Detection of Bacteriuria by Luciferase Assay of Adenosine

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Jn. 1975, p. 1-8 Copyright 1975 Amerin Soiety for Mirobiology Vol. 1. No. 1 Printed in U.S.A. Detetion of Bteriuri by Luiferse Assy of Adenosine Triphosphte A. THORE,* ANSAHN, A. LUNDIN, AND S. BERGMAN Ntionl Defense Reserh Institute, Deprtment 4, S Sundbyberg 4, Sweden, nd Deprtment of Clinil Bteriology, The Medil Shool, S Linkoping, Sweden Reeived for publition 4 September 1974 A seletive method for distinguishing bteril nd nonbteril denosine triphosphte (ATP) in linil bteriologil speimens ws studied. The method involved inubtion of smples with the detergent Triton X-1 nd the ATP-hydrolyzing enzyme pyrse. The inubtion seletively destroyed ATP in suspensions of vrious humn ells while not ffeting the ATP ontent in mirobil ells. ATP remining in the smple fter inubtion ws extrted in boiling buffer nd ssyed by the firefly luiferse ssy. Applition of the method to 469 linil urine speimens showed tht the ATP level fter tretment with Triton/pyrse ws orrelted to bteril ounts nd tht the sensitivity of the ssy ws suffiient for the detetion of 15 bteri/ml. The ATP levels per bteril ell remining in the urine speimens fter tretment with Triton/pyrse were lose to vlues observed in lbortory-grown ultures. The speifiity nd sensitivity of the luiferse ssy for the detetion of urinry bteri nd its possible use s bteriuri sreening method re disussed. Reently the ssy of denosine triphosphte (ATP) with the firefly luiferin/luiferse system hs been disussed in onnetion with mirobiologil work. The most frequently proposed pplition hs been the use of ATP ssys s mens of quntittion of mirobil ells in vrious smples (4, 1-14, 18; G. L. Piiolo nd E. W. Chppelle, personl ommunition). Numerous studies hve been mde to determine ellulr levels of ATP in both bteri nd other miroorgnisms (1, 3-5, 7, 9, 1, 12, 15), demonstrting tht vritions in ellulr ATP ontent rrely exeed one order of mgnitude (1, 6, 1, 12). Thus, it would seem tht, theoretilly, the ury of the ssy is dequte for mny mirobiologil pplitions. The luiferse ssy hs been shown to hve sensitivity llowing the detetion of pproximtely 1-14 M ATP, orresponding to the ATP ontent of fewer thn 1 bteri/ml (1, 1). This extreme sensitivity, ombined with the simple nd rpid ssy proedure, would mke the luiferse ssy n ttrtive lterntive to existing methods for quntittion of bteri. A mjor problem in the pplition of ATP ssys to quntittive bteriology hs been the frequent ourrene of lrge mounts of nonbteril ATP emnting from ellulr mteril present in mny types of bteriologil speimens. If nonbteril ATP nnot be removed prior to the ssy, erroneous results will be obtined (13, 14). The present study ws performed to investigte the ourrene of nonbteril ATP in urine nd possible wys of its elimintion. It hs been suggested tht the tion of the detergent Triton X-1, seletively lyzing nonbteril ells, ombined with the ATP-hydrolyzing enzyme pyrse, might be used for this purpose (Piiolo nd Chppelle, personl ommunition). The proedure ws worked out in model system inluding severl types of humn ells representtive of ells likely to our in urine speimens. The nlytil system rrived t through these studies ws pplied on urine speimens sent for routine ulture to linil bteriologil lbortory. MATERIALS AND METHODS Anlytil equipment. The nlytil equipment used in the luiferse ssy of ATP ws modifition of devie originlly desribed by Chppelle et l. (2). It onsisted of drk hmber into whih the smple to be nlyzed ws introdued, so tht it fed photomultiplier, RCA 931 A, operted t 1, V. The light signl obtined upon mixing luiferse regent nd smple ws reorded on potentiometri hrt reorder (Eletroni 194 Lb/Test Reorder, Honeywell), nd the pek light emission ws used in the lultions. The totl time required for ssy nd registrtion of the result ws in the order of 15 s per smple. Downloded from on Otober 2, 218 by guest

