Staining Techniques

and Biochemical Methods 19

for the Identification of Fungi

Jeyabalan Sangeetha and Devarajan Thangadurai

Abstract
In the past, conventional identification of fungi relied on the combination of
morphological and physiological properties. In recent years, morphological
studies, supplemented with staining techniques and biochemical methods,
still play an important role in the overall identification of fungi in the molec-
ular era. In most instances, these tools are widely used to determine the
correct identity of yeasts and molds at the genus and species levels.

Keywords
Identification • Molds • Yeasts • Staining techniques • Biochemical
methods

classification are also useful in fungal

Introduction
In general, fungal identification requires greater
visual acuity than bacteria. Unlike other impor-
tant microorganisms such as bacteria and viruses,
the identification of fungi heavily relies on mor-
phological criteria. The characteristics of fungal
structure are identified by observing colonial
growth both macroscopically and microscopi-
cally. These morphological features and other
classical methods that are routinely used in
J. Sangeetha (*)
Department of Zoology, Karnataka University,
580003, Dharwad, Karnataka, India
e-mail: drsangeethajayabalan@gmail.com
D. Thangadurai
Department of Botany, Karnataka University,
580003, Dharwad, Karnataka, India
identification. The correct identity of fungal taxa
is of great practical relevance in clinical mycology,
plant pathology, biodeterioration, and biotech-
nology. A recent review illustrates the inability to
identify fungi at the species, or even at the genus,
level in many cases [1]. Many species of fungi,
ascomycetes, basidiomycetes, and zygomycetes
in particular, have different microscopic and
macroscopic characteristics in each stage of their
life cycle. Moreover, they are synonymous to
each other with many names used to describe the
same organism [2].
The ever-increasing number of yeasts and
molds that are frequently impossible to identify
using morphological criteria due to lack of sporu-
lation has driven the need for the design and
development of rapid and robust biochemical and
molecular identification tools [3–7]. In recent
years, the ability to accurately and reproducibly

V.K. Gupta et al. (eds.), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology, 237
Fungal Biology, DOI 10.1007/978-1-4614-2356-0_19, © Springer Science+Business Media, LLC 2013
238
identify fungi has been greatly enhanced through
automated biochemical methods and comparative
DNA analysis [8–14]. The procedures for the
identification of yeasts and molds are different.
Identification of molds to the species level has
been difficult, because of the amount of experi-
ence required to accurately identify these
filamentous fungi. In many cases, molds are
identified based upon colony and microscopic
characteristics. The microscopic structures and
morphological features like type, size, shape, and
arrangement of spores, and size, color, and septa-
tion of hyphae usually provide definitive
identification for molds. However, species-level
mold identification has not been held to the same
standards as bacterial and yeast identification. In
yeast, morphological criteria and biochemical
tests are generally used to determine genus and
species level, respectively.
Direct microscopic examination of the fungal
specimen provides a clear view and valuable
information about the fungal structure. Yeasts
and molds can be identified based on a combina-
tion of macroscopic, microscopic, and biochemi-
cal analysis. Macroscopic evaluation of fungi
provides information about the probable region
of the presence of fungi in a specimen, whereas
microscopic examination reveals the important
features like type and color of hyphae, conidia,
septae, spores, and also the concentration of
fungi. A definitive fungal stain is an important
tool that is needed to begin the identification of
fungal specimens. Detection of fungi using direct
microscopy with various stains is quick, simple,
and can be optimized through the use of ready-to-
use staining solutions. The selection of staining
method is primarily based on the sample used. A
number of stains are routinely available to visual-
ize fungi; some of these are special fungal stains
and others are more general in use [15]. Achieving
a successful identification comes from the use of
appropriate stains and further microscopic exam-
ination. The major growth forms of the fungi that
help in identification after staining are the yeast
cells, hyphae, pseudohyphae, arthroconidia, chla-
mydoconidia, and endosporulating spherules [16,
17]. On the other hand, visualization of fungi in
smear often helps in identification; in some cir-
J. Sangeetha and D. Thangadurai
cumstances, it is essential to establish further
study on fungi. Even more, the evaluation of
staining is useful when multiple organisms are
cultured [18].
Additionally, fungal identification requires
biochemical tests that will distinguish genera
among families and species among genera.
Further, strains within a single species are usually
distinguished by genetic or immunological crite-
ria. The identity of certain fungi depends on their
ability or inability to grow in the absence of nutri-
tional substances such as carbon and nitrogen
sources. In addition, several routine biochemical
tests, including urease production and proteoly-
sis, are available for the identification of many
molds. Moreover, the presence of various
enzymes as determined by the biochemical tests
is also useful in identifying fungi. When grown in
selective liquid or solid media, fungi ferment car-
bohydrates and produce acids, alcohols, gases,
and metabolic and enzymatic products in patterns
characteristic of their genus and/or species. These
fermentation products are commonly used in the
differential identification of fungi [19].
Most frequently, these instant and incubated
biochemical tests and other “expert systems”
monitor the aptitude of the isolate to assimilate
and ferment various sugar and nitrogen sources.
In addition, the identification of yeasts has now
been regularized by a variety of commercially
available strips and kits that can be used to exam-
ine rapidly the absorption of carbon as well as
nitrogen. The most reliable commercially avail-
able yeast identification kits are API 20C AUX,
ATB 32C, MicroScan, and Vitek systems. In gen-
eral, biochemical tests are not important in iden-
tifying molds as they are specific for yeasts and
dermatophytes [20]. Many yeast-like fungi such
as the genera Geotricum and Trichosporon, which
form arthrospores, require a series of biochemi-
cal tests for their definitive identification. Most
recently, fungal DNA analysis has been consid-
ered a powerful identification tool that requires
specialized equipment and is impractical in much
of routine laboratory work. Although there is an
increasing move towards molecular diagnostic
approaches, often use of the more easily available
and still fundamental staining techniques and
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
other biochemical tests are the choice of mycolo-
gists for much of the day-to-day fungal
identification. The goal of this chapter is to
acquaint mycologists with various staining tech-
niques and differential biochemical tests avail-
able for detecting fungal specimens.
Materials
See Note 1.
1. A pure culture of 24- to 48-h old yeast cells
(growing on Sabouraud or other nonselec-
tive agar)
2. Microscopic slides
3. Sterile saline or water
4. Flame source
5. Fume hood
6. A straight nichrome wire with a long handle
for stabbing inoculation
7. A bent nichrome wire with a long handle for
handling the mycelia types of fungal
cultures
8. Pair of short, stiff teasing needles helpful in
pulling apart dense masses of mycelium on
the slide for better microscopic examination
9. Scalpel with blades
10. Pair of forceps
11. Light microscope, phase contrast micro-
scope, and fluorescence microscope with
filters
12. 20% KOH-Glycerol solution—20 g KOH,
20 mL glycerol, 80 mL distilled water (see
Note 2)
13. Lactophenol Cotton Blue stain—20 mL lac-
tic acid, 20 mL phenol, 40 mL glycerol,
0.05 g cotton blue or aniline blue. Dissolve
phenol in lactic acid, glycerol, and distilled
water, finally add cotton blue and mix well
14. India ink
15. Crystal violet
16. Gram’s iodine
17. Ethanol
18. Absolute alcohol
19. Safranin
20. Giemsa stain
21. Wright stain—9.0 g (0.3% w/v) powdered
Wright’s stain, 1.0 g (0.033% w/v) powdered
239
Giemsa stain, 90 mL glycerin, and 2,910 mL
absolute acetone-free methanol (see Note 3)
22. Weigert’s solution A—0.6 g ferric chloride,
100 mL distilled water, 0.75 mL hydrochlo-
ric acid
23. Weigert’s solution B—1.0 g hematoxylin,
100 mL 95% ethanol; combine equal parts
of solution A and B
24. Weigert’s iron hematoxylin solution
A—1.0 g of hematoxylin, 100 mL 95%
ethyl alcohol
25. Weigert’s iron hematoxylin solution
B—4.0 mL 29% aqueous ferric chloride,
95 mL distilled water, 1.0 mL concentrated
hydrochloric acid
26. Weigert’s iron working solution—mix equal
parts of Weigert’s iron solution A and B
27. Acridine orange
28. Schiff’s reagent—0.025 g pararosaniline,
100 mL distilled water, sulfur dioxide gas
(see Note 4)
29. Aldehyde fuchsin—2 g basic fuchsin, 1 mL
paraldehyde, 1 mL concentrated hydrochlo-
ric acid, 200 mL 70% ethanol; ripen at room
temperature for 48–72 h
30. Chromic acid—5 g chromic trioxide,
500 mL distilled water (see Note 5)
31. Metanil yellow—0.25 g metanil yellow,
100 mL distilled water, 0.25 mL glacial
acetic acid
32. 15% Potassium hydroxide solution—15 g
potassium hydroxide, 20 mL glycerol,
80 mL distilled water; store at 25 °C and
discard if precipitation occur
33. 0.1% Calcoflour White (CFW) solution
34. Mucicarmine stain—1.0 g carmine, 0.5 g
aluminum chloride (anhydrous), 2 mL dis-
tilled water (see Note 6)
35. Formal–ethanol mixture—10 mL 40%
formaldehyde, 90 mL absolute alcohol
36. 5% Periodic acid (see Note 7)
37. Basic fuchsin solution
38. Sodium metabisulphate solution—1.0 g
sodium metasulphite, 10 mL hydrochloric
acid, 190 mL distilled water (see Note 8)
39. 0.2% Light green solution—0.2 g light
green, 100 mL distilled water, 0.2 mL gla-
cial acetic acid
240 J. Sangeetha and D. Thangadurai

40. Xylene (see Note 5) 70. Peptone disc

41. 1% Sodium metabisulphite 71. Potassium nitrate
42. Hexamine, preheated in a water bath to 72. Durham’s tube
56 °C for 1 h 73. Skim milk agar
43. Ferric chloride 74. Casein agar
44. 5% Sodium thiosulphate solution 75. Microgranular cellulose
45. 5% Silver nitrate, store in dark-colored bot- 76. Inoculation chamber
tle at 4 °C (see Note 9) 77. Autoclave
46. 3% Aqueous methenamine (hexamethylene 78. Petriplates
tetramine) (see Note 9) 79. Incubator
47. Aniline–acetic acid (see Note 5) 80. Mycosel agar with cyclohexamide plate
48. Thiosemicarbazide 81. 10% Tween solution
49. Schmorl’s solution—30 mL ferric chloride 82. Cellulolysis Basal Medium (CBM)—5 g
(1% aqueous), 4 mL potassium ferric cya- C4H12N2O6, 1 g KH2PO4, 0.5 g MgSO4·7H2O,
nide, 6 mL distilled water; make immedi- 0.1 g yeast extract, 0.001 g CaCl2.2H2O,
ately before use and do not reuse 1000 mL distilled water
50. Mellor bleach solution A—1% potassium 83. 0.5% Esculin
permanganate 84. 0.5% Arbutin
51. Mellor bleach solution B—1% sulphuric 85. 2% Ferric sulfate
acid 86. Sabouraud agar plates
52. Mellor bleach working solution—mix 87. Christensen’s urea agar
Mellor bleach solution A and B 88. API 20C Yeast Identification Kit
53. Mellor bleach solution C—1% oxalic acid 89. Biomerieux Vitek Yeast Biochemical Card
54. Ammonium silver stock solution—25 mL 90. Abbott Yeast Identification Kit
10% silver nitrate, add ammonium hydrox- 91. Colorimeter (Vitek colorimeter, 52–1210)
ide drop by drop, until solution precipitates 92. Filling stand (e.g., Vitek 52–0700)
and clears again 93. Sterile wooden applicator sticks
55. Ammonium silver working solu- 94. Sterile Pasteur or plastic pipettes (5 mL)
tion—12.5 mL ammonium silver stock solu- 95. Squeeze bottle
tion, 37.5 mL distilled water (see Note 10) 96. Water bath (48–50 °C)
