WO1989010193A1

WO1989010193A1 - Improved method and apparatus for pipetting liquids

Info

Publication number: WO1989010193A1
Application number: PCT/US1989/001826
Authority: WO
Inventors: John R. Wells; Robert Caveney
Original assignee: Cavro Scientific Instruments, Inc.
Priority date: 1988-04-29
Filing date: 1989-04-28
Publication date: 1989-11-02

Abstract

An improved method for pipetting multiple aliquots of liquid employs a preliminary backsip prior the first expression of an aliquot of the liquid; an adjustable backsip between each expression of aliquots of liquid; and a blow out volume of air, aspirated into the pipette prior to the initial aspiration of liquid, for blowing out a retained volume of liquid after the expression of the last measured aliquot. The improved method for pipetting liquid may be performed on an improved apparatus which includes a computer or microprocessor having within its memory a schedule or correlation for the optimal backsip to execute after the expression of any given aliquot. The computer or microprocessor may also include means for driving the apparatus so as to perform the preliminary backsip and the aspiration of the blow out volume.

Description

IMPROVED METHOD AND APPARATUS FOR PIPETTING LIQUIDS

Specification The invention relates to methods for pipetting liquids. More particularly, the invention relates to improved pipetting methods which enhance the accuracy of pipetting by minimizing the occurance of unintended dripping.

Background of the Invention The employment of computers or microprocessors and step motors or positional servo-motors for automating and controlling the pipetting process has greatly enhanced the convenience of pipetting. In a typical automated pipetting apparatus, the liguid is aspirated or dispensed by means of a piston pump driven by a step motor. The displacement of the piston within the piston pump is proportional to the number of steps executed by the step motor. When the piston pump is pneumatically connected to a pipette, displacements of the piston can be employed to aspirate or express liquids into or out of the pipette. To a first approximation, the volume of liquid which is aspirated or expressed into or out of a pipette is directly proportional to the displacement of the piston and to the number of steps executed by the step motor. The computer .or microprocessor may be programmed so that inputting a request for aspirating or expressing a given volume of liquid causes an output which drives the step motor to - advance the appropriate number of steps. A method for programming a computer for driving an automatic pipetting device is disclosed in the Cavro RS232C Primitive Protocol Manual (Cavro Scientific Instruments, Inc., Sunnyvale California, P/N 015-5864 Rev. B, August 1984). However, the volume of aspirated or expressed liquids is. not strictly equal to the displacement of the piston. Inequality arises from the expansion of the air within the piston, tubing, and pipette due to the weight of the liquid column within the pipette and the resultant reduction of air pressure therein. Mezei et al (U.S. Pat. No. 4,586,546) disclose the use of a microprocessor within a pipetting apparatus to compensate for the inequality caused by the reduction of air pressure within the pipette due to the weight of the liquid column. Accuracy and precision were further improved by the introduction of a fixed backsip function (e.g., IQ 190 DS Sample Processor, manufactured by Cavro Scientific -Instruments, Inc., Sunnyvale California). A backsip function diminishes unintended dripping from a pipette. - After^* the desired volume of liquid has been pipetted from a pipette,- liquid which remains within the pipette is partially withdrawn into the pipette by means of the backsip function. If a drop of liquid remains hanging from the tip of the pipette after the desired volume of liquid 5 is dispensed therefrom, the withdrawal of this hanging drop back into the pipette reduces the possibility that the drop will accidently fall from the pipette. After dispensing liquid from a pipette, the IQ 190 DS Sample Processor can reverse the direction of the piston stroke and displace the 0 piston over a small fixed volume in the opposite direction. This causes unexpressed liquid to withdraw back into the tip of the pipette and reduces the occurance of unintended _.dripping from the pipette.

Although the accuracy and precision of automatic 5- pipetting processes are improved by the use of the fixed backsip function, it was unrecognized in the prior art that new sources of inaccuracy and imprecision were introduced by the fixed backsip function itself. The optimal magnitude of the fixed backsip function, i.e. the magnitude 0 which minimizes untended dripping and maximizes the accuracy and precision of the pipetting process over the entire range of the pipette, is a compromise. The avoidability of this compromise was unrecognized in the pπior art. 5" What was needed was both a recognition of the specific factors and causes of the poor performance of the prior art backsip function and a remedy for this problem. Summary of the Invention The invention described herein identifies some of the factors which detract from the performance of the fixed backsip function of the prior art and provides a solution for these problems.

