In this article we will discuss about the extracellular enzymatic activities of bacteria: 1. Starch Hydrolysis 2. Lipid Hydrolysis 3. Gelatin Hydrolysis 4. Casein Hydrolysis.
1. Starch Hydrolysis:
Starch is composed of glucose molecules linked together by glucosidic bonds. This is hydrolysed by amylase into dextrins and then maltose. Maltose, a disaccharide is finally hydrolysed by maltase to yield soluble glucose molecules which can be absorbed by microorganisms to produce energy by glycolysis.
Starch is hydrolysed and the hydrolytic activity of the organism is tested by I2.
a. Test organisms (Escherichia coli and Alcaligenes faecalis) grown on nutrient agar.
b. Starch agar plates.
c. I2 solution.
d. Inoculating loops.
f. Bunsen flame.
1. Spot inoculate test organisms on starch agar plate and incubate at 37°C for 24- 48 hours.
2. Flood the plates with I2 solution and keep it for 30-40 seconds.
3. Pour out I2 and observe. If the organism has hydrolysed starch, there will be a clear zone around the colony and if it is dark blue around the colonies, it is a negative test.
2. Lipid Hydrolysis:
Like triglyceride, lipids are degraded by lipases (esterases) which cleave the ester bonds in these molecules by addition of water, leading to production of glycerol (an alcohol) and fatty acids. These are assimilated into the cell to produce ATP.
The opaque medium, Triglyceride tributyrin produces a clear zone around the colony if the organisms produce lipase.
Dissolve peptone and beef extract by heating, cool and then add tributyrin.
Nutrient agar slant of test organism.
1. Inoculate test organism on Tributyrin agar plates and incubate for 24-48 hours at 37°C.
2. Examine plates for lipolysis which will be seen as a clear area surrounding bacterial growth. This loss of opacity is due to hydrolytic reaction yielding glycerol and fatty acids and is a positive reaction for lipolytic enzyme that hydrolyses lipids.
3. Gelatin Hydrolysis:
Gelatin is a major component of the connective tissue of tendons in human beings and animals. Gelatin is an incomplete protein lacking tryptophan and hence its value as a nutritional source is doubtful. However, it is useful in identifying bacterial species, since it remains as a gel below 25°C and forms liquid above 25°C.
Some microorganisms capable of producing gelatinase, hydrolyse gelatin to amino acids. This hydrolysed gelatin remains as liquid even at 4°C.
1. Tubes with gelatin medium:
2. Culture of test organism.
3. Bunsen flame.
4. Inoculation loop.
5. Glass marking pencil.
1. Inoculate test organism on gelatin medium and incubate for 24-48 hours.
2. Keep the tubes in refrigerator (4°C) for 30 minutes.
3. If the medium becomes liquid after incubation and if it remains so even after keeping it at 4°C the reaction is positive.
4. Casein Hydrolysis:
Casein which is the major protein in milk is composed of amino acid subunits linked together by peptide bonds (CO-NH). This has to be degraded into—>polypeptides—> dipeptides—>and amino acids before it can be assimilated by cells. This is achieved by proteases which cleave the peptide bonds by introducing water into the molecule. The low molecular weight amino acids are then transported to cells for the synthesis of structural and functional cellular proteins.
Milk gives an opaque medium due to its colloidal nature. Following inoculation, if the organism produces proteases, a clear zone around the colony is formed.
1. Milk agar (pH 7.2)
Skimmed milk powder 100.0 g
Peptone 5.0 g Agar 15.0 g
Distilled water 1000.0 ml
2. Cultures of test organisms.
3. Inoculation loop.
4. Bunsen flame.
6. Glass marking pencil.
1. Spot inoculate test organisms on milk agar plates and incubate at 37°C for 24 hours. Clear areas around the colonies indicate positive test.