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Microhiological cbaracteristics of natural minerai water ()
W. SCHMIDT- LORENZ
Lt~boratory of Pood- J i irrobiology in the Institute of Food cience ul the S toiss Federa/ l nstitute of T eclmology (ETI-l), Z rtrich.
Carbon dioxidc- frec minerai water, like an y otbt: r natura! spring-water , is ncver entirely free from hacteria; but it is always vcry low in bactcria. After the bottles are fìlled, in a few days or wccks, depending upon thc en vironmental conditions, t bc few prcsent bacteria incrcase markedly . Often, thc number of bacteria is as high as lO,OOO per mi, and cven as high as 100,000 per mi. In this rl'gard, a large number of quantitutive analyses bave alrcady been carried out [1- 7]. Quantitativc analyses hased on the v arious typcs of bacteria and on their sp ecific propcrt ies, are presently sti li scarcr in numbcr [8, 9].
For the microbiological characteriza t ion of carbon dioxide-free minerai water , the specifìc microbiological anJ <'cological conditions in this spccial substrate, mus t be chiefly considered. But since no analyses of minerai water exist that are oriented towards ecologica) fac tors, it wiU be necessary to use as a term of comparison the results relative to other waters low in nutrients, which < xist in nature. Qui te similar ecologica) conditions are found in free- flowing underground waters, in spring- water , in ali oligotrophic waters anù also in tbc sca watcrs tbat are ex tremely low in nutricnts.
Comparable ecologica) conditions are also found in drinking water supplied b y public plants, a lthough tlrinking water , unlike na tura! minerai water, is a technologically processcd product. Not cven drinking water is a lways totally sterile; it is comparatively low in bacteria, but they are almost always present in an amount l1igher than in minerai water.
The known values indicative of the number of acrobic colonics in th<' order of magnitudc of 20 to 100 per ml, are mcrely technological qualita tive features for 1 he qualitative contro! of tbc drinking watcr supply plant. Qui te oftcn, evcn if the quality is cxcellcnt a t the time of supply, the drinking water that reachrs the consumer's tap is bactcriologically of a much lower quali ty.
(* ) Ger man to>xt transluted iut o Euglish IJy the Istituto Sup(• rioro> rl i Sauità.
.4nn. l kl . .'>uptr. S!llli l4 (1970) 12, 93- l12
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_\ .. in tlw <'Ut- t· uf tlw hottlt·:- fìJI('(l with mint·ral watcr . uh·n iu that of
drinkiug wat,·r a l'ter a protrndctl r-.ta~ 111 tllt' pipe:- irH~vi t uiJI~ Lring:; alwul n
~··•·ondar~ iru' rt ' H ~>t' in the uumi,C'r of hal'lt'ria prl''t' lll in i t. l n aJI tl11• mn..,t
irnportnnl pijwluw :.yl'i tl'ln .... !Joth pri' ati' a01l in<lu~> triul. 111an~ p ipt· :;t·c tion~
<''-i~ t iu '' hidt '' alt'r s t ay.., for lou g JWrioù.., jii. 10 l~J. A partirularly rua~o:-h· t · iocrnuw in lhc unml11·r of bo:H'll'ria i:- l'I'JWaledly
IIU!' t'T\'I'd in int•·•·nal watl'r pol' t- treatrnr nt plant,., a• in filt Pr~. plw:::phat•·
fet·dn,.. , itm- t•:-.chan gt•r,. a11tl prt·~;, urt• 1nr1k" [5, 13- 16). Thi:- ;.t;condar)
growth of' !Jar·tC'ria in llw pipl'lin<' syt>Lt'lll ma~ e\ t•n slart from within tiH' loC'nl di ., triiiUtiOII nt' l\•tlfl :md continue in thl' C'fll&,.,umer,.' nrt,,urb [lì- 221. ·\ traajor rolt· il' r••rt uinly playcd h y tlw g<'llt'ral t•utroplaizatinn of tlri11k ing \\alt•r tbat i' a <'tHnparatiq· f' llridwwnt in Hilralt· ami plws phat•· - hul
rhit·fly uf son 11· tract• IHnouu ts of mguni(' !'uln•tnn l't'~o> [10. 23).
