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107 Verticillium Nees ex Link
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Members of this genus are often isolated from the environment. Verticillium wilt is a fungal disease of many
economically important plants, especially tomatoes. Verticillium dahliae, V. albo-atrum, V. longisporum,
V. nubilum, V. tricorpus and Musicilliium theobromae (formerly V. theobromae) and are the principal plant
pathogens. Verticillium has been reported as a rare agent of mycotic keratitis in humans.
RG-1 organism
Morphological description: Colonies are fast growing, suede-like to downy, white to pale yellow in colour,
becoming pinkish-brown, red, green, or yellow with a colourless, yellow, or reddish-brown reverse.
Conidiophores are usually well differentiated and erect, verticillately branched over most of their length,
bearing whorls of slender awl-shaped divergent phialides (Fig. 107.1). Conidia are hyaline or brightly
coloured, mostly one-celled, and are usually borne in slimy heads (glioconidia).
Molecular identification: ITS, ACT, TEF-1 GAPDH and tryptophan synthase genes have been used to
identify all recognised Verticillium species (Inderbitzin et al., 2013).
Key features: Verticillate branched conidiophores bearing whorls of awl-shaped, divergent phialides.
References: McGinnis (1980); Rippon (1988); Samson et al. (1995); Domsch et al. (2007); de Hoog et al.
(2015).
20 ˜m
Fig. 107.1. Verticillium spp. microscopy showing verticillately branched conidiophores bearing whorls of slender
awl-shaped divergent phialides and conidia.
DOI: 10.1079/9781800622340.0107
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis) 296
(E.C. Hansen) Kurtzman et al.
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Synonymy: Candida pelliculosa Redaelli.
Wickerhamomyces anomalus is widespread in nature. It has been reported from cases of candidaemia and
catheter-related infections in humans and has been isolated from soil, grains, fruit and warm-blooded
animals.
RG-1 organism
Culture: Colonies (SDA) white to cream coloured, smooth, glabrous, yeast-like.
Microscopy: Spherical to ellipsoidal budding blastoconidia, 2-4 x 2-6 μm. Pseudohyphae may be present.
Asci when present, contain one to four hat-shaped ascospores.
India ink preparation: Negative - no capsules present.
Dalmau plate culture: Spherical to ellipsoidal budding yeast cells and abundant pseudohyphae in most
strains.
Molecular identification: ITS sequencing is recommended for species identification.
MALDI-ToF MS: Able to accurately identify this species.
Germ tube formation: Negative.
Physiological tests: (+ Positive, - Negative, v Variable, w Weak, s Slow, n not done).
Fermentation:
Glucose + Sucrose + Lactose Galactose v Maltose v Trehalose -
Growth reactions:
Glucose + L-Sorbose – myo-Inositol –
Sucrose + L-Rhamnose – DL-Lactate +
Raffinose + D-Xylose v D-Gluconate v
Melibiose - L-Arabinose v 2-Keto-D-gluconate –
Galactose v D-Arabinose – D-Glucosamine –
Lactose – D-Ribose v N-Acetyl-D-glucosamine –
Trehalose + Glycerol + D-Glucuronate n
Maltose + Erythritol + Nitrate +
Melezitose + Ribitol v Urease –
Methyl-α-D-glucoside + Galactitol – 0.1% Cycloheximide –
Soluble starch + D-Mannitol + Growth at 30oC +
Cellobiose + D-Glucitol + Growth at 37oC v
Key features: Germ tube negative yeast and sugar assimilation pattern.
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
297
Antifungal susceptibility: (Table 108.1).
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Table 108.1. Wickerhamomyces anomalus (Diekema et al., 2009; and Australian national data); MIC μg/mL.
Antifungal N o. ≤0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 ≥64
AmB 44 3 14 21 5 1
FLU 44 3 10 24 7
ISAV 2 2
VORI 44 1 1 1 25 13 3
POSA 44 1 2 8 5 14 12 2
ITRA 4 1 3
ANID 18 15 2 1
MICA 18 6 10 2
5FC 4 2 2
References: Kurtzman (2011e).
DOI: 10.1079/9781800622340.0108
Descriptions of Medical Fungi 298
et al.) van der Walt & von Arx.
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Synonymy: Candida lipolytica (F.C. Harrison) Diddens & Lodder.
Yarrowia lipolytica is widespread in nature and is commonly found on substrates high in lipids. It is a rare
cause of candidaemia.
