COMMENT AND CONTROVERSY

Edited by Stephen P. Stone, MD

Origins of the KOH technique

Trisha Dasgupta MD
a
, Joya Sahu MD
b,

a
Albert Einstein Medical Center, 5501 Old York Rd Philadelphia, PA 19141
b
Dermatology and Cutaneous Biology, Jefferson Medical College of Thomas Jefferson University, 833 Chestnut St, Ste 740,
Philadelphia, PA 19107
The potassium hydroxide (KOH) procedure is an integral
diagnostic procedure, commonly used in daily dermatology
practice but with vague origins. Given the many eponyms
that exist in dermatologyboth for the discovery of a
disease and for the pioneering of a techniqueit is
surprising that no historical consensus exists on the inception
of this procedure, nor much scholarly commentary on
notable evolutions in technique or utilized materials.
Modern dermatology textbooks are notably vague on the
matter. Is it because the KOHexamination has been considered
a largely apprenticed practiceevading widespread descrip-
tion in print and rather kept alive through anecdotal teachings
from mentor to trainee? Alternatively, have we technically
evolved such that this procedure, with individual adaptations,
is largely accepted and practiced in a uniform and systematic
manner? The answers seem yes, for contemporary text-
book editors provide little detail about the KOH preparation.
After its introduction, the KOH examination was received
with great aplomb as a novel technique and acquired
subsequent modifications to mature into an efficient,
diagnostic method of choice. A retrospective exploration of
the literature appears to confirm the former, although the
occasional homage to this largely unheralded examination
technique is noted herein.
The beginnings
It behooves this discussion to begin with the crucial
recognition that certain dermatologic conditionsthose
classified today as dermatophytoseswere, in fact, caused
by fungi. With the advancements of microscopy, particularly
the evolution of achromatic lenses from 1810 through 1830,
visualization of larger-sized parasites (eg, fungi) became
possible.
1
According to Erasmus Wilson,* Robert Remak
first appreciated the appearance of fungoid filaments in
the crusts of favus infection in 1836, followed by con-
firmatory efforts by Johann Schoenlein in 1839 and Reinhart
Fuchs and Bernhard Langenbeck shortly thereafter.
2
David
Gruby's contribution
3
was the discovery of the fungus
responsible for what ultimately became known as tinea
capitis,
4
having visualized the following:
[I]mmediately within the epidermis is a thin layer of amor-
phous substance, composed of minute molecules; this layer
is dense, of a sulphur-yellow colour, and forms a capsule,
which is in contact by its external surface with the epi-
dermis, and by its internal surface with a fungous growth.
5

Editors comments: The following contribution provides an

outstanding review of the history of mycology by focusing on the
development of a diagnostic technique that has persistedbut has perhaps
not evolved a great dealthroughout many generations of physicians. At
the conclusion of this contribution, I shall mention a few newer
modifications of the technique and solicit suggestions from readers for
alternative means of demonstrating fungal organisms ex vivo.

Corresponding author.
E-mail address: joya.sahu@jefferson.edu (J. Sahu).
* It is somewhat ironic that the great Englishman had disdain for the
fungal etiology of ringworm and even offered a prize if this could be
disproved. (Ainsworth CG. The history of veterinary and medical
mycology. Cambridge: Cambridge University Press; 1986:40).
0738-081X/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.clindermatol.2011.11.020
Clinics in Dermatology (2012) 30, 238242
Pehr Malmsten further elucidated this pathogen, naming it
tricophyton tonsurans, in 1846.
4
Taken together, these
early discoveries warrant further enquiry into the origins and
evolution of the KOH procedure.
The earliest primary documentation we have obtained of
technique for the microscopic examination of dermatophytes
comes from John Bennett in 1842, in which there is no
mention of KOH use.
