Counting of Spores

Spore counting was done in the Microbiology Department using haemocytometer. For spore counting 0.1ml of spore suspention was poured on to heamocytometer under the cover slip. Using low power of microscope various regions of heamocytometer were observed spores were counted in mdium squares (each have 16 small squares.


3.2 Sugarcane molasses

Molasses obtained from Crescent Sugar Mill Faisalabad was used as the nutrient source and the substrate for citric acid production.

3.2 Carriers/inert support materials for solid state fermentation

Substrates used ascarriers for SSF is corn cobes. The substrate was dried to costant weight in vaccum oven at 70oC and groud through waley mill in department of soil and environmental sciences, UAF to get uniform particle size of 40mm mesh. The powdered substrate was stored in airtight plastic jars to keep them free of moisture.. .

3.3 Solid state Fermentation

Solid state fermentation was carried out in 250 ml conical flask. 5g of corn cobs was taken in separate duplicate flasks. 10 % molasses solution was prepared in the distilled water adjusted to pH 5.5 using 1MNaOH /1MHCl. The substrate in each flask was moistened by using molasses solution to 60% moisture level and autoclaved (121oC) for 15 min under 15 psi pressure.

Composition of medium for solid state fermentation of molasses medium using carrier substrates for citric acid production by A. niger.



Substrate Corn cobs

S No. T1a T1b


Substrate (g) 5 5

Molasses solution(10%) 15 15

pH, 5.5; temperature, 37oC.


After sterilization 5ml inoculum were added aseptically in each flask under the laminar flow with the help of sterilized pipette. The flask was incubated at 30oC for 3 days under still culture medium.