Effect of a Sputum Digestant on the Viability of

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1 APPLED AND ENVRONMENTAL MCROBOLOGY, May 1976, p Copyright ) 1976 American Society for Microbiology Vol. 31, No. 5 Printed in U.SA. Effect of a Sputum Digestant on the Viability of Mycobacterium fortuitum DAVD C. BAKER' AND EDWARD J. HSU* Department ofbiology, University of Missouri-Kansas City, Kansas City, Missouri Received for publication 28 October 1975 A microcolony technique has been demonstrated as being useful for the rapid determination of the viabilities of single cells of Mycobacterium fortuitum. Cultures of M. fortuitum grown to early logarithmic phase in broth were treated with the sputum digestant N-acetyl-L-cysteine-sodium hydroxide (NALC- NaOH) for periods of 10 to 40 s. After growth for three generations (7.5 h) on agar films, viabilities were determined by counting under a phase contrast microscope. The viable mycobacteria grew into microcolonies that exhibited extensive branching, whereas the nonviable mycobacteria remained as single cells. Sputum was mixed with some broth cultures before treatment to simulate the digestion process in a clinical laboratory. When broth cultures were treated with sputum digestant for 40 s, only 2.8% of the cells remained viable. When the broth cultures were mixed in a ratio of 1:4 with sputum and then treated for 40 s, 16.4% of the cells remained viable. The results indicate that M. fortuitum is very sensitive to the digestant. The results also indicate that a microcolony technique could be used for the assessment of the viability of mycobacteria. When isolating mycobacteria from patients with pulmonary disease, sputum is commonly treated with a digestant (digestion decontamination agent). Although digestants do liquefy the sputum and destroy the rapidly growing organisms, they also destroy some mycobacteria. To help elucidate this problem concerning the adverse effects of digestants on mycobacteria, we determined the effect of sputum digestant on the viability of Mycobacterium fortuitum. This species is easily cultured, widely encountered (1), and rarely pathogenic; thus it was regarded as the ideal organism for our study. We chose to use the N-acetyl-L-cysteinesodium hydroxide (NALC-NaOH) sputum digestant of Kubica et al. (3, 4). t is a recommended digestant in two laboratory manuals (2, 7) because the 2% NaOH results in the destruction of fewer mycobacteria than 4% NaOH or other agents, and because of the short treatment time (15 min). The dilution-plate count method initially used to determine viability was abandoned. Excessive clumping of the mycobacteria caused this method to be inaccurate due to the inability to determine the number of cells that initiated each colony. A microcolony technique that detected the viability of single cells and avoided the interference of cell clumps was adopted. This method, developed by Postgate et al. (6) Present address: Program in Medical Technology, Uni- ' versity of Maryland, Baltimore, Md and modified by Postgate (5), proved to be simple to perform and the results were reproducible. MATERALS AND METHODS Organism and culture conditions. M. fortuitum (ATCC 9820) was used. Stock cultures were maintained on nutrient agar slants under sterile mineral oil at 5 C. To grow the cells, 0.75 ml of sterile Tween 20 (Atlas Chemical ndustries) was added to 100 ml of sterile nutrient broth (Difco) in a 250-ml Erlenmeyer flask. The detergent enabled us to obtain uniform cell suspensions in the broth. An appropriate volume of a culture of M. fortuitum, previously grown in the same medium, was then added. The culture was incubated on a New Brunswick Scientific Co. gyrotory water bath shaker at 37 C, at a setting of 5.0. The culture was incubated until the optical density (OD), measured on a Bausch & Lomb Spectronic 20 at 600 nm, was 0.10 to 0.30, which indicated early logarithmic phase (determined from a growth curve obtained by readings of OD). Cultures of this OD range, after being digested and in the process diluted, resulted in 20 to 40 cells per field at x400. Before being digested, the cultures were examined with an Olympus phase contrast microscope at x400 for purity and the approximate percentage of single cells. The percentage of particles that were single cells was usually 50 to 70% (the rest of the particles were cell clusters containing two to several hundred cells). Digestion procedures. The NALC-NaOH digestant was prepared according to the formula of Kubica et al. (3, 4). The digestant was sterilized by 773