RG-1 organism
Culture: Colonies (SDA) white to cream coloured, smooth, glabrous, yeast-like.
Microscopy: Spherical, ellipsoidal to elongate budding blastoconidia, 3-5 x 3-15 μm.
India ink preparation: Negative - no capsules present.
Dalmau plate culture: Pseudohyphae and true hyphae are produced.
Germ tube formation: Negative.
Physiological tests: (+ Positive, – Negative, v Variable, w Weak, s Slow, n not done).
Fermentation: Absent.
Growth reactions;
Glucose + L-Sorbose v myo-Inositol –
Sucrose – L-Rhamnose – DL-Lactate +
Raffinose – D-Xylose – D-Gluconate v
Melibiose – L-Arabinose – 2-Keto-D-gluconate –
Galactose v D-Arabinose – D-Glucosamine –
Lactose – D-Ribose v N-Acetyl-D-glucosamine +
Trehalose – Glycerol + D-Glucuronate n
Maltose – Erythritol + Nitrate –
Melezitose – Ribitol v Urease –
Methyl-α-D-glucoside – Galactitol – 0.1% Cycloheximide –
Soluble starch – D-Mannitol + Growth at 35oC v
Cellobiose w,- D-Glucitol + Growth at 37oC –
Molecular identification: ITS sequencing recommended.
MALDI-ToF MS: Able to accurately identify this species.
Key features: Germ tube negative yeast and sugar assimilation pattern.
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
299
Antifungal susceptibility: (Table 109.1).
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Table 109.1. Yarrowia lipolytica limited data (Diekema et al., 2009; and Australian national data); MIC μg/mL.
Antifungal No. ≤0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 ≥64
AmB 20 1 1 8 5 4 1
FLU 20 1 1 10 6 2
VORI 20 1 8 8 2 1
POSA 20 2 3 10 4 1
ITRA 4 2 2
ANID 14 2 4 5 2 1
MICA 14 7 5 2
5FC 4 1 1 2
References: Kurtzman (2011f).
DOI: 10.1079/9781800622340.0109
Descriptions of Medical Fungi 300
110 Microscopy stains and
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techniques
110.1. KOH with Calcofluor white or Blankophor BA
For the direct microscopic examination of skin scrapings, hairs, nails and other clinical specimens for fungal
elements. This is a very rapid and sensitive method, however a fluorescence microscope fitted with filters to
give an excitation with ultraviolet light below 400 nm wavelength is required. Calcofluor white (M2R powder, Polysciences Inc.) or Blankophor BA (Indulor Leverkusen GmbH & Co. KG) are used as whitening
agents by the paper industry and selectively bind to cellulose and chitin. The dye fluoresces as it is exposed
to ultraviolet light (Fig. 110.1).
Prepare two solutions: (A) Dissolve 10 g of KOH in 90 mL distilled water and then add 10 mL of glycerol;
(B) Dissolve 0.5 g of Calcofluor white powder in 50 mL of distilled water by gentle heating. Store this solution in a light proof glass bottle; some precipitation will occur over time. Evans blue (0.02 g) may be added
to solution B to enhance contrast.
Method for making microscopic mounts: Mix one drop of each solution on the centre of a clean microscope
slide. Place the specimen in the solution and cover with a coverslip, squash the preparation with the butt of
the inoculation needle and then blot off the excess fluid. Gently heat the slide and examine microscopically
for the presence of fungal elements that fluoresce a chalk-white or brilliant apple-green colour, depending on
the filters used.
References: Hageage and Harrington (1984); Harrington and Hageage (2003).
(a) (b)
20 ˜m 20 ˜m
Fig. 110.1. Direct microscopy of fungal hyphae (a) KOH Parker Quink ink stain and (b) KOH with calcofluor white
showing fungal hyphae that fluoresce a chalk-white colour.
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
301
110.2. KOH with Chlorazol Black
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For the direct microscopic examination of skin scrapings, hairs, nails and other clinical specimens for fungal
elements. Note: Parker Quink permanent ink with solvex is no longer available, fluorescence microscopy
using Calcofluor white (or Blankophor BA) with 10% KOH is now the method of choice for direct microscopy of skin, hair and nail samples.
Method: Dissolve 10 g of potassium hydroxide (KOH) in 80 mL of distilled water and add 10 mL of glycerol
and 10 mL of Chlorazol Black E (Polysciences, Inc). The glycerol prevents crystallisation of the reagent and
prevents the specimen from drying out.
