This paper reports the preliminary findings of an investigation

using Medium V for culture of M. leprae. The medium was

prepared adhering to the procedures adopted by Veeraraghavan.

Tissues from 7 cases of lepromatous leprosy (Bacteriological

Index of 3 or more) were inoculated into Medium V and incubated

at 8–10°C for 72 hours. Quantitative estimation of leprosy

bacilli were made employing the enumeration technique of

Veeraraghavan. There was no evidence of multiplication in any

of the cultures. The findings are in conformity with those of

Kato (1983) and of Katoch and Desikan (1983)

Hari, Lalitha

Herbert, Daniel

Prabhakar, R

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Vol. 55 No. 3 LEPROSY IN INDIA July 1983OBSERVATIONS ON THE CULTURE OF M. LEPRAE INMEDIUM VA Preliminary ReportR. PRABHAKAR 1, LALITHA  HARI 2 AND DANIEL  HERBERT 3This paper reports the preliminary findings of aninvestigationusing Medium V for culture of M. leprae. The medium wasprepared adhering to the procedures adopted byVe raraghavan.Tissues from 7 cases of lepromatous leprosy (BacteriologicalIndex of 3 or more) were inoculated into MediumV and incuba-ted at 8–10°C for 72 hours.Quantitative estimation of leprosybacilli were made employing the enumeration technique ofVeeraraghavan.There was no evidence of multiplication in anyof the cultures. The findings are in conformity with those ofKato (1983) and of Katoch and Desikan (1983).INTRODUCTIONM. leprae, the causative organism of human leprosy, has eludedcultivation in vitro for more than a century since its discovery in 1873 bythe Norwegian Leprologist Dr. Armauer Hansen. Medical Scientists allover the world have failed in their attempts to cultivate the organismin vitro. None of the earlier claims of successful cultivation of M. lepraehas been confirmed.Failure to cultivate the organism in the laboratoryhas been a major hurdle to the progress of laboratory studies in leprosywith the result the research has been tardy. Recently, Veeraraghavan(1982) reported successful in vitro cultivation of M. leprae using a purelysynthetic medium, Medium V, formulated by him (1983).The claim has,however, been questioned by Kato (1983) and by Katoch and Desikan(1983). This paper presents our observations on the culture of M. lep aeusing the Medium V.MATERIALS AND METHODSMedium V for culture of M. leprae was prepared according to themethods described by Veeraraghavan (1983). From each of 7 patientswith lepromatous leprosy, skin scrapings from five different active siteswere collected by slit and scrape method and were inoculated into themedium. The cultures were incubated at 8–10°C in the lower shelf of a1. Dr. R. Prabhakar, M.D., Deputy Director,2. Mrs. Lalitha Hari, M.Sc., Assistant Research Officer,3. Mr. Daniel Herbert, M.Sc., Research Assistant,Tuberculosis Research Centre, Chetput, Madras 600 031.450Vol. 55 No. 3 LEPROSY IN INDIA July 1983domestic refrigerator. Smears were made from cultures as described byVeeraraghavan at 0 hr and at the end of 72 hrs of incubation using0.01 ml of the culture, accurately pipetted out using a micropipette, withequal volume of formal-milk-serum mixture.Smears were air-dried, heatfixed on the lid of a boiling water bath for 2 minutes and stained bystandard cold Ziehl-Neelsen’s method.The bacilli in the smear were enumerated using the method of count-ing suggested by Veeraraghavan (1983). They were classified in thefollowing manner, namely, single bacillus (SB), small groups (SG 2-10bacilli), big groups (BG 11-20 bacilli) and very big groups (VBG>20bacilli). The bacilli in the entire smear containing 0.01 ml of the culturewere counted.The counts at 0 hr and 72 hrs were compared to computethe degree of multiplication.Bacteriological index and morphologicalindex by Ridley’s scale were determined for each patient.Mouse foot-pad inoculations were carried out using CBA strain ofmice to confirm viability of M. leprae in the tissues of patients includedin the investigation. 