INFORMATION TO USERS
This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted.
The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction.
1.The sign or “target” for pages apparently lacking from the document photographed is “Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity.
2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame.
3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of “sectioning” the material has been followed. It is customary to begin filming at the upper left hand corner of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete.
4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department.
5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed.
University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106
8222056
Bums, Michael Charles
DIFFERENTIATION OF BIOCHEMICALLY SIMILAR BACTERIA BY GAS- LIQUID CHROMATOGRAPHY
The Ohio State University Ph.D. 1982
University Microfilms International 300 N. Zeeb Road, Ann Arbor, MI 48106
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V .
1. Glossy photographs or p a g______e s
2. Colored illustrations, paper or print______
3. Photographs with dark background______
4. Illustrations are poor copy______
5. Pages with black marks, not original copy_____
6. Print shows through as there is text on both sides of page______
7. Indistinct, broken or small print on several pages ^
8. Print ex ceed s margin requirem ents______
9. Tightly bound copy with print lost in spine______
10. Computer printout pages with indistinct print______
11. Page(s)______lacking when material received, and not available from school or author.
12. Page(s)______seem to be missing in numbering only as text follows.
13. Two pages numbered______. Text follows.
14. Curling and wrinkled pages______
15. Other______
University Microfilms International
DIFFERENTIATION OF BIOCHEMICALLY SIMILAR
BACTERIA BY GAS-LIQUID CHROMATOGRAPHY
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Michael Charles Burns, B.S., M.Sc.
X'X"K X X X
The Ohio State University
1982
Reading Committee: Approved By
Melvin S. Rheins
Bruno J. Kolodziej
George J. Baixwart Adviser Department of Microbiology James I. Frea ACKNOWLEDGEMENTS
The writer wishes to express sincere appreciation to Dr.
M. S. Kheins for his help and guidance throughout the course of this investigation.
The writer also wishes to thank Dr. J. I. Frea and Dr. G.
J. Banwart for their time and interest in this work as well as their valuable technical assistance. VITA
May 6, 1943 Born - Bellefontaine, Ohio
1967 B.S., The Ohio State University
1967-present Microbiologist, Ohio Department of Health, Columbus, Ohio
1976 M.Sc., The Ohio State University
PUBLICATIONS
Bums, M.C, 1977. "Trees on Campus Maps", p. 10-13. In R.L. Stuckey, T.F. Stuessy, and J.N. Brunken, Local Floral Reference Manual. The Ohio State University, Columbus, Ohio.
FIELDS OF STUDY
Major Field: Pathogenic Microbiology
Studies in Pathogenic Bacteriology. Professors Melvin S. Rheins and Matthew C. Dodd
Studies in Food Microbiology. Professor George J. Banwart
Studies in Mycology. Professors Roland Seymour and John A. Schmitt
Studies in Parasitology. Professors Joseph N. Miller and Julius P. Kreier TABLE OF CONTENTS
Page ACKNOWLEDGMENTS...... ii
VITA...... iii
LIST OF TABLES...... vi
LIST OF FIGURES...... ix
INTRODUCTION ...... 1
LITERATURE REVIEW:
1. Biochemical characterization of bacterial cultures ...... 3
2. Experiments with different types of chromatography ...... 8
3. Use of gas chromatography in identification of bacteria .. 13
MATERIALS AND METHODS:
1. Microorganisms ..... 23
2. Confirmation tests ...... 23
3. Culture media ...... 23
4. Preparation of methyl esters ...... 27
5. Acid standards ...... 28
6. Gas chromatography ...... 29
7. Schema ...... 30
■ 8. Statistical analysis ...... '...... 30
EXPERIMENTAL RESULTS:
1. Analysis of non-volatile/volatile products ...... 33
2. Examination of stationary phases ...... 37
3. Culture media ...... 47
4. Comparison of methylation procedures ...... 49
5. Quantitation of CHCl^ in extractions ...... 53
iv Page
6. Internal standard/external standard ...... 50
7. Study of Bacillus cereus/Bacillus anthracis ...... 53
8. Study of Bacillus subtilis/Bacillus cereus ...... 64
9. Study of Bacillus species/Lactobacillus species ...... 69
10. Study of Bacillus anthracis/Baci1lus subtilis ...... 74
11. Study of Escherichia coli/Enterobacter agglomerans ...... 86
12. Study of Vibrio cholerae/non-cholera vibrios ...... 87
13. Study of Salmonella gal1inarum/Salmone11a pullorum ...... 94
14. Study of Salmonella typhi/Salmonella typhimurium ...... 104
15. Study of Staphylococcus species/Micrococcus species ..... 104
16. Study of Staphylococcus epidermidis/Staphylococcus aureus. 110
17. Study of Staphylococcus species/Streptococcus species .... 118
DISCUSSION ...... 127
SUMMARY ...... 137
BIBLIOGRAPHY ...... 140
v LIST OF TABLES
Table Page
1. List of Cultures and Their Sources...... 2k
2. Standard Tests for Identification of Cultures...... 25
3. Formula for Anaerobic Culture Medium...... 26
1+. Organic Acid Standards...... 28
5. Non-Volatile Products of Selected Bacillus Species...... 3^
6. Volatile Products of Selected Bacillus Species...... 36
7. Peak Heights of Acids SP 1200 Stationary Phase with Selected Bacillus Species...... 1+7
8. Methylation Procedures Using & BF^ on Selected Bacillus Species...... 1+9
9- Comparison of Methylation Procedures with Methyl Lactate Standard...... 53
10. Optimum Amount of CHCl^ in Extraction of Selected Bacillus Species...... 55
11. Standard Plate Counts at 2k & k8 Hour Incubations of Representative Test Cultures...... 6l
12. Lactic/Succinic Acid Peak Heights at 2k & 1+8 Hour Incubations of Representative Test Cultures...... 6l
13- Differential Characteristics of Bacillus cereus and Bacillus anthracis Test Cultures...... 62
ll+. Lactic/Succinic Acid Peak Height Ratios of Bacillus anthracis and Bacillus cereus Isolates...... 63
15. Statistical Analysis of Lactic/Succinic Acid Peak Height Ratios...... 67
vi Table Page
16. Biochemical Differentiation of Bacillus cereus/ Bacillus subtilis Isolates ...... 68
17. Lactic and Succinic Acid Peak Height Ratios of Bacillus subtilis and Bacillus cereus Test Cultures...... 72
18. Statistical Analysis of Lactic/Succinic Acid Peak Height Ratios...... 73
19. Biochemical Differentiation of Lactobacillus sp. and Bacillus sp...... 77
20. Lactic & Succinic Acid Peak Height Ratios of Bacillus sp. and Lactobacillus sp...... 78
21. Statistical Analysis of Lactic/Succinic Acid Peak Height Ratios...... 79
22. Biochemical Characteristics of Bacillus subtilis and Bacillus anthracis Isolates...... 83
23. Lactic/Succinic Acid Peak Height Ratios of Bacillus subtilis and Bacillus anthracis Test Cultures...... 84
24 • Statistical Analysis of Peak Height Ratios of Lactic and Succinic Acids...... 85
25 . Biochemical Characteristics of Escherichia coli & Enterobacter agglomerans Isolates...... 91
26„ Lactic & Succinic Acid Peak Height Ratios of Escherichia coli & Enterobacter agglomerans Test Cultures...... 92
27. Statistical Analysis of Lactic & Succinic Acid Peak Height Ratios...... -93
28. Biochemical Characteristics of Vibrio cholerae & Non-cholera vibrio Isolates...... 97
29. Lactic & Succinic Acid Peak Height Ratios of Vibrio cholerae & Non-cholera vibrio Test Cultures...... 98
vii Table Page
30. Statistical Analysis of Peak.Height Ratios of Lactic & Succinic Acids...... 99
31. Biochemical Characterization of Salmonella gallinarum & Salmonella pullorum Isolates...... 103
32. Biochemical Characterization of Salmonella typhi & Salmonella typhimorium Isolates ...... 107
33* Lactic & Succinic Acid Peak Height Ratios of Salmonella typhimorium & Salmonella typhi...... 108
3k. Statistical Analysis of Peak Height Ratios of Lactic & Succinic Acid...... 109
35- Lactic & Succinic Acid Peak Height Ratios of Staphylococcus sp. & Micrococcus sp. Test Cultures...... 115
36. Statistical Analysis of Lactic & Succinic Acid Peak Height Ratios...... 116
37- Biochemical Characterization of Staphylococcus aureus & Staphylococcus epidermidis Isolates...... 117
38. Lactic & Succinic Acid Peak Height Ratios of Staphylococcus aureus & Staphylococcus Epid ermidis Test Cultures...... 119
39- Statistical Analysis of Lactic & Succinic Acid Peak Height Ratios...... 120
1*0. Biochemical Characteristics of Staphylococcus sp. & Streptococcus sp. Test Cultures...... 12^
Ul. Lactic & Succinic Acid Peak Height Ratios of Staphylococcus sp. & Streptococcus sp. Test Cultures...... 125
1*2. Statistical Analysis of Lactic & Succinic Acid Peak Height Ratios...... 126
viii LIST OF FIGURES
Figure Page
1. Schema for Chromatographic Analysis...... 31
2. Non-volatile acids: lactic and pyruvic from Bacillus laterosporus 73 ...... 35
3. Non-volatile acids: Lactic and succinic from Bacillus subtilis 87 ...... 35
U. Volatile acids: acetic, propionic, and unknown products from Bacillus pumilus 82 ...... 37
5. Volatile acids: acetic, propionic, and unknown products from Bacillus subtilis 8 7...... 37
6. Methylated standard on Carbowax 1500 stationary phase...... 39
7. Methylated standard on OV-1 and OV-17 stationary phases...... ^0
8. Methylated standard on D E G A stationary phase...... k2
9. Methylated standard on Lacl-R-296stationary phase...... h-2
10. Methylated standard on F F A P stationary phase...... ^3
11. Methylated standard on SP 1000 stationary phase...... ^
12. Methylated standard on SP 1220 stationary phase...... Mt-
13. Lactic and succinic acid standard on SP 1200 stationary phase...... ^6
lU. Culture extract of Bacillus subtilis 87 on SP 1200 stationary phase...... ^6
15. Peptone-yeast extract-glucose medium on D E G A stationary phase...... ^8
ix Figure Page
16. Yeast extract-glucose medium on D E (J A stationary phase...... k8
17. Bacillus sphaericus 36U H2 SO1* methylation on Lacl- R-296 stationary phase...... 50
18. Bacillus sphaericus 36U BF3 methylation on Lacl- R-296 stationary phase...... 50
19. Methylated acid standard on F F A P stationary phase...... 51
20. Methyl-lactate standard on F F A P stationary phase...... 51
21. I^SOii methylated lactic acid on F F A P stationary phase...... 52
22. BF3 methylated lactic acid on F F A P stationary phase...... 52
23. Bacillus cereus VI at 3 hours with pyruvic, lactic, and succinic acids on Lacl-R-296 stationary phase...... 5!+
2k. Bacillus cereus VI at 2k hours with pyruvic, lactic, and succinic acids on Lacl-R-296 stationary phase...... :^k
25. Bacillus megaterium 236 with 0.5 nil chloroform extraction showing lactic and succinic acids on Lacl-R-296 stationary phase...... 56
26. Bacillus megaterium 236 with 1.0 ml chloroform extraction showing lactic and succinic acids on Lacl-R-296 stationary phase...... 56
27. Bacillus cereus VII with benzoic acid showing suc cinic/benzoic peak on Lacl-R-296 stationary phase...... 57
28. Bacillus cereus VII without benzoic acid showing succinic peak on Lacl-R-296 stationary phase...... 58
29. Bacillus cereus 1^579 at 2k hours with lactic and succinic acids...... 65
30. Bacillus anthracis 33^-8a at 2k hours with lactic and succinic acids...... 65 Figure Page
31* Bacillus cereus 1^579 at ^8 hours with lactic and succinic acids...... ,,6 6
32. Bacillus anthracis 33^-8a at k8 hours with lactic and succinic acids...... 66
33. Bacillus subtilis III at 2k hours with lactic and succinic acids...... 70
3k. Bacillus subtilis III at U8 hours with lactic and succinic acids...... 70
35- Bacillus cereus 9&3^ at2k hourswith lactic and succinic acids...... 71
36. Bacillus cereus 963^ at U8 hours with lactic and succinic acids...... 71
37. Bacillus megaterium at 2k hours with lactic and succinic acids...... 75
38. Bacillus megaterium at U8 hours with lactic and succinic acids...... 75
39. Lactobacillus casei ss. rhamnosus at 2k hours with lactic and succinic acids...... 76
Uo. Lactobacillus casei ss. rhmnnoRiis at U8 hours with lactic and succinic acids...... 76
Ul. Bacillus subtilis II at 2k hours with lactic and succinic acids...... 8l
k2. Bacillus anthracis 0SU-1 at 2k hours with lactic and succinic acids...... 8l
U3. Bacillus subtilis II at U8 hours with lactic and succinic acids...... 82
kk. Bacillus anthracis 0SU-1 at U8 hours with lactic and succinic acids...... 82
U5. Enterobacter agglomerans 2a at 2k hours with lactic and succinic acids...... 88
xi Figure Page
1*6. Enterobacter agglomerans 3b at 2k hours with lactic and succinic acids...... 88
1*7. Enterobacter agglomerans 2a at 1*8 hours with lactic and succinic acids ...... 89
1*8. Escherichia coli K--.61 at 2k hours with lactic and succinic acids...... 89
1*9* Enterobacter agglomerans 3b at 1*8 hours with lactic and succinic acids...... 90
50. Escherichia coli K'6l at 1*8 hours with lactic and succinic acids...... 90
51. Vibrio cholerae 9 at 2l* hours with lactic and succinic acids...... 95
52. Vibrio cholerae 9 at 1*8 hours with lactic and succinic acids...... 95
53* Non-cholera vibrio I at 2l* hours with lactic and succinic acids...... 96
5!*. Non-cholera vibrio I at 1*8 hours with lactic and succinic acids...... 96
55* Salmonella nullorum H at 2k hours with the absence of succinic acid...... 100
56. Salmonella gallinarum 1* at 2l* hours with suc cinic acid...... 100
57. Salmonella nullorum H at 1*8 hours with the absence of succinic acid...... 101
58. Salmonella gallinarum 1* at 1*8 hours with suc cinic acid 101
59* Salmonella typhimurium I at 2l* hours with lactic and succinic acids...... 105
60. Salmonella typhi 2v at 2l* hours with lactic and succinic acids...... 105
61. Salmonella typhimurium I at 1*8 hours with lactic and succinic acids...... 106 xii Figure Page
62. Salmonella typhi 2v at 48 hours with lactic and succinic acids...... 106
63. Staphylococcus epidermidis 550b:4 at 24 hours with lactic and succinic acids...... I l l
6k. Staphylococcus epidermidis 550b:4 at 48 hours with lactic and succinic acids...... I l l
65. Micrococcus luteus I at 2k hours with lactic and succinic acids...... 112
66. Micrococcus luteus X at 48 hours with lactic and succinic acids...... 112
67. Staphylococcus epidermidis 721 at 2k hours with lactic and succinic acids...... 113
68. Staphylococcus epidermidis 721 at 48 hours with lactic and succinic acids...... 113
69. Staphylococcus aureus 717 at 2k hours with lactic and succinic acids...... 114
70. Staphylococcus aureus 717 at 48 hours with lactic and succinic acids...... Il4
71. Streptococcus pyogenes at 2k hours with lactic and succinic acids...... 122
72. Streptococcus pyogenes at 48 hours with lactic and succinic acids...... 122
73. Staphylococcus epidermidis 511b:2 at 24 hours with lactic and succinic acids...... 123
74. Staphylococ cus epidermidis 5Hb:2 at 48 hours with lactic and succinic acids...... 123
xiii INTRODUCTION
Metabolic products identified and measured by gas-liquid chroma tography have proved extremely useful in rapidly distinguishing bio chemically similar microorganisms, particularly anaerobes (4), (48),
(59), (63), (65), (73), (85). The characterization of organic acids produced by bacteria is a procedure that can be accomplished rapidly and efficiently (52), (70).
In this study the suitability of this technique for the chracter- ization of aerobic and facultative-anaerobic bacteria was investigated to determine if a similar procedure for differentiating these groups of bacteria could be achieved. The aerobic and facultative-anaerobic bac teria studied were selected because little information is available concerning gas-liquid chromatographic analysis of their metabolic pro ducts. In addition, their established importance in clinical micro biology requires reliable procedures for identification.
The standard identification procedures are limited in character izing the clinically important bacteria (20), (36), (119). Biochemi cally related bacteria are often distinguished by only a single testing parameter (36). Strains of a particular species may differ in biochem ical characteristics especially with the presently used identification systems. In order to obtain reliable results, a number of tests must be performed requiring involved procedures (24), (44). The biochemical tests, in order to be statistically significant, often demand a long incubation of cultures and after periods of up to ten days the bio chemical differences may prove to be inadequate for differentiating re lated bacteria.
Members of the genus Bacillus were chosen for the preliminary ex aminations because of their diversity of metabolic products (44).
Following these studies, standardized procedures were formulated for the study of subsequent test cultures. The major divisions of bacteria analyzed included the gram positive rods, gram negative rods, and the gram positive cocci. The various species were chosen because of their biochemical similarities with other members of the same group of bac teria. For example, Staphylococcus aureus and Staphylococcus epidermidis are differentiated by one biochemical test, and share most major bio chemical pathways. These similarities lead to problems in their iden tification.
In all eleven experiments comparing related bacterial genera, species, and groups, the standardized chromatographic procedure effec-- tively differentiated the microorganisms which are often identified incorrectly.
This modification of existing chromatographic methods is a new procedure devised to facilitate the differentiation of related bacterial groups. The procedure specifically has relevance to aerobic and facul tative-anaerobic bacterial identification. LITERATURE REVIEW
Previously, the only tests available for the identification of bac teria and their subsequent separation into groups or genera were morpho logical, serological, and biochemical procedures, such as testing for products by color reactions produced mainly by pH changes (119). It is appropriate to point out that early efforts to distinguish biochemically similar groups of bacteria depended on subtle differences in the inabil ity to utilize a wide variety of substrates. These differences in groups or genera of bacteria led to an array of biochemical procedures which could be used in the testing of a specific organism.
The following groups of bacteria are arranged taxonomically accord ing to various standard biochemical procedures, and represent examples of the numerous techniques required for differentiation into defined categories.
Bacillus species are identified by a combination of biochemical tests and physical parameters, among these are the Voges-Proskauer (VP) reaction, growth at 50°C, catalase reaction, anaerobic growth, starch hydrolysis, and carbohydrate utilization (44). The test for hemolytic activity is an important characteristic, as the primary isolation on sheep blood agar can aid in the speciation (20). From a public health standpoint, the most important species in the genus are Bacillus pereus and'Bacillus anthracis and they are identified by tests such as an aerobic growth, and the W-phage reaction (27), _B. cereus is among the 4
most commonly encountered species of this genus in human disease and
food poisonings (27), (82), (86).
The genus Lactobacillus is difficult to separate from other gram
positive rods, since the tests commonly employed are limited mainly to
carbohydrate fermentation (20), (119). Gram positive rods, which are
catalase negative and produce lactic acid, are grouped into this genus
(20). The utilization of esculin, production of gas from glucose, and
starch hydrolysis are all ancillary tests for the identification of a
large number of Lactobacillus species (119). In the past the genus was
considered to be comprised of saprophytes, but recently some species
have been associated with human disease. The separation of Lactobacillus
from other genera such as Bacillus, Corynebacterium, and other gram pos
itive rods is based on standard biochemical tests. More than one test
parameter can result in variable reactions depending on the strain in
volved (20), (119). These results may pose a problem in the definitive
identification of the lactobacilli and related bacteria.
Escherichia coli has been considered an enteric pathogen, while
Enterobacter agglomerans has been found only in association with other
bacteria in enteric disease (34) , (119) . These two species can be found
together, and their differentiation is difficult in that there exists
not many biochemical test parameters which permit a separation with a
confidence level of 90% or greater (36). The testing of Ei. coli for urease production, gas produced from glucose, indole production, motil
ity, oxidase production, and H^S production will usually identify a member of the species (33). Variant strains produce H^S, and some
isolates are non-motile, resulting in an incorrect identification (37).
The triple sugar, iron (TSlJtest with production of acid and gas from glucose, with no E^S production, is useful in identifying the species.
The serological identification of _E. coli with somatic (0) antigen and capsular (K) antigen is useful, as this facilitates comparison with other genera (35). E_. agglomerans is characterized by the lack of en zymes to degrade lysine, arginine, or ornithine (34), (36). There is no reliable serological identification scheme available for the species.
The biochemical tests that serve to identify _E. agglomerans are glucose fermentation, production of gas from glucose, malonate utilization H^S production, urease production, motility, and production of indole (36).
The comparison of _E. agglomerans with other members of the Enterobacter- iaceae using the tests provide variable results. For E_. coli and _E. agglomerans these identification tests have recently been published and exhibit a limited reliability in the clinical laboratory (35).