2 774 BAKER AND HSU membrane filtration, decanted into a sterile flask, and used the same day. Sterile 1 M HCl was used to neutralize the ph of the culture-digestant mixture. Five milliliters of a broth culture of mycobacteria (OD = 0.10 to 0.30) was placed in each sterile screwcap tube (16 by 150 mm) and mixed for 2 min at the fastest speed on a Vortex Genie to help break up any clumps of organisms. An equal volume of sterile digestant was added at time zero, and the mixture was mixed for approximately 5 s. One molar HCl was added after the digestion period to neutralize the action of the NaOH and thus stop the digesion process. The preparation was immediately mixed to insure complete neutralization. The broth cultures were digested for 0, 10, 20, 30, and 40 s, respectively. The treated and untreated cultures were allowed to remain in the tubes for 20 to 30 min before inoculation onto microslides. All digestion was done at 22 C. For the sputum experiments, sputum was collected, sterilized by vacuum filtration, and stored at 5 C. Amounts of 2.5 and 0.5 ml, respectively, of a broth culture (OD = 0.10 to 0.30) were placed into tubes and mixed. Then 2.5 and 2.0 ml, respectively, of filtered sputum were added to each of the tubes and spun on a Vortex Genie to blend the sputum and cells. The preparations were allowed to stand for about 30 to 35 min to allow the sputum to react with the mycobacteria. Digestion was then carried out. As a control (digestion time = 0 s) for each digestion experiment, the appropriate volumes of digestant and 1 M HCl were mixed, and this neutralized mixture was added to the proper volume of broth culture or broth culture-sputum. As an additional control for each experiment, a volume of broth culture with nothing added was placed in one tube. For the sputum experiments, in addition to zero digestion time and a tube of untreated broth culture, the appropriate volume of the respective broth culturesputum mixture was added to one tube. This enabled us to determine the effect of sputum, by itself, on the mycobacteria. Microcolony technique. Sterile microscope slides were aseptically placed in sterile petri dishes, one slide per dish. The petri dishes were used to help prevent contamination and to facilitate the handling of the microslides. Nylon gaskets (Dorman Products nc., Cincinnati, Ohio; 17-mm internal diameter by 2.5 mm deep) were aseptically placed on the slides (one ring per slide), and 0.25 to 0.30 ml of molten nutrient agar (Difco), precooled to 57 C, was pipetted inside the ring to a height of approximately 1.5 mm. The microslides were allowed to cool at room temperature (22 C) for 30 to 60 min. A 4-mm loopful of treated or untreated culture was placed on the cooled agar surface of a microslide. After the inoculum had dried for 20 to 30 min, a sterile cover slip (22-mm diameter) was aseptically placed on the ring. The cover slip was sealed to the ring with a drop of water-glycerol mixture (1:1), which created a sealed chamber with an air space of about 1.0 mm. Four microslides were made for each digestion tube and control tube. One microslide of each group was put into a 5 C refrigerator immediately and used as a control to determine the appearance of the mycobacteria on the microslides without APPL. ENVRON. MCROBOL. incubation. The microslides were placed in a 37 C incubator and incubated for 7.5 h (three generations as determined by growth curves of broth cultures). A 10-min incubation time was added to allow for warming to the 37 C incubation temperature, resulting in a total incubation time of 7.66 h. After incubation, the microslides were removed from the incubator, cooled at 5 C for at least 30 min, and then examined with an Olympus phase contrast microscope at x400. For each microslide, approximately 300 units were counted. A unit consisted of a cell cluster that contained 1 to 16 cells. A cell cluster (microcolony) containing 4 to 16 cells was counted as 1 viable unit, and a cluster of less than 4 cells was counted as 1 nonviable unit. Microcolonies of over 16 cells were disregarded because they had grown from initial cell clusters containing more than one viable cell. For each microslide, the viability percentage was derived directly from the ratio of viable units to the total number of viable and nonviable units. The recorded percentage of viable and nonviable units for each digestion tube and control tube was the average of three microslides. RESULTS Morphology of microcolonies. The cells in Fig. 1 were treated with the digestant for 10 s and placed on microslides. The cells in Fig. 1A, which were inoculated and then refrigerated immediately, displayed the characteristic morphology of mycobacteria (long, slender, curved rods). Single cells and cell clusters were easily distinguished. The cells in Fig. 1B had been incubated for one generation, and two viable cells had elongated (compared with the cells in Fig. 1A). The cells in Fig. 1C had been incubated for two generations. The viable cell had elongated and had branched at two points. These two short branches were easily seen. A cell cluster that had originated from two or more viable cells was seen on the left side. The cells in Fig. 1D had been incubated for three generations. A microcolony produced by a viable, single cell was seen; this viable cell had branched twice, sending out extensions in two directions at each fork or branching point. A nonviable, single cell was also seen. Also present were two large clusters of cells, each containing more than 16 cells. At the end of three generations (7.5 h), the number of viable, single cells and the number of nonviable, single cells on each microslide was counted. A normal cell can produce eight daughter cells after growth for three generations. To take into account any cells that might have divided just before orjust after inoculation onto the microslide, we considered any branching microcolony that contained 4 to 16 cells to have originated from one viable, single cell.

