Rapid methods for counting mycobaacteria—comparison of methods for extraction of mycobacterial adenosine triphosphate (ATP) determined by firefly luciferase assay

Tubercle 06 (1985) 99-108 0 Longman Group Ltd.

RAPiD MEfHODS FOR COUNTING MYCOBACTE#UA-COUlPAf#SON METHODS FOR EXTRACTION OF MYCDBACTBRiAL ATRIPHOSPHATE (ATP) DEIERMlNED BY FIREFLY LUCIFERASE ASSAY R. Department

P. Prioli,

of Medical

A.

Tanna

Microbiology,

OF

and I. N. Brown*

St. Mary’s

Hospital Medical

School, London

Summary A comparison of 5 different methods of extraction of adenosine 5’-triphosphate (ATP) from mycobacterial cells was carried out using Mycobacterium bovis, BCG as a model. ATP was measured using the luciferin-luciferase bioluminescence reaction. Boiling buffer extraction was the best method. The amount of ATP extracted correlated with the number of colony forming units over a wide range of count. Although great sensitivity in terms of number of bacilli detectable was not achieved the method was rapid and appears suitable for drug sensitivity testing of tubercle bacilli. R&urn6 L’auteur a utilise Mycobect&ium bovis, BCG comme modele pour comparer cinq methodes differentes d’extraction du 5’-triphosphate d’adenosine (ATP) a partir des cellules mycobacteriennes. L’ATP a 6th mesuree par la reaction de bioluminescence luciferine-luciferase. La methode d’extraction par dbullition en milieu tamponne s’est montree la meilleure. La quantite d’ATP extraite donnait une bonne correlation avec le nombre d’unites formant colonies, pour une large gamme de chiffres de comptage. Bien que I’on n’ait pas obtenu une sensibilite elevee en terme de nombres de bacilles decelables, la methode dtait rapide et elle semble convenir pour tester la sensibilite du bacille tuberculeux vis-a-vis des medicaments. Resumen Se compararon cinco metodos diferentes de extraction de adenosina-5’-trifosfato (ATP) a partir de celulas micobacterians utilizando coma modelo Mycobecterium boris, BCG. El ATP fue medido usando la reaction de bioluminiscencia luciferinluciferasa. El mejor metodo fue el de extraction por ebullicion en medio tamponado. La cantidad de ATP extraida daba una buena correlation con el ntimero de unidades que forman colonias sobre una amplia gama de cifras de recuento. A pesar de no haberse obtenido una sensibilidad elevada en terminos de nlimero de bacilos detectables, el metodo result6 rapid0 y parece convenir para probar la sensibilidad a 10s medicamentos, del bacilo tuberculoso. Introduction Colony counts

on suspensions

of mycobacteria

take a long time and are liable to error

*Correspondenceto: Dr I. N. Brown, Department of Medical Microbiology, St. Mat-y’s Hospital Medical School, Paddington, London W2 1PG

