1. The Basic Skills of the Biotechnology Workplace
Chapter 3

2. Learning Outcomes
Determine the most appropriate tool for measuring specific volumes of masses
Describe how to select, set, and use a variety of micropipets within their designated ranges to accurately measure small volumes
Convert between units of measure using the B <- -> S rule and appropriate conversion factors
Recognize the different expressions for units of concentration measurements and use their corresponding equations to calculate the amount of solute needed to make a specified solution
Describe what pH is and why it is important in solution preparation

3. 3.1 Measuring Volumes in a Biotechnology Facility
Volume is a measurement of the amount of space something occupies
Volume is measured in
Liters (L)
Milliliters (mL)
Microliters (mL)
Different tools are used to measure volume
Graduated cylinder
Pipet
Micropipet

4. Converting Units
Often volumes are measured in one unit of measurement and reported in another
Converting between metric units
Conversion factor
To measure volumes larger than 10 milliliters, technicians usually use a graduated cylinder

5. GRADUATED CYLINDERS

6. Reading a graduated cylinder
Reading a graduated cylinder. Before using a graduated cylinder, make sure you know the total volume it will hold and the value of each of the graduations. In the lab, common graduated cylinders include 10 mL, 25 mL, 100 mL, 250 mL, 500 mL, and 1 L.
Pipets are available that measure volumes between 0.1 mL and 50 mL. Shown from left to right are 25-, 10-, 5-, and 1-mL pipets.

7. MICROPIPETTE

8. Picking and Using the Appropriate Micropipet
Using Pipets
Picking and Using the Appropriate Micropipet
Measuring units smaller than 10 mL requires a pipet.
“Never mouth pipet!”
P-100 or P-200 micropipet
P-10 or P-20 micropipet
P-1000 micropipet
Using Micropipets
When measuring tiny volumes, less that 1 mL, a micropipet is used.
A micropipet has four parts
Plunger button
Ejector button
Volume display
Dispensing tip

9. READING A GRADUATED CYLINDER

10. Labeled micropipet. Learning to use each part of a micropipet correctly is essential. On the micropipet shown, the plunger has two “stops.” Pressing to the first stop evacuates air to the volume in the display. Pressing to the second stop evacuates that volume plus another 50% or so. To ensure accurate measurement, feel the difference between the first and second stop before using the pipet. Inaccurate measurement could waste costly reagents and cause invalid experiment results.
P-100 Micropipet. This micropipet will measure volumes as small as 10 mL and has precision to 0.2 mL.

11. P-10 Micropipet. P-10 micropipets are common in biotechnology labs
P-10 Micropipet. P-10 micropipets are common in biotechnology labs. A P-10 micropipet will measure volumes as small as 0.5 mL and has precision to 0.02 mL. A P-10 uses tiny tips that are usually white.
P-1000 Micropipet. A P-1000 micropipet will measure up to 1000 mL, or 1 mL, and uses large tips that are usually blue or white in color.

12. READING A MICROPIPETTOR

13. READING A MICROPIPETTOR

14. A multichannel pipet allows several samples to be measured at the same time, a feature that saves time during an experiment with multiple replications and repetitive pipeting.

15. Vocabulary
Volume – a measurement of the amount of space something occupies
Mass – the amount of matter (atoms and molecules) an object contains
Liter – abbreviated “L”; a unit of measure for volume, approximately equal to a quart
Milliliter – abbreviated “mL”; a unit of measure for volume; one one-thousandth of a liter (0.001 L) or about equal to one-half teaspoon
Microliter – abbreviate “mL”; a unit of measure for volume; equivalent to one-thousandth of a milliliter or about the size of the tiniest teardrop
Graduated cylinder – a plastic tube with marks (or graduations) equally spaced to show volumes; measurements are made at the bottom of the meniscus, the lowest part of the concave surface of the liquid in the cylinder
Pipet – an instrument usually used to measure volumes between 0.1 mL and 50 mL
Micropipet – an instrument used to measure very tiny volumes, usually less than a milliliter
Unit of measurement – the form in which something is measured (g, mg, mg, L, mL, mL, km, cm, etc.)
Conversion factor – a number (a fraction) where the numerator and denominator are equal to the same amount; commonly used to convert from one unit to another
Metrics conversion table – a chart that shows how one unit of measure relates to another (for example, how many milliliters are in a liter)

16. 3.1 Review Questions
What instrument would you use to measure and dispense the following volumes? Pick the instrument that is likely to give you the least error for each measurement.
23.5 mL 6.5 mL 125 mL 7mL 2.87 mL 555mL
Convert the following units to the requested unit:
1.7 L = _____ mL mL = _____ mL 2.37 mL = _____ mL
What numbers should be dialed into a P-10 display if a volume of 3.7 mL is to be measured?
What instrument should be used if a technician wants to fill 40 sets of 16 tubes all with identical volumes?

