DIAGNOSTIC TISSUE

MICROBIOLOGY METHODS
Nelson S. Brewer, M.D.,* aml Lyle A. Weed, M.D.~

Abstract

Tissue specimens, when processed properly, nmy yield important microbi-

ologic information. Various techniques have evolved lor specimen collection and
transport, processing of tissue specimens, and demonstration of organisms b)'
staining histologic material that enhance tim identification of infectious agents.
Carefifl attention to these details may yield diagnostic information that might
otherwise be missed.

One important flmction of tim diag- other diagnostic procedures, information

nostic microbiology laboratory is the micro-
biologic study of tissues. A number of tech-
niques have evolved that enhance the
demonstration of organisms b)' Ifistologic
staining and by recovery in cultures. With
attention to details of specimen collection
and transport, special s.taining procedures,
prepm'ation of tissue for culturing, and
inoculation of the processed specimen onto
the appropriate media, it is possible to ob-
tain valuable diagnostic information that
might otherwise be missed. These tech-
niques are especially important in the eval-
uation of chronic lesions in whicli only a
few viable organisms may r e m a i n .
A rt~view o f the patient's clinic record
reveals important information regarding
the type and duration of symptoms, physi-
cal findings, laboratory data, and resuhs of
that frequently gives clues as to the nature
and extent of organ involvement. Other
helpfltl information needed includes occu-
pational and travel histories, exposure to
animals or animal products, and disease in
close associates or family members.
l'articular note slmuld be made of any
serologic studies or skin test results. Assess-
merit of the patient's immune status re-
garding both humoral and delayed hyper-
sensitMty is important. For example,
C~3'ptococcus neoJormans and Nocardia infec-
tions are more likely to occur in patients
with Hodgkin's disease or sarcoidosis, two
diseases in which there is delayed hyper-
sensitMty nmlfimction. Pneumococcal in-
fections are not unconllllOn anlong patients
with myeloma, cirrhosis, or sickle cell dis-
ease. Children with chronic granulomatous

*Insnuctor in Internal Medicine, Ma)o Medical School. Consultant, Division of Intectious Dis-
eases, and Section of Clinical Microbiology,MayoCliificand Mayo Foundation, Rochester, Min-
nesota.
tEmerinls Professor of Microbiology, Mayo Graduate School of Medicine (Universityof Min-
nesota). EmeritusConsultant,Section of ClinicalMicrobiology,MayoClinicand MayoFoundation,
Rochester, ~linnesota.