100

Prioli

and others

because of clumping and carry over. Also, they require special media. In the case of the non-cultivable mycobacteria, Mycobacterium leprae and M. lepraemurium, viability is usually assessed by staining reaction (I-21 injection into animals [3-41 or by the uptake and metaboli‘sm of a suitable radiolabelled substrate [5-71. These methods are either indirect, time consuming or require relatively large numbers of bacilli. Reliable estimates of the viability of living cells can be made by measuring their ATP content using the luciferin-luciferase bioluminescence reaction. The method has had wide application in microbiology and related fields. Two particular aspects of the application of the ATP bioluminescence reaction to mycobacteriology have been examined here: 1. the extraction of mycobacterial ATP and 2. the sensitivity of the method. Several methods have been described for the extraction of bacterial ATP. These methods include heat [B-9], a combination of heat and chloroform treatment [IO], exposure to chemicals such as dimethylsulphoxide [ll], or trichloroacetic acid 1121, or exposure to commercially available reagents (e.g., NRB, Extralight, Picolite) which probably contain detergent. We have compared 5 extraction procedures using M. bovis, BCG as a model. BCG grows readily in culture and colony counts can be made in 14-21 days; moreover, special laboratory facilities are not required. Preliminary studies have also been carried out, using two of the methods and M. tuberculosis, with a view to the development of a drug sensitivity assay. Materials and methods Organisms Mycobacterium bovis, BCG. The Glaxo substrain was used. Lyophilised BCG in ampoules was reconstituted and maintained by transfer every 3-4 weeks on Middlebrook 7Hll agar (Difco Laboratories). Dispersed liquid cultures in Middlebrook 7H9 broth (Difco Laboratories) containing Tween 80 were prepared from the solid growths and maintained by serial transfer at l-2 week intervals. The number of bacteria present in culture suspensions was estimated indirectly by measuring opacity in a Spekker absorptiometer (Hilger and Watts, London), and reference to a previously constructed curve relating colony forming units (CFU) and opacity. Colony counts were made by plating out suitable dilutions of culture on Middlebrook 7HlO agar (Difco Laboratories) containing 0.5 % glycerol, after exposure for 20 seconds to mild ultrasonic vibration in an immersion type sonic bath, Luziesa, Paris, France (13). Plates were incubated in plastic bags at 37 “C for 2-3 weeks. The counts given in results are the mean of duplicate samples. Mycobacferium tuberculosis. Young growths of reference strains or recent clinical isolates on Lowenstein Jensen slopes were kindly provided by the PHLS Tuberculosis Reference Laboratofy, Dulwich Hospital, London and the MRC Unit for Experimental Studies on Tuberculosis, Royal Postgraduate Medical School, London. In order to prepare dispersed suspensions, tubercle bacilli from the slopes were transferred to glass bijou containing 1ml 7H9 broth, a nail and glass beads. The suspensions were gently agitated on a magnetic stirrer for about two minutes and diluted appropriately in 7H9 broth. Reagents

for extraction

Unless otherwise

procedures

stated reagents

and bioluminescence were purchased

from

reactions BDH.

TRIS-EDTA buffer solution (0. IM, pH 7.75). 6.06 g TRIS (hydroxyl methyl) amine and 0.372 g ethylene diamine tetra-acetate (EDTA) were dissolved in 500 ml distilled water and the

Extraction of mycobacterial ATP

101

solution titrated to pH 7.75 with glacial acetic acid. This buffer was kept at 4 “C when not in use. Chloroform 23% v/v. 0.23 ml chloroform was added to 0.77 ml distilled water. This reagent was made up as required. Dimethylsulphoxide

(DMSO)

90%

73.9 mo///. 0.1 ml TRIS/EDTA buffer was added to 0.9 ml

DMSO. The solution was stored at 4°C. NRB and Extralight extraction reagents. Vials were purchased ready for use from Comber and Son, Stockport (NRB) or Sterilin Instruments, Alton and Analytical Luminescence Laboratories, California, USA (Extralight). reagent. Kits containing 5 vials of ATP monitoring reagent and one vial of ATP standard were purchased from LKB Wallac, and stored at -70 “C until required. Before use each vial of monitoring reagent was reconstituted according to the manufacturers instructions in 10 ml water for injection (Antigen Limited). Repeated thawing and freezing decreased the potency of this reagent and when this decrease was greater than 10 % as assessed by the ATP standard control it was discarded.

ATP monitoring

solution. Each vial was reconstituted with 10 ml water for injection to give a 10T5 M ATP solution and frozen at -70 “C in 2 ml aliquots. Samples were thawed once and subsequent dilutions of thawed samples were made in water for injection.

ATP standard

Extraction

procedures

(see also Table II).