The biochemical characterization of the salmonella differs only slightly from that of the other members of Enterobacteriaceae, but the major separation of these species is in the serological identification tests of the somatic (0) antigen and flagellar (H) antigen (33). Use of triple sugar, iron agar for H^S production, glucose fermentation, lactose and sucrose fermentation, and lack of gas production from the glucose are the fundamental tests employed for members of the genus
(9), (34). The biochemical differences between the species are some times limited as for example, with some of the Salmonella species in which utilization of dulcitol is the principal test which permits their separation (34).
Identification procedures formulated at the Centers for Disease
Control are useful in the characterization of streptococci found in clinical situations (38), (39). These procedures explore the serological differences between the beta hemolytic streptococci and the
biochemical differences between the alpha and gamma hemolytic groups.
Originally, the use of different growth temperatures was of primary
concern in the identification of members of the streptococci (20), but
these tests have been replaced for the most part with a large number
of carbohydrate fermentations, amino acid utilization tests, as well as
tolerance to bile salts and 6.5% NaCl (39).
The genus Vibrio contains various bacteria that produce disease in
the intestinal tract (8). Cholera is caused by the bacterium Vibrio
cholerae while milder forms of the disease can be caused by the group
known as the non-cholera vibrios (23). Studies of the speciation of the
groups in the genus Vibrio were performed. The identification of these
bacteria is based on a few tests such as sucrose fermentation, 42°C
growth, and growth in 7-10% NaCl (8), (40). This genus is easily sep
arated from a number of common gram negative fermenters by the oxidase
test. The Vibrio species are all oxidase positive while the members of
the Enterobacteriaceae are all oxidase negative. Vibrio parahemolyti
cus is differentiated from V. cholerae and the non-cholera vibrios by
the ability to grow in relatively higher NaCl concentrations (8). The
major problem with biochemical differentiation resides in the separa
tion of V. cholerae and the non-cholera vibrios; therefore, separation
is accomplished at present by serological tests (23).
Cohen (24) has assembled a splendid compendium on the genus
Staphylococcus which outlines a scheme for differentiating the
Micrococcus species from other gram positive cocci. The biochemical
tests that separate the species in the genus Staphylococcus also dif
ferentiate the micrococci, planococci, and the aerococci. These tests 7 include glucose fermentation, motility, and catalase production (20),
(27). The separation of the genera Micrococcus and Staphylococcus is performed basically with one major biochemical test, the anaerobic fermentation of glucose (9).
Members of the genus Staphylococcus compose a varied collection of similar species, and most of these have been associated with disease
(62). The separation of these species is based on the biochemical and environmental examinations used for most gram positive cocci. The coagulase test and anaerobic mannitol fermentation are both used to dif ferentiate between _S. aureus and _S. epidermidis (24) . A set of bio chemical tests to separate and identify all groups aerobic to facul tative-anaerobic, of all the members of the Micrococcaceae is presently included in accepted standard procedures (20).
The streptococci are involved in human diseases such as pneumonia, meningitis, arthritis, endocarditis, and septicemia (38). The genus is divided into the beta hemolytic and viridans groups (39). These groups are composed of various species that are separated by biochemi cal and serological examinations, which include Lancefield tests, carbohydrate fermentations, and growth tolerance tests. The separation of these species into recognized taxonomic groups is critical to the management of disease syndromes.
The literature is replete with groupings of bacteria according to measurements of their biochemical activities. However, limitations of this method of classification were recognized and prompted contin uing research for better analytical methods which would serve to dif ferentiate similar microorganisms. The methods and techniques eval uated were various types of chromatography; namely paper, column, thin layer, glass capillary column, high performance liquid, and gas-liquid.
The use of all types of chromatography is widespread in the sep aration and identification of fatty acids, other organic acids, and some types are employed in the differentiation of certain groups of bacteria. Dyer (31), for example, proposed the original procedure for the separation and identification of volatile fatty acids by steam distillation. The technique is based on the fact that volatile acids distill at a constant rate, and a mixture of acids distill as if they were present singly. The acids were identified by a series of color imetric qualitative reactions.
From the basis suggestion that partition chromatography could be useful in analysis of chemical compounds by Martin and Synge (72),
James and Martin (55) formulated a procedure to separate mixtures of volatile fatty acids. This analysis was performed by compressing the stationary phase in a column of small diameter and increasing the pressure, thus removing the compounds at a much faster rate through the column. Ramsey and Patterson (96) separated and identified vola tile fatty acids by partition column chromatography, employing a sil icic acid column packing. The products were characterized by micro scopic examination of their crystalline mercurous salts. Ramseu (95) also separated organic acids found in human blood by partition chroma tography. This procedure also involved identification by comparison with known mercurous salts. By using thin layer chromatography,
Teichman, Tekei, and Cummins (115) were able to detect the presence of fatty acids, fatty aldehydes, phospholipids, glycolipids, and choles terol on chromatograms stained with malachite green. 9
Paper chromatographic separation of volatile acids and the attempts to identify them were studied by a number of investigators (53), (58),
(97), (111). The procedure was tested for the separation and identi fication of volatile short chain acids and was satisfactory in that chemical mixtures could be separated into individual components, but the precise identifications often fell short of the anticipated results.
Paper chromatography still remains an involved procedure.
From the techniques of preparing and employing glass capillary columns (2), (11), (45), (108), Niskanew, Kintame, Raisanen, and
Raevueri (88) developed a rapid gas chromatographic method for separ ating and identifying the cellular fatty acid composition of B. cereus, Bacillus mycoides, and Bacillus thuringiensis. The technique was developed in response to the problems encountered in using packed columns. The use of short six foot packed columns is limited in the study of fatty acids, and the column separation is time consuming and relatively inefficient (88). Much longer columns can be employed in fatty acid analysis, but the high cost and shorter life span are of major economic significance (55).
Gas capillary columns have been used to study the characteriza tion of bacteria on their cellular fatty acid components (11) . How ever, thermal decomposition of compounds in packed columns is a prob lem not encountered with glass capillary column chromatography (117) .
Etching of the glass surface can increase the sample carrying capacity
(2). The advantages of glass capillary columns over steel columns have been emphasized and documented (11), (108), (116). The main disadvantage to glass column chromatography is the fairly short life span in which the column can be employed in organic acid analysis 10
(88). The coating on the glass interior is consumed after a few samples have been tested.
The basic analytical procedures of gas-liquid chromatography have been organized into a coherent group of techniques and procedures (63),
(65), (71). These basic techniques served previously in quantitating volatile short chain fatty acids (45). The utilization of gas- liquid chromatography for the analysis of microbial fermentation pro ducts has been attempted by a few investigators (45), (54), (61), since a procedure that increases the efficiency of separating and identifying metabolic products of a bacterial culture is very valuable to commer cial interests as well as to the research scientist. For example,x the finite characterization of the lower carbon number free fatty acids in microbial products can be very important to the quality of commercial foods (43). Many commercial products such as exist in the food indus try are dependent on flavor quality, which is in part produced by certain organic acids. The quantitative determination of chemical con stituents in food by gas chromatography is important to the agricul tural chemist (109).
The procedures of identifying and quantifying fatty acids were simplified by Shelby, Salwin, and Horwitz (109), James (54), Kaneshiro and Marr (61), and Miwa, Mikelajczak, Earle, and Wolfe (77) using the mono and dicarboxcylic methyl esters. This procedure required the derivatization techniques to be simple and precise in order to chroma tography the acid mixtures. Methods of derivatization that have been used with varying success are the bromination and hydrogenation pro cedures described by James (54) and Kaneshiro and Marr (61), respec tively. The methylation of the non-volatile acids has been somewhat 11 useful in the chromatography of these compounds (55), particularly the non-volatile lower fatty acids that produce a very high viscosity may pose certain problems to the manipulation of the chromatography of the acids (20).
Formic, acetic, propionic, and butyric acids have been identified and quantified by comparison of peak heights with the peak heights of a known concentration (112), (113). This research technique gave the chromatographers a usable method of not only identifying by comparison of the retention time of an unknown with a known entity, but also offered a method of quantitation by comparison of peak heights of a known concentration. Derivatized butyl esters were employed in the technique to distinguish organic acids by GLC in order to quantify the compounds under study.
The non-volatile acids are of necessity converted to methyl esters for chromatographic analysis (32). The acids acetic, propionic, and butyric plus non-volatile acids such as lactic, pyruvic, and succinic were studied, and it was found that non-volatile acids were rendered more volatile by reacting them with f^SO^ plus methanol to make the methyl esters (79). The high volatility of the acetic, propionic, and butyric acids was sufficient to permit their extraction by ethyl- ether and forego derivatization for chromatography.
It was reported recently that results from GLC analyses could be made more useful by employing a dual column machine and combining the results in a data system to eliminate manual analysis of the results
(46). In a study comprising many specimens and numerous organic com pounds, a method to simplify the screening of results can be of great value to the researcher. The computers for organization scientific 12 material have proven of great significance in the field of chroma tography and are greatly superior to manual evaluation techniques (46).
High performance liquid chromatography (HPLC) has been utilized in the rapid separation of fatty acids and lipids (25), (28). The potential of HPLC for the identification of fatty acid methyl esters has been investigated (107). The technique promises a faster method of separation of organic acids, as the specimen needs no longer to be derivatized but can be injected directly into the chromatograph for analysis as shown by Scholfield (107). HPLC analysis is performed under higher pressures than occur in GLC, and therefore less time is necessary to pass the specimen through the column (106).
These methods of separation of organic acids and other organic compounds are being employed by microbiologists in defining the basic biochemical character of many microorganisms, and also in the separa tion and identification of biochemically similar groups of bacteria
(29), (48), (52), (63), (64), (87), (89).
The use of gas chromatography in the separation and speciation of various groups of bacteria has been a relatively recent development.
The testing of bacterial cellular constituent organic acids by the use of pyrolysis techniques has been used with some success (2), (51),
(81), (104), and the additional testing parameter of comparing cellu lar organic acids of different bacteria is a possible method to confirm or negate standard biochemical test results.
Methods of preparing pure samples of bacterial cellular components for pyrolysis have been recently developed (91), and are necessary in the use of pyrolysis gas-liquid chromatographic technqiue, in that the purest samples of the bacterial cells to be tested are required for 13 analysis if reliable results are to be obtained. The pyrolysis pro cedure requires that cells be harvested from agar or broth cultures in quantity; these, then, are washed and collected in large numbers for analysis. The sample prepared in this manner often contains small amounts of agar or other biological organic compounds (95). The analy sis can be simplified by using membrane filters upon which to grow the bacteria, and with the subsequent harvesting of the cells elimination of organic contaminants is accomplished (91). This purification pro cedure for many types of samples has increased column efficiency.
The technique of pyrolysis GLC has proven to be so successful that claims of differentiation of species even to the strain level have been noted (96).
Gram negative bacteria such as V. cholerae and Salmonella species have been investigated by the pyrolysis technique (47), (98), (99),
(100), (101). V. cholerae profiles of cellular constituents have been used to differentiate these organisms from other gram negative, glu cose fermenting bacteria (47). Separation of other gram negative bacteria such as Salmonella species has been investigated by the pyrol ysis gas-liquid chromatographic process with limited success (99).
Other gram negative bacteria of the most common types found in human disease have been studied by this chromatographic procedure (98). The newly discovered legionnaires' disease bacterium has been tested for cellular organic acid components and was subsequently placed into a distinct genus Legionella, (41), (85). Neisseria species, important in various pathological conditions in humans have been analyzed by pyrolysis techniques (17), (67), (84), (121), The organic acid con stituents of the cell walls of Neisseria species contain many short and long chain compounds (67), (121), and these have been used in species characterization. These cellular organic acids are particular in their occurrence according to the genus, and in many cases, the species (17),
(84).
Various gram positive rods such as Bacillus species, Propioni- bacterium species, and Listeria monocytogenes were analyzed for cellu lar components amenable to separation by pyrolysis chromatography (56),
(57), (60), (85), (88), (94) . The pyrolysis of the genus Bacillus in volved not only the standard separation and isolation of many straight chain organic acids, but members of this genus produced many branched chain acids in their cellular composition (56), (60). Furthermore, the genus Bacillus produces a mixture of straight and branched chain organic acids, and the procedures for detection and separation of these compounds require further manipulation (57). The analysis of the genus was expanded to include the food poisoning pathogen B_. cereus (88).
It was hoped that pyrolysis chromatography would provide a more rapid method of identification of _B. cereus isolated from contaminated food samples. The taxonomic state of the propionibacteria has been somewhat clarified by analyzing the cellular acids by pyrolysis chromatography
(85). Cellular organic acids of jL. monocytogenes have been character ized and provide an additional testing procedure for the identifica tion of this pathogen (94),
Gram positive cocci such as the peptococci and peptostreptococci were investigated using pyrolysis chromatography (63). Other microor ganisms such as the mycobacteria and fungi also have been subjected to pyrolysis, followed by gas chromatographic analysis in numerous stud ies (102), (103), (118). In summary, the use of pyrolysis gas-liquid 15 chromatographic techniques in differentiation and identifying cellu
lar organic acids and other components is of limited usefulness since many similarities exist between groups of unrelated bacteria, and the cellular constituents are of such a nature that their identification requires complicated comparison studies for a positive identification.
Many microbiologists feel that the study of the metabolic products may prove to be the most helpful technique in the identification of bacteria.
Investigation as to the feasibility of using metabolic products of microorganisms as biochemical markers were reported in experiments with species of' Bacillus, E_. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and anaerobic bacteria such as Clostridium species,
Bifidobacterium species, and Bacteriodes species. These studies pro vided the basis for the use of gas-liquid chromatographic analysis in the differentiation and identification of aerobic and facultative- anaerobic bacteria.
Because of the problem involved with pyrolysis chromatography, numerous investigators have employed GLC in the analysis of metabolic products produced by gram negative bacteria (14), (30), (63), (87).
_E. coli was evaluated for the production of formic and acetic acid
(30). This study was one of the few in which ratios of metabolic products were measured for a quantitative distinction between strains.
The differentiation of groups of gram negative bacteria based on the chromatographic analysis of products of glucose metabolism was pro moted as a viable method for identification (87) . This procedure involved the analysis of ether extracts of bacterial cultures. Anal ysis of amines and nitroamines of Proteus mirabilis cultures for use 16 in identification has shown the procedure to be a valuable microbio logical technique (14). Some general microbiological experiments in cluding carbohydrate fermentation utilizing chromatographic analysis for separation and identification of bacterial products were per- fomred by Drucker (29), Gray and Stevens (45), and Mitruka and Alex ander (75). Drucker (29) used the chromatographic analysis of meth ylated extracts of bacterial fermentations to study the acid produc tion of the lactic acid bacteria and members of the enteric group.
This investigation employed an isothermal detector instead of the flame ionization detector. The glucose fermenting bacteria were the focus of chromatographic techniques when Gray and Stevens (45) used an argon ionization detector to separate metabolic products. The problem with this technique is that the detector fails to register and record the passage of the non-volatile lactic acid (29). Increas ing the sensitivity of the chromatographic analysis has been of major importance, as seen in the work if Mitruka and Alexander (75). The sensitivity of the analytical technique using flame ionization de tectors makes the procedure a useful one in the study of many bacter ial species and groups.
The gram positive bacterial research programs are also important in the use of gas-liquid chromatographic analysis as an identification aid and are as numerous as the studies performed in anaerobic bacter iology. Drucker, Colland, Bostock, and Chapman (30), used the vola tile acetic acid in conjunction with formic and lactic acid in compar ison of acid ratios produced in bacterial cultures. Both f:. coli and
Streptococcus faecalis were studied in relation to the conditions 17 governing the difference in ratios of products (30). The lactic acid bacteria were investigated for possible ratio differences in acetic and lactic acid production.
Bacteria difficult to manipulate in the laboratory because of long and involved identification procedures such as the mycobacteria were examined in hopes of reducing the time involved for the specified char acterization (87), (93). The production of niacin in the testing of
Mycobacterium species is one of the most important tests used to sep arate Mycobacterium tuberculosis from the other mycobacteria (119).
Niacin is produced along with niacinamide and 3-pyridine-methanol and these compounds give a positive niacin test. The ratios of these pro ducts may be useful in separating mycobacterial strains. Prosser and
Shepard (93) developed a procedure for the detection of niacin and niacinamide by chromatographic analysis but omitted the important my cobacterial metabolic product 3-pyridine-methanol. The useful infor mation gained by chromatographic analysis of the products of mycobac terial metabolism is limited, but more research may provide pertinent data.
The recent epidemics of gonorrhea and the importance of rapidly identifying Neisseria meningiditis in disease outbreaks prompted studies involving GLC (3), (80), (84). The intermediate products dis covered in a recent study of N. meningiditis (3) can be of signifi cant value in the comparison of this species with Neisseria gonorrhea.
Epidemics of gonorrhea require a rapidly analytical schema to aid in the treatment and prevention of the disease, With the use of electron capture detectors, the metabolic products of N, gonorrhea have now 18 been elucidated (84). These products include acetylmethylcarbinol and other uncharacterized organic acids.
Some viruses have been investigated in hopes of more rapid identi fication through the use of chromatographic analysis (76). Viruses of canine origin have been subjected to chromatographic analysis in at tempts to characterize the separate entities. It was theorized that the viruses interacting with the host would produce compounds that could be detected by chromatographic procedures and then lead to a rapid diag nostic technique. Over a decade has passed since this study and very little has been discovered about viral infections that would aid in a rapid diagnosis of disease.
Mycologists have attempted chromatographic studies of metabolic products to identify Candida species and Cyrptococcus species (105).
A fungal metabolite, d-arabinitol, produced by Candida albicans has been detected in human serum by GLC (59) . This procedure is possibly useful in the diagnosis of candidiasis infections. Rapid diagnosis of cyrptococcosis in humans using GLC to analyze spinal fluid has been investigated (105).
Analysis of spinal fluid provides a possible diagnostic tool for diagnosis of a number of types of meningitis, namely tuberculosis, cryptococcal, and viral infections (26) . The as yet unidentified compounds are present in patients exhibiting the classic symptoms of meningitis, and are absent in the spinal fluid of normal controls.
A mass of data from chromatographic analysis of metabolic pro ducts appears in the experimental investigations performed on anaero bic bacteria (79), (89). Lewis, Moss, and Jones (66) used chromatographic analysis to determine the volatile acid production of
Clostridium species, and subsequently tried to classify the individual
species on differences in products. Limitations exist since volatile
butyric acid is produced by most members of the genus (52) . A study
of Clostridium botulinum producing F. toxin was performed to see if
the volatile acid products were unique among the species (83).
Clostridium sporogenes isolates were examined for adaption to different
culture conditions, and were found to produce volatile products at
only certain times of the growth phase (4). The amines produced by
certain species of the clostridia have been isolated and identified by
chromatography (18). Clostridium sordelli and Clostridium bifermentans
have been characterized by the organic products (19). Holdeman and
Moore (52) were the first investigators to characterize numerous an
aerobic bacteria in a number of genera. Mayhew and Gorbach (73) used
an internal standard instead of the external standard of Holdeman and
Moore (52). For the volatile standard they used 2-methyl-pentanoic
acid, and for the non-volatile standard they used benzoic acid, both
useful in the identification of anaerobic bacteria. Carlsson (21)
used a unique method of analysis of volatile acids and lactic, pyru
vic, and succinic acids. A cation exchange resin was used as a pre
filter before chromatographic analysis. This method purified the sam ple to a great extent. The chromatography of clinical specimens
directly to show the presence of organic acids, such as succinic acid, produced by certain invasive pathogenic bacteria has been shown to be
a possible technique of laboratory diagnosis of anaerobic infections
(43). Phillips, Teale, and Miller (92) also claimed rapid diagnosis 20 of anaerobic infections by chromatographic analysis of clinical mater ial .
Blood extracts were tested in the diagnosis of anaerobic infec tions in humans (120). This procedure was expanded to include aerobes such as Listeria species, facultative-anaerobes such as Klebsiella species, and strict anaerobes like Bacteriodes species (110).
The genera Peptococcus and Peptostreptococcus were separated and characterized by comparison of volatile and non-volatile acid products
(63). Formic, lactic, and acetic acid production differences were in vestigated and found to be a possible basis for separating the genera.
Food microbiologists have attempted to use chromatographic analy sis of products in the investigation of food spoilage (112), (113),
(122). The organic acids present in whole eggs are useful in deter mining the quality of the final product. The quantitation of butyric acid in eggs was performed by comparison of the peak heights of the acid with those of butyric acid standards (113) . York and Dawson
(122) inoculated eggs with known cultures such as Si. faecalis, E_. coli,
Salmonella cholerae-suis, Pseudomonas fluorescens, Achromobacter xerosis, and S. aureus and they extracted and chromatographed all the contents of the eggs to determine if any difference in metabolites could be shown. The study shows promise for use in the food industry as a means of assessing food quality, especially shelf life of a product,
The determination of lactic and succinic acid in food products, especially eggs, is important since these organic acids are related to the taste and odor quality of the product. The quantitation of 21 lactic and succinic in certain foods has been investigated in order to provide a standardized, reliable procedure for testing food quality
(112). This objective testing procedure has a better scientific basis for comparing food quality than do the organoleptic tests. Gas-liquid chromatographic analysis of organic acids is a standard procedure for estimating food quality in many foods such as butter, numerous fruits and vegetables, and certain fermented products like sauerkraut (6).