A principal problem with the fixed backsip function of the prior art can be most easily illustrated with an example where several aliquots of liquid are dispensed from a loaded pipette. It can be experimentally observed that, O after expressing the first aliquot of multiple aliquots from a loaded pipette, the magnitude of fixed backsip may be too little. When a large mass of liquid remains within the column of a pipette after dispensing the first aliquot, the fixed backsip function may raise the lower miniscus of 5 the liquid column too little. In fact, if a droplet remains pending from the pipette tip after the expression of the aliquot, a backsip of fixed magnitude may not retract the droplet all the way back into the pipette, i.e. a portion of the droplet may actually remain pending from 0 the tip of the pipette. Under these circumstances, the risk of unintended dripping is insufficiently reduced. On the other hand, it may also be experimentally observed¹ that, ^"when expressing the penultimate aliquot of multiple aliguots from a pipette, i.e. when very little ; mass remains within the liquid column of the pipette, the magnitude of a fixed backsip is some times too great. Under this circumstance, there is a risk that the lower miniscus of the liquid column may rise too high into the tip of the pipette, through the narrow bore region of the tip and into the main body of the pipette. If the main body of the pipette has a relatively wide bore, as is the case with conventional serological pipetts, the intr.o_duct±σn of air into this region, may cause the formation of a bubble. If a bubble forms and rises through the liquid column, the microprocessor will loss track of the true location of the lower miniscus. Hence when the next aliquot is expressed from the pipette, its volume will be inaccurate. The error of the volume of this last aliquot will correspond approximately to the volume of the air bubble. The invention described herein recognizes that the magnitude of a backsip function can sometimes be too great and can sometimes be too little. The invention described herein solves these problems by disclosing the use of an adjuatable backsip function. An adjustable backsip 0' function can be adjusted with respect to each aliquot of multiple aliquots. The backsip function is adjustable with respect to its magnitude, with respect to its speed of execution, and with respect to the delay between the execution of the backsip and the execution of the preceding 5 expression step. Furthermore, it is recognized that the speed of execution of the preceding expression step will effect the backsip step. However, of all of these backsip parameters, the adjustment of the magnitude is particularly important with regard to its contribution to the

20 improvement of the accuracy and precision of pipetting.

A schedule of the optimal backsip may be constructed with respect to any or all of the above parameters as a function of the volume of liquid remaining within the pipette. If the piston is displaced by means of a step

25. motσr, the liquid volume dispensed or remaining within the pipette may be correlated with the step count of the step motor:.. Each step count displaces the piston a constant volume and correponds to arefincremental increase or decrease of liquid within the pipette. Hence, the backsip

30 schedule may include a listing of the optimal number of steps for the backsip function as a function of the volume of liquid remaining within the pipette. The volume of liquid remaining within the pipette may be determined by the computer or microprocessor according to the amount of

35 liquid which has been aspirated and dispensed. Since the entire range of the step motor is countable, only a countable number of backsip schedules need to be determined. Construction of the backsip schedules may be facilitated by the use of considerable interpolation.

Since size and shape of the pipette and the surface interactions between the pipette and the liquid influence the optimal backsip, a backsip schedule must be determined for each type of pipette which may be employed with the apparatus. Additionally, since the viscosity, surface tension, and other physical properties of the liquid will effect the backsip schedule, a different schedule should be determined for each type of liquid with which the pipetting apparatus will be used. Fortunately, the optimal backsip schedule for most weak agueous solutions are similar and may be approximated by water. It has been found most expeditious to determine the optimal parameters for the backsip function empirically, i.e. by measurement. Once these parameters are determined they can be loaded into the memory of the computer or microprocessor which controls the apparatus. The invention further recognizes the need for a preliminary backsip. A preliminary backsip occurs after aspirating liquid into the pipette, but prior to the expression of the first aliquot. Since the preliminary backsip follows an aspiration step, it is not a true "backsip," i.e. the direction of the piston is not reversed. However, the preliminary backsip function shares at least one important similarity with the regular backsip, viz. both types of backsips serve to minimize the risk of dripping. In order to achieve good accuracy and precision with the expression of the last aliquot, it is also preferred to retain a small volume of liquid within the pipette after -the expression of the last aliquot. Accordingly, when liquid is aspirated into a pipette for subsequent pipetting, the total volume of liquid being aspirated should include a retained volume as well as the total volume of liquid which is to be expressed. The retained volume may be relatively small. Prior to aspirating new liquid into the pipette for further pipetting, the entirety of the retained volume should be discharged from the pipette, i.e. the pipette needs to be cleared of all residual liquid.

