Micromethod for the Methyl Red Test

Transcription

1 101 COWN, S. T. (1953). J. ga. M~CTOMO~. 9, Micomethod fo the Methyl Red Test BY S. T. COWN National Collection of Type Cultues, Colindule venue, London, N.W. 9 SUMMRY: micomethod fo the methyl ed (MR) test was woked out fo suspensions of bacteial cells. The most impotant factos in the test ae (1) the density of the cell suspension; (2) the atio of glucose to buffe concentations. daptation of the cells to glucose is essential fo the success of the test, and cells fom glucose-fee media ae not satisfactoy. t the optimal glucose : buffe atio Bacteium coli and Kkbsie2lu ueogenes suspensions have diffeent effects on the final ph value attained in the system. Because the MR micotest is dependent on a fine balance between glucose and buffe concentations, and is so geatly affected by even twofold diffeences in cell concentation, it is not eally suitable fo outine wok in the chaacteization of bacteia. The methyl ed (MR) test was developed by Clak & Lubs (1915) who athe optimistically said that ' Simple conditions have been found unde which metabolism can be so contolled that the hydogen ion concentations of cultues of one goup can be made to divege widely fom those of the othe goup' (p. 173). The test measues the final acidity of a cultue in a buffeed medium containing glucose and peptone, but the ph value ecoded at the time of the test is not necessaily the lowest value attained duing gowth. In the ealy stages of glucose fementation sufficient acid is poduced by both BacteizCm coli and Kkbsiella aeogenes (syn. Bad. aeogenes, eobacte mogm8) to tun methyl ed indicato oange o ed (MR-positive), but on futhe incubation K. aeogenes beaks down the pyuvic and othe acids, and poduces a evesion in the eaction of the cultue towads neutality; methyl ed indicato becomes yellow (ME-negative). The timing of this sequence, stating ph (c. 7) +low ph (c. 5) +highe ph (6 o moe), will be affected by the medium, paticulaly the buffeing powe of the peptone and phosphate, by the tempeatue and duation of incubation, and by the stain. It is supising, theefoe, that a geate attempt has not been made to standadize the test. t fist sight it did not appea that the MR test could be fitted into the ange of micotests on suspensions developed by Clake & Cowan (1952). In cultues gowing in a complex medium such as glucose phosphate peptone both, moe than one enzyme system will be in action and each may have some effect on the ph value of the cultue; in paticula NH, poduction fom peptone can be expected to pevent the development of an extemely low ph value. On the othe hand, in living but non-multiplying (static) suspensions, glucose fementation has a simple effect on the ph value; the

2 102 S. T. Cowan intoduction of phosphate buffe slows the initial fall and may pevent the ultimate inactivation of the enzyme system. Contay to expectation, a wokable MR micotest was developed fom the modified fementation method (Cowan, 1958). MTERILS ND METHODS Stains. Classical stains of Bact. cozi, including Escheich s oiginal stain (NCTC 86), and of K. aeogenes, including two eputed descendants of Pfeiffe s Bacillus capsulatw (NCTC 248 and 418), wee supplemented by othe well-authenticated stains fom D E. Windle Taylo of the Metopolitan Wate Boad s laboatoy, vaious seotypes of the Escheichia and Klebsiella goups fom D F. Kauffmann, stains fom D T. Richads of Reading Univesity, and a seies of colifom bacteia collected fo anothe pupose. Media. The basal medium (L) had the following composition: Lemco 1 yo, NaCl 0.5 %, Evans peptone 1 %, New Zealand aga 1 %; ph 7.6. Glucose aga (G) was L + 1 % glucose. Cultues. Except whee stated othewise, cultues wee gown fo h. at 30 on G. Suspensions. Cultues wee washed off solid medium into steile distilled wate, centifuged and the deposit esuspended in distilled wate. Bz@e. Ssensen s phosphate buffe, ph 6.8, was used at vaious mola concentations. Indicatos. Methyl ed (0.04 yo) and bomcesol puple (0.04 yo) wee made up in 98 yo ethanol. MethyZ ed (MR) test. Tests wee made in 65 x 10 mm. tubes, to which wee added 0.5 ml. phosphate buffe ph 6.8, 0.05 ml. glucose, and 0.05 ml. suspension. Concentations of buffe, glucose and cell suspension vaied in diffeent expeiments. Except whee stated, the mixtues wee kept at 37O (wate-bath), and at intevals tubes wee emoved fo test; to these ml. methyl ed solution was added, the tubes shaken and the colous compaed with a standad. In many expeiments duplicate sets wee made, and to each tube of one set nl. bomcesol puple was added, and ph values wee ead at intevals. Capillato sets of standads (Bitish Dug Houses, Ltd., London) fo methyl ed and bomcesol puple wee used fo expeiments in which the esult was expessed as a ph value. RESULTS Change in ph value duing glucose fementation In the fementation of glucose by Bud. coli suspensions thee was a pogessive incease in acidity and fall in ph value; on the othe hand, with K. aeogenes thee was a quicke initial fall in ph, and late a gadual evesal of eaction, pesumably because of the beakdown of pyuvic acid. Table 1 shows the esults of an expeiment in which M-glucose was femented in the pesence of M-phosphate buffe, ph 6.8. Within the fist hou K. aeogenes had loweed the ph much moe than had Bact. coli, but afte the second hou the evesal of eaction was appaent. Fom this expeiment it is seen that the

