IN ERNATIONAL JOURNAL OF LEPROSY^ Volume 47, Number 3

Printed in the U.S.A.

Pyridine Extractability of Acid-Fastness fromMycobacterium leprae 1

Gregory T. McCormick and Rita M. Sanchez'

It is a well-known fact that leprosy bacillipossess acid-fastness as well as an enzyme,o-diphenoloxidase, which converts 3,4-di-hydroxyphenylalanine (") to a coloredproduct that is readily discernible. Ob-viously, differentiating M. leprae from oth-er mycobacteria using acid-fastness alonecannot be done because this is also an in-herent trait of other mycobacterial species.On the other hand, the o-diphenoloxidaseactivity of leprosy bacilli harvested fromlepromatous tissues (human as well as ex-perimentally infected armadillo) (". 7) is spe-cific and differentiates M. leprae from oth-er mycobacteria (")). Pyridine extraction ofacid-fastness from the non-cultivatable my-cobacterium M. leprae was reported as anadditional means of identification becauseother species of mycobacteria retain theiracid-fastness ( 1 . 2 .''. 1 . 5).

Recent reports suggest that the pyridinetest is not as specific as was first thought( 12 .9. Cultivatable mycobacterial specieswere reported to lose their acid-fastness.Slosarek, et al. ( 15) have claimed recentlythat only M. leprae in histologic sectionsexposed to pyridine show a loss of acid-fastness.

The present results indicate that the pyr-idine test, when properly done, differen-tiates ,11. leprae from cultivatable myco-bacteria.

MATERIALS AND METHODS

M. leprae to be tested were provided pri-marily from tissues of 20 experimentally in-fected armadillos (''• 7). The 18 differentspecies of cultivable mycobacteria were ob-tained from the American Type CultureCollection, Rockville, Maryland, and thecultivatable strain H1-75 was obtained from

' Received for publication on 23 February 1979.2 Gregory T. McCormick, B.S., Biological Labora-

tory Technician (Microbiology); R. M. Sanchez, B.S.,Microbiologist, Laboratory Research Branch, U.S.Public Health Service Hospital, Carville, Louisiana70721, U.S.A.

the Pharmacology Research Department,USPHS Hospital, Carville, Louisiana(which received the culture from Dr. OlafK. Skinsnes, Hololulu, Hawaii, who gavehis permission for use of the culture) ( 15).These cultivatable bacteria were main-tained on Lowenstein-Jensen slants, andsmears were prepared after 2 and 10 weeksof incubation at 37°C. Cultivatable bacteriawere also separated from the lymph nodeof a feral armadillo that had not been ex-perimentally infected. Bacilli from the skinbiopsy of a lepromatous patient and knowncultivatable acid-fast organisms were usedas controls. Bacilli contained in armadillotissues (e.g. spleen, liver, and lymph node)were separated by mincing the tissue intosmall pieces, followed by homogenation in0.2 M sucrose, filtration, and differentialand density gradient centrifugation (").

The mycobacterial suspensions from ar-madillo tissue were prepared for testing byplacing an inoculation loopful of each onnumbered slides and heat fixing. Smears ofthe ATCC mycobacteria were prepared byfirst placing an inoculating loopful of 5%serum phenol on the slides, followed by thebacterial suspension and heat fixation.Slides of a known mycobacterial cultureand the M. leprae from lepromatous biopsytissue were prepared similarly.

Four separate slides of each organism tobe tested together with the controls wereprepared. Direct smears also were madefrom tissues of armadillos experimentallyinfected with M. leprae and from the onenaturally infected with a cultivatable my-cobacterium. Utilizing bacilli from liver andlymph nodes, four slides of each were alsoprepared. The smears were heat fixed. Twoslides from each set of four were processedby the following method of Convit and Pi-nardi ( 2): a) fixing in Bouin's fixative for 1hour; b) treating in 70% ethanol for 5 min-utes; c) treating in 50% ethanol for 5 min-utes; d) washing in running tap water for 2minutes; e) laying the slides horizontally in

495

496^ International Journal of Leprosy^ 1979

TABLE 1. Retention M . acid-Istness by 18 species of cultivable mycobacteria.

