DILUTIONS AND STANDARDS

Many of the laboratory procedures involve the use of dilutions. It is important to understand the concept of dilutions, since they are a hand tool used throughout all areas of the clinical laboratory. These dilutions have to be considered as they make a quantitative difference in what is going on. First, there are several terms used in expressing dilution: 1. "Dilution: - Dilutions are expressed as the ratio of the quantity of a desired solute (serum, urine, chemical solution, etc.) contained in a solvent (diluent). A 1:10 dilution of serum was made by adding one part serum to nine parts diluent to make a total of ten parts. If 1.0 milliliter of serum is added to 9.0 milliliters of H20, a total volume of 10.0 milliliters is obtained. Therefore, the dilution is expressed according to the following equation. volume of serum/volume of solution = [1.0 mL serum ]/[1.0 ml serum + 9.0 mL H 20] 1.0 mL serum/10 mL solution = 1:10 total This means that each milliliter of solution contains 1/10 as much serum as each milliliter of the original serum. Another way to say this a serum sample was diluted 1:10 with H 20. One precaution: Some people write ratio meaning the amount of solute in proportion to the amount of solute. If you are unsure of someone's intent, ask to clarify. 2. "Diluted to" - This is essentially the same as "dilution." If 1 milliliter is diluted to 10 milliliters, enough diluent is added to the original volume to yield a final, total volume of 10 milliliters. For example, if a 1 milliliter of serum is diluted to 10 milliliters of solution, 9 milliliters of H 20 is added to the original serum sample. Using this information, one can see why "diluted to" is the same as "dilution" using the following equation: volume of serum/total volume of solution = 1 mL/10mL = 1:10 One milliliter of serum was diluted to 10 milliliters. 3. "Added to" - This expression is usually a hang-up since it is not the same as "diluted to." "Added to" refers to the volume of the solute added to a specified volume of solvent. For example, if 1 milliliter of serum is added to 10 milliliters of H 20, this means 1 mL + 10 mL yields a total volume of 11 mL. If you expressed this using one of the above terms you must say 1 mL was added to 10 mL or 1 mL was diluted to 11 mL. This means a 1/11 dilution was made since: volume of serum/total volume of solution = 1 mL serum/[1 mL serum +10 mL H 20] = 1:11 4. "Serial dilution" - This term is frequently used and refers to a "multiple" dilution problem. In other words, an initial dilution is made and then this dilution is used to make a second dilution, and so on. For example, a 1:2 serial dilution is made using a 1 mL volume of serum. This expression indicates that 1 mL of serum is added to 1 mL of H 20 and then mixed. This initial dilution is 1:2. Then, 1 mL of this dilution is added to 1 mL of H 20 further diluting the sample. This same process is continued. Dilutions must be used carefully and the calculation of dilution factors must be done accurately, since an error may seriously affect a test result. Read the following material closely in order to understand the variety of ways dilutions are used.

Preparation of Dilutions

The first rule in performing dilutions is careful reading of procedural instructions. For example: "A 1 mL serum specimen diluted to 50 mL is not the same as: "A 1 mL serum specimen added to 50 mL." If several dilutions are made in succession of one another, the final dilution can be calculated by simply multiplying each dilution factor involved. To illustrate, consider the following situations: A serum specimen was diluted 1:20 and then 2 mL of that was diluted to 10mL, and 1 mL of that was added to 4 mL using H20. Exactly 2 mL of this last dilution was discarded, and the remainder was diluted to 30 mL. What was the final dilution of the serum specimen? Read each portion carefully. 1/20 x 2/10 x 1/5 x 3/30 = 6/30,000 = 1/5,000 or 1:5000 Always reduce the final answer to a fraction with the lowest possible denominator. In the Immunology or Serology area serial dilutions, sometimes referred to as serological dilutions, are extensively used in the test procedures. Serial dilutions usually refer to dilutions of the same proportion made repeatedly from the previous dilution. The directions for preparing these serial dilutions may be written several ways. To illustrate, consider the following two examples:

Example 1:
Eight test tubes are placed in a rack. To the first tube add 3 mL of saline. To each of the remaining seven tubes add 2 mL of saline. To the first tube add 1 mL of serum and mix well. Transfer 2 mL of tube # 1 to tube # 2 and mix well. 2 mL of the contents of tube # 2 is then transferred to tube # 3, and the procedure is repeated for the remaining tubes, finally discarding 2 mL from the last tube. What is the dilution of serum in tube # 8?

Dilution 1/4 x 2/4 x 2/4 x 2/4 x 2/4 x 2/4 x 2/4 x 2/4 = 1/512

Example 2:
One mL of serum is added to 3 mL of saline in the first tube of a series of eight. A 1:2 serial dilution is then made. What is the dilution of serum in the 8th tube? 1/4 x 2/4 x 2/4 x 2/4 x 2/4 x 2/4 x 2/4 x 2/4 = 1/512 Both example 1 and example 2 illustrate how the same serial dilution problem may be expressed in two different ways. In the first example a step-by-step procedural outline is provided. Go back and look at example 1. In the first tube 1 mL of serum was added to 3 mL of saline. To calculate the dilution contained in this tube, apply the following. volume serum/total volume of solution = 1mL/l mL serum + 3 mL saline = 1/4 In the second tube 2 mL of the first dilution was added to 2 mL of saline: 1/4 x 2/4 or 1/4 x 1/2 = 1/8 in 2nd tube

