increase after the first day and then increased slowly. This may be explained by the fact that the competing reaction rate of an amine with isocyanate is greater than the hydrolysis reaction rate to give carbon dioxide. The isocyanate concentration can therefore decrease with only a small increase in carbon dioxide concentration. As the amine concentration decreases, the substituted urea and urethane molecules that have been formed can react further with isocyanate to give biurets and allophanates, respectively. Regrinding and repressing a KBr disk liberated the trapped carbon dioxide since its absorption intensity at 2334 cm-’ decreased markedly. A KBr disk that was stored i n a desiccator over drierite for 100 days showed only a pronounced shoulder at 2334 cm-I. This indicated that the hydrolysis reaction is diffusion controlled, and is due to the diffusion of atmospheric water into the KBr disk to react with isocyanate to give COz which is trapped within the disk. The overall reactions (hydrolysis and amine) of isocyanate were found to obey second order kinetics (Figure 2). The reaction between isocyanate and alcohol has been found to be mainly second order (10-13) but (10) H. A. Smith, J. Polymer Sci., 6, 1299 (1968). (11) L. Willeboordse, J. Phys. Chem., 74, 601 (1970). (12) A. E. Oberth and R. S. Bruenner, ibid., 72, 845 (1968). (13) A. Farkas and P. F. Strohm, Ind. Eng. Chem. Fundam., 4, 32
(1965).
zero (14) and pseudo first orders (15) have also been observed The rate constants have been calculated by a least squares computer analysis and are given in Table 11. The activation energy for the isocyanate reactions in a KBr disk was 7.8 i: 0.6 kcal/mole (Figure 3). ACKNOWLEDGMENT
The author thanks George B. Wilmot and Doug Ayers for helpful discussions. RECEIVED for review August 16, 1971. Accepted December 7, 1971. Presented a t the 23rd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1972. The opinions or assertions made in this paper are those of the author and are not t o be construed as official or reflecting the views of the Department of the Navy or the Naval Service at large.
(14) N. D. Ghatge, S. D. Yadav, and A. C. Ranade, J. Appl. Polymer Sci., 12, 447 (1968). (15) M. E. Bailey, V. Kriss, and R. G. Spaunburgh, Znd. Eng. Chem., 48,794 (1956).
Rapid Characterization of Salmonella Organisms by Means of Pyrolysis-Gas-Liquid Chromatography E. Reiner, Judy J. Hicks, Mary M. Ball, and William J. Martin Microbial Chemistry Laboratory, Center f o r Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, Ga. 30333 FORSEVERAL YEARS the technique of pyrolysis-gas-liquid chromatography (PGLC) has been effectively used to differentiate genera, species, and, in many instances, subspecies of pathogenic bacteria (1-5). Successful use of this technique has stimulated investigations of other pathogens. The present report deals with differentiation and classification of the genus, Salmonella.
