E3 LAB: BASIC TECHNIQUES ON FUNGAL ISOLATION Flashcards
Media and Reagents
- Potato Dextrose Agar (PDA)
- Agar
- Lactophenol cotton blue
- Sterile distilled water
- Plated media (PDA and Water Agar)
Equipment and Materials
- Autoclave
- Dissecting microscope
- Compound microscope
- Sterile Petri dish
- Sterile Test Tubes (Loeffler’s)
- Sterile slides and coverslips
- Alcohol lamp
- Scalpel, blade 15A
- Agar block
- Filter paper
- Glass pasteur pipette
- Erlenmeyer flask
- Micropipettor
- Bent glass rod
Guide template for Agar block
5 mm x 5 mm
Specimen
- Soil
- fungal sample in PDA (Aspergillus niger ATCC 16404)
Method 1
Agar Blocking
Agar Blocking
Scalpel needle must cut down the medium touching the?
bottom part of plate
Agar Blocking
Lift the agar block and transfer it on a slide, placed inside a?
petri dish
Agar Blocking may be used to?
purify fungi in a mixture of microbial elements
Agar Blocking
Small block is usually cut from the?
outermost part of fungal growth
T/F: Agar block may also be used in slide-agar culture technique
True
Other term for slide culture technique
Modified Riddell Technique
Slide Culture Technique
Inoculate small fragment of sample to?
four edges of the block
Slide Culture Technique
Incubate at what temp, where, and for how many days?
room temperature (22-25C), locker for 10 days
Slide Culture Technique
Once sporulation has developed, transfer cover slip on slide with a?
drop of Lactopheral cotton blue
Spore Touch Isolation Technique
Pasteur pipet tip elongation
Pasteur pipet tip elongation
Place forcep at?
tip of pipet
Pasteur pipet tip elongation
Heat what part of the pipette?
middle part of thin portion of pipette
Pasteur pipet tip elongation
Once it softens, you must?
pull apart the end
Pasteur pipet tip elongation
When the tip cools, make sure it is?
straight
HOMOKOJIC
Spore isolation technique allows?
purification of fungal isolate in a given medium with mixed microbial growth
Spore Isolation Technique
This can be examined microscopically in a dissecting microscope.
Aerial spores
Soil sprinkling technique
Scoop soil sample and sprinkle using?
long handle square head spoon
Soil sprinkling technique
Incubate at?
room temperature
Soil sprinkling technique
Observe growth after?
1 week
Brush paint technique
Preparation of soil suspension
- 1g of soil in 50mL beaker
- 3mL distilled water
- mix using a stirring rod
Brush paint technique
Dip what paint brush in suspension?
paint brush #6
Brush paint technique
Spread ________ layer on plated media
3-single layer
This decreases number of microorganisms and allow distinct microbial growth.
Serial dilution
Serial dilution favors what organisms?
rapidly growing
Examples of rapidly growing organisms
Fungi Imperfecti
- Penicillium
- Aspergillus sp.
Serial dilution
Preparation of soil suspesion
- 1g of soil
- 100mL of distilled water
- shake for 15 minutes
Serial dilution
Soil suspension will give a?
10-2 dilution
Serial dilution
Set up what tubes?
Loeffler’s test tubes
Serial dilution
Add how many mL of distilled water?
9.0mL
Serial dilution
Transfer how many mL of the soil supernatant fluid of last dilution, and where?
0.1mL, center of agar medium
Serial dilution
Incubate at what temperature for how many days?
37C for 3-5 days
Serial dilution
Further dilution allows isolation of?
Actinomycetes
Aerial spore isolation
Cellophane Technique
Cellophane Technique
First step
Place drop of Lactophenol Cotton Blue on a slide
Cellophane Technique
Extend a?
2 inch adhesive tape
Cellophane Technique
Touch adhesive side onto the?
upper surface of old fungal culture
Cellophane Technique
What are expected to stick to the tape?
Aerial spores and hyphal elements
Cellophane Technique
To secure tape in the slide, you must flip?
both ends of the tape
For keratinophiles
Hair bait technique
Hair bait technique
_____ a sterile petri dish with soil specimens.
Half-fill
Hair bait technique
Moisten soil specimen with?
15mL of sterile distilled water
Hair bait technique
Place short tufts of what on soil surface?
autoclaved hair samples