In Llw phorrnat't' u t iral indublf) , for a numiJcr of n·a~-Cm'. an additiunal l't'W'IH'ra ti o n of c.l rin k ing wuter ì;, al n·ady llt 'l't'~su ry [25]. l n ,·ie'' of t Ili'
dctl'rioralion nf t h•· bar tl'fit,logiral quali t~ of drinking '' a tf'r tlaat will JJt' iu cr!'asi ngl~ ft·IL in tlu· futuri' , s imilnr prohlr·rns ''ili rtc<·ur in rnan~ oth<'l'
fit•ld .. of tllf' fuori indu;: t r~ . Fur t bi~ r1·ason. tlw footl micruhiolo~~ mu«l
alsc• dc·ul "ith tltt · l'rnt.lc·na '" a largt· r rxlcnt. Tht• cn riJI\Il dioxitll'- fn·t· millt·ral ''att>r i;: 11111~ parlly t'll lllJlaraiJit·. "lwn )H)ttlt•tl. with l'ti ll dri11kin l! "ah• r. fur tlw t'Uiltf' ll t in nutri t'll t ;, j .; n ·marl..al,h lo" rr. Ho\\ t• n · r. owì11~
t u tue t•mnparatì' •· l~ s tt•atl~ t ' Il\ irnnuwntal coud ition~. thi ~- n·prt';.l'nt;. a
~ootl mudl'l for tlw mi<·rul,inlo~iral pru<'<'Sl>e~ - 't · r~ lllll t h alikt· in prjrH·i
pl<' - thut takt• pia•··· in s t ili drinking wall•r. whid1 is alneost always tmhject
I n notahl t• changt•io in t'Il' iron m r ntal cundition~.
T o tlw m icrnllor11 nf carlwn dioxic.Jc fn•P, Lotti ed mim·ral ''ate r. then • t~o-cxist al all t inw~ l\\'o groups of baf'lt•ri~a , much ditTc·rrnt from unt' anothcr
Loth in origiu and propcrtil'!o:
l . Allochthonous or c·cmtamiuation ruicroflora:
2. Au tllchtltotlous mirrollora. or micmflora pmp•·r of lht· Euh~tratt· .
l . 11/ocltthorwu ... or contami rwt i o n mùro.flom
Tbc· allul'htbotltlll " haclf•ria that get into tlw minerai wat1:r during
hott liog actually play a I:H'Cnndary rolt·. \'\'•· musi howpvf· r d .. al with t hem bri('fl y in orÙl'T that u clt·ar- cut dis tinr tion may IH~ ma d t· L et" t•cn tL1•m ami
th c au t urhtbouou~ flnra p mper of the ~ubstrall·.
During th1· mirwral watl'r bntùiug. tlll' fuiJm,ing pollutinn :oiHUC'l'"
enter tJw p lay: a) t·outaiurr!-: b) <'up s. r ) uir l'li\ iroument. aud d) lwttling
plani ~.
Conflrim•r.~. Curlw11 dio:-.idl' frl' t' minerai "ate rE un· nowaday~ almost
alwn)>< Lnttkd inlo t hrowa\\ay pl a~-tit• bottl t·"· Tlw U " l' of gla!:iS bnttle~> i ~o.
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:- teadily losing ground. 'fhis is qui te a satisfactory c le\ elopmcnt fro m the sanitary angle, fo.r tbe wasbed glass hottles in particular ancl their caps, rc:prc:.entrc.l the main pollution source [2, 1t, 25-28).
As a rulc, tbc plastic bottles are producetl frorn granulate ;;hortly bcfore bottling. In the oxtrusion stage, t empcraturcs as high as 230 oc are used fo r some minutes. In thjs way vegetative bactcria, conic.lia and, for the rnost part, a lso bactNial cndospores, are kiUed. Thc experinncc acquired with dry s teriJization by means of heat shows th at such temperalure-timc conditions are not sufficient to kill endospores, or to make them inactive with a hi gh dcgrcc of ccrtainty (291. But thi.s point is stili debatcd. Some uuthors [30-32] succeeded in p.roving a total inactivation of :.pores in the ··xtrusion phase, whilst otbcrs wcre unsuccessful [33, 34]. lt is quite certain that, as soon as they are produced, extruded plastic bottles are very low m bacteria and are contaminated only by a few hacillus endosport!S.
