Annals of Clinical & Laboratory Science 39:51-54 (2009)
© 2009 Association of Clinical Scientists
Maintenance of Mycobacterium tuberculosis on Glass Beads
Carmen Maria Saraiva Giampaglia,
Artemir Coelho de Brito,
Maria Conceição Martins,
Suely Yoko Mizuka Ueki,
Fabio Oliveira Latrilha,
Rosângela Siqueira de Oliveira,
Jonas Umeoka Yamauchi and
Maria Alice da Silva Telles
Mycobacteriology Section, Instituto Adolfo Lutz, São Paulo, Brazil
Address correspondence to Dr. Carmen Maria Saraiva Giampaglia, Instituto Adolfo Lutz, Avenida Dr. Arnaldo 355, CEP 01246-902 São Paulo, Brazil; tel 55 11 3068 2986; fax 55 11 3865 0450; e-mail hrgiampa{at}uol.com.br.
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Abstract
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The intent of this study was to estimate the shelf life of Mycobacterium tuberculosis strains, and to observe the loss of viability in some of them from year to year. From 2000 to 2004, 10,015 cultures of M. tuberculosis were preserved by freezing on glass beads at –70°C. With the expectation that the loss of viability might be around 5–10%/yr of storage, 730 strains were analyzed in order to establish the prevalence of recovery within a 5% margin of error. This study shows that 94% of the strains preserved at –70°C on glass beads could be recovered within 30 days. The recovery rates for drug-susceptible and drug-resistant strains showed no significant differences. The growth rates and the number of strains that showed abundant growth before the 30th day of incubation represent important features, since the subculture of a strain preserved for future use ought to quickly produce abundant growth in order to avoid misinterpretation of the tests. Our experience indicates that storage of M. tuberculosis on glass beads at –70°C is a suitable procedure for an active culture collection in a public health laboratory like ours, where maintenance of M. tuberculosis cultures is a complementary activity and must be quick, practical, effective, and economical.
Keywords: Mycobacterium tuberculosis, mycobacterial cryopreservation, library of microbial strains
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Introduction
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Reference laboratories for mycobacteria are supposed to maintain isolates of Mycobacterium tuberculosis (MT) and other mycobacteria (NTM) in order to establish a collection of strains that can be used as reference strains both for routine testing and research. A library of cultures that exhibits typical reactions is also very important for teaching purposes. The collections are of importance to the development of novel technologies and the evaluation of new and existing diagnostic methods. Moreover, they supply reference materials used in quality control and proficiency testing programs.
The preservation of microorganisms continues to be a difficult task, although today it is possible to be successful in this endeavor. A large number of excellent preservation techniques have been developed and good results can be achieved when they are correctly conducted [1]. Although cell death occurs during the preservation process and also during storage, resulting in low viability levels, the choice of a correct preservation method allows the maximal maintenance of viability of cultures during long-term storage [2].
Lyophilization has been successfully used for culture storage of Mycobacterium bovis BCG [3–5]. However, this method must be avoided for M. tuberculosis strains because it involves very intensive aerosol-producing steps. Cryogenic conservation at –60 to –80°C has been successfully employed for the preservation of viability and virulence of mycobacteria [2,6]. The damage caused by repeated freezing and thawing when subcultures are required is a disadvantage of storing bacteria this way. To overcome this problem, Feltham et al [7] developed a method of storage at –70°C on small glass beads in glycerol-suspension medium. This technique allows individual beads to be removed without thawing the whole sample. Various laboratories use this method, which has proved to be a safe, reliable and simple procedure for the storage of a wide diversity of bacteria [7,8].
This report describes our experience with M. tuberculosis strains stored at low temperature on glass beads. We intended to estimate the shelf life of our strains, observing the loss of viability in some of them from year to year. No previous reports analyzed the loss of viability of M. tuberculosis strains when stored by this method. The efficacy of preservation and storage of broth-suspended M. tuberculosis on glass beads was evaluated by calculating the recovery rate of preserved strains with different patterns of resistance after years of storage at –70°C.
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Materials and Methods
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The M. tuberculosis strains used for the evaluation of viability after storage were those received by the Mycobacteria Laboratory at Instituto Adolfo Lutz for identification and susceptibility testing. From 2000 to 2004, 10,015 cultures of M. tuberculosis were analyzed and preserved by freezing on glass beads at –70°C.
The steps involved in preparing the bacterial suspension before freezing were as follows:
1. Preparation of beads.
Glass beads (2 mm embroidery beads) were washed in tap water with a detergent and then washed several times in tap water to eliminate the detergent. The beads were subsequently washed in distilled water and finally dried in a 37°C oven.
2. Preparation of plastic cryovials.
About 6 to 12 washed glass beads were placed in plastic cryovials with capacities ranging from 1 to 2 ml. The plastic cryovials were sterilized at 121°C for 15 min. After sterilization, 500 µl of sterile Sauton’s medium* containing 10% (v/v) glycerol was aseptically dispensed.
The bacteria were harvested from the surface of Löwenstein-Jensen (LJ) medium with either a swab or a plastic loop. The colonies were directly emulsified in the plastic cryovial containing glass beads and Sauton’s medium, and care was taken to ensure that all the beads were thoroughly covered with mycobacteria. The plastic cryovial was allowed to stand for a few min before the excess of Sauton’s medium was completely removed with a pipette.
3. Freezing of plastic cryovials.
The plastic cryovials were placed in appropriate boxes. The boxes were placed in a commercial freezer capable of maintaining a temperature of –70°C. The contents of each box were recorded in a map to easily find the strain to be recovered in the future.
Recovery of frozen mycobacteria.
