july, no. detection and identification bacteria by gas ... · gas chromatography apparatus. the...

APPLIED MICROBIOLOGY, JUlY, 1966Copyright @ 1966 American Society for Microbiology

Vol. 14, No. 4Printed in U.S.A.

Detection and Identification of Bacteriaby Gas Chromatography'

Y. HENIS,2 J. R. GOULD, ANJD M. ALEXANDER

Laboratory ofSoil Microbiology, Department ofAgronomy, Cornell University, Ithaca, New York, and ElectronicsLaboratory, General Electric Company, Syracuse, New York

Received for publication 13 January 1966

ABSTRACTHENIS, Y. (Cornell University, Ithaca, N.Y.), J. R. GOULD, AND M. ALEXANDER.

Detection and identification of bacteria by gas chromatography. Appl. Microbiol.14:513-524. 1966.-Ether extracts of cultures of 29 strains representing 6 speciesof Bacillus, and of individual strains of Escherichia coli, Aerobacter aerogenes, andPseudomonas aeruginosa were examined in a gas chromatograph by use of flameionization and electron capture detectors. Among the products detected were com-pounds with the chromatographic characteristics of acetic, propionic, and butyricacids, ethyl alcohol, diacetyl, acetoin, and 2,3-butanediol. The differences in peakareas of the various products formed by the bacteria were determined statisticallyfor the chromatograms obtained with the two detectors, and the peaks were arrangedin order of decreasing areas to yield a signature for each bacterial strain. Differentsignatures were obtained for the various genera and species and for strains of thesame species. B. licheniformis, B. subtilis, and A. aerogenes formed significant quanti-ties of a number of volatile compounds, and qualitative and quantitative differ-ences between strains were noted. The electron capture detector was particularlysensitive to diacetyl and acetoin as well as to unknown compounds. By use of thisdetector, the presence of 5 pg of diacetyl and 20 pg of acetoin could be demon-strated. The quantity of acetoin detected in B. subtilis and B. licheniformis cultureswas present in as little as 6.3 X 10-3 Mliters of medium.

Detecting the presence of a small populationof an individual bacterial species in a samplefrom a natural environment or in clinical speci-mens is commonly a time-consuming processinvolving growth in selective media and often thesubsequent isolation and identification of theorganism by suitable procedures. The identifica-tion step is also frequently a time-consumingoperation, because, unlike higher organisms,morphology cannot be used as the sole criterionfor identification of the true bacteria.Gas chromatographic techniques have been

employed to some extent for the detection oridentification of microorganisms. Thus, Oyama(6) proposed a technique involving a chromato-graphic characterization of the pyrolysis productsof bacteria or substances of biological origin, todetermine the possible existence of life in extra-terrestrial environments. Pyrolysis coupled withgas chromatography has also been used for thepurposes of differentiating between bacterial

1 Agronomy Paper No. 699.2Present address: Faculty of Agriculture, Hebrew

University, Rehovot, Israel.

strains (8; Garner and Gennaro, Abstr. 150thMeeting Am. Chem. Soc., p. 11Q, 1965), andAbel et al. (1) and Yamakawa and Ueta (12)demonstrated the feasibility of employing gaschromatographic characterizations of cellularfatty acids and carbohydrates in bacterial classi-fication.An approach to the rapid detection or differ-

entiation of microorganisms not hitherto inves-tigated involves use of the gas chromatographwith highly sensitive detectors for the examina-tion of bacterial products which are either vola-tile or can be converted to volatile derivatives.Many volatile compounds are synthesized bymicroorganisms during their growth (3, 10), andthe sensitive detectors might be useful in deter-mining the presence of small numbers of bacteriaby detecting the formation of specific products.Such techniques might have an application inbacterial diagnostics as well. Many species ofmicroorganisms, possibly most, form uncommonor possibly unique compounds. Knowledge of theexistence of such substances is derived from ob-servations of biological responses to the products

513

on May 24, 2020 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

HENIS, GOULD, AND ALEXANDER

of individual microbial genera, species, or strains,for example, compounds which in low concen-trations have a toxic effect upon animals orplants, produce an odor, or which inhibit micro-organisms. Thus, many antibiotics or animaltoxins are synthesized by only selected strains ofan individual species or by a few species of asingle genus, and the typical earthy odor is char-acteristic of certain actinomycetes. Because ofthe sensitivity of certain detectors employed withthe gas chromatograph, it may be possible todevise a method for the detection of as yet un-known unique compounds which might serve asan additional or rapid means for bacterial detec-tion and classification.The present investigation was undertaken to

determine the feasibility of employing gas chro-matography for the detection and differentiationof bacteria by analysis of their metabolic prod-ucts.

MATERIALS AND METHODS

Microorganisms. The following bacteria were used:Escherichia coli; Aerobacter aerogenes; Pseudomonasaeruginosa; Bacilluspolymyxa ATCC 842; B. coagulansATCC 7050; B. pumilus ATCC 7061; B. megateriumATCC 14581; B. subtilis ATCC strains 7058, 6633,9799, 6051, 11838, 13933, 12100, 12711, 7067, 11774,and 10774; B. licheniformis ATCC strains 12759,13438, 11946, 12713, 9259, 8190, 8187, 6598, and14580; and B. circulans ATCC strains 61, 4513, 4515,4516, and 9966. All cultures designated ATCC werekindly provided by the American Type Culture Col-lection, Rockville, Md.

