurement region, thus causing the interference it is desired to remove. This could be overcome by pulsatile operation, although a continuously operating device would be preferable.
All these devices are either unduly complex and expensive, or insufficiently effective, or both.