Caps. - As a rulc, caps are also made in m ost cascs of soft plastic materials, and are •nanufactured shortly before they are used . As in thc case of bottlcs, thcy may at worst he con taminatcd by some endospores. When the bottles are conveyed to the op.:n tank of the bottling eq uipment, as is most commune, a continuous secondary po lluticm may take piace either through the air or through thc parts of the machinery. In <·asc> of a mechanit>al brcakdown, the caps should not lw tou checl with the bare hunds.
Air. - The maio pollutiou of bottles a nd caps uccurs viu tbc en vironment air and via the comprcssed unfilterec.l ai r. whcn , bhortly bcfore bottling, air is forcihly Jet into the bottles and then l1•t out of them. Such a pollution may be uotably reduced by means of a ste rile 6Jtcring nf compressed ai.r [35]. The polluting bacteria are chiefiy thc spores of bacilli, conidia and gram- positivc bacteria most resistant to ùry conditions, such as micrococci. Experience sbows that the gram- negative bacteria are scarce in the comparatively humid air of' bottling plants (35, 36].
Bottling plants. The fast-action bottling heads must be always wcU luhricatcd. In the thin layer of oil, bacteria may surv ive and, undcr given conJitions, tbey may even multiply. Thcy may evcn gct into the minerai water during bottling. A higber contarnination risk cxists if thc equipment is to be repaired or if t h e lwads to be changcd are handled with unprotccted hanrls.
Transitory contamination jlom.
Ln the modero processes fo r the bouJjng of carbon clioxidc-frcc minerai wat cr in throwaway plastic containers, microhi<Ù contamination is much lower t h an hf'fore, wlwn gluss botti es were used. J n some cases, pollution
l 1111. }st. 'iuper. S ullild (l U76) 12, 93- 112
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96 BATTJ::RIOLOCIA DELLE ACQL'J:: llfi"'ERALI NATURALI
may b e furth cr reduced by adopting protection m <'asures . Unquestionably. aseptic bottling would be lecbnically fl'asibl!' today, but for mic robiolugical rea ons the requests put forth at timi'S in this direction appear lo bi' unrcalisti<". On the one band. thl' product to be hottled is not free from bat·· teria, on the other hand, the dct ermining factor is not so much constitutcd by the absolu te ordt·r of magnitudt' of thr microbial contamination , as by th <' specifir behaviour of thc· con tamination flora in the bottled substratt•.
The carbon- dioxide- frt•(· minerai water is a substratc which is mu ch unfavourab](' to exarting microorganis ml'. 1t only contains trace amount :Qf organi c matter in concentrationl' much lower than the IO\\ est limits of thc most sensi}Jit' cht·m.icaJ Y('rification ID(' thods, namcly. more or l es~ iu tb c ppb rangt'. In this substratl' , a more or less notablc percentage of polluting microfiora. rapidly dif'S. A furthr·r perccntagt\ cither is fl lowly madl' inactive, or rf•tains for a loug tint(' thf• ability of r<'producing. But a r eproduction propcr vcry seldom occur:;. H cncr. it may be rcgarded as a prevailingly trunsitory pnlluting fiora.
Tht· numher of exacting: bacteria. kill('d or made inactivc in substratc~
-extremely poor in nutrienti- , is iu relation to a largl' number of factors ami may range between widf• limit s. As far as l could establish , no analyscs of thc carbon dioxidc- fn·c minerai water han• been madc available so far . But it has emcrged from rnany works that a largc number of bacteria arP made 50 % inactiYc within 20 min, and 90 % inactive within l h , in distill ed watcr, phosphate buO'er solution , physiological solution of common salt, Rjnger 's solution , and a lso tap water. This does not apply m(•rely t o the various spccies of bacilli, for enterobacteriaccae and for many othcr bacteria. hut a lso t o Pseudonumas fluorescens and aeruginosa [37-42].
The cells of tbc bacteria that survive this environmental changc, which is similar to a shock, are slowly mad(' inactivc and may b e capaiJie of r epro.c:Jucing eYen for a lon g timc. Thorough studies havl' been carried out to try and clear up th(' debatt•d question of the indicating value of E. coli ancl the coliforms, with regarcJ to tlw fccal pollution of water. Thr resultto are partly contradictory. lo most cas<"s, hnwever. it ·was founJ that al.so in the cas(' of Entrrobactaiaceaf' in natura] water a r educt ion takt•s piace (morP or k ss graduai according lo tl11• enYironrnt•ntal conditions) in tlw numher of tlw cells capah le of reproduring. whils t no rcproduction l'\·rr OCl'UTS ( 43- 50).
