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4 S1-Us A-Bo-Le Gist'y March 31, 1970 G. ZWEG ET All 3,504,185 APPARATUS FOR MEASURING AND CONTROLING CEL POPULATION DENSITY IN A LIQUID MEDIUM Filed May 9, 1968 2. Sheets-Sheet :9 O - 32. 33 : o SAHH 3. ?m WeavroAs, GUNT.R Z. We G ROBERT e. PP-ER JOAn . - 4 S1-us a-bo-le Gist'y. March 31, 1970 G. ZWEG ET All- 3,504,185 APPARATUS FOR MEASURING AND CONTROLLING CELL POPULATION DENSITY IN A LIQUID MEDIUM Filed May 9, 1968 2. Sheets-Sheet 2 N /Ayyam AOes. GUnTER ZWEG RO2ERT E. PPHER JOAN. E. HTT 3,504,185 United States Patent Office Patented Mar. 31, 1970 2 3,504,185 medium to dilute the cultured medium until the cultured APPARATUS FOR MEASURING AND CONTROL medium becomes less turbid again and the output of the LING CELL POPULATION DENSITY IN A LIQUID comparator is no longer energized so that the switch is MEDUM no longer operated and the valve is shut off. Gunter Zweig, Syracuse, Robert E. Pipher, Cortland, and A constant fluid level is maintained in the chamber Joan E. Hitt, Syracuse, N.Y., assignors to Syracuse Uni versity Research Corporation, Syracuse, N.Y., a corpo in which the cultured medium is contained and the over ration of New York flow resulting from dilution is collected for use. Usually Filed May 9, 1968, Ser. No. 727,910 continual agitation of the fluid in the chamber is required nt. C. G01n 21/26 to keep the cells in suspension in the medium and physical U.S. C. 250- 218 S Claims 10 conditions which are necessary for optimum growth speed are maintained in the usual manner. ABSTRACT OF THE DISCLOSURE BRIEF DESCRIPTION OF THE DRAWINGS A glass chamber for cell growth in a liquid medium has FIGURE 1 is a diagrammatic view of the culture means for adding medium controlled by a solenoid valve. 5 chamber and associated apparatus of the invention; and One photocell faces a light source through the medium FIGURE 2 is a circuit diagram of the electrical con and another photocell faces the source directly. The pho trol portions of the apparatus of FIGURE 1. tocells are connected with resistances in a Wheatstone bridge whose midpoints are connected to a voltage com DESCRIPTION OF THE PREFERRED parator whose output operates the valve for diluting the 20 EMBODIMENT medium when cell growth causes increased turbidity in Referring to FIGURE 1, a growth chamber 10 has a the chamber and changes voltage in the bridge. A strip glass sidewall 11 in the form of a water jacket with chart recorder potentiometer connected across the bridge entrance 12 and exit 13 through which water is con records changes in voltage in the bridge. 25 stantly pumped, the water being maintained at a chosen constant temperature by conventional means not shown. The chamber is stoppered at top 14 and bottom 15, as BACKGROUND OF THE INVENTION shown, the top of the chamber being vented to atmos This invention relates to the growth of single cell organ phere by the tube 16. isms in a liquid medium and more particularly to a device 30 Chamber 10 contains a liquid medium, usually nutri for measuring such growth and for controlling the cell ent, containing organic and inorganic salts and other sub density in the medium. stances and a population of living cells such as algae, In growing single cell organisms, such as algae, bac bacteria, mold or animal cells in suspension in the medium teria, mold and yeast, in a liquid medium difficulty has and capable of growth therein. A tube 17 leading from a been experienced in that, as the medium becomes more 35 Source, not shown, through the top 14 extends substan densely populated, a point is reached where the rate of tially to the bottom of the chamber for supplying gas growth is slowed because the medium is too densely popu which bubbles up through the medium. Nitrogen, oxygen, lated. Usually there is an optimum population density, or or carbon dioxide or a mixture may be used and one or a range of densities, where the cell reproduction is fastest. more of the gases may be necessary for cell growth. The As the medium becomes more densely populated, it be 40 continuous stream of bubbles from tube 17 agitates the comes more turbid or opaque so that attempts have been cultured medium to keep the cell population in suspension. made to measure the density by optical means so that the Alternatively or additionally, a mechanical agitating de optimum density may be obtained by adding more medi vice may be used. um to dilute the populated or cultured medium in which A sampling tube 18 with shut off valve 19 is provided growth takes place. Such optical equipment has been ex 45 through the bottom 15 and