Fungi Basic laboratory methods

1. Chapter 7
Basic Laboratory Methods

2. 7.1. Culture technique and media
• Cultivation of microorganisms requires sterile techniques.
by employing aseptic techniques.
• Sterilization implies the complete destruction of all unwanted
microorganisms including spores.
 accomplished by the combined usage of most heat,
UV light, chemicals, radiation, and filtration.

3. Cont.
• Microorganisms- grown in either liquid, solid or semi-
solid media forms.
• Solid media are useful mainly for observations of
characteristic colonies, for isolation of pure cultures and
for short-term maintenance of cultures.
• Cultures usually grow well on agar slopes in test-tubes
(also called agar slants) or culture bottles.
• e.g. medical fungi grow well on Sabouraud’s medium.

4. Cont.
• In the laboratory, fungi normally grow on chemically
undefined medium.
• Commonly used media- Potato Dextrose Agar, Cornmeal
Agar, Czapek Agar (CZ) and Malt Extract agar (MA)
media
• Different fungi prefers different media formulation
• Most soil isolated fungi thrive well on Cornmeal Agar
(CMA).

5. Isolation of fungal strains in pure culture
• In natural habitats microorganisms usually grow in complex,
mixed populations containing several species.
• Common approaches to prepare pure cultures are: pour
plate, spread plate, and streak plate.
oAfter plates are prepared, they should be incubated.

6. Preservation and Storage of Cultures:
• Cultures are usually maintained by several methods like
sub culturing, under liquid paraffin, by lyophilization and
storage under liquid nitrogen,etc.
• Refrigeration: Most of the micro-fungi survive at 5-10oC.
• Subculturing: serial transfer from staled to fresh solid or
liquid media and store in suitable conditions.
• Lyophilization: is effected by suspending the metabolic
activities of fungi at -70oC.
It expensive but needs a very little storing space and cultures
remain viable for many years.
• Liquid Nitrogen: cultures are preserved in liquid nitrogen
(-196oC).
• Mineral Oil: cultures under oil for many years give very
good results.
advantage of putting under the oil is that the mites could not
penetrate the layer of the oil.

7. 7.2. Microscopy techniques and stains
• The direct microscopic examination of specimens is an important first step
for several reasons.
fungi take days to weeks to grow on agar media,
Provide rapid report to the physician, which may allow early treatment to begin.
• A number of different stains used to detect and characterize fungi from
clinical material.
10%KOH is used for direct examination of specimens, but other methods can be used like:
 (smear in iodine, Gram stain, Giemsa stain or acid fast stain, silver stain, India ink, etc.)

8. 7.3. Specimen collection and Processing and Mushroom Cultivation
• Mushrooms are good cash crops; due easy to grow and are rich in protein,
vitamins and minerals.
Time between spawning and harvesting can be as short as three weeks.
after the cultivation, the substrate used as a good soil fertilizer.
• In the practice of edible mushroom cultivation no use is made of spores.
Their small size makes them difficult to handle and their genetic characteristics may differ
from those of their parent.
 it takes some time for mushroom spores to germinate, whereas other fungi such as green
moulds germinate and spread much faster.
The desired mushroom must be able to colonize the substrate before other fungi or bacteria
do so. :-To achieve this, pre-grown mycelium spawn is used.