E3 LAB: BASIC TECHNIQUES ON FUNGAL ISOLATION

Question 1

Media and Reagents

Answer 1

• Potato Dextrose Agar (PDA)
• Agar
• Lactophenol cotton blue
• Sterile distilled water
• Plated media (PDA and Water Agar)

Question 2

Equipment and Materials

Answer 2

• 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

Question 3

Guide template for Agar block

Answer 3

5 mm x 5 mm

Question 4

Specimen

Answer 4

• Soil
• fungal sample in PDA (Aspergillus niger ATCC 16404)

Question 5

Method 1

Answer 5

Agar Blocking

Question 6

Agar Blocking

Scalpel needle must cut down the medium touching the?

Answer 6

bottom part of plate

Question 7

Agar Blocking

Lift the agar block and transfer it on a slide, placed inside a?

Answer 7

petri dish

Question 8

Agar Blocking may be used to?

Answer 8

purify fungi in a mixture of microbial elements

Question 9

Agar Blocking

Small block is usually cut from the?

Answer 9

outermost part of fungal growth

Question 10

T/F: Agar block may also be used in slide-agar culture technique

Answer 10

True

Question 11

Other term for slide culture technique

Answer 11

Modified Riddell Technique

Question 12

Slide Culture Technique

Inoculate small fragment of sample to?

Answer 12

four edges of the block

Question 13

Slide Culture Technique

Incubate at what temp, where, and for how many days?

Answer 13

room temperature (22-25C), locker for 10 days

Question 14

Slide Culture Technique

Once sporulation has developed, transfer cover slip on slide with a?

Answer 14

drop of Lactopheral cotton blue

Question 15

Spore Touch Isolation Technique

Answer 15

Pasteur pipet tip elongation

Question 16

Pasteur pipet tip elongation

Place forcep at?

Answer 16

tip of pipet

Question 17

Pasteur pipet tip elongation

Heat what part of the pipette?

Answer 17

middle part of thin portion of pipette

Question 18

Pasteur pipet tip elongation

Once it softens, you must?

Answer 18

pull apart the end

Question 19

Pasteur pipet tip elongation

When the tip cools, make sure it is?

Answer 19

straight

HOMOKOJIC

Question 20

Spore isolation technique allows?

Answer 20

purification of fungal isolate in a given medium with mixed microbial growth

Question 21

Spore Isolation Technique

This can be examined microscopically in a dissecting microscope.

Answer 21

Aerial spores

Question 22

Soil sprinkling technique

Scoop soil sample and sprinkle using?

Answer 22

long handle square head spoon

Question 23

Soil sprinkling technique

Incubate at?

Answer 23

room temperature

Question 24

Soil sprinkling technique

Observe growth after?

Answer 24

1 week

Question 25

Brush paint technique Preparation of soil suspension

Answer 25

- 1g of soil in 50mL beaker - 3mL distilled water - mix using a stirring rod

Question 26

Brush paint technique Dip what paint brush in suspension?

Answer 26

paint brush #6

Question 27

Brush paint technique Spread ________ layer on plated media

Answer 27

3-single layer

Question 28

This decreases number of microorganisms and allow distinct microbial growth.

Answer 28

Serial dilution

Question 29

Serial dilution favors what organisms?

Answer 29

rapidly growing

Question 30

Examples of rapidly growing organisms

Answer 30

Fungi Imperfecti - Penicillium - Aspergillus sp.

Question 31

Serial dilution Preparation of soil suspesion

Answer 31

- 1g of soil - 100mL of distilled water - shake for 15 minutes

Question 32

Serial dilution Soil suspension will give a?

Answer 32

10-2 dilution

Question 33

Serial dilution Set up what tubes?

Answer 33

Loeffler's test tubes

Question 34

Serial dilution Add how many mL of distilled water?

Answer 34

9.0mL

Question 35

Serial dilution Transfer how many mL of the soil supernatant fluid of last dilution, and where?

Answer 35

0.1mL, center of agar medium

Question 36

Serial dilution Incubate at what temperature for how many days?

Answer 36

37C for 3-5 days

Question 37

Serial dilution Further dilution allows isolation of?

Answer 37

Actinomycetes

Question 38

Aerial spore isolation

Answer 38

Cellophane Technique

Question 39

Cellophane Technique First step

Answer 39

Place drop of Lactophenol Cotton Blue on a slide

Question 40

Cellophane Technique Extend a?

Answer 40

2 inch adhesive tape

Question 41

Cellophane Technique Touch adhesive side onto the?

Answer 41

upper surface of old fungal culture

Question 42

Cellophane Technique What are expected to stick to the tape?

Answer 42

Aerial spores and hyphal elements

Question 43

Cellophane Technique To secure tape in the slide, you must flip?

Answer 43

both ends of the tape

Question 44

For keratinophiles

Answer 44

Hair bait technique

Question 45

Hair bait technique _____ a sterile petri dish with soil specimens.

Answer 45

Half-fill

Question 46

Hair bait technique Moisten soil specimen with?

Answer 46

15mL of sterile distilled water

Question 47

Hair bait technique Place short tufts of what on soil surface?

Answer 47

autoclaved hair samples