The first variable that was evaluated was the effect of medium manufacturer on the results of the salt tolerance test. This study shows that medium source does not contribute significantly to the variability of the test when tests are incubated at 35°C in ambient air (Table ). The formulations of the three brands of medium tested were essentially identical. The differences in the results noted among the media from the various manufacturers were most significant with the dilute suspension, suspension C (Table ). It appears that these differences are more likely related to inoculum concentration than to medium manufacturer.
An important variable in the salt tolerance test is the concentration of organisms in the inoculum. It appears that the inhibitory nature of the salt-containing medium, combined with low concentrations of organisms in the inoculum, could lead to false-negative results, especially for M. abscessus. The best results are obtained when slants are inoculated with a high concentration of organisms. Overall, suspension A gave the best results at all temperatures, while suspension B gave only slightly fewer incorrect results (Tables and ). Suspension C gave the largest number of incorrect results (Tables and ) and also produced the only tests which were considered uninterpretable due to low inocula (fewer than 50 colonies on the control slant).
It was useful to standardize the suspensions used here to ensure that sufficient numbers of organisms were present in the inoculum. In practice, organism suspensions prepared by visual comparison to a 1 McFarland standard suspension appear to be less dense than those prepared spectrophotometrically. We therefore recommend the suspension standardization procedure described herein. More than half of the organism suspensions reached the target percent T within 6 days of incubation at 28°C. Some suspensions of M. abscessus and M. abscessus variant, although displaying heavy growth in TAB, did not attain the target percent T by day 8, perhaps because of the tendency of the organisms to grow in clumps. Control slants inoculated with suspensions A and B of these organisms did show adequate growth at 35°C. In nine tests inoculated with suspension C, adequate growth was not achieved on the control slant. This failure to grow on the control slant illustrates the need for the use of a heavy inoculum prepared from a suspension which was vigorously vortexed with glass beads to distribute the organisms more uniformly.
Another important variable in the salt tolerance test is the incubation temperature. All isolates of M. fortuitum and M. mucogenicum gave the expected results at all temperatures tested (Tables , , and ). Only one test (with slants from one manufacturer) with M. peregrinum gave a false-negative result with suspension B at 35°C (Table ).
Two isolates of M. fortuitum biovar 3 (ATCC 49403 and ATCC 49404) also gave the expected positive results with inocula from a 1 McFarland suspension incubated at 35°C in ambient air (data not shown).
is generally expected to grow on salt-containing media (3
), a characteristic which separates this species from M. chelonae
. Overall, the results of the salt tolerance test for M. abscessus
and the M. abscessus
variant showed minimal deviation from the expected results when incubation was at 28 and 35°C with inocula containing high concentrations of organisms. The tendency for this organism to grow in clumps requires that inocula be prepared carefully; an adequately dense inoculum suspension will decrease the possibility of false-negative results. One of the 11 M. abscessus
and M. abscessus
variant isolates tested failed to grow on salt-containing media at either 28 or 35°C from any inoculum concentration (Tables and ). Wallace et al. (11
) noted that some M. abscessus
isolates are inhibited at temperatures higher than 28°C; it appears that some M. abscessus
isolates may also be inhibited on salt-containing media at 28°C. This isolate also did not grow on salt-containing medium at 35°C in a CO2
-enriched atmosphere when suspension A was used (Table ). A fine film of growth was noted on media incubated at 30°C with an inoculum from suspension A (Table ). The control LJs showed adequate growth at all temperatures for all inoculum concentrations. None of the M. abscessus
or M. abscessus
variant isolates studied failed to grow at 35°C when a large inoculum was used.
is generally expected to be salt tolerance test negative (3
). With inocula from suspensions A and B, all M. chelonae
isolates tested gave false-positive reactions when they were incubated at 28°C. Even with the very dilute suspension, suspension C, 9 of 12 tests gave false-positive results at 28°C. All M. chelonae
isolates were also salt tolerance test positive when they were tested at 30°C, and one of five isolates was positive when tested at 35°C in 5% CO2
(Table ). In contrast, all tests incubated at 35°C in ambient air (Table ) gave the expected negative results. Kusunoki and Ezoki (5
) previously noted these effects with a reference strain of M. chelonae
. Silcox et al. (8
) describes the profuse growth of M. chelonae
at 28°C, and from the data presented here it appears that the stimulating effects of a lower temperature, and possibly those of a higher CO2
content as well, encourage the growth of M. chelonae
and override the inhibitory effect of the salt-containing medium for this species.
The interpretation criteria for salt tolerance test results are also unclear. In this study positive reactions showed mostly confluent or nearly confluent growth on the salt-containing media with the more dense inocula. The criterion for positivity recommended by Kent and Kubica (3
) (at least 50 colonies on the salt-containing media) appears to be appropriate for accurate test result interpretation. We would like to add that the presence of greater than 50 colonies of any size should be considered a positive reaction. Some M. abscessus
or M. abscessus
variant strains incubated at 35°C grew as pinpoint or tiny colonies growing over the surface of the slant. Slants should therefore be carefully examined to detect weakly positive strains.
The use of the LJ control is an important part of the performance of this test since the amount of growth that it shows gives an accurate estimation of the adequacy of the inoculum. Criteria for adequate growth on the control slant as specified by others include growth (1
), numerous colonies (3
), and positive (7a
) to greater than 50 colonies (12
). Since inoculum density appears to be a critical factor in test performance, we recommend that the control slant show more than 50 colonies for the test to be considered interpretable, because this number is also the recommended minimum needed on the salt-containing slant for a positive result (3
). If 50 or fewer colonies are visible, the inoculum density is probably inadequate and an apparently negative salt tolerance test result may merely be due to an insufficient inoculum and not to inhibition of the isolate.
Several investigators have recommended the use of the carbon utilization test because it more reliably distinguishes between M. abscessus
and M. chelonae
). Isolates of M. chelonae
utilize citrate as a carbon source, while M. abscessus
does not. This test medium appears to be used mostly in reference laboratories and until recently has not been commercially available, limiting its usefulness for many clinical laboratories. In our hands, several isolates identified as M. chelonae
by REA have given negative or weakly positive citrate reactions (data not shown); more study of the parameters affecting the results of the citrate utilization test seems warranted.
The performance of the salt tolerance test can be improved with the use of well-dispersed inocula with concentrations spectrophotometrically equal to a 1 McFarland turbidity standard (or the use of an inoculum incubated for 8 days for organisms which do not attain a high enough concentration otherwise). Test and control slants should be incubated at 35°C in ambient air for 4 weeks. Salt-containing media and control slants should be examined carefully for the presence of greater than 50 colonies of any size. Isolates of M. abscessus
that fail to grow in the salt tolerance test, such as the one that we encountered, are presumably rare. However, the routine use of a citrate utilization test may be helpful to increase the likelihood of the correct identification of such isolates. For those laboratories with the capability of performing them, molecular methodologies such as PCR and REA (1a
) are probably the most unambiguous methods for the rapid and accurate identification of these organisms.