Figure 4. Effects of heatup rate and mode on weight loss histories at 350 “C
TIME, MINUTES
for these was used as input to the Arrhenius equation. Integration of the latter provided the weight-time curves shown in Figures 3 and 4. From the foregoing, it can be seen that the heatup mode is important in determining the amount of weight that is lost after a given time, but is relatively unimportant in its influence on the weight loss rate and subsequent kinetic analysis. This treatment has provided an estimate of the influence of constant thermal errors and of fluctuations about the programmed temperature level. It is noteworthy that symmetrical fluctuations do not result in a cancellation of errors when the rate behavior is an exponential rather than linear function of temperature. Although all of these findings would be anticipated without the computer study, this technique provides a graphic, reasonably accurate picture of the magnitude of such effects.
JOHNB. GAYLE CARLT. EGGER~ Laboratories Division National Aeronautics and Space Administration John F. Kennedy Space Center Kennedy Space Center, Fla. 32899 RECEIVED for review June 9, 1971. Accepted September 9, 1971. This paper is based on material contained in NASA Technical Memorandum X-53106, August 13, 1964: Cross Check Study of Thermal-Vacuum Weight Loss Determinations for Selected Polymers, by C. T. Egger and J. B. Gayle. Present address, Director of Development, Grain Processing Corporation, Muscatine, Iowa.
Computer Matching of Pyrolysis Chromat ogra ms of Pathogenic Microorganisms SIR: Since ancient times man has been preoccupied with problems of classification and differentiation. Attempts to solve these problems have often been hampered by subjective judgments. Thus, the taxonomic relationships of bacterial cells have been obscured by the nonquantitative nature of traditional methods of classification such as morphology and serology. For example, the well known Voges-Proskauer test for enteric bacteria relies on the development of a red color as seen by a technician. Chemotaxonomic methods using modern analytical techniques provide a more objective approach to the problem of classification and differentiation. The pyrolysis-gas-liquid chromatography (PGLC) method for identifying bacteria
(1-5) is a good example. Pellets of dry bacteria are subjected to high temperatures in the absence of air, and the resulting cellular breakdown products are separated by gas-liquid chromatography. Each bacterial strain produces a characteristic chromatogram or “fingerprint” which identifies the microorganism. Even closely related cells, such as the subtypes of the same species, may be differentiated in this way. (1) E. Reiner, Nature, 206, 1272 (1965). (2) E. Reiner, J . Gas Chromafogr.,5, 65 (1967). (3) E. Reiner and W. H. Ewing, Nature, 217,191 (1968). (4) E. Reiner and G. P. Kubica, Amer. Rec. Resp. Dis., 99, 42
(1969). (5) E. Reiner, R. E. Beam, and G. P. Kubica, ibid.,p 750.
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Table 1. PGLC Data for Two Unknowns Unknown Accepted Rejected bacterium library bacterium library bacterium RT" PAb RT PA RT PA No. 1 9 3 11 3 9 3 17 6 17 6 123 3 53 29 53 32 140 4 75 3 74 3 170 3 102 3 115 3 194 9 131 4 130 4 238 6 181 9 180 8 273 16 221 6 219 5 254 16 252 14 No.2 9 3 11 3 19 3 18 15 18 14 123 3 66 3 114 3 139 5 122 3 130 4 192 10 140 6 180 10 237 6 192 9 218 5 27 1 17 237 6 251 13 270 15 a Retention time in minutes = 0.118 RT 4 - 18.7. Peak amplitude in mm.
One of the outstanding characteristics of the PGLC technique is its reproducibility. We decided, therefore, to test the feasibility of comparing bacterial pyrochromatograms by means of computer rather than by the visual method used in earlier work (1-5). Pyrochromatograms were obtained with the aid of a Barber-Colman Series 5000 gas chromatograph equipped with a flame ionization detector and a 20-ft copper column packed with 5 % Carbowax 20M (terephthalic acid terminated) coated by the vacuum distillation method on Anakrom ABS, 110/120 mesh. We performed the analyses using a 5-mV recorder and an initial column temperature of 20 "C; temperature was increased at a rate of 12"/minute until an upper value of 175 "C was reached. Chromatograms required 35 to 40 minutes (6). A computer program was written which directed an RCA Spectra 70/55 to read peak heights and retention times characteristic of an unknown bacterium, to search a file of PGLC parameters of known bacteria, and to produce as output the identity of the unknown if the library of knowns contained the unknown. The library of knowns was comprised of retention times and amplitudes of six to nine peaks from six bacteria each belonging to a different Kauffman-White serological group of Salmonella spp. As in previous work (4), we ignored the complicated initial portion of all chromatograms ; the few peaks of heights 3 mm or greater which emerged toward the end of the PGLC tracings sufficed for identification purposes. Library and unknown pyrochromatograms were all secured under identical conditions from 90-100 pg dry weight of bacteria. Peaks were divided into two groups: those with amplitudes of 3 or 4 mm and those with amplitudes greater than 4 mm. Within each group, the peaks were arranged according to increasing retention time. The larger peaks of an unknown bacterium were then compared one for one with those of ( 6 ) E. Reiner, J. J. Hicks, M. M. Ball, and W. J. Martin, un-
published work.
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0
library bacteria by means of both retention time and amplitude. If any pair of values differed by more than 5 % , the library bacterium was rejected. The sets of 3- and 4-mm peaks were compared by retention time only. Often the total number of small peaks differed, even for repeat runs, because minor changes in amplitude caused peaks to fall below or to rise above the arbitrary 3-mm cut-off point. In such an event, the computer would eliminate a 3-mm peak whose retention time did not fall within the 5 % error limit. When the entire library was searched, the error limit was doubled to lo%, and the process repeated. After two more such error increases, the error limit was returned to 5 %, the sets of small peaks were enlarged to include all those of 5 mm or less, and the search was continued as before. Typical data from unknown bacteria and from corresponding accepted and rejected library bacteria are shown in Table I. Ten unknowns were examined, and nine of these were identified correctly. One unknown was at first incorrectly reported as not existing in the file of knowns because a single comparison of peak amplitudes exceeded the allowable error limits. When the pair of peaks was deleted, the unknown was identified correctly. The agreement between unknown and accepted library bacteria in Table I is better for the first example than for the second one. This is because the time span between PGLC runs was much smaller in the first case; deterioration of the chromatography column led to changes in the absolute values of the PGLC parameters. We are currently investigating methods (such as using internal references and relative PGLC parameters) which will preclude this problem. To our knowledge, the prototype program demonstrates for the first time the feasibility of comparing pyrochromatograms automatically. Although the program has the virtue of simplicity, there are disadvantages which we hope to overcome with additional instrumentation and more sophisticated statistical techniques. The search program as described above gives only a "yes" or "no" answer; a program which quantitatively assesses the degree of bacterial differenceswould be more powerful. Of course, the computer-PGLC method need not be restricted to bacteria. We plan to use it for a wide variety of other purposes such as determining the difference between normal and pathogenic tissue cells. ACKNOWLEDGMENT
We thank Judy Hicks (CDC) for technical assistance and Robert Williams (Emory) for helpful advice. Supported in part by the National Science Foundation. FMM thanks the Camille and Henry Dreyfus Foundation for a Teacher-Scholar Award and the NIH for a Career Development Award. Department of Chemistry Emory University Atlanta, Ga. 30322 Microbial Chemistry Laboratory Center for Disease Control Atlanta, Ga. 30333
F. M. MENGER G. A. EPSTEIN D. A. GOLDBERG E. REINER
RECEIVED for review June 23, 1971. Accepted September 10, 1971.
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