141
 HUMAN I'ATHOLOGY-VOLUME 7, NUMBER 2 March 1976
disease have no difliculty witll pneunlococci
but are susceptible to staphylococci and
gram negative organisms. Besides inllu-
encing the etiologic agent involved, the
patient's underlying disease may determine
the nature of the inflammatory lesion. Ne-
crotizing nonsuppurative lesions may be
found in gram negative infections iil pa-
tients with m,dignant hematologic disease,
whereas similar organisms in patients with-
out malignant hematologic disease would
produce I)olynmrpllonuclear exudation
and abscess formation. ~
T h e demonstration of micro-organ-
isms in draining wounds and tissue speci-
mens gives important clues to the etiologic
agent and may enable therap)'to be planned
and started on a rational basis. However,
the morphologic appearance can be mis-
leading, and recovery of the organism by
cultural techniques provides tile most
accurate method for identification of the
infectious agent.
SPECIMEN COLLECTION
AND TRANSPORT
Draining sinuses should be curetted
with a sterile curene to recover organisms
tlmt might be found only in the sinus wall
or in the site of origin, such as suppurative
lymph nodes or a lesion of osteomyelitis.
Collection of purulent material fi'om the
sinus cavity with a swab is not adequate.
Often only secondary invaders are recov-
ered by this technique, and not infre-
quently the swab has dried by the time it
reaches the laboratory. The curettings may
be placed into a sterile bottle with 2 to 3 nil.
of nutrient broth for transport to the lab-
oratory. A specimen collection kit, such as
~he one described by Start and Weed3
makes tile procedur e more convenient.
For anaerobic cultures, samples of pu-
l'ulent drainage should be aspirated with a
syringe and injected into an anaerobe trans-
port vial containing l)rercduced medium, a
When feasible, biopsy of an ulcer wall or
surgical removal of a sinus tract provides a
more representative specimen for histo-
logic as well as microbiologic study. (;ran-
ular elements resembling sulfnr granules
in purulent drainage should be noted
especiall }' and collected. If multiple fistulas
Ol- sinus tracts are i)resent, sl)ecimens from
142
several sites should be obtained to enhance
recovery of tile etiologic agent.
Closed lesions may be aspirated with a
syringe using a large needle to permit re-
covery of larger chnnps of organisms and
necrotic debris3 Local anesthesia may be
necessary with very l)ainfld lesions; how-
ever, one shoukl be judicious in using these
agents because the antibacterial activity of
procaine ires been demonstrated. 4 Use of
general anesthesia in the operating room
may be required in some cases. Sterile mi-
croscopic slides may be smeared with the
purulent material at the time of collection
for Gram, acid fast, or 1)otassium hydroxide
preparations.
Biopsy specimens may be placed into a
sterile, wide mouthed bottle. If biopsy of a
large lesion is contemplated, it is important
to obtain specimens from several parts of
the lesion to ensure adequate sampling.
Also, if there are muhil)le lesions, several
specimens should be obtained fi'om dif-
ferent sites. Sterile, wide mouthed bottles
should be available in tile operating room
so that the surgeon can place the material
for culture into them directl}', tints mini-
mizing the possibility of contamination of
the specimen in the pathology laborator)'
or inadvertent fixation. If an abscess is
surgically removed, a generous portion of
tile abscess wall should be sent lot micro-
biologic study; some organisms, like No-
cardia astemMes, are usually found in the
abscess wall, whereas others, such as Ac-
timmoces israelii, are more likely to be in
purulent drainages. The surgeon can bisect
a lesion, sending half for histopathologic
study and placing the remainder of tile
specimen into the sterile container for
microbiok)gic stud)'.
Sometimes, when one lympli node is
sent to the histology labor,ttory~and an ad-
jacent node is sent for microbiologic study,
the histologist identifies organisms in the
stained sections but no organisms are ctd-
tured from the other node. Because the
distribution of organisms in a lymph node
cimin may not be uniform, study of muhi-
pie nodes by both histologic stains and cul-
ture provides tile best opportunity for
identifying the etiologic agent.
Abdominal exploration in cases of
fever of unknown origin is a sEecial in-
stance ~:equiring communication "among
the nficrobiologist, the internist, and the
 DIAGNOSTIC TISSUE MICROBIOLOGY METHODS--BREwER, WEEo
surgeon. When collecting surgical speci-
inens, one must keep in mind that addi-
tional material will not be available once
the chest or abdomen is closed. Therefore,
as much material as is reasonable should be
collected for study at the time of the proce-
dure. If a wedge liver biopsy is desired, the
surgeon should be so informed. If multi-