Accordingly, the invention further recognizes the need for a blow out volume of air for clearing out the pipette of: the retained volume after the expression of the last

1O-¹ aliquot. Hence, a "blow out" volume of air should be aspirated into the pipette prior to the first aspiration of liquid. At the conclusion of the expression of the last aliquot, this blow out volume of air remains within the pipette and is employed for blowing out the retained volume

15 of liquid from the pipette.

In the preferred mode, the improved method for pipetting liquids is performed by means of an improved apparatus. The improved apparatus shares many similarities with prior art pipetting apparatus. It employs a piston

20 ump, step motor, and a computer or microprocessor. However, a principal difference between the improved apparatus and the prior art apparatus is the content of the memory of the computer or microprocessor, i.e. the memory of the computer or microprocessor includes one or more

^ correlations or schedules for the backsip function. These correlations or schedules describe the step counts which should be performed in order to optimize the backsip parameters as a function of the height of the liquid column within the pipette. Additionally, the computer or

30 microprocessor of the improved apparatus includes the means to execute the adjustable backsip function according to the information contained within these correlations or schedules.

Furthermore, the improved pipetting apparatus includes

35 the option of employing a variety of pipettes. In addition to the standard serological pipette, the improve apparatus can employ a multichannel pipette and a bag type pipette. In the preferred embodiment, each channel within a multichannel pipette is pneumatically attached to its own ' piston pump. All of the multiple piston pumps may then be driven by a single step motor.

The bag type pipette includes a bag suspended within a pneumatic chamber. A tip portion is connected to the bag and projects out of the chamber. The pneumatic chamber is pneumatically connected to a piston pump which is then driven by a step motor. In use, the tip of the pipette is submerged into a source of liquid and air is drawn from the pneumatic chamber. As air is drawn from the pneumatic chamber, liquid is aspirated into the bag. Liquid can then be expressed from the bag by releasing air back into the pneumatic chamber. In an alternative embodiment, the air within the pneumatic chamber may be partially or whole replaced by a hydraulic fluid.

It is contemplated that each of the pipettes described above are may be operated pneumatically. However, the air within the pump may be completely replaced with hydraulic fluid and the air within the tubing which connects the pump to the pipette may be partially replaced by hydraulic fluid. On the other hand, if all of the air is replaced by an incompressible hydraulic .fluid, then the need for a variable backsip is obviated. .Accordingly, when any system is described herein as pneumatic, it is contemplated that the system will include at least one air pig, i.e. a small quantity of air as would be sufficient to separate the sample liquid from hydraulic fluid. Brief Description of the Drawings

Figure 1 is a perspective view of the improved pipetting apparatus showing all of the major components joined together, including a handle for a serological pipette. Figure 2 is a plan view of an alternative handle and micro-pipette which may be substituted for the handle and_. serological pipette in Figure 1.

Figure 3 is a plan view illustrating the serological pipette of Figure 1 as it is employed in a partial sequence of the improved pipetting method. The sequence illustrates the expression of liquid and subsequent backsipping of the liquid into the pipette.

Figure 4 is a plan view which illustrates a conical shaped micro-tip which may be employed as a pipette and which may be inserted onto the end of the handle illustrated in Figure 2. The preferred conical shaped micro-tip need not include an internal annulus.

Figure 5 is a perspective view of a portion of an alternative embodiment of the improved pipetting apparatus illustrating a multichannel pipette, a handle which is adapted to accept the attachement of the multichannel pipette, multiple piston.pumps, and multiple pneumatic hoses connecting the multiple piston pumps to the handle. Figure 6(a) and 6(b) are plan views of a further alternative embodiment of the pipette and handle illustrating a handle having a pneumatic chamber and illustrating a bag type pipette. Figure 6(a) illustrates a closed pneumatic chamber and Figure 6(b) illustrates an opened pneumatic chamber. The opened pneumatic chamber may be employed to insert or remove the bag type pipette.