2 2 THORE ET AL. J. CLIN. MICROBIOL. Anlytil regents. Firefly luiferse ws purhsed from Sigm Chemil Co., St. Louis, Mo. Of linil urine speimens with > 15 TABLE 1. Qulittive yield of miroorgnisms in 123 the rude firefly lntern extrt, 5 mg ws mde up miroorgnisms/ml in 2 ml of.1% bovine serum lbumin ontining 1 mm MgSO4 nd 1 mm ethylenediminetetrette (EDTA), nd the ph ws djusted to 7.4. The speimens Type of miroorgnism suspension ws entrifuged t 27, x g for 15 min to remove prtiulte mtter. To redue bkground 54 Esherihi oli light emission, the solution ws llowed to 16 Proteus sp. ge overnight in refrigertor before use. The luiferse 12 Streptoous sp. regent ould be kept t room temperture for severl 9 Klebsiell/Enterobter hours without ppreible loss of tivity nd ould 4 Pseudomons sp. be used for 2 to 3 dys if kept refrigerted. 3 Stphyloous sp. Apyrse, grde II (rude), ws purhsed from 2 Cndid lbins Sigm Chemil Co. Stok solutions ontining 1% 14 Mixed infetions pyrse mde up in distilled wter were kept frozen 9 Unidentified bteri until use. Other hemils were of nlytil grde. After heting for 9 s, the extrts were ooled nd Miroorgnisms nd humn ells. In model studies, strins of Esherihi oli, Proteus mirbilis, This extrtion proedure gve reproduible results kept on ie or frozen until the ssy ws performed. Stphyloous ureus, Streptoous sp., nd Cndid lbins reovered from linil urine speimens to 99% ompred with id extrtion proedures suh with ll bteri tested. The reovery of ATP ws 76 were used. Cultures were grown in sterile urine t 37 C s KCIO4 or trihloroeti id extrtions. Nevertheless, the Tris-EDTA extrtion ws preferred be- overnight for use in the experiments. Humn blood ells (red blood ells, mononuler white blood ells use of its gret tehnil dvntges. [16], nd polymorphonuler grnuloytes) nd tissue Assy proedure. The ssy ws performed on ulture ells (HeL [epitheloid rinom] ells nd 1-ml portions of the boiling buffer extrts whih were humn embryoni lung fibroblsts) were lso used in trnsferred to 4-ml polystyrene Ellermn tubes nd model studies. The tissue ulture ells were grown in pled in the drk hmber of the luminometer. bsl medium (Egle; Flow Lbortories) supplemented with 1% humn nd 5% lf serum. The ells extrt by mens of 1-ml syringe driven by liner Luiferse regent (.4 ml) ws then injeted into the were dethed with trypsin (rystllini,.25%) nd motor. Stndrds with known mounts of ATP nd wshed twie in phosphte-buffered sline before use. regent blnks were ssyed in eh series. Clinil speimens. Urine speimens were obtined from 469 ptients, inluding lini s well s the urine smples were lulted by using ssys of Clultion of ssy results. The ATP levels of hospitlized ptients. Of the 469 speimens, 29 stndrd mounts of ATP s referene nd orreting yielded < 14 miroorgnisms/ml nd were designted for regent bkground vlues. The ATP onentrtion rrived t in this wy hd to be further orreted s negtive, 56 showing 14 to 15 miroorgnisms/ml were designted s borderline speimens, nd the for effets of interfering omponents present in the remining 123 speimens with ounts of > 15 miroorgnisms/ml were designted s positive. The qulitril ultures nd humn ells, orretion ftor ws extrts. In the model experiments with pure btetive yield of these speimens is summrized in Tble obtined by the ddition of known mount of ATP 1. to the extrt followed by repeted