Boiling TRWEDTA buffer. 0.1 ml suspension was pipetted into a three by half inch glass test tube and 0.2 ml TRIS/EDTA buffer, preheated in a boiling water bath, was added. The contents were thoroughly mixed and the tube placed in a boiling water bath for 5 minutes. The extract was allowed to cool (2-5 minutes) before the ATP assay was carried out. Heat and Chloroform. 0.03 ml 23 % chloroform was added to 0.1 ml bacterial suspension and after mixing, heated in a boiling water bath for 5 minutes. The pellet was rehydrated with 0.4 ml TRIS/EDTA buffer for ATP measurement. Dimethylsulphoxide (DMSO). 1 .O ml bacterial suspension was filtered onto a 0.45~ 25 mm filter pad by vacuum suction. The bacteria were washed with 1.0 ml TRISIEDTA buffer and then, using a collection tube, treated with 0.2 ml 90 % DMSO for 30 seconds, washed with two lots of 1.0 ml TRISIEDTA buffer and the ATP content of the filtrate measured. This procedure was carried out at room temperature. NRB Lumac. Equal volumes of NRB and bacterial suspension were mixed and left at room temperature for 10 minutes before sampling for ATP content. For some experiments the reaction mixture was heated to 60 “C. Extralight. Equal volumes of extralight reagent and bacterial suspension were mixed and allowed to react at room temperature for 60 seconds before sampling.

102

Prioli and others

Measurement

of ATP bioluminescence

Light output was measured using a LKB 1250 luminometer. Unless otherwise stated, 0.2 ml monitoring reagent and 0.2 ml bacterial extract or ATP standard solution were thoroughly mixed in a 4 ml disposable polystyrene cuvette which was then loaded into the luminometer. For the purpose of this study, the peak light output was recorded in millivolts (mV) either on a digital printout or a standard chart recorder (LKB Bromma 2210). ATP loss In order to monitor the effect of the extraction procedures on ATP, simulated extractions were carried out using ATP standard (10-7M) as a substitute for bacterial suspension. ATP loss was defined as the difference between the peak light output readings before and after the extraction procedure expressed as a percentage of the before extraction reading. Results Preliminary

experiments

Colony counts. BCG cultures in 7H9 broth maintained as described contained between IO7 and lo8 CFU/ml after 1 week. Earlier work [13] had shown that mild sonication increased the colony count 3 to 11 fold (mean 6.65) over untreated suspensions by partially dispersing clumps of bacilli. This treatment was therefore routinely employed to obtain more accurate estimates of viable count. It was important to find out whether, under certain conditions, sonication might have a detrimental effect on the count. Figure 1 illustrates a typical experiment and shows the counts obtained from a 7H9 BCG suspension before and after sonication for periods ranging from 5 to 120 seconds. The increase in count obtained after

Figure 1. Effects of sonication on the colony forming ability of BCG. A 14 day old 7H9 BCG culture was aliquoted in 1 ml amounts and each sample was submitted to mild ultrasonic treatment for the different times indicated. The points in the graph are the mean of four determinations of colony count on each suspension. The counts varied from 8 x 10* to 2 x 10’. The zero time point represents the untreated suspension.

Extraction of mycobacterial

ATP

103

20 seconds was about 10 fold. There were two other important observations. Firstly, the increase in count was detectable after only a 5 second burst of sonication and, secondly, sonication in this machine for a full two minutes did not affect the viable count. Bioluminescence reaction. The volumes of reagent recommended by LKB Wallac were, 0.2 ml ATP standard or extract, 0.2 ml luciferin-luciferase monitoring reagent and 0.6 ml TRIS/EDTA buffer, making a total of 1 ml in the cuvette. When the working volume was decreased by gradually eliminating the buffer, the light output increased. The highest reading was obtained with equal volumes of ATP standard and monitoring reagent (see Table I). The sensitivity of the method was thus increased. ATf standard and heat. Since two of the procedures employed involved heating the bacterial suspensions in a boiling water bath, it was important to establish at the onset that heating in this way did not significantly destroy ATP. Duplicate samples of ATP standard 10V7M were assayed before and after heating in a boiling water bath for 5 minutes. The ATP loss was 6.2 %, an order of loss confirmed in subsequent studies (see below). Inhibition

of the bioluminescence

reaction

Extracts of BCG prepared by treatment with nitric acid, trichloracetic acid or the detergent benzalkonium chloride, reacted poorly with luciferin-luciferase. Control tests using ATP standard solution revealed that the luminescence reagents themselves were inhibited and these extraction methods were therefore abandoned. Comparison of extraction procedures. Details of the 5 methods chosen for this study are summarized in Table II. The major differences among the methods were in the extraction Table I.