Growth medium. The bacteria were grown in amedium similar to that devised by Proom and Knight(7). This medium contained three sets of ingredients.Solution A contained: KH2PO4, 1.5 g; (NH4)2HP04,7.0 g; MgSO4.7H20, 0.5 g; CaCl2-2H20, 0.3 g;MnSO4-4H20, 40 mg; FeSO4-7H20, 25 mg; (NH4)2-MoO4, 2.0 mg; and distilled water, 780 ml. The pH ofthis solution was adjusted to 7.0, and the liquid wasboiled, filtered, and then autoclaved. Solution Bhad the following components: DL-alanine, 380 mg;DL-aspartic acid, 890 mg; L-arginine hydrochloride,300 mg; L-cyStine, 20 mg; L-glutamic acid, 1,400 mg;glycine, 170 mg; L-histidine hydrochloride, 240 mg;DL-isoleucine, 760 mg; L-leucine, 570 mg; L-lysinehydrochloride, 240 mg; DL-methionine, 60 mg;L-proline, 10 mg; DL-serine, 120 mg; DL-threonine,100 mg; L-tyrosine,60 mg; DL-valine, 150 mg; anddistilled water, 100 ml. Solution C contained: biotin,1 ug; folic acid, 2 ,.ug; riboflavine, 100 ,Ag; 500 jg eachof thiamine (aneurin), nicotinic acid, pyridoxin, andcalcium pantothenate; and distilled water, 20 ml.Solutions B and C were sterilized by filtration throughsintered glass. To prepare the complete medium,solutions A, B, and C were combined asepticallywith 100 ml of 30% (w/v) autoclaved glucose solu-tion. Because not all the bacteria grew well in thismedium, peptone (0.01%) was added.

Growth conditions. The bacteria were grown onslants of nutrient agar at 35 C for 24 hr. The cellswere washed from the agar with saline, and the tur-bidity of the suspension was adjusted to an absorbancyof 0.50 at 550 ml,. To 16 tubes each containing 5.0ml of modified Proom and Knight's (7) medium wasadded 0.1 ml of the inoculum. The tubes were incu-bated in an inclined position at 35 C for 40 hr.

Extraction procedure. To each tube were added 0.1ml of 5 N HCI and 1 ml of 0.2 M HCI-KCl buffer,pH 2.0. The solutions were then centrifuged at 4 Cfor 15 min at 8,000 X g. Samples (5 ml) were satu-rated with Na2SO4, and the liquid was extractedthree times with 10 ml of ether. From the combinedether extracts, 1 ml was removed; the remainder wasconcentrated in a flash evaporator at room tempera-ture to 0.5 ml and then freed from water by additionof anhydrous Na2SO4. The Na2SO4 was washedtwice with 1.0 ml of ether, and the combined etherfractions were concentrated again to 0.5 ml.

Samples were stored at 4 C in 5-ml screw-cappedvials. To eliminate from the chromatograms im-purities originating from the caps of the vials, Teflonliners were inserted into the caps. Samples wereanalyzed either immediately or within 24 hr aftercollection, during which time they showed no chemi-cal changes.

Gas chromatography apparatus. The instrumentused was an Aerograph model 204 dual-channelgas chromatograph (Wilkens Instrument and Re-search, Inc., Walnut Creek, Calif.) fitted with flameionization (FID) and electron capture detectors(ECD). The apparatus was fitted with a Wilkens H2generator, model 650, and a model LDllA dual penrecorder (Westronics, Inc., Fort Worth, Tex.) operat-ing at a speed of 40 inches (101.6 cm) per hr. Eachpen was adapted to swing freely along the full 11-inch (27.9 cm) scale of the recorder with an inputsignal of 2.2 mv. The column was stainless-steel,6 ft (1.8 m) long, 18 inch (0.3 cm) in outer diameter,and packed with 10% Carbowax 4000 terminatedwith terephthalic acid (CW-TPA) on ChromosorbW HMDS 60/80 mesh (Wilkens Instrument andResearch, Inc.).The flow rate of the carrier gas, N2, was 40 ml/min

through the column and 20 ml/min through eachdetector. The flow rate of H2 through the FID was20 ml/min and that of air, 400 ml/min. A 3.0-,ulitersample was injected into the instrument, each de-tector thereby receiving 1.5 ,uliters. The standing cur-rent of the ECD, measured by multiplying the re-corder shift when switching the cell voltage from onto off by the attenuation, corresponded to 64 and45 mv at 70 and 110 C, respectively.The ratio of the ECD response to the FID re-

sponse, the 0 value (5), of various compounds wasdetermined for the highest sensitivity of the instru-ment, namely, a range of 1 and an attenuation of 1for ECD and a range of 0.1 and an attenuation of 1for FID.