ssy. Quntittive ulture. Quntittive ulture ws In the linil urine speimens, n verge orretion ftor ws determined to be (95% performed on ll bteriologil speimens by plting on blood gr pltes.2 ml of 1-fold seril dilutions onfidene level), orresponding to n inhibition of the in sterile sline. In Fig. 4 nd 5, results re lso inluded from experiments in whih bteril ounts were orreted by multiplition with this ftor. nlytil retion of bout 6%, nd ll ssy results were only pproximtely estimted by gross exmintion of the plte from the first dilution. urine speimens inlude sttistil error of ±4% Thus, the ATP onentrtions determined for the Pretretment of urine speimens nd bteril (95% onfidene level). ultures before ssy. When nonbteril ATP ws to be removed, smples were inubted with pyrse in the presene of Triton X-1. The proedure finlly RESULTS dopted ws s follows: 1 ml of the bteriologil In the first series of experiments, the Triton/ speimen ws mde up to ontin.1% Triton X-1, pyrse tretment ws developed nd pplied 2 mm CSO4, nd.1% pyrse (.4 units) in totl to model systems of humn nd bteril ells. volume of 2 ml. The mixture ws then inubted for In 1 min t 37 C, fter whih 1-ml smple Fig. 1, the time ourse of the effets of the ws pipetted into 4 ml of boiling.1 M tris(hydroxymethyl)minomethne (Tris) buffer, ph 7.75, ontining ells nd E. oli is shown. The tretment tretment on ATP levels in humn red blood 2 mm EDTA to extrt ATP. From non-pretreted resulted in the omplete elimintion of ATP smples,.5-ml portions were tken diretly from the from the red blood ells within 2 min. The rte smple nd extrted in 4.5 ml of boiling Tris buffer. of ATP brekdown ws identil to tht ob- Downloded from on Otober 2, 218 by guest

3 VOL. 1, 1975 DETECTION OF BACTERIURIA : < _1-q o,i _ ~v "u C - - o 1 - o E' 5 ui } Time (min) FIG. 1. Time ourse of hnges of ATP levels during tretment of red blood ells nd E. oli with Triton/pyrse. Wshed red blood ells (), 5 x 16 ells/ml, nd E. oli (), 19 ells/ml, were inubted in sterile urine (diluted 1:1 in distilled wter) ontining pyrse (.2 unit/ml) nd Triton X-1 (.1%) t 37 C. At the times indited, 1.-ml portions were extrted nd ssyed. served with free ATP; i.e., the detergentindued lysis of red blood ells ws not rte limiting. The upper urve in Fig. 1 demonstrtes the effet of the sme tretment on E. oli. With the bteri, essentilly no effet on the ATP level ws observed, exept for slight derese t the strt of the inubtion, possibly resulting from metboli disturbne used by 'the trnsfer of the bteri to the inubtion medium. In the following experiments, n inubtion time of 1 min ws used. In the experiment shown in Fig. 2, four different types of humn ells were treted t vrious onentrtions of Triton X-1 in the presene of onstnt mount of pyrse. For omprison, strin of E. oli ws inluded. At.1% Triton X-1, the loss of ATP ws omplete with ll types of humn ells, wheres E. oli ws not ffeted even t higher onentrtions. A onentrtion of.1% Triton X-1 ws hosen for use in ll of the following experiments. In Tble 2, the effet of Triton/pyrse tretment on vrious ells of humn nd mirobil origin is summrized. The tretment resulted in n lmost omplete loss of ATP in ll humn ells, wheres the mirobil ells were more or less resistnt to the tretment. In the bsene of Triton X-1, no effet of the pyrse ws observed on either type of ells. Thus, in ll of the model experiments, the Triton/pyrse tretment resulted in the desired differentil effet. Subsequent experiments were performed to evlute the effet of Triton/pyrse tretment in linil mteril. To determine whether nonbteril ATP in Oos i 15 Conentrtion of triton X- 1 (perent) FIG. 2. Influene on