Lab Safety and diagnosis of Fungal Infection

What are some safety instruments do we use?

• Class II biosafety cabinet

• Enclosed electric incinerator

• Use screw-top tubes to prevent aerosols/airborne spores

  • trrrryyyyyyy to avoid petri dishes :(

What are the common specimens in fungi infections?

• Respiratory secretion, hair, skin, nail, tissues, blood, bone marrow, CSF

What do we use to inhibit bacterial growth?

Antimicrobial containing media since not all sites are sterile

How do we collect and check hair samples?

• Use a wood lamp and if it glows? shit is infected bro LOL

• Sterile forceps should be used to pull affected hair to place directly into a sterile petri dish then inoculated onto fungal medium and incubate at 22 to 30C

How do we collect and check skin and nails?

• Clean the area with 70% alcohol before sampling

• Skin —> Outer edge is scraped

• Nail —> Cut or removed

• Mount via KOH wet mount onto the sample (everything but fungal will dissolve)

• Sample will inoculate directly onto agar (but you need a new sample cause you cant shove KOH sample onto a plate cause yknow— its dead lmfao)

How do we collect and check blood / bone marrow samples?

• Lysis centrifugation system

  • Release organisms from cells

  • and then check what popped out :)

How do we collect and check CSF?

• Concentrate via centrifugation before inoculation

• Always test with India ink to test for C. neoformans (Halo around the cell)

• Plate directly

What is a quick test for C. neoformans?

India Ink in CSF sample —> Will produce a halo effect

How do we collect and check for abscess, wound or tissues?

• Mince and grind tissues

• Plate directly or perform other tests

How do we collect and check respiratory specimens?

Plated directly or digested with mucolytic prior to plating

How do we collect and check urogenital or fecal specimens?

• Culture

• If urine —> USE THE FIRST MORNING URINE TO CONCENTRATE IT

How do you prepare KOH?

• 10-20% of solution of KOH

• Equal parts sample to KOH

• Cover slipped and heated gentle then cooled for 15 minutes

What is the purpose of KOH in a KOH stain?

Dissolve the keratin to visualize dermatophytes

What is the mode of action of KOH w/ calcofluor white?

Dye binds to cellulose or polysaccharides present in chitin that appears apple-green or blue-white in UV light

How does India ink help will direct examination?

Visualize capsules of yeast or bacteria

What are different types of tissues stains?

1) Periodic acid-schiff (PAS)

2) Gomori methenamine-silver (GMS)

3) Hematoxylin and eosin (H&E)

4) Giemsa

5) Fontana-Masson

What are the 3 types of Media used?

1) Primary plating media

2) Media to induce sporulation

3) Biochem media for ID

What are some of the culture media?

1) Sabouraud dextrose agar (SDA)

2) Potato dextrose agar (PDA)

3) Brain-Heart infusion (BHI) agar w/ blood and antimicrobials

4) plates or tube media

What usually happens when you have nutrient poor agar?

Will induce sporation

What are some characteristics of sabouraud dextrose agar (SDA)

• Non-selective, general purpose medium

• Nutritionally poor medium but grows most fungi

• Acidic pH 5.6

What usually happens when you have a nutrient rich agar?

Will induce yeast formation

What is the most common temperature to grow fungi at?

Room temperature or 30C

What is the temperature to grow dimorphic?

37C

How many weeks do you keep the fungi and how many times do you examine them?

Keep them for 4-6 weeks and examine twice a week for growth

What are some macroscopic fungi ID categories?

1) Colony growth time

2) Color

3) Texture

4) Pigment on reverse of colony

What are some microscopic fungi ID categories?

1) Septate versus sparsely septate hyphae

2) Hyaline or dematiaceous hyphae

3) Fruiting structures

4) Types, size, shape and arrangement of conidia

How do you do a tease mount?

• Place drop of lactophenol cotton blue (LPCB) on a glass slide

• Grab a sample and slap it on

• Cover with a coverslip and examine at 100x or 400x

What is the disadvantage of tease mount?

Some of the overall morphology maybe be lost in the teasing process

How do you do a cellophane tape preparation?

• Press cellophane tape gently but firmly to surface of the colony

• Press one side of tape to slide w/ drop of LPCB

• Stretch tape across slide, lowering it into the stain

• Pull taught and affix another side of tape, avoiding air bubbles

How to do slide culture?

• Agar block is placed on agar plate —> Inoculated w/ fungal culture

• Sterile coverslip placed on top and then the whole plate is incubated

• lactophenol cotton blue is added to slide

What is LPCB?

Lactophenol cotton Blue

What are miscellaneous test for molds?

• Hair perforation test

• Urease test

• Thiamine requirements

• Trichophyton agars

• Growth on rice grains

What are miscellaneous test for yeast?

• Germ tube productions

• Carbohydrate assimilation

• Chromogenic substrates

• Cornmeal agar

• Potassium nitrate assimilation

• Temperature studies

• Urease

• (1-3) beta-D-Glucan detection

• Galactomannan

• T2MR

• Cryptococcal antigen

What is advantage of tease mount?

1) Stains

2) Preserves

3) Kills the organisms

What are some species you can find on cornmeal agar?

• Blastoconidia

• Chlamydoconidia

• Pseudohyphase

• Arthroconidia

What are some immunodiagnosis of fungal infections?

• Skin test reactivity to fungal antigens

• Antibody detection assays

What is the main agent of antifungal agents?

Amphotericin B

What are the CLSI standards for yeast testing?

M27-A3

What are the CLSI standards for mold testing?

M38-A2

What are the CLSI standards for disk diffusion testing for yeasts?

M44-A

What color does potassium nitrate assimilation turn when its positive?

Blue

What color does potassium nitrate assimilation turn if its negative?

Medium Yellow

What is T2MR used ffor?

Diagnosis of invasive candidiasis

What are some direct examinations of specimens?

1) KOH Prep

2) KOH with calcofluor white

3) India Ink

4) Tissue stain