Sensitivity

of the firefly reaction with different volumes of the reactants.

ATP standard Buffer Monitoring reagent Working volume Light signal (mV)

0.2 0.6 0.2 1.0 13.5

0.2 0.5 0.2 0.9 14.9

0.2 0.4 0.2 0.8 15.9

0.2 0.3 0.2 0.7 24.7

0.2 0.2 0.2 0.6 27.9

0.2 0.1 0.2 0.5 29.7

0.2 **0.2 0.4 31.1

**System with the highest output light signal/unit ATP concentration. ATP standard solutron concentration = 10es M The working volume and all other quantities of reactants are expressed in ml.

Table II.

Description of extraction procedures. Volume of culture used

Final working volume

Dilution factor to

Extrection time lminutes)

Temperature (Cl

5 5 10 0.5 1

100 98 20 20 20

mV/ml+ TRISEDTA boiling buffer Heat and chloroform NRB Lumac reagent DMSO Extralight

0.1 0.1 0.25 1.o 0.25

0.3 0.53 0.5 2.2 0.5

15 26 10 11 10

* Readings were made on 0.2 ml extract and converted to /ml equivalents

104

Prioli and others

Table III.

Comparison

Culture

A B C D Range

C.F.U./ml

5x 2 x 1 x 6x

10’ IO’ lo7 10s

of extraction procedures. TRWEDTA boiling buffer

Heat and chloroform

NRB Lumac

3765 1575 832.5 391.5

3482.5f 75 1592.5-tl57.5 730 t 32.5 380 + 10

100

k 256.5* f 120.9 t 87 f 12

DMSO

Extralight

667 f 14.6 328 f 7.0 124+ 4 59t 2

Not Not 30.8 18.7

194 106 26.8 13

16-21

4-7

reagent Done Done f 4.4 + 1.1

I5.0 f 5.8 + 1.5 ? 2.1

of**

Efficiency (%)

99-114

5-10

ATP measured in mV/ml * Arithmetic mean of 5 determinationskstandard deviation ** Heat and chloroform was chosen as the point of comparison

time and the temperature of the reaction. All the methods involved some dilution of the bacterial.suspension under test and the dilution factor has been taken into account in the calculation of results. The extraction methods were compared on 4 separate occasions using 7H9 broth cultures containing approximately lo7 BCG/ml (see Table Ill). Each procedure was carried out on five samples of culture, and the mean + S.E. mV/ml obtained are shown in Table Ill. All the methods were reproducible and produced a ranking which agreed with the ranking subsequently made according to colony count. There were considerable differences in the amount of ATP extracted because the hot methods were more efficient than the cold methods. The heat and chloroform method proposed by Dhople and Hanks [lo] for M. lepraemurium, was chosen as the point of comparison. TRWEDTA boiling buffer proved to be the better method of extraction on three occasions and of similar efficiency on the fourth occasion. It was also a safer and simpler method, and for these reasons it was chosen for further studies.

E a

-7

=

-8

0” -

-9 -1 0

0

3

2

1 log

mV ml

4

5

-1

Figure 2. Correlation between ATP standard concentration and output light signal in millivolts tmV). ATP standard was diluted in TRWEDTA buffer. Light output is expressed as mV ml-’ peak reading. Correlation coefficient r* = 0.99233. See text for further details.

.