ATP level of onentrtion of Triton X-1 on tretment of vrious types of ells with Triton/pyrse. Experimentl onditions s in Fig. 1 exept tht vrious onentrtions of Triton X-1 were used nd the inubtion ws for 1 min. The following numbers of ells/ml were dded to the inubtion medium: red blood ells, 5 x 16 (); mononuler white blood ells, 15 (); HeL, 15 (A); humn embryoni lung fibroblsts, 3 x 15 (A); E. oli, 19 (C]). TABLE 2. Cellulr ATP levels of vrious mirobil nd humn ells before nd fter tretment with Triton/pyrse Cellulr ATP level (moles x 118/ell) Cell Before After tretment tret- ment E. oli Proteus mirbilus S. ureus Streptoous sp Cndid lbins Red blood ells... 6 Mononuler white blood ells Polymorphonuler grnuloytes 17 HeL ells... 8,8 Humn embryoni lung fibroblsts The experiment ws performed s in Fig. 2 with Triton X-1 onentrtion of.1% nd n inubtion time of 1 min. The following numbers of ells/ml were dded to the inubtion medium: bteri, 5 x 18 to 1 x 18; C. lbins, 2 x 16; polymorphonuler grnuloytes, 1.5 x 16; other humn ells s in Fig. 2. urine ws free or intrellulr, we treted 26 ulture-negtive speimens with pyrse in the presene nd bsene of.1% Triton X-1. The results re presented in the histogrm of Fig. 3A, depiting perentge ATP remining fter Downloded from on Otober 2, 218 by guest

4 4 THORE ET AL. o Ln 4) I- C IL - n J. CLIN. MICROBIOL. In this experiment, the differene between posi- A tive nd negtive urine speimens is pronouned nd most of the negtive speimens fll below the detetion limit of the ssy, whih ws pproximtely 1-1 M ATP. Positive speimens re not ffeted or even show slightly inresed ATP levels, s lso shown in Fig. 3B nd severl of the model experiments. The 7-T--T--Tmedin ATP levels in the Triton/pyrse fl 4 < treted urine speimens were <1-1 nd 1.5 x 1-7 M in the negtive nd positive speimens, B respetively. The dignosti utiliztion of the differene in ATP level between ulture tegories neessittes the determintion of the ATP onentrtion limit resulting in the most effetive disr n S rimintion between ulture-positive nd -neg-, II~ 1, I,Ii [1I.III tive speimens. To filitte the determintion of this limit, the dt of Fig. 4B were replotted Remining ATP ( perent) s shown in Fig. 5, whih lso inludes 15 FIG. 3. ATP ssy in 26 ulture-negtive (A) nd dditionl urine speimens. The ordinte de- (B) linil urine speimens. After sribes the perentges of eh of the three 14 ulture-positiyve 1:1 dilution inx distilled wter speimens were sub- ulture tegories whih would be lssified s jeted to the fo trols (1% remining.owingatp); tretments: inubted nontreted with on- "luiferse positive" if the ATP pyrse onentrtion limit is rbitrrily vried long the bsiss. (.2 unit/ml), 1 mmn, 37 C (open brs); s bove but in mt sbrrlyvreonthss. the presene of.1% Triton X-1 (filled brs). In the present mteril, the bility of the ssy to identify the ultivtion-positive speithe tretment;s. Both types of tretments re- mens is dequte up to n ATP onentrtion sulted in deresed levels of ATP in ll spei- limit of pproximtely 4 x 1-9 M, s shown in mens. HoweveZr, in most speimens the tret- Fig. 5. When this ATP onentrtion is defined ment ws o] nsiderbly more effiient when s the lower limit for smple to be regrded s Triton X-1 ws inluded. Thus, it ppers "luiferse positive," 96% of the ulture-posispeimens mjor prt of the tive speimens re lssified s "positive" by tht in mny nonbteril AiTP my be ellulr sine deter- luiferse ssy, wheres only 2% of the ul- for the brekdown of ATP by ture-negtive speimens hve ATP levels ex- gent is requiretd pyrse. eeding the vlue of 4 x 1-9 M. The perentge As shown in Fig. 3B, the effets of the sme of borderline speimens