lab_append (2024)

Appendix C

Measurement of liquids

Accurate, meaningful data for the laboratory exercises in this manual require that you develop both the knowledge and skill to measure and transfer very specific quantities of liquid with both accuracy and precision. Although the use of instruments for the measurement of volume is widespread, few students have been exposed to the correct techniques for volume measurement. Even though many students may say "I know how to pipette", few can distinguish between a "to deliver" and a "to contain" pipette, determine the appropriate type of pipette for a particular job, or properly use a pipette pump. Instruction in the use of volumetric glassware may seem rudimentary, but their proper use really requires considerable sophistication, and your success in gathering data during laboratory experiments is dependent on your competence with a pipette. Proper measurement of liquids requires choosing the proper glassware and using it correctly.

Laboratory glassware

The most commonly used types of laboratory glassware used in the measurement of liquid volumes are graduated cylinders, volumetric flasks, pipettes and burettes. However, there is a difference between graduated and volumetric glassware. Graduated beakers, flasks, cylinders, and pipettes have a series of marks indicating different volumes. The volumes indicated are generally only approximations, sometimes close approximations, but approximations nevertheless. Each graduate (mark) is not calibrated separately, rather, one volume is calibrated and then a standard scale is applied to the glassware using the calibration mark as the starting point. Thus, on a 100 ml graduated cylinder, only the hundred ml volume may be calibrated and then a scale with standard number of equidistant divisions is applied below the calibrated volume. Clearly, this assumes an absolutely true (straight) tube with no variations in diameter. Any variation in diameter between milliliters 50 and 60 would mean that between divisions 52-53 there may actually be more or less than 1 ml, even if the total volume of the cylinder is exactly 100 ml. Inexpensive (cheap) graduated glassware is often not even calibrated at one reference point, but rather just has a scale of graduated values painted on it. The variations due to tube diameter variations, etc. are merely accepted as being within a specified range (100 ml plus or minus 1 ml, etc.). This glassware is never used where accurate measurement is essential.

On the other hand, each volume indicated on volumetric glassware has been carefully calibrated. Thus, a 100 ml volumetric flask has had exactly 100 ml of water or mercury placed in it, then the volume mark scribed on it. As with other measurement equipment, it is extremely important to know which type of glassware should be used for each specified task. If you need about 100 ml of water, a graduated cylinder will do quite nicely and is relatively fast and easy to use. However, if you need exactly 100 ml to make up a "standard" solution, only a volumetric flask would be appropriate.

The types of glassware most commonly used in liquid measurement include the following:

Graduated cylinders
: used to measure and transfer various volumes of liquids. The smaller the cylinder diameter, the more accurate the volume measured. Accuracy of graduated cylinders range from 0.5% to 1% of the total volume.
Graduated flasks and beakers: used to obtain an estimate of the volume of liquid in the container. Graduated flasks and beakers may be off by as much as 10% of the total volume. They are for gross estimates only.
Volumetric flasks: used to obtain precise volume measurements. A high quality volumetric flask can have an accuracy of plus or minus 0.05%. They are normally used in the preparation of solutions and also for the precise dilution of solutions (i.e. diluting a solution 1:100 would require the transfer of 1 ml of original solution to a 100 ml volumetric flask, and then filling the flask to its 100 ml calibration mark with solvent).
Graduated pipettes: pipettes are used for the transfer of solutions and solvents from one vessel to another. As with other graduated glassware, they have an approximate scale consisting of a series of equivolume units that subdivide the maximum volume of the pipette. Their accuracy is similar to that of a graduated cylinder, 0.5 to 1% of the total volume . Again, the smaller the diameter, the more accurately it can be subdivided. Although not extremely accurate, a whole series of approximate volumes can be quickly and easily transferred. For instance, you could rapidly transfer 0.1 ml of solvent to each of 10 test tubes using a 1.0 ml pipette graduated in 0.1 ml subdivisions by merely filling to the maximum (1.0 ml) line, and then releasing 0.1 ml into each test tube
Volumetric pipettes: carefully calibrated pipettes used to transfer a single volume of liquid. The liquid is drawn into the pipette until the miniscus is exactly even with the calibration mark. Release of the liquid from this point delivers a single accurate volume of liquid. Accuracy of volumetric pipettes can exceed 0.05% of their total volume
Burettes: used to deliver approximate volumes of liquid. Because there is a stopco*ck at the bottom and a tapered, small diameter tip, liquids can be released in very small, accurately measured amounts. These are especially useful for titration. The accuracy of burettes is similar to that of a high quality graduated pipette (0.5%)
Syringes: used to transfer liquids, particularly if these need to be delivered by injection (penetration of the delivery tip into the organism or through a rubber septum). Most syringes are graduated, and thus deliver only approximations, even though they are close approximations. There are also glass volumetric syringes whose internal diameters are ground to have extremely small variations and whose plungers are mechanically driven. Because these can deliver extremely accurate volumes of liquids and the rate of delivery can be precisely controlled by a delivery (plunger depressing) screw, they can be substituted for burettes, especially when delivering small total volumes.<