Extraction of mycobacterial Correlation between ATP concentration

ATP

105

and light output

Dilutions of ATP standard in 0.2 ml volumes were added to 0.2 ml monitoring reagent. Figure 2 shows that there was a linear relationship between log ATP concentration and log mV/ml light output over the range lo-” to lop5 M ATP. Correlation between colony count and light output Twenty two samples of neat or diluted BCG culture were extracted using the TRIS/EDTA method and the ATP measured. Colony counts were made on sonicated suspensions. The results are shown in Figure 3. Using the formula: log,,[ATP] = A + B. logIo[CFU], where A = -2.7797 and B = 0.87255, a linear correlation was obtained between the light output and colony count (correlation coefficiency r2 = 0.97933). ATP loss The two hot procedures resulted in higher ATP loss than the cold procedures. The maximum loss obtained with boiling TRIS/EDTA buffer or heat and chloroform was 15 % and it was usually less (range 6-15 %). The loss obtained with the cold extractants rarely exceeded 7 % (range 0.5-7.3 %). Despite the higher ATP loss, the hot methods were much more efficient as judged by the overall amount of ATP extracted. Once extracted, the ATP was remarkably stable. The level of ATP extracted from a BCG culture by heating at 98 “C for 30 minutes remained constant over 5 days of storage in a refrigerator (data not shown). Studies with human tubercle bacilli Cold procedures were more attractive because of the increased safety hazard associated with this organism. A direct comparison was made therefore between the boiling TRIS/EDTA buffer method and NRB extraction. Two slight modifications to the methods already described were included: 1) the time of exposure was varied; and 2) the NRB extraction was carried out also at 60 “C.

log C.F.U. mi’ Figure 3. Correlation between light output in mV and BCG colony forming units (C.F.U.). C.F.U. were determined for 22 sonicated samples of 7H9 BCG cultures. ATP was extracted by the boiling buffer method and is expressed as mV ml-’ light output. Correlation coefficient r* = 0.97933. See text for further details.

106

Prioli and others

A preliminary study of the boiling buffer method using recent clinical isolates of human tubercle bacilli showed that heating in this way removed the colony forming ability of the bacilli. The amount of ATP obtained by exposure of representative BCG and M. tuberculosis suspensions to boiling buffer and NRB for different times is given in Table IV. The results showed that both methods of extraction were applicable to tubercle bacilli but confirmed the superiority of the boiling buffer method. The conditions already established appeared to be optimal. A further more detailed study was carried out using the standard methods but also including extractions with NRB at 60 “C (Table V). The boiling buffer method was more efficient than NRB either at room temperature or at 60 “C. In the case of BCG, extraction with NRB at 60°C was consistently twice as efficient as NRB at room temperature but with tubercle bacilli the results were variable. We do not know the reason for this difference. It may reflect the degree of aggregation of the suspensions or differences in the cell wall structure of the bacilli.

Table IV. Extraction of BCG and tubercle different lengths of time.

bacilli

with

5 51765 10953

10 47775 10549.5

buffer

and

NRB for

NRB ATP in mV/ml

TRWEDTA buffer ATP in mV/ml

Time (minutes) 7H9 BCG (sonicated) Tubercle (untreated)*

TRIS/EDTA

15 45510 7468.5

5 7238 873

10 12070 2047

15 11230 2043

* The TB suspension, prepared from L. J. slopes was not sonicated and the viable count was probably an underestimate. BCG = 5 x 10’ C.F.U. ml-’ Tubercle bacilli = 2.2 x 10’ C.F.U. ml-’ Results given represent the arithmetic mean of triplicate determinations

Table V. Comparison and tubercle bacilli.

between

TRISIEDTA

C.F. U./ml

buffer and NRB extraction

TRWEDTA

38970*

NRB 60C

13290 8049

NRB Room

8174

7788 4194 4217

29925 1176 3453

168 1083

4137 118 499

6 x 10’ 1.9 x 10s 1.1 x lo*

57645 29685 17970

11730 11140 4299

7720 12380 4211

1.5 x 10s 2.4 x lo8 9 x 10’

23775 29385 14098

6442 7122 5095

3486 6290 4168

3.1 x IO8 2.5 x 10s 1.9 x 108

31470 30885

1 x IO8 5x lo6 2 x 10’

procedures

temp.