Detailed Description The pipetting apparatus includes a piston pump (1 ) and a step motor for driving the piston pump (1 ) . Additionally, a microprocessor is employed for controlling the step motor. Data and programming may be entered into the microprocessor by means of an input box (2). The input box (2) may include a display which requests instructions, echoes the response, and displays the status of the apparatus. A first electronic cable (3) connects the input box (2) with the microprocessor. Furthermore, a set of electronic controls (4) may be incorporated into a pipette handle (5) for initiating various commonly employed functions, including aspiration, expression, and mixing functions. The electronic controls (4) are connected to the microprocessor by means of a second electronic cable (6) . The pipette handle (5) may be hand held with the electronic controls (4) conveniently located for operation _ by the user's fingers or thumb. The pipette handle (5) includes a connector to which a pipette (7) may be attached. A pneumatic hose (8) connects the piston pump (1) with the handle (5) so that displacements by the piston pump (1) displace air within the pneumatic hose (8) and cause liquid to rise and fall within the pipette (7).

In use, a pipette (7) is attached to the handle (5) by means of the connector, the step motor and microprocessor are energized, and the step motor goes through an initialization procedure. During the initialization procedure, the step motor drives the piston pump (1) through it^'s full range and establishes a zero reference point. The range of the piston pump (1) and zero reference point are then recorded within the memory of the microprocessor. The zero reference point is the reference point from which the step motor is driven and from which the step count is measured. The display on the input box (2) may then prompt the user to enter data with respect to the particular pipette (7) which is to be used and with respect to the liquid volumes which are to be aspirated and expressed or dispensed. Alternatively, the user may enter a new program into the microprocessor or may enter other data into the memory of the microprocessor relating to the viscosity, temperature, and other information. After the microprocessor has been instructed, the electronic controls (4) on the handle (5) can then be employed to initiate the pipetting process. Typically, a user will wish to aspirate liquid into the pipette (7) and then express one or more aliquots. In this case, the user may employ the electronic controls (4) to instruct the microprocessor to drive the step motor so as to aspirate a specified volume of liquid into the pipette (7) and then to express aliquots of liquid in specified volumes. The aliquots may be identical or may differ in size.

In a preferred mode, the memory of the microprocessor includes a correlation which.relates the aspirating step count by which the step motor is drive with the precise volume of liquid which is aspirated into the pipette (7). This correlation may vary from one pipette to the next and from one liquid to the next. Accordingly, the memory of the microprocessor may include data for this correlation for each pipette and liquid which may be employed with the apparatus.

It has been found to be easiest to determine these correlations empirically. Hence, for a given pipette and liquid, the volume of the aspirated liquid is determined aver the complete range of step counts through which the piston pump (1) may be driven. Typically, water is the most important liquid for which aspiration correlations are obtained. The aspiration correlations for other dilute aqueous solutions will approximate the aspiration correlation for water. Hence, the standard memory may include an aspiration correlation for water only. However, if a user has a need for pipetting other liquids having viscosities and other properties which significantly differ from that of water, aspiration correlations for these liquids may also be empirically obtained and entered into the memory of the microprocessor.

In a preferred mode, prior the actual aspiration of liquid into the pipette (7), the microprocessor causes the step motor to drive the piston pump (1) so as to aspirate a blow out volume of air into the pipette (7) . The blow out volume of air is drawn into the pipette (7) prior to the submersion of the pipette (7) tip into a source of liquid. The blow out volume of air is employed during the dispensing portion of the pipetting method in order to blow out residual liquid remaining within the pipette (7) after the all of the various aliquots have been expressed therefrom. The blow out volume of air allows the piston pump (1) to blow out the last portion of liquid from the pipette (7). Without the blow out volume of air, residual liquid may remain within the pipette (7) due to expansion within the pipette (7) caused by heat expansion, degassing, liquid adherence to the inner wall surface of the pipette (7), or other reasons. Without the aspiration of a blow out volume of air into the pipette (7), the piston pump (1) would be unable to blow out residual liquid from the pipette (7) once step motor had reached the zero reference step. Hence, the blow out volume of air is an extra volume of air, which is drawn into the pipette (7) prior to the aspiration of liquid and which allows the piston pump (1) ' to blow residual liquid from the pipette (7) after the completion of the pipetting process.