Pyridine ExtractionA'I'CC^ Age of Culture

Mycohacterial species^ No.^2 weeks^10 weeks

M ycobacterium microti^

19530^

Acid-fast^Acid-fast,Mycobacterium gastri

^15754

ycobacterium i^

13950Mycobacterium t errae^ 15755Mycobacterium sp. (Duval)

^ 4233Mycobacterium chelonei

^19977

Mycobacterium chitae^ 19627Alycobacterium nonchromogenicum^19530,ilycobacterium burls^ 19015M ycobact erne m phlei^

11758Mycobacterium sme,imiatis^ 11468Mycobacterium fi wittit um^ 68741Mycobacterium vaccae^ 15483Mycobacterium diernholeri

^19340

Mycobacterium (triton^ 25291Mycobacterium mari num^ 927Afvcobacterium tuberculosis

^ 25177BCG (Chicago)Known acid-fast staining organism"

Bacilli separated from a skin biopsy^ Lost acid-fastnessof a lepromatous patient*

^(—)^(—)

* Used as controls during all tests.

a large staining dish and treating with pyr-idine for 2 hours at room temperature (ini-tially spectrophotometric grade pyridineobtained from J. T. Baker Chemical Co.,Phillipsburg, New Jersey; thereafter dis-tilled pyridine was used for the procedure);f) washing in running tap water for 2 min-utes: g) placing all four slides (the two un-treated and the two treated slides) of eachset in formol-calcium solution for 1 hourand washing in running tap water for 2 min-utes. The pyridine was rejuvenated by plac-ing the used solvent in a closed containerthat had a one inch thick layer of potassiumhydroxide pellets (KOH) on the bottom.The solvent remained in contact with theKOH pellets for approximately 2 weeks.Refluxing of the solvent was then per-

formed at a temperature of 115.5°C. Theinitial distillate was discarded, and the restcollected and placed in tightly sealed bot-tles for future use. After treatment withpyridine, all smears were stained with amodified Ziehl-Neelsen method: a) theslides were laid horizontally on a stainingrack over a sink; b) carbol fuchsin solutionwas poured over the slides; the staining so-lution was then heated for 4 seconds witha Bunsen burner, and the solution allowedto remain there for 10 minutes; c) the slideswere then washed in running tap water for15 seconds; d) decolorized for 2 minuteswith n HCl acid-alcohol solution; e) andthen washed in running tap water for 15seconds. Slides were read microscopicallywithout counter-staining.

TABLE 2. Pyridine e.vtractability of acid-JUstness of Skinsnes' cultivatable bacterium(14) and M. leprae in direct smears.

Sample^ Acid-fast staining results

Skinsnes' Strain 111-75 2-week old cultureSkinsnes' Strain 111-75 10-week old cultureDirect smears from lepromatous armadillo tissueKnown acid-fast staining organism*Bacilli separated from a skin biopsy of a lepromatous patient*

Retained acid-fastnessRetained acid-fastnessLost acid-fastnessRetained acid-fastnessLost acid-fastness

* Used as controls during all tests.

47, 3^McCormick & Sanchez.: Pyridine and NI. leprae Acid-Fastness^497

TABLE 3. Comparison of the DOPA o.vulase test results with those of the pyridinee.vtraction procedure.

Acid-fast^

DOPA oxidaseSource of bacilli^

staining results^ results*

Armadillos numbers 1-20^ Lost acid-fastness^Positive

Cuhumble mycobacterium from a feral armadillo:

Separated from lymph node^ Retained acid-fastness^NegativeGrown in culture^ Retained acid-fastness^Negative

Skin biopsy of a lepromatous patient**^Lost acid-fastness^

Quantity insufficientfor DOPA testing

* Provided by Dr. K. Prahhakaran.** Used as a control on all tests.

RESULTSThe results showed that pyridine extrac-

tion leads to no loss of acid-fastness in allcultivatable mycobacteria tested. These re-sults were the same both in the older (10weeks old) and in the younger (2 weeks old)mycobacterial cultures (Table 1). Skinsnes'cultivatable bacteria (") also produced thesame results, i.e., no loss of acid-fastnessafter pyridine extraction (Table 2). Smearsof cultivatable mycobacteria in a suspen-sion prepared from feral armadillo tissueand grown in culture retained their acid-fastness as shown in Table 3.

