Question

Inoculation and Sterilization Experimental Set Up for Microbiology: I was wondering how I can do an experiment inoculating different age classes of a plant species with fungi. For example seeds can be sterilized and then inoculated and put into a sterile growth medium, but what if I wanted to do an older plant? Would that plant need to be grown in a sterilized environment up until inoculation to the age I want? Would it need to be sterilized again for extra measure?

Answer 1

It is possible to inoculate fungi from plant parts at any stage you want, though it is grown in a non sterilized environment.

Fungal pathogens are able to infect various plant parts such as roots, stems, leaves, flowers and fruits, with characteristic visible symptoms like spots, blights,wilts, rots etc.

After washing the tissues thoroughly in sterile water, fungi are isolated from plant tissues exhibiting clear symptoms. The infected tissues along with adjacent small unaffected tissue are cut into small pieces (2–5 mm squares) .
By using flame-sterilized forceps, they are transferred to sterile petridishes containing 0.1% mercuric chloride solution used for surface sterilization of plant tissues.

The plant parts were transferred to PDA (Potato Dextrose Agar),which is the selective media for fungi.plates and Incubated for 5-7 days for the complete growth of fungi.

Potato Dextrose Agar (PDA) is a general purpose medium for yeasts and molds that can be supplemented with acid or antibiotics to inhibit bacterial growth.

Dextrose is a carbohydrate source which serves as a growth stimulant, and potato infusion that provides a nutrient base for luxuriant growth of most fungi.

The resulted fungi were purified using the hyphal tips technique on Rose Bengal medium and then subculture of each isolated fungus on slant medium for future studies. The fungi were identified according to cultural characteristics

Rose Bengal Agar is a selective medium to detect and enumerate yeasts and moulds. The use of media with an acidic pH that selectively inhibits the growth of bacteria and thereby promotes the growth of fungi .Addition of Chloramphenicol will increase its selectivity for fungi.

To isolate fungi from the internal tissue,it is washed in sterile water and then swabbed with cotton wool dipped into 80% ethanol, followed by exposure to an alcohol flame for a few seconds.

The outer layer of tissues are quickly removed by a flame sterilized scalpel. Small pieces from the central core of tissues in the area of infections are removed by a sterilized scalpel or scissors.

Sterilized by dipped into 90% alcohol then flaming for a few seconds. The tissues thus sterilizes, are transferred to PDA plates and incubated for 5-7 days.