lssified s "luiferse tretments on ulture-positive urine speimens positive" is lwys higher thn tht of the re quite different from the effets on negtive ulture-negtive speimens. speimens. Grn the verge, these speimens In Tble 3, verge nd medin ATP levels inrese rtherr thn derese their ATP ontent per ell remining fter tretment with Triton/ s result of tiie tretments, nd there is lso no pyrse re shown for different bteril speies obvious differ( ene between tretment with or in ulture-positive urine speimens. As omwithout Triton X-1. prison, results from lbortory experiments The histogrnm of Fig. 4A presents the ATP with bteril strins grown in sterile urine re levels before Triton/pyrse tretment in inluded. In spite of lrge vritions in ellulr lrger prt of the mteril studied, 319 spei- ATP ontent within eh group of orgnisms, mens in tot] l. It is evident tht there re there is striking similrity between the verdifferenes in ATP ontent between negtive ge ATP levels per ell in the linil speimens nd positive urine speimens. Medin levels of nd those obtined in the model studies. In the ATP were 2.' 5 x 1-8 nd 1.4 x 1-7 M, se of Proteus, there is tendeny to low respetively. IHowever, onsiderble overlp- vlues, nd the lower ATP ontent of this ping ours, ind the ATP ssys under these orgnism ws lso observed in the model experinot give relible indition of ments. onditions do the ourrene of bteri. In Fig. 6, the ATP ontent of the 86 positive In Fig. 4B, the sme linil mteril ws urine speimens shown in Tble 3 is plotted nlyzed fter tretment with Triton/pyrse. ginst the number of vible bteri deteted Downloded from on Otober 2, 218 by guest

5 so E z < :Jf <i L1 mlmi I -8 I Li[ 1 II -7 1 A DII II id-6 'U I D ATP onentrotion in urine ( M ) FIG. 4. Effet of Triton/pyrse tretment on ATP levels in 319 linil urine speimens. (A) ATP levels mesured without Triton/pyrse tretment. (B) Speimens treted with Triton/pyrse s desribed in Mterils nd Methods. The speimens were lssified ording to ulture: positive (>15 ells/ml), filled brs; negtive (<14 ells/ml), open brs; borderline (14 to 15 ells/ml), hthed brs. The totl numbers of speimens in eh of these tegories were 94, 187, nd 38, respetively. The proportions of speimens in eh tegory ontining vrious levels of A TP re represented in the histogrm. In LJ.)._ o- U.) U)l o i d n. B Downloded from on Otober 2, 218 by guest -4- L) Limit set for positive tuiferose ossy (M ATP) FIG. 5. Speifiity of the luiferse test. Limits of ATP onentrtion set to signify "positive" luiferse ssy results re given on the bsiss; the ordinte represents the orresponding perentges of "luiferse positives" in eh of the three ultivtion tegories:, ulture positives (123 speimens);, ulture negtives (29 speimens);, borderline speimens (56 speimens). The mteril is tht presented in Fig. 4B with the ddition of 15 other speimens ssyed only fter tretment with Triton/pyrse. 5

6 6 THORE ET AL. TABLE 3. A TP ontent in some bteri identified in urine speimens nd grown in the lbortory in sterile urine Cellulr ATP level (moles x 118/ell) No. of Urine Model ptients speimens expt Avg + Me- Expt Expt SEM din 1 2 E. oli ± Proteus sp ± Strep.tooDus sp ± Misellneousb ± Totl ± The urine speimens nd lbortory ultures were treted with Triton/pyrse s in Tble 2 nd Fig. 3 nd 4B. Only speimens re inluded in whih ulture ounts were urtely determined. One ulture-positive speimen (E. oli) not ontining mesurble mounts of ATP ws exluded. b Inluding Klebsiell (4), Stphyloous sp. (3), Pseudomons sp. (4), vrious mixed infetions (1), nd unidentified bteri (7). by quntittive ulture. The rnge of vrition in ATP ontent enlosed by the broken lines