STUDENT INDUSTRIAL WORK

EXPERIENCE SCHEME
UNDERTAKEN AT BIOLOGICAL
SCIENCES LABORATORY,
AFE BABALOLA UNIVERSITY
ADO- EKITI
OGUNTUASE OLUWASEUN MERCY
MAT NO: 168867175
 OUTLINE OF WORK DONE

 Microbial isolation and purification

 Media preparation for isolation of organisms'
 Preparation and extraction of microbial
metabolites
 Preparation of mineral medium for
biosurfactant production.
 Antimicrobial susceptibility test
 Post and pre
Microbial Isolation
SERIAL DILLUTION PROCESS
PREPARATION OF MINERAL MEDIUM FOR BIOSURFACTANT
PRODUCTION.
Detection of biosurfactant production by Aspergillus niger
 Mineral medium that consisted of:

was prepared in 200ml quantity in 250ml conical flask and was sterilized
 Sterile medium was inoculated with 1ml broth culture of the fungi and incubated at
25°c .
 10ml of sample was aseptically withdrawn from flask and used for the pour plating
method and biosurfactant dilution.
 Biosurfactant detection was carried out using the following techniques:
Drop collapse Test
Oil displacement
Surface tension and Emulsification index (E 24) measurements
• Drop collapse Test: In the case of drop collapse test, 5ul of cell-free broth
culture of the test organism was placed on the lid of microtitre plate and allowed to
equilibrate before checking for biosurfactant potential. A positive test was regarded
recorded when there was a collapse of drop of a supernatant.
• Oil displacement: Oil displacement and drop collapse tests were
carried out as reported by phulpoto et al,(2020). For oil displacement test,
on the surface of a clean Petri dish that contained 30mL of distilled water,
100ul of vegetable oil was placed before adding 10ul of cell-free broth
culture of the test organisms. The presence of zone of displacement was
regarded a positive test.
• Surface Tension and Emulsification index measurement:
The surface tension measurement of cell-free supernatants was determined
using a Tensiometer. Prior to measurements, medium was centrifuged at
5000 rpm for 15min. Supernatant was separated from pellets and the
required measurement taken.

To determine E24, 2ml of vegetable oil was added to equal volume of cell free
supernatant. The mixture was vortexed for 2 min and allowed to stand for
24h. The E24 was taken as percentage of height (mm) of layer that is
emulsified divided by the total height (mm) of the liquid column.
 Media preparation for isolation

• Preparation of media for isolation of organisms

To prepare agar slants-
Nutrient agar powder was weighed into a conical flask, following the
manufacturer’s instruction& dissolved in the required volume of distilled water.
The solution was allowed to dissolve in a water bath heater.
After dissolving, 10ml was dispensed in McCartney bottles and autoclave. After
autoclaving, the bottles containing the nutrient agar were slanted and allowed to
solidify

prepared Agar Slant

 Preparation and extraction of microbial
metabolites
Microbial metabolites
 Metabolites re the intermediate and end products of cellular
regulatory process.
 Microbial metabolites are products of metabolisms produced by the
action microbes which enhances growth.
Bacterial isolation was grown in mineral medium that consist of:
5g of glucose
3.8g of yeast extract
5g of peptone
1g of Ammonium sulfate
1ml of metal solution
0.2g of magnesium sulfate.
• Medium was sterilized, allowed to cool and 1ml of pure broth
culture was used for inoculation of respective flask containing
media.
• Growth was determined on a daily basis by pour plating techniques.
• After 72hours of incubation at which growth was observed to be
stationary, medium was centrifuge to separate cells from
supernatant.
• Cell free supernatant was extracted with acetone at ratio 1:1and
incubated at 4°c for 12 hours.
• After incubation the acetone was separated from the solution by
placing the solution in a water bath heater at 70°c.
• At the end of evaporation, brownish pellet was recovered, otherwise
known as the METABOLITES.
• Metabolites was used for anti microbial potential and characterized
using gas chromatography.
 Antimicrobial susceptibility testing
For the test, nutrient agar was prepared in 100ml conical flask
and allowed to cool a portion of broth culture of bacteria
(Salmonella typhi, Pseudomonas aeruginosa, staphylococcus
aureus) where added to there respective flask.
The flask were then swirled to archive homogenization. Each of
the agar was then poured into well labeled plate and allowed to
solidify.
Using a sterile cork borer, four holes were bored into the agar
plate containing the microorganisms to accommodate the known
concentration of the metabolites solution. To the bored holes, few
drops of metabolites were added using micro pipette the plate were
left for the metabolites to diffuse into the agar after which they were
incubated for 37° for 24 hours zone of inhibition were measured in
millimeters.
 ZONE OF INHIBITION MEASUREMENTS
ORGANISMS USED SAMPLE A SAMPLE B SAMPLE C SAMPLE D

Salmonella typhi 8mm 13mm 13mm 10mm

Pseudomonas aeruginosa 7mm 11mm 14mm 11mm

Staphylococcus aureus 8mm 14mm 12mm 9mm