3 Methyl ed micomethod 103 changes in ph value closely paallel those of cultues gowing in glucose phosphate peptone both. Tanslated into tems of the MR test a eaction of ph 5.0 o less is MR+, 5.8 o above is MR-, and values between 5-0 and 5.8 ae MR & o doubtful. With a doubtful ange extending so widely it may be advisable to use bomcesol puple to supplement the infomation given by methyl ed. Table 1. Changes in ph value duing the beakdown of glucose by Bacteium coli and Klebsiella aeogenes suspensions M-buffe, ph 6.8, 0.5 ml. ; M-glucose, 0.05 ml. ; suspension, 0.05 ml, Time (h Bact. coli K. aeogem (NCTC 86) (NCTC W) ph values Impotance of bufle in test Bact. coli (NCTC 8169) and K. aeogenes (NCTC 8848) wee gown on L and G and suspensions wee made fom each cultue, Suspensions wee added to 0-1 na-glucose and diluting fluid which, in thee cases was phosphate buffe ph 6.8, and in the fouth was distilled wate. Houly tests up to 6 h. and at 24 h. made by the addition of methyl ed showed (Table 2) that both stains wee always Ma-positive (MR+) when wate was the diluent and Table 2. Effect of gowth medium, bufle concentation and duation of incubation on methyl ed micotest Buffe ph 6.8, o wate, 0.5 ml. ; 0-1 M-glucose, 0.05 ml. ; suspension, 0.W ml. Incubation at 37' Suspension fom gowth on I -I L G Buffe concen- Time of eading (h.) tation stain (M) % NCTC 8169, 0.07 Bact. COZ~ Wate NCTC83418, 0.07 E. WOgmS * * * - Wate h 7 I \ _ MB test : + =ed, 4 = oange, - =yellow. > I

4 T. Cowan mt-negative (MR - ) with stong buffe (0-07 M); that the distinction between the stains was only seen in suspensions made fom gowth on G; and that it was best seen in buffe at M concentation, and afte 24 h. incubation. Table 3. Effect of suspension concentation and volume of M-glucose in the test M-phosphate buffe, ph 6-8,0-5 ml. ; M-glucose, ml. ; suspension 0.05 ml. Suspension Stain x 10s/ml. NCTC 86, Buct. mli NCTC 243, K. mogenes M-glucose ml ml ml =ph 5-0 o less; & =ph c. 5.6; - =5.8 o moe. Readings afte 24 h. at 37". Othe peliminay expeiments showed that, at one buffe concentation, the esult was influenced by suspension concentation and by the volume of M-glucose added (Table 8). It became clea that in the application of the test to outine wok, oughly calibated pipettes, adequate fo the othe micotests, would not be eliable and that coectly gaded pipettes wee. essential. Relation between glucose and buffe concentations Phosphate buffe, ph 6.8, was kept at a constant volume of 0.5 ml., glucose at 0.05 ml., and suspension at 0.05 ml., and the concentations wee vaied. The geneal plan was to set up the expeiment in duplicate; bomcesol puple was added as an intenal indicato to one set and eadings wee taken at intevals by compaing the colou with the capillato colou cad fo that indicato. fte 24 h. methyl ed-was added to the second set and the colous matched against the appopiate capillato cad. In the ange in which the indicatos ovelapped, the eadings wee aveaged. In most expeiments the suspension was kept constant and both the buffe and glucose concentations wee vaied, in a few expeiments all thee wee vaied (Table 4). When the suspension is constant and is at least equivalent in density to 8 x 109 Bact. cotilml., thee is a atio of glucose : buffe concentations at which the distinction between Bact. coli and K. mogenes can be seen. When glucose is popotionately geate both species poduce moe acid and ae indistinguishable; popotionately moe buffe again masks the diffeences. The optimal glucose : buffe atio geneally holds good between 0-1 and 0.8 M-glucose and and M-phosphate buffe; ignoing the diffeence in volumes and expessing the atio in tems of mola concentations, it is 4 :,I; when allowance is made fo the diffeence in volume the optimal popotion