REsuLTs

Comparative chromatography of bacterial ex-tracts. Preliminary experiments showed that en-

514 APPL. MICROBIOL.


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

VOL. 14, 1966 BACTERIAL IDENTIFICATION BY GAS CHROMATOGRAPHY 515

TABLE 1. Retention times and areas ofpeaksa obtained with the flame ionization detector

Peak.... A B C D E F G H I J K(+LM) N

Organism Rt .... 39 60 80 95 106 150 205 287 359 418 495 799SD 3.1 2.3 2.2 1.1 2.3 ob 9.2 7.2 14 3.4 26 2.1CV ... 8.0 3.7 2.7 1.1 2.1 ob 4.5 2.5 3.8 1.6 5.4 3.7

Bacillus subtilis7058 .................. 38 3,305 T 1,401 534 946633................ T T 38 2,975 T T 135 829799................ T 60 4,365 T T 951 1786051................ T 71 4,721 34 50 (1) 2,671 1,46111838................ T 73 5,440 87 1,406 T 151 49113933................ T 104 582 87612100................ T 72 11,107 25 T 55 (3) 1,217 806

T (1)12711 ................ T 55 3,680 71 24 1657067 ................ 32 940 T 341 2,882 43611774................ T T 36 8,508 21 T 2,198 1,35510774 .................. 47 10,335 49 617 2,377 633

B. Uicheniformis12759.................. T T 29 T 94 11,800 T T T 1,242 1,04113438.................. T T T T 37 4,000 1,715 21511946.................. T T T T 74 5,795 209 306 1,670 49412713.................. T T T T 72 9,415 T 51 T 67 890 1,1019259.................. T T T 457 259 1398190.................. T T 59 5,300 T 713 T 2,024 1,6248187.................. T T T T 33 3,223 195 330 1,480 5286598.................. T T T 130 T 5,480 1,013 95 2,520 38714580.................. 790 79 13 33 57 3,454 50 (1) 1,930 262

T (3)

B. circulans9966 .................. 234513.................. 177 T 56761 .................. 85 19 860 T3(2)4515P...udo242 19 8344516........... T T 676

B. pumilus7061 ......... T 250 372 99 37

B. coagulans7050..........T 130 981

B. polymyxa842...........1,040 T T 843 T 46 1,954 T

B. megaterium14581..........T 501 46

Escherichia coli...... 140 T 681

Aerobacter aerogenes....-1,470 350 565 703 3,200

Pseudomonas aeruginosa..-

a Peak areas (results) are expressed as square millimeters.' All other values corrected according to F peak (acetoin).


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

HENIS, GOULD, AND ALEXANDER APPL. MICROBIOL

RETENTION TIME (MINUTES)

16 14 12 10 8 6 4 2 O 16 14 12i 1 6 4 2 016 14 12 10 8 6 4 2


FIG. 1. Representative chromatograms of extracts of several bacteria as determined with the flame ionizationdetector. F refers to the range on the detector and Att is the attenuation.

516

z0

LfLJ

LLJ

LU)

z

LXJa-

LLJ

LX

LJ

CL


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

VOL. 14, 1966 BACTERIAL IDENTIFICATION BY GAS CHROMATOGRAPHY

tirely different conditions were required for theseparation and detection of bacterial productscausing responses in ECD as compared with FID.Thus, it was not possible to obtain simultaneousdual chromatograms in the manner described byOaks et al. (5). For example, a column tempera-ture of 110 C and 10-fold concentrated extractswere employed for the FID system, whereasanalysis with the ECD was run at 70 C with eitherunconcentrated or diluted extracts.To minimize errors arising from biological

variability and from variations in the extractionand chromatographic operations, four replicatecultures of each bacterial strain were prepared,extracted, and examined separately. A decreasein retention time (Rt) of the acetic acid and ace-toin standards was noted when the chromato-graph was operating continuously; to correct forthe consequent change in retention times, theobserved Rt values of all peaks were multipliedby a correction factor equal to the ratio of theRt for acetoin at the beginning of the study tothe Rt observed for the same compound at thetime of testing of the particular bacterial extract.The Rt values and corresponding peak areas

recorded by the chromatograph fitted with theFID are summarized in Table 1 for the extracts.The peaks are designated by letters correspond-ing to the order of their appearance. For eachpeak, the designated Rt value is the mean of thecorrected retention time, in seconds, for all testbacteria; for each mean Rt value, the standarddeviation (SD) and coefficient of variation (CV)have been calculated. Considerable variability inRt values and in FID response was noted amongthe replicate cultures with those peaks designatedK-L-M, and no differentiation was possible.Peak areas were generally measured by use of aplanimeter, and products with peak areas of lessthan 10 mm2 were regarded as traces (T). Allvalues are calculated for an FID range of 0.1 andan attenuation of 32. In occasional instances, aproduct was observed in fewer than the four rep-licates; the number of replicates showing thepresence of the compound is designated in paren-theses. The operating temperatures for the gaschromatograph in the FID system were: column,110 C; detector, 120 C; and injector, 160 C. Inthe tabular presentation, a blank signifies that noproduct with the designated retention time wasobserved. Representative chromatograms of ex-tracts of several of the bacteria are presented inFig. 1; the designation of peaks is the same asthat used in Table 1.The Rt values and peak areas for the sub-