7H9 BCG

Tubercle bacilli

* ATP readings are the arithmetic mean of 3 determinations and are expressed in mV/ml.

for BCG

Extraction of mycobacterial ATP

107

Discussion The luciferin-luciferase bioluminescence reaction provides a sensitive assay for ATP and light output is directly proportional to the amount of ATP present. This study has shown that the best ATP extraction procedure for BCG is to boil the bacilli in TRWEDTA buffer for 5 minutes. The method is applicable also to tubercle bacilli. Using the apparatus available, it was possible to detect as few as 5 x IO4 BCG obtained from broth cultures. A sensitivity of this order would be adequate for drug susceptibility testing and it may therefore be possible to develop a rapid procedure using this technique. Estimates of the number of bacilli present in a suspension were obtained within 30 minutes of sampling. The efficiency of the extraction procedure is a critical requirement in the preparation of samples for the bioluminescent assay. The methods compared in these studies either required high temperature or were based on the physico-chemical properties of the extractants and could therefore be carried out at room temperature. Extracts of BCG prepared using acids or detergent-like substances such as benzalkonium chloride inhibited the bioluminescent reaction. The extracts could be diluted to overcome the inhibitory effect but dilution reduced the sensitivity of the methods. Hot procedures were more effective than cold procedures and also more convenient because they killed the bacilli. The difference in efficiency may have been due to aggregation of the bacilli or possibly the cell wall structure of mycobacteria. Cold procedures are extremely effective with organisms of other bacterial genera. ATP proved to be a stable molecule and degradation was low even during hot extraction procedures. The amount of ATP extracted from a BCG colony-forming unit (1.014 fg) agreed well with the value of 1.2-1.5 fg obtained previously for BCG (Pasteur substrain) by Gheorghiu and Lagranderie [14]. For the studies on M. tuberculosis a simple cold chemical extraction procedure would have been preferable because it could have been carried out inside a Class 1 microbiological safety cabinet. Boiling buffer extraction could not be carried out in the cabinet but safety requirements were met by capping the tubes during the heating step. As with BCG the tubercle bacilli were killed during the extraction procedure as judged by their lack of growth on solid medium (data not shown). A comparison of hot and cold methods showed that, as for BCG, the boiling TRWEDTA buffer method was the best method for extracting ATP from tubercle bacilli. Extraction particularly of BCG with the Lumac NRB reagent was improved by increasing the temperature to 60°C. NRB is a detergent like substance and probably acted on the cell membrane to alter the stoichiometric structure of its lipids (Dr S. Bascomb, personal communication). Heating at this temperature would affect the permeability barrier of the cell and facilitate the penetration of the extraction agent and consequently the release of ATP. The same principle of membrane alteration by heat is used when mycobacteria are stained by Ziehl-Neelsen’s method [15]. Mild sonication breaks up aggregates of mycobacteria and increases the colony count. The degree of dispersion varies according to the growth medium [13]. BCG colony counts increased after mild sonication, but there was no increase when stronger sonic treatment, obtained from a probe-type ultrasonic disintegrator, was applied (date not given). In contrast, mild ultrasonic treatment caused a decrease in the overall ATP content of the suspension whereas treatment in the disintegrator caused an increase in ATP level. Possibly some bacilli ruptured when subjected to probe sonication, thus reducing the viable count while maintaining ATP levels. Intense sonication may also release ATP molecules which are closely bound to intracellular organelles and possibly resistant to standard extraction procedures. One criticism of the ATP bioluminescence technique as a viability assay concerns its