In order to aspirate liquid into a pipette (7), the data is first entered into the microprocessor with respect to the pipette (7) into which the liquid will be aspirated and the the volume of liquid to be aspirated is then specified. After this data has been entered into the microprocessor, the tip of the pipette (7) is submerged into a source of the liquid. The aspiration control on the handle (5) is then activated and the microprocessor employs its aspiration correlation in order to determine how many step counts to send to the step motor. The appropriate number of step counts are then sent to the motor. The step motor executes the appropriate number of step counts, thereby driving the piston pump (1) and cause liguid to be drawn into the pipette (7). Once the liquid is drawn into the pipette (7) , the tip of the pipette (7) is withdrawn from the liquid source. If the tip of the pipette (7) was significantly submerged within the liquid source, its withdrawal will cause a small loss of pressure within the pneumatic hose (8). This may result in the formation of a small droplet of liquid, hanging from the tip of the pipette (7) . If the pipette (7) were then vertically accelerated sharply, such acceleration could cause the droplet to separate and fall from the pipette (7). The precise volume of liquid within the pipette (7) would then become unknown. Precise pipetting would then become impossible.

Accordingly, in the preferred mode, the microprocessor may be programmed to backsip the liquid into the pipette (7) subsequent to the aspiration of liquid. The backsip occurs after the tip of the pipette (7) is withdrawn from the source of liquid. A backsip causes liquid to be partially withdrawn into the pipette (7). If a droplet of liquid is pending from the tip of the pipette (7) of if the liquid protrudes in a convex fashion from the tip of the pipette (7), a backsip after the aspiration step will cause the droplet or convex bulge to be withdrawn into the pipette (7). If the tip of the pipette (7) is elongated and includes a narrow bore, then the backsip may cause air to enter into the tip. Alternatively, the backsip may merely reduce the convexity of the droplet or may cause the liquid air interface to become concave instead of convex.

In any event, when backsipping liquid into the pipette (7), it is critical to backsip only within an allowable range. A backsip which is too small will not sufficiently draw the liquid into the tip of the pipette (7) to prevent it from being shaken off during an unintended vertical jolt. On the other hand, a backsip which is too great, may cause ^'bubbling within the pipette (7). Bubbling will occur if air is drawn too far into.the pipette (7). If the tip has a wide bore, bubbling will readily occur if the liquid is drawn into the region of the wide bore; but if the tip has a narrow bore, bubbling will not readily occur so long as the liquid is not, withdrawn beyond this narrow bore.

The optimal magnitude of the backsip will depend upon the size and shape of the pipette (7), upon the volume of liquid which is aspirated into the pipette (7), and upon the nature of the liquid which is aspirated, i.e. its viscosity, surface tension, its attaction to the surface material of the pipette (7), and other factors. The optimal magnitude of the backsip is most easily determined empirically. Hence, the memory of the microprocessor is loaded with a backsip coorelation which relates the optimal backsip to each of these factors.

After the liguid is aspirated into the pipette (7) and, if desired, after the backsip has occurred, the aliquots of the liquid may be expressed from the pipette (7). Once again, the volume of the aliguot which is expressed from the pipette (7) should be empirically correlated with the step count of the step motor. This correlation will depend upon both the pipette (7) and the liquid which is being expressed. The correlation will also depend upon the speed with which the step count is executed.

During the expression process, the liquid within the pipette (7) will behave similar to a mass on a damped spring. The liquid is the mass; the compressed air is the spring; and the resistance to fluid flow through the tip of the pipette (7) is the damping. If the damping is low, i.e., if the resistance to fluid flow through the tip of the pipette (7) is low, the system may be under-damped. Under such circumstances, if the step count is executed quickly, the liquid within the pipette (7) may over shoot the desired volume. Over shooting the desired volume may be prevented by increasing the resistance to fluid flow at the tip of the pipette (7), by slowing the execution of the step count, and by executing a backsip at the precise moment that the desired volume of liquid has been expressed from the pipette (7) .

Hence, the backsip subsequent to the first expression step, will depend not only on the pipette (7), the volume of aspirated liquid, and the nature of the aspirated liquid, but will also depend upon the volume of expresses liquid and upon the velocity of the liquid column within the pipette (7) at the moment of the backsip. Once again, the velocity of the liquid column, is dependent upon the

10 speed with which the step count was executed for the expression step, upon the mass of the liquid column, and the resistance to fluid flow at the tip, viz. its bore size.

Accordingly, when determining the empirical

15 correlation between the volume of expressed liquid and the step count for the backsip, all of these factors should be taken into account. Fortunately, these factors are reducable to the size and shape of the particular pipette, the physical properties of the liquid, and the number and

20. speed of the aspiration step and the expression step.

