In a biological laboratory, a variety of chemical solutions are necessary to properly perform any given set of experiments. If these solutions are too concentrated or too dilute, the experiment may not work correctly and the results may not be accurate. For this reason, it is important to understand how to mix a solution to the proper concentration. It is also important to understand how to choose the equipment for mixing solutions and how to care for that equipment.

Since volumes and concentrations can vary from one experiment to the next, the metric system can be used to compare measurements over a wide range of values for grams, liters and molarity. Table 1 provides you with a range of commonly used metric prefixes and the power of 10 that is associated with each one.

There are many different “tricks” for converting from one metric unit to another. One of those methods is described here.

Solving a Problem

1. Determine what units we “have” and what units we “need”

1 ml = ______ μl

have need

2. Write down the exponents (power of 10) associated with those prefixes

1 ml = ____ μl

-3 -6

3. Subtract the exponent of what we need from the exponent of what we have

-3 – (-6) = + 3

4. If the resulting number is positive, move our decimal that many places to the right. If the resulting number is negative, move our decimal that many places to the left.

1 ml = 1.000 = 1000 μl

There are a number of tools that can be used to measure volume in a laboratory. We should choose the equipment based on the volumes being dispensed, the accuracy required, and the number of times the measurement must be made. For large liquid volumes, graduated cylinders and beakers can be used for measurement with graduated cylinders being more accurate. However, very small volumes, less than 25 milliliters require the use of graduated pipettes and micropipettes.

Micropipettes

Micropipettes are special pipettes that are able to dispense liquids in the microliter range. Although they come in a variety of shapes and sizes, there are three common types:

p20 1 μl – 20 μl

p200 20 μl – 200 μl

p1000 200 μl – 1000 μl

Note: ** For volumes greater than 1000 µl (=1 ml),a graduated pipette should be used.

We can determine the volume that a micropipette is set to deliver by reading the numbers in the display from top to bottom. Although the display may read the same numbers on three different pipettes, they dispense very different volumes depending on the type of micropipette that is being used.

Note: It is important to note that pipettes are very different depending on the model. You will need to note what kind of pipettes we are working with in our lab and learn how to use those.

Follow the directions below on how to use the micropipettes. Practice using them until comfortable handling them and adjusting or reading the volume.

All of the solutions made in the lab consist of specific molecules suspended in a solvent (generally water). To determine the concentration of a given solution, we must first have information about the properties of the molecules in that solution.

Basic Chemistry Review

Molecules are composed of atoms (the smallest unit of matter that still retains the properties of an element). Atoms are composed of protons and neutrons (in the nucleus) and electrons (in orbit around the nucleus). All of these components contribute to the atom’s atomic mass. When a molecule is formed, the atomic masses of all of the atoms in the molecule are added to calculate the molecular weight of the molecule. For example, the atomic mass of sodium (Na) is 23 and the atomic mass of chlorine (Cl) is 35, so when a molecule of salt (NaCl) is formed, the molecular weight of that molecule is 58 atomic mass units.

When mixing solutions, it is necessary to know how many molecules are present so the desired reaction will occur. The number of molecules in a solution is measured in moles. A mole (mol) is equal to 6.02 x 10²³ molecules (Avogadro’s number) and is also equal to the molecular weight of the molecule in grams.

For our lab, we will need to make various solutions in order to perform experiments. Typically, these will have two types: one in which we have to dissolve a specific amount of solid into a solvent in order to make a solution and the second involves disolving a liquid in more solvent. We'll showcase both examples below. When looking at specific problems, try to determine which type it is before solving.

Example:

1 mol NaCl = 6.02 x 10 molecules of NaCl (sodium chloride).

1 mol NaCl = 58 g NaCl

For solutions, we use a value called molarity to express the number of moles of a molecule per liter of water in a given solution. So, a 1 Molar (M) solution of NaCl contains 1 mol (=58 g) of NaCl in 1 L of water.

Another commonly used form of measurement is expressed as percentage, %. The definition of a percentage is either as weight per 100 ml (g/100 ml), or as volume per 100 ml (ml/100ml).

Example:

1 % = 1 g in 100 mL distilled water

1 % = 1 mL in 100 mL distilled water

Practice Problem:

Make 100 mL of a 0.5 M solution of sucrose (Formula Weight of Sucrose = 342.3 g).

1 M = 342.3 g / 1 L

0.5 M = 0.05 mol/1 L = (0.5 x 342.3 g)/1L = 171.15 g/1L

So, to make 1 L you would add 171.15 grams of sucrose in 1 liter of water. However, we only want 100 ml (0.1L). So we would only need 0.1 times as much sucrose for 100 ml.