Method for making direct microscopy mounts: Mount a drop or small portion of the specimen in a drop of
KOH-based stain, cover with a coverslip, squash the preparation with the butt of the inoculation needle and
then blot off the excess fluid. Note: Specimens may take from 20 minutes to several hours for nail clippings
for the specimen to clear so that fungal elements can be detected. Larger pieces of specimen such as nail clippings or fibrous tissue, should be digested in 10% KOH overnight prior to mounting on the slide. Negative
specimens should be kept and re-examined the next day to avoid reporting false negative results due to
delayed clearance and staining of the specimen.
110.3. India ink mounts
For the direct microscopic examination of CSF or bronchoalveolar lavage for Cryptococcus neoformans and
C. gattii. India ink is a negative stain that provides a black, semi opaque background to enable the capsule
of the yeast to be clearly visible.
Method: Place a drop of the specimen on a glass slide, followed by a drop of India ink, mix well with
a loop, and cover with a coverslip. Note: The best quality brands are “Pelikan” or “Talens” India ink,
which is what is used by tattooists. The slide should be viewed immediately by bright field (not phase
contrast) microscopy. Beware of artefacts such as white blood cells that can look similar to cryptococcal cells.
110.4. Lactophenol cotton blue (LPCB)
For the staining and microscopic identification of fungi. This stain contains phenol which will kill the fungus
and lactic acid that acts as a clearing agent and helps preserve the fungal structures. Cotton blue is an aniline
dye that stains the chitin in the fungal cell walls, which adds colour to the fungal preparation thereby enhancing and contrasting the structures. The stain can be purchased commercially, or it can be prepared in the laboratory, although this requires two days.
Method: This stain is prepared over two days.
On the first day, dissolve 0.05 g of Cotton blue (Aniline blue) in 20 mL distilled water. Leave overnight to
eliminate insoluble dye.
On the second day, wearing gloves add 20 g of phenol crystals (C
(CH
CHOH COOH) in a glass beaker and place on a magnetic stirrer until the phenol is dissolved. Then
3
) to 20 mL of lactic acid
6H5O4
add 40 mL of glycerol.
Filter the Cotton blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and
store at room temperature.
110.5. Microscopic mounts and squash preparations
Using sterile technique, remove a small portion of the colony with an inoculation needle and mount in a drop
of lactophenol cotton blue on a clean microscope slide. Cover with a coverslip, squash the preparation with
the butt of the inoculation needle and then blot off the excess fluid. Note: Cellotape flag preparations or slide
culture preparations are superior methods for the examination of fungal structures.
Descriptions of Medical Fungi 302
110.6. Cellotape flag preparations
tape, near stick
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An excellent technique for the rapid mounting of sporulating fungi because it keeps more of the reproductive
structures intact (Fig. 110.2).
Square of cellotape
Stick
Drop of lactophenol cotton
blue on slide
Touch sticky side to colony
Ethanol
Place tape sticky side up on stain
Add a drop of 95% ethanol to the
Remove stick. Add another
drop of stain to tape. Place
coverslip over tape.
Fig. 110.2. Diagrammatic instruction for making a cellotape mount.
Method: Using clear 2 cm wide cellotape and a wooden applicator stick (orange stick) make a small cellotape
flag (2 x 2 cm). Gently press the sticky side of the flag onto the surface of the culture. Remove and apply a
drop of 95% alcohol to the sticky side of the flag, this acts as a wetting agent and dissolves the adhesive glue
holding the flag to the applicator stick. Place the flag onto a small drop of lactophenol cotton blue on a clean
glass slide (sticky side up or down, doesn’t matter), remove the applicator stick and add another drop of stain.
Then cover with a coverslip and gently press, mopping up any excess stain. Note: Best viewed within 2 hours;
flag mounts only last for a few days before the phenol/lactic acid destroys the cellotape.
110.7. Slide culture preparations
To accurately identify many fungi, it is essential to observe the precise arrangement of the conidiophores and
the way in which the conidia are produced (conidial ontogeny).
Riddell’s (1950) simple method of slide culturing permits fungi to be studied virtually in situ with as little
disturbance as possible (Fig. 110.3). A simple modification of this method using a single agar plate is
described below (Fig. 110.4).