The foods are tested for acids and these acids are identified by gas chromatography.
One of the most recent and promising uses of organic acid analy sis has been in testing body fluids such as serum, spinal fluid, and synovial fluid of ill individuals and comparing the results to normal asymptomatic individuals (74). Analysis of body fluids by electron capture chromatography has been postulated as a valuable aid in diag nosis, as compared to the accepted method employing flame ionization detectors (12), (15), (26). The diagnosis of arthritis has been at tempted by chromatography of synovial fluid from arthritic patients
(16). Differentiation of the causal agents, namely staphylococci, streptococci, gonococci, and traumatic arthritis has been shown to be a clinal procedure possibly useful in diagnosis.
To date, there have been no studies on the use of peak height ratios of intermediate metabolic products in the differentiation of biochemically similar bacteria. The presence or absence of parti cular products has been employed in the identification of anaerobic bacteria, fungi, and gram negative glucose fermenting bacteria. Cell wall constituents have been used to characterize the mycobacteria, gram positive rods, and gram positive cocci. The use of a peak height ratio comparison of products from similar bacteria should prove to be a valuable addition to the working laboratory knowledge in clini cal microbiology. MATERIALS AND METHODS
Microorganisms: The cultures studied and their sources are listed
in Table 1. The stock cultures of all isolates were cultivated on
tryptic soy agar (Difco Laboratories, Detroit, MI) for 18 hours at
37°C and stored at room temperature until needed (51). The cultures
were grown on tryptic soy agar slants (DIFCO) for 18 hours at 37°C
when required for experimentation, except the Streptococcus species,
and these were cultivated and stored on brain heart infusion agar
(DIFCO) slants and transferred to fresh media every 72 hours.
Confirmation Tests: The battery of tests for confirming the
identity of the organisms to be used in the chromatographic analyses
are listed in Table 2. These standard tests were utilized previous to
the plate count estimation and extractions for analysis of metabolic products. The media used in the tests are standardized for each pro
cedure as listed in the references.
Culture Media: Media employed in this study were peptone-yeast
extract-glucose broth (PYG), yeast extract-glucose broth (YG), yeast
extract-dulcitol broth (YD), yeast extract-mannitol (YM), yeast
extract-rhamnose (YR), and yeast extract-glucose with 10% NaCl (YT).
The anaerobic culture medium of Holdeman and Moore (52) was used in the preliminary studies to determine which metabolic products were
23 TABLE 1
List of Cultures and Their Sources
Bacillus anthracis 2,l)-,5 Lactobacillus plantarum 1+ Bacillus cereus 1,2,3,*+ Lactobacillus salivarius ss. Bacillus circulans *+ salivarius k Bacillus globinii 1 Micrococcus cryophilus 2 Bacillus laterosporus 2 Micrococcus luteus 1,2,3 Bacillus licheniformis 2 Micrococcus roseus 2 Bacillus megaterium 1 Micrococcus species 2 Bacillus pantothenticus 1 Salmonella gallinarum 2,6 Bacillus pumilus "5 Salmonella pullorum 2,6 Bacillus sphaericus U Salmonella typhi 2,3,8 Bacillus stearothermophilus 1 Salmonella typhimurium 1,2 Bacillus subtilis 1,2,3,^+ Staphylococcus aureus 1,2,3,*+ Bacillus thuringiensis 3 Staphylococcus epidermidis 1,2,3 ,*+ Enterobacter agglomerans 2,h Streptococcus agalactiae 2 Escherichia coli 2,k Streptococcus anginosus 2 Lactobacillus acidolphilus h Streptococcus bovis h Lactobacillus buchneri"^ Streptococcus equisimilis k Lactobacillus bulgaricus 3 Streptococcus faecalis 2 Lactobacillus casei h Streptococcus mg 2 Lactobacillus casei ss. rhamnosus 2 Streptococcus mutans 2 Lactobacillus . cellobiosus 4 Streptococcus pneumoniae 2 Lactobacillus curvatus 2 Streptococcus pyogenes 3 Lactobacillus fermentum k Streptococcus salivarius 2
1...American Type Culture Collection, 12301 Park Lawn Dr., Rockville, Md. 2...0.io Department of Health, Division of Laboratories, Columbus, Ohio 3...The Ohio State University, Microbiology Department, Columbus, Ohio 4...Department of Health and. Human Services, Center for Disease Control, Atlanta, Ga. 5...0.lahoma State University, Department of Veterinary Science, Stillwater, Okla. 6 ...United States Department of Agriculture, Animal and Plant Inspection Service, Ames, Iowa 7...University of Maryland, Microbiology Department, College Park, Md. 8...Analytab Products Inc., 200 Express St., Plainview, N.Y. TABLE 2
Standard Tests for Identification of Cultures
Test for Alpha, Beta, Gamma Hemolysis (l6) Cellular Morphology (5 ), (10) Gelatin Hydrolysis (l), (21) b-Phage Susceptability (i) Catalase Production (20) Glucose Fermentation (l4), (15), (17), (18) Urease Production (12), (17), (50) Oxidase Production (21) Cellobiose Fermentation (21) Malonate Utilization (21) Lysine Utilization (6), (12) Arginine Utilization (6), (12) Ornithine Utilization (6), (12) Motility (21) H2 S Production (8), (12), (21) Anaerobic Growth (10), (11) Lethicinase Production (U) Jordan's Tartrate Utilization (20) Serological Characterization (13), (19) Coagulase Production (20) Mannitol Fermentation (15), (20) Dnase Production (15) Phenylalanine Deaminase Production (20) Indol Production (3 ), (12) 26
present in cultures of selected aerobic and facultative-anaerobic
Bacillus species. The formula for this medium is shown in Table 3.
After the preliminary investigations, this medium was altered by omit
ting the peptone component and modifying the salts content. The new
medium was supplemented with the following salts solution, as recom-
TABLE 3
Peptone-Yeast Extract-Glucose Broth (PYG)
Peptone 1.0 g Cysteine HCl-lLO 0.05 g Yeast Extract 1.0 g Glucose 1.00 g Salts Solution 4 ml Distilled Water 100 ml
Salts Solution:
CaCl? (anhydrous) 0.2 g KH2PO4 1.0 g MgSC>4 0.2 g NaHC03 10.0 g K2HP04 1.0 g NaCl 2.0 g
mended by Kaneda (56): MgS04 • 7H20 - 0.1 g; ZnS04 • 7H20 - 1.0 mg;
FeS04 * 7H20 - 1.0 mg; MnCl2 * 4H20 - 0.45 mg; CuS04 • 5H20 - 0.05 mg;
K2HP04 - 1.5 g; NH4H2P04 - 0.5 g. The new medium contained the follow
ing in addition to the salts solution, YG : 10.0 g glucose; YD : 5.0 g
dulcitol; YM : 10.0 g mannitol; YR : 5.0 g rhamnose; YT : 1.0 g glucose with 100 g NaCl, all in 1000 ml as the total volume for each medium,
and each also contained 1.0 g yeast extract per liter. The aerobic plate count examinations to determine the most useful growth period
for evaluation of metabolic products were performed with standard plate
count agar and the standard procedure (7). This technique involves preparing dilutions in buffered dilution water of the pure culture under
study with subsequent spreading of 0.1 ml of each suspension onto an
agar plate with a sterile glass rod. After incubation of duplicate sets 27 at 35°C for 24 and 48 hours, the colonies were counted on a Quebec colony counter and plates with 30-300 colonies/plate were examined for determining the number of bacteria in the original sample. The cultures were plated from the PYG medium. These plate counts were used to de termine which incubation, 24 or 48 hours, would be most useful for exam ination of organic acid production.
Preparation of Organic Acid Methyl Esters: The organic acids can be analyzed either as the free acids such as in the instance of the volatile products acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, and caproic acids by extraction with ethyl-ether, or as the methyl esters of the non-volatile products pyruvic, lactic, oxalacetic, oxalic, methyl-malonic, malonic, fumaric, and succinic acids. In the majority of routine analyses, acids were converted to volatile esters before chromatography (68). The non-volatile acids must be converted to methyl esters in order to be chromatographed (52).
The creation of methyl esters used in the experiment on comparison of methylation procedures was accomplished by adding 1.0 ml BF^ in methan ol to 1.0 ml of the acidified culture after centrifugation and heating for 30 minutes at 80°C (73). 0,4 ml of 50% plus 2.0 ml methanol added to 1.0 ml of culture at 559C for 30 minutes was the procedure used to methylate the products in this and all subsequent experiments
(52). The extractions were performed with 0.5 ml CHCl^ per speciment
(52), (73). The procedure using H^SO^ and methanol served to methylate the culture supemates, and the portion used in chromatographic analy sis was 2.0 ul per specimen. 28
Acid Standards: The organic acid standards included pyruvic,
lactic, oxalacetic, oxalic, methyl-malonic, malonic, fumaric, and suc
cinic as the non-volatile acids; and acetic, propionic, isobutyric,
butyric, iso-valeric, valeric, iso-caproic, and caproic as the volatile
acids (52), (73). One milliequivalent of each acid was added per 100
ml of distilled water. These quantities appear in Table 4 for the non
volatile standard acids and for the volatile standard acids. The appro
priate standard mixture was chromatographed with each bacterial group
analyzed (52). The major metabolic products of the bacterial isolates
examined were compared with the organic acid standards and identified
by elution profiles (73). The benzoic acid internal standard was
TABLE 4
Organic Acid Standards
Non-volatile Standard: Pyruvic 0.068 ml Lactic 0.087 ml Oxalacetic 0.06 g Oxalic 0.06 g Methyl-malonic 0.06 g Malonic 0.05 g Fumaric 0.06 g Succinic 0.06 g
Volatile Standard: Acetic 0.057 ml Propionic 0.075 ml Isobutyric 0.092 ml Butyric 0.091 ml Isovaleric 0.127 ml Valeric 0.125 ml Isocaproic 0.126 ml Caproic 0.126 ml prepared as suggested by Onderdank (personal communication) by adding
12.5 millimole of the compound per specimen. This was accomplished by adding 0.1 ml of the stock solution, which contained 125 millimole 29 of benzoic acid or 15.26 grams/liter to 0.9 ul of sample.
Gas Chromatography: The instruments that were utilized in the gas- liquid chromatographic analyses were the Beckman GC-5 (Beckman Instru ment Co., Fullerton, CA), Varian 2740 (Varian Instrument Group, Palo
Alto, CA), Hewlitt-Packard 5880 (Hewlitt-Packard Inc., Los Angeles,
CA), and Perkin-Elmer 900 (Perkin-Elmer Corp., Norwalk, CN) chromato graphs. All chromatography was performed with a flame ionization de tector at 150°C, and the recording speed was 5 mm/minute. The chromato graphic parameters were as follows: A) Varian 2740: Oven 110°C and -4-4 120 C; sensitivity 8 x 10 amps and 16 x 10 amps; output lmv; nitro gen at 60 ml/min flow rate; column packing 10% F F A P (Free Fatty
Acid Product) on 80/100 chromosorb W A W (Supelco, Bellefonte, PA) and
10% SP1200/1% H-jPO^ on 80/100 chromosorb W A W (Supelco, Fellefonte, PA) -4 B) Perkin-Elmer 900 : Oven 100 C; sensitivity 4 x 10 amps; output lmv; air, N^, at 400, 75, 50 ml/min flow rate; column packing 10%
D E G A (diethylene glycol adipate) on 80/100 chromosorb W A W (Supel co, Bellefonte, PA) C) Beckman GC-5: Oven 90°C; sensitivity 64 x 10-4 amps; output lmv; air, N^, at 300, 50, 45 ml/min with Lacl-R-296
(Supelco, Bellefonte, PA) and 300, 60, 55 ml/min with SP 1000 (Supelco,
Bellefonte, PA) and 325, 60, 50 ml/min with SP 1220 (Supelco, Belle fonte, PA); column packing Lacl-R-296; 10% SP 1000/1% H3P04 on 80/100 chromosorb W A W, and 15% SP 1200/1% H..P0. on 80/100 chromosorb W A W O T D) Hewlitt-Packard 5880: Oven 135°C and 105°C; sensitivity 64 x 10~^
-12 amps and 256 x 10 amps; output lmv; air, N^, at 300, 65, 45 ml/ min; column packing 0.2% Carbowax 1500 on 80/100 carbopack C (Supelco,
Bellefonte, PA) E) Perkin-Elmer 3920: Oven 130°C and 105°C; 30 -4 -4 sensitivity 8 x 10 amps and 16 x 10 amps; output lmv; air, N2> H?
at 375, 70, 55 ml/min; column packing 3% OV-1 on 80/100 gas chrom q
(Analabs, New Haven, CN) and 3% OV-17 on 80/100 chromosorb W H P (Ana-
labs, New Haven, CN). The stationary phases F F A P , D E G A , SP 1000,
SP 1220, Lacl-R-296, OV-1, and 0V-17 were used in methyl ester analysis.
The SP 1200 staionary phase was used in free acid analysis, Lacl-R-296
and SP 1200 were both used in 2 ul quantities. Carbowax 1500 stationary
phase was examined for possible utilization in identification of organic
compounds in the bacterial cultures, including free acids and methyl
esters. The glass columns containing the stationary phases were all 6
feet in length with an outer diameter of 1 inches and inner diameter of
2 millimeters.
Schema: Figure 1 illustrates the basic procedure- for determination
of peak height ratios of products of the test cultures. The isolate is
cultured on tryptic soy agar (DIFCO) before inoculation into the appro priate medium for analysis. After incubation of 24 or 48 hours the cul
ture broth was centrifuged on a Sorvall angled centrifuge at 3500 xg
for 5 minutes. The methylation procedure of Holdeman and Moore (52) was
employed for the analysis. The extractions were performed and followed by analysis on the appropriate column. The resultant chromatogram was
compared to a standard of organic acids in order to identify the compon
ents. Peak heights were measured and ratios calculated for the major products, lactic and succinic acids.
Statistical Analysis: The product ratios of bacterial isolates in the experiments were statistically analyzed by the "t" test for Pure culture
inoculated onto
Tryptic Soy Agar slants
Inoculation into test
medium
Centrifugation
3500 x g - 5 tnin
Methylation of supernate: HgSOU/methanol 55°C for 30 min
Extraction with 0.5 ml CHClo
Injection of CHCI3 extract into chromatograph
Comparison with methyl ester standard
Computation of organic acid peak height ratio
Fig. 1 Schema for Chromatographic Analysis determining the significance of the difference between two independent means (51). The mean values for the product ratios of the bacterial isolates of each group were compared. Student's "t" test is the most appropriate procedure with these numbers of observations (42). The standard deviation and standard error were calculated, and a "t" value then was derived. From a standard table of "t" numbers, the values or degrees of freedom were used to obtain the "p" value, or the level of significance (51). The criteria served as a basis for confidence lev els and was greater or equal to 90%, so a "p" value of 0.10 or less was within acceptable limits. The "t" values for each experiment were calculated and listed with the standard deviation, standard error, num ber of samples, and the "p" value giving the significance level for the results in each chromatographic study. EXPERIMENTAL RESULTS
Since the purpose of this investigation was to examine the poten tial of gas-liquid chromatographic analysis procedures for differentiat ing and identifying aerobic and facultative-anaerobic bacteria, it seemed appropriate to test these procedures with biochemically similar bacter ial groups or species. The criterion employed was peak height ratios of the organic acid metabolic products. To this end, certain tests were undertaken early; included were determination of the class of microbial products which could be suitably employed in this analysis, the media which would serve in this purpose, and other parameters of the procedure that would permit conclusions to be drawn about the characteristics of the bacteria under study.
Bacillus species were examined since many are biochemically similar and they produce many metabolic products (44). In this regard, it was postulated that analysis of the products from these species would pro vide evidence that volatile or non-volatile acid products could be used to characterize or identify some, if not all, of the isolates because of the latitude of the metabolic compounds associated with members of the genus.
The Perkin-Elmer 900 chromatograph was employed in the analysis of the Bacillus species with D E G A as the stationary phase, and results
33 34
are shown in Table 5. All isolates were obtained from the Ohio Depart ment of Health and were analyzed for peak height ratios of their major
organic acid products: lactic, methyl-malonic, and succinic acids. The
isolates were incubated for 48 hours in the PYG medium, as previously
described. Bacillus macerans and Bacillus sphaericus produced acids not
seen in the other cultures; they were malonic and oxalacetic acids, which
separate these species from the other isolates. _B. anthracis, Bacillus oumilus. and Bacillus subtilis are separated from the others by production of methyl-malonic and succinic acids, and from each other by the differ
ences in peak height ratios of these products. Bacillus laterosuorus. B.
cereus, and Bacillus firmus show differences in pyruvic/lactic ratios and
separated by them. Pyruvic acid was detected only in these three species, distinguishing them from all other isolates tested. Chromatograms of
B_, laterosnorus and 3.. subtilis and they are seen in Figures 2 and 3* respectively, as representative isolates showing these differences in metabolic products. The different products and peak height ratios de tected in the cultures analyzed illustrate how the species can be dis tinguished from each other based solely on chromatographic analyses.
TABLE 5
Non-volatile Products of Selected Bacillus Species
1. B. macerans I presence of malonic acid 2. B. sphaericus 292 presence of oxalacetic acid methyl- lactic/succinic succinic/malonic
3. J3. anthracis 9651 13.5/1 2/1 4. _B. pumilus D87 14.0/1 4/1 5. B. subtilis D87 6. 2/1 pyruvic/lactic 6. _B. laterosporus 0.05/1 7. jB. cereus 883 0.17/1 8. B. firmus I 0.57/1 35
c h c i 3
0) Pyruvic CO oe O. Lactic co 0) Pi o 4-> o CD 4-1 QJ o
18 16 14 12 10 8 6 4 Time (Min)
Fig. 2 Non-volatile acids: lactic and pyruvic from Bacillus laterosporus 73 .
CHC13
Lactic
Succinic Detector Response Detector
18 16 14 12 10 8 6 4 2 Time (Min)
Fig. 3 Non-volatile acids: lactic and succinic from Bacillus subtilis 1)87 . 36
In studies of volatile organic acid products such as acetic, pro pionic, and butyric acids, Drucker (29) found differences adequate to
characterize members of the enteric group of bacteria. A study of the volatile products of Bacillus species under investigation seemed appro priate as a possible method of chromatographic chracterization.
Ethyl-ether extracts of Bacillus species cultures previously studied were chromatographed on the GC-5 chromatograph, with Lacl-R-296 as the stationary phase, for the examination of volatile metabolic products and the results are recorded in Table 6. The product differences and simi larities between the species are illustrated. The isolates were incu bated for 48 hours in PYG medium and show little separation by volatile acid product analysis. Only _B. firmus and B, cereus both were easily differentiated by comparison of peak height ratios.
TABLE 6
Volatile Products of Selected Bacillus Species
(mm) acetic/propionic ratio 1 . B. firmus I 4/0 2. B, laterosporus 73 14/3 4.6/1 3. B. macerans I 9/2 4.5/1 4. B. pumilus D87 15/3 5.0/1 5. B. subtilis D87 16/2.5 6.4/1 6. B. anthracis 9651 11/1.5 7.3/1 7. B. sphaericus 292 9/1 9.0/1 8. B. cereus 883 18/1 18.0/1
The results of similar analyses with Bi. •pumilus and also that of B, subtilis can be seen in Figures 4 and 5, respectively, as the represen tative isolates, with unknown and known products.
The volatile acids in the representative cultures were identified as acetic and propionic, while the non-volatile products were lactic and CHCI3
Propionic. Acetic
+ 5 V «Q) 16 r? 12 30 B S TT 2 Time (Min)
Fig. 4 Volatile acids: acetic, propionic, and unknown products from Bacillus pumilus 1)82 .
CHC1
Propionic A.ceti c
IS i5 12 10 B S £ 2 Time (Min)
Fig. 5 Volatile acids: acetic, propionic, and unknown products from Bacillus subtilis D37 . 38 succinic acids. In view of these results it was decided to examine var ious test cultures for the presence of non-volatile metabolic products.
In order to identify the appropriate non-volatile acids in the cul tures examined, stationary phases were scrutinized to determine which were most suitable for the procedure. This evaluation of stationary phases began with Carbowax 1500 stationary phase.
A methyl ester standard composed of the non-volatile acids, after methylation and extraction, was chromatographed on the Hewlitt-Packard
5880 machine, as were the methylated products of selected Bacillus species such as jL laterosporus and _B. cereus.
The chromatogram of the methylated standard shown in Figure 6 demon strates the masking effect of the CHCl^ and the subsequent failure to separate the methyl esters from the solvent. The analyses of both
Bacillus species exhibited the same results, i.e. these produced lactic acid which did not appear on the chromatogram with Carbowax 1500.
