2.

2 SAMPLE COLLECTION
The method of sample collection by Oguche et al (2021) will be adopted. Maize
cobs infected with molds will be collected from IMAP premises and surrounding
into sterile polythene bags using precise aseptic techniques. The samples will be
transported to the microbiology laboratory for analysis.

2.3 SAMPLE PREPARATION

Method of sample processing/ preparation as described by Oguche et al (2021) will
be used. The maze cobs will be scraped or dusted using a sterile spatula into a
sterile beaker to obtain sample from the maize cobs.

2.4 SERIAL DILUTION

Six-fold serial dilution technique will be used as describe by Vijay et al (2016). 1g
of the powdered sample will be aseptically weighed on an analytical weighing
balance placed n a test tube containing 9ml of distilled water. It will be allowed to
stand for 5mnutes to obtain stock solution. 1ml of the stock solution will be
introduced into first test-tube to get 10 -1 dilution. 1ml of the content from 10 -1 tube
will be transferred into the second tube using sterile pipette to get 10 -2 dilution.
Subsequent step wise transfers will be carried out using a sterile pipette for each
transfer until dilution 10-6 s reached. Vigorous shaking to mx content of the test
tubes will be done before any transfer to next.
2.5 MEDIA PREPARATION
The media to be used for the cultivation and isolation of the fungi are Photo
Dextrose Agar (PDA) and yeast extract Agar which will be prepared according to
manufactures instructions / specifications.

2.5.1 PREPARATION OF PHOTO DEXTROSE AGAR (PDA)

39g of dehydrated powder of PDA will be added to 100mls of distilled water. This
will be heated to boiling and water bath in order to dissolve the medium
completely. The dissolved medium will be autoclaved at 15psi or 121 0c for 15ms
after which he conical flask will be taken out and cooled to a temperature of 45 0c,
then, chloramphenicol will be added to the medium and mixed properly before
dispensing into plates.
2.5.2 PREPARATION OF YEAST EXTRACTT (YEPDA)
65g of the yeast extract peptone dextone powder will be suspended n 100ml of
distilled water. This will be mixed thoroughly. The mixed medium will be heated
with frequent stirring to boiling for 1mnuteto completely dissolved the powder.
The medium ell be autoclaved at 1210c (or 15psi) for 15mnutes after which t will
be removed and allowed to cool to a temperature of 45 0c. The medium will be
dispensed into sterile petri-dish.

2.6 MICROBIOLOGICAL SAMPLE

2.6.1 INOCULATION OF SAMPLE
1ml of dilutions 10-2, 10-4 and 10-6 of the sample will be transfer into petri-dishes n
duplicates using pour plates method. The 10-2, 10-4 and 10-6 dilutions will be
selected, and an aliquot (0.1ml) of the dilutions will be inoculated n well-labelled
petri-dishes in duplicates using spread plates method. Each of the planting or
culturing will be done on both Photo dextone Agar and Yeast Extract Agar media.
The plates will be in incubated at 25-300c for 3-5 days and will be monitored for
fungal growth.

2.6.2 TOTAL FUNGAL COUNT (TFC)

The fungal colonies on the culture plates will be counted manually and with a
colony counter. The results will be expressed n CFU/ml.

2.6.3 PURE CULTURES OF THE FUNGAL ISOLATES.

A sterile wire loop will be used to pick discrete colonies on the culture plates and
will be incubated by streaking on freshly prepared PDA and YEA plates. The
incubated plates will then be incubated at temperature of 25 0c for 3-5days to obtain
pure fungal isolates.

2.6.4 CHARACTERIZATION AND IDENTIFICATION OF THE FUNGAL

ISOLATES.
The fungal isolates will be characterized based on pigmentation, typhae formation,
growth rate and spore type, also they will be microscopically examined under X40
microscope objective lens using lacto-phenol cotton blue stain. The results of their
cultural and morphological characteristics will be compared with those of the
standard fungal morphological atlas as described by Geo et al (2013).
2.7 SCREENING OF FUNGAL ISOLATES FOR CELLULASE ACTIVITY
The fungal isolates screened from the sample will be cultured on carboxy-methal
cellulose agar, for the investigation of cellulose hydrolyzing activity. The fungal
isolates that show a distinct zone around fungal out-growth on staining with Congo
Red indicates cellulose hydrolyzing activity of respective fungal stains.