Basic Laboratory Techniques Dilutions
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Dilution is a process of making a weaker or a less concentrated solution. Substances which are highly concentrated can be problematic to carry out tests. For instance, when a blood sample is placed on a slide it would be difficult to count cells due to overlapping. As a result dilutions are carried out so that the cells will be more spread. Moreover a large amount of reagent would be needed to react with a highly concentrated substance. This is inadequate since large amounts of reagent would be wasted. In addition it is impractical to use large volumes of substances example buffers. Generally a concentrated form is present and then the necessary dilutions are made for different tests. A manual of how the dilutions are made is typically present with the reagent.
Adequate apparatus must be used for dilutions. Basically, there are two types of dilutions. One, where the final specific concentration only is significant and the other where both the final volume and concentration are significant.
Dilution symbols are significant.
1/10 refers to 1ml sample with 9mls diluents for a total volume of 10mls. This is the same as 1+9.
1:10 refers to 1ml sample with 10mls diluents for a total volume of 11mls. This is the same as 1+10.
Serial dilution is a method used to dilute a substance into solution stepwise with a constant dilution factor in each step. The dilution factor is the volume of stock / total volume. The first step in making a serial dilution is to take a known volume (example 1ml) of stock i.e. the original sample and place it into a known volume of water (example 9ml). This produces 10ml of dilute solution. The dilute solution has 1ml of original sample / 10ml. The technique used to make a single dilution is repeated using the previous dilute solution. At each step, 1ml of the previous dilution is added to 9ml of distilled water. This is repeated sequentially until the required dilution is achieved. The volumes of substances used vary accordingly. During dilutions it is essential to pipette the larger volume first and then the smaller volume.
Diagram of how serial dilutions are made:
Rule : Original concentration = New concentration
Since the dilution-fold is the same in each step, the dilutions are a geometric series i.e a constant ratio. Example: 1/3, 1/9, 1/27, 1/81. Each dilution is a three-fold. A two-fold and a five-fold also exist where it is multiplied by 1/2 and 1/5 respectively.
Serial dilutions are principal for several situations. In the lab there are a number of volumetric flasks however there is not a lot of 1000ml flasks. Hence serial dilutions are the only way to get the desired concentration. Serial dilutions are essentially used for calibration curves to ensure the accuracy of the measurements. This is useful since if a minor mistake is done, it is not noticed since the mistake will be repeated in all the dilutions and as a result there will be no effect. Furthermore serial dilutions are used for antibody titres. A test can be quantitative example the concentration of glucose in blood is 6 or qualitative if the test is positive or negative example when testing for the human immunodeficiency virus if it is present in the blood sample, the result is either positive or negative. However there is another test known as the semi-quantitative test where the result is neither numerical nor positive or negative. When serial dilutions are carried out, the result can be that the antibody titre is positive up to 1/320. It shows that the patient is immune up to a certain limit. This is often used to monitor treatment.
Six test-tubes were placed in a rack. To the first tube 500µl of water were added. To each of the remaining tubes 100µl of water were added. 20µl of solution B were added were transferred to tube 1 and mixed well. 100µl of tube one were transferred to tube 2 and mixed well. 100µl of the contents of tube 2 were transferred to tube 3, and the procedure was repeated for the remaining tubes. The dilution of the serum in tube 6 is 1/832 since the dilution of tube 1 is 20/520 = 1/26. The dilutions are 2-fold. As a result 1/26 x 1/2 x 1/2 x 1/2 x 1/2 x 1/2 = 1/832