pie, enlarged lymph nodes are found, lilts
of several of them should be submitted for
microbiologic study. If an enlarged spleen
is removed, all the tissue not needed for
pathologic examilmtion should be sent for
microbiologic study. If only a few granulo-
111atotls lesions or abscesses a r e present,
these areas can be studied microbiological-
ly, increasing the likelihood of recovering
the infecting agent.
At our institutiolL frozen sections are
prepared from ahnost all surgical speci-
luens. Granulontatous lesions, both caseous
and noncaseous, are cuhured for mycobac-
teria, flmgi, Brucella, and general bacteria.
Other specimens are placed on appropriate
media t"o1"culture according to the request
of the patient's physician.
Tral~sport of tissue specimens to the
laboratory in sterile glass containers is prob-
ably satisfactory tor recovei'y of anaerobic
organisms; however, this subject deserves
further study.
Weed and Baggenstoss 5 have shown
that reliable Imctcriologic information can
be obtained fiom embahned tissues in
many instances. They isolated such organ-
isms as Escherichia coil, Kh'bsiella pneumoniae,
various species of streptococci, F'semlomonas
aeruginosa, Proteus vulgaris, Haemophilus in-
fluenzae, Nocardia asteroides, tlistophmmt cap-
sulalum, and M)'cobacterimn tuberculosis from
tissue obtained three to 60 hours after the
bodies hild bcen embalmed, collecting their
study niateri,d with sterile scissors after
sterilizing the tissue surface by quick-heat-
ing gynecologic cautery.
P R O C E S S I N G OF
TISSUE SPECIMENS
Tissue specimens shovld be proccssed
as soon as possible upon their arrival in the
microbiology laboratory. Therefore, at our
institution these specimens are bandied on
an emergenqy basis a f t e r regular laboratory
hours.
Tissue specimens are minced with ster-
ile scissors into a sterile mortar and then
ground with a sterile pestle and a sterile
abrasive (Alundum). Sufficient nutrient
broth is added to produce a 10 to 20 per
cent suspension. T h e material is then
placed into ix sterile dropper bottle and
inoculated onto tile appropriate media.
Then the tissue suspension is stored in a
refi'igerator at ,t~ C. for four weeks.
In our laboratory, if no diagnosis is
made with gralmlomatous lesions witlfin
two weeks after the time of accession, on
the basis of either special histologic stains
or preliminary culture results, the tissue
suspension is recultured for mycobacteria,
general bacteria, and fungal organisms
and the surgical specimen is retrieved ibr
additioiml microbiologic study. On several
occasions fimgi that were demonstrable in
tissue sections but did not grow in the initial
ctllture attempt were finally grown after re-
culture. Also Listeria monocvtogenes some-
times can be recovered only after storage at
4 ~ C. according to procedures previously
described. 6
D E M O N S T R A T I O N OF
ORGANISMS BY STAINING
Tile use of special stains for histologic
specimens can be a valuable adjunct to cul-
turing in demonstrating infectious agents.
Also these stains may help define tile role
of some cultured organisms as primary
pathogen, secondary invader, or contami-
nant. For example, biopsy specimens of
necrotic lesions not infrequently grow
Staphylococcus aureus when cuhured, and
tiffs organism is capable of causing tissue
necrosis. However, it also may proliferate
in tissue that is necrotic for some other
reason. The presence of these gram posi-
tive cocci in necrotic tissue without an in-
flammatory reaction indicates the need to
look furtl~er for the cause of the tissue ne-
crosis.
We. have found that in most cases a
Gram stain, an acid fast stain, and a Gomori
methenamine silver stain suffice for screen-
ing of tissue sections fi)r micro-organisms.
Fungai organisms are readily demonstrable
by the Gomori methenamine silver tech-
nique, and for screening purposes this
stain is prefelved over Other stains such as
143
 IlUMAN PATHOLOGY--VOLUME 7, NUMI?,ER 2 March 1976
tire periodic acid-Schiffand Gridley fungus
stains; 7 however, the latter stains occasion-
all}" may be hellfful. Protozoa such as Leish-
mania donovani or Toxoplasma gomlii do not
stain by the periodic acid-Schiff, Gridley,
or Gomori methenamine silver technique, s
and this negative reaction helps to differ-
entiate these organisms fi'om fungi. Pneu-
moc)'stis carinii, whose taxononty is uncer-
tain, is readily demonstrable with the
Gomori methenamine silver stain. Tiffs
stain imparts the same black-brown colora-
tion to the filantents of Nocardia astemides
and Actinomyces israelii. Mycobacteria may
be stained by the Gomori metllenanaine
silver stain if the time in silver is increased, s
.Acid fast organisms can be demon-
strated by a variet)" of staining techniques.
Weed et al? fi)und the night blue stain to
be better than the ZiehI-Neelsen method
for screening of tissue sections. Fluorescent
staining teclmiques for delnonstrating