Soil hac tt·ri a a!' wt'l l a!' E. coli mar also n·produc(' in extrrmd~ clilutt·d lahoratory media [51 s:~J. Tlw lowest limit- conce·ntration" for ~rowth for E. coli w<•re nwasurt•d al 0.28- 0.5 mg organil' m atlt'r fl (43. 54- 56). Zobdl antl Grnnt [58] haYe founù growth of K coli. Stapll:ylococcu..~ citreu.s, Hacillu .~
mcgatherium. Proteus vul~nris and also of Lacwbacilhts lactis ('\'en in solutions with only 0.1 mg of glucosi" fl.
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SCRMI DT- LORENZ 97
Similar experiments on minerai water have so far only been performed hy Buttiaux and Boudier (8]. The AA. having Jet to st and E. coli and Klebsiella nerogenes in water passed through an autoclave or filtered and moùerately mineralized, did not fìnd any signs of reproduction, but oniy a slow rt·duct ion of the viable cclls after 3 months at -L 140C. Converseiy, a surprising net increase was observcd in highly rnineralized water.
T n some expcriments l ha t we carri ed out, person ally after placing E. coli, Pseudomonas aeruginosa, S a/monella, staphylococci and strains of bacilli in an initial concentration of 104 cellsfml in untrcated and bottled minerai water, we fo.und no growth, during four wecks a t + 20 °C, but only a r<low decrease in the number of viable cells. Some given effeets, antagonistie or competitive in respect of the autochthonous flora, probably enter thc play.
Summing up, we must state tbat the poUuting microllora tbat gcts into the carhon dioxide-free minerai watcr during bottling, is prevalently transitory, i.e. no multiplication nccurs as a ru le h ut only a more or Iess graduai inactivation of poUutants.
Pt>rmanent contamination flo ra.
From thc sanitary angle, the problems involving the bactcria that might he dc(ined « pcrmancnt contamination microflora », are much more difficult to solve than those involving the bacteria of t he t.ransitory contamination rnicroflora. The formcr are a specifie group of gram- negative bacteria that may dcvclop rapidly and ahundantly in substrates extremely poor in nutriente. They are, so to say, « oligocarho-tolerant ». Th eir most important rcpresentative is Pseudomonas aeruginosa. Such species of Pseudornonas, as, for instanec, Pseudomonas cepacia (multivorans) anc.l certainly also an (•ntirc group of strains of P seudonumas JluorPscens, also faU within this group. They usu ally originate from man anc.l from animals aud thcy are ali clcariy mcsophilic. Therefore, they can dcvelop at f- 37 °C but aiso at higher tem pcratures, up to + 42 °C. Prf'valcntly, howev•·r , they are not psycbrotropbic.
The potcntially pathogenic speci('s of P seudomonas, ueruf{inosa and ct>pacìa (rnultivorans), are acquiring an increasing importancc as dreaded pathogt'nic agents of secondary infections rcsistant to antibiotic tlrugs in thc hospita l sector. Pseuclorrwnas neruginosa is also likely to be the pathogenic agent of unspecifi c food inloxications. In rccent years, a la rge numbcr of analyses havo shown that its reduccd requiremcnts anù its marked ahility of rcproducing and surviv ing in any type of water ]ow in nutrients, as weH as its strong resistance to disinfectants, makP possible its widesp rcad use in ali clinical sectors.
.l nn. lrt . ,,,,tr . . ~a11ità Il !1;6) 1%. !13 112
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9!! UATT ER IQLOt: IA DELLI:: ACQlfE Ml~ F.II AT.I :'>I ATU RAI.l
A massive incrcase of Pseudomonas aerugirwsa, and a protraete<] pcriod of survival in a high number of cell:s, were provcù: in distilled watc r (57-61) , in dcionized water ( l 6J and in pure minera i salts solutions [61 , 62 j. The sam<· also applics to Pseudornonas cepacia (63-66) which was found even in disinfectant solutions [64, 67, 68].