Measuring Volume

Once you have chosen the proper piece of glassware for the job, there are several important points to be kept in mind when measuring liquid volumes. One point that may seem obvious is to be sure all glassware is clean and dry before beginning a volume measurement. A second point is to be certain to read the volume properly. As the liquids that you will commonly use have a rather high surface tension, they will tend to rise higher along the sides of a container and be lowest in the center. The lowest point in the center is termed the miniscus, and it is with respect to this point that all liquid measurements are made. Thus a correct volume would be obtained by adding solvent until the miniscus is exactly at the level of the calibration mark (usually a ring scribed in the neck of the flask), not when the highest rim of solvent reaches the calibration mark. Finally, to be redundant, it is extremely important that the proper piece of glassware is used to perform the desired measurement.

Pipetting

There are a few things that you should know before you start pipetting. On each pipette is given its total volume for measurement, the size of its smallest division, the temperature at which it was calibrated (as liquids change volumes with changes in temperature), and the letters T.D. (to deliver), T.C. (to contain), an upper and sometimes a lower calibration line.

SelectionSelecting the proper pipette is half the battle in accurate pipetting. A few basic rules will help you to reach for the most appropriate one.Always use the smallest pipette that will contain all of your sample. The smaller the pipette more accurate it will be. Do not, however, use repetitive deliveries from a too small pipette. That is to deliver 3.2 ml use a 5ml pipette not a 10 ml or four deliveries with a 1 ml. The size information is critical for selection of the proper pippette, while the TD or TC designation and the calibration lines will help you select the proper delivery technique.FillingThere are two basic ways to draw liquid into the pipette. One is "by mouth" or using the mouth for suction, and the other is using a "pipette pump". Mouth suction is tempting. It is quick and relatively easy to control and you always have one with you. However, for the pipetting of noxious, poisonous or just plain disgusting liquids it is at best a risky business. A mouthful of concentrated acid, rat urine, or even frog spit, is not recommended. Therefore, for any solution or solvents, the use of a pipette pump is Mandatory! Pipette pumps come in a variety of forms. One is merely a rubber ball with three (intake, exhaust, and suction) check valves. A more contemporary form consists of a cylinder and piston with a thumb wheel control over the position of the piston.

The pipette is fitted into the pipette pump with a twisting motion and the liquid is aspirated in to the pipette (either by releasing the rubber squeeze bulb or by pulling back on the piston). Do not use pipettes with rough or chipped ends in a pipette pump or you will damage the rubber seal. If you cannot get a seal easily, check both the end of the pipette and the rubber seal to be sure that both are smooth. Do not force the pipette or you may break it. If the syringe pump is equipped with control valves, it can be filled to the upper calibration mark with the pump. If the pump is not equipped with control valves, it must be over filled so that you have time to place your finger over the pipette top before the liquid drains past the upper calibration mark.

Delivery

If your pipette pump is equipped with a delivery valve, you can deliver your measured volume by opening the valve and allowing air into the top of the pipette. The flow of liquid from pipettes equipped with pumps without valves must be controlled with your forefinger: Overfill the pipette, remove the pump, and put your finger over the end of the pipette. You can not accurately control the flow of liquid from a pipette with your thumb. Never use your thumb to control release of liquid from a pipette.

Hints to better pipetting

Select a proper pipette.
Always read the pipette. Is it a T.D or a T.C? Does it have both upper and lower calibration marks or upper marks only and graduations into the tip? Each uses a different delivery technique so it is critical to know.
Whenever possible have your sample as close to the calibration temperature as possible.
Always touch the tip of a pipette to the receiving vessel (not the liquid in it) at the completion of delivery.

The measurement of very small volumes

Contemporary biological laboratory practices have in many cases been scaled down to minimum sizes. This miniaturization allows the conservation of expensive reagents and reduces the amount of potentially hazardous wastes generated. this miniaturization, however, require the transfer of very small volume of liquids (0.01 to 1000ul).

Volume Setting

The volume indicator consists of three number dials and is read from top to bottom. The three digits indicate the volume selected and are colored black and/or red.

The black digits on P-10, and P-20 pipettes show microliters; the red digits show tenths and hundredths of microliters. P-100 pipettes have all-black dials. For the P-1000 pipettes, the digits in red represent milliliters and the digits in black microliters. For each model, the smallest incremental setting can be set between the digits by referencing the vernier marks.