56. 0.1% Gold chloride 97. 0.45–0.50% Saline
57. Neutral red stain 98. Sterile tubes
58. Sulfation reagent—45 mL glacial acetic 99. Fine-tip markers
acid, 15 mL concentrated sulphuric acid 100. 0.05% Noble agar
(see Note 11) 101. Sterile inoculating loop
59. Toluidine blue O—3 g toluidine blue O, 102. Vortex mixer
60 mL distilled water, 2 mL concentrated 103. Polyester film
hydrochloric acid, 140 mL absolute ethyl
alcohol
60. Basal salt medium Methods
61. 0.1% Carbohydrate
62. Bromocresol purple Wet Mount Techniques
63. Sugar disc
64. Whatman filter paper Fungal specimens can be visualized using wet
65. Sugars mount techniques through suspension of culture
66. Hot-air oven in either water or saline, mixed with alkali to dis-
67. Yeast Nitrogen Base (YNB) medium solve background material [21] or mixed with a
68. Molten agar combination of alkali and contrasting dye (e.g.,
69. Yeast Carbon Base (YCB) medium lactophenol cotton blue or India ink) [22, 23].
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
The dyes nonspecifically stain the fungal mate-
rial, which increases contrast with the background
and permits examination of the detailed struc-
tures. A variation is the India ink method, in
which the ink darkens the background rather than
the fungi.
Potassium Hydroxide Wet Mount
Potassium hydroxide (KOH) is used to dissolve
proteinaceous material and facilitate detection of
fungal elements that are not affected by strong
alkali solution. It is a strong alkali used as a clear-
ing agent to observe fungi in a wet mount prepa-
ration. The concentration of KOH is usually based
on the specimen that is being used. Normally,
10–20% KOH is used; occasionally, 40% is used
when the specimen is not cleared by 10–20% of
KOH. In this method, the fungal structures, such
as hyphae, large yeasts (Blastomyces), spherules,
and sporangia, are well distinguished. In unstained
preparations (KOH without stain), the fungal
structures may be enhanced by using a phase con-
trast microscope [18, 24]. The clearing effect
throughout the specimen can be accelerated by
gently heating the KOH preparation.
Visualization of fungi can be further enhanced
by the addition of dyes to the preparation. This
method is quick, simple, and inexpensive [25, 26].
1. Place a large drop of KOH solution with a
Pasteur pipette.
2. Transfer small quantity of the culture with a
loop or the tip of a scalpel into the KOH drop.
3. Put a clean coverslip over the drop gently so
that no air bubble is trapped.
4. Clearing can be hastened by gentle heating of
the slide, but it is best avoided.
5. Observe under 20× and 40× objective of light
or phase contrast microscope.
6. Look for budding yeast cells; branching
hyphae; type of branching; and the color, sepa-
ration, and thickness of hyphae (see Note 12).
Lactophenol Cotton Blue Wet Mount
Lactophenol cotton blue (LCB) is a mounting
medium commonly used in microbiology labora-
tories for preparing mounts of fungal cultures.
LCB is used as both mounting fluid and stain. In
this method, phenol will kill the organisms, and
241
the lactic acid preserves fungal structures; chitin
in the fungal cell wall is stained by the cotton
blue. It can be used alone or in conjunction with
KOH. Library slides may be made by allowing
the mount to dry for 3 weeks and then sealing
with collodion [24, 27, 28].
1. Put a large drop of LCB with a Pasteur
pipette.
2. Transfer a small quantity of the culture to the
drop.
3. Tease the culture well with teasing needles, so
as to get a uniform spread.
4. Put on a coverslip gently to avoid entrapment
of air bubbles.
5. Examine under the 20× and 40× objectives of
light microscope.
6. Observe the morphological features carefully.
7. Fungal elements will stain deep blue against a
clear pale-blue background.
India Ink Wet Mount
India ink can be added to specimens to provide
dark background that will highlight hyaline yeast
cells and capsular material. This method is used
to detect microorganisms that are surrounded by
capsules. The dye is excluded by the capsule, cre-
ating a clear halo around the yeast cell. It is a
rapid method for the preliminary detection and
identification of specimens containing species of
Cryptococcus [29].
1. Add a small drop of India ink on a smear.
2. Place a coverslip over the smear and press it
gently to obtain a thin mount.
3. If India ink is too thick (dark), dilute it by 50%
with saline.
4. Allow the preparation to stand for few minutes
to settle.
5. Scan under low power in reduced light; switch
to high power, if necessary.
6. Organisms possessing a capsule appear highly
refractile, surrounded by a clear zone against a
dark background.
Staining Techniques
Direct microscopic examination without stain
lacks sensitivity, especially when hyphae are
242
sparse in the specimen. A variety of differential
stains are commonly used like Gram, Giemsa,
Wright stain, toluidine blue O, and Weigert’s
iron hematoxylin to stain fungi [30]. The sensi-
tivity of microscopic examination is improved
when fungus-enhancing stains like Mayer’s
mucicarmine, periodic acid Schiff, Gomori’s
methenamine silver, acridine orange fluorescent,
calcoflour white, thiosemicarbazide, Fontana-
Masson, and Gridley’s stains are used. Since the
stain is immediately taken up by the fungal cell
wall in the scraping, the staining usually becomes
much brighter after 5–10 min [31]. Some speci-
mens need alkali pretreatment. In that case, it is
important to make sure that they do not react for
a long period of time; otherwise, a gelatinous
consistency will form, and the specimen should
be neutralized with 10% lactic acid before stain-
ing and adjusted to pH 3.0–5.0 [32]. The method
of preparation of smear for staining is as
follows:
1. Take a clean grease-free glass slide.
2. Place a large drop of saline solution.
3. Transfer a small quantity of the culture with a
loop or the tip of a scalpel into the saline drop.
4. Make a smear over the surface of the slide.
5. Fix by heat, if necessary.
Gram Staining
Gram stain is a key starting point to identify
microbial species. The stain differentiates mem-
brane structures between gram-positive and
gram-negative microorganisms. Gram-positive
microbes have a thick cell wall made up of pep-
tidoglycan (50–90%), which are stained purple
by crystal violet, whereas gram-negative
microbes have a thinner layer (10% of cell wall),
which are stained pink by the counter-stain
safranin [24, 30, 33–36].
1. Apply two drops of crystal violet on smear
for 30 s.
2. Wash with tap water.
3. Add two drops of Gram’s iodine for 30 s.
4. Repeat step 2.
5. Add 95% ethanol.
6. Repeat step 2.
7. Add two drops of safranin.
8. Repeat step 2.
J. Sangeetha and D. Thangadurai
9. Observe in microscope under oil immersion.
10. Yeasts are gram-positive, but poorly stained;
Cryptococcus neoformans is a notable excep-
tion (gram-negative).
Giemsa Staining
A variety of “Romanowsky-type” stains with
mixtures of methylene blue and azure eosin com-
pounds have been used successfully for many
years on diverse fungi with various procedures
and modifications. Giemsa stain is a member of
the Romanowsky group of stains, which are
defined as being the black precipitate formed
from the addition of methanol [37]. In this stain,
eosin ions are negatively charged and stain basic
components of cells orange to pink. It was also
originally designed to incorporate cytoplasmic
(pink) staining with nuclear (blue) staining and
fixation as a single step for smears and thin films.
This stain has widely been used to examine
Pneumocystis jiroveci, Rhinosporidium seeberi,
and Histoplasma capsulatum [38–40].
1. Flood the smear with methyl alcohol and leave
for 3–5 min for fixation.
2. Add prepared Giemsa stain and leave for
45 min.
3. Wash slide thoroughly with running tap
water.
4. Blot dry with absorbent paper.
5. Observe under oil immersion.
6. Look for intracellular budding yeasts; fungi
stain with purplish-blue.
Wright Staining
The Wright stain is an alcoholic solution of meth-
ylene blue, azure A, thionin, and eosin Y. Methyl
groups are activated and react with charged com-
ponents of the cell to produce coloration. It is
used to detect blood parasites, viral and chlamyd-
ial inclusion bodies, yeast cells, and species of
Pneumocystis. Eosin ions are negatively charged
and stain basic components of cells orange to
pink, whereas other dyes stain acidic cell struc-
tures to various shades of blue to purple [41, 42].
1. Cover the smear with freshly filtered Wright
stain and leave for 1–3 min.
2. Without removing the stain, pour on buffer
solution (pH 6.4).
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
3. Gently mix buffer and stain; upon proper mix-
ing, metallic green sheen (green scum) rises to
the surface of the fluid.
4. Leave for 3 min or longer.
5. Wash the slide gently with flowing tap water
and wipe the bottom of the slide with a clean
filter paper.
6. Air-dry the slide and observe under the
microscope.
7. Intracellular yeast cells are typically stain blue
and species of Pneumocystis stain purple.
Weigert’s Iron Hematoxylin Staining
This stain can be used with fixatives that include
polyvinyl alcohol, sodium acetate, and formalin.
The staining method involves application of hae-
malum, which is a complex formed from alumi-
num ions and oxidized hematoxylin. This stains
nuclei of cells blue. Counterstain eosin Y may
also be used to color other structures in various
shades of red, pink, and orange [28, 43–47].
1. Add staining solution on a smear and leave for
1–2 h.
2. Rinse with tap water.
3. Add 1% HCl.
4. Add 70% ethanol.
5. Repeat step 2.
6. Counterstain with eosin Y, if necessary.
7. Dehydrate with ethanol.
8. Clear with xylene and observe under
microscope.
9. Yeast cells stain blue–gray to black.
Acridine Orange Staining
Acridine orange is a fluorochromatic dye that
binds to nucleic acids of fungi. Under UV light,
acridine orange stains RNA and single-stranded
DNA orange, while double-stranded DNA
appears green. At neutral pH, fungi and cellular
materials stain reddish orange. At acid pH, fungi
remain reddish orange but background material
stains greenish yellow [31, 48–53].