Stationary phases OV-1 and OV-17 were examined with the set of methyl ester standards. The results of the trial study (Figure 7) at a temperature of 130°C revealed no separation of the esters on either column stationary phase. The second trial was performed at a lower tem perature, 105°C, in an effort to achieve better separation. A partial separation was accomplished on the OV-17 and somewhat more on the OV-1 phase. The CHCl^ was detected nearly at the same time as were the com ponents of the methyl ester standard, illustrating little component separation. Carbowax 1500 stationary phase. stationary 1500 Carbowax Fig Detector Response CHCI .,6 Methylated standard on on standard Methylated 3 ie (Min) Time 10 CHC13 CHCI3
OV-17
-p -p
OV-1
— CHC1
Time (Min)
Pig. 7 Methylated standard on OV-1 and OV-17 stationary phases. 41
D E G A was tested with a methyl ester standard and the results are
in Figure 8 . Excellent separation of the components of the standard mix
ture, with good column performance as shown by the sharpness of each in
dividual peak, was recorded. Thus, this stationary phase is well suited
for separation of the non-volatile methyl esters.
Stationary phase Lacl-R-296 was employed in chromatographing the
standard mixture on the Beckman GC-5 machine. The resulting chromato
gram is seen in Figure 9 with good performance and separation of all
components with the exception of the oxalacetic/oxalic peaks. These two
peaks are not completely separated as they were in the F F A P station
ary phase. This could pose a problem if the products involved in an
analysis are critical to an identification and/or separation of biochem
ically related bacteria.
The stationary phase, F F A P , was utilized in the Varian 2740
chromatograph. The standard composed of methylated esters was separated
into each peak corresponding to the appropriate compound. The resolution
into separate peaks and performance of the column were very satisfactory
since each methyl ester can be seen as a distinct, sharp peak in Figure
10. This suggests a useful stationary phase for separating non volatile methyl esters.
Another stationary phase tested for use in the separation of methyl esters of organic acids was SP 1000. This was carried out on the Beck man GC-5 chromatograph and the results in Figure 11 illustrate the ex cellent separation achieved with good column performance. The peaks are sharp and well separated. This phase is comparable to SP 1220, seen chci3
Oxalacetic Oxalic Methyl-malonic
Malonic Lactic i Fumaric Succinic
Time (Min)
Fig. 8 Methylated standard on D E G A stationary p
CHC1 Oxalacetic Oxalic \ I ' Pyruvic Methyl-malonic iactic I Malonic
Fumaric
Succinic. Detector Detector Response jjf Detector Response
12 10 8 Time (Min)
Fig. 9 Methylated standard on Lacl-R-296 stationary phase Detector Response i. 0 ehltdsadr nFFAP stationary phase. Methylatedstandard on F FP A Fig. 10 2 Pyruvic Lactic k Oxalacetic
6 Oxalic 8 ie (Min) Time 10 Methyl-malonic : Malonic
12
Ik
16 Fumaric
18
20 Succinic Oxalacetic Oxalic CHC1 Pyruvic
Methyl-malonic
Lactic
Malonic
Succinic
-p
20 IS IB l5 12 10 8 6 k 2 Time (Min)
Fig. 11 Methylated standard on SP 1000 stationary phase.
Lactic Oxalacetic Pyruvic Oxalic 1 Methyl-malonic
Malonic
Fumaric
ou -p O)V -p0) Q
16 l5 12 10 8 Time (Min)
Fig. 12 Methylated standard on SP 1220 stationary phase, 45
in Figure 12, in that the peaks are also well separated with excellent
resolution and peak sharpness. The components are slightly less separ
ated when compared with the SP 1000 phase, but their differentiation can
be observed readily.
In reviewing the examination of the different stationary phases,
the most satisfactory for detecting and separating methyl ester compon
ents proved to be F F A P , DEGA, Lacl-R-296, SP 1000 and the SP
1200 phases. There were minor differences in column performance and
resolution, as noted earlier, and all five phases were acknowledged as
useful in chromatographic analysis of the organic acid products examined.
The stationary phases least reliable in the studies were Carbowax 1500,
OV-1, and OV-17. These stationary phases could not separate the indiv
idual components, and thus were rejected for further consideration. To
further prove the identity of the compounds detected in the bacterial
test cultures examined, a stationary phase was investigated that has been used in detecting free, underivatized acids, SP 1200 (90). Figure 13
shows the chromatogram of a lactic and succinic acid standard chromato
graphed on the Varian 2740 machine, with SP 1200 as the stationary phase. Figure 14 shows the chromatogram of the culture supemate ex tract of the representative culture _B. subtilis D87 with resultant peaks of lactic and succinic acid. The identities were determined by compar
ing retention times with the known compounds (52) . This comparison establishes that the products found in the J3. subtilis culture were
lactic and succinic acids. In Table 7 is listed the test cultures and peak heights of lactic/succinic acid detected on the SP 1200 stationary phase. The culture medium employed in analysis of anaerobic bacteria k 6
■p Lactic
■p Succinic
8 10 12 Time (Min)
Fig. 13 Lactic and succinic acid standard on SP 1200 stationary phase.
Lactic
-p
12 Time (Min)
Fig. lU Culture extract of Bacillis subtilis D87 on SP 1200 stationary phase. 47
such as Clostridium species, Propionibacterium species, and Bacteriodes
species, for metabolic products is PYG medium (52) . The chromatogram of the uninoculated medium sometimes displays the peaks identified as lac tic and succinic acid as can be seen in Figure 15. Since these compounds may cause confusion as to their origin, whether they are naturally pres ent or produced by the bacteria metabolically, a new medium devoid of the compounds was required.
TABLE 7
Peak Heights of Acids SP 1200 Stationary Phase with Selected Bacillus Species
lactic/succinic (mm) B. subtilis D87 78/10 jl. pumilus 8779 23/115 J3. thuringenesis 946 7/4 _B. laterosporus 73 18/5 B_. megaterium 236 50/44 _B. circulans D8447 100/23 B. licheniformis 447 15/7 B. globinii 556 15/10
A chromatogram of the uninoculated medium is shown in Figure 16.
Using the YG medium, any production of lactic and/or succinic acid can be attributed to the microorganisms under study.
In the search for the most efficient procedure to illustrate the products useful in separation and identification of bacterial groups, aspects such as type of products, stationary phase, CHCl^ quantities, and other pertinent parameters of chromatographic analysis procedures employed in the investigation were examined.
The methylation of the non-volatile organic acids was studied to determine the most efficient method available for methylation, and also 48
20 Time (Min) Pig. 15 Peptone yeast extract, glucose medium on D E G A stationary phase. 20 Time (Min) Pig. 16 Yeast extract, glucose medium on D E G A stationary phase. 49 the most economical of the two methods currently in use, that of H?SO4/methanol, and BF^/methanol. They were chromatographed using the Beckman GC-5 chromatograph with stationary phase Lacl-R-296. The method of employing F^SCH/methanol (52) was contrasted with the other method using BF^/methanol (73). The results are shown in Figures 17 and 18, respectively, showing few differences between the two methylation procedures. These chromatograms are of the extract of the representative culture B_. sphaericus 364 in Figure 17 and Figure 18. Table 8 shows the comparison of the methylation procedures on a group of Bacillus species, and display similar results. The methylation procedure should react with a large amount of the bacterial products, thus a comparison was undertaken to measure the rel ative amounts recovered with each methylating agent. Lactic acid was used as was the methylated form, methyl lactate, and the quantity was 1 TABLE 8 Methylation Procedures Using F^SCH 5 BF^ on Selected Bacillus Species H2S04 bf3 JB. sphaericus 364 *19/6 23/3 jl. circulans A285 18/5 19/3 B. laterosporus K777 26/6 26/5 B_. licheniformis R778 40/4 44/3 B. cereus A2685 41/3.5 42/2 *Peak heights in millimeters of lactic/succinic acids milliequivalent of each compound. Figures 19 and 20 show the peak heights of methyl lactate compared to the known standard. Figure 21 and 22 are the results of methylating lactic acid with H2SO4/methanol and BFj/methanol, respectively. The methylations recovered a majority 50 Lactic Succinic 20 18 16 ll+ 12 10 8 6 2 Time (Min) Fig. 17 Bacillus sphaericus 36U HgSOLj. methylation on Lacl-R-296 stationary phase. Lactic -p Succinic 20 18 16 lb 12 10 8 6 ■1+ 2 Time (Min) Fig. 18 Bacillus sphaericus 36b EF3 methylation on Lacl-R-296 stationary phase. stationary phase. stationary Detector Response i. 9 ehltdai tnado P F A F on standard acid Methylated 19 Fig. CHCl' Pyruvic Lactic Oxalacetic Oxalic ie (Min) Time Malonic Methyl-malonic 10 12 Ilf. Succinic 16 tionary phase. tionary tnado sta P A F F on standard -p -p « i I 1 + Methyl lactate 6 51 Methylated Lactic Acid Methylated Lactic Acid (1) w c o ft w « 2 k 6 8 10 12 4 6 8 10 12 Time (Min) Time (Min) Fig. 21 HpSOl* methylated lactic Fig. 22 BF3 methylated lactic acid acid on F F A P stationary phase. on F F A P stationary phase. 53 of the product, but not as much as anticipated. However, there were sufficient amounts to permit measurements of acid product ratios. Demonstrated in Table 9 is the similarity in methylation procedures as contrasted with a chemically pure methyl lactate standard. The cultures were chromatographed on the Beckman GC-5 chromatograph with stationary phase Lacl-R-296. TABLE 9 Comparison of Methylation Procedures with Methyl Lactate Standard Peak height (mm) *Methyl lactate 134 **Lactic acid (^SCM/methanol) 83 Lactic acid (BF^/methanol) 87 *Eastman Chemical **Matheson, Coleman, § Bell Chemicals Analysis of the CHCl^ extracted products should take place as soon as possible after extraction since the volume of succinic acid will in crease if the CHCl^ is allowed to remain in contact with the culture supemate. This increase in succinic acid was studied to determine quantitatively the uptake rate of the product. Figure 23 shows a B. cereus VI chromatogram three hours after extraction from the Beckman GC-5 chromatograph with stationary phase LaclrR-296. Figure 24 shows JB. cereus VI analyzed after twenty four hours, and demonstrates an in crease in succinic acid when compared with that recorded for the three hour extraction. The lactic acid peaks are of similar height. During the extraction procedure, the amount of CHCl^ used can play a significant role in an efficient chromatographic analysis. The small est quantity that allows reasonably good results, is the optimal quantity. Pymvic Lactic • Succinic 20 Time (Min) Fig..23 Bacillus cereus VI at 3 hours with pyruvic, lactic, and succinic acids on Lacl-R-296 stationary phase. Pyruvic Lactic Succinic Detector Detector Response Detector Response Time (Min) Fig. 2k ■ Bacillus cereus VI at 2k hours with pyruvic.', lactic, and succinic acids on Lacl-R-296 stationary phase. 55 Figure 25 is a chromatogram of the extraction using 0.5 ul of CHCl^ while Figure 26 is of an extraction in which 1.0 ul of CHC1„ was used. o One can see the effects of the additional CHCl^ with smaller peaks re sulting. Table 10 displays peak variations with different species of Bacillus. TABLE 10 Optimum Amount of CHCl^ in Extraction of Selected Bacillus Species 0 .5 ml 1.0 ml J3. megaterium 236 *24/5 6/3 B. thuringiensis 946 36/3 11/1 jL globigii 556 39/3 19/1 B. pumilus D7996 38/3 19/2 B. cereus D8749 69/5 15/1 *Lactic/succinic peak heights measured in millimeters In order to identify the organic acid peaks a mixture of known acids was chromatographed and compared with elution characteristics of all unknown peaks. The use of an internal versus external standard, namely benzoic acid, has been proposed (73) for addition to the bacter ial supemate to provide comparison with a known compound. The chemi cal is added prior to methylation and extraction and should appear as a predetermined elution peak. With EL cereus VII as the test culture, two supemates were used, one contained 12.5 mmole benzoic acid, and the compound was omitted from the other as a control; both were analyzed on the GC-5 chromatograph with stationary phase Lacl-R-296. The chromato gram seen in Figure 27 is the supernate with the benzoic acid, while Figure 28 is the supemate without the chemical. The benzoic acid could not be separated from the succinic acid with this procedure. 56 0) Lactic a c o mp< (D « U $ o Succinic 0) -p (D « 20 18 16 lU 12 10 8 6 U 2 Time (Min) Fig. 25 Bacillus megaterium 236 with 0.5 ml CHCI3 extraction showing lactic and succinic acids on Lacl-R-296 stationary phase. Lactic Succinic Time (Min) Fig. 26 Bacillus megaterium 236 with 1.0 ml CHCI3 extraction showing lactic and succinic acids on Lac-R-296 stationary phase. 57 | Succinic + Benzoic Lactic Detector Detector Response Oxalacetic Fig. ?7 Bacillus ccreus VII vith benzoic acid shoving succinic/benzoic peak on Lacl-R-?96 stationary phase. 58 Pyruvic Lactic Oxalacetic Detector Detector Response Succinic > Time (Min) Fig. 28 Bacillus cereus VII without benzoic acid showing succinic peak on Lacl-R-?96 stationary phase. 59 Chromatography of non-volatile products, with an external standard as lactic and succinic acid, was the method chosen to attempt separa tion and characterization of similar bacterial species and/or groups based on differences in peak height ratios. One large division of bacteria studied was the gram positive rods, namely selected Bacillus species and Lactobacillus species. Two well recognized Bacillus species were investigated because of their close biochemical relationships. It has been proposed that B. anthracis is actually a subspecies or variant of jl. cereus (27). This idea is based on their nearly identical biochemical character (27) . To be tested was whether gas-liquid chromatographic procedures could separate the species and provide biochemical data useful in their identification, such as the nature of the organic acid products produced under specific conditions. The optimum incubation proved to be 48 hours at 35°C, and, as seen in Table 11, the standard plate count shows a decrease in viable cells which precludes further production of organic acids. The peak heights of the lactic and succinic acid methyl esters (Table 12) illustrate a high level of organic acid production by 48 hours. The results of biochemical tests for confirmation are listed in Table 13.. The species can be separated only by o-phage susceptibility. This is an involved procedure (44). Listed in Table 14 are all cultures showing the peak heights of their lactic/succinic ratios. The ratio range for each species was _B. cereus 11.0 to 42.0 and _B. anthracis 3.7to 10.0. This difference in ranges suggests a major variation in the biochemical pathways of 6o TABLE 11 Standard Plate Counts at 2k & 1+8 Hour Incubations of Representative Test Cultures 2k Hours 1+8 Hours (ATCC) Bacillus cereus 11+579 1.5 x 107/ml 0.85 x 107/ml (ODH) Bacillus anthracis 33l+-8a 1.0 x 107/ml 0.60 X 107/ml (CDC) Escherichia coli K6l 1.6 x 107/ml 0.1+0 x 107/ml (ODH) Enterobacter agglomerans 1.3 x 108/ml, 0.U5 x 108/ml, 2a,3b 1.2 x 10°/ml 0.1+3 x 108/ml (ATCC) Bacillus cereus. 9&3k 1.5 x 0.80 X (ODH) Bacillus subtilis III 1.0 x i $ £ 0.51 x (ATCC) Bacillus megaterium 2.3 x 107/ml 1.20 x 107/ml (ODH) Lactobacillus casei ss. 1.1+ X lO^/ml 0.65 x 105/ml rhamnosus (u s d a ) Salmonella gall inarum 1+ 1.3 X 107/ml 5.50 X 106/ml (u s d a ) Salmonella pullorum H 7.3 X 107/ml 3.00 X 107/ml (ODH) Staphylococcus aureus 717 8.0 x 101/mi 3.10 X 107/ml (ODH) Staphylococcus epidermidis 1.1 X 108/ml 5.80 X 107/ml 721 (UM) Vibrio cholerae 9 0.75 X 107/ml 0.33 X 107/ml (ODH) Non-cholera vibrio I 0-90 X 107/ml o.l+l x 107/ml (OSU) Micrococcus luteus I 1+.2 x 106/ml 1.50 X 106/ml (ODH) Staphylococcus epidermidis 2.0 x 108/ml 0.75 x 108/ml 550:B!+ (ODH) Salmonella typhi 2V 3-1 x 107/ml 1.30 X 107/ml (ODH) Salmonella typhimurium I 1.6 x 107/ml 0.50 X 107/ml (OSU) Streptococcus pyogenes 8.2 x 105/ml 1+.1+0 x 105/ml (ODH) Staphylococcus epidermidis 1.0 x 107/ml 0.30 X 107/ml 511:B2 (ODH) Bacillus subtilis II 1.0 x 107/ml 0.51 x 107/ml (OkSU) Bacillus anthracis 0SU-1 1.0 x 107/ml 0.60 X 107/ml TABLE 12 Lactic/Succinic Acid Peak Heights at 2k 8c k& Hour Incu bations of Representative Test Cultures 2k Hours H8 Hours L/S (mm) L/s (mm) (ATCC) Bacillus cereus l*+579 2U.5/1.5 50.0/3.0 (ODH) Bacillus anthracis 33*+-$a 8 .0/2.0 28.0/3.0 (GDC) Escherichia coli K6l 32.0/16.0 95.0/37.0 (ODH) Enterobacter agglomerans hk.O/29.O, 98.0/71.0, 2a, 3b 56.0/13.0 88.0/21.0 (ATCC) Bacillus cereus 963*+ lU.0/1.0 23.0/1.0 (ODH) Bacillus subtilis III 18.0/2.5 25.0/1+.0 (ATCC) Bacillus megaterium 30.0/6.0 39.0/5.5 (ODH) Lactobacillus casei ss. 30.0/U.0 88.O/3 .O rhamnosus (USDA) Salmonella gallinarum U 0 .0/6.0 *+.0/33-5 (USDA) Salmonella pullorum H 0 .5/0.0 1. 0/0.0 (ODH) Staphylococcus aureus 717 36.O/U.O 8U.0/10.0 (ODH) Staphylococcus epidermidis 1.0/4.0 2 .0/9.0 721 (UM) Vibrio cholerae 9 12.0/11.0 15.0/lL.O (ODH) Non-cholera vibrio I 2.0/5.5 3 .0/6.0 (OSU) Micrococcus luteus OSU 5.0/2.0 3.0/h.O (ODH) Staphylococcus epidermidis 6k.O/k.5 102.0/5.0 550:Bk (ODH) Salmonella typhi 2V 12.0/3.0 16.0/5.0 (ODH) Salmonella typhimurium I IO.5/1U.O 13.0/37.0 (OSU) Streptococcus pyogenes 11.0/3.5 20.0/U.5 (ODH) Staphylococcus epidermidis 26.O/U.O U5.O/3 .O 511:B2 (ODH) Bacillus subtilis II 23.0/U.O 27.0/6.0 (OkSU) Bacillus anthracis OSU-1 8 . 0/2. 0 28.0/3.0 6 2 TABLE 13 Differential Characteristics of Bacillus cereus and Bacillus anthracis Test Cultures B. cereus: Spores Gelatin Gram + Rod -Phage ODH III + + + R OSU 123 + + + R ATCC 11*579 + + + R ATCC 706U + + + R osu 285 + + + R ODH XI + + + R CDC KC + + + R ODH VIII + + + R ODH I + + + R ODH II + + + R B. anthracis: CDC M -36 + - + S CDC 76-11 + - + S CDC 10lU-I + - + S CDC Vi-a + - + S ODH 33!*-8b + - + S CDC 76-1 + - + S OkSU 0786U + - + S CDC 965-11 + - + S OkSU 5 + - + s ODH 33h-8a + _ + s 63 TABLE 1*+ Lactic/Succinic Acid Peak Height Ratios of Bacillus anthracis and Bacillus cereus Isolates B. anthracis' Lactic/Succinic (mm) Ratio CDC M-36 39/8 U .9 CDC 76-11 19/3 6.3 CDC 101U-I 37/6 6.2 CDC VI-a 59/16 3-7 ODH 33U-8b 13/2 6.5 CDC 76-1 lU/2 7.0 OkSU 0786U 17/2 8.5 CDC 965-II 23/3-5 6.6 OkSU 5 30/3 10.0 ODH 33^-8a U2/5 8.U B. cereus: ODH III 6U/3-5 18.3 ODH 123 ll/l 11-0 ATCC 1U579 63/3-5 31-5 OSU 706k 27/2 13-5 osu 285 100/7 1U.3 ODH XI 68/2 3U.1 CDC KC 23/2 11.5 ODH VIII 28/2.5 11.2 ODH I 15/1 15-0 ODH II 8U/2 1+2.0 64 the two species. The growth medium for the production of organic acids was YG medium. Figures 29 and 30 are representative chromatograms of _B. cereus 14579 and _B. anthracis 334-8a, with analysis performed at 24 hours. Both of the organisms were analyzed on the Beckman GC-5 chromatograph with stationary phase Lacl-R-296 stud. conc. Figures 31 and 32 are chromatograms of these cultures at 48 hours incubation which were used in the calculation of peak height ratios. At 48 hours obser vation, the peak heights almost doubled, so this was judged the opti mum incubation period for analysis. The results (Table 15) are statis tically significant, and separation could be achieved as based on the lactic/succinic ratios. The chromatographic study of E5. cereus led to the comparison of this species with another often found in close associ ation, _B. subtilis. _B. subtilis, like _B. cereus, is a common soil contaminant as well as being present in dust, water, and the air (119). The separation and identification of these species are important to the clinical lab oratory, since J3. cereus is regarded as a pathogen and _B. subtilis as a saphrophytic bacterium (44). The separation of these species is not easily accomplished because of their similar physiology. The gas- liquid chromatographic analysis provides a reliable method of separa tion with a chromatographic chracterization that did not previously exist. The biochemical test results are in Table 16 displaying few physi ological differences. J3. cereus may be separated from J3. subtilis by the anaerobic growth test and lecithinase production, but variable re sults are a very common occurrence (44). Standard plate count results 65 Lactic Detector Response Detector Succinic 20 18 16 lh 1? 