5 Methyl ed micomethod 105 is 4 : 10. Below 0-1 M-glucose this atio does not hold, and even at M- glucose an occasional stain will show an anomalous esult. When the suspension density is less than 8 x 100 Bact. colilml. the distinction between Bact. coli and K. wogenes is lost, even at the optimal glucose : buffe atio. Table 4. Expeiment to show elation between concentations of glucose and buffm, and the effect of szlspension concentation stain NCTC 86, Bact. coli Buffe ph 6-8, 0.5 ml. ; glucose solution, 0.05 ml. ; suspension, 0-05 ml. Test ead afte 24 h. at 37" Suspension concentation (cells/ml.) Glucose concentation ( \ 16 x x lo9 h 7 I > Concentation of buffe (M) 005 * NCTC K. aeog& St&ll Glucose concentation (M) NCTC 86, Bact. co2i t Suspension concentation (cells/ml.), 4x lo9 2 x 109 I Concentation of buffe (M) NCTC 248, K. aeogenes 0.05 ph values estimated with methyl ed and bomcesol puple ; a mean was taken in the ange of ovelap. Squaed figues have the same glucose : buffe atio.

6 S. T. Cowan E'eci! of tempeatue Cultues wee gown at 80 and 87', and suspensions made fom each wee tested in wate-baths at 30 and 37'. The tempeatue of the test had little effect on the final ph value attained in 24 h. at the optimal glucose : buffe atio, but the tempeatue at which the cultue had been gown occasionally had some influence. Some stains, such as NCTC 8835, when gown at 80' yielded suspensions which poduced a ph of about 6.0 (MR-) and when gown at 37' the SUSpensions poduced a ph of , that is in the MR doubtful ange. Othe stains gown at the two tempeatues yielded suspensions that hadly diffeed fom each othe; any effect was a slightly lowe ph fom cultues gown at 37". What diffeences thee ae seem to elate to the optimal tempeatue fo gowth which, fo most colifoms, is neae 30' than 37". Eflect of gowth medium daptation of the cells to glucose seems to be a peequisite fo the MR micotest; without such adaptation, as with suspensions fom gowth on L, K. aeogenes emained MR+ (Table 2). The effect of dieeent cabohydates was compaed by gowing Bad. coli and K. aeogenes on L +glucose, lactose, maltose o mannitol, and pepaing suspensions fom the esulting gowths. In the micotest made at diffeent glucose and buffe concentations, K. ueogenes suspensions fom L +glucose poduced a ph of (i.e. MR - ) in all tubes at the optimal glucose : buffe atio. On the othe hand, the final ph value attained by suspensions fom the othe suga media was not unifom, even at the optimal atio, and it appeaed that adaptation to glucose was incomplete (Table 5). Most expeiments wee made on suspensions gown on L and G, but simila esults wee obtained when the oganisms wee gown on 'iche' media such as Hatley digest aga and the same medium +glucose. Hoogeheide (1940) showed that klebsiellas wee less mucoid on nitate media, and because of difficulties in spinning out the cells tials wee made of suspensions fom L+ 1 % KNO, and L+ 1 % KNO,+ 1 yo glucose. Table 6 shows that L + KNO, gave suspensions that behaved as those fom L, i.e. a K. ueogenes suspension maintained a low ph (MR+) and did not poduce a evesal of eaction; L + KNO, + glucose gave suspensions that behaved like G, and the ph eveted to the alkaline side of methyl ed (MR-). DISCUSSION It is hadly appopiate to call a test the ' MR ' when bomcesol puple may be used as an altenative indicato, but the application of the MR test to cultues of colifom bacteia is so well established that to change the name of the test would only cause confusion. The MR test meely indicates the ph value of a cultue gown in a glucose phosphate peptone medium fo an unspecified time at an unspecified tempeatue, and individual wokes have set thei own

