Question

7. The bacteria Lactococcus are widely used in industrial biotechnology to produce lactic acid. There is intense research to create new strains that produce more lactic acid. You have created a library of UV-light mutagenized Lactococcus. Describe or draw a way to screen them for increased lactic acid production? Show what would be the “output” of your screen.

Answer 1

To solve this question, you need to understand the principle of lactic acid estimation based on a colorimeteric method.

Principle -

Lactic acid is oxidized by sulphuric acid to acetaldehyde which gives a purple-red colour with p-hydroxydiphenyl in the presence of metallic ions, of which copper. Higher the intensity of purple red colour, greater will be the value of absorbance by colorimeter.

Method -

1. You have 2 samples, sample A which is normal and produce normal amount of lactic acid and sample B which is mutant and produces more amount of lactic acid.

2. Culture cells of both the cells on a nutrient medium. This is a bacteria, you can use nutrient agar for culturing them.

3. Now pick up one colony from both the samples and make a suspension in 1 ml water in an eppendorf.

4. Now kill the cells and extract all the cytoplasm from them by using a sonicator (sonicator will lyse the cells at high vibration).

5. Now, add sulphuric acid in the extract and then add p-hydroxydiphenyl. This will then change the colour to purple to red.

6. Now, measure the absorbance of the coloured extract using a colorimeter.

7. Record your observations in two tables, one for sample A and the other for sample B. Sample B will have more values for absorbance.

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