acid fast organisms have been available fi~r
many years. ~~ ~ The lluorescent technique
allows rapid scaiming of tissue suspensions
and histologic sections.
For examination of broth suspensions
of tissue, the auranfine-rhodantine stain is
used. T h e sntears are then scanned under
the • objective. For tissue suspensions,
urine, sputum, and exudates, this method
is reliable and much faster than the Ziehl-
Neelsen or night blue technique in which
scanning witlt a • objective or oil im-
mersiou is required. TM
It is unustml to find acid fast orgauisms
in histologic sections stained with night
blue or carbolfuchsin tlmt were not demon-
strated by fluorocllrome dye staining of
the ground tissue suspension. One should
keep in mind tlmt demonstration of an acid
f~st organism is no ntore diagnostic of tu-
b~erculosis than demonstration of a gram
negative organism is diagnostic of Pseudo-
moats aerugim~sa. It may be highly sugges-
tive, however, if the stained organisnts fit
with the clinical and histologic pictures, ht
the Mayo Clinic laboratory, for the two
}'ear period Noventber 1972 througll Oc-
tober 197,t, both smears and cultures of
tissue specimens were positive for myco-
bacteria in 27 instances; 21 of the 27 myco-
bactel:ia were Mycobacterium tuberculosis. A
negative auranfine-rhodamine sntear of
the ti,ssue concentrate certainly does not
rule ou( mycobacterial infection, htdeed,
144
of the 45 positive Mycobacterimn tuberculosis
tissue cultures during this same two year
period, smears for acid fast organisms were
negative in 24 (53 per cent). ~2 Previous
authors Imve shown that nontuberculosis
mycobacterial infections may account for
histopathologic findings that are indis-
tinguishable from those of the infectious
lesions of Al~'cobacterium tuberculosi.O3, 14
A Gram stain technique, such as the
one recently described l)y Brown and
Hopps, ~5 should be adequate for the dem-
onstration of most gram positive and
gram negative bacteria in histologic sec-
tions. Members of the order Actinomyce-
tales, which includes the mycobacteria,
Nocardia, and Actinomyces, demonstrate
their kinship in sharing the property of
being acid fast. The nature of the de-
colorizing solution is important and de-
termines whether Nocardia or Actinomyces
retains the carbolfuchsin stain. 16 Unless a
weak organic acid is used for decolorization,
Actinono'ces usually does not show up as
acid fast. Cultural differentiation between
the gram positive filamentous Nocardia and
Actinomyces israelii is not difficult, however.
Nocardia is aerobic and grows on most
laboratory inedia, whereas Actinomyces is
anaerobic and fastidious.
Nocardia asteroides is fi'equently iso-
lated from sputuna specimens cultured for
Mycobacterium tuberculosis. Specimens to be
cultured for mycobacteria are treated with
only a 2 per cent sodium hydroxide solu-
tion prior to culture, rather than a higher
concentration as used in some other lab-
oratories, which probably accounts for the
fi'equent recover)' of Nocardia on mycobac-
terial cultures in our laboratory. Actino-
myces often appears in purulent drainage
as small granules resembling elemental
st, lfur (Figs. 1,2). However, chronic drain-
ing sinuses due to Staphylococcus or Pseudo-
monas may occasionally discharge clumps
of organisms and necrotic debris (botryo-
mycosis). Gram stains of these ntacroscopic
cluntps ntay depict gram positive filanten-
tous organisms characteristic of Actinomyces
or other organisms such as gram positive
cocci, gram negative bacilli, or fimgi.
A scheme for tissue identification of
fungi similar to the one proposed by An-
thony tr is helpfifl. Detailed descriptions of
fimgi and their appearance in Ifis.tologic
sectiolrs are available, s We find it helpfid to
 DIAGNOS'I'IC TISSUE MICROBIOLOGY ME'I'HODS-B~mvvR, V~'EED
Figure I. Sulfur granules in purulent
drainage from patient with Actimmo'cesin-
fection.
keep illustrative histologic sections that
have been prepared in our laboratory
readily accessible for comparison. Usuall)'
)'east forms (round bodies) found in vis-
ceral organs prove to be Histoplasma cap-
sulatum, Co'ptococcus neofimna,s, Coccidi-
oMes immitis, or Blastomyces dermatitidis.
Co'ptococcus neoformans often appears
in hematoxylin and eosin stained sections
Figure 2. Sectionof sulfur granule in tissue, showi,lgradiating clubl~e projection around peripher)" of
granule. (}tematoxylinand eosin stain. •
9 O
-, ..~
-'o
:77"
t" . . ' t
: I
with a clear space surrounding tile faint
cell wall of tire organism. The periodic
acid-Schiff stain also demonstrates the
surrounding clear space, often with a small
amount of stained material around the
periphery of the organism that perhaps
represents shrinkage of the thick polysac-
charide capsule (Fig. 3). The organisms
may show considerable variation in size,
145
 HUMAN I'ATIIOLOGY--VOLUME 7, NUMBER 2 March 1976