The strains of Pseudomonas aerugino,~a and cepacia grown in distillcd water prcsent significant m orphological and physiological difl"erences in rcspect of similar culturcs in conventional nutrient media [60, 66, 69]. In distilled watcr, cells are much smaller and unfiagellated. With a number of cells highc· r than 107 /m], water is s tili perfectl y clear. After the transfer into distilled water , without any « Jag » phasc, a rapid multipli cation occurs. Aftcr th(· inocula tion from standard laboratory cultures into distilled water , there takcs piace, instead , a d ecrease in tbc number of bactcria for some decimai potencirs beforr reproduction begins. The cells from distilled watcr are liablc to grow further up to + 12 oC or -L 18 oc. Thc cells from riclwr nutrients do not grow, ins tead. Furthcrmore, the distilled waler cells clcarly show a higher rcsistancc to disinfectants .
These pcrmanent poUutants « oligocarbo- tolerant » an: clearly by far more dangerous, from tb<· sanitary viewpoint, than the transitory contaminating fiora , for they can prcsumably also incrcase in minerai water . lt is thus necessa ry to adopt aU the suitablc measurcs to prevent a contamiuation , particularly of the bottling-plant resulting from the operators working bare- handed. In case of a shut-down of the plant, baclt>ria may enter the pipclines and multiply right in the t erminals, in the tanks and in other places where watcr stays for a whilc. It is in these placcs that the running minerai water roight undcrgo a conslant contamination prior to bottling.
lt is difficul t to ascertain whethcr, and to what extent, such a danger actuaUy exist s in practic<". At any rate, and as far as I know, the numbcrless bacteriological controls havc never shown the presencc of Pseudonwnas aeruginosa or cepacia in bottled minerai water, in signjfìcant amounts. lt is likcly that the autochthonous reproducing bactcrial flora produce an inhibition by competition with antagonistic effcct.s. Contrary to untreated minerai watcr, distilled watcr and other t ypcs of water , in thc clinical sector , are primarily stt>rile and after infcction, a lmost always a pure culture of these P seudomonas is present. I t is qui t e possiblr, therefore, that the autochthonous bactc rial fiora in carbon dioxide- frer minerai water, afrords an effective protection againsl the reprodu ction of such p crm anent pollutants.
An effectivc bact eriological contro! for detect ing this spccifi c contaminating fiora is alrcady possible by counting thc cultura! colonies on a rich nutricnt medium and by incubating at + 37 °C. If thc indicativ e valuf• of « less than l colony/ml » , recommcnùed by thr Scientific Commission of thc
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3rd International Symposium on Food }ficrobiology, held in Evian in 1960, is complied with, then an adequate security from thc sanitary angle should be guarantecd.
2. Autochthonous microflora, or microjlora proper of the Sttbstrate
The minerai water coming direc~ly from the spring and immediately after b ottling, is always very low in bacteria. The aerobic colonie count is in the order of magnitude of l-2/ml [2], if the colonie counting methods are used, such as, for instance, pour plating with standard nutritive media an d an incubation at 20°.<or + 22 °C or filtering by means of a membrane filter with pores 0.5 !J.ID large and filter incubation, still on standard nutritive media. Using cultural media very low in nutrients, with surface culture procedures and longer periods of incubation, or using enrichment media low in nutrients in MPN procedures, also those bacteria are considered that are sensitive to higher substrate concentrations and to the beat shock in pour plating procedure. As a rule, the number of colonies is higher, it is in the range of lO to 20 ml, and should correspond to the actual number of bacteria.
If membrane filters with pores 0.1 !J.ID large are used, the number of colonies increases remarkably. Obviously, a large number of cells are smaller than 0.5 (J.ID· In point of fact , the unusually small size of cells is a special feature of all autochthonous microflora. Cells are usually large only l /3 to l /4 of the norma] bacterial cells, with a diameter comprised between 0.2 and 0.5 !J.ID. They are irregularly coccoid and it is often difficult to recognize them at the microscope as bacterial cells . Even in the case of an intense increase in bottled water, the cclls become only slightly larger, and thus even if the number of cells is very high no water clouding occurs. These so-called degenerated forms, that are however stili capable of reproducing, are characteristic of many biotopes extremely low in nutrients in nature [70, 71]. In the long term, the mean number of cells capable of reproducing in continuously drawn spring water, is rather constant. In the meanwhile, however, longer or shorter oscillations may certainly occur, with bacterial numbers much lower or even higher and with the composition of species temporaneously changed to a marked extent.