Extractability of acid-fastness by pyri-dine was found in the smears of .11. lepraeseparated from infected armadillo tissues(Table 2) and from the lepromatous patient.Direct smears of the liver and the lymphnodes of experimentally infected armadillosshowed complete loss of acid-fastness afterexposure to pyridine for 2 hours. As shownin Table 3, there was a perfect correlationbetween pyridine extraction of acid-fast-ness and DOPA oxidation.

DISCUSSIONIt has been reported by Skinsnes (i 2) and

by Skinsnes, et al. ( 13 ) that the pyridine ex-traction procedure was only effective on"aged, probably nonviable bacilli.'' Slosa-rek, et al. ( 15) reported that "the results ofthe experiments showed that, under theconditions used, pyridine extraction leadsto a loss of acid-fastness in M. /eprae inhistologic sections only. —

Smears prepared from suspensions of .11.leprae separated from organs of experimen-tally infected armadillos contain an astro-

nomical number of bacilli. Such suspen-sions contain mycobacteria of various agesand in various states of viability. When ex-posed to pyridine for 2 hours and stainedby a modified Ziehl-Neelsen technic, thebacilli in smears prepared from M. lepraesuspensions consistently lost their acid-fastness. The cultivatable mycobacteriaand the cultivatable bacilli separated fromthe feral armadillo consistently retainedtheir acid-fastness. It seems impossible toexplain this as being related to "stages inthe life cycle of the leprosy bacillus'' ( 12.13) .

Experiments dealing with the possiblepyridine extraction of acid-fastness fromcultivatable mycobacterial species (TableI) were not confirmed in our laboratory. Allcultivatable mycobacteria were grown onstandard Lowenstein-Jensen slants, andsmears were prepared at growth periods of2 weeks and 10 weeks. The smears presum-ably contained mycobacteria of variousgenerations. Staining with the Ziehl-Neel-sen technic demonstrated that all of theseorganisms remained acid-fast after 2 hoursexposure to pyridine. Dr. Skinsnes' allegedM. leprae culture ( 14 ) also retained its acid-fastness.

The claim of Slosarek, et al. ( 15) that pyr-idine extractability of acid-fastness from M./twit(' occurs only after histochemical re-actions during histological section prepa-ration and staining was not confirmed. .11.leprae in direct smears prepared from ex-perimentally infected armadillo tissues losttheir acid-fastness after exposure to pyri-dine. The bacterial controls were from alepromatous patient's skin biopsy and aknown cultivatable acid-fast staining organ-

498^ International Journal of Leprosy^ 1979

ism. Leprosy bacilli from the skin biopsylost their acid-fastness while the cultivat-able mycobacteria retained theirs.

Convit and Pinardi's pyridine extractionprocedure ( 2) stated that pyridine of thehighest quality must he used. The solventthat has been recycled in our laboratorymet this criterion when compared to pyri-dine received from the manufacturer. Atwo hour limit is sufficient to show the ex-tractability of pyridine. Exceeding this timeperiod will only demonstrate the eventualloss of acid-fastness in all mycobacteria.Counter-staining is not necessary becauseafter exposure to pyridine, methylene bluemay impart a purplish color to the slidewhich may be confusing.

Experiments in our laboratory haveshown that the pyridine extraction methodin conjunction with the D-DOPA oxidasetest are reliable tests for the identificationof ;V. leprae.

SUMMARYVarious mycobacteria were tested for

their ability to retain acid-fastness aftertreatment with pyridine: a) Alycobacteriumleprae separated from organs of 20 experi-mentally infected armadillos (which weresacrificed); b) leprae separated from abiopsy of a lepromatous patient; c) directsmears of lepromatous tissues from arma-dillos; d) eighteen cultivable mycobacteriaobtained from the American Type CultureCollection (ATCC); e) cultivatable myco-bacteria separated from the lymph nodes ofa wild-caught armadillo and also the sameorganism grown in culture and Skinsnes'alleged M. leprae culture.

A loss of acid-fastness was observed mi-croscopically from ,11. leprae separatedfrom experimentally infected armadillo tis-sues, M. leprae separated from a leproma-tous patient biopsy, and I. leprae foundin direct smears prepared from infected ar-madillo tissues. The eighteen cultivatablemycobacteria from ATCC, cultivatable my-cobacteria separated from the tissue of awild-caught armadillo (and also grown inculture) and Skinsnes' alleged lepraeculture retained their acid-fastness. Testingof pyridine extractability of acid-fastnesscombined with those of D-DOPA oxidasetesting proved to be extremely reliable in

our laboratory in differentiating Al. lepraefrom other mycobacteria.

