30-06-2012, 01:23 PM
Chemicals Materials and methods
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Materials and methods
Materials
Maintenance of fungal culture
Culture of Aspergillus spp. from the laboratory collection was used for the studies. Spore suspension of O.D 0.1 were used. The strain was maintained in the Sabouraud medium and used. Inoculation of the culture was done by standard sterile technique. Medium and all the glassware used were properly sterilized autoclaved at 15lb/in2 pressure and 121° C for 30minutes.
Chemicals
DNSA (3,5-Dinitrosalicylic acid ) for estimation of reducing sugars is obtained from Loba chemie Pvt. Ltd. The Biuret Reagent is made of sodium hydroxide and copper sulfate for Biuret test.
K2Cr2O7 and Pb(NO3) 2 are obtained from Loba Chemie Pvt ltd, India. Sabouraud medium and Sabouraud broth for sub culturing Himedia, India. Hydrochloric acid (HCl) and sodium hydroxide (0.5N NaOH ) for adjusting pH. Concentrated Nitric acid used for digestion from Loba chemie pvt. Ltd Mumbai
Media Composition
Potato dextrose broth (PDB) from Potato infusion can be made by boiling 300 grams of sliced potatoes in ~ 1 liter water for 30 minutes and then decanting or straining the broth through cheesecloth. Distilled water is added such that the total volume of the suspension is 1 liter 20 grams dextrose powder is then added and the medium is sterilized by autoclaving at 15lb/in2 pressure for 15 minutes. New formulation of media Potato Jaggery Broth( PJB) was also made by replacing dextrose to Jaggery under same conditions.
Methods
Effect of media composition on biomass production
There were three different media’s used namely Sabouraud dextrose broth , Potato dextrose broth and Potato Jaggery broth for cultivation of Aspergillus. These were incubated on rotary shaker for 5 days .
Chemical Estimation is done by Biuret and DNSA methods for protein and reducing sugar respectively.
Estimation of Dextrose by DNSA method Principle :
Certain sugars can be estimated based on their ability to reduce certain compounds or elements like potassium ferricyanide , 3,5- dinitrosalicylic acid (DNSA) , copper and hence called as reducing sugar. These compounds are generally reduced by reducing sugars in presence of an alkali .An alkaline solution of DNSA is reduced by sugars to 3-amino-5-nitrosalicylic acid .The chemistry of the reaction is complicated and gives a reddish – brown product which provides the basis of a simple spectrophotometric assay :