Descriptions of Medical Fungi 303
(c)
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(d)
(b)
(a)
Fig. 110.3. Riddell’s (1950) slide culturing method required using an agar block of media transferred to a glass slide
and placed in a moist chamber (a) petri dish, (b) bent glass rod to support the slide above some water, (c) agar
square cut out and inoculated on 4 sides, (d) cover slip.
(c)
(a)
Fig. 110.4. A simple slide culture method using a single agar plate (a) square agar block is cut out and flipped up onto
the agar surface, (b) all 4 sides are inoculated with the mould and (c) a coverslip is placed on top.
(b)
Method: One plate potato dextrose is recommended; however, some fastidious fungi may require less
nutrient-rich media to induce sporulation like cornmeal agar or Czapek dox agar.
Using a sterile blade cut out an agar block (7 x 7 mm) small enough to fit under a coverslip. Flip the block
up onto the surface of the agar plate. Inoculate the four sides of the agar block with spores or mycelial fragments of the fungus. Place a flamed coverslip centrally upon the agar block. Incubate the plate at 26
o
C until
growth and sporulation have occurred.
Remove the coverslip from the agar block. Apply a drop of 95% alcohol as a wetting agent. Gently lower
the coverslip onto a small drop of lactophenol cotton blue on a clean glass slide. The slide can be left overnight to dry and later sealed with nail polish. When sealing with nail polish use a coat of clear polish followed
by one coat of coloured polish.
DOI: 10.1079/9781800622340.0110
Descriptions of Medical Fungi 304
111.1. Bird seed agar
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For the selective isolation of Cryptococcus neoformans and C. gattii.
Ingredients: Guizotia abyssinica (niger seed) 50 g; Glucose 1 g; KH
(potassium dihydrogen orthophos-
2PO4
phate) 1 g; Creatinine 1 g; Bacto-agar (BD) 15 g; Distilled water 1000 mL.
Additives: To each 500 mL bottle add Penicillin G (20 units/mL) 1 mL; Gentamicin (40 mg/mL) 1 mL.
Method: Grind seeds of Guizotia abyssinica as finely as possible with an electric grinder and add to 1000 mL
distilled water in a stainless-steel jug. Boil for 30 minutes, pass through filter paper and adjust volume to
1000 mL. Add remaining ingredients except Bacto-agar to filtrate and dissolve. Cool to room temperature
and pH to 5.5. Dispense into 500 mL bottles. Add 7.5 g Bacto-agar to each 500 mL reagent bottle. Autoclave
o
at 121
C for 20 minutes. Cool to 48oC and add 0.5 mL Penicillin G and 0.5 mL Gentamicin to each 500 mL
bottles of agar. Mix gently and pour into 90 mm plastic petri dishes.
111.2. Bromocresol purple milk solids glucose agar (BCP)
For the differentiation of Trichophyton species (Fischer and Kane 1971; Summerbell et al.,1988).
Solution A: Distilled water 1000 mL; skim milk powder (Carnation brand is best) 80 g; bromcresol
(or bromocresol) purple (1.6% solution in ethanol) 2 mL. Dissolve in 2 litre flask and autoclave 121
o
C for
10 minutes.
Solution B: Glucose 40 g; distilled water 200 mL. Dissolve and autoclave at 121
o
C for 10 minutes.
Solution C: Bacto-agar 30 g; distilled water 800 mL. Soak for 15 minutes in 3 litre flask and autoclave at
o
121
C for 10 minutes.
Final mixture: To make media add solution A and B to solution C. Adjust final pH to 6.6. Aseptically
dispense for slopes.
111.3. Creatinine dextrose bromothymol blue thymine (CDBT) agar
For differentiation of C. neoformans serotypes A and D (Irokanulo et al., 1994).
Solution A: Creatinine 1 g; dextrose 0.5 g; KH
distilled water 980 mL. Dissolve ingredients in small beaker and adjust pH to 5.6. Store at 4
Solution B (aqueous bromothymol blue): Bromothymol blue 0.4 g; 0.01N NaOH 64 mL; distilled water
36 mL. Dissolve the bromothymol blue in the NaOH and add to the water.
Final mixture: To prepare a litre of medium, mix 980 mL of solution A with 20 mL of solution B. Add 20 g
of Bacto-agar and autoclave to 121
o
C for 15 minutes, cool to 48oC and dispense as plates.
1 g; MgSO4 7H2O (epsomite) 0.5 g; thymine 0.1 g;
2PO4
o
C.
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
305
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