Origin of autochthonous microjlora.
The question repeatedly put fonvard as to the origin of bacteria in spring water, cannot be answcred in a clear- cut way if experimen tal methods are used. According to all the microbiological-ecological experiences carried
.l 1111. ht. S"per. Srmitll (1976) IZ, 93-112
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100 RATTERIOLOC IA DE!.LE ACQliE lfll'iERAl.l NATli RAU
ljQ far, it SIIC)uld h«· rathcr certain that this mieroflora multiplics rurectly in thc water of tlu; cntirc spring, and that it renews continuously. lt sccm!:ò therrforc quitc unlildy that tht· Lactaia are dragged in a continuous fluw from the surface of th e soil. and that tlwy are eonlinuously carried forward through crack,. crf'va sses and clcfts in the stone. Thib ccrtainly orcur;. sporadically all(l rcsults in a subscqu(•nt poUution of thc pring watf'r ami nothing else.
Tlw wholt• sprill l! is an op t•n no\1 - syst em with 311 inOO\\' and outOow of 11 at!'r. \\ ho~r ral'<' nf flow may hl' mon· or lcss variaiJI (• do>pending upvn t!H· sizt· and capacit y of tlw entirr. spring. As a wholc. a more or l e~F; uniform ralt• of flow should n·suh . B actcria ma) be in suspensi()n in free wal!·r ami ndht·n· to F;o]itl surfa rt• (72, 73]. Tht· C<'lll' of the hnct t•ria suspcnded in wat t· r can m ePt their nutritional r«'fJUÌre mrnts almost cxclusi,·ely through tl11• uplake of dis"oh !'tl lowf·r mol«~cul nr organie suiJSlanccs. A decomposi tiou of the higlwr moh·cular substane«•s, whieh is elfective for thc sing!P. ccii, il" possible. al tlw lwst. by mrans of enzymes hound lo the celi surfacr wall and not b y nwan>- nf t')o.Oenzymt•>.. P art icularly tiH' higltc·r moleeular ;;;uhsla ncc:: are prderabl~· abr-orhed al surfa<'f',> of ali t yprs . Thc s urfacr flora . tltat u~ually JOf•!- no t adhcr f' vcry s lrougl). is in a po~ition to exploit tn bel Ll'r
ach antagc· 1m cl1 lUI «· n;~,ymatiC' dccomposition, in vicw of LlH· fact that tl11· routf•s of difT'uRiou of the low molce.ular scission pruùucts are vt•ry short. lt is huwen-1· p O!:'siblc to cxperimentally obtain, in water extrcmely low in nutricnts. a signifi cant speeding- up of bacterial growth by simply cnlarg ing thc interna l surface of tbc aqucous boùy [54.. 77]. The lowcr in nutrients a natura! water. thc greatc·r will usnally hc thc amoun t of attachcd bactt'ria in relation to thost' in free susprnsion f70]. Thc~ attachcd flora also prcvail~ in undcrground water [78]. lt is difficult to cstablish in which phasr th t· greatest multiplicatinn of bacteria takes piace in underground sprin~s.
This is la rgrly dcpendcnt upon thr type of spring. The attachcd flora 1s t'«·rtainly the rnost i m por tant in the majority of casr s.
The h eterog1'11eous open flow- syst ems prescnt in nature are often co•n parcll, from thc microhiological anglc, to a continuous culture, such as, for instance, in chcmostalc . In tbc chcmnstate with a s ll:ady nutricnl" Oow and att equa] discharge, there rupidly occurs a Oow balanet: (stcady !'; tal(•) , provided that ali ot lwr conditions r«·maiu unchangNI. Ali thr cclls aro· in the logarithm growth phase. Nowadays, cxpcriment nl ecolog~· surress full y utilizcs chcmoslatc fo r ecology-ori(·utr d experiml'n lal modf'ls [79-8 ~].