Figure 3. Coptococcusm'oJbrmans,showing clear space surrounding organisms. (Periodic acid-Schiff stain.

x.t00.)

from 4 to 20 p.m., and may be associated
with a g r a n u l o m a t o u s reaction; in some
cases t h e r e may be a minimal inflammatory
reaction or n o n e at all. *layer's mucicar-
mine stain may be used to stain the poly-
saccharide capsule.
l!istoplasma capsulatum, on the o t h e r
hand, appears much m o r e u n i f o r m in size
and shape; the organisms usually are 3 to 4
p.m. in d i a m e t e r (Fig. 4). A granulonmtous
response, usually caseous, is found, and the
darkly staining cell wall o f the u n e n c a p s u -
lated organism stands out readily with the
Gomori m e t h e n a m i n e silver stain. T h e or-
ganisms as seen inside macrophages in
hematoxylin and eosin stained sections
may resemble Toxoplasma or Leishmania;
however, organisms o f these two g e n e r a do
not stain readily with the (,omori methena-
mine silver stain. C o m m o n l y p u l m o n a r y
granulomas contain structures resembling
Ilistoplasma capsulatum, but no growth is

Figure 4. llistoplasmacap.~ulatum,demonstrating uniform si.,,Y'cand shape in silver chromate stained tissve

1-'t6 set,i,,,,. ~•
 D I A G N O S T I C TISSUE M I C R O B I O L O G Y METIIOI)S-B~EwER, WF.Et)

Figure 5. B&shmo'cesdermatitidL~,showing considerable variatiou in size and the characteristic wide base
:~ttachmel]t it] budding organisms. (Silver chromate stain. •

o b t a i n e d o n f i m g a l cultures. T h e s e struc- f o r m s usually can be seen a n d show the

tures o f t e n a p p e a r d i s t o r t e d a n d st:fin ir- characteristic b r o a d base a t t a c h m e n t . T h e
regularly in histologic sections; t h e y a r e i n f l a m m a t o r y r e s p o n s e to these o r g a n i s m s
t h o u g h t to r e p r e s e n t n o n v i a b l e fimgiY is fi'equently a c o m b i n a t i o n o f s u p i ) u r a t i o n
Bla.~tomyces dermatitidis yeast a r e l a r g e r and granuloma formation.
than llistophmna (8 to 15/tin. in d i a m e t e r ) T h e r o u n d bodies o f Coccidioi~h's ira-
a n d f i e q u e n t l y s h o w c o n s i d e r a b l e variation mills m a y be l a r g e r t h a n a n y o f the o t h e r
in size in tissue sections (Fig. 5). I f t h e r e o r g a n i s m s p r e v i o u s l y m e n t i o n e d , with g r e a t
are m a n y o r g a n i s m s p r e s e n t , b u d d i n g variability in size f r o m 10 to 70 v m . (Fig.

,-.~,~![7~" "-"7;,; "27: :.:.~: ia[ T lyCJtD It~, d i ~ , Z s:,. : 7 ~ . "~:~ i

.- .-...,~., .. . . . . . -.~ ' - 9 , . ..:- i..,..~ .--. -

L' , ~~ t. . ." ~
, 9 ~9 w,"
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".... 9 ,. -, . '.", : ~....,~:~..- ..-~."
.-~,: ~. 7 ~ 7.
9 . 9 ~ :. "~ . 7 ~ . , , ~ ; " " .- . ...-. t,.,'~'. .... .:.
17, .. . .
~:~ '. :. ~ , ' , . '~. ' ~ "a,., :; , , ' . . . ' i ~ 99 , / . ',..;:..":~ . ' ~ '. ~ ..

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F i g u r e 6. Cocchliohles imm#i~, s h o w i n g an e n d o s p o r u l a t i n g s p h e r u l e . I I " o n l ) ' t h e e n d o s p o , e s are seen, t h e y