3 VOL. 31, 1976 VABLTY OF M. FORTUTUM 775 \N t~n. -%, r:t O, Air B 0., "", f.4 '.. F 11t if, \ t N el.-.001/ -.40 V ow W %,. D j,. >,1~~~~~~~~~ 4 FG. 1. Morphology of M. fortuitum after treatment with NALC-NaOH for 10 s, followed by inoculation onto microslides. (A) Cells that were not incubated, showing single cells and a cell cluster. (B) Cells after incubation for one generation, showing elongated cells (arrows). (C) Cells after incubation for two generations, showing a viable cell that had begun to branch (arrow). (D) Cells after incubation for three generations, showing a single, viable cell that had branched twice (arrow) and a nonviable, single cell (double arrow). Marker indicates 10 gm. ik As the single, viable mycobacteria cells began to grow on the microslide, they elongated and began to branch. These branching microcolonies resembled the branches of a tree. Single, nonviable cells remained as single cells. The cytoplasm of all cells, viable and nonviable, was quite granular, and sporelike refractile bodies were sometimes observed. t was not difficult to determine the approximate number of cells in each branching microcolony; however, it was tedious to make a precise count of the cells in each microcolony because the cells /l were not separated by obvious walls. Microslides with up to 40 objects (dead bacteria, single-cell microcolonies, and a few multicell microcolonies) per microscope field at x400 were relatively easy to count. The observer had to look for true branching, since some clusters of dead cells looked somewhat like the microcolonies of viable, single cells. Approximately 10 min was needed to count the microcolonies on each microslide. Effect of digestant on viability. Figure 2 shows the death curves of exponentially grow-

4 776 BAKER AND HSU LU z LU TME N SEC. FG. 2. Death curves of exponentially growing broth cultures of M. fortuitum when treated with the NALC-NaOH digestant. Symbols: (0) Plain broth culture; (E) broth culture mixed in a ratio of1:1 with filtered sputum before treatment; (A) broth culture mixed in a ratio of 1:4 with filtered sputum before treatment. ing broth cultures of M. fortuitum treated with NALC-NaOH digestant. The viability of cells at zero digestion time (treated with digestant preneutralized with 1 M HCl) for all experiments was about 88 to 93%. The viability of the untreated cells for all experiments was also about 88 to 93%. Therefore, digestion time zero on the graph represents both the percent viability of 0-s-treated cells and the percent viability of untreated cells. After digestion of a broth culture for 10 s, the viability of the mycobacteria was about 66%; after digestion for 20 s, the viability was about 46%; after 40 s of digestion, the viability was about 2.8%. n the broth culture mixed with sputum in a ratio of 1:4, the viability was about 16.4% after 40 s of digestion. The D value (death of 90% of the single cells) of the mycobacteria in the broth culture was estimated to be 33 s. n the determination of a D value, a straight line is drawn that connects the various points (viability versus time) on the graph. The point on this line that intersects 10% viability (90% death) is extrapolated to find the time at which 10% viability occurred. This time is D value. n the determination of the D value of the mycobacteria in the broth culture, the first part of the curve was disregarded because it showed a lower D value, perhaps due to a permeation APPL. ENVRON. MCROBOL. barrier. The curve representing the death of a broth culture of M. fortuitum, when mixed in a ratio of 1:1 with filtered sputum and then treated with the digestant, suggests that the sputum at this concentration was slightly inhibitory (D value about 23 s) to the treated bacteria. The D value was again determined by disregarding the first part of the curve. The curve representing the death of a broth culture of M. fortuitum, when mixed in a ratio of 1:4 with filtered sputum and then treated with the digestant, suggests that the sputum at this higher concentration had a slightly protective effect (D value about 52 s) for the treated bacteria. )SCUSSON The evidence shows that the commonly used sputum digestant NALC-NaOH has a rapid, lethal effect on M. fortuitum. This study also demonstrates