stances recorded by the ECD are presented inTable 2. The operating temperatures in the in-

strument were: column, 70 C; detector, 80 C;and injector, 140 C. Because of the sensitivity ofthis detector to the bacterial products, the ex-tracts of all cultures had to be diluted up to 80-fold so that the peak heights did not exceed 30%of the standing current. Peak areas are expressedin terms of the values calculated for the undilutedextract at a range of 1 and for attenuation 32.The letters A to N designate the order of peaksobserved with the ECD and do not correspondwith the letters used in Table 1 for FID. In Fig.2 are representative ECD chromatograms of ex-tracts of six strains.A signature or fingerprint for each bacterium

was established by arranging the various letters,designating the peaks, in an order correspond-ing to decreasing peak areas. Such signaturesfor the 32 strains investigated are listed in Table3. Only peaks having areas greater than 10 mm2are ranked in this way; metabolites elaboratedin trace amounts are designated by lower-caseletters and are arranged in alphabetical order.The statistical significance of the differences be-tween the peaks making up the various bacterialsignatures was determined by use of either theDuncan multiple range test at the 5 % confidencelevel or, for signatures having less than 10 de-grees of freedom, the hsdb5 (9). The differencesbetween letters enclosed by the same brackets arenot statistically significant; the differences be-tween those not so enclosed are significant at the5% confidence level. The peaks less than 10 mm2in size were not considered in the statisticalanalyses.By means of the signatures thus obtained, the

strains could be distinguished from one anothersimply by considering the presence or absence ofpeaks. Alternatively, the bacteria could be dis-tinguished by considering the quantities of thevarious products formed. Thus, a comparisonwas made among the 32 bacteria to determinethe FID- or ECD-positive metabolites whichcould be used to distinguish among the strains onthe basis of statistically significant differences inproduct yield. For this purpose, the bacteriawere considered to be distinguishable if any ofthe following applied. (i) The two strains beingcompared yielded peaks having identical reten-tion times but statistically different areas. (ii)One of the two strains produced the metabolite,either in significant or trace amounts, whereas asubstance with the same Rt value was not foundin any of the replicates of the second organism.(iii) One of the organisms produced the com-pound in significant quantities, whereas the otherproduced only traces; however, if the peak wasin the lowest homogeneous group in the Duncan

517


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

518

z

HENIS, GOULD, AND ALEXANDER APPL. MICROBIOL.

-°4 °°°q°°W°W°a NN mm "t,-4N tn-0 "I e' a00O 000000 00RC'00 000 e t t-00 - t

C t 0 0t- 0e00t-0a OO-tOO-' e 't

-- - -1N \o N "t O O)e t) e1 t- t- en t) 00 tn 61 N 1C14 - N --I -_ _ _- -4tT -_' 4 _4

gooo

0o

> |a H HHE-<HHHHHH~E~H HE-H Hu H Hz H

; or°I H Ht H HHHHHHHE~E EHHH Hz Hz H H

W oY H H HHHHH HH HHH H W H HO~

t-00 0o% 000000 000001 Nn tr tcn-

Y ~~~~~~~~o C1 00 en oo o - O - ON n t- W) O' 00 °°4 C 4 (-O00 W) "-q0_I000 0oo0J en oo-4

"4

a HH'-HF HH H-HHHHHHHe HH HHH HH Hs er FH H

oo 0-00r000000 00 00 Ini 0C0I0o~ e

t3 00(O I In4CR4) 00

4)00~~~~~~~~~~~~~~~~~000c: N ^ W t N b b so ob mooC 0 o

000n0o 0000 000 ' O QQQ 0 tQC1'.Q 00 Cnq6) - - - ~~- - ,- ~

.s~~~~~~~~~~~O N CN O "T cz- OnN1. ON o0 £ enNuqt

m C

S~~~~~~~~~~~~~W en0ooVPHF 4WI HFFFFcw) twn noFco F

O t-O O~~~~~~~~~~~OW Oc Q O O O~O1-44en

0 t- 1t 00Vcu -fo-w

zEHHHHHHHHHHHHHHHHHH HHHHHHHHH HHE -(~

H_ o^ mE*° ~~~~~~~~00e~" H - H rNoooooOOo\ Q0m o0 rtff 00 tnt£400en -00 t- d0tO e 0 a Ftn A V) m r-t-q l tn m -otfI o cyO £ r0.08b O 8 b t t C N -

t_ aN 8 I" en t1 t t_~ 4 a 61 o) _ N 08 00 00 "_4 "- _" I " "4 "-I"4 r_.

0 -4 tn -o0 -00 I'D0 -" *00 c Cd

dt as 'In It °N °F °°'f

L I<"- r.

L~4)

0

Qz

00

4)

64

0d

4)

>

cs

0.