5 x 103 acid-fast bacilli in 0.03 ml of Hank’s balancedsalt solution was used as inoculum per foot-pad.Harvesting of foot-padswill be done at the end of 4 and 8 months of inoculation.HISTORY OF PREVIOUS TREATMENT IN PATIENTSOf the 7 lepromatous leprosy patients, one patient (No. 7) had nohistory of previous chemotherapy for leprosy, one (No. 6) had nativetreatment for 3 years, 3 patients had specific treatment with DDS for lessthan 3 months, and 2 patients had DDS for 2 years and 3 years, respec-tively.RESULTSThe results of cultures from 7 lepromatous leprosy patients are pre-sented in the Table. According to Veeraraghavan (1983a), a rise in countsof all groups, particularly the single bacillus and groups of 2-10, is evidenceof multiplication.None of the 7 cultures showed an increase in allgroups.Four showed an insignificant increase in the single bacillus count,namely, 1%, 6%, 20% and 29%, respectively.The value of Bacteriological Index and Morphological Index are alsopresented in the Table.It may be observed that all patients had a Bacte-riological Index of 3.00 or more and Morphological Index ranged between0 to 2.08%.The results of mouse foot-pad inoculations are awaited.451PRABHAKAR ET AL—OBSERVATIONS ON THE CULTURE OF M. LEPRAETABLEResults of Cultures from Lepromatous TissueBACT. MORPH. GRADINGS OFCULTURESINDEX INDEX VBG BG SG SBPatient1:0–hr 4.172–hrsPatient2:0–hr 4.572–hrsPatient3:0–hr 4.372–hrsPatient4:0–hr 4.072–hrsPatient5:0–hr72–hrs3.0Patient6:0–hr 4.572–hrsPatient7:0–hr 5.072–hrs0%0.33%0%0%0%2.08%0.86%277 103780 124105 1590 5008 602 481 90 112 644 341 163 270 41 39226 706391706 24749241 527984400 8700944 4962720 440881 62386 804848 3207536 38602400 175932065 17833135 695198 7371VBG = Very big groups (21 or more bacilli);BG = Big groups (11-20 bacilli);SG = Small groups (2-10 bacilli);SB = Single bacillus.DISCUSSIONAn organism can be considered as successfully cultivated in vitroif there is definite evidence of significant increase in the number of orga-nisms in the culture medium, and the organisms thus grown are confirmed452Vol. 55 No. 3 LEPROSY IN INDIA July 1983to be identical with the organism originally inoculated into the medium.The claims made by many workers of having successfully cultivatedM. leprae could not be substantiated since the organisms grown could notbe identified with the original organisms.Skin nes et al. (1975) reportedthe cultivation of M. leprae from lepromatous leprosy patients but theresults were regarded inconclusive without establishing infection in animals.The isolate of Skinsnes (H1-75) was subsequently identified as M. marinum(M. scrofulaceum) by Pattyn (1976). Stanford et al. (1977) in Londonalso examined a subculture of this isolate and came to the same conclusion.Kato (1976) had examined the isolate and showed that it was a sub-species of M. scrofulaceum. Another group working on in vitro cultivationof M. leprae had reported spheroidal bodies of leprosy (SPBL) from plasmaof lepromatous patients (Barksdale et al. 1973).Attempts at fulfilment ofKoch’s postulates with SPBL were unsuccessful since negative results werereported for normal mouse foot-pad inoculations and inconclusive resultswere produced in experiments with Armadillo.Serodiagnosis of M. lepraeis not sufficiently stringent since it has been shown by Kronvall et al.