Operation

To set a volume: hold the Pipetman in one hand and turn the volume adjustment knob with the other hand until the correct volume shows on the digital indicator. The friction ring "locks" the volume. Turn the knob 1/3 revolution above the desired setting then slowly down to the setting, to prevent mechanical backlash from affecting accuracy. If you pass the desired setting, turn the dial to 1/3 revolution above the desired setting and reset the volume.
Attach a new disposable tip to the pipette shaft. Press firmly to ensure a positive airtight seal.
Depress the plunger to the first stop. This part of the stroke is the calibrated volume displayed on the digital volume indicator.
Holding the Pipetman vertically, immerse the disposable tip into the sample liquid to the proper immersion depth.
Allow the pushbutton to return slowly to the UP position. Never let it snap up!
Wait a few seconds to ensure that the full volume of sample is drawn into the tip.
Withdraw the tip from the sample liquid. Should any liquid remain on the outside of the tip, wipe it carefully with a lint- free tissue, taking care not to touch the tip orifice.

Pipetting Guidelines and Precautions

Consistency in all aspects of pipetting procedure will significantly contribute to optimum reproducibility. Attention should be given to:

1. Consistent speed and smoothness when you press and release the pushbutton.

2. Consistent pushbutton pressure at the First stop.

3. Consistent immersion depth.

4. Minimal angle (from vertical).

If an air bubble is noted within the tip during intake, dispense the sample to the original vessel, check the tip immersion depth, and pipette more slowly. If an air bubble appears a second time, discard the tip and use a new one.

Prevent liquids from being drawn into the Pipetman shaft (with possible resultant damage) by observing the following precautions:

1. Pipette slowly, holding Pipetman no more than 20o from vertical.

2. Never invert or lay Pipetman on its side with a tip installed. There is a great a chance that liquid in the tip will run in to the pipettet and damage it permanently.

3. For the Model P-5000 Pipetman, use the special safety filters supplied.

Proper tips

The requirments for disposible pipette tips are demanding. The proper function of the pipettor is dependant on matching the tip exactly to the instrument. Disposable pipette tips are usually color coded to make this task easier. As we use tips from many different suppliers the color code will be provided at the time of the laboratory.

Dilution and Concentration

It is frequently necessary, for many reasons, to dilute biological samples, and one must know the concentration (at least the relative concentration) of the diluted sample. The simplest example is a 1:2 dilution; the 1:2 designation means 1 part stock (the thing you want to dilute) to 2 parts total. If you wanted to make 10 ml of a 1:2 dilution, you would add 5 ml of the stock to 5 ml of diluent (the thing you dilute with - usually water, buffer or medium). It makes intuitive sense, in this simple example, that the concentration in the diluted sample is one half that in the stock; if the stock is 10 mM, the diluted sample is 5 mM.

In may instances, the concentration of the diluted sample will not be so intuitively obvious, and you will need to calculate it. A valuable relationship to know is:

concentration(stock) x volume(stock) = concentration(diluted)

x volume(diluted).

In our first example:

10 mM (5 ml) = X mM (10 ml)

X = 5 mM

Suppose you made a 3:5 dilution of a 45 mM solution:

45 mM (3 parts) = X mM (5 parts).

X = 27 mM.

When making a 1:10, 1:100, 1:1000 etc. dilution, it is more conve nient to use scientific notation to indicate the dilution factor. You can dilute something 10-fold (a 1:10, or 10-1 dilution) by mixing one volume of the solution to be diluted with nine volumes of the appropriate solvent, e.g., 0.5 ml of yeast stock plus 4.5 ml medium, or 0.1 ml of yeast stock plus 0.9 ml medium. You could get a 100-fold dilution (1:100, or 10-2) by mixing 1 volume of yeast stock with 99 volumes of medium, e.g., 0.05 ml into 4.95 ml.

For many applications, the concentration you need is close, within a couple of orders of magnitude, to that of the stock. In such cases, a single dilution will bring your sample into the needed concentration range. But other samples are much too concentrated, and you'd need to dilute them drastically. For example, what do you do if you want to dilute a yeast suspension that has 109 cells/ml to make one that has 103 cells per ml? Volumes smaller than about 0.05 ml cannot be pipeted accurately without special equipment, so if you were to do it in a single dilution, you would need to add the 0.05 ml of yeast stock to 5x104 ml or 50 l of sterile water! That would be impractical.

If you want to dilute a solution by more than about 10-2 without using huge liquid volumes, you must employ a technique known as serial dilution. That is, you must dilute by a factor of ten or a hundred, and then take this new solution and dilute it again (Figure E-1).

Figure E-1. Using serial dilution to make a 10-5 dilution of a yeast stock. A 0.5 ml aliquot from the stock is transferred to the second tube, and the contents are thoroughly mixed. Thus, the second tube represents a 10-1 (10-fold) dilution of the stock. Then, a 0.05 ml aliquot from the second tube is added to the third tube. After mixing, the third tube contains a 10-2 dilution of the contents of the second tube, which is equivalent to a (10-1) Ñ (10-2) = 10-3 dilution of the original stock. Another 10-2 dilution brings the total dilution to 10-5 in the last tube. Note that the 10-1 dilution and the two 10-2 dilutions can be done in any order with the same result.

Walter I. Hatch
[email protected]

August 12, 2012