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

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?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

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/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. connected as an emitter follower to the inverting input When the turbidity of the medium reaches a level 44 of the comparator and the emitter of transistor 43, which indicates the desired cell density, as indicated by also as a follower, to the non-inverting input 45. the recorder or as indicated by samplings withdrawn The integrated circuit comparator 41 is available as through tube 18, the comparator 41 may be reconnected model A710C of the Semiconductor Division of the or switched on and variable resistor 37 may then be Fairchild Camera and Instrument Corporation and has a again set so that midpoint 39 is positive with respect to, terminal 46 which is grounded and terminals 47 and 48 25 but near to, the voltage of midpoint 40, to start the adapted to be connected to about -6 volts and about operation of valve 26 by means of the comparator 41. --12 volts respectively. Whenever the turbidity of the cultured medium in The power source for the bridge and comparator is a chamber 10 increases, as sensed by an increase in re transformer 50 whose primary winding is connected to sistance in photocell 35, relay 73 is operated, as herein an ordinary house current line source at 51 through the 30 after described, valve 26 opens and fresh medium from usual switch and fuse. An indicator lamp 52 may be reservoir 27 flows into chamber 10 diluting the cultured connected across the primary. The same source at 51 medium and reducing the turbidity. An equal amount of may be connected by lines x and y to recorder 31 for cultured medium is automatically withdrawn from the powering its chart motor. bottom of chamber 10 through the constant level device The secondary of transformer 50 is connected to the 35 21 and the overflow from device 21 is collected in the input terminals of a diode bridge rectifier 53 whose out tank 24. put terminals a and b are connected to a respective plate . As indicated above, the comparator 41 is a known of a capacitor in a current limiting arrangement featur device and has maximum ratings of supply voltage of ing a Zener diode 55. Output terminal a of rectifier 53 is -- 14 and -7 with optimum operating voltages of about also connected through a resistor 56 to the emitter of an 40 --12 and -6 volts. The rectifier, current-limiter, voltage PNP transistor 57 whose base is connected through the regulation arrangement described above supplies a -- 12.6 anode and cathode of Zener diode 55 to the junction of voltage at terminal 48 and a -6 volts at terminal 47. terminal a and capacitor 54, the base being also con The state of output 67 of the comparator is controlled nected through another resistor 58 to the other output by the voltage relationship of inverting input 44 and non terminal b of the rectifier. inverting input 45. When input 44 is more positive than This latter output terminal b is also connected to a input 45 the output 67 is approximately zero volts. When voltage regulation arrangement comprising two Zener input 44 is less positive than input 45 the output is ap diodes 60 and 61 in series in the order of anode-cathode, proximately --3 volts. anode-cathode, the cathode of the latter Zener diode When the variable resistance 37 is first set, the balance being connected to the collector of transistor 57. The point 39 is positive with respect to point 40. As the junction of the cathode of diode 60 and the anode of turbidity in chamber 10 increases, the resistance of photo diode 61 is connected to ground. cell 35 increases, decreasing the voltage difference be The junction of the cathode of Zener diode 61 and the tween points 39 and 40. Continuing increase in turbidity collector of transistor 57 supplies a positive 12.6 volts causes point, 40 to become positive with respect to point and is connected by a line 63 to the terminal 48 of the 39. This condition is coupled through emitter followers comparator, to the collectors of transistors 42 and 43 42 and 43 to the inputs 44 and 45. Hence, input 44 is now and to one input point of the Wheatstone bridge arrange negative with respect to input 45 and the output 67, as ment, the other bridge input point being grounded. described above, changes from 0 to -4-3 volts. The junction of the connection of output terminal b The emitter follower arrangement of the transistors with the anode of Zener diode 60 supplies a negative 6 60 42 and 43 between the midpoints 39 and 40 of the Wheat volts to a line 64 which is connected to terminal 47 of stone bridge and the inputs 44 and 45 of the comparator the comparator and to the input terminals 44 and 45 isolates the bridge and comparator. through resistors 65 and 66, respectively. The feedback through resistance 68 ensures that posi The output 67 of the comparator is connected through tive Switching occurs at the threshold point of the sensing a high resistance 68 to the non-inverting input 45 to 65 by the Wheatstone bridge arrangement that excessive tur provide a small amount offeedback. bidity exists in the chamber 10. The feedback is kept small The comparator output 67 is also connected through to minimize the hysteresis effect that it produces. a resistor 69 to the base of a switch or driver NPN The plus 3 volts at output 67 is communicated through transistor 70, the junction of resistor 69 and the base resistor 69 to the base of transistor 70 causing