1. Add Weigert’s iron hematoxylin on smear for
5 min.
2. Wash well with tap water.
3. Place few drops of acridine orange solution
for 2 min.
4. Repeat step 2.
243
5. Observe smear on the fluorescence
microscope.
6. Fungi stain bright orange and the background
appears greenish yellow.
Gridley Staining
Gridley staining method is used to identify fungi,
based on Bauer chromic acid leucofuchsin stain
with the addition of Gomori’s aldehyde fuchsin
stain and metanil yellow as counterstains. Against
a yellow background, hyphae, conidia, yeast cap-
sules, elastin, and mucin appear in different
shades of blue to purple. It can be used to identify
Rhiosporidium seeberi and Histoplasma capsu-
latum [28, 47, 51, 54, 55].
1. Place chromic acid on smear for 1 h.
2. Wash well with tap water.
3. Treat with sodium metabisulphite bleach for
1 min.
4. Repeat step 2.
5. Rinse with distilled water.
6. Place in Schiff’s reagent for 20 min.
7. Repeat step 2.
8. Rinse with 70% ethanol.
9. Place in aldehyde fuchsin for 30 min.
10. Rinse off excess with 95% ethanol.
11. Repeat step 2.
12. Counterstain with metanil yellow for 1 min.
13. Rinse well with distilled water.
14. Dehydrate and observe under fluorescence
microscope.
15. Fungi show purple color with yellow back-
ground (see Note 13).
Calcoflour White Staining
Calcoflour White (CFW) stain is used to detect
fungal elements, particularly Pneumocystis spe-
cies. The fluorophore shows a high affinity for
chitin-forming hydrogen bonds with free hydroxyl
groups and stains fungal cell walls blue. The use
of CFW staining requires the addition of KOH,
which helps to dissolve keratinized particles and
emulsify solid, viscous material and enhance the
visualization of fungal elements in microscopic
examination. Positive results are indicated by a
bright green to blue fluorescence using a
fluorescence microscope (see Note 14) [56]. A
bright yellow-green fluorescence is observed
244
when collagen or elastin is present. KOH-CFW
preparations may be preserved for several days at
4 °C. This stain can be used to identify Fusarium
solani, Aspergillus fumigatus, and Candida
albicans [24, 56–59].
1. On smear add a drop of 15% KOH and a drop
of the CFW solution or mix in equal volumes
before processing.
2. Mix and place a coverslip over the material.
3. If necessary, allow the KOH preparation to
remain at room temperature (25 °C) for a few
minutes until the material has been cleared;
the slide may be warmed to speed up the clear-
ing process.
4. Observe the slide by UV microscopy.
5. Fungal cell walls fluorescence apple green to
blue.
Mayer’s Mucicarmine Staining
Mucicarmine is a red stain that contains alumi-
num chloride and carmine. Aluminum is believed
to form a chelation complex with the carmine and
change the molecule to a positive charge, allow-
ing it to bind with the acid substrates of low den-
sity, such as mucins. It is used to detect
mucin-secreting fungi and capsules of
Cryptococcus neoformans and Rhinosporidium
seeberi. It will also stain the walls of the spores
and the inner surface of the sporangia. However,
the cell walls of yeasts and Blastomyces dermati-
tis may stain weakly with mucicarmine
[55, 60–64].
1. Stain the smear with a working solution of
Weigert’s hematoxylin for 7 min.
2. Wash well in tap water.
3. Add metanil yellow for 1 min.
4. Repeat step 2.
5. Place in mucicarmine stain for 45 min.
6. Rinse quickly in distilled water.
7. Dehydrate in 95% ethanol and absolute alco-
hol (two changes of each).
8. Clear with two changes of xylene.
9. Mount in DPX and view under the
microscope.
10. Mucopolysaccharide capsule stain deep rose
to red, nuclei are black, and the other debris
stain yellow.
J. Sangeetha and D. Thangadurai
Periodic Acid–Schiff Staining
Periodic acid–Schiff (PAS) reactions are effective
stains for demonstrating fungal elements of essen-
tially all fungi. Periodic acid attacks some carbo-
hydrates containing 1,2-glycol or OH group with
the conversion of this group to 1,2-aldehydes,
which then react with the fuchsin-sulfurous acid
to form the magenta color [25, 32]. Identification
of fungal elements can be enhanced if a counter-
stain such as light green is used. Species of
Coccidioides, Cryptococcus, Histoplasma,
Candida, Malassezia, and Aspergillus can be
stained with this stain [65].
1. Immerse the smear in ethanol for 1 min.
2. Place 5% periodic acid for 5 min.
3. Wash gently in running tap water.
4. Place basic fuchsin for 2 min.
5. Repeat step 3.
6. Add sodium metabisulphite (0.5%) for
3–5 min.
7. Repeat step 3.
8. Counterstain with dilute aqueous light green
(0.2%) for 2 min.
9. Dehydrate with 70%, 80%, 95%, 100% etha-
nol and xylene, each for 2 min.
10. Observe under microscope.
11. Fungi stain bright pink-magenta or purple
against green background when light green
is used as a counterstain.
Grocott-Gomori Methenamine Silver
Staining
Grocott-Gomori methenamine silver (GMS)
staining is preferred for screening degenerated
and nonviable fungi because it provides better
contrast. The fungal cell wall contains mucopoly-
saccharides that are oxidized by GMS to release
aldehyde groups, which later react with silver
nitrate. Silver nitrate is converted to metallic sil-
ver, which becomes visible in the silver stains;
this is useful in detecting fungal elements. Fungi
stain in black against a pale-green background.
Pneumocystis jiroveci, Cryptococcus neofor-
mans, Coccidiodes immitis, Histoplasma capsu-
latum, Aspergillus fumigatus, and Candida
albicans can be detected by this staining tech-
nique [18, 24, 55, 64, 66, 67].
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
1. Add two drops of absolute ethanol for 5 min.
2. Wash in distilled water.
3. Flood the smear with 4% chromic acid for
45 min.
4. Repeat step 2.
5. Add 1% sodium metabisulphite for 1–2 min.
6. Repeat step 2.
7. Add working solution of hexamine (smear
becomes dark brown).
8. Wash with distilled water or if smear turns
black, wash with 0.1% ferric chloride.
9. Add 5% sodium thiosulphate for 2 min.
10. Repeat step 2.
11. Wash with 1% light green solution for 1 min.
12. Dry and view under oil immersion.
13. The slide with fungal elements stains black;
inner part of micelle or hyphae stains pink
with background in pale green.
Double-Oxidation Thiosemicarbazide,
Schmorl
The hydrazine group (H2NNH-) combines with
any aldehyde generated by periodic acid oxida-
tion. The thiocarbamyl group (−CSNH2) is a
more powerful reducing agent than aldehydes
and rapidly reduces ferricyanide to ferrocyanide,
which immediately forms a prussian blue deposit
at the site. The mallory bleach lightens back-
ground staining and improves contrast. It may
also produce some aldehyde, which is removed in
step 6. This method is widely used to identify
fungal colonies in tissues [39, 68].
1. Do mallory bleach (place mellory working
solution for 10 min and rinse with water;
then place mallory solution C for 2 min until
the tissue is bleached).
2. Wash with a running tap water.
3. Oxidize in periodic acid for 10–20 min.
4. Repeat step 2.
5. Place aniline-acetic for 30 min.
6. Repeat step 2.
7. Repeat step 3.
8. Repeat step 2.
9. Place into thiosemicarbazide for 10 min.
10. Repeat step 2 to remove all traces of
thiosemicarbazide.
11. Place into freshly made Schmorl’s solution
for 10 min.
245
12. Repeat step 2.
13. Optionally, counterstain with eosin.
14. Repeat step 2.
15. Dehydrate with ethanol, clear with xylene,
and mount with a resinous medium.
16. Fungi stain with blue, nuclei with red, and
background is pink.
Fontana-Masson Staining
The Fontana-Masson (FM) stain can be used to
detect the presence of melanin in cell walls of
dematiaceous fungi such as species of Bipolaris,
Curvularia, Exophiala, and Phialophora. FM
stain is often believed to be a diagnostic tool to
differentiate dematiaceous fungi from
Aspergillus sp. and some Zygomycetes. Also, it
is particularly useful for distinguishing capsule-
deficient Cryptococcus neoforamans from
Histoplasma capsulatum and Blastomyces der-
matitis. Melanin has the ability to reduce solu-
tions of ammonical silver nitrate to metallic
silver without the use of an external reducing
agent. The intensity and amount of staining may
reflect differences in melanin deposition owing
to growth rate, age, availability of precursors, or
loss of pigment staining associated with hyphal
death and destruction. Extent of stain intensity
and its distribution in fungal elements in tissue
were evaluated by means of intensity; for exam-
ple, dark brown (strong intensity), medium
brown (moderate intensity), and pale brown
(weak intensity) [69].
1. Treat smear with ammonical silver nitrate
solution for 20 min at 60 °C.
2. Check microscopically after 15 min and
repeat step 1 if necessary.
3. Wash well in distilled water.
4. Tone with 0.1% gold chloride for 2 min.
5. Repeat step 3.
6. Fix in 2% aqueous sodium thiosulphate for
2 min.
7. Repeat step 3.
8. Counterstain with neutral red stain for
1 min.
9. Repeat step 3.
10. Rapidly dehydrate well in absolute alcohol,
clear, and mount.
11. Observe under microscope.
246
12. All dematiaceous fungi show strong inten-
sity (black); species such as Bipolaris,
Exophiala, Fonsecaea, and Phialophora are
darkly pigmented because of melanin.
Toluidine Blue O Staining
This stain is primarily used for the detection of
Candida albicans, Rhinosporidium seeberi, and
Pneumocystis carinii. Background staining is
removed by sulfation reagent. Yeast cells get
stained differentially and are difficult to distin-
guish from Pneumocystis cells. The stain can be
replaced with specific fluorescent stains.
Toluidine blue O gives polychromatic staining
for all the fungal structures (such as conidia,
germ tubes, haustoria, and hyphae) as well as
cells [55, 64, 70–73].
1. Add sulfation reagent for 10 min.
2. Wash with tap water.
3. Add toluidine blue O for 3 min.
4. Add 95% ethyl alcohol, absolute ethyl alcohol,
and xylene, each for 10 s for decolorizing.
5. Place a coverslip on the slide.
6. Observe with 20× and 40× objectives.
7. Fungi stain reddish blue to dark purple on
light-blue background.
Manual Biochemical Methods
Biochemical tests have been used to classify and
identify various groups of fungi. Because fungi
grow rapidly in pure culture, it is possible to use
biochemical methods to identify and classify
them [74]. The classical broth methods were
originally developed by Wickerham for utiliza-
tion and fermentation testing of yeasts [75–77].