108 Prioli and others ability to distinguish between live and dead organisms. Actively growing cultures were used for most of the experiments described here. In such cultures, a high proportion of bacilli would be live and ATP measurement would therefore reflect fairly accurately the number of live bacilli present capable of producing colonies. However in the presence of effective drug, the metabolic status of the bacilli would be less predictable. Almost certainly it would be more variable as the culture would contain a higher proportion of moribund or dead bacilli. The ATP method in its present form measures total extractable ATP. It would not therefore differentiate between a bacterial suspension containing 50 % live and 50 % dead bacilli and a suspension of moribund bacilli each containing half the amount of ATP normally found in live bacilli. Also, dead bacilli with an intact permeability barrier might still contain ATP and errors could thus be made in the estimation of cell death. Whether these factors would give rise to problems in the interpretation of drug susceptibility assays is not yet known.

Acknowledgements This work was supported by the Brazilian Government Agency CAPES, The Joint Standing Research Committee of St. Mary’s Hospital and the North West Thames Regional Health Authority through their Local Organising Committee. References 1 Rees,

2 3 4 5 6

7 8 9 10 11

12 13

14 15

R. J. W., 81 Valentine, R. C. (1964). Application of quantitative electron microscopy to the study of Mycobacterium lepraemurium and Mycobacterium leprae. In Leprosy in Theory and Practice (R. C. Cochrane and T. F. Davey, eds.). pp. 26-35, Bristol, John Wright and Sons Ltd; 2nd Edition. Rees, R. J. W. (1971). The impact of experimental human leprosy in the mouse on leprosy research. international Journal of Leprosy, 39, 201. Shepard, C. C. (1960). Acid fast bacilli nasal excretion in leprosy, and results of inoculation in mice. American Journal of Hygiene, 71, 147. Shepard, C. C. (1960). The experimental disease that follows the injection of human leprosy bacilli into the foot pads of mice. Journal of Experimental Medicine, 112, 445. Ambrose, E. J., Antia, N. H., & Khanolkar, S. R. (1974). Uptake of radioactive DOPA by M. leprae. Nature, 249, 854. Camargo, E. E., Kertcher, J. A., Larson, S. M., Tepper, B. S., &Wagner Jr., H. N. (1979). Radiometric measurement of differential metabolism of fatty acids of Mycobacterium lepraemurium. International Journal of Leprosy, 47. 126. Khanolkar, S. R., Antia, N. H., & Ambrose, E. J. (1976). Autoradiographic studies of Mycobacterium leprae. Leprosy Review, 47. 267. Holm-Hansen, O., 81 Booth, C. R. (1966). The measurement of adenosine triphosphate in the ocean and its ecological significance. Limnology and Oceanography, 11, 510. Thore, A., Lundin, A., & Ansehn, S. (1983). Firefly luciferase ATP assay as a screening method for bacteriuria. Journal of Clinical Microbiology, 17, 218. Dhople, A. M., & Hanks, J. H. (1973). Quantitative extraction of adenosine triphosphate from cultivable and host-grown microbes: calculation of adenosine triphosphate pools. Applied Microbiology, 26, 399. Lee, Y. S., & Crispen, R. G. (1977). Rapid quantitative measurement of drug susceptibility of mycobacteria. In Proceedings of the Second Biannual ATP-Methodology Symposium (G. Borun, ed.). pp. 219-235, SAI Technology, San Diego, California. Lundin, A., & Thore, A. (1975). Comparison of methods for extraction of bacterial adenine nucleotides determined by firefly assay. Applied Microbiology, 30, 713. Brown, C. A., & Brown, I. N. (1982). Mycobacterium bovis, BCG, modulation of murine antibody response: influence of dose and the degree of aggregation of live and dead organisms. British Journal of Experimental Pathology, 63, 133. Gheorghiu, M., & Lagranderie, M. (1979). Mesure rapide de la viabilite du BCG par dosage de I’ATP. Anna/es de Microbiologic, 130 B, 147. Riddel, R. W. (1964). The acid fast bacteria. In Leprosy in Theory and Practice, (R. C. Cochrane and T. F. Davey, eds.). pp. 4149, Bristol, John Wright and Sons Ltd; 2nd Edition.