Volume measurement Pasteur pipettes

Fig. 3.1. C02 delivery systems. On the left is a simple arrangement where air (from an air pump) may be mixed with C02 to produce 5% C02 in air for gassing bottles or feeding to an incubator. On the right is an arrangement for measuring out fixed volumes of C02 and delivering them through a three-way tap (at the bottom of the syringe) and a sterile Pasteur pipette to a bottle of cells.

Modem Erlenmeyer flasks and beakers have approximate volume calibrations fused into the glass, but these are very approximate. Somewhat more accurate volumetric measurements are made in the 10-mL graduated cylinders. For volumes less than about 4 mL, use a graduated pipette. Never apply suction to a pipette by mouth. The pipette can be fitted with a small rubber bulb. A Pasteur pipette can be converted into a calibrated pipette with the addition of a plastic syringe body [see Fig. 11(d)] or you can calibrate it at 0.5, 1.0, and 1.5 ml and put three file scratches on the tube this eliminates the need to use a syringe with this Pasteur pipette in the future. Also see the Pasteur pipette calibration marks in the back of this book. You should find among your equipment a 1-mL pipette, calibrated in hundredths of a milliliter [Fig. 11(a)]. Determine whether it is designed to... 

A volumetric flask is used to prepare accurate volumes of solution. These flasks are pear-shaped with long, thin necks that allow the operator to dilute accurately to the mark with solvent. Volumetric flasks are available in all sizes from 1 mL up to 10 litres, but the most common sizes are 20,50 and 100 mL. When selecting which size of flask to use, a compromise should be reached between the desire to use a small-volume flask and so save on expensive reagent, and the desire to use a large-volume flask to minimise dilution errors. The usual procedure is to pipette in a known volume of concentrated solution, add solvent until just short of the mark, shake or invert the flask to mix the contents and then make up to the mark, as accurately as possible, with a Pasteur pipette. Volumetric flasks should be used for all accurate dilutions. Use of measuring cylinders or (even worse) beakers to dilute solutions should be avoided. 

Assay. The standard assay mixture for determination of the activator consists of 1 nmol ganglioside Gmi, [ H]-labeled in the terminal galactose moiety ( 100,000 cpm), 5 mU p-galactosidase and up to 25 p,l of the suitably diluted activator sample, in a total volume of 50 jil of 50 mM citrate buffer, pH 4.5. The assays are incubated for 1 h at 37°C and then transferred to an ice-bath, and 1 ml of an ice-cold 1 mM galactose solution is added. The mixtures are loaded onto small (0.5—1 ml) columns of DEAE-cellulose (in Pasteur pipettes) that have been washed with distilled water. Liberated [ HJgalactose is washed out with 2 x 1 ml of 1 mM aqueous galactose solution, the combined effluents are collected in scintillation vials, and, after addition of 10 ml of scintillation fluid, their radioactivity is measured in a liquid scintillation counter. Blanks run with water instead of activator solution are subtracted. 

In general, Pasteur pipettes should not be used to measure volumes of reagents needed for organic reactions, because they are not accurate enough for this purpose. 

After partial evaporation the pressure inside the rotary evaporator is equilibrated with the atmosphere through a 5 A molecular sieve trap. The solution is transferred into a 10 mL volumetric flask with a Pasteur pipette and made up to volume. This solution is ready for measurement in the UV spectrofluorimeter. Should volumetric flasks not be available, the voliunes of individual extracts may be determined by weighing on a top-loading balance. The densities are 0.6603 g/cm (n-hexane) and 0.77855 g/cm (cyclohexane), both at 20 °C. 

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