Salmonellosis, a common worldwide disease, is most often associated with food poisoning. Difficulties in diagnosis may stem from three sources: use of inadequate isolation procedures, use of time-consuming biochemical tests which often rely on subjective judgments (e.g., the development of a certain color or the evolution of a gas), and failure to use reliable, well characterized standard reference sera for serological procedures which are used to confirm the identity. In addition, the serologist may be confronted with confusing cross-reactions from other enteric bacteria. We have compared results from the PGLC technique with those of two methods currently in use. One, a serological method, is exemplified by the Kauffmann-White Scheme (6) ; (1) E. Reiner, Nature, 206, 1272 (1965). (2) E. Reiner, J . Gas Chrornatogr., 5 , 65 (1967). (3) E. Reiner and W. H. Ewing, Nature, 217, 191 (1968). (4) E. Reiner and G. P. Kubica, Amer. Rer;. Resp. Dis., 99, 42 (1969). ( 5 ) E. Reiner, R. E. Beam, and G. P. Kubica, ibid., p 750 (6) P. R. Edwards and W. H. Ewing, in “Identification of Enterobacteriaceae,” 2nd ed., Burgess Publishing Co., Minneapolis, Minn., 1962. 1058
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
the other, a biochemical method, is exemplified by the distribution of sugars in Salmonella cell walls (chemotypes) (7). EXPERIMENTAL
Apparatus. All pyrochromatograms were recorded from Barber-Colman Series 5000 gas chromatographs, each equipped with a flame ionization detector and a Model 5180 pyrolysis module. The latter was attached to the gas chromatograph inlet by means of Swagelok fittings. BarberColman (5 mV) and Beckman (1 mV) strip chart recorders were used simultaneously. We also used a n Infotronics Digital Integrator, Model CRS-1l H , to record retention times and peak areas. Column Preparation. Refrigerator grade copper tubing (20 ft long; o.d., 0.125 in.; i.d., 0.065 in.) was cleaned by running through a series of solvents of increasing polarity (methylene chloride last). A stream of nitrogen was passed through the empty column, and its entire length was flamed with a torch. The usual support consisted of 5 Carbowax 20M, terephthalic acid terminated, coated by the vacuum distillation method on Anakrom ABS, 110/120 mesh. To eliminate fines, the packing was carefully sieved and fluidized. Columns prepared in this manner achieved efficiencies of 600-800 plates/foot. Carrier flow, as measured by a bubble meter, was 16 ml/minute. Sample Preparation. In preparing samples for PGLC study, the enterobacteriologists used standard biochemical and serological procedures which were developed a t the (7) F. Kauffmann, 0. Luderitz, H. Stierlin, and 0. Westphal, Zentralb. Bakteriol. Parasitenik., Abt. I Orig., 178, 442 (1960).
ana-u
II
1 1 1 8 1
1
,
S. bredeney. B
Figure 1. Representative pyrochromatograms of frequently encountered Kauffmann-White serological groups
National Salmonella Center, Center for Disease Control, Atlanta, Ga. (3, 6, 8, 9). The National Salmonella Center is affiliated with International Salmonella Center, Paris, France, and WHO. Large (150-mm diam.) Petri dishes, which had been filled beforehand with 240-250 ml of meat infusion agar, were inoculated with 1 ml of a young (4-6 hr) broth culture. The inoculum was spread over the entire surface of the agar medium. Cells were grown for 18-20 hours at 37 "C. Aseptic techniques were used throughout. Mature cultures thus produced were harvested by scraping the cells with a glass rod into 100 ml of 0 . 5 z phenolized physiological saline solution (0.85z). Care was exercised in harvesting to be sure that a minimum of particles from the medium were included. The "wash-kill" procedure was designed to eliminate all such extraneous material. Cells suspended in saline were spun in an angle-head centrifuge at 16,000 rpm fcr 20 minutes. The supernatant was discarded and the procedure was twice more repeated. Finally, the wet, packed cells were labelled with a code number and sent to the chemists for lyophilization and subsequent PGLC analysis. There were 54 samples encompassing 47 of the most important species. Procedure. A pellet of dried bacteria (ca. 80 pg) was weighed on a Cahn Electrobalance, transferred to a platinum pyrolysis ribbon, and sealed in a gas-tight chamber filled with nitrogen or helium. To eliminate residual gases, we vented the chamber to the atmosphere just before pyrolysis. All analyses were carried out at an initial column temperature of 20 "C; temperature was increased at a rate of 12"jminute until an upper temperature of 175 "C was reached. Electrometer settings were 1 X a.f.s. with no attenuation of signal. The chart speed on the 5-mV recorder was 0.33 in./min and on the I-mV recorder, 0.5 in./min. Each analysis required 35-40 minutes for all of the peaks to emerge. All analyses were performed at least twice to eliminate the possibility of instrumental or sample variation. To classify the (8) W. J. Martin and W. H. Ewing, Appl. Microbioi., 17, 111
(1969). (9) W. J. Martin, W. H. Ewing, A. C . McWhorter, and M. M. Ball, J . Public Health Lab., 27, 61 (1969).