17. 3.2 Making Solutions
Solution preparation is one of the most essential skills of a biotechnology lab employee.
Solutions are mixtures in which one or more substances are dissolved in another substance.
Solid solutes are measured on balances or scales.
Concentration is measured in several ways:
Mass/volume
Volume/volume
% mass/volume
Molarity
Normality

18. Most analytical balances measure down to milligrams, even though they usually report in grams.

19. ELECTRONIC BALANCE

20. Vocabulary
Positive displacement micropipet – an instrument that is generally used to pipet small volumes of viscous (thick) fluids
Solution – a mixture of two or more substances where one (solute) completely dissolves in the other (solvent)
Aqueous – describing a solution in which the solvent is water
Solute – the substance in a solution that is being dissolved
Balance – an instrument that measures mass
Weight – the force exerted on something by gravity; at sea level, it is considered equal to the mass of an object
Gram – abbreviated “g”; the standard unit of mass, approximately equal to the mass of a small paper clip
Solvent – the substance that dissolves the solute
Molarity – a measure of concentration that represents the number of moles of a solute in a liter of solution (or some fraction of that unit)
Normality – a measurement of concentration generally used for acids and bases that is expressed in gram equivalent weights of solute per liter of solution; represents the amount of ionization of an acid or base

21. 3.2 Review Questions
What instrument should be used to measure and dispense the following solutes? Choose the instrument that is likely to give you the least error for each measurement.
3.5 g of salt 6.5 mg of DNA 12.5 g of gelatin
What happens to the ratio of solute molecules to solvent as a solution becomes more concentrated?
Which of the following are concentration units?
mi/hr g/mL mM °F/°C
4. Describe how glassware should be prepared before using it to prepare or store solutions.

22. 3.3 Solutions of Given Mass/Volume Concentrations
Mass/Volume Solution. Solvent is added until a volume of 10 mL is reached. A protein solution that has a concentration of 1 g/mL is considered fairly concentrated.

23. Making Mass/Volume Solutions
Mass/Volume Concentration Equation
____ g/mL X ____ mL = ____ g of solution
concentration volume to be weighed out,
desired desired dissolved in the solvent

24. 3.3 Review Questions
Which of the following are mass/volume concentration units?
mg/mL g/mg L/mg mg/mL g/l
What mass of the protein, gelatin, is needed to make 0.5 L of a 3 g/L gelatin solution?
What mass of sugar is need to make 25 mL of a 25 mg/mL sugar solution?
What mass of salt is needed to make 150 mL of a 100 mg/mL salt solution? Describe how the solution is prepared.

25. 3.4 Solutions of Differing % Mass/Volume Concentrations
A percentage represents something that is part of 100.
Mass/Volume Concentration Equation
____ % = ____
percent value decimal value of the g/mL
____ X ____ = ____ g of solute to
decimal total volume be measured and
value desired (mL) added to the volume desired of solvent

26. Vocabulary
Percentage – a proportion of something out of 100 parts, expressed as a whole number

27. 3.4 Review Questions
What is the decimal equivalent of the following percentages?
10% 15% 25% 2% 1.5% 0.5%
What mass of gelatin (a protein) is needed to make 0.5 L of a 3% gelatin solution?
What mass of sugar is needed to make 25 mL of a 2.5% sugar solution?
What mass of salt is needed to make 150 mL of a 10% salt solution? Describe how the solution is prepared.

28. 3.5 Solutions of Differing Molar Concentrations
Molarity Concentration Equation
volume molarity molecular the number of grams to be
wanted (L) X desired X weight of the = dissolved in solvent, up to
(mol/L) solute (g/mol) the total volume of
solution desired

29. Periodic Table. The Period Table of Elements shows the elements (atoms) found in compounds (molecules). Each element is listed along with the atomic weight (mass) of each atom in the element. A NaCl molecule has a molecular weight of about 58.5 amu (atomic mass units) because the Na atom weighs about 23 amu, and the Cl atom weighs about 35.5 amu. Together, in the NaCl molecule, the atoms total approximately 58.5 amu. The mass of a hydrogen atom equals 1 amu.

30. This instrument is a mass spectrometer
This instrument is a mass spectrometer. Scientists use it to determine the molecular weight of a compound. A “mass spec” can also determine if a sample is contaminated with molecules of different molecular weights.

31. Vocabulary
Mole – the mass, in grams, of 6 X 1023 atoms of molecules of a given substance; one mole is equivalent to the molecular weight of a given substance, reported in grams
Molecular weight – the sum of all the atomic weights of the atoms in a given molecule
amu – abbreviation of atomic mass unit; the mass of a single hydrogen atom
Mass spectrometer – an instrument that is used to determine the molecular weight of a compound

32. 3.5 Review Questions
What is the molecular weight of each of the following compounds?
NaOH MgCl2 MgO HCl
What mass of NaCl is needed for 0.5 L of a 0.5 M NaCl solution?
What mass of MgO is needed for 200 mL of a .025 M MgO solution?
What mass of sodium hydroxide (NaOH) is needed to make 750 mL of a 125 mM NaOH solution? Describe how to prepare the solution.

33. 3.6 Dilutes of Concentrated Solutions
Concentrating 1 L Solution. Many chemical and biological reagents are purchased in concentrated form. Concentrated solutions can be prepared initially with a greater amount of solute to solvent, or a solution can be concentrated by removing water. A diluted solution can be prepared by adding solvent to a concentrated one.

34. Diluting a 100 mg/mL Stock Solution to 1 mg/mL.

35. AGAROSE GEL ELECTROPHORESIS

36. Vocabulary
Dilution – the process in which solvent is added to make a solution less concentrated
Stock solution – a concentrated form of a reagent that is often diluted to form a “working solution”
Buffer – a solution that acts to resist a change in pH when the hydrogen ion concentration is changed
TRIS – a complex organic molecule used to maintain the pH of a solution
TAE buffer – a buffer that is often used for running DNA samples on agarose gels in horizontal gel boxes; contains TRIS, EDTA, and acetic acid

37. 3.6 Review Questions
How do you prepare 40 mL of a 2 mg/mL protein solution from 10 mg/mL protein solution?
How do you prepare 200 mL of 2X enzyme buffer from 10X enzyme buffer solution?
How do you prepare 500 mL of 10 mM NaCl solution from 5 mM NaCl solution?
How do you prepare 3 L of 1X TAE buffer from 50X TAE buffer stock solution?

38. Questions and Comments?