forward being connected to ground through a resistor 71. A 70 bias in the transistor. The transistor being connected be capacitor 72 is connected between this same junction and tween ground and through relay coil 73 to line 63 at ground in parallel with resistor 71. The emitter of tran -- 12.6 volts, its forward biased condition causes transistor sistor 70 is also connected to ground. The collector of 70 to draw heavily through coil 73 to operate the relay, transistor 70 is connected to one terminal of the coil of As relay 73 is operated, its contacts 75 are separated, a relay 73 and to the anode of a diode 74, The other 75 opening valve 26, 3,504,185 5 6 As fresh medium is admitted to chamber 10 the tur As will be apparent to those familiar with the art, the bidity is reduced and the resistance of photocell 35 de invention may be embodied in other specific forms with creases until balance point 39 becomes positive again with out departing from the spirit or essential characteristics respect 40. At that time input 44 becomes positive with thereof. The embodiment disclosed is, therefore, to be respect to input 45 and output 67 again becomes Zero. considered in all respects as illustrative rather than re Transistor 70 is thereby switched off and relay 73 returns 5 strictive, the scope of the invention being indicated by to normal, closing valve 26. the appended claims. After each opening and closing of valve 26 and conse What is claimed is: quent dilution of the contents of chamber 10, the Wheat 1. In apparatus for growing living cells in a liquid stone bridge arrangement continues sensing until subse medium, a growth chamber containing cells suspended quent increased turbidity causes another opening of the 10 in the medium therein and having at least a portion of valve. Thus it will be seen that means have been provided its sidewalls transparent, means for continually agitat for maintaining the cell density at a substantially con ing the medium in the chamber, a light source adjacent stant level. the chamber, a pair of matched photocells including Since the potentiometer of recorder 31 remains con a first photocell arranged to receive light directly from nected in the circuit 30, a continuous record of additions the source and a second photocell arranged to receive of the medium through valve 26 is maintained. light from the source through a portion of the medium Comparator 41 does not require highly regulated supply in the chamber, a fixed resistance and a variable re voltages and the parallel arrangement of the resistances sistance arranged in a Wheatstone bridge with the photo in the bridge is likewise relatively immune to voltage fluc 20 cells, the bridge having one midpoint between the first tuations so that simple Zener regulation of the voltages photocell and the fixed resistance and the other mid to the control circuitry by the Zener diodes 60 and 61 is point between the second photocell and the variable sufficient. resistance, a stylus and strip chart recorder having a The arrangement of capacitor 54, Zener diode 55, potentiometer for operating its stylus, the potentiometer resistances 56 and 58, and transistor 57, of course, pro 25 being operatively connected to the bridge midpoints, and vides means for limiting the current supplied to the con a source of electric current operatively connected to trol circuitry and also provides means for protecting the the bridge for energizing the bridge, whereby changes power supply components, transformer 50, rectifier 53 in turbidity of the medium in the chamber are sensed and Zeners 60 and 61. by the second photocell and the consequent changes in A constructed embodiment of the invention was Suc 30 voltage differential between the midpoints are recorded cessfully operated using the following circuit components, on the chart. listed by way of example only: 2. Apparatus defined in claim 1 in combination with means for automatically maintaining the turbidity of the Comparator 41-Fairchild "A710C medium in the chamber at a substantially constant level, Photocells 35 and 36-Clairex Cdse iCL904 35 comprising: a reservoir of medium for diluting the Resistors 69, 71, 58, 38 and potentiometer 37-1K cultured medium in the chamber, conduit means leading Resistor 68-510K from the reservoir to the chamber through a normally Resistors 65 and 66-15K. closed solenoid controlled valve, the chamber having Resistor 56-100 ohms overflow means, a voltage comparator having a refer Recorder-High point impedence, potentiometer type, 40 strip chart recorder with balanced input capabilities and ence input connected to the bridge midpoint between minimum sensitivity of 100 mv. full scale deflection. the first photocell and the fixed resistance and a measur Transistors 42, 43 and 70-2N706 ing input connected to the other bridge midpoint, the Transistor 57-2N1486 comparator having an output which changes state when Rectifier-10DB2A the voltages at the midpoints of the bridge are reversed Zener 55-5.6 v. from normal in their quantitative relation and delivers Zener 60-6.2. v. current, switch means responsive to current at the output Zener 61-12.6 v. to operate the solenoid controlled valve for opening Relay 73-W102PCX the valve and diluting the cultured medium in the cham Diode 74-1N270 ber until the voltages at the midpoints of the bridge Capacitor 72-47 f. 50 are again reversed back to normal, and a source of electrical current operatively connected to the comparator Capacitor 54-1100 f. for energizing the comparator. Experiments using the constructed embodiment lasting 3. Apparatus for growing