I n natura! watrrs, cxtremely low in nutrient s, thr m ctabolic r a l<'!> of organic mal tcr are excecdingly low, and do not allow clircct m eas urr mr nt,.: of the mctabolic uctiviti.es and of tlu· productiou of bacterial subslanct·. l n the « stcady state» cxpcrimcnt.al chemostatc, rven in v<>ry low nul'ricnt eoncentrations, it is possible to drt1•rmine the ration (irrespcctivcly of the
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!lCiiMI DT- LORENZ 101
time factors) bctwccn cultura! conditions and the growth characteristics of an organism. In this way, some strains of bacteria with exceedingly low spccific rates of growth, usually unidentifiable, can be isolatcd and analyzed. Only in chemostate, it is p ossible to make a closer s tudy of both growth and metabolism at submaximal rates of · growth, as they are prevalently found in nature, in substrata low in nutrients and at the same time cool, also an alyzing the mutuai competition among such spccies [84].
In open natura} systems certainly no microbiological fiow balance cxists. 'l'hey do not in any way correspond to a chemostate. The growth of natura} bactcrial populations in such an open heterogeneous flow- system can take piace, dcpending upon the locnl conditions, in part as a static culture and in part as a continuous culture. But it is certainly possible that in the very « n a turai spring » system, a fio w balance of bacterial reproduction may be established at least temporarily. Quite often, notable fluctuation in the number of bacteria, with an initially high bactcrial expulsion, are followed by periods almost entirely free from b acteria. In given occasions, the washing-out effects are similar to the processes occurring in a chemostate.
On the hasis of the present, chielly indirect experience, acquired in the field of experimental ecology, it is possible to assume with a certain degree of certainty that a true and significant multiplication of the autochthonous microflora takes piace in the underground springs area .
;\-~ultiplication of the autochthonous microflora in the dosed system.
In the water bottled dircctly from natural springs, low in nutrien ts, the cnv ironmental conditions for bacterial growth undergo, along with the Jimitation in volume, a dras tic change, passing from an open flow system to a closed system. Shortly afterwards, a sizable multiplication of bacter ia occurs. The natura! mixed populations show the same regularity of growth as that of p ure cultures in a static culture, with a lag, a logarithm and stationary phase. The onc clifference lies in the fact that in water vcry low in nutricnts, a rapid decrease in the number of cclls capable of reproducing occurs after the attainment of the s tationary phase. Oftcn, a further increase in the number of cells is attained in a second exponent ial p hase. Tbis may cven occur more than once during a long pcriod of stay. As it seems, the products of the autolysis and disgregation of the ùead cells are utilized as nutrients for t he new popuJations with a specics composition that is almost always differcnt.
Such a rapid rnultiplication of Lactcria after bottling is typical of aH llutrient-low waters. This was repcatedly confirmed by a large number of analyscs, as, for example, in the case of river water, unclcrground water, oligotrophic lakes water, sea water [5, 57, 85- 88] and also in the case of drin-
.1>111. l•t. i>llptr. i>llltità (1976) lZ, 93- 112
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l O~ DATTERIOLOG IA DELLF. AC()UE Ml~E IIAL I "'ATU RALI
king water [5, 7, 89], of d eionized and dist iJl,,d water [6, 90- 93J, and finall y also in t hr ca!'>r of carbon dioxidt·- fr..c: minerai water [6, 7, 8- 28] .
It is worth noting, ltowe,·r r , th a t this rapid bactnrial growt.h aftt·r bottling also occurs when ali the otiH:' r environmental conditions (tempe· raturc in particular) are maintaincd perfectly similar to thosc prcvailing in th(• open system whencr tlw water was takcn [77, 86, 94]. Tbis only applie~ ,
however, to water exceedingly low in nutrients . The internai surfaces seemingly allow a surface adsorption of the few orgauic matters present. Thi;; fact alone actually allows a better exploitation by th e bacteri a [54, 57, 95] which probably scttle on thc surfaccs. According to thc studics carried out by Barhesier [96] the inner wall of the plastic bottles looks quite rough under a microscopic examination. After a long period of time from bottling, crystalclear and fìlamcntous stratifications are found, that are irregular and with interposed hacterial colonies.
In bottled minerai water, two other growth-promoting factors becomr relevant. During bottling, water is more or less suhstantially enriched with oxygen. Furthermore, temperatures during the period of storage are usually much higher than at the spring.