Additionally, the speed of execution of the step count for the backsip and the delay between the execution of the step count for the expression step and the step count for backsip step may be adjusted. For large masses, the speed

25 of execution of the step count for the backsip may be quite fast, and the delay may be rather long. However, for smaller masses, the speed of execution of the step count for~ the backsip may be somewhat slower and the delay may be raths-r: short. In the finally analysis, however, it is

30 eas-±est to .empirically determine he,optimal correlation between the optimal step count, speed, and delay of the backsip.

Example The variable backsip may be illustrated by an example.

35 Assume that it is desired to use an automated pipetting apparatus to pipette 10 aliquots, 0.5 milliliters each, from a conventional 10 milliliter serological pipette. An schedule for the optimal preliminary backsip, subsequent backsips, blowout volume, and retained volume may be observationally determined to be as follow:

Function Volume Steps

Volume of each aliquot: 0.500 milliliter 84 steps

Total volume of aliquots: 5.000 milliliter 840 steps

Retained volume: 0.600 milliliter 100 steps

Blowout volume: 0.100 milliliter 17 steps Preliminary backsip: 0.050 milliliter 8 steps

1st backsip: 0.100 milliliter 17 steps

2nd backsip: 0.095 milliliter 16 steps

3rd backsip: 0.090 milliliter 15 steps

4th backsip: 0.085 milliliter 14 steps 5th backsip: 0.080 milliliter 13 steps

6th backsip: 0.070 milliliter 11 steps

7th backsip: 0.060 milliliter 10 steps

8th backsip: 0.050 milliliter 8 steps

9th backsip: 0.040 milliliter 7 steps 10th backsip: 0.030 milliliter 5 steps

In order to perform the above described pipetting task with an automatic pipetting apparatus, each of the above gas or liquid volumes have been converted to the corresponding number of steps which must be performed by a step motor. In order to calculate the appropriate number of steps, it has been assumed that the automated pipette employs a 12.5 milliliter syringe as its pump and employs a step motor which can drive the syringe over its entire 12.5 millimeter range with precisely 2000 steps, so that each 1 millimeter displacement of the syringe requires 160 steps from the step motor. It has been further assumed that the density correction is 1.045 for the particular serological pipette and liquid being pipetted therewith, so that the number of steps for pipetting any given volume may be approximated by the following equation:

Motor Steps = (Desired volume) x (Density Correction) x (Motor Scale factor) = (Desired volume) x (1.045) x (160) = (Desired volume) (167.2) steps/milliliter

Accordingly, the pipetting task described above may be performed by programing a computer or microprocessor to direct a step motor to execute the following steps, each at a uniform conventional speed: Function Motor Steps

Aspirate blowout: +17

Aspirate 10 aliquots + retained volume: +940

(10 x 84) + 100 Aspirate preliminary backsip: +8 Express preliminary backsip + 1st aliquot

+ 1st backsip: -109

Wait backsip delay of 0.1 seconds Aspirate 1st backsip: +17

Express 2nd aliquot + 2nd backsip: -100 Wait backsip delay of 0.1 seconds

Aspirate 2nd backsip: +16

Express 3rd aliquot + 3rd backsip: -99

Wait backsip delay of 0.1 seconds Aspirate 3rd backsip: +15 Express 4th aliquot + 4th backsip: -98 Wait backsip delay of 0.1 seconds Aspirate 4th backsip: +14

Express 5th aliquot + 5th backsip: -97 Wait backsip delay of 0.1 seconds Aspirate 5th backsip: +13

Express 6th aliquot + 6th backsip: -95

Wait backsip delay of 0.1 seconds Aspirate 6th backsip: +11

Express 7th aliquot + 7th backsip: -94 Wait backsip delay of 0.1 seconds

Aspirate 7th backsip: +10 Express 8th aliquot + 8th backsip: -92

Wait backsip delay of 0.1 seconds Aspirate 8th backsip: +8

Express 9th aliquot + 9th backsip: -91 Wait backsip delay of 0.1 seconds

Aspirate 9th backsip: ₊7

Express 10th aliquot + 10th backsip: -89

Wait backsip delay of 0.1 seconds Aspirate 10th backsip: +5 Express retained volume + Blowout volume to waste -117

Claims

Claims What is claimed is: 1. In an improved method for expressing an aliquot of liquid from a pipette, the method including the following steps:

Step A: submerging the tip of the pipette into a source of the liquid, then

Step B: loading the pipette with an initial volume of liquid by means of a piston pump driven by a step motor, the piston pump being pneumatically connected to the pipette, the initial volume of liquid including both an aliquot and a retained volume; then

Step C: removing the tip of the pipette from the source of the liquid, and then

Step D: expressing the aliquot of liquid from the pipette by means of the piston pump; the improvement comprising the following additional steps:

Step A' : prior to said Step A, aspirating * blow out volume of air into the pipette and.