7 Methyl ed micomethod 107 Table 5. Tk egect of adaptation to difment sugas on ph value eached in the femnentation of glucose by Klebsiella aeogenes (NCTC 8848) and by Bacteium coli (NCTC 8169) Suspension fom gowth on Glucose concentation (4 I K. aeogenes Bad. coli L I \ I -l Buffe concentation (ph 6.8) M 0.05 M M M 0.05 M 0025 M ph value afte 24 h. at 37' (bomcesol puple) -l L +glucose L +lactose L+ maltose L + mannitol Table 6. Eflectof gowth mdium on. ph value attained by Klebsiella aeogenes NCTC 243 in 24 h. at optimal glucose : kffm atio Medium on which cultue was gown > Glucose L + KNO, L + KNO, +glucose G concentation ph value (M) time and tempeatue standads. Clak & Lubs (1915) used 30", incubated cultues fo at least 3 days and ecommended 5 days ; in England the Ministy of Health method (Repot, 1939) specified 3 days at 37". The indicato used has always been methyl ed, but the citical ange between 5.2 and 5.8 also lies at the acid end of the bomcesol puple ange. 6-2 \

8 S. T. Cowan Bomcesol puple has a distinct advantage ove methyl ed: it can be included in the micotest as an intenal indicato and eadings can be taken at any time; methyl ed is apidly decoloized by many colifom bacteia and can only be used at the conclusion of the test. Duing the fementation of glucose by bacteial cells the hydogen-ion concentation depends pimaily on the glucose : buffe atio; fo coli om bacteia in an excess of glucose the final ph value will be below 5, but in excess of buffe it will be above 6. t the optimal glucose : buffe atio suspensions of Bud. coli and K. aeogenes have diffeent and distinctive effects; afte 24 h. Bud. coli shows a ph value of 5.0 o less, K. wogenes 5-8 o moe. These diffeences ae not seen with cell suspensions fom glucose-fee media; glucose adaptation seems to be essential fo a successful test of this type. The optimum glucose : buffe atio geneally applies between and 0.8 M-glucose and and M-phosphate buffe, but a few stains of Buct. coei poduced anomalous esults with the lowest concentations. Consequently, as a test fo the chaacteization of colifom bacteia M-glucose and 0.05 M-phosphate buffe o 0-4 M-glucose and M-phosphate buffe ae ecommended; these concentations maintain the optimal glucose : buffe atio and give easonably consistent esults. The tanslation of ph values into the MR test is lagely abitay; at the extemes thee is no difficulty in fixing positive and negative values; I have used ph 5-0 as MR+ and ph 5.8 as MR-, with a doubtful o & ange between these values. Haold (1936), on the othe hand, suggested that the line of demacation between MR+ and MR- should be 5.8; at 5-8 and above the test is negative and at 5.6 and below it is positive. The advantage of eliminating the f o doubtful zone is obvious, but without it epeated tests may suggest an instability that is much less obvious with an intemediate zone. The micotest developed is not eadily applicable to outine wok; unlike ou othe micomethods a degee of pecision in the measuement of volumes is essential if the suspension : glucose : buffe atios ae to be maintained. vey dense suspension is necessay; suspensions adequate fo othe micotests may give iegula esults in the MR. The usual MR test on glucose peptone both cultues is not without its difficulties, many of which can be ovecome by the use of defined media (Clak& Lubs, 1917; Richads & Fouad, 1952). Suspensions in place of gowing cultues, and glucose instead of a glucose-peptone mixtue, simplify the conditions of the test but they do not emove all the souces of iegulaity. I wish to thank au those who supplied me with cultues of colifom bacteia, especially Ds Kauffmann, Richads and Windle Taylo. REFERENCES CLRK, W. M. & Lvss, H.. (1915). The diffeentiation of bacteia of the colonaeogenes family by the use of indicatos. J. infect. Dis. 17, 160. CLRK, W. M. & LUBS, H.. (1917). Impoved chemical methods fo diffeentiating bacteia of the wli-aemgenes family. J. hl. Chem. 30, 209.

9 Methyl ed micomethod 109 CLRKE, P. H. & COWN, S. T. (1952). Biochemical methods fo bacteiology. J. gen. Micobhl. 6, 187, COWN, S. T. (1953). Fementations : biochemical micomethods fo bacteiology. J. gen. MicTobiol. 8, 391. HBOLD, C. H. H. (1986). met~. Wat. Bd. NO. 80, pp HOOGEREEIDE, J. C. (1940). Studies on capsule fomation. 11. The influence of electolytes on capsule fomation by KlebsielZa pneunwnh. J. Bact. 39, 649. REPORT (1989). The bacteiological examination of wate supplies. Z&p. p&l. HZth me&. Subj.,Lond. no. 71. RICHRDS, T. & FOUD, M. T.. (1952). The use of glucose inoganic media in the classihation of coli-aeogenes bacteia. Roc. SOC. appz. Bmt. 15 (in the Pess). (Received 26 Febzcay 1953)