'00o

I- 4)*4)00

4) 4)0bl

I- .~ 0

- 4)

0'0

000=lo ..4

0<0.0dc)4) 0'-


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/


e SLb7ll4s774

9. swtlisi21000 14S~~~~~~~~~~~~~~~~~~~~~

i6 i4 t2 10 8 6 4 2 6 1'614

8 lichsniforrms659S

B Bchenit r'sa8190

/

9ul

A.carogents

I0'lsJaA~~~~~~~~~~~~~~~~

12 10 8 6 4 2 O l 14 12 10 8 6 4 2


FIG. 2. Representative chromatograms of several bacteria as determined with the electron capture detector.The ether extract of the medium was either undiluted (Escherichia coli) or diluted 4- (Aerobacter aerogenes),40- (Bacillus licheniformis), or 80-fold (Bacillus subtilis) before a sample was injected into the instrument.

test, a difference with a trace quantity was notconsidered significant. In instances where thereplicates of a single organism gave some peaksgreater and some less in area than 10 mm2, theyield was designated as trace. Peaks which didnot appear in all replicates were not taken intoaccount. Metabolites K, L, and M in the FIDchromatograms were considered as a single peak.All 32 strains were compared with one another, atotal of 496 comparisons for each of the two de-tectors. For the sake of brevity, however, onlyillustrative comparisons are presented in Table 4.The data indicate that the signatures could be

employed to distinguish between all the pairs ofstrains examined. In general, fewer differentiatingpeaks were observed when comparing strains ofthe same species among themselves than whencomparing strains of one species to those ofanother. Thus, ATCC strain 12100 differs fromATCC strain 9799 of B. subtilis only by peak Fin FID, whereas it differs from E. coli in peaksA, C, E, F, H, I, K, and N. It was not possible,however, to differentiate between B. circulansstrains with FID, whereas the strains differedfrom one another in their ECD chromatograms.

Chromatography of standard compounds. Tofacilitate identification of the volatile metabolites,a number of known products of bacterial metabo-lism were examined by use of a CW-TPA column.The quantity of each compound reaching thedetector ranged from 3 ng to 7.5 ,ug. In Table 5

are given the retention times, peak areas, andvalues of eight such compounds. Formic acid

exhibited severe tailing when tested with FIDat a column temperature of 110 C, but it had aretention time, after correction, of 710 sec. TheRt values were corrected as described above, andthe peak areas were calculated for FID range of0.1 at attenuation 32 and ECD range 1 at attenua-tion 32. The sensitivity limit was estimated as thequantity of the chemical necessary to give a peakarea of 50 mm2 for the ECD at range 1 andattenuation 1 and for FID at range 0.1 andattenuation 1, although operations were usuallyperformed at attenuations 4 to 32; the calculatedsensitivities, which are estimates, indicate theremarkable sensitivity of these detectors.The high 0 values for diacetyl and acetoin show

the extreme sensitivity of the ECD to these twocompounds, the results suggesting that it is

100-

801

60

40-

20-

1001

80

60

40

20

E.coli

Ci

I~~~~~~~~^ 1H~~~~~~J~~~~ 519

i

4

ii1'.. a

J.,.!i

L

tiI

..---, - //\'."


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

TABLE 3. Flanme ionization (FID) anid electroni capture detector (ECD) signtatures of various bacteria

Signaturesb

FID

F H K'N E GIF KN E G: cdh

F KIN E: chi

FFKN E G: c

F1H N K G E1: cf

N K F: c

F'K N E H': cij

F NiH E K: c

K F'N H E1: g

FFKN~EG: bch

FK'IHNGEFIK N E C': abdghi

F KiN E: abcd

F KIN J H EI: abcdF1N K E J H1: abcdgi

FrH N': ace

F'K N H E G': aci

FK J H~E: abcd

F1K H N D Gl: abce

F KIA N E D B C': h

FH A-l: fHAF'HA'

H A F

H: af

H FFKNi: e

N K: h

J'A F H': (legh

HNA. f

FH Al: f

K AHF

ECD

N CJ G FE A: bdi

N C J E G A: bdfhi

FN--V~JG~E Z bdfhikliii

N G E Al: bdfhliC'NTJVA: bdfil

CN J AGD: Ibehikm

7TTN-C GFE-A': bdfhlmli

N J C G A D: bdfhikmN: lr~dfhikm

N CJ G A El: bdtikliir NllG Al: bdefhiklIn

N1C J G A1: bdeflhiklm

C'J G NE FA' : bhklmrFV

C J N G E A: bdfilmr-EN---iCF''"G : bdflhikliim

N J C G F A: bdfhik

NYJC G Ab:dfhiHWH:~bdfhikFN C'5 GF A: bdlhiklmi

NrTCG A: bdfhiklm

N7 C A: bdfhiklm

FN C1J --'F: bdfhiklk

N1C J G A': bdhi

CIN J E G7Al: bdhi

CIN G J A': be

CiN J G E Al: bfhi

CNJtA: bi

N CJGE K F1: abdhilm

C EN GA: bdf

N C7GFi: bdehiklinC A E N1: bdfghi

C1NEAl: bdgj

N1iFC G: bdhiklnC: abde

520

Strain"

7058

6633

9799

6051

11838

1393312100

12711

7067

11774

10774

12759

13438

11946

12713

9259

8190

8187

6598

14580

9966

4513

61

4515

4516

7061

7050

842

14581

Ec

Aa

Pa

a Ec, Aa, and Pa refer to Escherichia coli, Aerobacter aerogenies, and Pseudomonas aeruoginiosa, re-spectively.