living cells in a liquid as long as five days have been conducted in the produc medium and for automatically controlling the cell popu tion of algae cells such as Chlorella pyrenoidosa, bacteria lation density in the medium, comprising: a growth cells such as Escherichia coli, Chlorobium thiosulfatophil chamber containing cells suspended in the medium there um, and Rhodospirillum rubrum, mold cells such as yeast, in, at least a portion of the chamber walls being trans and animal cells such as Hela cells. Constant turbidity parent, means for continuously agitating the medium in was maintained, using the apparatus described above, at a the chamber, a reservoir of medium for diluting the level varying as little as +0.02% O.D. 60 cultured medium in the chamber, conduit means leading The apparatus of FIGURE 1 is shown diagrammati from the reservoir to the chamber through a solenoid cally and it will be understood that the sidewall of cham controlled valve, the chamber having overflow means ber 10 might be of stainless steel or other material so long and liquid level maintaining means connected to the as a window or windows were provided therein. Either overflow, a light source adjacent the chamber, a pair light source or photocells might be contained within the 65 of matched photocells including a first photocell arranged chamber so long as one photocell views the source directly to receive light directly from the source and a second and the other views it through a portion of the cultured photocell arranged to receive light from the source medium. Suitable protection capable of withstanding auto through a portion of the medium in the chamber, a claving, of course, would be necessary for any of the com fixed resistance and a variable resistance arranged in ponents in the chamber. 70 a Wheatstone bridge with the photocells, the bridge The control circuitry shown in FIGURE 2 may also having a first midpoint between the first photocell and be used to measure and control the mixing or maintaining the fixed resistance and having a second midpoint be of a constant turbidity or opaqueness in any mixture tween the second photocell and the variable resistance, where a relatively translucent liquid is mixed with a a voltage comparator having a non-inverting input more turbid or opaque liquid. 75 coupled to the first bridge midpoint and an inverting in 3,504,185 7 8 put coupled to the second bridge midpoint, the com through a portion of the mixture, a fixed resistance and parator having an output which is at substantially zero a variable resistance arranged in a Wheatstone bridge voltage when the inverting input is at a greater positive with the photocells, the bridge having a first midpoint voltage than the non-inverting input and which output is between the first photocell and the fixed resistance and positive when the inverting input is negative with re a second midpoint between the second photocell and spect to the non-inverting input, a transistor switch oper the variable resistance, a voltage comparator having a able by the output when it is positive, the transistor non-inverting input coupled to the first midpoint and an switch being adapted to open the solenoid operated valve inverting input coupled to the second midpoint, the com when the switch is operated, the solenoid operated parator having an output which is at Substantially zero valve being adapted to close when the transistor switch 0. voltage when the inverting input is at a greater positive is not operated, and a source of electric current operative voltage than the non-inverting input and which input is ly connected to the bridge and comparator for energiz positive when the inverting input is negative with re ing them, whereby cell growth in the chamber produces spect to the non-inverting input, switch means operable increased turbidity of the cultured medium and increased by the output when it is positive for opening the valve, resistance in the second photocell to open the valve and 15 and a source of electric current for energizing the bridge dilute the cultured medium causing the chamber to over and the comparator, whereby, when the variable re flow, and dilution ceases when the resistance of the sistance is set so that the second midpoint is substantial second photocell is reduced because of the dilution of ly at the voltage of the first midpoint, any increase in the medium in the chamber. opacity of the mixture energizes the comparator output 4. Apparatus as defined in claim 3 having a potentiom 20 and causes dilution of the mixture. eter operated, strip chart recorder to record changes in the turbidity in the chamber, the potentiometer of the References Cited recorder being operatively connected to the midpoints of UNITED STATES PATENTS the bridge. 5. Apparatus for controlling the opacity of a liquid 25 2,843,756 7/1958 Wise et al. ...------250-210 mixture having one component of less opacity than the 2,872,588 2/1959 Barton ------250-210 mixture, comprising: a container for the mixture hav 2,990,339 6/1961 Frank et al. ------195-103.5 X ing at least a portion of its sidewall transparent, a reser 3,172,235 3/1965 Bjorklund ------47-1.4 voir containing a dilution supply of the less opaque com ponent, conduit means connecting the reservoir and con 30 WALTER STOLWEIN, Primary Examiner tainer, an electrically operated normally closed valve U.S. Cl. X.R. in the conduit means, a light source adjacent the con tainer, a first photocell arranged to view the light Source 47-1.4; 195-103.5, 143; 250-210; 356-240 directly, a second photocell arranged to view the Source