6
Bennett, in his endeavor to confirm
and extend the observations and experiments of Gruby
concerning the mycodermatous vegetations found in the
crusts of the disease named Tinea favosa, describes his
study of the similarly crusted lesions of a boy's scalp. On the
12th day of observing the boy's lesions, Bennett removed
from the posterior scalp one of several bright yellow spots,
the size of a pin's head, somewhat depressed below the
surface. With a lancet tip, he detached the lesion, and with
microscopic examination, he located a:
smooth, cupped-shaped, bright yellow capsule, the
diameter of which was about 1/20 of an inch... . On
placing this capsule in a drop of water, pressing it
between two slips of glass, and examining it with a
magnifying power of 800 diameters, it was found to be
composed of an amorphous mass, in which were
numerous long-jointed filamentous tubes... seen coming
from the edge of the capsule, as M. Gruby has
described... The further development of the plants, and
of the disease, appeared to be exactly as M. Gruby has
described it.
Of note, Bennett made reference to solvent use only,
when needing to destroy mycotic lesions inoculated upon his
own scalp, using frequent application of caustic to remedy
the affliction.
6-8
Are fungi pathogens?
There followed a period during the mid-19th century in
which there was not a consensus on the fungal etiology of
ringworm infections, and hence (amidst such dispute), it
would have been difficult for the KOH procedure to become
the established standard for diagnosis.
9
Yet, those who
remained steadfast in their support of a mycological etiology
continued to develop methodology to visualize best the
pathogenic fungi.
Among the earliest accounts highlighting the utility of
KOH in the microscopic examination of dermatophytes is
that by Tilbury Fox in The Lancet in 1878. He writes that in
the case of ringworm of the body... it is very important that
thin layers of tissue should be taken for examination, and that
these should be well soaked in weak potash solution, and
carefully flattened out without any rougher manipulation.
10
George Thin advocated similarly for the use of KOH in
identifying fungal elementsspecifically of T tonsurans
in scalp hairs suspicious for dermatophyte infection,
specifying in 1882 that the ringworm hairs were picked
out, macerated in potash solution, and examined in his
investigative methodology.
11
It may have been Raymond Sabouraud who popularized
the use of KOH in microscopic examination of dermato-
phytes through a more detailed description of his technique
in his 1894 work, Les Trichophyties Humaines.
12
His
writings spawned a flurry of acceptance and praise, as well
as detailed modifications from contemporaries in Europe and
the United States. In an 1895 paper in The British Journal of
Dermatology, Horatio Adamson leaned on Sabouraud's
establishment of the superiority of the KOH technique to
promote his own modification of a staining technique that
would allow for preservation of the KOH-soaked specimen:
Under the influence of the potash solution the horny
structures swell up and become softened, and at the same
time lose their keratin nature, so that they subsequently
decolorize readily, as do other non horny epithelial
tissues. The previous use of potash, moreover, enables
one to first examine the specimen in the ordinary way,
and afterwards to secure a permanent record of any
appearance desired. Specimens of hair, or of scales,
are placed on a slide and covered with a drop of a 5 to
10 per cent solution of caustic potash and a cover glass
applied.... They may remain here for from ten minutes to
half an hour, according to the rapidity with which the
clearing takes place, or according to the particular
appearances one may wish to record... for staining the
elements of the fungus simply, without regard to their
relations to the hair, a longer period is required, and the
specimen may also be flattened out by pressure of the
cover glass.
13
In the same journal, the following year, T. Colcott Fox
offered alterations to the potency of and duration for which
the KOH solution should be applied to collected specimens:
In preparing for the microscopical examination of the
lesions we found the most convenient course was to
collect material from each case in tiny bottles of ether.
The specimens could then be retained for subsequent
examination, as it was impossible to devote the
necessary time in the out-patient room. The ether was
subsequently poured off, and replaced by Liquor potasse
of about 7 percent strength. Sabouraud recommends that
hairs should be placed in Liquor potasse of 40 per cent
strength, and then carefully warmed. This proceeding
may be useful for a hurried immediate examination,
especially with dark hairs, but we have found the time-
honoured soaking in Liquor potasse of about seven per
cent strength very effective. The length of time for which
the hair should soak in this reagent varies from a few
hours to a few days, according to the size, quality, and
pigmentation of the hairs. It is often desirable to watch
the clearing of the hair under the influence of Liquor
potasse, but as a rule it is perfectly satisfactory to make
the examination after the soaking. Beautiful specimens of
the fungus, apart from the details of its localization, may
be obtained by a prolonged soaking until the hair
structure is completely disintegrated. The softened
specimens should be floated on to the slide, and care
239 Origins of the KOH technique
taken not to disarrange the position of the fungus by
pressure of the cover-glass.