With the expectation that the loss of viability might be around 5–10%/yr of storage, 730 strains were selected for inclusion from among the preserved strains. The data for the 4th quarter of each year were selected. We had 794, 577, 602, 360, and 639 isolates that were frozen, respectively, in the last quarter of 2000, 2001, 2002, 2003 and 2004. To establish the prevalence of recovery within a 5% margin error, we randomly selected 228, 163, 173, 100, and 66 strains for 2000, 2001, 2002, 2003, and 2004, respectively. The frozen mycobacteria were recovered in the 4th quarter of 2005 and in the first quarter of 2006.
One plastic cryovial of each strain was removed from the freezer and one glass bead was removed from the vial using a sterile loop. The glass bead was transferred to one tube of Löwenstein-Jensen medium. The plastic cryovial was immediately replaced to prevent the remaining contents from thawing. The bead was rubbed over the surface of the medium with the loop, so that the bacterial inoculum could be released. The inoculated tube was incubated under appropriate conditions for growth.
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Results
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Mycobacterial growth of each subcultured strain was analyzed by morphological characteristics and the abundance of colonies observed in the tubes. The result was reported as negative (–) no growth after 40 days, (+) <20 colonies, (++) 20/100 colonies, or (+++) >100 colonies. Table 1
lists the observed growth rate and the amount of growth of the frozen strains after thawing, inoculation of LJ medium, and incubation at 37°C. Thus, 160 (22%) strains grew after 10 days of incubation, 462 (63%) after 20 days, 63 (8.6%) after 30 days, and 9 (1.2%) after 40 days. Meanwhile, 36 (4.9%) strains failed to grow. Table 2 shows the recovery rates of the preserved M. tuberculosis strains stratified by year of storage. Table 3 shows the recovery rates and drug susceptibility patterns of 636 (87%) strains preserved on glass beads at –70°C. The susceptibility of the remaining 94 strains was not tested.
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Table 1. Growth rate of recovery process of 730 M. tuberculosis strains preserved on glass beads at –70°C, subcultured after varying durations of storage.
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Table 2. Recovery rate of preserved M. tuberculosis strains subcultured on LJ medium, after 40 days of incubation at 37°C.
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Table 3. Recovery rates and drug susceptibility patterns of M. tuberculosis strains stored on glass beads at –70°C for 1 to 5 yr.
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Discussion
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A variety of methods are available for preserving bacterial strains while maintaining their viability and authenticity [1,10]. The main difference among these methods is the length of time the samples can be stored without losing their viability. Cryogenic conservation is a method by which specimens can be preserved unchanged for decades [6,10].
Different taxonomic groups and even different strains within a species exhibit significant differences in their responses to the stresses imposed by culture preservation and recovery [1]. Kim and Kubica [6] showed that the storage temperature, instead of the suspending medium, is more important for the prolonged survival of M. tuberculosis and M. bovis BCG. That study concluded that all tested fluids except distilled water maintained the viability of mycobacteria for several years at –70°C. It concluded also that cultures stored at –20°C show a gradual decrease in numbers of viable cells, in contrast with cultures stored at –70°C, which showed virtually no loss in viability after prolonged storage periods.
The present study shows that 94% of the strains preserved at –70°C on small glass beads could be recovered within 30 days. Since only 18 strains that were frozen in the year 2000 could not be recovered, the loss of viability was less than expected after 5 yr of storage. In accordance with the observations of Huang et al [11], the recovery of drug-susceptible and drug-resistant strains preserved on glass beads showed no significant differences.
The main advantage of the cryopreservation method as demonstrated by our study is that almost 100% of the 730 strains remained viable after 1 to 5 yr of frozen maintenance. The number of strains that showed abundant growth before the 30th day of incubation represents an important feature since the subculture of a strain preserved for future use ought to be able to quickly produce abundant growth, representative of the original population, in order to avoid misinterpretation of the tests. Abundant growth without subculturing is important to minimize handling and to limit the selection of undesirable mutants. Subculturing can produce clonal selection, leading to predominance of a non-representative portion of the bacterial population.
Other advantages of the method are that minimal preparation of materials is required, the method is simple to perform, many hundreds of strains can be stored for long periods in small spaces, recovery of cultures is quick with little or no disturbance of other stored cultures, only a portion of the culture is removed and thawed, the bulk of the stock culture remains frozen, and the beads thaw rapidly when placed on solid or liquid growth medium, allowing for immediate recovery.
Our experience with almost 730 strains indicates that storage of M. tuberculosis on glass beads at –70°C is a suitable procedure for an active culture collection in a public health laboratory like ours where, besides diagnosis, culture maintenance is a complementary activity that must be quick, practical, effective, and economical. Preserved strains are an essential resource for research to answer questions about microbial growth, physiology, and molecular epidemiology. They are also valuable for validating new diagnostic tests, quality control measures, and teaching methods.
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Acknowledgments
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We thank Dr. Andrew Ramsay for advice on calculating the necessary sample size to be analyzed. We thank the ICOHRTA AIDS/TB, FIC/NIH#5U2R TW006883-02 project, which supported the course "Leitura Critica e Redação de Artigos Científicos" and stimulated the preparation of this manuscript.
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Footnotes
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* Sauton’s medium [9]: KH2PO4, 0.5 g; MgSO4 7H2O, 0.5 g; citric acid, 2.0 g; ferric ammonium citrate, 0.05 g; glycerol, 60 ml; sodium glutamate, 4.0 g; distilled water to 1 L. The reagents, except glycerol, were dissolved in 300 ml of water using heat. Glycerol was added and the volume brought to 1 L with distilled water. The pH was checked and, if necessary, adjusted to pH 7.4 with 1 M NaOH. The solution was then autoclaved at 121°C for 15 min. 
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References
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Abstract
Introduction
