Basic Serologic Laboratory Techniques
PIPETTING TECHNIQUES Manual Pipettes
With practice, it is important to develop a good technique for han- dling pipettes (Fig. 8-2). The same general steps apply to pipet-ting with all manual pipettes (Box 8-1), with few exceptions.
Laboratory accidents frequently result from improper pipet-ting techniques. The greatest potential hazard is when mouth pipetting is done instead of mechanical suction. Mouth pipet-ting is never acceptable in the clinical laboratory.
After the pipette has been filled above the top graduation
Automatic Pipettes
Automatic pipettes allow fast, repetitive measurement and delivery of solutions of equal volumes. The sampling type mea-sures the substance in question. The sampling-diluting type measures the substance and then adds the desired diluent. The graduationTop
mark
Capacity mark Etched
band
Ostwald Volumetric Figure 8-1 Types of manual pipettes. TD, To deliver. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.)
or specimens. The most common type of micropipette used in many laboratories is one that is automatic or semiautomatic, called a micropipettor. These are piston-operated devices that allow repeated, accurate, reproducible delivery of specimens, reagents, and other liquids requiring measurement in small amounts. Many micropipettors are continuously adjustable so that variable volumes of liquids can be dispensed with the same device. Delivery volume is selected by adjusting the settings.
Different types or models are available, which allow volume delivery ranging, for example, from 0.5 to 5000 µL. The cali-bration of these micropipettes should be checked periodically.
The piston, usually in the form of a thumb plunger, is depressed to a stop position on the pipetting device. The tip is placed in the liquid to be measured, and then the plunger is slowly allowed to rise back to the original position (Fig. 8-4).
This will fill the tip with the desired volume of liquid. The tips are usually drawn along the inside wall of the vessel from
1.
Using mechanical suction
2.
Wipe off outside of pipette with
gauze
3.
Adjusting
the meniscus 4.
Drain into receiving vessel
Figure 8-2 Pipetting technique. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.)
Eye level
Calibration mark Meniscus
Figure 8-3 Reading the meniscus. (From Turgeon ML: Linné &
Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby.)
Box 8-1 Pipetting With Manual Pipettes
1. Check the pipette to ascertain its correct size, being careful also to check for broken delivery or suction tips.
2. Wearing protective gloves, hold the pipette lightly between the thumb and the last three fingers, leaving the index finger free.
3. Place the tip of the pipette well below the surface of the liquid to be pipetted.
4. Using mechanical suction or an aspirator bulb, carefully draw the liquid up into the pipette until the level of liquid is well above the calibration mark.
5. Quickly cover the suction opening at the top of the pipette with the index finger.
6. Wipe the outside of the pipette dry with a piece of Kim-Wipe tissue to remove excess fluid.
7. Hold the pipette in a vertical position with the delivery tip against the inside of the original vessel. Carefully allow the liquid in the pipette to drain by gravity until the bottom of the meniscus is exactly at the calibration mark. To do this, do not entirely remove the index finger from the suction hole end of the pipette; rather, by roll-ing the froll-inger slightly over the openroll-ing, allow slow drainage to take place.
8. While still holding the pipette in a vertical position, touch the tip of the pipette to the inside wall of the receiving vessel. Remove the index finger from the top of the pipette to permit free drainage. Remember to keep the pipette in a vertical position for correct drainage. In TD (to deliver) pipettes, a small amount of fluid will remain in the delivery tip.
9. To be certain that the drainage is as complete as possi-ble, touch the delivery tip of the pipette to another area on the inside wall of the receiving vessel.
which the measured volume is drawn, so that any adhering liquid is removed from the end of the tip. These pipette tips are not usually wiped, as is done with the manual pipettes, because the plastic surface is considered nonwettable. The tip of the pipette device is then placed against the inside wall of the receiving vessel and the plunger is depressed. When the manufacturer’s directions for the device being used are fol-lowed, sample delivery volume is judged to be extremely accurate.
The pipette tips are usually made of disposable plastic, so no cleaning is necessary. Various types of tips are available.
Some pipetting devices automatically eject the tip after use.
These will also allow the user to insert a new tip and remove the used tip without touching it, minimizing infectious bio-hazard exposures.
The problems encountered with automatic pipetting depend largely on the nature of the solution to be pipetted. Some reagents cause more bubbles than others and some are more
viscous. Bubbles and viscous solutions can cause problems with the measurement and delivery of samples and solutions.
Micropipettors contain or deliver 1 to 500 µL of solution. It is important to follow the individual manufacturer’s instruc-tions for the device being used; each may be slightly different.
In general, the following steps apply for use of a micropipettor:
1. Attach the proper tip to the pipettor and set the delivery volume.
2. Depress the piston to a stop position on the pipettor.
3. Place the tip into the solution and allow the piston to rise back slowly to its original position (this fills the pipettor tip with the desired volume of solution).
4. Some tips are wiped with a dry gauze at this step and some are not. Follow the manufacturer’s directions.
5. Place the tip on the wall of the receiving vessel and depress the piston, first to a stop position where the liquid is allowed to drain and then to a second stop position where the full dispensing of the liquid takes place.
6. Dispose of the tip in the waste disposal receptacle.
Some pipettors automatically eject the used tips, minimizing biohazard exposure.
Automatic Dispensers or Syringes
Many types of automatic dispensers or syringes are used in the laboratory for repetitively adding multiple doses of the same reagent or diluent. These devices are used for measuring serial amounts of relatively small volumes of the same liquid. The volume to be dispensed is determined by the pipettor setting.