10 8 6 U 2 Time (Min) Fig. 29 Bacillus cereus ll+579 at Ph hours vith lactic and succinic acids. Lactic Succini c De'-ector Response De'-ector 26 ?k 22 20 18 16 lU 12 10 8 6 U 2 Time(Min) Fig. 30 Bacillus anthracis 33U--8a at 2h hours vith lactic and succinic acids 6 6 Lactic Succinic Time(Min) Fig. 31 Bacillus cereus 3^579 at 1*8 hours vith lactic and succinic acids. Lactic Succinic Time(Min) Fig. 32 Bacillus anthracis 33U-8a at U8 hours vith lactic and succinic acids. 67 Table 15 Statistical Analysis of Lactic/Succinic Acid Peak Height Ratios Bacillus anthracis Bacillus cereus U.9 18.3 6.3 ' 11.0 6.2 31.5 3.7 13.5 6.5 1^.3 7.1 3^.1 8.5 11.5 6.6 11.2 10.0 15.0 8.U U2.0 N = 10.000 N = 10.000 M = 6.820 M = 20.2^0 SD = 1.8l8 SD = 11.293 SE = 0.570 SE = 3.570 T = -3-710 (18) P<0.005 6 8 TABLE 16 Biochemical Differentiation of Bacillus cereus/Bacillus subtilis Isolates B. cereus; Anaerobic Growth Lecithinase Gram + Rods Spores ODH VI + ODH 1390 + + + + CDC KC + + + + CDC D88 + + + + ODH 236 + + + + ATCC 963U + + + + ATCC W579 + + + + ODH IV + + + + ODH V + + + ATCC 11778 + + B. subtilis: CDC A25 ODH 503 OSU UH-2 + ATCC 6051 + OSU 001 + + ODH 128 + + ODH I + + ODH II + + CDC D87 + + OSU UH-1 + + 69 in Table 11 and the product peak heights in Table 12 indicate the op timum incubation period to be 48 hours. These results indicate the possibility of using the gas-liquid chromatographic analysis for characterization in a time period as short as 48 hours. The biochemical characterization of both species by conventional methods may require a time period of up to 10 days (44). This comparison of the time required to characterize the two species illustrates how efficient chromatographic analysis techniques can be in the investigation of aerobic and facultative-anaerobic bacteria. In Table 17 are the ratios of lactic/succinic acid products of each species. The ratio ranges were 3.0 to 7.2 for _B. subtilis and 11.5 to 31.5 for _B. cereus, thus offering a chromatographic separation of the two species. The cultures were grown in YG medium and the stationary phase used was Lacl-R-296. Figures 33 and 34 are of chromatograms of J3. subtilis III at 24 and 48 hours incubation, and jB. cereus 9634 represented in the Figures 35 and 36. The statistical analysis displays a significant difference in the peak height ratios of their major products and this is listed in Table 18. The separation and subsequent identification of members of the genus Lactobacillus from those of the genus Bacillus are involved procedures and are usually accomplished by the catalase test and spore formation. The similarities between the genera provide problems in the separation of the two groups. Spores are not always produced by Bacillus species, and the members of both genera are large gram posi tive rods (44). With these few procedures available in the laboratory, the identity of an unknown isolate may be difficult to determine. 70 Lactic Succinic Time (Min) Fig. 3? Bacillus subtilis III at ?U hours with lactic and acids. Lactic Succinic Detector Response Response Detector Response Detector ?? ?0 18 16 lU 1? 10 8 6 U ? Time(Min) Fig. 3^ Bacillus subtilis III at b8 hours with lactic and succinic acids. Lactic Succinic Time(Min) Fig. 35 Bacillus cereus 9^3^ at Pk hours with lactic and succinic acids. Lactic Succinic PO 5B JS lU IP 10 8 6 1 ? Time(Min) Fig. ?6 Bacillus cereus 9^3^ at i+8 hours with lactic and succinic acids. TABLE 17 Lactic and Succinic Acid Peak Height Ratios of Bacillus subtilis and Bacillus cereus Test Cultures B. subtilis: Lactic/Succinic Ratio CDC A25 22/3.5 6.2 ODH 503 12/3.5 3.4 OSU UH-2 12/3 7.0 ATCC 6051 12/3.5 3.4 OSU 001 12/2 6.0 ODH 128 18/5 3.6 ODH I 18/4 4.0 ODH II 22/6 3.0 CDC D87 14/4 3.5 OSU UH-1 18/2.5 7.2 B. cereus: ODH VI 32/2 l6.0 ODH 1390 22.5/1.5 15.0 CDC KC 23/2 11.5 CDC D88 24/2 12.0 ODH 236 20/1.5 13.3 ATCC 9634 36/2 18.0 ATCC 14579 63/2.5 31.5 ODH IV 58/2.5 29.0 ODH V 70/5 14.0 ATCC 11778 83/3 21.3 73 Table l8 Statistical Analysis of Lactic/Succinic Acid Peak Height Ratios Bacillus cereus Bacillus subtilis 16.00 6.20 15-00 3.U0 11.50 7.00 12.00 3-^0 13-30 6.00 18.00 3-60 31.00 1+.00 29.00 3.00 Ik.00 3.50 21.30 ' 7.20 N = 10.000 N = 10.000 M = 18.110 M = U.730 SD = 6.913 SD = 1.663 SE = 2.186 SE = 0.526 T = 5.951 (18) P <0.0005 74 Chromatographic analysis of these two genera can aid in recogniz ing the appropriate taxonomic category in which to place a particular bacterial isolate. The differences in lactic acid production between the genera are registered on the chromatograms during analysis, and the peak height ratio ranges are readily seen as being dissimilar. Since the laboratory analysis time required for characterization of these bacteria can extent for up to 7-10 days (44), the comparatively short time period of 48 hours for chromatographic analysis can be of great value. The biochemical tests to confirm the generic designations are in Table 19. Tables 11 and 12 list the plate counts and peak heights of representative isolates. The dramatic increase of lactic acid in the 48 hour incubation versus the 24 hour time period is readily apparent. Figures 37 and 38 are chromatograms of Bacillus megaterium cultures at 24 and 48 hours, and Figures 39 and 40 of cultures of Lactobacillus casei ss. rhamnosus. The difference in product ratios can be seen in Table 20, where are listed peak height ratios of both genera with ranges of 0.25 to 10.67 and 17.5 to 34.0 for Bacillus species and Lactobacillus species, re spectively. YG was the culture medium used in the analysis and the stationary phase was F F A P . A statistically significant difference between the product ratios of Bacillus species and Lactobacillus species can be seen in Table 21. The chromatographic differentiation of the major Bacillus species should include the separation of jl. anthracis and _B. subtilis because of the close relationship in biochemical similarities, and the fact that both may be found in the same environments (42), (27). Lactic a> CO c coa ft) K P O •Po ft) -pft) Succinic « Time (Min) Fig. 37 Bacillus megaterium at 2b hours vith lactic and succinic acids. Lactic Succinic Time(Min) Fig. 38 Bacillus megaterium at hours vith lactic and succinic acids. 76 «ID C o (0p< 2 U 6 8 HO 12 ill 16 18 Time(Min) Fi g. 39 Lactobacillus casei ss. ’ rhamnosus at ?h hours vi+.h lactic and succinic acids. Lactic 0) (0 c o (A CO ID P h U O ■P CJ ID 4 Time(Min) Fig Lo Lactobacillus casei ss. rhamnosus at U8 hours vith lactic and succinic acids. IA3LE 19 Biochemical Differentiation of LactobaciHus sp. and Bacillus sp. Lactobacillus sp: Catalase Scores Gram + Rods ODH L.casei ss. rhamnosus CDC L.fermentum CDC L. easel CDC _L, buchnerl CDC L. plant arum + OGU l . bulgarlcus + CDC L. sallvarlus ss. + sallvartus ODH L. curvatus + CDC _L. eelloblosus CDC L_. acidophilus + 3aclllus sp: CDC B.clrculans + + + CDC B.pumllus + + + ATCC B.globlnll + + + ATCC B.megaterium + + + o d h _b , lishsnlEOTls + + + ODH _b . laterosporus + + + ATCC _B. pantothenticus + + + CDC _b . sphaerl cus + + + ATCC _b . s tearo thermoph 1 lus -r + + OSU j b . thuringiens 1 s + + + 78 TABLE 20 Lactic & Succinic Acid Peak Heigrt BatioS of Bacillus sp. and Lactobacillus sp. Bacillus sp. : Lactic/Succinic (®m) Ratio CDC B. eirculans 8 5/IA . 5 -5*86 CDC B. pumllus 61/6 10.17 ATCC B. globlnll 7A/9 8*2.0 ATCC B. megaterium 27/12 2.25 ODH B. llchenlformls 32/3 10.67 ODE B. laterosporus A6/9 5*^ ATCC B. pantothentlcus 25/16.5 1.52 CDC 3. sphaerlcus 2/6 0*33 ATCC B. s t eapo thermo Philus A3/11 3*92 OSU B. thurlngjensls 1/A 0.25 Lactobacillus sp. ODH L. easel ss. rhamnosus 20A / 6 3A.00 ODH L. curvatus 80/A 20.00 CDC L. plantarum 213/9 23*67 OSU L. bulgarlcus 205/7 29.29 CDC L. easel 206/10 20.60 CDC L. celloblosus 82/2.5 32.80 CDC L. sallvarlus ss. 87/3.5 2A.85 "* sallvarlus CDC L. fermentum 212/9 23*56 CDC L. acidophilus 70/A 17*50 CDC L. buchnerl 209/10 20.90 79 •TABLE 21 Statistical Analysis of Lactlc/Succlnlc Acid Peak Height Hatios Bacillus species vs. Lactobacillus species L/S L/S 5.86 3^.00 10.17 23.56 8.22 20.60 2.25 20.90 10.67 23.67 5.11 29.29 1.52 2 4 , 8 5 0.33 20.00 3 . 9 1 32.80 o»25 1 7 ^ 0 N a 10.0000 N a 10.000 M a A.829 M “ 2A.717 SD = 3.872 SD = 5.585 SEM = 1.224 SEM a 1.766 T a -9,25A3 (18) P a <0.0005 80 The differentiation of these species is useful since B. anthracis is a virulent human and animal pathogen while J3. subtilis is most usu ally found as a common saphrophytic contaminant in soil (27). The separation of the species by biochemical means can be done usually by the anaerobic growth test and b-phage test, as seen in Table 22. The 24 and 48 hour plate counts and peak height measurements of the repre sentative cultures are in Tables 11 and 12, respectively. The index cultures used were _B. subtilis II, Figures 41 and 43, and J3. anthracis OSU-1, Figures 42 and 44. The culture medium utilized was YG and pro vided a measurable amount of metabolic products for the chromatographic separation. The stationary phase used was Lacl-R-296. The incubation period selected was 48 hours based on results in Tables 11 and 12. The chromatographic results are in Table 23 and show a ratio range of 3.0 to 7.2 for _B. subtilis, and a range of 3.7 to 10.0 for JB. anthracis. The statistical analysis in Table 24 shows a significance between the ratios, despite some results showing that certain of the ratios are very close. The chromatographic chracterization of these Bacillus species under investigation provides an efficient separation and identification based on the product peak height ratios, namely those of lactic and succinic acid. The separate species _B. cereus, EL anthracis, and _B. subtilis, which include the human pathogens and the most commonly encountered saprophyte, can be characterized in a comparatively short time using chromatographic procedures, as compared to conventional methods. Lactic Succinic 20 18 16 lU 12 10 8 6 1+2 Time(Min) Fig. 1+1 Bacillus subtilis II at 2b hours with lactic and succinic acids. Lactic Succinic Time (Min) Fig. 1+2 Bacillus anthracis OSU-1 at 2h hours with lactic and succinic acids. 8 2 Lactic Succinic 22 20 18 16 lit 12 10 8 6 U 2 Time (Min) Fig. 1*3 Bacillus subtilis II at U8 hours with lactic and succinic acids. Lactic Succinic Time(Min) Fig. kk Bacillus anthracis OSU 1 at U8 hours with lactic and succinic acids. 83 TABLE 22 Biochemical Characteristics of Bacillus subtllls and Bac illus anthracis Isolates Anaerobic B. subtllls: Growth Spores Gram + Rod B-Phage Catalase ODH II + 4* R 4- ODH I 4- 4- R + CDC A25 4- 4- R 4- CDC D8? 4- 4- R 4- OSU 001 4- 4- R 4- ATCC 6051 4- 4- R 4- ODH 128 4- 4- R 4- ODH 503 4- 4- R 4- OSU U H 1 ■4- 4- R 4- OSU u h 2 4- 4- R 4- B. anthracis: OkSU OSU- 5 4- 4- S CDC M-36 + 4- 4- s 4- OSU OSU-1 + 4- 4- s 4- CDC 101A-I + 4- 4- s 4- CDC 76-11 + 4- 4- s 4- OkSU 07864 4- 4- s 4- CDC 76 I 4- 4- s + ODH 334-8A 4- 4- s 4- ODH 334-8B 4- 4- s 4- CDC VI-A 4- 4* s 4- 84 TABLE 23 Lactic/Succinic Acid Peak Height Ratios of Bacillus subtilis and Bacillus anthracis Test Cultures B. subtilis: Lactic/Succinic (mm) Ratio ODH II 27/6 ' 3/1 ODH I 18/4 4/1 CDC A25 22/3.5 6.2/1 CDC DS7 14/4 3.5/1 OSU 001 12/2 6/1 ATCC 6051 12/3.5 3.4/1 ODH 128 18/5 3 .6/1 CDH 503 12/3.5 3.4/1 OSU UE1 18/2.5 7.2/1 osu u?:2 ' 12/3 7/1 B. anthracis: OkSU osu-5 30/3 10/1 CDC K-36 39/8 4 .9/1 OSU OSU-1 23/3.5 6 .6/1 CDC 1014-1 37/6 6.2/1 CDC 76-II 19/3 6 .3/1 OkSU 02864 17/2 8 .5/1 CDC 761 14/2 7/1 CDH 334-SA ’ A2/5 8.4/1 ODH 334-55 13/2 6.5/1 CDC VI-A 59/16 3 .7/1 85 TABLE 2k Statistical Analysis of Peak Height Ratios of Lactic & Succinic Acid Bacillus subtilis Bacillus anthracis 3.0 10.0 4.0 - 4.9 6.2 6.6 3.5 6.2 6.0 6.3 3.4 8.5 3.6 7.0 3.4 8.4 7.2 6.5 M. 2*1 H = 10.000 N •= 10.000 M = 4.730 M = 6.810 SD = 1.66 SB = 1.82 SE = 0.53 SE = 0.57 T = -2.670 (18) P =<0.01 86 The complicated biochemical character of many gram negative enteric bacteria poses a problem in the laboratory, as various clinically impor tant members of this group are so similar the identification and dif ferentiation may be a difficult process demanding extensive time periods for analysis. Chromatographic chracterization can reduce the time and procedures required to separate and identify certain gram negative bac teria, such as E. coli, Enterobacter agglomerans, selected Salmonella species, and the V. cholerae group. These bacteria are often encountered in enteric disease (119). Both EL coli and EL agglomerans are found in close association with other enteric microorganisms in the human intestinal tract (119), and most of the other gram negative bacteria can be easily separated from EL coli and EL agglomerans; but the separation of these two bacteria is difficult. Most biochemical test results are variable, and too simi lar for a clear separation. EL coli and EL agglomerans both have been isolated from sick, as well as healthy individuals (27). The confused state of taxonomy of E. agglomerans adds to the difficulty in separat ing these two species. The genus has been referred to as a "garbage genus" by scientists. Thus the examinations of the chromatographic analyses seemed appropriate as a possible means of delineating differ ences between the bacteria. After a 48 hour incubation in YG medium, the product peak heights of the representative cultures of EL coli and EL agglomerans show at least a two-fold increase over the 24 hour incubation period. Thus the 48 hour incubation was chosen for study. The stationary phase used was F F A P. These results (Table 12) and the plate count listed in 87 Table 11 signify the loss of live cells by the 48 hour period. The results of biochemical testing appear in Table 25 as do the serological identification of the _E. coli isolates. The utilization of tartrate aids in the separation of the genera. The range of the ratios of lactic and succinic acid for E_. coli was 1.71 to 3.30 and the ratios for _E. agglomerans were divided into two separate ranges: 0.29 to 1.38. I and 4.28 to 10.70 II, listed in Table 26. The Figures 45 thru 50 are chromatograms at 24 and 48 hours of _E. coli K61 and JE. agglomerans 2a, 3b respectively. The analysis results imply that there are two differ ent groups of _E. agglomerans isolates, a fact substantiated by the bio chemical results that indicate group I indeed differs from group II. Group I isolates were all malonate and cellobiose positive. The statis tical results in Table 27 suggest that significant differences exist be tween _E. coli and both groups I and II of E. agglomerans. This gas-liquid chromatographic analysis has proven to be a useful procedure for identifying both species while providing data concerning the basic biochemical character of each bacterium. V. cholerae and the non-cholera group of enteric pathogens are the important agents in the cause of cholera and the cholera-like disease (27). They are glucose fermenting, gram negative, rod shaped bacteria and are biochemically inseparable, but do not share the 01 antigen which is found exclusively in V. cholerae (40). Virulent V. cholerae infection results in the cholera syndrome while the non-cholera vibrio produces a mild disease of lower pathogenicity, which sometimes resembles a typical cholera infection (23). Lactic a> ca a> K Succinic u B vQ> -P 0) (=) Time (Min) Fig. U5 Enterobacter agglomerans 2a at 2b hours vith lactic and succinic acids. Lactic +5 Succinic Time(Min) Fig. U6 Enterobacter agglomerans 3b at 2b hours vith lactic and succinic acids. Lactic Succinic 0) COc o up* v PS u $V a> +3 0) Time(Min) Fig'. U7 Enterobacter agglomerans 2a at h8 hours vith lactic and succinic acids. Lactic Succinic 2 b 6 8 10 1? 1^ 16 18 Time(Min) Fig. h8 Escherichia coli K6l at 2b hours vith lactic and succinic acids. Lactic CQ c a caa> K u o -po 0) -p (!) « Succinic 2 U 6 8 10 12 ll+ l6 18 Time(Min) Fig-. U9 Enterobacter agglomerans 3b at H8 hours with lactic and succinic acids. Lactic Succinic a) £CQ (D K P O -P v 0) -p0) « Time(Min) Fig. 50 Escherichia coli K6l at U8 hours with lactic and succinic acids. 91 TABLE 25 Biochemical Characteristics of Escherichia coli & Enterobacter agglomerans Isolates E. coli: Malonate Tartrate Lysine Ornithine Arginine Cellobiose CDC K69 - + + + ~ CDC K6l - + + + + CDC K63 - + + - + CDC K59 - + + + - - CDC K77 - + + + + ODH K67 - + + + ~ ~ ODH K71 - + + - + ODH K73 - + + + ■ ODH K7U , - + - - + - ODH K19 - + + + - E. agglomerans: CDC la + - - - + CDC 2b -- - - CDC 5b + -- -- + CDC lb - -- - ODH 3b + -- - ODH kb + - - - + CDC 6b + - -- ODH 2a + - - - + CDC 7b + - - - - CDC 3a _ _ _ M. + 92 TABLE 26 Lactic & Succinic Acid Peak Height Ratios of Escherichia coli E. coli: Lactic/Succinic (mm) Ratio CDC K69 6U/23 2.80 CDC K6l 128/52 2.50 CDC K63 81/U8 1.71 CDC K59 10U/U5 2.30 CDC K77 1+9/15 3.30 ODH K67 95/37 2.60 ODH K71 33/15 2.20 ODH K73 156/52 3.00 ODH K7U llU/38 3-00 ODH K19 70/29 2 .1+1 E. agglomerans: CDC la 61/67 0.90 CDC 2b 32/3 10.70 CDC 5b 1+/10 0.29 ODH lb 33/5 6.60 ODH 3b 1+8/6 8.00 CDC kb 12/19 0.63 CDC 6b 1+1/6 6.83 ODH 2a 98/71 1.38 CDC 7b 30/7 1+.28 CDC 3a 89/13 6.81+ 93 TABLE 27 Statistical Analysis of Lactic & Succinic Acid Peak Height Ratios Escherichia coli Enterobacter agglomerans 1.70 1-0.29 II- 4.28 2.20 0.63 6.60 2.30 0.90 6.83 2.41 1.38 6.84 2.50 K = 4.000 8.00 2.60 M = 0.800 10.70 2.80 SD = 0.460 N = i 3.00 SE = 0.230 M = ' 3.00 T = 6.4809 (12) s d = : P = <0.0005 SE = I N = 10.000 The only accepted method for separation employs serological pro cedures (40). Here, however, chromatographic analysis of both groups revealed a significant difference in their biochemical products. YG was the medium utilized in the analysis and it proved satisfactory, and the stationary phase used was F F A P. The biochemical tests for con firmation as shown in Table 28 include the test results of identification with antisera to V. cholerae strains. The representative isolate chromatograms after 24 and 48 hours are shown in Figures 51 and 52, 53 and 54, respectively, of the representative V. cholerae and non cholera vibrio cultures. Table 29 lists the peak height ratios based on the production of lactic and succinic acids and the Tables 11 and 12 show the plate counts and peak heights at 24 and 48 hours, respectively. After 24 hours, the product peak heights are sufficient for calculating ratios for separating the groups of bacteria. The representative cul tures were V. cholerae 9 and non-cholera vibrio I, and the ratios ranged from 0.29 to 0.50 for the non-cholera vibrio and from 0.58 to 1.50 for the V. cholerae cultures. The statistical analysis results confirmed that the ratio differences were significant, and permitting the inter pretation that the ratios, indeed, differentiate the two groups in question (Table 30). Salmonella pullorum and Salmonella gallinarum were investigated because of their importance in the food industry and relation to human food poisoning cases. S^. pullorum is a common contaminant of various chicken meat products, and £>. gallinarum is also commonly found in poultry meat (119) . The subsequent chracterization of each bacterium from contaminated food is difficult since few differences exist in a> Lactic CQ C a> P5 ' Succinic $v 0) •p Time (Min) Fig. 51 Vibrio' cholerae 9 at ?U hours with lactic and succinic acids. Lactic ECQ o & 0) K CJ 0) Succinic pd) p 2 k 6 8 10 12 3^ l6 38 Time(Min) Fig. 5? Vibrio cholerae 9 at U8 hours with lactic and succinic acids. 