(1976) that one of the eight antigens associated with M. leprae was alsopresent in three slow growing and eight fast growing mycobacterial speciesand further, an antigen 21A was shared by all mycobacterial strainsexamined. Thus immunological evidence for M. leprae is not sufficientlystringent. Dr. Veeraraghavan (1982) reported successful in vitro cultureof M. leprae in a purely synthetic medium and had stated that therewas a 2-6 fold multiplication in 8-60 hrs.D armendra (1982), comment-ing on the findings of Veeraraghavan noted that the multiplication claimedin 18-40 hrs was only 2-6 fold; the claim of rapid growth could not bereconciled with well documented long generation time of M. leprae,namely, at least 12 days, as computed from the data from mouse foot-padexperiments.Further, the guidelines prescribed by Veeraraghavan (1983a)for computation of growth in cultures appear to be purely arbitrary andas such could lead to variations between any two readers or even with thesame reader with the same smears.N vertheless, the counts may beconsidered to be reasonably valid if there has been a uniform increasein large numbers in all the groups and single bacillus at 72 hours. Theresults of investigation carried out at the Tuberculosis Research Centre,Madras on 7 patients have shown no growth of M. leprae in Medium Vduring 72 hrs of incubation at 8-10°C and the findings are in conformitywith those of Kato (1983) and of Katoch and Desikan (1983). Veera-raghavan (1983) has also stated that the Medium V with some modifica-tions gives very good growth of M. tuberculosis in 48 and 72 hrs.It isvery well known that M. tuberculosis can be grown in simple liquid media453PRABHAKAR ET AL—OBSERVATIONS ON THE CULTURE OF M. LEPRAEsuch as Tarshis, Youman and Karlson and Kirschner’s medium. Thesemedia do not require very expensive ingredients as found in Medium Vand as such are better for cultivation of M. tuberculosis than using a veryexpensive medium like Medium V.REFERENCES1.  BARKSDALE ,  L . ,  CONV IT ,  J . ,  KIM ,  K.S. AND PINARDI ,  M, E D E (1973) .Spheroidal Bodies and globi of human Leprosy. Biochemical and BiophysicalResearch Communications, 54 : 290-296.2. DHARMENDRA (1982). Book Review. Leprosy in India, 54 : 591-594.3. KATO, L. (1976). In vitro grown Mycobacterium leprae probably a member ofthe Mycobacterium scrofulaceum species. Int. J. Lepr., 44 : 385-386.4. KATO, L. (1983). No growth of Mycobacterium leprae t +5°C LeprosyScientific Memoranda-Memo- L-1 1182/l, March, 1983.5. KATOCH, V.M. AND DESIKAN. K.V. (1983). Observations on the cultivationof M. leprae in Medium ‘V’ (Veeraraghavan).Lepr. India, 55 : 292-298.6.  KRONVALL ,  G. ,  STANFORD,  J .L .  AND WALSH.  G.P.  (1976).  Studies ofmycobacterial antigens, with special reference to M. leprae. Infection and Immunity,13: 1132-1138.7. PATTYN , S.R. (1976). Belgium researcher tests Skinsnes sample. Star, 36,2-3.8. SKINSNES, O.K., MATSUO, E.. CHANG, P.H.C. AND ANDERSON, B. (1975).In vitro cultivation of leprosy bacilli on hyaluronic acid based medium.1. Preliminaryreport. Int. J. Lepr., 43 : 193-2039. STANFORD, J.L., BIRD, R.G., CARSWELL, J.W., DRARER, P., LOWE, C., MCDOUGALL ,  A.C.,  MC INTYRE ,  G. ,  PATTYN ,  S.R.  AND REES,  R. J .W. (1977).  Astudy of alleged leprosy bacillus strain HI-75.Int. J. Lepr., 45 : 101-106.IO. VEERARAGHAVAN, N. (1982). Studies on Leprosy. Research Publication,Voluntary Health Services Medical Centre, Madras 600113, p. 121.11. VEERARAGHAVAN, N. (1983). Method of cultivation of M. leprae. CurrentScience. January 20, 52 : 60-63.12.  VEERARGHAVAN,  N.  (1983a) . C o m m e n t  o n  a r t i c l e  “ N o  g r o w t h  o fMycobacterium leprae t +5°C” and essential guidelines for cultivation of M. lepraein Medium V. Leprosy Scientific Memoranda – Memo-L-l 199/1, June, 1983.454

Observations on the culture of M.leprae in medium V.

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Observations on the culture of M.leprae in medium V.

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