Biochemical methods like utilization of carbon
and nitrogen, fermentation of carbon, and enzyme
activity like caseinase, cellulase, gelatinase, glu-
cosidase, fatty acid esterase, lipase, urease, and
so forth are currently in use to assist in the dif-
ferentiation of fungi.
Utilization of Carbon Sources
Assimilation tests are extremely important in the
taxonomy of yeasts. They should be performed
before tests involving chemical analysis of the
J. Sangeetha and D. Thangadurai
fungus for the very simple reason that they are
easier to perform and generally require no special-
ized apparatus. An assimilation test is performed
based on the fact that nutritional factors are capa-
ble of differentiating fungi. Storage of carbohy-
drates fulfills multiple functions in fungi; they not
only constitute a source of carbon and energy but
also protect fungi against a variety of environmen-
tal stresses, such as desiccation and frost. Fungal
metabolism dominates the assimilation of exoge-
nous carbohydrates into tissues. Several sugars
can be used as a carbon source. In physiologic
characterization, tests for the ability of a fungi to
utilize various carbon substrates (see Note 15) as
the sole source of carbon by employing a basal
medium such as yeast nitrogen base that contains
ammonium sulfate (universal nitrogen source),
vitamins, amino acids, and trace elements are
required for growth of yeasts. Assimilation tests
are read by growth and turbidity. For each test or
organism, negative controls without a carbon
source should be maintained [78–82].
Liquid Auxanographic Method
1. Prepare basal medium with bromocresol pur-
ple (0.5 g/L).
2. Adjust pH to 5.4 by adding NaOH or HCl.
3. Sterilize at 121 °C for 20 min.
4. Add filter-sterilized 1% (w/v) selected
carbohydrate.
5. Pour the medium into test tubes.
6. Inoculate with fungal mycelium.
7. Incubate at 20 °C for 14 days.
8. Change in color of the medium to orange or
yellow is taken as positive, whereas a change
to pink or purple is negative.
Pour Plate Auxanographic Method
1. Sugar discs can be obtained commercially or
prepared manually (steps 2–5).
2. Punch 6-mm diameter discs from Whatman
no. 1 filter paper.
3. Sterilize the discs by placing them in a hot-
air oven for 1 h.
4. Allow to cool, and then add one drop of 10%
filter-sterilized sugar solution to each disc.
5. Dry the disc at 30 °C in incubator and store
at 0 °C.
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
6. Prepare yeast nitrogen base (YNB) medium.
7. Prepare a yeast suspension from 24- to 48-h
old culture in 2 mL of YNB by adding heavy
inoculum.
8. Add this suspension to the 18 mL sterilized
Molten agar mix well.
9. Pour the entire medium into Petri dish.
10. Allow the media to solidify at room
temperature.
11. Now place the various carbohydrate-impreg-
nated discs onto the surface of the agar
plate.
12. Incubate at 30 °C for 4–7 days.
13. Positive reactions can be noted by growth
and color change around the disc.
Utilization of Nitrogen Sources
Simple basal media to which single nutrients
(vitamins, amino acids) could be added will be
used as the bases of these tests. For testing vari-
ous nitrogen substrates (see Note 16) as the sole
source of nitrogen, one can use yeast carbon base
that contains glucose (universal carbon source)
and the vitamins, amino acids, and trace elements
required for growth. In general, if yeast can uti-
lize nitrate, it can also use nitrate as a nitrogen
source. For each test or organism, negative con-
trols without nitrogen source should be main-
tained [83–89].
Liquid Auxanographic Method
1. Prepare basal medium along with bromocre-
sol purple (0.5 g/L).
2. Adjust pH to 4.5 with HCl or NaOH.
3. Sterilize the medium at 121 °C for 20 min.
4. Add filter-sterilized nitrogen compound
(2 g/L).
5. Inoculate with fungal mycelium.
6. Incubate at 20 °C for 4–7 days.
7. Hyphal mat in liquid media is the positive
result.
Pour Plate Auxanographic Method
1. Peptone discs can be obtained commercially
or prepared manually (steps 2–5).
2. Punch 6-mm diameter discs from Whatman
no. 1 filter paper.
247
3. Sterilize the discs by placing them in a hot-
air oven for 1 h.
4. Allow to cool, and then add one drop of 3%
filter-sterilized potassium nitrate or peptone
solution to each disc.
5. Dry the disc in 30 °C in incubator and store
at 0 °C.
6. Prepare yeast carbon base (YCB) medium.
7. Prepare a yeast suspension from 24- to 48-h
old culture in 2 mL of YCB by adding heavy
inoculum.
8. Add this suspension to the 18 mL sterilized
molten agar and mix well.
9. Pour the entire medium into Petri dish.
10. Allow the media to solidify at room
temperature.
11. Now place the various nitrate-impregnated
discs onto the surface of the agar plate.
12. Incubate at 30 °C for 4–7 days.
13. Positive reactions can be noted by growth
and color change around the disc.
Carbohydrate Fermentation
This method is a powerful tool for definitive
characterization and taxonomy of yeasts.
Carbohydrate fermentation tests whether a cer-
tain microbe has the capability to ferment differ-
ent carbohydrates. Fungi able to ferment a
particular sugar are also able to assimilate the
same sugar; however, the reverse is not always
true. To test fermentative abilities, a different
basal medium is employed. Normally, 2% sugar
solution is added to the basal medium in a test
tube that also contains an inverted Durham tube
in order to observe production of CO2 and etha-
nol as the by-products of sugar fermentation.
Because most yeasts are also able to assimilate
ethanol as the sole source of carbon, it is neces-
sary to incubate assimilation tests separately from
fermentation tests, as ethanol vapor produced by
fermentation can dissolve in assimilation tests
and cause false-positive results [90–98].
1. Prepare basal medium and sterilize at 121 °C
for 20 min.
2. Add filter-sterilized sugar at the concentration
of 2% (w/v) to the medium aseptically.
3. Pour the medium to the test tubes.
248
4. Insert inverted single sterile Durham’s tube in
each and close the lid.
5. Incubate at 20 °C for 7 days.
6. Gas accumulation in Durham’s tube is indica-
tive of a positive result.
Casein Hydrolysis
Caseinase is an exoenzyme that is secreted outside
of the cells into the surrounding media. It has the
ability to break down milk protein, called casein,
into small peptides and individual amino acids for
their energy use or as building material. The
hydrolytic reaction creates a clear zone around the
cell as the casein protein is converted to soluble
and transparent end products, like small chains of
amino acids, dipeptides, and polypeptides. This
test can be used to identify some species of yeast
and fungi like Citeromyces matritensis, Aspergillus
dimorphicus, A. ochraceus, Fusarium illudens, F.
moniliforme, F. solani, Penicillium citrinum, P.
brevicompactum, P. chrysogenum, P. fellutanum,
and P. waksmanii [99, 100].
1. Prepare Petri plates with autoclaved skim milk
agar or casein agar in sterile conditions.
2. Inoculate fungal mycelia onto the center of the
plate and incubate at 20 °C for 14 days.
3. Examine for the presence of a clear zone.
4. The appearance of a clear zone around the
fungal colony is the positive result.
Cellulose Hydrolysis
Cellulase is produced chiefly by fungi, bacteria,
and protozoans that catalyze cellulolytic activity.
In the most familiar case of cellulose activity, the
enzyme complex breaks down cellulose to b-
glucose. This type of cellulase is produced by
symbiotic bacteria and fungi in the ruminating
chambers of herbivores. Three hydrolytic
enzymes, such as five endo-1,4-b-glucanases,
one exo-1,4-b-glucanase, and one or several
1,4-b-glucosidases, are involved in cellulolysis.
This can be used to identify Sporotrichum poul-
verulentum, Trichoderma viride, Aspergillus
niger, Peziza sp., Fusarium sp., and Penicillium
sp [101–103].
1. Prepare basal salt medium with the addition of
1% (w/v) microgranular cellulose (sigma) and
1.2% (w/v) agar.
J. Sangeetha and D. Thangadurai
2. Autoclave at 121 °C for 30 min, disperse into
Petri dishes.
3. Inoculate fungi onto the center of the agar
plates.
4. Incubate at 20 °C for 14 days.
5. Appearance of a clear zone around the fungal
colony is a positive result.
Cyclohexamide Resistance
This technique is to confirm the presence of a
possible dimorphic fungus or dermatophytes.
Determination of the resistance of isolates to
cyclohexamide is useful when screening cultures
for Blastomyces dermatitidis, Coccidioides immi-
tis, Epidermophyton floccosum, Histoplasma
capsulatum, Microsporum sp., Paracoccidioides
brasiliensis, Sporothrix schenckii, and
Trichophyton sp. All these fungi will grow in the
presence of cycloheximide at 30 °C or less, while
fungal species such as Absidia, Aspergillus,
Mucor, Rhizopus, Scedosporium and many more
are inhibited by cyclohexamide [104–106].
1. Inoculate a small portion of mold colony onto
mycosel agar with cyclohexamide and
Sabouraud agar plates.
2. Incubate at 30 °C for 7–10 days.
3. Observe for the growth of the colonies on
plate.
4. Growth on Sabouraud agar and mycosel agar
indicates resistant, and growth on Sabouraud
agar but no growth on mycosel agar indicates
sensitive. Repeat the test if there is no growth
on Sabouraud agar or mycosel agar.
Fatty Acid Esterase Activity
Fatty acid esters are cleaved by enzymes includ-
ing esterase, cutinase, and lipases, which can
release free fatty acids from several sources,
including lipids, phospholipids, sterol esters,
waxes, cutin, and suberin. In this method,
Rhizopus circinans, R. microspores, Fusarium
oxysporum, R. boreas, R. thermosus, R. usamii,
R. stolonifer, R. fusiformis, and Pseudomonas
cepacia are screened by its enzyme activity
[107–112].
1. Prepare basal medium with bromocresol pur-
ple as indicator at pH 5.4.
2. Prepare 10% tween solution.
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
3. Autoclave the medium and tween solution
separately at 121 °C for 30 min.
4. Add the tween solution in a ratio of 1:9 by vol-
ume to the cooled medium.
5. Pour the medium into petri dishes.
6. Inoculate the fungal mycelia onto the center of
the medium.
7. Incubate at 20 °C for 14 days.
8. Change in the color of the medium to purple is
a positive result.
Gelatin Hydrolysis
Gelatin, a protein derived from collagen, is too
large to enter the cell as a whole and, hence, the
exoenzyme gelatinase cleaves gelatin to poly-
peptides and then further degrades polypeptides
to amino acids, which are taken up and utilized
by the fungi. This test can be performed to dif-
ferentiate between fungi that produce gelatinase
and those that do not produce the enzyme
[113–116].
1. Inoculate culture into a nutrient gelatin tubes
with a straight needle.