14
At a societal meeting of physicians in Boston in 1897,
Charles J. White credited Sabouraud with entirely revolu-
tioniz[ing] our former simple ideas regarding ringworm and
subsequently offered his own commentary on KOH
examination method:
For the quick examination of the [hair] specimens I
should recommend placing a few drops of ether over the
hair or the scale to remove the fat, and secondly a few
drops of a 40 per cent solution of caustic potash to
dissolve the keratin. Place a cover-slip gently over the
specimen and in a few minutes examine the slide with
most of the light excluded. Don't mount in glycerine, for
the refraction of the fungus and the glycerine is so nearly
alike that the plant will be scarcely discernible.
15
By this era, scholarly sentiment clearly favored the fungal
etiology of ringworm infection and the use of a simple,
clinic-based microscopic technique involving KOH for rapid
confirmation of the diagnosis.
KOH in the 20th century
With the utility of the KOH examination established,
commentary on the technique in the 20th century was
sporadic and largely illustrative of individual clinicians
(often-nuanced) preferences in specimen preparation.
16
For example, the Journal of Investigative Dermatology
featured a symposium in 1940 regarding the management of
common fungal infections, in which survey responses were
compiled from several prominent dermatologists across the
United States.
17
Arthur Schoch of Baylor University suggested that all
cases which fail to present a typical clinical appearance
are subjected to microscopic examination for fungi,
using 20 percent potassium hydroxide and allowing at
least twelve to twenty four hours to elapse before
examining the slide for fungi.
Jacob Swartz of the Massachusetts General Hospital
likewise offered his version: The skin scale taken from
the border of an active area or from the roof or a vesicle,
or the nail material is treated with 1040% potassium
or sodium hydroxide, slightly heated and allowed to
clear. Further cautioning against mistaking mosaic-
like structures, potassium hydroxide crystals and other
artifacts for fungal elements, Swartz advocated the use
of a lactophenol cotton blue stain to minimize the
appearance of confounding structures.
Similar themes emerged in subsequent writings, with
dispensing of tips and tricks to improve yield, accuracy, or
expediency of the KOH examination. As Walter Green and
Maurice Shepard warned in the Archives of Dermatology
and Syphilology, the novice in the field of medical
mycology may be readily deceived by potassium hydroxide
preparations of shavings of skin which extend down into the
corium,
18
discussing the potential confusion that elastic
fibers might generate when lesional skin samples are
examined for fungi:
Such specimens contain elastic tissue fibers which
withstand digestion by potassium hydroxide and heat.
The usual types of specimens examined in dermatologic
practice, such as scales, scrapings and peelings of
epidermis alone, naturally do not contain elastic tissue.
Under low power, high dry or oil immersion magnifica-
tion, the elastic tissue fibers appear to be a dense
mycelium of fine, nonseptate, intertwining and branch-
ing hyphae. Although elastic tissue fibers are known to
vary in thickness from a fraction of a micron to as much
as 11 microns in diameter, they appear deceptively
uniform in potassium hydroxide wet mounts. The
fluorescence of the elastic tissue fibers under ultraviolet
radiation may serve further to confuse the observer.
Nothing resembling spores is present.
Even mid-20th century luminaries of dermatology, such
as Albert M. Kligman of the University of Pennsylvania,
contributed modifications to the KOH examination, observ-
ing in 1951 that the enthusiasm and vigorousness of the
person doing the scraping
19
of affected skin highly
influenced the yield of mycotic elements, especially when
contending with the hyperkeratosis of acral surfaces.
Kligman's suggested practice of applying a few drops of
10 percent potassium hydroxide such as is ordinarily used to
mount scrapings... directly on the skin over the peripheral
portion of the lesion was among those whichlikely and
understandably for reasons of patient comfortdid not gain
widespread acceptance.