organisms, we superimposed their respective pyrochromatograms over a light box and compared them visually. RESULTS AND DISCUSSION
Although no standard reference cultures had been provided beforehand, the analysts correctly sorted and classified all 54 coded samples by comparing their PGLC tracings. All duplicate samples gave identical profiles. Figure 1 shows representative pyrochromatograms of the most frequently encountered Kauffmann-White serological groups. Close inspection reveals differences in all the pyrochromatograms illustrated. Starting with S. panama, Kauffmann-White serological group D, we see a peak labeled 3 with retention time 59 or 60 seconds greater than that of peak 2 (e.g., S. newport-C2). At first glance, S. cubana and S. paratyphi var. Durazzo may appear similar to each other in the lower, 5-millivolt (mV) tracings. However, in the upper, 1-mV tracings, peaks have been expanded in amplitude five times, and we now can see differences at peak 5. In addition, in the S. paratyphi (1-mV recording), we see a peak of greater amplitude between peaks 6 and 7. For S. illinois, the 1 peak appears to be somewhat elevated, and even in a I-mV tracing, the pyrochromatograms show no peaks of corresponding magnitude to the 2, 3, 4, and 61/2peaks present in other organisms. S. newport and S. bredeney are both characterized by a prominent peak a t 2. Both S. typhimurium and S. Chester have a discernible peak at 2 as well as a prominent peak at 4. In Table I, the last three columns, Kauffmann-White (K-W) group, chemotype, and PGLC characteristic peaks, represent three different approaches to a classification scheme for Salmonella. Yet, when we compare characteristic PGLC peaks with the of her two, we see some interesting correlations. First, with each member of a Kauffmann-White (IC-W) serological group, we observe a consistent interrelationship between distribution of hexose sugars and PGLC peaks characteristic of this group. For example, the K-W group A is associated only with chemotype XV, a chemotype having among its complement of sugars the unusual member, paraANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
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Table I. Comparative Serology and Chemistry of Salmonella
Name Paratyphi A var. Durazzo Heidelberg Chester Derby Bredeney Paratyphi B Java Paratyphi B var. Odense Typhimurium Typhimurium var. Copenhagen St. Paul Cholerae Suis Cholerae Suis var. Kunzendorf Bareilly Montevideo Braenderup Tennessee Oranienberg Infantes Thompson Newport Manhattan Blockley Kentuckey Muenchen Typhi H901W Typhi 0901W Typhi 2v Typhi Watson Typhi GS, Rough Typhi Vi I, Rough Typhi R2,Rough Enteritidis Gallinarum Pullorurn Panama Typhi Lact+ Javiana Typhi TP Anatum Senftenberg Illinois Newington Give Cubana Worthington Poona a
Somatic (0)Antigen (6) 2,12
H Antigen ~(6) Phase 1 Phase 2 a ...
1,4,5,12 4,5,12 1,4,5, 12 1,4,12,27 1,4,5,12 1,4,5,12 1,4,12
r e, h f, g 1, v b
PGLC Kauffmann ( 6 ) Characteristic White Group Chemotype (7) Peaks" A xv 6l/2 B B B B B
b
152 e, m, ... 1,7 1,2 (1,2) 132
1,4,5,12 1,4,12
i i
1,4,5,12 67 67
e, h C
6,7 67 6,7 6 7 67 6,7 67 68 6,8 68 (8),20 68 9,12 9,12 9, 12(Vi) 9, 12(Vi) (Vi) (91, (Vi)
... 1,9, 12 1,9,12 9,12 1,9,12 9,12, (Vi) 1,9,12 9, (121, (Vi) 3, 10 1,3,19 (31, (15), 34 3, 15 3,lO 1,13,23 1, 13, 23 13,22
2,4 2,4 2
B
XIV XIV XIV XIV XIV XIV XIV
192 132
B B
XIV XIV
2,4
B Cl Cl
XIV 111 111
2,4 ...