is of bout one order of mgnitude t eh level of mgnitude. In spite of the lrge vrition, reltionship between bteril level nd ATP onentrtion my be inferred. However, the reltionship does not pper to onform to the diret proportionlity desribed by the theoretil solid line lulted from the verge ATP level per ell (3.6 x 1-18 mol; f. Tble 3). It my be noted tht in speimens ontining high levels of bteri there is tendeny towrds ATP levels lower thn the verge, in ordne with results on lbortory-grown bteri (3). DISCUSSION A serious obstle in the pplition of the luiferse ssy to mny mirobiologil problems hs been the inbility to differentite between bteril nd nonbteril ATP. The present study shows tht in model systems this my be hieved by simple hemil tehnique bsed on the ombined tion of detergent. Triton X-1, nd n ATP-hydrolyzing enzyme, pyrse. When pplied to urine speimens, the Triton/ pyrse tretment strongly enhnes the differene in ATP level between ulture-positive nd -negtive speimens. Furthermore, the mount of ATP remining in the treted urine spei- J. CLIN. MICROBIOL. mens is reltively well orrelted with bteril ounts, nd the ATP ontent per bterium is similr to results obtined in model experiments. This is n indition tht the differentiting effet of the Triton/pyrse tretment obtined in model systems my be representtive lso of the effet in the linil mteril. Thus, it ppers likely tht the ATP remining in the Triton/pyrse treted urine speimens is minly of mirobil origin. Vritions in ATP level per vible unit observed in treted urine speimens my, mong other things, be used by differenes in metboli rte nd bteril ell size, ffeting ellulr ATP pools (3, 1, 12). Furthermore, ell ggregtion, using erroneous ulture results, would be expeted to be ommon in this type of mteril. A level of 15 bteri/ml of urine hs been shown to be n importnt riterion for the -identifition of ses of signifint bteriuri (8). The verge ATP ontent in single vible bteril ell ws shown to be pproximtely 3.6 x 1-18 mol/ell. Thus, level of 15 bteril ells/ml of urine would orrespond to n ATP onentrtion in the urine of 3.6 x 1- ie M, pproximtely fourfold higher thn the lowest ATP onentrtion detetble in the ssy s set up by us. For prtil purposes, however, the riterion for "luiferse-positive" ssy my be shifted towrds somewht higher levels of ATP, resulting in lower number of "flse positives," s shown in Fig. 5. Thus, in ny prtiulr linil mteril it my be worthwhile to estblish empirilly the onentrtion limit of ATP resulting in the highest degree of speifiity. "Flse-positive" results obtined with the luiferse ssy might prtly be used by filure to detet ll bteri by ulture beuse of unusul nutrient or nerobi requirements or beuse of the presene of ntibiotis in the urine. In the borderline group, the reson for the reltively high numbers of "flse positives' might, in ddition, be lk of preision in the referene method, mking the orret lssifition of these speimens somewht unertin. The present study hs shown tht luiferse ssy of ATP my be potentilly useful method for bteriuri sreening. The sensitivity is suffiient to detet the presene of 15 bteri per ml of urine, nd the speifiity llows the elimintion of mjority of the ulture-negtive speimens, in totlly unseleted linil mteril. The ssy proedure is rpid, inexpensive nd simple. Commeriil equipment for the bioluminesene ssy is vilble, nd the tehnique my be further Downloded from on Otober 2, 218 by guest