Dispensers are available with a variety of volume settings.
Some are available as syringes and others as bottle top devices.
Most of these dispensers can be cleaned by autoclaving.
Diluter-Dispensers
In automated instruments, diluter-dispensers are used to pre-pare a number of different samples for analysis. These devices pipette a selected aliquot of sample and diluent into the instru-ment or receiving vessel. They are primarily of the dual-piston type, with one used for the sample and the other for the diluent or reagent.
DILUTIONS
It is often necessary to make dilutions of specimens being ana-lyzed or to make weaker solutions from stronger solutions in various laboratory procedures. Clinicians must be able to work with various dilution problems and dilution factors. They often need to determine the concentration of antibody in each solu-tion, the actual amount of material in each solution, and the total volume of each solution. All dilutions are a form of ratio.
Dilution is an indication of relative concentration.
Diluting Specimens
In most laboratory determinations, a small sample is taken for analysis and the final result is expressed as concentration per some convenient standard volume. In a certain procedure, 0.5 mL of blood is diluted to a total of 10 mL with various reagents,
A B
Filling Emptying
C
Figure 8-4 Steps in using piston-type automatic micropipette. A, Attaching proper tip size for range of pipette volume, and twisting tip as it is pushed onto pipette to give an airtight, continuous seal. B, Holding pipette before use. C, Follow instructions for filling and emptying pipette tip. (From Kaplan LA, Pesce A: Clinical chemistry: theory, analysis, correlation, ed 5, St Louis, 2010, Mosby.)
and 1 mL of this dilution is then analyzed for a particular chemical constituent. The final result is to be expressed in terms of the concentration of that substance per 100 mL of blood.
Dilution Factor
A dilution factor is used to correct for having used a diluted The preceding material may be summarized by the follow-ing statement and equations. In reportThe preceding material may be summarized by the follow-ing results obtained from laboratory determinations, one must first determine the amount of specimen actually analyzed in the procedure and then calculate the factor that will express the concentration in
the desired terms of measurement. Thus, in the previous exam-ple, the following equations may be used:
0.5 mL
(volume of blood used) 10 mL
(volume of total dilution)
=
x mL
(volume of blood analyzed) 1 mL
(volume of dilution used) x= 0.05 mL (volume of blood actually analyzed) 100 mL (volume of blood
required for expression of result) 0.05 mL (volume of blood actually analyzed)
= 2000 (dilution factor)
Single Dilutions
When the concentration of a particular substance in a speci-men is too great to be determined accurately, or when there is less specimen available for analysis than the procedure requires, it may be necessary to dilute the original specimen or further dilute the initial dilution (or filtrate). These single dilutions are usually expressed as a ratio, such as 1:2, 1:5, or 1:10, or as a fraction, ½, 1⁄5, or 1⁄10. These ratios or fractions refer to 1 unit of the original specimen diluted to a final volume of 2, 5, or 10 units, respectively. A dilution, therefore, refers to the volume or number of parts of the substance to be diluted in the total volume, or parts, of the final solution. A dilution is an expression of concentration, not volume; it indicates the relative amount of substance in solution. Dilutions can be made singly or in series.
To calculate the concentration of a single dilution, multi-ply the original concentration by the dilution expressed as a fraction.
Example of Calculation of Concentration of a Single Dilution
A specimen contains 500 mg of substance per deciliter of
Use of Dilution Factors
A 1:5 dilution of a specimen is prepared and an aliquot (one of a number of equal parts) of the dilution is analyzed for a 0.5 mL blood
10 mL solution= 1 mL blood x mL solution x=1 mL blood× 10 mL
0.5 mL = 20 mL
1 mL blood
20 mL solution= x mL blood 1 mL solution x=1 mL× 1 mL
20 mL = 0.05 mL
100 mL
(volume of blood desired) 0.05 mL
(volume of blood used)
=concentration desired concentration used
or determined
Concentration desired=
100 mL× concentration determined
0.05 mL Concentration desired= 2000 × value determined
particular substance. The concentration of the substance (C) in the aliquot is multiplied by 5 to determine its concentration in the original specimen. If the concentration of the dilution is 100 mg/dL, the concentration of the original specimen is:
C= 100 mg/dL × 5 (dilution factor) = 500 mg/dL in blood
Serial Dilutions
to as serial dilutions (Table 8-1). A complete dilution series usually contains 5 or 10 tubes, although any single dilution may be made directly from an undiluted specimen or substance. In calculating the dilution or concentration of a substance or serum in each tube of the dilution series, the rules previously resulting in a 1:16 dilution (⅛ × ½ × 1⁄16). One milliliter of the final dilution is discarded so that the volumes in all the tubes are tube is calculated by multiplying the previous concentration dilutions in such a series would be 1:2, 1:20 (½ ×
1⁄10 × 1⁄20), 1:200 (1⁄20 × 1⁄10 × 1⁄200), 1:2000, 1:20,000, and 1:200,000. preceding dilution (mL)
1 1 of 1:2 1 of 1:4 1 of 1:8 1 of 1:16 1 of 1:32 1 of 1:64 1 of 1:128 1 of 1:256 1 of 1:512
Final dilution 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024
Dil. of tube 1
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6
1/2
Dil.
made1/2
Dil.
made1/2
Dil.
made1/2
Dil.
made1/2
Dil.
made1/2 Dil. of
SV = Sample volume (e.g., serum) DV = Diluent volume (e.g., saline)
Figure 8-5 Schematic of a twofold serial dilution. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby, p. 166.)