(C)
1, 2 1,5 195
Y
1,5
c 1
I11 I11 111 I11 I11 I11 I11 XIV XIV XIV XIV XIV XVI XVI XVI XVI XVI XVI XVI XVI XVI XVI XVI XVI XVI XVI XI11 XI11 XI11 XI11 XI11 VI VI VI
b
g, m, s e, h
...
m, t r k e, h d k
e, m, z ... ... 1,5 1,5 1,2 1,5 1,5
1
26
229
d d
1,2
...
d d
(4 (4 d g, m
...
... 1, v
d l,Z28
d e, h g, s, t z10
e, h e, v 229
2 2
... ... ... ... ...
...
... ... .
I
.
... 195 ... 1,5
...
1,6 , . .
1,5 196 1,7
...
1, w 1,6
B
c 1
c 1
C1 Cl C1 Cl
cz
c 2 c 2
cz cz D D D D D D D D D D D D
D D E E4 E3 E2
E1 G G G
2
2,4 2,4 2
2
...
...
...
...
... . .
...
... 2
2 2 2 2 3 3 3
3 3 3 3 3 3 3 3 3 3
3 ...
...
... ...
...
... .. .
...
See Figure 1.
tose. The corresponding pyrochromatogram has a distinguishing peak, 6l/2, and this fact enabled us to select that organism from all the others. In like manner, K-W groups B and C2, chemotype XIV have the rare sugar, abequose, and organisms comprising this chemotype all have a distinguishing peak 2. In addition, the D serological (K-W) group and its chemotype, XVI, invariably have a peak of greater magnitude a t 3. Chemotype XVI has in its array of cell wall hexoses, the unique one, tyvelose. The three sugars, paratose, abequose, and tyvelose are 3,6-dideoxyhexoses. On the other hand, chemotypes 111, XIII, and VI have no 3,6-dideoxyhexoses in their cell wall structures (10) and we (10) 0. Luderitz, A. M. Staub, and 0. Westphal, Bact. Reo., 30,192 (1966). 1060
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
find no 2, 3, 4, or bl/* peaks in their pyrochromatograms. It is curious and perhaps coincidental that, in this study, PGLC peaks appear to match with a particular chemotype as well as a particular K-W group. PGLC-mass spectrometry studies now in progress may lead to a n identification of these characteristic fragments (peaks) and may allow us to decide whether a true association exists. Second, inspection of Table I shows that the chemotypes give little information concerning somatic (0)and flagellar (H) antigenic structures, but the characteristic pyrolysis peaks do indicate a certain correspondence with serotypes. For example, when the somatic 0 9 antigen is present, a PGLC peak always occurs a t 3, and when an 0 5 antigen is present, in five out of six cases a peak occurs a t 4. In eight cases where this antigen is absent, the peak a t 4 is also lacking. We can see
how well this correlation holds by comparing S. paratyphi B and S. parutyphi B var. Odense and again by comparing S . typhimurium and S . typhimurium var. Copenhagen. At present, we are trying to identify chemically the definitive peaks in pyrochromatograms derived from various types of cellular matter. Simmonds and his associates have identified pyrolysis peaks from geological samples and from bacteria by means of mass spectrometry (11, 12). We conclude that PGLC may lead to a more direct, determinative classification and a more rapid means of cellular identification. In this (11) P. G. Simmonds, G. P. Shulman, and C. H. Stembridge, J. Chromatogr. Sci., 7, 36 (1969). (12) P. G . Simmonds, Appl. Microbial., 20,567 (1970).
respect the compatibility of the PGLC technique with modern computer methods could have practical significance. ACKNOWLEDGMENT
The authors thank W. H. Ewing, Consulting and Research Microbiologist, CDC, for his expert advice and encouragement. RECEIVED for review September 3,1971. Accepted December 14, 1971. Use of trade names is for identification only and does not constitute endorsement by the Health Services and Mental Health Administration or by the U S . Department of Health, Education, and Welfare.