96 a co c COa Lactic a> « Succinic $v Q) -P0) o ? . h 6 8 10 1? jk l5 l5 20 22 ?E Time(Min) Fig 5? Non-cholera vibrio I at 2k hours -with lactic and succinic acids. 0) cCO Lactic Succinic Time(Min) Fig. 5U Non-cholera vibrio I at U8 hours vith lactic and succinic acids. 97 TABLE 28 Biochemical Characteristics of Vibrio cholerae & Non-cholera vibrio Isolates V. cholerae; Motility Lysine Gram - Rods eholera antisera UM h + + + + UM 6 + + + + DM 10 + + + + UM 7 + + + DM 8 + + + ODH VCI + + + UM 9 + + + UM 1 + + + + UM 2 + + + + UM 3 + + Non-cholera vibrio: UM J + UM C + UM D + + + ODH NCVI + + + UM H + + + UM G + + + UM I + + + UM B + + + UM F + + UM A + v+ 98 3ABLE 29 Lactic & Succinic Acid Peak Height Ratios of Vibrio cholerae & Non-cholera vibrio Test Cultures UM k V3 1.33 UM 6 3A 0.75 UM 10 k/6 0.67 UM 7 9/8.5 1.06 UM 8 7/12 0.58 ODH VCI 7/9 0.78 UM 9 lU/12 1.09 UM 1 3/2 1.50 UM 2 11/11 1.00 UM 3 fc.5/7.5 0.60 Non-cholera vibrio: UM J 5/10 0.50 UM C 1/2.5 0 .1*0 UM D 1/3.5 0.29 ODH NVCI 2/5-5 0.36 UM H 5/12 0 .1*2 UM G 1 0 * 0.29 UM I 1.5/16.5 0.36 UM B 5/11 0.1*5 UM F 5/12 0 .1*2 UM A 6/12 0.50 99 TABLE 30 Statistical Analysis of Peak Height Ratios of Lactic & Succinic Acids 1.33 0.50 0.75 0 .U0 0.67 0.29 1.06 0.36 O .58 0 .1*2 0.78 0.29 1.09 O .36 1.50 0.1*5 1.00 0 .1*2 0.60 0.50 N = 10.000 N = 10.000 M = 0.936 M = O .399 SD = 0.31^ SD = 0.080 SE = 0.099 SE = 0.027 T = 1*. 97l* (17 ) P =<0.0005 1 0 0 'ft) Succinic -p k 6 8 10 12 1 4 1 6 Time(Min) Fig. 55 Salmonella pullorum H at 24 hours vith the absence of succinic acid. 10o> c toa ft) K P o ■pc Succinic 0) •p'0) Time(Min) Fig, 56 Salmonella gallinarum 4 at 24 hours with succinic acid. 1 0 1 0) cto « K fn » V 0) ■p (1) Succinic k 6 8 10 12 lU 16 18 Time (Min) Fig. 57 Salmonella -pullorum H at U8 hours vith the absence of succinic acid. 0! ctO I « U Succinic q u w •pa> O 2 k 6 8 10 12 1U 16 18 Time(Min) Fig. 58 Salmonella gallinarum U at U8 hours vith succinic acid. 102 their biochemical activity. The gas-liquid chromatographic analysis offered here may provide a method which will be valuable in an identi fication technique. The biochemical confirmation tests used in the chracterization of the species are sparse in number and include the tartrate utilization test, ornithine utilization, and H^S production (34). The serological examination of the species also aids in the final definitive identifi cation (33). The results of which are seen in Table 31 as are the bio chemical identifications. The serological examinations are employed in characterizing each Salmonella species. The table lists the antigenic factors according to species, and there is a factor present in S_. gallinarum that is absent in _S. pullorum. Chromatograms of cultures of jS. pullorum H and S^. gallinarum 4 incubated at 24 and 48 hours are shown in Figures 55, 56, and 57, 58, respectively. These chromatograms show the difference in the produc tion of succinic acid. _S. gallinarum produced large amounts of the product while none was produced by SL pullorum. In Tables 11 and 12 are listed the plate counts and peak heights at 24 and 48 hours incu bation of the representative cultures. It would appear then, that at least one difference between the species is the production or non production of succinic acid. The stationary phase used was F F A P , The YG medium proved unsatisfactory for separating these species and was modified by replacing the glucose component with the sugar alcohol, dulcitol. The results of the analysis showed all isolates of S_. gallinarum produced a significant amount of succinic acid, while none of the S_. pullorum isolates produced it. 103 TABLE 31 Biochemical Characterization of Salmonella gallinarum & Salmonella pullorum Isolates S, gall inarum: Ornithine Motility Serology Gram - Rod USDA C - - 1,9,12 + ODH I -- 1,9,12 + ODH 26 - - 1,9,12 + USDA 3 - - 1,9,12 + USDA H -- 1,9,12 + USDA E - - 1,9,12 + USDA 2 - - 1,9,12 + USDA k - - 1,9,12 + USDA 7 -- 1,9,12 + USDA A - - 1,9,12 + S. pullorum: USDA 6 + 9,12 + USDA D + - 9,12 + USDA 3 + - 9,12 + ODH 86hl + - 9,12 + USDA H + - 9,12 + ODH 28 + - 9,12 + USDA E + - 9,12 + USDA 5 + - 9,12 + USDA 2 + - 9,12 + USDA 1 + _ 9,12 + 104 The clinical importance of both gram negative species. Salmonella typhi and Salmonella typhimurium, is well documented and requires no additional comments here (34), (35), (49), (119). The separation of these species biochemically, is not easily accomplished by conventional test procedures, with ornithine utilization playing a primary role. The test parameter and serological test offer the basis for the differentia tion of these two species (Table. 32). This paucity of biochemical test procedures makes it difficult to differentiate the species routinely with a high degree of accuracy. In Table 11 and 12 are the plate counts and product peak heights of cultures of the representative species at 24 and 48 hours incubation. Different media were tested with YG and YD both exhibiting no major differences in product ratios. YG medium was examined and proved to be capable of separating the two species by ratio comparisons. The stationary phase used was F F A P. Figures 59, 61 and 60, 62 are chromatograms of the representative cultures at 24 and 48 hours incubation with the peaks of the major products easily seen. Table 33 lists the ranges of ratios for each species with S_. typhi at 0.17 to 0.56 and _S. typhimurium at 0.63 to to 2.73. The statistical analysis results, as shown in Table 34, show a significance level that supports the premise of differentiation by peak height ratio analysis. The separation and characterization of Staphylococcus species and Micrococcus species are important procedures in the clinical lab oratory. Even though these genera are usually differentiated by a few procedures, including oxidation/fermentation of glucose, there are many instances in which strain variations render these tests useless. YG Lactic 4)co c 01 K U Succinic $ V0) ■p0) ft 2 U 6 8 10 12 lU l6 18 20 Time (Min ) Fig. 59 Salmonella typhimurium I at 2h hours vith lactic and succinic acids. 0) CO ac Vto Lactic K u o •pt) 0! •P Succinic ai e Time(Min) Fig. 60 Salmonella typhi 2V at 2U hours vith lactic and succinic acids. 1 0 6 Lactic a> COc I Succinic Time (Min) Fig. 6l Salmonella typhimurium I at U8 hours vith lactic and succinic acids. Lactic to0) c 8. Succinic 09o P5 u o •po 0) •p p Time (Min) Fig. 62 Salmonella typhi 2V at U8 hours vith lactic and succinic acids. 107 TABLE 32 Biochemical Characterization of Salmonella typhi & Salmonella typhimnrium Isolates S.typhi: Lysine Motility Ornithine Serology APi APi500 + + - 9,12,VI,D ODH I + + - 9,12,VI,D APi APi540 + + - 9,12,VI,D ODH 4l8M + + - 9,12,VI,D CDS 2V + + - 9,12,VI,D OSU M.M.I + + - 9,12,VI,D CDC DIO + + - 9,12,VI,D ODH II + + - 9,12,VI,D ODH III + + - 9,12,VI,D ODH 80S + + - 9,12,VI,D S . typhimnrium: ODH 891 + + + 1,4,5,12:1:1,2 ATCC 13311 + + + 1,4,5,12:1:1,2 ODH I + + + 1,4,5,12:1:1,2 ODH 2Ul + + + 1,4,5,12:1:1,2 ODH II + + + 1,4,5,12:1:1,2 ODH 721 + + + 1,4,5,12:1:1,2 ODH 861 + + + 1,4,5,12:1:1,2 ODH 611 + + + 1,4,5,12:1:1,2 ODH 551 + + + 1,4,5,12:1:1,2 ODH 901 + + + 1,4,5,12:1:1,2 1 0 8 TABLE 33 Lactic & Succinic Acid Peak Height Ratios of Salmonella typhimurium & Salmonella typhi ODH 2kl 11/15 0.73 ODH II H1/15 2.73 ODH 891 55/37 I.U9 ODH 611 13/15 0.87 ODH 861 12/13 O .92 ATCC 13311 lU/10.5 1.33 ODH 721 55/36 1.53 ODH 551 5/8 0.63 ODH I 13/13 1.00 ODH 901 15/5.5 2.73 S.typhi: ODH Ul8M 6/13 0 .U6 ODH III 2/6 0.33 ODH APi5^0 3/12 O .25 ODH I k/2k 0.17 CDC 2V 3/9 0.33 ODH 801 3/15 0.20 ODH II H/8 0.50 CDC DIO 5/12.5 O.HO ODH APi500 10/25 o.Ho OSU M.M.I 10/18 0.56 109 TABLE 3k Statistical Analysis of Peak Height Ratios of Lactic & Succinic Acid Salmonella typhimurium Salmonella typhi 1.36 0»46 2.73 0.33 1.49 0.25 0.87 0.17 1.33 O .33 0.92 0.20 1.53 0.50 O .63 0.40 1.00 0.40 2.73 ' 0.56 N = 10.000 N = 10.000 M = 1.459 M = 0.360 SB = 0.730 SB = 0.128 SE = 0.231 SE = 0.041 T = 4.689 (18) P = <0.0005 110 was employed as the growth medium and proved to be useful as representa tives of both genera grew well and significant differences in metabolic products could be observed after analysis. The stationary phase used was F F A .P . After 24 hours (Tables 11 and 12), the difference in peak height ratios was sufficiently significant so that this incubation period was used for examination of the test cultures, the results may be seen in Figures 63, 64 and 65, 66 at 24 and 48 hours, respectively, of SL epi- dermidis 550 B:4 and Micrococcus luteus I, Table 35 lists the peak height ratios of lactic/succinic acid pro duced in YG and shows the ratio ranges of 1.14-2.67 for Micrococcus species, and 8.40 to 38.33 for Staphylococcus species. Statistically (Table 36), the results illustrate the high level of reliability that the peak height ratio analysis provides in differentiating and character izing these two genera. The analysis is rapid and offers a biochemical differentiation in the short time of 24 hours, as seen in the results. In contrasting S^. aureus and _S. epidermidis, it is acknowledged that Sh aureus is a clinically important organism responsible for a variety of disease entities, while _S. epidermidis usually causes a more modest skin problem, such as a post-surgical abscesses (24). The basic tests which separate these species are coagulase production and mannitol fermentation (20), as seen in Table 37, Chromatograms of products at 24 and 48 hours incubation are shown in Figures 67, 69 and 68, 70, re spectively. The production of lactic acid is seen as the differentiating factor. The medium used was YM, and the organisms were incubated under anaerobic conditions. The basis of this test was the anaerobic 1 1 1 Lactic / 2 U 6 8 10 12 lfc 16 18 Time(Min) Fig. 63 Staphylococcus epidermidis 550 h:U at 2k hours vith lactic and succinic acids. CO C o & 0) P5 o •p v Time (Min) Fig. 6U Staphylococcus epidermidis 550 b:U at U8 hours •with lactic and succinic acids. 1 1 2 Lactic o> CO o c & O) K u o •p a a> •P Succinic 0) « 2 b 6 8 10 12 3.6 18 20 Time (ML n) Fig. 65 Micrococcus luteus I at 2h hours with lactic and succinic acids. Lactic 4) CO c M8. 2 h 6 8 10 12 lU 16 18 Time(Min) Fig. 6 6 Microcoecus luteus I at U8 hours with lactic and succinic acids. 113 v n c o & cj « ft o Lactic -p o 0) Succinic •p0) n Time(Min) Fig. 67 Staphylococcus epidermidis 721 at 2U hours ■with lactic and succinic acids. 0 w c o (0p< 0 K Lactic o 0) Succinic ■p 2 U 6 8 10 12 3 U 16 18 Time(Min) Fig. 6 8 Staphylococcus epidermidis 721 at U8 hours vith lactic and succinic acids Lactic &a> K ft su « Succinic ■p Time (Min) Fig- 69 Staphylococcus aureus 717 at 2 ^ hours vith lactic and succinic acids. Lactic Time(MLn) Fig. 70 Staphylococcus aureus 717 at U8 hours ■with lactic and succinic acids. TABLE 35 Lactic & Succinic Acid Peak Height Ratios of Staphylococcus sp. Micrococcus sp. Test Cultures Staphylococcus sp.: Lactic/Succinic (mm) Ratio OSU S. aureus (Camp) 6UA-5 1U.20 OSU S . aureus 502A 67/8 8.38 ATCC S.aureus 25923 115/3 38.33 CDC S.aureus 29 b2/5 8.U0 CDC S.aureus 55 V7/5.5 8.55 ATCC S.epidermidis 27626 58/2.5 23.20 ODH S .epidermidis 551B2 25/1.5 16.67 CDC S .epidermidis B3 88/5 17.60 OSU S.epidermidis B1 52/6 8.67 ODH S. epidermidis 550Blt- 96/6 16.00 Mierococcus sp.: ODH M.luteus III 5/2.5 2.00 OSU M. luteus I 5/2 2.50 ATCC M. luteus U698 5/2 2.50 ODH M. roseus I 5/3.5 1.U3 ODH M.roseus II 5/1.5 3.30 ODH M. species I V 3 . 5 1.1k ODH M. species 107 k/2 2.00 ODH M. species 513G 5/3 1.67 ODH M.cryophilus I 5/1.5 3.33 ODH M. cryophilus 513C k/l.5 2.67 1 1 6 TABLE 36 Statistical Analysis of ^Lactic & Succinic Acid Peak''Height Ratios Staphylococcus species vs. -.Micrococcus species L/S L/S 14.2 0 - 2.00 8.38 2.50 38.33 2.50 8.40 1.43 8.55 3.30 23.20 1.14 16.67 2.00 17.60 1.67 8.67 3.33 16.00 iiil N = 10.0000 N = 10.0000 M = 16.000 M = 2.254 SP = 9.320 SP = 0.741 SEM = 2.947 SEM = 0.234 P = <0.0005 T = 4.6493 (18) 117 TABLE 37 Biochemical Characterization of Staphylococcus aureus & Staphylococcus epidermidis Isolates S. aureus: Gram + Cocci Coagulase Anaerobic Mannitol Dnase ATCC £5923 + +' + ODH 7k0 + + + CDC 29 + + + + ODH PEACH + + + + ODH 733 + + + + ODH 1558 + + + + ODH 717 + + + + CDC hi . + + + + CDC Dll + + + + CDC 75 + + S. epidermidis: ATCC 27626 + ODH 721 + ODH 51^ B:2 + ODH 581 + OSU OSU B:1 + ODH 1071 + ODH 1079 + OSU G.T. + ODH 595 + CDC CDC B: 3 + utilization of mannitol by S^. aureus and the lack of utilization by _S. epidermidis (24). The results of production of lactic and succinic acid and the differences between the species are easily seen in the chromatograms. Table 11 and 12 list the plate counts and product peak heights at 24 and 48 hours incubation of the representative cultures S*. epidermidis 721 and S. aureus 717, respectively, after incubation in the YM medium. Table 38 shows the peak height ratios of the isolates examined. The ratios ranged from 1.5 to 9.0 for _S. aureus and from 0.2 to 1.0 for S. epidermidis. Based on peak heights at 24 and 48 hours, 24 hours was chosen as the optimum test parameter. Here the large difference between the quantities of lactic acid can be seen. Separation of the species was effected by comparing the lactic/succinic acid products from the YM medium. The use of the YG medium also was attempted, but no clear distinc tion between the species was noted. The stationary phase used was F F A The statistical analysis in Table 39 indicates the level of signif icance of the differences between the peak height ratios of the species. Streptococcus species and Staphylococcus species, both being gram positive cocci and facultative-anaerobes, can be incorrectly identified. And with rare biochemical differences these genera can be confused be cause of the number of strains encountered with variations in testing results. The usually dependable mannitol, deoxyribonuclease, catalase, and pigment production tests often are variable as to the species. These two genera are important in clinical infections, necessitating a rapid, accurate identification to facilitate early and propitious treatment of the disease (39), (69), (119), The biochemical tests 119 TABLE 38 Lactic & Succinic Acid Peak Height Ratios of Staphylococcus aureus & Staphylococcus epidermidis Test Cultures S. aureus: Lactic/Succinic (mm) Ratio ATCC 25923 1+1/5 8.20 ODH 7k0 9/1+ 2.25 CDC 29 7.5/5 1.50 ODH Peach 10/h 2.50 ODH 733 10/1+ 2.50 ODH 1558 36/1+ 9.00 ODH 717 25/5 5.00 CDC .1+7 16/1+ 1+.00 CDC Dll . 20/6 3.33 CDC 75 22/1+ 5.50 S. epidermidis: ATCC 27626 3/3 1.00 ODH 721 3/6 0.50 ODH 511+B2 2/1+.5 0.1+1+ ODH 581 2/3 0.67 OSU B1 1/5 0.20 ODH 1071 2/1+ 0.50 ODH 1079 1/U 0.25 OSU G.T. 1/1+ 0.25 ODH 595 2/1+ 0.50 CDC B3 2/1+ 0.50 1 2 0 TABLE 39 Statistical Analysis of Lactic & Succinic Acid. Peak Height Ratios Staphylococcus aureus Staphylococcus epidermidlB L/S LZ§ 8.20 1.00 2.25 °«50 1.50 0.44 2.50 0.67 2.50 0.20 9.00 0.50 5.00 0.25 4.00 0.25 3.53 0.50 5.50 0 ^ 0 N = 10.0000 N = 10.0000 M = 4.378 M = 0.481 SL = 2.554 SD = 0.254 SEM = 0.808 SEM = 0.074 P = <0.0005 T = 4.8050 (18) 121 utilized in the confirmation of the isolates are shown in Table 40. The similarity of biochemical characteristics can readily be seen. The cat- alase test is the standard procedure to categorize a gram positive, coccus shaped bacterium (38) . The possibility of a variable catalase test is seen with the other standard testing parameters. This test is employed in separating the genera, but positive reactions have been noted with the normally negative streptococci (20). Both of the genera ex amined have been reported in cases of septicemia, pneumonia, meningitis, septic throat, and inner ear infections (24), (38). Tables 11 and 12 list the plate counts and peak heights at 24 and 48 hours, respectively, for the representative cultures Streptococcus pyogenes and _S. epidermidis 511 B:2. The medium employed in this study was YT. No satisfactory chromatographic differences between the test cultures were observed by using the YG medium. After 24 hours incuba tion, sufficient metabolic products were measured. Figures 71, 73 and 72, 74 are chromatograms of the representative cultures at the 24 and 48 hour test periods. In Table 41 are shown the ratio ranges of Strep tococcus species at 0.25 to 6.00 and Staphylococcus species at 6.50 to 36.00. The statistical analysis in Table 42 shows the significance level for the differences between the peak heigh ratios of the genera examined. The stationary phase used was F F A P. These ratio differences were of a level which permitted a rapid and reliable differentiation of the genera under study. The genera are often difficult to characterize because of the variant strains that appear in each genus, and attempts at a rapid identification may or may not prove successful (39), 122 i K BV V •pa> Succinic P Time (Min) Fig. 71 Streptococcus pyogenes at 2U hours with lactic and succinic acids. Lactic a0) c o & K u o ■pc 0) -p 0) p Succinic Time(Min) Fig. 72 Streptococcus pyogenes at U8 hours with lactic and succinic acids. 123 Lactic v K U $ V 0) ■p 20 Fig. 73 Staphylococcus epidermidis 511 h:2 at 2k hours with lactic and succinic acids. Lactic 0) ta c o & 0) K (h O ■P V 0) Time(Min) Fig. ?k Staphylococcus epidermidis 511 h:2 at 1*8 hours with lactic and succinic acids. 