that sputum provides some protection for the mycobacteria from the effects of the digestant. The evidence presented suggests that many patients infected with M. fortuitum may receive negative culture reports from clinical examinations of their sputum specimens. Positive culture results are obviously obtained in cases of pulmonary infections caused by M. fortuitum, as shown by numerous reports in the literature. t is quite possible that these positive results are obtained only when the patients are heavily infected. The microcolony technique can be used as a rapid, reliable method for the assessment of the viability of a mycobacteria species. The most obvious advantage of the technique is the time factor; only 7.66 h is needed to determine the viability of M. fortuitum. The worker has the added benefit of being able to actually see the viable and nonviable organisms. t is possible to determine the viability of single cells only, thus avoiding the interference of any cell clumps. Since mycobacteria species are well known for their clumping characteristics, the microcolony procedure offers the researcher a tool for dealing with the problem. Other advantages include the small inoculum (a 4-mm loopful per microslide) and small amount of medium needed (0.25 to 0.30 ml per microslide). The growth medium that is used must be sufficiently translucent so that enough light can come through it to register an image on a phase contrast microscope. n this respect, a clear agar medium such as 7H10 or 7H11 could be used; darker media such as Lowenstein-Jensen medium could not be used. We were not able to determine, based on present experience, whether this procedure

5 VOL. 31, 1976 could be used to grow any of the mycobacteria species that, compared with M. fortuitum, have lengthy generation times. The thin agar films may possibly dry out under prolonged incubation. This procedure probably did not detect any mycobacteria that were injured but still capable of recovery and growth after prolonged incubation. This problem was due to the short incubation period of only 7.66 h. Such cells can be detected by the regularly used culture methods in tuberculosis laboratories, which allow for long incubation periods. This method has been successfully used to grow various gram-negative and gram-positive bacteria and to study the killing effect of various bacteriocides (6). We believe that this procedure will prove adaptable to other mycobacteria and other agents, both chemical and physical. Perhaps microslides, along with regularly used culture media, could be inoculated directly from patients' digested sputa. Because of the unique morphology of mycobacteria microcolonies, detection of some mycobacteria infections could probably be accomplished in a few days instead of 1 to 2 months. The microcolony technique could have application to a variety of tests applied to mycobacteria, such as antibiotic susceptibility testing, and to biochemical tests. VABLTY OF M. FORTUTUM 777 ACKNOWLEDGMENTS This study was supported in part by a University of Missouri faculty research grant. The technical assistance of Steve Brash and Damini Mehta is gratefully acknowledged. LTERATURE CTED 1. Edwards, L. B Current status of the tuberculin test. Ann. N.Y. Acad. Sci. 106: Kubica, G. P., and W. E. Dye Laboratory methods for clinical and public health mycobacteriology, p Public Health Service, Government Printing Office, Washington, D.C. 3. Kubica, G. P., W. E. Dye, M. L. Cohn, and G. Middlebrook Sputum digestion and decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am. Rev. Respir. Dis. 87: Kubica, G. P., A. J. Kaufmann, and W. E. Dye Comments on the use of the new mucolytic agent, N- acetyl-l-cysteine, as a sputum digestant for the isolation of mycobacteria. Am. Rev. Respir. Dis. 89: Postgate, J. R Viable counts and viability, p n J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 1. Academic Press nc., New York. 6. Postgate, J. R., J. E. Grumpton, and J. R. Hunter The measurement of bacterial viabilities by slide culture. J. Gen. Microbiol. 24: Sommers, H. M., and J. P. Russell The clinically significant mycobacteria: recognition and identification, 2nd ed. American Society of Clinical Pathologists, Chicago. Downloaded from on October 2, 2018 by guest