Of note, there were evolutions to the KOH examination in
this period that had broader appeal and are used currently in
modern practice. For example, Nardo Zaias and David
Taplin of the University of Miami observed through study of
T rubrum-infected scales of tinea corporis in 1966 that the
addition of dimethyl sulfoxide (DMSO) to [a 20% KOH]
solution dramatically shortens the time necessary for
clearing, does not dry out, clears thick scales and nail
samples, and imparts a transparency to the horny cells which
aids in the identification of the fungal elements.
20
DMSO is
frequently still used in preparation of fungal specimens for
microscopic examination because of its benefits, as noted
nearly a half-century ago.
Contemporary concepts
Upon examination of modern seminal textbooks of
dermatology, the discussion of KOH examination for cases
of suspected dermatophytosis is remarkably scant with
240 T. Dasgupta, J. Sahu
respect to sampling technique and slide preparation. The
Bolognia text, in fact, altogether avoids discussing clinical
specimen collection and notes simply, Tinea [infections] . .
. are usually diagnosed via KOH examination.
21
The Lever
book forgoes mention of the KOH examination, whereas
Weedon's treatise focuses on appropriate solvent selection:
It is our practice to use a solution that combines [10%] KOH
with glycerol (to prevent drying out) and calcofluor white, an
agent that imparts a bright fluorescence to fungi when
examined with a fluorescence microscope.
22,23
In contrast,
the Rook, Fitzpatrick, and Andrews books do offer advice for
high-yield sampling of lesional skin:
Rook: Disposable scalpel blades... held vertically to
the skin are used to obtain scrapings.... If the lesion has
a definite edge, the material should be taken from the
active margin, otherwise a general scraping is ade-
quate.... When blisters are present, a pair of fine
scissors may be used to cut off a blister roof.
24
Fitzpatrick: Skin samples should be taken by scraping
with a dull edge of a scalpel outward from the
advancing margins of a lesion.... For vesiculo-bullous
lesions [of tinea pedis/manuum], examination of the
roofs yields the highest rate of positivity on KOH
examination.
25
Andrews: [Obtain] copious dry scale from the instep,
heel, and sides of the foot . . . bullae should be unroofed
and either the entire roof mounted intact or scrapings
made from the underside of the roof.... A drop of a 10%
to 20% solution of KOH is added to the material on the
glass slide.
26
Conclusions
The identity of the pioneer(s) of the KOH examination
remains unclear. A literature review reveals the depth of
resistance instigated by both the revolutionary idea of fungi
as the etiologic infectious agents in dermatophytosis and the
subsequent introduction, evolution, and acceptance of the
KOH examination as the diagnostic procedure of choice in
confirming dermatophyte infections.
Today, the KOH examination is accepted in dermatology
as a fundamental, requisite skill and is taught in training
programs world-wide, and as such, perhaps the Bolognia text
is wise in briefly alluding to the examination but not
elucidating further, because every dermatologist surely
develops his or her own unique technique, based upon
mentorship and personal experience.
References
1. Long ER. A history of pathology. 1st ed. Baltimore: Williams &
Wilkins; 1928.
2. Wilson E. The student's book of cutaneous medicine and diseases of the
skin. New York: William Wood & Co; 1865.
3. Gruby D. Comptes rendus de l'Academie des Sciences, Paris 1842.
tome XV, p 512; 1842 tome xvii, p 301; 1844 tome xviii p 583.
4. Duhring LA. A practical treatise on diseases of the skin. Philadelphia:
Lippincott; 1877.
5. Wilson E. On diseases of the skin. London: Churchill; 1842.
6. Bennett JH. On the parasitic vegetable structures found growing in
living animals. Trans R Soc Edinburgh 1842;15:277-94.
7. Biddle JB. Review of materia medica, for the use of students.
Philadelphia: Lindsay and Blakiston; 1852. p. 278-82.
8. Potassium-Hydroxide.com [Internet]. Available at: http://www.
potassium-hydroxide.com/HistoryPotassium.htm. Accessed Sept 26,
2011.
9. Wilson ER. The student's book of cutaneous medicine and diseases of
the skin. New York: William Wood & Co; 1865.
10. Fox T. On ringworm of the head, and its management. Lancet
1877;110:719-22.