RESUMENSe proki la habilidad de las siguientes micobacterias

para retener su acido resistencia despues de trata-

[Menlo con piridina: a) Mycobacterium leprae sepa-

rado de los organos de 20 armadillos infectados expe-

rimentalmente, h) leprae separado Lie una biopsia

de un paciente lepromatoso, c) M. leprae encontrado

en extendidos directos de tejidos de armadillos infec-tados, d) dieciocho micobacterias cultivables obteni-

das de la ColecciOn Americana de Cultivos 'hp°

(ATCC), e) micobacterias cultivables separadas de los

ganglios linfaticos de un armadillo "salvaje — captu-

rado, el mismo organismo crecido en cultivo, y el cul-

tivo del microorganism° procalamdo como .1/. leprae

por Skinsnes.Microscopicamente, se observo una perdida de Ia

acid° resistencia del M. leprae separado de tejidos de

armadillos infectados experimentalmente, del .1/. lep-

rae separado de Ia biopsia del paciente lepromatoso

y del .11. leprae encontrado en los extendidos directosde los tejidos infectados de armadillo. Las siguientes

micobacterias retuvieron su acido-resistencia: las 18

micobacterias cultivables de Ia ATCC, las micobac-terias cultivables separadas de los tejidos del armadillo

"salvaje — capturado, y el organismo crecido en el

medio de Skinsnes y proclamado comp M. leprae. En

nuestro lahoratorio, la combinaciOn de las pruebas deextractabilidad de Ia acido-resistencia con piridina y

de Ia D-DOPA oxidasa, ha resultado ser extremada-

mente confiable para la diferenciaciOn del .14. leprae

en relacitin a otras micobacterias.

RESUME

On a etudie diverses mycobacteries en ce qui con-cerne leur capacite a retenir l'acido-resistance apres

traitement par Ia pyridine. Ces mycobacteries ont con-

siste de: a) Mycobacterium leprae recoltes a partir des

organes de 20 tatous infectes experimentalement, qui

ont etc sacrifies; b) M. leprae isole a partir dune biop-

sie de malade lepromateux; c) des frottis directs de

tissus lepromateux de tatous; d) dix-huit mycobacter-ies cultivables obtenues a partir de ('American Type

Culture Collection (ATCC); e) des mycobacteries cul-tivables recueillies a partir des ganglions lymphatiquesdun tatou capture dans Ia nature, et egalement le

meme organisme apres croissance en culture, de

meme que des cultures pretendUment de lepre sur

milieu de Skinsnes.Une perte d'acido-resistance a etc observee micros-

copiquement au niveau de M. leprae recueilli a partirde tissus de tatous infectes experimentalement, de .11.

leprae isole a partir dune biopsie de malade lepro-

mateux, et egalement de .11. leprae mis en evidence

dans des frottis directs prepares a partir de tissus de

tatous infectes. Les dix-huit mycobacteries cultivables

de I'ATCC, de meme que les mycobacteries cultiva-

47. 3^McCormick (K7 Sanchez: Pyridine and M. leprae Acid-Fastness^499

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Ackno■iledgment. 'Phis work was supported in partby Intraagency Agreement #2-YOI-A1-60001, Nation-al Institutes of Allergy and Infectious Diseases, Na-

tional Institutes of Health. 'this investigation also re-

ceived support from the Immunology of Leprosy(ININILEP) component of the UNDP/World Bank/

WHO Special Programme for Research and Train-

ing in Tropical Diseases. The authors wish to express

their gratitude to the following persons who havecontributed substantially to the project: Dr. K. Prab-

hakaran, Chief, Biochemistry Research Department,

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hies recueillies de tissus de tatous captures dans Ia^5.

nature (et egalement de ccs bacilles de tatous aprescroissance en culture), de meme que les cultures pre-

tendtiment de M. leprae sur milieu de Skinsnes, ont

conserve lour acido-resistance. L'epretive d'extraction^6.

de l'acido-resistance par Ia pyridine, combinee aver

les epreuves de D-DOPA oxydase se sont reveleesextremement fiables dans notre laboratoire pour dif-

ferencier 3/. leprae d'autres mycobacteries.7.