2. Incubate for 48 h at 22 °C.
3. Observe for liquification.
4. Liquification of the gelatin is a positive test for
gelatin hydrolysis.
b-Glucosidase Activity
The hydrolysis of cellobiose to glucose is
achieved by b-glucosidase. b-glucosidase is pre-
dominantly a cell associated or intracellular
enzyme in many fungi. This enzyme is ubiqui-
tous among cellulolytic fungi producing hydro-
lytic endonucleases or cellobiohydrolases.
Activity of b-glucosidase can be detected by
growth of the test fungi on agar containing escu-
lin (6,7-dihydroxycoumarin 6-glucosidase) or
arbutin (hydroquinone-b-d-glucopyranoside) as
the sole carbon source. Splitting of the substrate
by the enzyme yields glucose and a coumarin
product that react with iron sulfate to produce a
black color in the growth medium [117–120].
1. Sterilize cellulolysis basal medium (CBM)
supplemented with 0.5% (w/v) esculin or arb-
utin and 1.6% (w/v) agar.
2. Add 1 mL of sterile 2% (w/v) aqueous ferric
sulfate solution per 100 mL of CBM agar.
249
3. Pour the medium to the Petri dishes.
4. Inoculate the fungal culture into the medium.
5. Incubate at 25 °C in darkness for 5 days.
6. Development of black color in the medium
indicates the production of b-glucosidase.
Lipase Activity
Lipase catalyzes the hydrolysis of the ester bonds
of triacylglycerols, thereby releasing free fatty
acids. Lipolytic activity has been associated with
survival and pathogenicity of several fungal
species. It can be used to detect species of
Malassezia and Candida [121–124].
1. Prepare Sabouraud agar medium and sterilize
at 121 °C for 30 min.
2. Add 0.1% (v/v) n-tributylin to the medium
and pour into the Petri dishes.
3. Inoculate fungal mycelia onto the surface of
the medium and incubate at 20 °C for 7
days.
4. Occurrence of clearance in the medium is a
positive result.
Urease Activity
Urea may be hydrolyzed by some fungi with the
help of urease so that the ammonia that is liber-
ated can be used as a nitrogen source. Phenol red
indicator is added to the broth or solid medium at
pH 8.4, and the media will turn from red color to
pink color owing to ammonia production. Urea
hydrolysis is primarily used to distinguish
Trichophyton mentagrophytes, T. sulphureum, T.
tonsurans, and T. verrucosum from T. gallianae
and T. rubrum and to identify Cryptococcus neo-
formans [125–128].
1. Prepare Christensen’s urea agar medium and
sterilize at 121 °C for 30 min.
2. Pour the medium into Petri dishes and inocu-
late fungal mycelium onto the center of the
medium.
3. Incubate the inoculated tubes at 25–30 °C for
8 days.
4. Examine the slants every 2–3 days for color
change.
5. Change in the color of the medium to pink or
purple can be taken as a positive result, while
a change to orange or yellow is considered as
a negative result.
250
Automated Biochemical Methods
Conventional methods of identifying fungi
require as long as 14–28 days for the completion
of all the biochemical tests. In recent years, it has
become important to develop rapid, automated,
and modern methods for species identification
and strain differentiation in fungi [129–138]. In
this context, the newer miniaturized fungal
identification systems such as the API 20C Yeast
Identification System (API Analytab Products,
Plainview, NY), Biomerieux Vitek Yeast
Biochemical Card (Hazelwood, MO), and Abbott
Yeast Identification System (Abbott Laboratories,
Irving, Texas) provide biochemical testing in 3–7
days with an acceptable degree of reliability [98,
139]. The biochemical tests designed in such
automated systems are those routinely followed
in conventional identification systems, except the
test for assimilation of mixture of r-hydroxyben-
zoic acid and protocatechuic acid, as in the Abbott
Yeast Identification System, which helps in the
identification of Candida parapsilosis and cer-
tain other yeasts [140]. These three fungal
identification systems are based on modifications
of the classic auxanographic technique of carbo-
hydrate assimilation [141, 142]. They are easy to
use, require less preparation of reagents, and
offer a significant saving of time compared to
conventional tube tests and other currently avail-
able biochemical approaches. These commer-
cially available kits have been widely used to
identify filamentous fungi and yeasts [143–145].
API 20C Yeast Identification System
The commercially available API 20C Yeast
Identification System is easy to use, requires less
preparation of reagents, and has been widely used
to identify both yeast and filamentous fungi. The
kit consists of 20 microtubes containing dehy-
drated substrates in which 19 assimilation tests
are performed. After inoculation and incubation,
the reactions are interpreted by comparison to
growth controls and use of a reference
identification table that is provided with each kit.
The system is based on modifications of the clas-
sic auxanographic technique of carbohydrate
J. Sangeetha and D. Thangadurai
assimilation. When the yeast is able to assimilate
a particular carbohydrate accompanied with a
color change, the system must also be supple-
mented with morphological studies. Germ tube
tests should be done in conjunction with them as
a means of obtaining a more complete profile of
the yeast cells being identified (see Note 17)
[143–145].
1. Melt the basal medium in the ampoules by
placing them in an autoclave for 2 min.
2. Place the ampules in a water bath at 50 °C and
allow to cool.
3. Dispense 20 mL of water into the incubation
tray.
4. Place the strips into incubation tray.
5. Open the ampules according to manufacturer’s
instructions and inoculate the molten medium
with an applicator stick that has touched one
or two colonies of >2 mm diameter.
6. Inoculate the strip (20 cupules of approxi-
mately 200 ml each) using a Pasteur Pipette
and then place the lid on the tray.
7. Incubate the trays at 28–30 °C for 24–72 h and
check for growth.
8. Record and compare the results with the
identification table to identify yeast (see
Note 18).
Biomerieux Vitek Yeast Biochemical Card
The disposable plastic Vitek 30-well Yeast
Biochemical Card contains 26 conventional bio-
chemical tests and 4 negative controls. The card
is one of several testing packages used with the
Vitek System, which includes a programmed
computer, a reader-incubator unit, a filling mod-
ule, a sealer module, and a printer. Identification
is generally based on conversion of biochemical
test results into nine-digit biocodes that will be
analyzed by the Vitek computer. This automated
system has been used in the identification of clin-
ically significant yeasts, particularly Candida
albicans [146–151].
1. Use 1–3 colonies to prepare yeast suspension
in 1.8 mL saline tubes (see Note 19).
2. Adjust the suspension to a McFarland no. 2
standard using colorimeter (46–56% transmit-
tance, 450 nm).
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
3. Label the yeast cards with a marker and place
in the filling stand with a transfer tube kept in
the yeast suspension.
4. Inoculate the cards via the filling module.
5. Seal the cards via the sealer module and incu-
bate at 30 °C for 24–48 h, depending on the
readings provided by the instrument.
6. Vitek computer converts biochemical test
results into nine-digit biocodes in printed form.
7. Most of the results are obtained after 24 h and a
few isolates may require additional incubation.
8. Identification can be accepted if the printed
result had a reliability of more than 85%.
If the reliability is less than 85%, an API
20C Yeast Identification System or any
manual biochemical tests in addition to mor-
phological features can be used to identify
the yeast (see Note 18).
Abbott Yeast Identification System
The Abbott Yeast Identification System is an
instrumental method based on matrix analysis of
19 biochemical reactions in addition to the germ
tube test for identifying yeast. It is a disposable
plastic unit of 20 chambers containing lyophilized
biochemical substrates such as arabinose, cello-
biose, dulcitol, erythritol, galactose, glucose,
inositol, lactose, maltose, melibiose, melizitose,
nitrate, r-hydroxybenzoic acid and protocate-
chuic acid, raffinose, rhamnose, sucrose, treha-
lose, urea, xylose, and negative control. This
rapid and automated system has now been widely
in use for the identification of yeasts within
20–24 h after test initiation and offers significant
saving in time compared to conventional bio-
chemical and other currently available manual
systems [152–157].
1. Isolate yeast pure colonies from Sabouraud
agar plates.
2. Prepare the inocula and incubate for 24–48 h
at 30 °C.
3. Select colonies of a test organism and sus-
pend in 0.05% Noble agar using a sterile
inoculating loop or cotton-tipped applicator
(see Note 20).
4. Mix the suspension on a vortex mixer and
deliver 0.2 mL into each chamber of the
Abbott IDS cartridge.
251
5. Seal the cartridge with a polyester film (see
Note 21).
6. Take the initial optical density of each reac-
tion chamber with the MS-2, which will be
automatically stored in the computer
memory.
7. Incubate the cartridges off-line at 30 ° C for
22–25 h.
8. Reinsert the cartridge into the MS-2 for final
reading.
9. Enter the results of several other morpho-
logical and additional tests (see Note 22).
10. Take the MS-2 data printout with a record of
positive and negative biochemical tests.
11. Identify the yeast with up to five species
listed in the printout in descending order of
likelihood based on the percent likelihood
value for each species.
Notes
1. Wear gloves, goggles, and lab coat. Nitrile
gloves are suggested when working with sol-
vents and acids. Avoid contact and inhalation
of dyes and chemicals. Reagents should be
prepared in a fume hood [72].
2. Addition of glycerol to KOH solution will
prevent crystallization from occurring in the
solution, thus enhancing the shelf life of this
reagent. It will also help in preserving KOH
preparation for a couple of days.
3. The components have to be mixed in a brown
bottle and allowed to stand for 1 month
before use. The stain must be stored at 4 °C;
otherwise the components may degrade.
4. Under fume hood, dissolve pararosaniline
with distilled water and slowly bubble sulfur
dioxide gas through the solution until solution
color begins to change. Stopper well and store
in the dark for one or two days. Add activated
charcoal and shake for about a minute if solu-
tion is not clear; filter and store at 0–5 °C.
5. Chromic acid is corrosive to skin and mucous
membranes, highly toxic, and carcinogen.
Aniline and xylene are moderate skin and
severe eye irritants, possible carcinogens,
and combustible liquids.
252
6. Mix stain in test tube and heat it in a water
bath for 2 min. Liquid becomes almost black
and syrupy. Dilute with 100 mL of 50% alco-
hol and let stand for 24 h and filter. Again
dilute to 1:4 with tap water for use.
7. Periodic acid solution and the stock of peri-
odic acid (a white powder) should be kept in
dark bottles.
8. Add HCl to distilled water in a brown bottle
before adding sodium metabisulphite, cool at
5 °C, and filter. Then add 20 mL diluted HCl
(83 mL concentrated HCl/l,000 mL distilled
water) and cool to 25 °C. Add 1 g sodium
bisulphite and store in screw-top bottle in
dark for 2 days. Add 0.5 g activated charcoal,
shake intermittently for 1 h, and filter. Store
in dark-colored, tightly closed bottle in
refrigerator (expiration 5 years); pour into a
Coplin jar for further use. Solution may be
reused until it turns pink, at which time it
must be discarded.
9. Silver nitrate is toxic, and skin contact should
be avoided; methenamine is a flammable
solid and an irritant. Any spill should be
mopped up immediately with water.