can be c o n f u s e d w i t h Coptocorcu., ne~#~rmam o r Ili.itoplasma. (Silver c h r o m a t e stain, x .t00.) 147
 IIUMAN P A T I t O L O G Y - - V O L U M E 7, NUMBER 2 March 1976
6). T h e s e spherules m a y be visible in he-
tnatoxylin a n d eosin stained sections a n d
definitely can be seen with the G o m o r i
tnethenanfine silver sthin. T h e sphernles
m a y a p p e a r hollow, or any n u m b e r o f en-
d o s p o r e s m a y be visible within tltem. T h i s
endbsporulatiota o f Coccidioides immitis is in
contrast to the b u d d i n g forth o f r e p r o d n c -
tion seen with Co'ptococcus new,tartans, llislo-
plasma capsulatum, anti Blastom)'ces dermati-
tidis. I f no s p h e r u l e s are f o u n d and only
small e n d o s p o r e s are seen, the organisnas
conld be confitsed with llistoplasma or
Cryptococctts. Rhinosporidium seeberi is an-
o t h e r o r g a n i s m with e n d o s p o r n l a t i n g
s p h e r u l e s d e m o n s t r a b l e in tissue sections;
however, tltese s p h e r u l e s tnay reach 200
/xm. in diatneter, stain with Mayer's muci-
' c a r m i n e stain, anti usnally infect only skin
a n d mucotts m e m b r a n e s ?
Sporothrixschenckii nmy rarely cansc dis-
s e m i n a t e d disease. T h e oval or spherical
bodies m a y o r ntay not be d e t n o n s t r a b l e in
the tissues on p r e p a r a t i o n s stained with
h e m a t o x y l i n a n d eosin a n d are usually not
seen even in metl~enamine silver stained
sections. T h e o r g a n i s m s grow rapidly on
artificial media, however, and f r e q u e n t l y
p r o d n c e significant growtlt in two to t h r e e
days. T h e Phialophora attd Cladosporittm
species that cause c h r o m o m y c o s i s usually
p r o d u c e ulcerative o r v e r r u c o u s skin le-
sions a n d the brownish, r o u n d sclerotic
bodies are visible in sections stained with
h e m a t o x y l i n a n d eosin.
T h e mnltiple b u d d i n g )'east o f l'ara-
coccidioides brasiliensis is a Sonth Antet-ican
p a t h o g e n and is seen in the United States
only in travelers. T h e multiple bnds p r o -
d u c e a "pilot's wheel" configuration that
usually is quite distinctive in tissue speci-
~lens. H e r e again, however, if only the
b u d s (the knobs on the pilot's wheel) are
seen, the organisnts tnay be misdiagnosed
as Histoplasma cap.~ulatttm in histologic sec-
tions.
A m o n g hyl~hal t o r m s that o c c u r in
sections o f visceral organs, Aspergillus usu-
all)' can be distinguished by its septate hy-
p h a e with d i c h o t o m o u s b r a n c h i n g , usually
at a 45 d e g r e e angle. T h e Phycontycetcs,
including Rhizopus, Mucor, and Absidia,
have b r o a d (6 to 50 ~m.), ribbon-like, n o n -
septate h y p h a e . Camlida o r g a n i s m s fre-
qtter~tly,,Sppear as both )'east a n d h y p h a l
f o r m s in tissue sections. T h e oval, b u d d i n g ,
148
yeastlike f o r m s o f Camlida m e a s u r e a b o u t 2
to 4 ~ m . a n d s o m e may exhibit g e r m tubes
or p s e u d o h y p h a e b r a n c h i n g off the oval
structnres.
Patients receiving i t n m u n o s u p p r e s -
sire t h e r a p y a n d suspected o f having Pneu-
mocystis carinii infection shottld u n d e r g o
h m g biopsy w h e n possible. T h i s is nsually
d o n e transbronchoscopically at o n r insti-
tution, a n d the technique generally has
been snccessful in d e m o n s t r a t i n g the or-
ganisms in sections o f the biopsy material
stained with Gontori tnethetmnfine silver. TM
L u n g i m p r i n t s o f the biopsy s p e c i m e n also
may be stained by this tnethod, circunlvent-
ing the time reqnired for tissue fixation
anti p r o c e s s i n g anti providing a m o r e r a p i d
diagnosis o f Pneumoo'stis carinii pnennaoni-
tis.
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Clinical and autopsy findings in 33 cases. Am.J.
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2. Starr, G. F., and Weed, I.. A.: The bacteriologic
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t.9 Schmidt, R. M., and Rosenkranz, H. S.: Antinfi-
crobial activit)" of local anesthetics: l.idocaine
and procaine. J. Infect. Dis., 121:597, 1970.
5. Weed, L. A., and Baggenstoss, A. If.: The isola-
tion of pathogens fiom tissues of embalmed
humall bodies. Am. J. Clin. l'athol., 21:11 !-t,
1951.
6. Bailey, W. R., and Scott, E. G.: Diagnostic Micro-
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l)Msion of Infectious Diseases
and Internal Medicine
Ma)o Clinic and Mayo Foundation
Rochester, Minnesota 55901 (Dr. Brewer)
149