12k TABLE 1+0 Biochemical Characteristics of Staphylococcus sp. & Streptococcus sp. Test Cultures Streptococcus sp.: Catalase Lancefield Gram + Cocci Coagulase ODH S.pneumoniae. - + ODH jS. faecalis ± D + ODH _S. salivarius - + ODH S. MG — — 4* — ODH J3_. agalactiae B + ODH S^. anginosus - + CDC £!. bovis D + CDC _S. equisimilis C + OSU jS. pyogenes A + ODH S. mutans - + Staphylococcus sp.: OSU _S. aureus (Camp) + ODH _S. epidermidis 511B2 + + ATCC _S. aureus 25923 + + ATCC _S. epidermidis 27626 + + ODH _S. epidermidis B1 + + CDC _S. aureus l8H + + + CDC _S^ aureus 80 + + + CDC J>. aureus 71 + + + ODH _S. epidermidis 550B1+ + + OSU S. epidermidis B1 + + TABLE kl Lactic & Succinic Acid Peak Height Ratios of Staphylococcus & Streptococcus sp. Test Cultures Streptococcus sp.: Lactic/Succinic (mm) Ratio ODH S. pneumoniae l/3 0.33 ODH S. faecalis 18/3 6.00 ODH S. salivarius 2/3 0.67 ODH S. MG l/k 0.25 ODH S.agalactiae 11/3*5 3.1^ ODH £>. anginosus 1/3-5 0.29 CDC S.bovis 2/3.5 0.57 ODH S. equisimilis l/2 0.50 OSU S. pyogenes V 2*5 1 .6 0 ODH S. mutans 2/2 1.00 Staphylococcus sp.: OSU S . aureus (Camp) 26/k 6.50 ODH S.epidermidis 511B2 27/2.5 10.80 ATCC _S. aureus 25923 ^0/2 20.00 ATCC S_. epidermidis 27626 29/2 11+.50 ODH _S. epidermidis B1 72/2 36.00 CDC S. aureus 18h 36/U 9-00 CDC S. aureus 80 3 5 A 8.75 CDC S. aureus 71 ko/k.5 8 .8 8 ODH S.epidermidis 550BU 31A 7.75 OSU S. epidermidis B1 72/5 lU.UO 1 2 6 TABLE 42 Statistical Analysis of Lactic & Succinic Acid Peak Height Ratios Streptococcus species vs. Staphylococcus species l/S L/S 0.53 6.50 6.00 10.80 0 .6? 20.00 0.25 14.50 ' 3.14 36.00 O .29 9.00 0.57 8.75 0.50 8.88 1.60 7.75 1.00 14.40 N = 10.000 N = 10.0000 V = 1.435 M = 13.658 SL = 1.829 SD = 8.842 SEM = 0.578 .SEM = 2.796 T = -4.2808 (18) P = < 0.0005 DISCUSSION Gas-liquid chromatographic analysis of bacterial products can be useful in the chracterization and separation of biochemically similar bacterial species or groups. Modifications of existing chromatographic techniques can enable microbiologists to apply these procedures to study the metabolites of aerobic and facultative-anaerobic bacteria. The growth medium is important since appropriate altering of the medium, such as the carbohydrate portion, permits the study of a wide range of bacteria that differ in their metabolic requirements. The peak height ratios reported here were found to be distinctive according to the species or group studied and could be utilized in com parison of closely related bacteria, resulting in their differentiation by these biochemical differences. The established use of volatile and non-volatile organic acid products in the characterization of anaerobic bacteria, yeasts, and viruses (59), (63), (76), prompted the investigation of products from aerobes and facultative-anaerobes with the examination of peak height ratios of these products. Certain gram positive and gram negative bacteria were employed to examine the possibility of discovering signif icant biochemical differences. The presence of metabolic products in varied quantities of non volatile versts volatile organic acids led to further investigation of 127 128 these metabolites of diverse groups of clinically significant bacteria. The volatile products present in the test cultures offered little promise as useful components in the chromatographic analysis of bacterial cultures. The non-volatile products, namely lactic anc succinic acid, were methylated and converted into the corresponding esters of the organic acids, methyl-lactate and methyl-succinate. The accepted method of Holde- man and Moore (52) to methylate the test cultures was used in the investi gations. The peak height ratios calculated from millimeter measurements of individual peak heights were logged for each culture in the comparisons. In order to discover if differences in metabolic products exist, a bacterial group that produces varied and measurable quantities of prod ucts was needed so selected Bacillus species were chosen for preliminary studies because of their wide range of biochemical processes (44). This genus employed in the preliminary investigations provided a working model for further studies with other diverse groups of aerobic and facultative-anaerobic bacteria. Different stationary phases were examined in order to evaluate them for the identification of metabolic products. These stationary phases have multiple uses in the characterization of organic compounds (114), and so it seemed appropriate to examine them for possible application to identification of bacterial products, Included in the testing of selected stationary phases were Carbowax 1500, OV-1, and 0V-17, and were found to be inadequate for testing be cause of their chemical natures. Carbowax 1500 is a polyethylene-glycol, OV-1 a dimethyl silicone gum, and OV-17 a phenyl-methyl silicone gum (114), and all being of low polarity, preclude the solution and 129 separation of highly polar organic acids such as lactic and succinic, for example. The polar stationary phases such as D E G A , Lacl-R-296, SP 1000, SP 1220, F F A P , and SP 1200 are capable of adequately separating or ganic acids. These phases performed well over a range of temperatures, and proved to be valuable in separating organic acid products found in bacterial test cultures. The PYG medium, having proved unreliable by containing organic acids, was altered by removing the peptone and adding a salt solution in order to promote bacterial growth (56). The new uninoculated medium exhibited no organic acid peaks, thus it was employed in all analysis experiments with selected modification in a few studies. For exmaple, the carbohydrate portion of YG medium was changed to rhamnose, in one experiment, and for selective test purposes NaCl was added to the medium. The comparison of methylation procedures involving l^SCH and BF^ with methanol displayed little difference in each set of results. Both proved satisfactory for the testing of organic compounds. The procedures were contrasted and the resultant organic acid methyl esters were chrom atographed to compare with the pure commercial esterified organic acid. In this instance, the lactic acid methyl ester served as the indicator to test the recovery of esterified organic products. From the results of this experiment, it was apparent that almost equal quantities were recovered, but both were less than the 100% yield anticipated. This discrepancy could be explained since all the lactic acid is not always converted to methyl-lactate because of variations in temperature, mixing of reactants in the tubes, and also the purity of 130 the reactants involved. Moreover, the reaction is reversible and water must be added to force the reaction toward the methyl-lactate product, while sufficient time must be allowed for the reaction to go to comple tion. The extraction step of the derivatized methyl esters with CHCl^ is critical in the comparison of bacterial product ratios. It was observed that the peak height ratios of some of the products could be altered if the bacterial culture was not analyzed shortly after extraction. The experiment involving extended periods of delaying the extraction of the cultures proved the optimal time period to be shortly after addition of the CHClg. The increase of succinic acid in these test cultures has made necessary the extraction of a bacterial culture within a few hours. The quantity of CHCl^ is important in the extraction as too much dilutes the products, and too little is difficult to manipulate in the procedure. The minimum amount of CHCl^ capable of extracting the or ganic acid components was 0.5 ml. Any less and the products could be lost on the surfaces of the test tubes or in the drying agents, such as MgS04, which is used frequently. The extraction of organic acid methyl esters requires the optimal amount of CHCl^ to be employed, and this step must be accomplished in a short period of time for successful results. In determining the usefulness of an external methyl ester standard or an internal standard for identification of products, the comparison of the two proved the external standard to be a reliable research tool. The benzoic acid test was not successful because of some as yet uniden tified aspects involved in the procedure. These results led to the 131 abandoning of the internal standard for use as an identification aid. Meanwhile, the use of an external standard has proven reliable for or ganic acid identification (52), (78). If the chromatograph is used daily, it is seldom necessary to use the standard mixture more than once weekly (78). The groups of bacteria compared for their close biochemical char acters included selected aerobic and facultative-anaerobic gram posi tive rods, gram negative rods, and gram positive cocci; important in clinical microbiology. The non-volatile metabolic products, namely lactic and succinic acids, were detected and identified in the bacterial cultures. These were detected in a newly devised basic medium, YG. This medium was altered in order to separate selected groups of bacteria. The biochem ical, morphological, and environmental similarities among members of the genus Bacillus provided an opportunity to test the procedure to peak height ratio characterization. The lactic/succinic acid ratios of the three species examined were each grouped separately, with a median of 6.82 for B_. anthracis, 4.73 for J3. subtil is, and 18.16 for B_. cereus. This difference was expected as different species should pro duce different proportions of metabolic products or possibly even dif ferent products. The significant difference between these product ratios allows not only a separation of the closely related species, but also offers a basis for biochemical characterization of each species in the studies. 132 Bacillus species and Lactobacillus species are often confused be cause of the cellular and colonial morphology of both genera and numer ous biochemical similarities (119). Difficulties in separation of the genera prompted the investigation into the basic biochemical character of the bacteria. The limitations of conventional test parameters are numerous. The calculation of peak height ratios of the major non volatile products provided a basis for chracterization and identifica tion as to the appropriate genus in which to place a specific bacterium. Thus, product ratios of various Bacillus species were compared to those of selected Lactobacillus species, and offered evidence that shows a wide separation of the two genera. The non-utilization of lac tate by the Lactobacillus is shown by large quantities of this product present upon analysis. This is not the case in the Bacillus species test results. Here exists a rapid separatory procedure to differentiate between these similar bacteria. The facultative gram negative rods, especially the enterics pose a difficult problem in their characterization by gas- liquid chromatographic analysis because of their similar biochemical characters. The species within the enteric group all utilize glucose to a great extent (34), and thus metabolic products would be expected to be similar, if not identical. This presumption was true, but the peak height ratios were distinctive, permitting separation of the closely related species examined in the chromatographic analyses. Different genera were selected, as well as species, often confused with each other to investigate the possibility of chracterization by peak height ratio comparison. It was found that the products are of 133 the same general types, namely lactic and succinic acids. There are limited differences between the general biochemically (34), and, in the separation of V. cholerae and the non-cholera vibrio group, no biochemical differences exist. The peak height ratios of the products provide the basic separation schema. Between the non-motile _S. gall inarum and S_. pullorum the production of succinic acid by S^. gall inarum is the differential factor. The categorization of EL typhi and S^. typhimurium into two separate species can be performed by anal ysis of peak height product ratios, as in V. cholerae/non-cholera vibrio and E. coli/E. agglomerans comparisons. The EL coli isolates could be separated easily from the EL agglomerans isolates within ap proximately 48 hours, for example. Among the E. agglomerans isolates were two separate groups based on peak height ratios of products, and this separation was substan tiated by biochemical tests. The characterization of these cultures was accomplished by analysis of basic biochemical metabolites and has not been reported previously. The chracterization of products was successfully performed in a relatively short period of time. The rapid separation of the species with a high level of accuracy shows the value of a chromatographic an alysis. The vibrios were examined biochemically and found to be identical within the existing test parameters. The separation of the non-cholera vibrios from V. cholerae is based on serological differences only. The chromatographic analysis provides a relatively rapid chracterization of both groups of organisms. This analysis also provides valuable 134 data as to the basic biochemical character of each group. The problems of separating and identifying the individual gram positive cocci are multiple in that the cellular and colonial morphol ogy are similar, along with pigment production, most growth character istics, and the ability to utilize many of the same substrates as car bon and energy sources are similar (119). Hypothetically, the metabolic products from bacteria with similar biochemical pathways should be the same or very similar. Again, this was found to be true with additional data as to the unique proportions of these products according to the individual genus or species. Various groups of clinically important gram positive cocci, in cluding aerobic micrococci and the facultative streptococci and staphyl ococci, are characterized by a few biochemical test parameters, and a rapid categorization is not always possible. The chromatographic analy sis offers a rapid, basic biochemical characterization of these major genera. The capability of producing metabolites in various proportions by each group of cocci allows a separation from other related bacteria. The micrococci that are often confused with the staphylococci are segregated by virtue of a different peak height ratio of lactic/succinic acid. The comparison is valid for _S. aureus and S_. epidermidis isolates and the differentiation of the staphylococci from the streptococci. The aerobic micrococci seemingly oxidize the intermediate products more completely than the staphylococci, as the quantity of lactic acid is lower. The peak heights of lactic acid in the culture of staphylo cocci are very high in comparison to the micrococci. The difference produces a marked separation of the two groups. 135 The peak height ratios of products from S_. aureus and S_. epidermidis isolates are vastly different, reflecting a major biochemical difference between the species. These chromatographic differences portray a basic identity of each species. The characterization of the streptococci as compared to the staph ylococci shows a similar biochemical identity, but a low tolerance to NaCl. This fact allows the separation of these genera since even Streptococcus species that tolerate moderately high NaCl levels do not produce any organic acid peak heights as large as those of Staphylococcus species examined. The analyses performed on various groups of bacteria apparently can be useful in categorizing and characterizing a wide variety of bacteria. These chromatographic characterizations of selected closely re lated and sometimes inseparable bacterial groups are rapid diagnostic procedures amenable to use with a wide variety of aerobic and facultat- tive-anaerobic microorganisms. The basic experimental procedures pro duced reliable results that are statistically significant in bacterial characterizations. These groups, genera, and species selected for separation and identification by chromatographic analysis of metabolites were chosen because of their importance in clinical microbiology, the minimal bio chemical procedures that effectively separate these closely related bacteria, and the need for basic biochemical characterization of each of the bacteria studied. To date, this has not been attempted using chromatographic analy sis of peak height ratios of the intermediate metabolic products. These experiment results prove the usefulness of gas-liquid chromatographic analysis of product peak height ratios in separating and identifying biochemically similar bacteria. SUMMARY The technique of comparing peak height ratios of metabolic products is useful in the characterization of various bacteria. The procedure is amenable to the study of non-volatile metabolic organic acids pro duced by various bacteria, gram positive and gram negative, which have not been previously examined utilizing these chromatographic analytical parameters. This method of gas-liquid chromatographic analysis is ap plicable to the characterization of different groups of bacteria by al tering the carbohydrate component of the growth medium thus allowing a chromatographic separation of biochemically similar organisms, specifi cally aerobes and facultative anaerobes, by the study of their inter mediate metabolic organic acid products. The incubation is important in the characterization of these selected bacteria as the incubation that allows production of maximal quantities of products in the shortest time is desired for analysis. Altering the growth medium selectivity, such as variations in salt content, can make this technique useful in differentiating groups of similar bacteria. Utilizing the basic medium, YG, the capability exists for calculating peak height ratios of different organic acid products found in bacterial cultures because of the absence of these products in the uninoculated medium. 137 138 Among the bacteria that can be differentiated with this procedure are _B. cereus, _B. anthracis, and B>. subtilis. These often confused species produced significantly different product ratios, grown in YG medium, allowing their separation into appropriate taxonomic groups. Bacillus species and Lactobacillus species are separated by dif ferences in lactic acid production which can be seen in lactic/succinic acid product ratios after incubation in the YG medium. Other gram positive bacteria such as the micrococci and staphylo cocci can be separated by ratios of products found in cultures of the growth medium. At the species level S_. aureus and S^. epidermidis, grown in YM medium, are separated by product ratios. Selected staphylococcal and streptococcal species grown in YT medium were differentiated by product ratios and placed into the appro priate genus. Various gram negative bacteria, including enteric species, were found to produce organic acids permitting their placement into differ ent groups. E. coli/E. agglomerans and V. cholerae/non-cholera vibrios were separated by peak height ratios. _S. gall inarum and _S. pullorum were contrasted by the production or lack of production of succinic acid. S.. pullorum isolates failed to produce this organic acid when grown in YD medium, while SL gallinarum isolates all showed production of the acid. S. typhi and S_. typhimurium, grown in YR medium, displayed ade quate differences in product ratios for reliable separation. These pairings of bacteria were chosen because of the lack of rapid and reliable biochemical testing procedures available. 139 The experimental data illustrate that the incubation period of each bacterial group, the type of organic products to be examined, the amount of CHCl^ used in extraction of organic products, the time period after extraction until chromatographing the sample, the method of methylating samples, the type of chromatographic stationary phase, and the external standard employed are all important factors that must be taken into consideration when performing these chromatographic investi gations . BIBLIOGRAPHY 1. Abel, K. , Deschmertzing, E., and Peterson, J. 1963* Classification of Microorganisms by Analysis of Chemical Components. I. Feasibility of Using Gas Chromatography. J. Bacterid. 