10. Neutralize the ammonium silver solution
immediately after use, as it can be explosive
when allowed to dry.
11. To avoid splashing while mixing sulphuric
acid with glacial acetic acid, place the jar in
a plastic tub filled with cold water.
12. For more precision, stains like methylene
blue or Parker blue-black fountain ink or
chlorazol may be used along with KOH.
13. If background fluorescence is too bright for
fungi to be distinguished, it may be quenched
with alum hematoxylin for 1 min or potas-
sium permanganate for 1 min. Quench imme-
diately before the final dehydration step. This
should be done with caution because it may
reduce fungal fluorescence.
14. An epifluorescent microscope equipped with
a mercury vapor lamp and either an ultravio-
let (UV) or a blue-violet (BV) excitation
filter to achieve radiation on the slide below
412 nm should be used, because the maxi-
mum absorbance of CFW is 347 nm. A
microscope with selective filters that will
J. Sangeetha and D. Thangadurai
prevent radiation below 490 nm should not
be used for CFW [56].
15. Glucose, sucrose, lactose, arabinose, galac-
tose, xylose, mannose, dulcitol, ethanol, etc.
16. Potassium nitrate, sodium nitrate, amino
acids, urea, glycine, ammonium sulfate,
asparagine, peptone, aliphatic amines, etc.
17. Germ tube tests and morphological studies
should be included, as API 20C Yeast
Identification System does not include rham-
nose and urea. The API yeast profiles some-
times give many different yeast identifications
for an individual isolate; this warrants sup-
plemental tests.
18. For quality control, include known isolates
of Torulopsis glabrata, Candida albicans,
and Cryptococcus laurentii.
19. Heavy encapsulated yeasts and isolates with
extensive mycelial growth are difficult to
suspend [146].
20. Prepare a slightly turbid suspension that
matches with the turbidity of 0.5 McFarland
standard.
21. To eliminate the adverse effects of volatile
metabolic end products of adjacent reactions,
if any.
22. To get a comprehensive record of all the
results, including morphological observa-
tions of hyphae, chlamydospores, arthro-
conidia, germ tube formation, capsules, and
phenol oxidase activity [156].
References
1. Jimenez L (2007) Microbial diversity in pharmaceu-
tical product recalls and environments. PDA J Pharm
Sci Tech 61:383–399
2. Meyer SAM, Payne RW, Yarrow D (1988) Candida.
In: Kurtzman CP, Fell JW (eds) The yeasts: a taxo-
nomic study. Elsevier Science Publishers,
Amsterdam, The Netherlands, pp 476–477
3. Borman AM, Linton CJ, Miles SJ, Johnson EM
(2008) Molecular identification of pathogenic fungi.
J Antimicrob Chemother 61:i7–i12
4. Williamson PR (1994) Biochemical and molecular
characterization of the diphenol oxidase of
Cryptococcus neoformans: identification as a lac-
case. J Bacteriol 176:656–664
5. Guo LD, Huang GR, Wang Y, He WH, Zheng WH,
Hyde KD (2003) Molecular identification of white
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
morphotype strains of endophytic fungi from Pinus
tabulaeformis. Mycol Res 107:680–688
6. Hinrikson HP, Hurst SF, Lott TJ, Warnock DW,
Morrison CJ (2005) Assessment of ribosomal large-
subunit D1-D2, internal transcribed spacer 1, and
internal transcribed spacer 2 regions as targets for
molecular identification of medically important
Aspergillus species. J Clin Microbiol 43:2092–2103
7. Flórez AB, Álvarez-Martín P, López-Díaz TM,
Mayo B (2007) Morphotypic and molecular
identification of filamentous fungi from Spanish
blue-veined Cabrales cheese, and typing of
Penicillium roqueforti and Geotrichum candidum
isolates. Int Dairy J 17:350–357
8. Nilsson RH, Kristiansson E, Ryberg M, Hallenberg
N, Larsson KH (2008) Intraspecific ITS variability
in the kingdom Fungi as expressed in the International
Sequence Databases and its implications for molecu-
lar species identification. Evol Bioinform Online
4:193–201
9. Rodriguez-Tudela JL, Diaz-Guerra TM, Mellado E,
Cano V, Tapia C, Perkins A et al (2005) Susceptibility
patterns and molecular identification of Trichosporon
species. Antimicrob Agents Chemother
49:4026–4034
10. Cooke DEL, Drenth A, Duncan JM, Wagels G,
Brasier CM (2000) A molecular phylogeny of
Phytophthora and related Oomycetes. Fungal Genet
Biol 30:17–32
11. Bertini L, Agostini D, Potenza L, Rossi I, Zeppa S,
Zambonelli A et al (1998) Molecular markers for the
identification of the ectomycorrhizal fungus, Tuber
borchii. New Phytol 139:565–570
12. Galagan JE, Calvo SE, Borkovich KA, Selker EU,
Read ND, Jaffe D et al (2003) The genome sequence
of the filamentous fungus Neurospora crassa. Nature
422:859–868
13. Brookman JL, Mennim G, Trinci APJ, Theodorou
MK, Tuckwell DS (2000) Identification and charac-
terization of anaerobic gut fungi using molecular
methodologies based on ribosomal ITS1 and 18S
rRNA. Microbiology 146:393–403
14. Ciardo DE, Schär G, Böttger EC, Altwegg M,
Bosshard PP (2006) Internal transcribed spacer
sequencing versus biochemical profiling for
identification of medically important yeasts. J Clin
Microbiol 44:77–84
15. Hussain Z, Martin A, Youngberg GA (2001)
Blastmyces dermatitides with large yeast forms.
Arch Pathol Lab Med 125:663–664
16. Chandler FW, Watts JC (1987) Pathologic diagnosis
of fungal infections. ACSP Press, Chicago, pp
193–263
17. Haque AK, McGinnis MR (2008) Dail and Hammer’s
Pulmonary Pathology. Springer, New York
18. Woods GL, Walker DH (1996) Detection of infec-
tion or infectious agents by use of cytologic and his-
tologic stains. Clin Microbiol Rev 9:382–404
19. Ryan KJ, Ray CG (2004) Sherris medical microbiol-
ogy. McGraw-Hill, New York
253
20. Kim SO (2003) Molds identification. Kor J Med
Mycol 8:97–102
21. Nag DR, Chatterjee S, Chatterjee S, Khan M (2002)
Role of potassium hydroxide mount in rapid diagnosis
of fungal corneal ulcers. J Ind Med Assoc 100:18–20
22. Shearer CA, Langsam DM, Longcore JE (2004)
Fungi in freshwater habitats. In: Mueller GM, Bills
GF, Foster MS (eds) Biodiversity of fungi, inventory
monitoring methods. Elsevier Science Publishers,
New York, pp 520–521
23. Parija SC, Prabhakar PK (1995) Evaluation of lacto-
phenol cotton blue for wet mount preparation of
feces. J Clin Microbiol 33:1019–1021
24. Larone DH (2002) Medically important fungi: a
guide to identification. ASM Press, Washington,
D.C
25. Chapin KC (2007) Principles of stains and media.
In: Murray PR, Baron EJ, Landry ML, Jorgensen JH,
Pfaller MA (eds) Manual of clinical microbiology.
ASM Press, Washington, D.C., pp 182–191
26. Kern ME, Blevins KS (1997) Laboratory procedures
for fungal culture and isolation in medical mycol-
ogy. F.A. Davis, Philadelphia
27. Gurr E (1966) Rational use of dyes in biology.
Williams & Wilkins, Baltimore, pp 276–277
28. Humason GL (1967) Animal tissue techniques. W.H.
Freeman, San Francisco
29. Murray PR, Baron EJ, Pfaller MA, Tenover FC,
Yolken RH (1999) Manual of clinical microbiology.