11. Thin G. Contributions to the pathology of parasitic diseases of the skin.
Br Med J 1882;2:301-5.
12. Sabouraud R. Les trichophyties humaines. Paris: Rueff & Cie; 1894.
13. Adamson HG. A note on the permanent staining of ringworm fungus.
Br Med J 1895;7:373-7.
14. Fox TC. An inquiry into the plurality of fungi causing ringworm in
human beings as met with in London. Br Med J 1896;VIII.
15. White CJ. Ringworm as it appears in Boston. J Boston Soc Med Sci
1897;1:1-5.
16. Ormsby OS, Mitchell JH. Ringworm of hands and feet. JAMA 1916;
67:711-7.
17. Investigations concerning actual methods employed in the management of
common dermatoses: II. Symposium on the practical management
of eczematous ringworm of the hands and feet. (Athlete's foot
dermatophytosis and dermatophytids). J Invest Dermatol 1940;3:523-62.
18. Green WS, Shepard MC. Semblance of elastic tissue to mycelium
in potassium hydroxide preparations. Arch Derm Syphilol 1945;52:115.
19. Kligman AM. Application of potassium hydroxide to the skin as an aid
in the direct examination of scales for fungi. Arch Derm Syphilol
1951;63:252.
20. Zaias N, Taplin D. Improved preparation for the diagnosis of mycologic
diseases. Arch Dermatol 1966;93:608-9.
21. Bolognia JL, Jorizzo JL, Rapini RP, editors. Dermatology. 2nd ed.
Philadelphia: Mosby; 2008.
22. Elder D, Elenitsas R, Johnson B, Murphy G, Xu X. Lever's
histopathology of the skin. 10th ed. Philadelphia: Lippincott Williams
& Wilkins; 2008.
23. Weedon D. Weedon's skin pathology. 3rd ed. Philadelphia: Churchill
Livingstone/Elsevier; 2009.
24. Burns T, Breathnach S, Cox N, Griffiths C, editors. Rook's textbook
of dermatology. 7th ed. Oxford: Blackwell Science; 2004.
25. Wolff K, Goldsmith L, Katz S, Gilchrest B, Paller A, Leffell D.
Fitzpatrick's dermatology in general medicine. 7th ed. New York:
McGraw-Hill; 2008.
26. James WD, Berger TG, Elston DM. Andrews diseases of the skin:
clinical dermatology. 10th ed. Philadelphia: Elsevier/Saunders; 2006.
E D I T O R ' S C O M M E N T A R Y
Within the last few decades, there have been some changesI would
suggest, improvementsto the old 5% to 20% KOH for the diagnosis of
fungal infections. Many have not been published in the mainstream
dermatologic literatureor at allbut have entered use by word of mouth.
For example, Dr. Stuart Brown, of Dallas, Texas, has recommended the use
of xylene to be added to the scales on the slide, and covered with a cover
slip. This provides three advantages: (1) the slide need not be warmed for
a rapid reading, (2) the keratinocytes are promptly cleared to view, though
not necessarily lysed, and (3) the xylene is both near at hand, being used
to clean the lenses of the microscope, and will not affect the surface of the
stage (or the glass of the lens) as does KOH.
241 Origins of the KOH technique
I personally use KOH in DMSO to which chlorazol black stain has been
added, The DMSO accelerates the lysis of the keratinocytes, and the stain
renders the fungal fibers much more visible, enhancing my ability to locate
them quickly.
For more superficial fungal specimens, such as suspicion of Tinea or
Pityriasis versicolor, I will use clear plastic adhesive tape to strip cells
from the surface, then invert the tape on a slide, and add a drop of
Paragon Multiple Stain, and the fungal elements appear to jump off
the slide, especially if the excess stain is gently rinsed off with water.
So now, I the Editor ask you, the Reader: have you a trick for confirming
the diagnosis of Tinea? Or do you prefer the time honored 5% to 20%
KOH? Do you still use an alcohol lamp? Or have you moved on to
DMSO or the microwave? Please let me hear from you, at sstone@
siumed.edu.
242 T. Dasgupta, J. Sahu