7 VOL. 1, 1975 DETECTION OF BACTERIURIA 7 1 o z _2 L- 1 1' A A A A O / I t Level of bteri (ells/mt ) FIG. 6. Urinry ATP levels s funtion of bteril ontent. The dt in this figure orrespond to the dt of Tble 3 obtined from 86 ulture-positive linil urine speimens treted with Triton/pyrse. The solid line is lulted from the verge ATP ontent per ell in ll of the 86 speimens; the broken lines onnet 95% onfidene intervls for lulted verge A TP levels in groups of speimens not further thn four times prt with respet to bteril ounts. Severl suh lultions were mde by regrouping the vlues, nd the lines onnet the end points of the intervls. Symbols:, E. oli; A, Proteus sp.;, Streptoous sp.; nd, misellneous bteri s defined in Tble 3. simplified by utomtion (17). Further studies on the luiferse ssy s bteriuri sreening method re underwy. They inlude the investigtion of lrger linil mteril nd the development of equipment nd ssy proedures suited for routine work. An investigtion is lso being mde to determine the use of remining disrepnies between ATP ssys nd ulture. ACKNOWLEDGMENTS This work ws supported by the Swedish Bord for Tehnil Development nd by grnt to one of us (A.L.) from Fdrsvrsmediinsk Forskningsdelegtionen, Stokholm. We grtefully knowledge the skillful tehnil ssistne of Ann-Chrlotte Erisson. LITERATURE CITED 1. Chppelle, E. W., nd G. V. Levin Use of the firefly bioluminesent retion for rpid detetion nd ounting of bteri. Biohem. Med. 2: ,'" A o l o /. - A 2. Chppelle, E. W., G. L. Piiolo, nd R. H. Altlnd A sensitive ssy for flvin mononuleotide using the bteril bioluminesene retion. Biohem. Med. 1: Forrest, W. W Adenosine triphosphte pool during the growth yle in Streptoous felis. J. Bteriol. 9: Hmilton, R. D., nd. Holm-Hnssen Adenosine triphosphte ontent of mrine bteri. Limnol. Oenogr. 12: Hobson, P. N., nd R. Summers ATP pool nd growth yield in Selenomons ruminntium. J. Gen. Mirobiol. 7: Holms, W. H., I. D. Hmilton, nd A. G. Robertsson The rte of turnover of the denosine triphosphte pool of Esherihi oli growing erobilly in simple defined medi. Arh. Mikrobiol. 83: Huzyk, L., nd D. J. Clrk Nuleoside triphosphte pools in synhronous ultures of Esherihi oli. J. Bteriol. 18: Kss, E. H Bteriuri nd the dignosis of infetions of the urinry trt. Arh. Intern. Med. 1: Knowles, C. J., nd L. Smith Mesurement of ATP levels of intt Azotobter vinelndii under different Downloded from on Otober 2, 218 by guest

8 8 THORE ET AL. J. CLIN. MICROBIOL. onditions. Biohim. Biophys. At 197: Levin, G. V., C.-S. Chn, nd G. Dvies Development of the firefly bioluminesent ssy for the rpid quntittive detetion of mirobil ontmintion of wter. Tehnil Report TR-67-71, Aerospe Medil Reserh Lbortories, Wright-Ptleron Air Fore Bse, Ohio. 11. Levin, G. V., J. R. Glendenning, E. W. Chppelle, A. H. Heim, nd E. Roek A rpid method for detetion of miroorgnisms by ATP ssys: its possible pplition in virus nd ner studies. Biosiene 14: Levin, G. V., E. Usdin, nd A. R. Slonim Rpid detetion of miroorgnisms in erospe wter systems. Aerosp. Med. 1: Shrpe, A. N., M. N. Woodrow, nd A. K. Jkson Adenosine triphosphte levels in foods ontminted by bteri. J. Appl. Bteriol. 33: Strnge, R. E Rpid detetion nd ssessment of sprse mirobil popultions, p In A. H. Rose nd D. W. Tempest (ed.), Advnes in mirobil physiology, vol. 7. Ademi Press, In., New York. 15. Strnge, R. E., H. E. Wde, nd F. A. Drk Effet of strvtion on denosine triphosphte onentrtion in Aerobter erogenes. Nture (London) 199: Trnvik, A Isoltion of lymphoytes from blood. At Pthol. Mirobiol. Snd. Set. B 78: Vn Dyke, K., R. Stitzel, T. MClelln, nd C. Szustkiewiz An utomted proedure for the sensitive nd speifi determintion of ATP. Clin. Chem. 15: Willims, M. L. B The limittions of the Du Pont luminesene biometer in the mirobiologil nlysis of foods. Cn. Inst. Food Tehnol. J. 4: Downloded from on Otober 2, 218 by guest