ASM Press, Washington, D.C
30. Brucker MC (1986) Gram staining a useful labora-
tory technique. J Nurse Midwifery 31:156–158
31. Clark G (1981) Staining procedure. Williams &
Wilkins, Baltimore
32. Clarridge JE, Mullins JM (1987) Microscopy and
staining. In: Howard BJ, Klass J, Weissfeld AS,
Tilton RC (eds) Clinical and pathogenic microbiol-
ogy. C.V. Mosby, St. Louis, pp 87–103
33. Macher AM, Shelhamer J, MacLowery JD, Parker
M, Masur H (1983) Pneumocystis carinii identified
by Gram stain of lung imprints. Ann Intern Med
99:484–485
34. Bottone EJ (1980) Cryptococcus neoformans: pit-
falls in diagnosis through evaluation of Gram-stained
smears of purulent exudates. J Clin Microbiol
12:790–791
35. Felegie TP, Pasculle AW, Dekker A (1984)
Recognition of Pneumocystis carinii by Gram stain
in impression smears of lung tissue. J Clin Microbiol
2:1190–1191
36. Mahan CT, Sale GE (1978) Rapid methenamine sil-
ver stain for Pneumocystis and fungi. Arch Pathol
Lab Med 102:351–352
37. Ward EWB, Ciurysek KW (1961) Somatic mitosis in
a basidiomycete. Can J Bot 39:1497–1503
38. Wilson AD (1992) A versatile Giemsa protocol for
permanent nuclear staining of fungi. Mycologia
84:585–588
39. Lillie RD (1965) Histopathologic techniques and
practical histochemistry. McGraw-Hill, New York
254
40. Lillie RD (1977) H. J. Conn’s biological stains.
Williams & Wilkins, Baltimore, pp 289–291
41. Domingo J, Waksal HW (1984) Wright’s stain in
rapid diagnosis of Pneumocystis carinii. Am J Clin
Pathol 81:511–514
42. Pattengale P (2005) Task for the veterinary assis-
tants. Lippincott Williams & Wilkins, Baltimore, pp
258–260
43. Kiernan JA (2008) Histological and histochemical
methods: theory and practice. Scion, Bloxham, UK
44. Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear
stains with soluble metachrome mordant lake dyes -
the effect of chemical endgroup blocking reactions
and the artificial introduction of acid groups into tis-
sues. Histochemistry 49:23–35
45. Llewellyn BD (2009) Nuclear staining with alum-
hematoxylin. Biotech Histochem 84:159–177
46. Puchtler H, Meloan SN, Waldrop FS (1986)
Application of current chemical concepts to metal-
hamatein and -brazilein stains. Histochemistry
85:353–364
47. Bancroft JD, Stevens A (1982) Theory and practice
of histological techniques. Churchill Livingstone,
London, UK, pp 188–201
48. Kronvall G, Myhre E (1977) Differential staining of
bacteria in clinical specimens using acridine orange
buffered at low pH. Acta Pathol Microbiol Scand
Sect B 85:249–254
49. De Brauwer E, Jacobs J, Nieman F, Bruggeman C,
Drent M (1999) Test characteristics of acridine
orange, Gram and May-Grunwald-Giemsa stains for
enumeration of intracellular organisms in bronchoal-
veolar lavage fluid. J Clin Microbiol 37:427–429
50. Lauer BA, Reller LB, Mirret S (1981) Comparison
of acridine orange and Gram stains for detection of
microorganisms in cerebrospinal fluid and other
clinical specimens. J Clin Microbiol 14:201–205
51. Culling CFA (1974) Handbook of histopathologi-
cal and histochemical techniques. Butterworth,
London, UK
52. Mote RF, Muhm RL, Gigstad DC (1975) A staining
method using acridine orange and auramine O for
fungi and mycobacteria in bovine tissue. Stain
Technol 50:5–9
53. Pickett JP, Bishop CM, Chick EW, Baker RD (1960)
A simple fluorescent stain for fungi: selective stain-
ing of fungi by means of a fluorescent method for
mucin. Am J Clin Pathol 34:197–202
54. Drury RAB, Wallington EA (1980) Carleton’s his-
tological technique. Oxford University Press,
Oxford, UK
55. Luna L (1980) Manual of histologic staining meth-
ods. Armed Forces Institute of Pathology,
Washington, D.C., pp 228–229
56. Hageage GJ, Harrington BJ (1984) Use of calcofluor
white in clinical mycology. Lab Med 15:109–112
57. Monheit JE, Cowan DF, More DG (1984) Rapid
detection of fungi in tissue using calcoflour white
and fluorescence microscopy. Arch Pathol Lab Med
108:616–618
J. Sangeetha and D. Thangadurai
58. McGowna KL (1987) Practical approaches to diag-
nosing fungal infections in immunocompromised
patients. Clin Microbiol Newsletter 9:33–36
59. Baselski VS, Robinson MK, Pifer LW, Woods DR
(1990) Rapid detection of Pneumocystis carinii in
bronchoalveolar lavage samples by using Celluflour
staining. J Clin Microbiol 28:393–394
60. Totty BA (2002) Mucins. In: Bancroft JD, Gamble
M (eds) Theory and practice of histological tech-
niques. Churchill Livingstone, Edinburgh, pp
163–200
61. Prophet EB, Mills B, Arrington JB, Sobin LH (1992)
Laboratory methods in histotechnology. Armed
Forces Institute of Pathology, Washington, D.C
62. Mikel UV (1994) Advanced laboratory methods in
histology and pathology. Armed Forces Institute of
Pathology, Washington, D.C
63. Mallory FB (1961) Pathological techniques. Hafner
Publishing Co., New York
64. Sheehan D, Hrapchak B (1980) Theory and practice
of histopathology. Battelle Press, Columbus, OH
65. Jackson JA, Kaplan W, Kaufman L (1983)
Development of fluorescent-antibody reagents for
demonstration of Pseudallescheria boydii in tissue. J
Clin Microbiol 18:668–673
66. Grocott RG (1955) A stain for fungi in tissue sec-
tions and smears. Am J Clin Pathol 25:975–979
67. Schumann GB, Swensen JJ (1991) Comparison of
Papanicolaou’s stain with the Gomori methenamine
silver (GMS) stain for the cytodiagnosis of
Pneumocyctis catrinii in bronchoalveolar lavage
(BAL) fluid. Am J Clin Pathol 95:583–586
68. Hayashi I, Tome Y, Shimosato Y (1989)
Thiosemicarbazide used after periodic acid makes
methenamine silver staining of renal glomerular
basement membranes faster and cleaner. Stain
Technol 64:185–190
69. Lazcano O, Speights VO Jr, Stickler JG, Bilbao JE,
Becker J, Diaz J (1993) Combined histochemical
stains in the differential diagnosis of Cryptococcus
neoformans. Mod Pathol 6:80–84
70. Witebsky FG, Andrews JWB, Gill VJ, MacLowry
JD (1988) Modified toluidine blue O stain for
Pnemocystis corinii: further evaluation of some
technical factors. J Clin Microbiol 26:774–775
71. Gosey LL, Howard RM, Witebsky FG, Ognibene
FP, Wu TC, Gill VJ et al (1985) Advantages of a
modified toluidine blue O stain and bronchoalveolar
lavage for the diagnosis of Pneumocystis carinii
pneumonia. J Clin Microbiol 22:803–807
72. Crookham JN, Dapson RW (1991) Hazardous chem-
icals in the histopathology laboratory: regulations,
risks, handling and disposal. Anatech Ltd., Battle
Creek, MI
73. Furtado JS (1970) Alcoholic toluidine blue: a rapid
method for staining nuclei in unfixed mycetozoa and
fungi. Mycologia 62:406–407
74. Paterson RRM, Bridge PD (1994) Biochemical tech-
niques for filamentous fungi. CAB International,
Wallingford, UK
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
75. Wickerham LJ (1943) A simple technique for the
detection of melibiose-fermenting yeasts. J Bacteriol
46:501
76. Wickerham LJ (1946) A critical evaluation of the
nitrogen assimilation tests commonly used in the
classification of yeasts. J Bacteriol 52:293
77. Wickerham LJ, Burton KA (1948) Carbon assimila-
tion tests for the classification of yeasts. J Bacteriol
56:363
78. Schwarz P, Lortholary O, Dromer F, Dannaoui E
(2007) Carbon assimilation profiles as a tool for
identification of zygomycetes. J Clin Microbiol
45:1433–1439
79. Hobbie EA, Watrud LS, Maggard S, Shiroyama T,
Rygiewicz PT (2003) Carbohydrate use and assimi-
lation by litter and soil fungi assessed by carbon iso-
topes and BIOLOG® assays. Soil Biol Biochem
35:303–311
80. Steadham JE, Geis PA, Simmank JL (1986) Use of
carbohydrate and nitrate assimilations in the
identification of dematiaceous fungi. Diagn
Microbiol Infect Dis 5:71–75
81. Middelhoven WJ, Hoog GS, Notermans S (1989)
Carbon assimilation and extracellular antigens of
some yeast-like fungi. Antonie Van Leeuwenhoek
55:165–175
82. Murray IG (1968) Some aspects of the biochemical
differentiation of pathogenic fungi: a review. J Gen
Microbiol 52:213–221
83. Sangtiean T, Schmidt S (2002) Growth of subtropi-
cal ECM fungi with different nitrogen sources using
a new floating culture technique. Mycol Res
106:74–85
84. Watkinson S, Bebber D, Darrah P, Fricker M, Tlalka
M (2006) The role of wood decay fungi in the carbon
and nitrogen dynamics of the forest floor. In: Gadd
GM (ed) Fungi in biogeochemical cycles. Cambridge
University Press, Cambridge, UK, pp 151–181
85. Crawford NM, Arst HN (1993) The molecular genet-
ics of nitrate assimilation in fungi and plants. Annu
Rev Genet 27:115–146
86. Krappmann S, Braus GH (2005) Nitrogen metabo-
lism of Aspergillus and its role in pathogenicity. Med
Mycol 43:S31–S40
87. Gorfer M, Blumhoff M, Klaubauf S, Urban A,
Inselsbacher E, Bandian D, et al. (2011) Community
profiling and gene expression of fungal assimilatory
nitrate reductases in agricultural soil. ISME J.
Accessed 12 May 2011; doi: 10.1038/ismej.2011.53
88. Keller G (1996) Utilization of inorganic and organic
nitrogen sources by high-subalpine ectomycorrhizal
fungi of Pinus cembra in pure culture. Mycol Res
100:989–998
89. Ye ZH, Garrad RC, Winston MK, Bhattacharjee JK
(1991) Use of a-aminoadipate and lysine as sole
nitrogen source by Schizosacharomyces pombe and
selected pathogenic fungi. J Basic Microbiol
31:149–156
90. Bhavan PS, Rajkumar R, Radhakrishnan S,
Seenivasan C, Kannan S (2010) Culture and
255
identification of Candida albicans from vaginal
ulcer and separation of enolase on SDS-PAGE. Int J
Biol 2:84–93
91. Freydiere AM, Robert R, Ploton C, Marot-Leblond
A, Monerau F, Vandenesch F (2003) Rapid
identification of Candida glabrata with a new com-
mercial test, GLABRATA RTT. J Clin Microbiol
41:3861–3863
92. Felek S, Asci Z, Kilic SS, Yilman M, Kokcamm I
(1989) Yeasts and yeast like fungi as causative agents
in diarrhea. J Islam Acad Sci 2:182–184
93. Fontes CO, Carvalho MAR, Nicoli JR, Hamdan JS,
Mayrink W, Genaro O et al (2005) Identification and
antimicrobial susceptibility of micro-organisms
recovered from cutaneous lesions of human
American tegumentary leishmaniasis in Minas
Gerais, Brazil. J Med Microbiol 54:1071–1076
94. Bonfante R, Barroeta S (1966) Development and
evaluation of a rapid identification test for Candida
albicans. Mycopathologia 34:33–39
95. Sandven P (1990) Laboratory identification and sen-
sitivity testing of yeast isolates. Acta Odontol Scand
48:27–36
96. Waltimo TMT, Sirén EK, Torkko HLK, Olsen I,
Haapasalo MPP (2003) Fungi in therapy-resistant
apical periodontitis. Int Endodont J 30:96–101
97. Land GA, Salkin IF, Zaatari ME, McGinnis MR,
Hashem G (1991) Evaluation of the Baxter-
MicroScan 4-hour enzyme-based yeast identification
system. J Clin Microbiol 29:718–722
98. Huppert M, Harper G, Sun SH, Delanerolle V (1975)
Rapid methods for identification of yeasts. J Clin
Microbiol 2:21–34
99. Merheb CW, Cabral H, Gomes E, Da-Silva R (2007)
Partial characterization of protease from a thermo-
phillic fungus, Thermoascus auranthacus, and its
hydrolytic activity on bovine casein. Food Chem
104:127–131
100. Rodarte MP, Dias DR, Vilela DM, Schwan RF
(2011) Proteolytic activities of bacteria, yeast and
filamentous fungi isolated form coffee fruit (Coffea
arabica L.). Acta Scientiarum Agron 33:457–464
101. Eriksson K (1978) Enzyme mechanisms involved in
cellulose hydrolysis by the rot fungus Sporotrichum
pulverulentum. Biotechnol Bioengineer 20:317–332
102. Hankin L, Anagnostakis SL (1977) Solid media con-
taining carboxymethyl cellulose to detect Cx cellu-
lose activity of micro-organisms. J Gen Microbiol
98:109–115
103. Tomme P, Warren RA, Gilkes NR (1995) Cellulose
hydrolysis by bacteria and fungi. Adv Microb
Physiol 37:1–81
104. Ali-Shtayeh MS, Jamous RMF, Abu-Ghdeib SI
(1999) Ecology of cycloheximide-resistant fungi in
field soils receiving raw city wastewater or normal
irrigation water. Mycopathologia 144:39–55
105. Crouzet M, Begueret J (1980) A new mutant form of
the ribosomal protein L21 in the fungus Podospora
anserina: identification of the structural gene for this
protein. Mol Gen Genet 180:177–183
256
106. Bagy MM, El-Shanawany AA, Abdel-Mallek AY
(1998) Saprophytic and cycloheximide resistant
fungi isolated from golden hamster. Acta Microbiol
Immunol Hung 45:195–207
107. Caldwell BA, Castellano MA, Griffiths RP (1991)
Fatty acid esterase production by ectomycorrhizal
fungi. Mycology 83:233–236
108. Brunke S, Hube B (2006) MfLIP1, a gene encoding
an extracellular lipase of the lipid-dependent fungus
Malassezia furfur. Microbiology 152:547–554
109. Byrde RJW, Fielding AH (1955) Studies on the acet-
ylesterase of Sclerotinia laxa. Biochemistry
61:337–341
110. Rapp P, Backhaus S (1992) Formation of extracel-
lular lipases by filamentous fungi, yeasts and bacte-
ria. Enzyme Microbiol Technol 14:938–943
111. Shishiyama J, Araki F, Akai S (1970) Studies on
cutin-esterase II. Characeteristics of cutin-esterase
from Botrytis cinerea and its activity on tomato
cutin. Plant Cell Physiol 11:937–945
112. Singh R, Gupta N, Goswami VK, Gupta R (2006) A
simple activity staining protocol for lipases and
esterases. Appl Microbiol Biotechnol 70:679–682
113. Valiente C, Quesada E (1991) Morphologic and
physiologic characteristics of Costa Rica pathogenic
fungi (Dermatiaceae). Rev Biol Trop 39:103–106
114. Kitancharoen N, Hatai K (1998) Some biochemical
characteristics of fungi isolated from salmonid eggs.