I Peaks significantly different from one another are not enclosed by the same bracket. Peaks so en-closed are not significantly different from one another. Traces are given as small letters.


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/


z z Z z ZZ

U- z o o U.m4 U. U L LL X;LLU.U.O<<< L< <:<U. mZ mUZ mZ0 Q 0LIw4 z ~c )4I.0WUUUUUWQUUUQ4QQ

kZZU.t=LI4LV.Z "«0O< Z<

z z

Y~~~~~~~~~~YYZ ZY-Z Z zY 0-4Os<<:<<<:<<<<<<mmmm mmm<UbUb<:<U<

«««««<~~===<UUUU«<UUUc<

~Z Z z ~ ZZZ-%4-~4 *n ~

O-

I

-O * -0~ *-O 00~4 .z~

Inz~~~~~

3~ -. -

=- -

m . U.0O0I"w0. xZ L. xo o= =" -0-~~Q-"

zn 0 0- -x m -4 zx .1 0 .1 Y".-n

X0 0_

"

Z.

0V0OLC00VQ4)V U

QQULLIQUQ4U 0 " t v . v " ". 0 0 z . . . v

U4.<<<U<<U<<oLu.uU W4U<UbUUUUUUUUUU

__U)Ubv <O<6<<<SO<<<_U<< <<<<<<< S<I

w.Wuu(u4Yz«40«<4.UUUU

521

-cn0

._

n

cL)

._

._

._

v)

~Q

._

uz

v.I

0

v.d.1Z)

.z;L.

0

li

C._c)

QC(,

SCCQ

:.CUC

OC_v -

boC)

0 o~

00

0

0c

m m a-, --o m m 8 --4 t-- ,:I- "T C)'. m .sc m a. r- m 0 I.-C m - tn 'IC - eq --4

tn en ON tn en eln -4 .,c r- r- W) M "zt - W) RN m Os m I'D - 1,0 - - .c R "Zt 00 f I cfs CdC) .c r- S m 0. - r- 0 t- t- r- 'IT O. t- " - - tn W) ON tn tn W) C) C) 00 tnr- 111.0 (21. - cn " cq t-- -" 0 " m I" " CN m m "D I'll CN ":t "Zt "Zt r- I- I- 4 < 4

4 I" I" _-o -.4 I" -.4 1-4 I" I" I" 1-

o11


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

HENIS, GOULD, AND ALEXANDER APPL. MICROBIOL.

TABLE 5. Retention time and sensitivity of the two detectors to several compounds

Charge Sensitivity limitCompound Column Retention time 0 valuetemp

FID ECD FID ECD

C sec pg Ag ng ng

Butyric acid.... 110 630 7.5 7.5 0.26 1.8 6.82,3-Butanediol ....... 110 418, 495a 7.5 7.5 0.24 1.7 7.4Propionic acid ....... 110 410 7.5 7.5 0.21 2.0 9.5Acetic acid .......... 110 280 7.5 7.5 0.45 3.8 8.4Acetoin.............. 110 150 0.75 0.003 17 2.4 0.15Acetoin.............. 70 800 0.3 0.003 60 1.5 0.022Diacetyl ............. 70 120 7.5 0.0075 130 2.7 0.022Ethyl alcohol ...... 70 64 7.5 7.5 0.018 3.1 170Ethyl alcohol ..... 110 40 7.5 7.5 0.0038 2.7 730Acetone.............. 70 49 7.5 7.5 0.011 2.9 270Acetone .............. 110 33 7.5 7.5 0.0030 2.9 960

a Two peaks present in commercial 2,3-butanediol; calculated as a single peak.

possible to detect quantitatively as little as 22 pgof these bacterial products (Table 5). Moreover,the sharpness of the diacetyl peak in ECD makesit possible to determine 5 pg at a column tem-perature of 70 C, a quantity that has been de-tected in separate trials. For metabolic productssuch as 2, 3-butanediol and acetic, propionic, andbutyric acids, the 0 values are near unity, whereasthe ECD exhibits little response to ethyl alcoholand acetone.On the basis ofa comparison ofRt and 0 values

of the commercially available chemicals with thecompounds observed in the bacterial cultures, itappears that FID peaks A, B, F, H, J, and M areethyl alcohol, diacetyl, acetoin, and acetic,propionic, and butyric acids, respectively; peaksK and L show Rt and 0 values identical withproducts appearing in commercial 2, 3-butanediol(K & K Laboratories, Plainview, N.Y.), andthey may represent isomers of this compound.The Rt values of ECD peaks B, C, and N aresimilar to those of ethyl alcohol, diacetyl, andacetoin, respectively.FID chromatography of a solution of com-

mercial acetoin (K & K Laboratories) in etherrevealed that the main component was accom-panied by several other constituents. On thebasis of peak areas, the minor componentsaccounted for about 5% of the acetoin. Thesechromatograms resembled the FID chromato-graphic pattern of the faster-moving productsformed by B. licheniformis strains (Fig. 1),corresponding to peaks A, B, C, D, and E.Moreover, a dilute ethereal solution of commer-cial acetoin showed an ECD pattern almostidentical with the ECD chromatogram of most ofthe acetoin-forming bacteria. The secondarysubstances are not decomposition products ofacetoin formed during chromatography, as no

100 I

LJU)z0toCL

a:U)

w

r-J

a

C-

-JL-iI-zLU.