8 5:1039-10^ 4.. 2. Alexander, G., Garzo, G., and Pali, G. 197k Methods for Preparing Gas Chromtographic Columns for Gas. Chromatography. J.. Chromat. 91:25-37. 3. Alley, Co, Brooks, J . , and Kellogg, D. 1979- Electron Capture Gas Liq uid Chromatography Identification of Acids Produced by Neisseria men- ingiditis in a. Defined Medina. J. Clin. Microbiol. 9:97-102. k. Anema, P.J., Eboiman, W.J., and Geers, J.M. 1973* Volatile Acid Prod uction of Clostridium suorogenes Under Controlled Culture Condit ions. J. Appl. Bacterial. 3 0:6 8 3-6 8 7. 5. Anhalt, J.P., and Moyer, T.P. 1980. Th& Bole of Gas Liquid Chromat ography and Liquid Chromatography in Theraputic Drug Monitering. Lab. Med. H : 143-^9* 6. Association of Official Analytical Chemists-Methods of Analysis. 1930. 13th. Ed. Washington, D.C. 7. Bacteriological Analytical Manual. 1978. 5th. Ed. Food and Drug Admin istrations. Washington, D.C. 8. Balows, A., Herman, G., and Dewitt, W. 1972. The Isolation and Ident ification of Vibrio cholerae. D.H.E.W. C.D..C. Atlanta, Ga. 9. Balzevic, D. and Edener, G. 1975. Principles of Biochemical Tests in Diagnostic Microbiology. Wiley Interscience. New York, N.Y. 10. Baumann, F., and Hadden, N. 1971. Basie Liquid Chromatography. 1st. .Ed. Varian Aerograph. Walnut Creek, Cal. 11. Blakesley, C., and Torline, P. 1975* Preparation of Stable Gas Chrom atographic Columns'. with Polar Stationary Haases. J. Chromst.105:35-7. 12. Brooks, J.B. 1975* Identification of Disease and Disease Agents by Analysis of Spent Culture Media and Body Fluids with Electron Capt ure Gas Liquid Chromatography. In D. Schleasinger (Ed.) Microbiol- ology. A.S.M. Washington, D.C. Ik) Ibl 13- Brooks, J.B., Alley, C.C., Heaver, J.W., Green, V„E., and Harkness, A.M. 1973. Practical Methodology for Barivitizing and Analyzing Bacterial Metabolites ■with a Modified Automatic Injector and Gas Chromatograph. Anal. Chem. U5:2083-2087. lk. Brooks, J.B., Cherry, W.B., Thacker, L . , and Alley, C.C. 1972. Anal ysis by Gas Chromatography of Amines and Mtrossa&nes Produced In- vivo and In-vitro by Proteus mirabilis. J. Infect. Dis. 126:lk3-153« 15. Brooks, J.B., Choudhary, G., Craven, R.B., Edman, B.C., Alley, C.C., and Liddle, J.A. 1977. Electron Capture Gas Chromatographic Detect ion and Identification of 3-(2*- ketohexyl) indoline in Spinal Fluid of Patients with Tubercular meningitis. J. Clin. Microbiol. 5:625-628. 16. Brooks, J.B., Kellogg, D.S., Alley, C.C., Short, H.B., Handsfield, H.H. and Huff, B. 197k. Gas Chromatography as a Potential Means of Diagnosing Arthritis. I. Differentiation Between Staphylococcal, Streptococcal, Gonococcal, and Traumatic Arthritis. J. Infect. Dis. 1 2 9 ‘6 6 0-6 6 8. 17- Brooks, J.B., Kellogg, D.S., Thacker, L . , and Turner, E.M. 1972. Analysis by Gas Chromatography of Fatty Acids Found in Whole Cult ure Extracts of Heisseria Sp. Can. J. Microbiol. 18:157-168. 18. Brooks, J.B., Moore, W.E.C. 1969- Gas Chromatographic Analysis of Amines and Other Compounds Produced by Several Species of Clost ridia. Can. J. Microbiol. 15:lk33-lkk7. 19- Brooks, J.B., Moss, C.W., and Dowell, V.R. 1 9 6 9. Differentiation Between Clostridium sordelli and Clostridium bifermentans by Gas Chromatography. J.Bacterio!. 100:525-530- 20. Buchanan, R.E., and Gibbons, H.E. 197k. (Co-odltors) Bergey's Man ual of Determinative Bacteriology. 8th. Ed. Williams and Wilkins Co. Baltimore, Md. 21. CarIsson, J. 1973- Simplified Gas Chromatographic Procedure for the Identification of Bacterial Metabolic Products. Appl. Micror biol. 25:287-289. 22. Christensen, M.D., Albury, M.H., and Pederson, C.S. 1958. Variat ion in Acetic Acid /Lactic Acid Ratios Among Lactic Acid Bacter ia. Appl. Microbiol. 6:316-3 1 8. 23- Carpenter, K.P. 1 9 6 6. Isolation and Identification of Cholera Vibrios. Monthly Bull. Minister of Health W.H.O. 25:58-63. 2k. Cohen, J.(Editor) 1972. The Staphylococci. 1st. Ed. Wiley Inter- science. New York, U.Y. 25. Cooper, M.J. and Anders, M.W. 1975- H.P.L.C. of Fatty Acids and Lipids. J. Chromat. 13:^07-^11. lk2 26. Craven, R.B., Brooks, J.B., Edman, B.C., Converse, J.B., Greenlee, J., Schlossberg, B., Furlow, T . , Gwaltney Jr., J.M., and Miner, W.F. 1977- Rapid Biagnosis of Lymphocytic Meningitis by Frequency Pulsed Electron Capture Gas Liquid Chromatography: Bifferentiat- ion of Tubercular, Cryptoeoccal, and Viral Meningitis. J. Clin. Microbiol.■6:27-32. 27. Bignostic Procedures. 1970. 5th. Ed. American Public Health Assoc iation. Hew York, H.Y- 28. Bistler, W. 1 9 7 8. Separation of Lactic, Glyeaeylic, Formic, Acetic, and Proorionic Acids by Reverse Phase H.P.l.C. J. Chromat. 152: 250-252^ 29. Brucker, B.B. 1969- Analysis of Lactic Acid and Volatile Fatty Acids Exrtacted from Bacterial Fermentations. J. Chromat. Sci. 8:k8 9-k9 0. 30. Brucker, B.3., Coilard, P.J., Bostock, A., and Chapman, K.B. 197k. Influence of Growth Conditions on Formic/Acetic Acid Ratio in Escherichia coli and Streptococcus faeealis. Microbios 106:39-kk. 31. Byer, B.C. 1917. A Hew Method of Steam Bistillation for the Det ermination of the Volatile Fatty Acids Including a Series of Coloraetric Quantitative Reactions for Their Identification. J. Biol. Che®. 28:W+5-k72. 32. Sdberg, S.C. 1 980. Gas Liquid Chromatography in Microbiology. Laboratory Management. 6:31-1+3. 33. Edwards, P.R. 1962. Serological Examination of Salmonella Cult ures for Epidemiology Purposes. B.H.E.W. C.B.C. Atlanta, Ga. 3k. Edwards, P.R., and Ewing, W.H. 1972. Identification of Entero- bacteriaceae. 3fd. Ed. 3urgess Co. Minneapolis, Minn. 35. Ewing, W.E., Bavis, B.R., and Martin, W.J. 1972. Escherichia coli. B.H.E.W. C.B.C. Atlanta, Ga. 36. Ewings W.H., and Fife, M.A. 1972. Snterobacter agglomerans. Int. J. Syst. Bacteriol. 22:k-ll. 37. Ewing, W.H., and Martin, W.J. 197k. Enterobscteriaceae. In Manual of Clinical Microbiology. 3?d. Ed. A.S.M. Washington,B.B. 3 8. Facklam, R.R. 1976. Isolation and Identification of Streptococci. Critical Rev. in din. Lab. Sci. 6:287-317. 39. Facklam, R.R. 1977. Viridans Streptococci. J. din. Microbiol. 5:l8k-201. ko. Finkelstein, R.A. 1973- Cholera. Crit. Rev. Microbiol. 2:553-623- Iks 4l. Fisher-Hoch, S., Hudson, M. J. , and Thompson, M.H. 1979* Identificat ion of a Clinical Isolate as Legionella pneumophila by Gas Chromat ography and Mass Spectroscopy of Cellular Fatty Acids. Lancet. Vol. n : 4 82-556. kZ. Garrett, H.E. 1972. Elementary Statistics. 2nd. Ed. D. McKay Co. Publ. Hew York, N.Y. 4 3. Gorbach, S.L., Mayhew, J.W., Bartlett, J.G., Thadepalli, H., and Onderdank, A.W. 1973. Rapid Diagnosis of Anaerobic Infections by Direct Gas Liquid Chromatography of Clinical Specimens. J. Clin. Invest. 57:4?8=484. 44. Gordon, R.E., Haynes, W.C., and Pang, C.H. 1973* The.Genus Bacillus. Agriculture Handbook #427. U.S.D.A. Washington, D.C. 4 5. Grey, T.C.and .Steyens, B.J. 1 9 6 6. Analysis of the Lower Free Fatty Acids from Microbial Fermentations. Anal. Chem. 38:724-729- 46. Gross, J., and Porter, L. 1976. Automatic Reduction of Multicolumn Chromatographic Data. J. Forensic Sci. 22:70-74. 47. Haddadin, J.M., Stirland, R.M., Preston, H.W., and Coilard, P. 1973- Identification of Vibrio cholerae by Pyrolysis-Gas Liquid Chromat ography. Appl. Microbiol. 25:40-43. 48. Henis, Y., Gould, J.R., and Alexander, M. 1 9 6 6. Detection and Ident ification of Bacteria by Gas Chromatography. Appl. Micrcfoiol.l4: 51-6 4 9. Hickman, F.W., and Farmer, J . J . m . 1978. Salmonella typhi : Ident ification, Antibiograas, Serology, and Bacteriophage Typing. Am. J. Med. Tech. 44:1149=2259- 50. Hickman, F.W., Farmer, J.J.III, Steigcrwalt, A.G., and Brenner, D..1 9 SO.Morganella raorganii.' J. Clin. Microbiol. 2:88-94. 51. Hill, A.B. 1966. Principles of Medical Statistics. 8th. Ed. Oxford University Press. New York, N.Y. 52. Holdeman, L.V., and Moore, W.E.C. 1972. Anaerobe Laboratory Man ual. Anaerobe Laboratory, V.P.I. Blacksburg, Va. 53* Isherwood, F.A., and Hanes, C.S. 1953- Separation Testing of Organ ic Acids on Paper Chromatograms. J. Biochea. 55:824-830. 54. Jamas, A.T. i9 6 0. Qualitative and Quantitative Determination of Fatty Acids by Gas Liquid Chromatography. Methods Biochea. Anal. 8:1-59- 55. James, A.T., and Martin, A.J.P. 1952. Gas Liquid Partition Chrom atography: The Separation and Micro-Estimation of Volatile Fatty Acids from Formic to Dodecanoic Acid. J.Biochea. 50:679-690. Ikk 5 6. Kaneda, T. 1 9 6 2. Isolation and Identification of Fatty Acids From Bacillus subtilis.-J. Biol. Chem. 238:1222-1228. 57. Kaneda, T. 1967. Fatty Acids in the Genus Bacillus. J, Bacterid. 93: 89U-CC3 . 5 8. Kennedy, E.P., and Barker, H.A. 1951. Paper Chromatography of Volatile Acids. Anal. Chem. 23:1033-103^. 59* Kiehn, T.E., Bernard, E.M., Gold, J.W.M., and Armstrong, S.D. 1979. Candidiasis: Detection by Gas Liquid Chromatography of D-Arabinitol, a Fungal Metabolite, in Human Serum. Science. 206:979-986. 60. Kineda, T. 1977. Fatty Acids of the Genus Bacillus: An Example of Branched Chain Preference. Bacteriol. Reviews. 41:391-^18. 61. Kineshiro, T . , and Marr, A.G. 1961. Cis 9>10 Methylene Ioxadecanoic Acid from the Phospholipids of Escherichia coli. J. Biol. Chem. 236:2615-2619. 62. KLoos, W.E., and Smith, P.B. 1980. Staphylococci. In Manual of Clinical Microbiology. 3rd. Ed. A.S.M. Washington, D.C. 6 3. Lambert', M.A.S., and Armfield, A.Y. 1979- Differentiation of Peptococcus and Peptostreptococcus by Gas Liquid Chromatography of Cellular Fatty Acids and Metabolic Products. J. Clin. Microbiol. 10:1+64-^76. 64. Lambert, M.A.S., and Moss, C.W. 1973- Use of Gas Chromatography for Detecting Ornithine and Lysine Dacarboxylase Activity in Bacteria. Appl. Microbiol. 26:517-520. 6 5. Leathard, D.A., and Shurlock, B.C. 1970. Identification Tech niques in Gas Chromatography. 1st. Ed. Wiley Interscience. New York, N.Y. 66. Lewis, V.J., Moss, C.W., and Jones, W.L. 1967. Determination of Volatile Acid Production of Clostridium by Gas Chromatography. Can. J. Microbiol. 13:1033-1036. 67. Lewis, V.J., Weaver, R.E., and Hollis, D.G. 1 9 6 8. Fatty Acid Comp osition of Neisseria Species as Determined by Gas Chromatography. J. Bacteriol. 96:1-5- 68. Littlewood, A.B. 1970. Gas Chromatography Principles, Techniques, and Applications. 1st. Ed. Academic Press. Inc. New York, N.Y. 6 9. MacFaddin, J.F. 1 9 8 0. Biochemical Tests for the Identification of Medical Bacteria. 2nd. Ed. Williams and Wilkins. Baltimore, Md. 1^5 70. McNair, H.M. 1970. Selecting a Gas Chromatography Column. 1st. Ed. Gow-Mae Instrument Company. Madison, N.J. 71. McNair, H.M., and Bonelli, E.J. 1971. Basic Gas Chromatography. 1st. Ed. Varian Aerograph Co. Walnut Creek, Cal. 72. Martin, A.J.P., and Synge, H.L.M. 19*H. Gas Liquid Partition Chromatography. J. Biochem. 35:358-360. 73- Mayhew, J.W., and Gorbach, S.L. 1977. Internal Standards for Gas Chromatographic Analysis of Metabolic End Products from Anaerobic Bacteria. J. Appl. and Environ. Microbiol. 33:1002-3* 7U. Miller, G.G., Witwer, M.W., Brause, A.I., and Davis, C.E. 197^. Gas Liquid Chromatography of Serum in Diagnosis of Clinical Infection. J. Clin. Invest. 5^:1235-12^3. 75- Mitruka, B.M., and Alexander, M. 1967. Ultrasensitive Detect ion of Certain Microbial Metabolites by Gas Chromatography. Anal. Biochem. 20:5^8-550. 76. Mitruka, B.M., Carmichael, L.E., and Alexander, M. 1 9 6 9. Gas Chromatographic Detection of in vitro and in vivo Activity of Certain Canine Viruses. J. Inf. Dis. 119:625-63^. 77. Miwa, T.K., Mikolajczak, K.L., Earle, F.R., and Wolfe, I.A. i9 6 0. Gas Chromatographic Characterization of Fatty Acid Identification Constants for Mono and Dicarboxyl Methyl Esters. Anal. Chem. 32:1739-17^2. 7 8. Moore, W.E.C. 1 9 8 0. Chromatography for the Clinical Laboratory -All You Wanted to Know (and Possibly More). A.P.I. Species* it: 21-25. 79- Moore, W.E.C., Cato, E.P., and Holdeman, C.V. 1 9 6 6. Fermentation Patterns of some Clostridium species. Int. J. of Syst. Bacteriol. 16:383-^15. 80. Morse, C.D., Brooks, J.B., and Kellogg, D.S. 1976. Identification of Neisseria by Electron Capture Gas Liquid Chromatography of Metabolites in a Chemically Defined Growth Medium. J. Clin. Microbiol. 3:3U-Ul. 81. Moss, C.W., and Dees, S.B. 1975. Identification of Microorganisms by Gas Chromatography-Mass Spectroscopy Analysis of Cellular Fatty Acids. J. Chromat. 112:595-60U. 82. Moss, C.W., Dowell, V.R., Farshichi, D., Raines, I.J., and Cherry, W.B. 1969. Cultural Characteristics and Fatty Acid Composition of Propionibacteria. J. Bacteriol. 97:561-570. 1 A 6 8 3 . Moss, C.W., Howell, R.T., Farshy, D.C., Dowell, V.R., and Brooks, J.B. 1970. Volatile Acid Production by Clostridium botulinum Type F. Can. J. Microbiol. 16:-421-430. 84. Moss, C.W., Kellogg, D.S., Farshy, D.C., Lambert, M.A., and Thayer, J.D. 1970. Cellular Fatty Acids of Pathogenic Neisseria. J. Bact eriol. 36:683—687. 8 5. Moss, C.W., Weaver, R.E., Dees, S.B., and Cherry, W.B. 1977. Cell ular Fatty Acid Composition of Isolates from Legionaires Disease. J. Clin. Microbiol. 6:140-143. 8 6. Mossell, D.A.A., Koopman, M.J., and Jongerius, E. 1 9 6 7. Enumeration of Bacillus cereus in Foods. Appl. Microbiol. 15:650-653. 8 7. Motomiya, M . , Takeda, S., Arai, H., Sato, H . , Yokomwa, A., and Oka, S. 1972. An Attempt in Identifying Mycobacteria Strains by Means of Gas Liquid Chromatography. Sci. Rep. Res. Inst. Tohoku Univ. 19:23-25. 8 8. Nskanen, A., Kiutarau, T., Raisanen, S., and Raevuori, J. 1978. Determination of Fatty Acid Composition of Bacillus cereus and Related Bacteria : A Rapid Gas Chromatographic Method Using a Gas Capillary Column. Appl. and Environ. Microbiol. 35:453- 455 . ' 8 9. O'Brien, R.T. 1 9 6 7. Differentiation of Bacteria by Gas Chromato graphic Analysis of Products of Glucose Catabolism. Food Tech. 21:78-80. 90. Ottenstein, D.M., and Bartley, D.A. 1971. Separation of Free Acids (C2 -C5 ) in Dilute Aqueous Solution Column Technique. J. Chromatt ogr. Sci. 9:673-681. 91. Qxborrow, G.S., Fields, N.D., and Puleo, J.R. 1976. Preparation of Pure Microbial Samples for Pyrolysis Gas Liquid Chromatographic Studies, Appl. and Environ, Microbiol. 32:306-309.. 92. Phillips, K.D., Tearle, P.V., and Willis, A.T. 1976. Rapid Diag nosis of Anaerobic Infections by Gas Liquid Chromatography of Clinical Material. J. Clin. Pathol. 29*428-432. 93. Prosser, A.R., and Sheppard, A.J. 1 9 6 8. Gas Liquid Chromatography of Niacin and Niacinamide. J. Pharm. Sci. 57:1004. 94. Raines, J . , Moss, C.W., Farshichi, D., and Pittman, B. 1 9 6 8. Fatty Acids of Listeria Monocytogenes. J. Bacteriol. 96:2175-2177. 95. Ramsey, H.A. 1963- Separation of Organic Acids in Blood by Part ition Chromatography. J. Dairy Sci. 46:480^483• 147 9 6. Ramsey, L.Le, and Patterson, W. 1945. Separation and Identificat ion of the Volatile Saturated Fatty Acids (Ch-Cn). J. Assoc.Off* Agr. Chem. 28:644-656. 97. Reid, R.L., and Lederer, M. 1952. Separation and Estimation of Saturated (C2 -C7) Fatty Acids by Paper Partition Chromatogr aphy. J. Biol. Chess*. 50:60-67. 9 8. Reiner, E. 1 9 6 5. Pyrolysis-Gas Liquid Chromatography of Gram Regative Fermenting Rods, nature. 106:1272-1278. . 99. Reiner, E. 1 9 6 7. Studies on Differentiation of Microorganisms by Pyrolysis-Gas Liquid Chromatography. J. Gas Chromat. 5 :6 5-6 7. 100. Reiner, E . , and Ewing, W.H. 1968. Chensotaxonomic Studies of Some Gram negative Bacteria by Means of Pyrolysis-Gas Liquid Chromatography, nature. 217:191-194* 101. Reiner, E . , Hicks, J.J., Ball, M.M., and Martin, W.J. 1972. Rapid Characterization of Salmonella Organisms by Means of Pyrolysis-Gas Liquid Chromatography. Anal. Chem. 44:1058-1061. 102. Reiner, E . , Hicks, J.J., Beam, R.E., and David, H.L. 1971. Recent Studies on Mycobacteria Differentiation by Means of Pyrolysis-Gas Liquid Chromatography. Am. Rev. Resp. Dis. 10^:656-660. 103. Reiner, E . , and Kubica, G.P. 1 9 6 9. Predictive Values of Pyro lysis-Gas Liquid Chromatography in the Differentiation of Mycobacteria. Am. Rev. Resp. Dis. 99:42-48. 104. Saito, K. i9 6 0. Chromatographic Studies on Bacterial Fatty Acids. J. Biochem. 47:699-719* 105. Schlossberg, D., Brooks, J.B., and Schulman, J.A. 1976. Poss ibility of Diagnosing Meningitis by Gas Chromatography. I. Cryptococcal Meningitis. J. Clin. Microbiol. 3:239-245. 106. Scholfield, C.R. 1974. High Performance Liquid Chromatography of Fatty Methyl Esters: Analytical Separations. J. Am. Oil Chem. Soc. 51:327. 107. Scholfield, C.R. 1975* High Performance Liquid Chromatography of Fatty Acid Methyl Esters: Preparative Procedures. Anal. Chem. 47:1417-1420. 108. Schomberg, G., Husmann, H . , and Weeke, F. 1974. Preparation, Performance, and Special Applications of Gas Capillary Columns. J. Chromat. 99:63-79* lU8 109* Shelley, R.H., Salwin, H . , and Horwitz, W. 1963* Quantitative Determination of Formic, Acetic, Proprionic, and Butyric Acids by Gas Liquid Chromatography. J. Assoc. Off. Agr. Chem. **:U86- **93. 110. Sondag, J.E., Ali, M. ,and Murray, P. 1980. .Rapid Presumptive Identification of Anaerobes in Blood Cultures by Gas Liquid Chromatography. J. Clin. Microbiol. U : 27*1—277. 111. Stark, J.B., Goodban, A.E., and Owens, H.S. 1951* Paper Chromat ography of Organic Acids. Anal. Chem. 23:*+13-**15. 112. Steinhauer, J.E., and Dawson,L»1969a. Quantitative Determination of Lactic and Succinic Acids in Frozen Whole Eggs by Gas Liquid Chromatography. J. Food Sci. 3*+:37-**2. 113. Steinhauer, J.E.,and Dawson,L. 1969b. Quantitative Determination of Formic, Acetic, Proprionic, and Butyric Acids in Frozen Whole Eggs by Gas Liquid Chromatography. J. Food Sci. 3*+:359- 36U. 11**. Supelco Inc. Publication # 1 8. 1979- Bellfonte, Pa. 115. Teichman, R.J., Takei, G.H., and Cummins, J.M. 197**. Detection of Fatty Acids, Fatty Aldehydes, Phospholipids, Glycolipids, and Cholesterol on Thin Layer Chromatograms Stained with Methyl Green. J. Chromat. 88:**25-**27. 116. VanHcut, P., Szafranek, J . , Pfaffenberger, C.D., and Horving, E.C. 197**. Thermostable Polar Phase Open Tubular Gas Capillary Chromatography. J.Chromat. 99:103-110. 117. Vertele, M . , Verstappe, M . , Sandra, P., VanLuchene, V., and Vuye, A. 1972. Gas Capillary Columns. J. Chromat. Sci. 10: 668-673. 118. Vincent, P.G., and Kulik, M.M. 1970. Pyrolysis-Gas Liquid Chrom atography of Fungi: Differentiation of Species and Strains of Several Members of the Aspergillis flavus Group. Appl. Microbiol. 20:957-963. 119. Wilson, G.S., and Miles, A. 1975- In Topley and Wilson's Princ iples of Bacteriology, Virology, and Immunology. Vol.l 6th. Ed. Wilkins and Williams Co. Baltimore, Md. 120. Wust, J. 1977. Presumptive Diagnosis of Anaerobic Bacteremia by Gas Liquid Chromatography of Blood Cultures. J. Clin. Microbiol. 6:586-590. 121. Yamakawa, T., and Veta, N. 196U. Gas Chromatographic Studies of Microbial Composition. I. Carbohydrate and Fatty Acid Constituents of Neisseria. Japan J. Exp. Med. 3^:361-37^. 122. York, L.R. and Dawson, L.E. 1972. Organic Acid Accumulation in Egg Products Inoculated with Known Cultures. Poultry Sci. 51:12W~12U7.