Understanding of Bacterial Endotoxin Test Calculation

Hello Friends,

This particular blog is about calculation in BET testing. In microbiology, BET is a type of test which requires lot of calculation. It become difficult to understand the calculation as every person has their own style of calculation. I have also done many efforts during the beginning of my carrier to understand the calculation in BET. I know what kind of difficulty a fresher/microbiologist face during the beginning of their carrier.

But such time has gone as I am here to help you with this blog. In this blog we will understand how to prepare the particular potency of endotoxin with labelled claim lysate sensitivity.

Let's start to understand the calculation of BET.

In BET you have requirement of three types of reagents.

1. Lysate

2. Controlled Standard Endotoxin

3. Lal Reagent Water

When you receive the material, you will get a CoA of Lysate and seperate CoA for LRW. In the CoA of lysate you will get the lot no. of lysate, its labelled claim sensitivity and direction of storage, direction of reconstitution. In the same CoA you will get the detail of endotoxin like its lot no., reconstitution volume of CSE, its potency etc. First thing, you should know the labelled claim lysate sensitivity and potency of controlled standard endotoxin. Both are mentioned on the same CoA.

For example if we have a vial of CSE and in CoA it is mentioned that one vial contains 100 EU (endotoxin unit). Reconstitution volume also mentioned in CoA and consider this 5.0 mL here. So, after reconstituting the vial of endotoxin you will get 20 EU/mL potency. 

Your labelled claim lysate sensitivity is 0.125 EU/mL. Labelled claim lysate sensitivity is also denoted by lembda (λ) and if it is 50 test vial then you have to reconstitute the vial with 5.2 mL LRW and if it is 10 test vial then you have to reconstitute the vial with 1.2 mL LRW. Please refer vendor CoA for that.

Preparation of 4 λ endotoxin concentration:

Here 4λ means 4 multiplied by λ (which is lysate sensitivity)

4 X 0.125 =0.5 EU/mL. So you required 0.5 EU/mL concentration of endotoxin which is equivalent to 4λ.

Your initial vial concentration is 20 EU/mL and you have to prepare 0.5 EU/mL. Here is a simple technique. What you have divided by what you required?

You have 20 EU/mL and you required 0.5 EU/mL. So, divide 20 EU/mL with 0.5 EU/mL and you will get the dilution factor.

20 EU/mL = 40
0.5 EU/mL

Here 40 is the dilution factor which means if you dilute 20 EU/mL endotoxin 40 times you will get the concentration of 0.5 EU/mL.

Now, how to dilute the 20 EU/mL concentration to 40 times?

You have to take one part of controlled standard endotoxoin and rest part lal reagent water.

In case of 40 take 1.0 mL Controlled standard endotoxin and 39 mL LRW. Further to reduce the consumption as controlled standard endotoxin is only 5.0 mL available we can do like this, take 0.1 mL controlled standard endotoxin and 3.9 mL LRW and you will get the concentration of 0.5 EU/mL which is also called 4λ.

Preparation of 20 λ endotoxin concentration:

Here 20λ means 20 multiplied by λ (which is lysate sensitivity)

20X 0.125 =2.5 EU/mL. So you required 2.5 EU/mL concentration of endotoxin which is equivalent to 20λ.

Your initial vial concentration is 20 EU/mL and you have to prepare 2.5 EU/mL. Here is a simple technique. What you have divided by what you required?

You have 20 EU/mL and you required 2.5 EU/mL. So, divide 20 EU/mL with 2.5 EU/mL and you will get the dilution factor.

20 EU/mL = 8
2.5 EU/mL

Here 8 is the dilution factor which means if you dilute 20 EU/mL endotoxin 8 times you will get the concentration of 2.5 EU/mL.

Now, how to dilute the 20 EU/mL concentration to 8 times.

You have to take one part of controlled standard endotoxoin and rest part lal reagent water.

In case of 8 take 1.0 mL Controlled standard endotoxin and 7.0 mL LRW. Further to reduce the consumption as controlled standard endotoxin is only 5.0 mL available we can do like this, take 0.1 mL controlled standard endotoxin and 0.7 mL LRW and you will get the concentration of 2.5 EU/mL which is also called 20λ.

Hope this calculation is clear to everyone!

Thanks!!!