LU

60 4

40

20

ECDN

''1

FIG. 3. Comparison of the chromatograms ob-tainedfrom extracts ofcultures ofBacillus licheniformis12713. A 3-,uliter sample wvas used. The operatingparameters were attenuation 32, range I for ECD andattenuation 16, range I for FID, except as otherwiseindicated. Operating temperatures: column, 70 C;detector, 80 C; and injector, 140 C. The culture extractexamined with ECD was 2,400-fold more dilute thanthat examined with FID.

additional peaks were observed upon rechro-matographing the main acetoin peak that hadbeen isolated by collecting the effluent from thecolumn.

522

12 10 8 6 4


2 0


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/


Relation between ECD and FID active sub-stances. Although the detection of low concen-trations of certain metabolites required differentoperating conditions of the chromatograph,some of the peaks appearing in both the FIDand ECD chromatograms may represent the samesubstances. To test this possibility, extracts of B.licheniformis 12713 were chromatographed at acolumn temperature of 70 C with the use ofFID, and the response was compared with ECDchromatograms of the same extracts afterdilution.The results of Fig. 3 show a number of peaks

recorded by the two detectors. The slight dis-crepancy in retention times between apparentlyidentical volatile compounds probably resultsfrom the fact that, because of the necessity to usetwo different concentrations of the extract, thechromatograms were obtained separately ratherthan by use of the dual channel detector systemon the instrument. The Rt values for peaks C, G,J, and N in ECD chromatograms essentiallycorrespond to those of FID peaks B, D, E, andF; the first of these four products has an Rtvalue identical to that of diacetyl although the 0value is higher than that of standard diacetyl, thesecond has a 0 value of 32,000, the third hasa 0 value of 6,000, and the fourth appears to beacetoin. The letters designating the several peaksrepresent metabolites having the same Rt valuesas before.

DIscussIoNIn view of the vast array of products which

microorganisms can form, no one method for theextraction or chromatography on a single columnof all the metabolites is possible. However, manyof these bacterial products are small, polarmolecules that are soluble in ether, and columnsof the Carbowax series would appear to beparticularly suitable for the necessary separa-tions. Moreover, the compounds represented bythe peaks observed in the chromatogramsundoubtedly represent only a portion of thetotal number of metabolic products, i.e., onlythose ether-soluble volatile substances whichhave retention times of 30 to 800 sec under thegiven conditions. Other methods and columnswill undoubtedly reveal the presence of numerousother products of microorganisms.For the purpose of examining extracts of

bacterial cultures by gas chromatography, thesterile growth medium should yield few peaks inthe instrument, yet it should support good growthof the test organisms and allow for the generationof metabolites detectable by gas chromatography.The modified Proom and Knight's (7) medium is

particularly suitable for this purpose. Othermedia, especially those containing yeast or beefextracts, were found to contain a variety ofsubstances readily detected by ECD. The culturemethods described were adopted because of theirsimplicity and the ease of preparing manyreplicates. It is of interest that, not only is themedium composition important, but also themethod of its preparation. For example, differ-ences in chromatographic patterns were obtainedfrom extracts of media in which the amino acidsolution was sterilized by means of a Seitz ratherthan with a sintered-glass filter.

Differences between chromatograms of thebacterial strains may be either real or apparent.The apparent differences may result from varia-tions between individual replicates, extractions,injections, and variations attributable to the gaschromatograph itself; e.g., fluctuations in columntemperature. For these reasons, the significanceof differences in chromatograms was determinedby statistical analysis of replicate cultures. Suchanalyses demonstrated that the genera, species,and strains tested could be distinguished on thebasis of either the qualitative or the quantitativemakeup of the chromatographic patterns, orboth. With increased replication, smaller differ-ences in signatures of the cultures or in peakareas could be distinguished.

It is likely that the ability to distinguish betweenorganisms will be increased if the cultures producea large number of peaks. Thus, the chromato-graphic patterns of B. subtilis and B. licheniformisstrains appear to be significantly different fromone another, whereas differentiation among B.circulans strains was not possible with FID.With P. aeruginosa and E. coli, which producedfew or no peaks despite their abundant growth,it will probably not be possible to differentiatebetween strains by the procedures or conditionsherein described. On the other hand, althoughmany components were observed in the ECDchromatograms, the patterns were relativelyuniform. Patterns of the FID chromatogramswere more distinctive. Nevertheless, it waspossible to differentiate among the bacteria byECD chromatography by making use of thequantitative differences in peak areas.