171.15 g x 0.1 = 17.115 grams in 100 ml of water.

Make sure to write the answer in statement, such as: "In order to make 100 mL of a 0.5 M solution of sucrose, add 17.115 grams of sucrose to 100 mL of water".

Psst: Want to know a cool equation that can make life soo much easier with molarity problems involving a solid into a solvent? Here it is:

M x MW x V = Grams needed to make the solution

M= Molarity of the solution we need (in units moles/liter)

MW= Molecular weight of the solid (in units grams/liter)

V= Volume of the solution we need (in units liter)

For example, in the same practice problem above we would have the following:

(0.5 M) x (342.3 g/liter) x (0.100 L) =17.115 g.

***When using this formula, there's several conversions that may need to be completed. Just ensure those are good to go and this equation can save alot of time and effort!

Solutions that are used for experiments are stored as concentrated stock solutions to prevent bacterial contamination and to prolong shelf life. These stocks must be diluted to “working concentrations” just prior to use.

One common solution that will be used in this course is Trypan blue dye, a viability stain for cells. The Trypan dye is stored as a stock solution at a 1.5% concentration. However, the dye needs to be diluted to a 0.4% concentration in order to stain the cells.

The formula used to calculate dilutions is: C₁V₁ = C₂V₂

where:

C₁=Concentration of a certain stock solution

V₁= Volume of Stock Solution Used

C₂= Concentration of the final solution

V₂= Final Volume

Example Problem:

Let's say we need to make up 200mL of 0.4% Trypan dye. Our final volume will be 200mL, and we need to figure out how much of that 200mL will be 1.5% Trypan dye. After setting up the formula, solve for the unknown variable which in this case is V₁.

1.5% x V₁ = (0.4%) x (200 mL)

V₁ = 53.33 mL

Next, we need to bring our solution up to final volume with our solvent, which in most cases, is water. Another common solvent is 1X PBS, which is used to dilute Trypan dye. We know that 53.33mL of our 200mL will be 1.5% Trypan dye so the remaining volume will be 1X PBS.

200 mL - 53.333 ml = 146.67 ml of 1X PBS.

So, to wrap it all up: Add 53.33 ml of 1.5% Trypan blue to 146.67 ml of 1X PBS, to make 200 mL of 0.4% of Trypan blue.

1. Atomic Mass: the mass of an atom, expressed in atomic mass units (amu)

2. Atoms: basic unit of matter, a central nucleus surrounded by negatively charged electrons

3. Colony-stimulating factor: secreted proteins that bind to receptors on the surface of hematopoietic stem cells to activate proliferation and differentiation pathways, allowing for a certain lineage of progeny cells

4. Cytokine: a term used to describe protein molecules that are secreted by cells (especially in the immune system) that serve as chemical messingers or regulator molecules, a molecule that allows cells to communicate and alter one another’s function

5. Extracellular matrix: a protein scaffold existing in the intercellular spaces which connects cells to one another and provides support

6. Fibronectin: an extracellular matrix glycoprotein that binds to the extracellular matrix and to integrins

7. Integrin: cell surface receptors that interact with the extracellular matrix and act as cell-adhesion receptors

8. Matrix Metalloproteinases: a group of enzymes that can break down proteins that are found in the extracellular matrix (such a collagen, gelatin, etc), to allow for processes such as migration, metastasis, and angiogenesis

9. Metric system: standardized system of measurement in which all units are evenly divisible by 10

10. Micropipette: accurately measures and dispenses liquid volumes less than one milliliter

P20- micropipette that measures and dispenses 1-20 microliters of liquid

P200- micropipette that measures and dispenses 20-200 microliters of liquid

P1000- micropipette that measures and dispenses 200-1000 microliters of liquid

11. Molarity: the number of moles of a molecule per liter of water in a given solution

12. Mole: equal to 6.02 x 1023 molecules (Avagadro’s Number) and is also equal to the molecular weight of the molecule in grams

13. Neutrons: subatomic particle located in the nucleus, with a charge of 0

14. pH: the negative log (base 10) of the molar concentration of dissolved hydrogen ions in a solution

15. Protons: subatomic particle located in the nucleus, with a charge of +1e

16. Tumor Necrosis Factor: an inflammatory cytokine that is produced by white blood cells (monocytes/macrophages), a cytokine that causes inflammation, apoptosis, inhibit tumorigenesis, and inhibit viral replication