Step E: subsequent to said Step D, expressing both the retained volume of liquid and the blow out volume of air from the pipette.

UTE SHEET 2. In an improved method for expressing an aliquot of liquid from a pipette, the method including the following steps:

Step A: submerging the tip of the pipette into a source of the liquid; then

Step B: loading the pipette with an initial volume of liquid by means of a piston pump driven by an electric motor, the piston pump being pneumatically connected to pipette, the initial volume of liquid including both an aliquot and a retained volume; then

Step C: removing the tip of the pipette from the source of the liquid; and then

Step D: expressing the aliquot of liquid from the pipette by means of the piston pump; the improvement comprising the following additional step:

Step D¹: after said Step C and prior to said Step D, executing a preliminary backsip for drawing the liquid further into the pipette so as to reduce the risk of dripping liquid from the pipette prior to said Step D.

3. In an improved method for expressing multiple aliquots of liquid from a pipette, the method including the following steps: Step A: submerging the tip of the pipette into a source of the liquid; then

Step B: loading the pipette with an initial volume of liquid by means of a piston pump driven by an electric motor, the piston pump being pneumatically connected to the pipette, the initial volume of liquid including both an aliquot and a retained volume; then

Step C: removing the tip of the pipette from the source of the liquid; then

Step D: expressing a first aliquot of liquid from the pipette by means of the piston pump; then Step E: backsipping the liquid into the pipette by means of a first backsip stroke of a piston pump; then

Step F: expressing a subsequent aliquot of liquid from the pipette by means of the piston pump; and then Step G: backsipping the liquid into the pipette by means of a subsequent backsip stroke; the improvement wherein: in said Step E, the first backsip stroke having a magnitude for maintaining the backsipping within an 0 allowable range which substantially reduces the risks of both unintendedly dripping liquid from the pipette and of bubbling air within the pipette; and in said Step G, the subsequent backsip stroke having a magnitude for maintaining the backsipping within an 5 allowable range which substantially reduces the risks of both unintendedly dripping liquid from the pipette and of bubbling air within the pipette; the magnitude of the first backsip stroke being greater than the magnitude.of the subsequent backsip stroke. σ

4.. In a^~r improved method for expressing multiple aliquots of liquid from a pipette as described in claim 3, the improvement being further characterized by: the first and subsequent backsip strokes differing from 5^j one_ another with respect to speed.

£.. ϋr. am improved method for expressing multiple aliquots o ¹ liquid from a pipette as described in claim 3, the method further characterized as follows: 0 in said Step D, the expressing occurring by means of a ^~first expression stoke by a piston pump; in. said Step F, the expressing occurring by means of a subsequent expression stoke; tha improvement being further characterized as follows: 5 in said Step E, the first backsip stroke following a first delay period after the first expression stroke; and

SUBSTITUTE SHEET Step C¹ : after said Step C and prior to said Step D, executing a preliminary backsip of the liquid into the pipette by means of a preliminary backsip stroke of the piston pump; in said Step E, the first backsip stroke having a magnitude for maintaining the backsipping within an allowable range which substantially reduces the risks of both unintendedly dripping liquid from the pipette and of bubbling air within the pipette; and Step E(1): prior to said Step E, determining a step count required by the step motor for performing the first backsip stroke; in said Step E, backsipping the liquid into the pipette by driving the step motor according to the step count of said Step E(1 ); in said Step G, the subsequent backsip stroke having a magnitude for maintaining the backsipping within an allowable range which substantially reduces the risks of both unintendedly dripping liquid from the pipette and of bubbling air within the pipette;

Step G(1): prior to said Step G, determining a step count required by the step motor for performing the subsequent backsip stroke; in said Step G, backsipping the liquid into the pipette by driving the step motor according to the step count of said Step G(1 ) ; the magnitude of the first backsip stroke being greater than the magnitude of the subsequent backsip stroke.

8. In an improved apparatus for expressing an aliquot of liquid from a pipette, the apparatus including: a piston pump pneumatically connected to said pipette, a step motor for driving said piston pump, and a computer or microprocessor for controlling said step motor, the improvement comprising:

SUBSTITUTE SHEET in said Step E, the subsequent backsip stroke following a subsequent delay period after the subsequent expression stroke; the first delay period differing from the subsequent delay period.