Seven common and structurally simple com-pounds were identified by their Rt and 0 values,namely, ethyl alcohol, acetoin, diacetyl, 2,3-butanediol, and acetic, propionic, and butyricacids. Bacteria that produced acetoin also re-leased many other volatile metabolites, thesecompounds appearing prominently in the ECDchromatograms as poorly separated peaks be-tween diacetyl and acetoin. On the other hand,

523


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

HENIS, GOULD, AND ALEXANDER

ECD chromatograms of bacteria producing littleor no acetoin exhibited fewer and smaller peaks.These findings suggest that the compounds maybe involved in the synthesis or degradation ofacetoin. Not only do these substances accompanyacetoin on the chromatograms, but they arefound in commercial acetoin and are produced instrikingly similar proportions by A. aerogenes andB. licheniformis. Intermediates which may appearduring acetoin synthesis and degradation includediacetyl and the various isomers of 2,3-butane-diol, diacetylmethylcarbinol, and acetyl butane-diol. In addition, racemizing enzymes maytransform the naturally produced 1 form ofacetoin to the d isomer (2, 4, 11).

Because of the sensitivity of the ECD detectorto diacetyl, acetoin, and several unknownmetabolites, culture extracts often had to bediluted prior to injecting samples into the chro-matograph; such extracts are frequently 2,400to 4,800 times more dilute than samples testedwith FID. With a 240-fold dilution of the extractand knowledge of the fact that each detectorreceives a 1.5-,uliter sample, each ECD chro-matogram represents 0.0063 uliter of the originalmedium. Inasmuch as the area observed foracetoin in the ECD chromatograms of thesediluted bacterial extracts was commonly about300 mm2 when calculated for attenuation 32, anarea of 50 mm2 at attenuation 1 would be expectedto result from the acetoin in 3 X 10-5, liters ofmedium. It may be expected that a smallerquantity of medium would be needed for thedetection of diacetyl. E. coli, B. circulans, and B.megaterium, organisms classified as acetoin-negative (Bergey's Manual), generated productswith similar Rt and 0 values to diacetyl andacetoin. In view of the frequency of these productsin bacterial cultures and the sensitivity of theECD unit to their detection, one of the mostsensitive methods for detecting the presence ofmicroorganisms might well be by examining fordiacetyl and acetoin formation.The need for standardized culture techniques

cannot be overemphasized. In addition, unlessthe population of bacteria obtained from aspecimen is known to be homogeneous, or thedistinctive chromatographic pattern is knownnot to be obscured by interfering organisms, pureculture techniques must first be employed.

ACKNOWLEDGMENTSWe thank Namie Tanaka for her valuable as-

sistance.This investigation was supported with funds pro-

vided by the U.S. Air Force Office of Scientific Re-search under contract AF49(638)-1418.

LITERATURE CITED1. ABEL, K., H. DESCHMERTZING, AND J. I. PETER-

SON. 1963. Classification of microorganismsby analysis of chemical composition. 1. Feas-ibility of utilizing gas chromatography. J. Bac-teriol. 85:1039-1044.

2. JUNI, E., AND G. A. HEYM. 1956. A cyclic path-way for the bacterial dissimilation of 2,3-butanediol, acetylmethylcarbinol, and diacetyl.I. General aspects of the 2,3-butanediol cycle.J. Bacteriol. 71:425-432.

3. LAMANNA, C., AND M. F. MALLETTE. 1959.Basic bacteriology: its biological and chemicalbackgrounds, 2nd ed. The Williams & WilkinsCo., Baltimore.

4. LEDINGHAM, G. A., AND A. C. NEISH. 1954.Fermentative production of 2,3-butanediol,p. 27-93. In L. A. Underkofler and R. J. Hickey[ed.], Industrial fermentations, vol. 2. Chemi-cal Publishing Co., Inc., New York.

5. OAKS, D. M., H. HARTMANN, AND K. P. DIMICK.1964. Analysis of sulfur compounds withelectron capture/hydrogen flame dual channelgas chromatography. Anal. Chem. 36:1560-1565.

6. OYAMA, V. I. 1963. Use of gas chromatographyfor the detection of life on Mars. Nature200:1058-1059.

7. PROOM, H., AND B. C. J. G. KNIGHT. 1955. Theminimal nutritional requirements of somespecies in the genus Bacillus. J. Gen. Microbiol.13:474-480.

8. REINER, E. 1965. Identification of bacterialstrains by pyrolysis-gas liquid chromatography.Nature 206:1272-1274.

9. STEEL, R. G. D., AND J. H. TORRIE. 1960. Prin-ciples and procedures of statistics. McGraw-Hill Book Co., Inc., New York.

10. STEVENS, R. 1960. Beer flavour. I. Volatile prod-ucts of fermentation. J. Inst. Brewing 66:453-471.

11. TAYLOR, M. B., AND E. JUNI. 1958. Mechanismsof formation of stereoisomers of 2, 3-butanediolduring microbial fermentation of sugars.Nature 181:1389-1390.

12. YAMAKAWA, T., AND N. UETA. 1964. Gas chroma-tographic studies of microbial components. I.Carbohydrate and fatty acid constitution ofNc vseria. Japan. J. Exptl. Med. 34:361-374.

524 APPL. MICROBIOL.


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

july, no. detection and identification bacteria by gas ... · gas chromatography apparatus. the...

Documents