Simultaneous Determination of Sample Concentration and Reagent Blank Max B. Kloster and Clifford C. Hach Water Analysis Research Laboratory, Hucli Chemical Company, Ames, Iowa 50010
THENEED FOR TESTING low concentrations of contaminants has increased in the areas of pure water and reagent grade chemicals. Although a great deal of work has been done on iron and a large number of good reagents are available for iron determinations, it remains difficult to separate the reagent blank from the iron content at low iron concentrations (less than 200 pg/l.). The same problem applies to silica. In many cases the reagent blank is larger or approximately equal to the iron or silica concentration being determined. In the case of ultrahigh purity water determinations, a sample of water is not available which has a lower iron or silica content than the sample being analyzed. It is then impossible to run a reagent blank without knowing the iron or silica content of the water being used. It is also impossible to determine the water concentrations without knowing the reagent blank. Formerly this dilemma could be resolved only by an independent analysis of either the sample or the reagent, but this procedure suffered from the inherent errors of a second analysis. A method has now been established for the simultaneous determination of reagent blank and trace iron in ultrapure water and is being extended to iron determinations in reagent grade chemicals using single buffer-reagent system and to trace silica determinations. EXPERIMENTAL
Apparatus. Colorimetric readings were taken with a Hach Model 1104 D R Colorimeter with a I-inch cell, a Bausch & Lomb Spectronic 20 spectrophotometer with a 1/2-inch cell, and a Hach Model 2400 Expanded Range Colorimeter. Reagents. Reagent grade chemicals were used to prepare the following solutions : ACID REAGENTSOLUTION.Dissolve 5.14 grams of disodium 3(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FerroZine) and 100 grams of hydroxylamine hydrochloride in a small increment of water. Add 500 ml of concentrated hydrochloric acid, allow to cool, and dilute to 1 liter with deionized water.
BUFFERSOLUTION.Dissolve 400 grams of ammonium acetate in water, add 350 ml of concentrated ammonium hydroxide, and dilute to 1 liter with deionized water. REAGENT-BUFFER SOLUTION.Dissolve 13.8 grams of disodium 3-(2-pyridyl)-5,6-bis (4-phenylsulfonic acid)-1,2,4triazine (FerroZine) in 1 liter of an iron buffer-reductant formulation (Hach Chemical Co. Cat. No. 2532-00). MOLYBDATE REAGENT.Dissolve 20 grams of ammonium molybdate [(NH4)6M01024.4H20] in deionized water, add 15 ml of concentrated sulfuric acid, and dilute to 100 ml. Do not store solution in glass as silica may leach out and cause high blanks. OXALIC ACIDSOLUTION.Dissolve 10 grams of oxalic acid (H2Cz04.2Hz) in deionized water and dilute to 100 ml. REDUCINGREAGENT.Dissolve 500 ml of l-amino-2naphthol-4-sulfonic acid and 1 gram of sodium sulfite (Na2SOa) in deionized water. Add 30 grams of sodium bisulfite (NaHS03) dissolved in deionized water. Dilute mixture to 200 ml and store in polyethylene. Procedure. DETERMINATION OF IRONIN NEUTRAL SOLUTIONS (1). To a 50-ml sample, add 1 ml of acid reagent solution followed by 1 ml of buffer solution and allow the color to develop for one minute; or to a 50-ml sample, add 1 ml of reagent-buffer solution and allow the color to develop for one minute. If iron is present, a purple color will develop which has a maximum absorbance at 562 nm. The color may be read and compared to a similarly treated standard. The concentration can then be determined by comparing the absorbance of the sample with that of the standard, or as an alternate method, a calibration curve may be prepared and the concentration of the sample may be read from it. The FerroZine-iron complex obeys the Beer-Lambert law between 5 pgjl. and 4 mg/l. If the sample contains magnetite (black iron oxide) or other highly refractory oxides, it may be necessary to heat the sample for 20 or 30 minutes after the addition of the acid reagent or the reagent-buffer solution to effect complete reduction. After heating, the sample is brought to its original volume with deionized water. (1) L. L. Stookey, ANAL.CHEM., 42, 779 (1970). ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
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