another tube 20 through the pensive and continuous attention to the measurement and bottom leads to a constant level overflow device 21 for addition of fresh medium is necessary. maintaining the medium in the chamber 10 at a chosen level 22. The drain 23 from the constant level device SUMMARY OF THE INVENTION carries the withdrawn cultured medium to a collecting The present invention provides means for continuously 50 tank 24. electrically scanning the cultured medium by means of a Another tube 25 through the top 14 is led through a photoelectric cell and measuring the voltage of this photo normally closed solenoid operated valve 26 from a reser cell. In order to obtain the measurement a second match voir tank 27 of fresh uncultured liquid medium for dilut ing photocell, in constant direct view of the same light ing the cultured medium in chamber 10 when the cell pop source is used together with two resistances connected in ulation in the chamber rises above a chosen population the well known Wheatstone bridge, one resistance being density. All of the above described apparatus is kept un variable so that the range of the bridge may be adjusted. contaminated as by autoclaving before assembly. A strip chart recorder may be used to record the measure Valve 26 is operated by a control circuit 30 which ment, the potentiometer which operates the stylus of the includes a moving strip chart recorder 31 having a recorder being connected in conventional manner to the 60 potentiometer-operated stylus 32 for marking the strip balance point of the bridge. chart 33. Furthermore, a known type of voltage comparator is A Source of light 34 is located adjacent the chamber also connected to the balance points of the bridge and 10 and two matched photo-electric cells 35 and 36 are the output of the comparator is energized by changes in connected in the circuit 30. The light source 34 may be voltage of the scanning photocell in the bridge. As cell 65 of the fluorescent type required for the growth of cer population density increases the turbidity of the medium tain photo-Synthetic cells but, in any event, is located increases resulting in increased resistance and lower volt on one side of chamber 10 while the photocell 35 is lo age in the Scanning cell as compared to the non-scanning cated on the other side of the chamber for receiving light cell. from the source through the cultured medium in the Automatic control of the cell density is accomplished 70 chamber. Care is taken to install the photocell 35 so that by using the output of the comparator for operating a gas bubbles from the tube 17 do not pass between the switch arrangement which opens a valve to admit fresh photocell and the light source. 3,504,185 3 4. The second photocell 36 is located, preferably Sub terminal of coil 73 is connected to the cathode of diode stantially equidistantly from the light source 34, for 74 and to line 63. receiving light directly from the source as indicated The normally closed contacts 75, 75 or relay 73 nor diagrammatically in FIGURE 1. mally complete a circuit from the line terminals at 51 Referring now to FIGURE 2, photo-electric cells 35 through the solenoid of valve 26 so that the valve is and 36 are wired in a Wheatstone bridge arrangement normally held closed by the solenoid. with resistors 37 and 38, resistor 37 being variable and In operation the comparator 41 may be disconnected in the form of a ten-turn potentiometer for controlling from the Wheatstone bridge arrangement or rendered the balance point of the bridge. inoperative by a suitable switch in the driver arrange The midpoints 39 and 40 of the Wheatstone bridge O ment. Recorder 31, then operates to graph the increas are connected to the respective terminals of the recorder ing turbidity of the cultured medium in chamber 10 as potentiometer and are also each connected through a the cells of the culture multiply. NPN transistor to a respective input terminal of a com The variable resistance 37 is set at a value to bring mercially available high-speed differential voltage comr the balance points 39 and 40 into approximate equality. parator 41. Point 39 between photocell 35 and resistor 5 As the resistance of photo-electric cell 35 changes due 37 is connected to the base of transistor 42 and point 40 to the increasing turbidity of the medium in chamber between photocell 36 and resistor 38 is connected to the 10 the voltage difference between the balance points 39 base of transistor 43. The emitter of transistor 42 is and 40 is recorded by the voltage sensitive recorder.
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