By transferring 2 mL of dilution from one tube to a second tube containing 2 mL of saline, and continuing to do so, this is actually making a 1:2 serial dilution. PROBLEMS: 1. You made a 1:2 dilution of serum. Then you added 2 mL of that to 4 mL of water. Then 1 mL of that was diluted to 8 mL. What was the final dilution of serum? 2. You took 4 mL of a glucose solution and diluted it to 10 mL. Then a 1:10 dilution was made of that. What was the final dilution? 3. In performing a serological procedure, you add 1 mL of serum to 5 mL of saline. A 1:2 serial dilution is then made. What is the dilution of serum contained in tube 6? 4. Six tubes are placed in a rack. To all six tubes you add 2 mL of water. Then 1 mL of serum is added to the first tube and mixed well. You then transfer 1 mL of that to the second tube and again mix well. You continue the transfer of 1 mL of mixture from one tube to each subsequent tube, finally discarding 1 mL from tube 6. What is the dilution of serum contained in tube 6? 5. How would you set up a series of tubes with a 1:4 dilution such that you end up with 3 mL of solution in each tube at the end of the procedure? 6. How would you set up a series of tubes for a 1:2 dilution so that you end up with 0.5 mL of solution in each tube at the end of the procedure?

Determining Concentrations of Dilutions

If you want to determine the concentration of a substance in a particular dilution, you multiply the original concentration times the dilution. To illustrate consider the following: Example 1: You had a solution with 4 g of glucose per mL. You dilute this original solution by adding 1 mL of it to 9 mL of water. What is the dilution you prepared? By adding 1 mL of solution to 9 mL of water, you have prepared a 1:10 dilution. Therefore: 4 g/mL x 1/10 = 4 g/mL/10 = 0.4 g/mL Example 2: If you had a 100 mg/dL solution of glucose and made a 1:5 dilution, what concentration of glucose is contained in the dilution? 100 mg/dL x 1/5 = 100 mg/dL/5 = 20 mg/dL Problems: 7. What is the final concentration of your solution when you took 1 mL of a 100 mg/dL solution and added 9 mL of water? 8. You had a 1,000 mg/dL solution of glucose. You took 1 mL of that and diluted it to 5 mL. Then you took 4 mL of that and added it to 12 mL of water. What is the final concentration of your glucose solution?

Preparation of Standards
Another common way we use dilution problems is in making standards for out tests. We dilute "stock" standards to make "working" standards. These working standards are then used in testing and calculating unknown concentrations. For example, we may have a stock glucose standard of 10 mg/mL. When we run glucose levels on patients we need to also run a standard to calculate the results. The working standard should be within the ball park of the normal values. The normal individual may have a glucose of between 60-90 mg/dL, so a

good glucose working standard would be about 100 mg/dL. We are now faced with preparing such a working standard. 1. Since the stock standard is expressed in mg/mL and the working standard is expressed in mg/dL, we must first convert the concentration of them both to the same units. Stock standard = 10 mg/mL x 100 mL/1dL = 1,000 mg/dL 2. To determine the dilution that must be made to yield a working standard of the desired concentration, we use the following equation: desired concentration of working std./concentration of stock std. = dilution required Using the information from our example, we now see: 100 mg/dL = 1/10 1000 mg/dL Therefore, a 1:10 dilution needs to be made of the stock standard to obtain a working standard of the desired 100 mg/dL concentration, i.e., one part (mL or whatever) of stock qs 10 parts total solution (qs = quantity sufficient to make). 3. To determine the actual amounts of stock standard and diluent needed to prepare the working standard, use the following equations. Vol wanted x desired conc/stock conc.. = vol of stock std needed [total vol working std needed] - [vol of stock std to use] = vol of diluent needed Suppose we wanted to prepare 50 mL of a 100 mg/dL working standard of glucose from the original stock standard of 10 mg/mL. 50 mL needed x 1/10 = 5 mL stock standard 50 mL needed - 5 mL stock std = 45 mL diluent Therefore, to prepare 50 mL of working standard, 5 mL of stock standard is added to 45 mL diluent (such as water). Problems: 9. How would you make 50 mg/dL working glucose solution from a 2.0 g/dL stock glucose solution? 10. How would you make a 4 mg/dL uric acid working standard from a 200 mg/dL stock uric acid standard? 11. You have a serum with 7 g/dL of protein. What is the concentration in mg/dL of protein in the 6th tube of a 1:2 serial dilution of the serum? 12. 5 mL of a stock urea nitrogen standard contains 1mg Nitrogen. 5 mL is diluted to 10 mLs. 1 mL of this is rediluted to 10mL. 2.5 mL of this is rediluted to 25 mL. What is the concentration of nitrogen per mL of the final dilution. 13. If you use 5 mL of stock glucose containing 10 mg/mL and dilute to 100 mL, what is the concentration of glucose per mL in the dilution? 14. How would you dilute a 1 mg/mL urea stock standard to be equivalent to a 5 mg/dL working standard? 15. How would you dilute a 2 mg/mL glucose stock standard to make 100 mL of a 50 mg/dL standard?