6. In an improved method for expressing multiple aliquots of liquid from a pipette as described in claim 5, the improvement being further characterized by: the first and subsequent backsip strokes differing from one another with respect to speed.

7. In an improved method for expressing multiple aliquots of liquid from a pipette, the method including the following steps:

Step A: submerging the tip of the pipette into a source of the liquid; then

Step C: removing the tip of the pipette from the source of the liquid; then Step D: expressing a first aliquot of liquid from the pipette by means of the piston pump; then

Step E: backsipping the liquid into the pipette by means of a first backsip stroke of the piston pump; then

Step F: expressing a subsequent aliquot of liquid from the pipette by means of the piston pump; and then

Step G: backsipping the liquid into the pipette by means of a subsequent backsip stroke; the improvement comprising the following additional steps: Step A' : prior to said Step A, aspirating a blow out volume of air into the pipette;

S said computer or microprocessor being programmed for causing said step motor to drive said piston pump so as to automatically aspirate a blow out volume of air into the pipette prior to the aspiration of liquid. 5

9. In an improved apparatus for expressing an aliquot of liquid from a pipette, the apparatus including: a piston pump pneumatically connected to said pipette, a step motor for driving said piston pump, and a computer or 10 microprocessor for controlling said step motor, the improvement comprising: said computer or microprocessor being programmed for accepting and executing a command to perform a preliminary backsip of liguid which has been aspirated into the pipette 15 but which has not yet been expressed from the pipette.

10. In an improved apparatus for expressing an aliquot of liquid from a pipette, the apparatus including: a piston pump pneumatically connected to said pipette, a 20 step motor for driving said piston pump, and a computer or microprocessor for controlling said step motor, the improvement comprising: said computer or microprocessor being programmed for determining an adjustable backsip and for causing said step 25 motor to drive said piston pump so as to automatically backsip the liquid into the pipette after each expression of liquid from, the pipette, the backsip being adjustable with respect to magnitude.

30 11. In an improved apparatus as described in claim 10, the improvement being further characterized by: the backsip being adjustable with respect to speed.

12. In an improved apparatus as described in claim 10, the 35 improvement being further characterized by: the backsip being adjustable with respect to timing.

SUBSTITUTE 13. In an improved apparatus for expressing an aliquot of liquid from a pipette, the apparatus including: a piston pump pneumatically connected to said pipette, a step motor for driving said piston pump, ^"and a computer or microprocessor for controlling said step motor, the improvement comprising: said computer or microprocessor being programmed for causing said step motor to drive said piston pump so as to automatically aspirate a blow out volume of air into the pipette, prior to the aspiration of liquid, and said? computer or microprocessor also being programmed for determining an adjustable backsip and for causing said step motor to drive said piston pump so as to automatically backsip the liquid into the pipette after each expression of liquid from the pipette, the backsip being adjustable with respect to magnitude.

1 . lit an improved apparatus for expressing an aliquot of liquid,; the apparatus including: a_^ pipette, a; p±ston pump.

Si step motor for driving said piston pump, said step motor being connected to said piston pump, a- computer or microprocessor for controlling said step motor, an input box having a display, a handle, a pneumatic hose for pneumatically connecting said piston; pump to said handle, an electronic control for controlling said computer or microprocessor, said electronic control being*"±ncorporated into said handle, a first electronic cable for connecting said input box tec the computer or microprocessor, and

S a second electronic cable for connecting said electronic control to said computer or microprocessor, the improvement comprising: said computer or microprocessor being programmed for 5 causing said step motor to drive said piston pump so as to automatically aspirate a blow out volume of air into the pipette prior to the aspiration of liquid, and said computer or microprocessor also being programmed for determining an adjustable backsip and for causing said 10 step motor to drive said piston pump so as to automatically backsip the liquid into the pipette after each expression of liquid from the pipette, the backsip being adjustable with respect to magnitude.

15 15. In an improved apparatus as described in claim 14, the improvement being further characterized by: said pipette being a serological pipette.

16. In an improved apparatus as described in claim 14, the 20 improvement being further characterized by: said pipette being a multichannel pipette.

17. In an improved apparatus as described in claim 14, the improvement being further characterized by: 5 said pipette being a bag type pipette.

UTE SHEET