Mycoscience 39:249–255
115. Reddy NG, Ramakrishna DPN, Gopal SVR (2011) A
morphological, physiological and biochemical stud-
ies of marine Streptomyces rochei (MTCC 10109)
showing antagonistic activity against selective
human pathogenic microorganisms. Asian J Biol Sci
4:1–14
116. Abrusci C, Martín-González A, Amo AD, Catalina
F, Collado J, Platas G (2005) Isolation and
identification of bacteria and fungi from cinemato-
graphic films. Int Biodeterior Biodegrad 56:58–68
117. Pointin SB (1999) Qualitative methods for the deter-
mination of lignocellulolytic enzyme production by
tropical fungi. Fungal Divers 2:17–33
118. Hayanko K, Tubaki K (1985) Origin and properties
of b-glucosidase activity of tomato-field soil. Soil
Biol Biochem 17:553–557
119. Zanoelo FF, Moraes MLT, Terenzi HF, Jorge JA
(2004) b-glucosidase activity from the thermophilin
fungus Scytalidium thermophilium is stimulated by
glucose and xylose. FEMS Microbiol Letters
240:137–143
120. Breuil C, Mayers P, Saddler JN (1986) Substrate
conditions that influence the assays used for deter-
mining the b-glucosidase activity of cellulolytic
microorganisms. Biotechnol Bioengineer
28:1653–1656
121. Kakde RB, Chavan AM (2011) Effect of carbon,
nitrogen, sulfur, phosphorus, antibiotic and vitamin
sources on hydrolytic enzyme production by storage
fungi. Recent Res Sci Technol 3:20–28
J. Sangeetha and D. Thangadurai
122. Iftikhar T, Niaz M, Hussain Y, Abbas SQ, Ashraf I,
Zia MA (2010) Improvement of selected strains
through gamma irradiation for enhanced lipolytic
potential. Pak J Bot 42:2257–2267
123. Kakde RB, Chavan AM (2011) Extracellular lipase
enzyme production by seed-borne fungi under the
influence of physical factors. Int J Biol 3:94–100
124. Mateos JC, Ruiz K, Rodriguez JA, Cordova J, Baratti
J (2007) Mapping substrate selectivity of lipases
from thermophilic fungi. J Mol Catal B: Enzymatics
49:104–112
125. Philpot C (1967) The differentiation of Trichophyton
mentagrophytes from T. rubrum by a simple urease
test. Sabouraudia 5:189
126. Shepard MC, Lunceford CD (1970) Urease color
test medium U-9 for the detection and identification
of “T” mycoplasmas in clinical material. Appl
Microbiol 20:539–543
127. Rosenthal SA, Sokolsky H (1965) Enzymatic stud-
ies with pathogenic fungi. Int J Dermatol 4:72–79
128. Mahmoud ALE, El-Shanawany AA, Omar SA
(1996) Factors affecting growth and urease produc-
tion by Trichophyton spp. Mycopathologia 135:
109–113
129. Wise MG, Healy M, Reece K, Smith R, Walton D,
Dutch W et al (2007) Species identification and
strain differentiation of clinical Candida isolates
using the DiversiLab system of automated repetitive
sequence-based PCR. J Med Microbiol 56:778–787
130. Healy M, Reece K, Walton D, Huong J, Frye S, Raad
II et al (2005) Use of the DiversiLab system for spe-
cies and strain differentiation of Fusarium species
isolates. J Clin Microbiol 43:5278–5280
131. Jackson CJ, Barton RC, Evans GV (1999) Species
identification and strain differentiation of dermato-
phyte fungi by analysis of ribosomal-DNA inter-
genic spacer regions. J Clin Microbiol 37:931–936
132. Shin JH, Sung JH, Park SJ, Kim JA, Lee JH, Lee DY
et al (2003) Species identification and strain differ-
entiation of dermatophyte fungi using polymerase
chain reaction amplification and restriction enzyme
analysis. J Am Acad Dermatol 48:857–865
133. Gherbawy Y, Voigt K (2010) Molecular identification
of fungi. Springer, Heidelberg
134. Bhardwaj S, Sutar R, Bachhawat AK, Singhi S,
Chakrabarti A (2007) PCR-based identification and
strain typing of Pichia anomala using the ribosomal
intergenic spacer region IGS1. J Med Microbiol
56:185–189
135. Lopes MB, Soden A, Martens AL, Henschke PA,
Langridge P (1998) Differentiation and species
identification of yeasts using PCR. Int J Syst Evol
Microbiol 48:279–286
136. Gupta AK, Kohli Y, Summerbell RC (2000)
Molecular differentiation of seven Malassezia spe-
cies. J Clin Microbiol 38:1869–1875
137. Hopfer RL, Walden P, Setterquist S, Highsmith WE
(1993) Detection and differentiation of fungi in clin-
ical specimens using polymerase chain reaction
19 Staining Techniques and Biochemical Methods for the Identification of Fungi
(PCR) amplification and restriction enzyme analysis.
Med Mycol 31:65–75
138. Odds FC, Abbott AB (1980) A simple system for the
presumptive identification of Candida albicans and
differentiation of strains within the species. Med
Mycol 18:301–317
139. Land GA, Harrison BA, Hulme KL, Cooper BH,
Byrd JC (1979) Evaluation of the New API 20C strip
for yeast identification against a conventional
method. J Clin Microbiol 10:357–364
140. McGinnis MR (1980) Laboratory handbook of med-
ical mycology. Academic, New York
141. Wadlin JK, Hanko G, Stewart R, Pape J, Nachamkin
I (1999) Comparison of three commercial systems
for identification of yeasts commonly isolated in the
clinical microbiology laboratory. J Clin Microbiol
37:1967–1970
142. Sand C, Rennie RP (1999) Comparison of three
commercial systems for the identification of germ-
tube negative yeast species isolated from clinical
specimens. Diagn Microbiol Infect Dis 33:223–229
143. Espinel-Ingroff A, McGinnis MR, Pincus DH,
Goldson PR, Kerkering TM (1989) Evaluation of the
API 20C Yeast Identification System for the differ-
entiation of some dematiaceous fungi. J Clin
Microbiol 27:2565–2569
144. Rath AC, Carr CJ, Graham BR (1995)
Characterization of Metarrhizium anisopliae strains
by carbohydrate utilization (API 50 CH). J Invertebr
Pathol 65:152–161
145. Wasfy EH, Bridge PD, Brayford D (1987)
Preliminary studies on the use of biochemical and
physiological tests for the characterization of
Fusarium isolates. Mycopathologia 99:9–13
146. Heelan JS, Sotomayor E, Coon K, D’Arezzo JB
(1998) Comparison of the rapid yeast plus panel
with the API20C Yeast System for identification of
clinically significant isolates of Candida species. J
Clin Microbiol 36:1443–1445
147. Aubertine CL, Rivera M, Rohan SM, Larone DH
(2006) Comparative study of the new colorimetric
VITEK 2 yeast identification card versus the older
fluorometric card and of CHROM agar Candida as a
257
source medium with the new card. J Clin Microbiol
44:227–228
148. Fricker-Hidalgo H, Vandapel O, Duchesne MA,
Mazoyer MA, Monget D, Lardy B et al (1996)
Comparison of the new API Candida System to the
ID 32C system for identification of clinically impor-
tant yeast species. J Clin Microbiol 34:1846–1848
149. Ahmad S, Khan Z, Mustafa AS, Khan ZU (2002)
Seminested PCR for diagnosis of Candidemia: com-
parison with culture, antigen detection, and bio-
chemical methods for species identification. J Clin
Microbiol 40:2483–2489
150. Moghaddas J, Truant AL, Jordan C, Buckley HR
(1999) Evaluation of the RapID Yeast Plus System
for the identification of yeast. Diagn Microbiol Infect
Dis 35:271–273
151. Costa AR, Silva F, Henriques M, Azeredo J, Oliveira
R, Faustino A (2010) Candida clinical species
identification: molecular and biochemical methods.
Ann Microbiol 60:105–112
152. Rohm H, Lechner F, Lehner M (1990) Evaluation of
the API ATB 32C System for the rapid identification
of foodborne yeasts. Int J Food Microbiol 11:
215–223
153. Sekhon AS, Padhye AA, Garg AK, Pruitt WR (1987)
Evaluation of the Abbott Quantum II yeast
identification system. Mycoses 30:408–411
154. Qadri SMH, Flournoy DJ, Qadri SGM, Ramirez EG
(1986) Rapid identification of yeasts by semi-auto-
mated and conventional methods. Med Microbiol
Immunol 175:307–316
155. Kiehn TE, Edwards FF, Tom D, Lieberman G,
Bernard EM, Armstrong D (1985) Evaluation of the
Quantum II yeast identification system. J Clin
Microbiol 22:216–219
156. Salkin IF, Schadow KH, Bankaitis LA, McGinnis
MR, Kemna ME (1985) Evaluation of Abbott
Quantum II yeast identification system. J Clin
Microbiol 22:442–444
157. Cooper BH, Prowant S, Alexander B, Brunson DH
(1984) Collaborative evaluation of the Abbott
yeast identification system. J Clin Microbiol 19:
853–856