The multiplication of bacteria in bottled water very low in nutrients, is a perfectly norma! biologica! process . lt may be prevented only through a sufficient water pasteurization or sterilization.
Geldreich and Clark [93] point out that in the absence of bacteria in any watcr unsterilized and low in nutrients , such as, for instance, in distilled water , the doubt always exists of the presence of toxic substances.
Bacterial sp ecies of the autochthonous microflora and their properties.
Well grounded analyses of the bacteria l species of the autochtonous microflora in natura! minerai waters, and of t heir specific physiological properties, are not yet much developed. This also applies, as it is obvious, to all other minerai watcrs low in nutrients . In the absence of more accuratr data, oniy rather summary statements a re possible.
In waters low in nutrients, gram- ncgativc bacteria usually prevail. In decp undcrground water, microftora prevalently consists of Achrobacter and Flavobacterium species. Therc also exist gram- positive species of M icrococcus and N ocardia [78]. A microfiora like this prevails in all spring8, in streams an d in oligothrophic lakcs [97, 98]. Sue h Pseudomonas as P seudomonas jluorescens in particular, are usually prescnt only in tbc case of a s lronger eut rophization [98]. Also fouml in natura! minerai waters wcrc A chromobacter and Flavobacterium species aud also P seudom.onas a nd Xero· monas species [1 , 8] . Ali these data must he vi t~wrd , ho"·ever, with some reserve, eithcr because a more accurat•~ description of such specics is not
SCIIMIDT- LORE:\Z 103
avai1able, or bccause the descriptions at hand are so incomple te as to make it quite difficult to defìne also thc genera according to the new taxonomic viewpoints (100] .
• \ s it results from personal experiences, in the autochthonous microflora of minerai waters, the gram- negative species prevail to an extent of over 60- 70 %, with formation of yellow colonies belonging to the Flavobacterium- C)'tophaga group. The comparative small number of species with no more than 3 or 4 ùifferent types in a changing relationship, was quite striking. Among the species that formed the yellow colonies, there were also gramvariable Arthrobacter species present. The remaining microflora chiefly consis ted of Alcaligenes species, defined so far as A chromobacter. Typical Pseudomonas were found less frcquently and always in a comparatively low amount.
The composition of species, after their cultivation on mediums low in nutrients, consisting of meat bouillon or minerai agar, that always gave the highest colonie count were determined. On the othe·r culture media rich in nutrients, selectiveiy species that may grow also at higher nutrient concentrations, are favoured. Many species of the autochthonous microflora proper, either show an insufficient growth when cultivated on that medium, or do not grow there at ali. The often mentioned prevaience of Pseudomonas in bottled minerai water is thus only methodic, and in way reflects not the actual situation.
The major physiologicai feature of the autochthonous microflora is the marked oligocarbophilie, being the preference for solutions extremeiy low in nutrients. Oligocarbophilic bacteria may grow in minerai salts ·without any addition of organic matter. Such organic matters as those present in tap water , are in trace amounts andare quite sufficient. The minimal limit-concentrations for the organic matter are notably lower than the chemo- analytical verification limit (101].
It is repeatedly stated that during the period of bottling in plastic containers, the low molecular substances coming from the polymer, may be used hy the bacteria present in minerai water as additional nutrients. Comp arable experiments witb tap water and minerai water however show no ;;;ignifìcant difference in tbe devf>lopment of bacteria betwecn bottles made of glass or plas tic. The traccs of organic substances, prcsent in minerai watPr off tbe source are evidently suffici<'nt for Lhe growth of bacteria.
T he defìnition of « autothrophic » (2] should be replaced by the word « oli gocarbophilic ». No true autotrophie was found so far in any of these bacteria, and it is quite likely that it does not evcn exist. As far as we know now, a ll the bacteria of the autochthonous flora a re clearly chemoorganotrophic. An optional chemolithotrophy, with H 2 and CO as a source of energy, cannot be however excluded - as it is known in the case of some P seudo-
.IIm. hl. Su~r. Snr1ità (1976) 12, 93- 112
101 BATI'Io:IIIOI,Ot;IA DF.I,U:: ACC) I IF. MlNEltA!.l :-IATU IIA I,f
mollaS specie!'. A hc lcrotrophic fìxin~ of co2 1 ha t , a ccording lo K usnctSO\' an<l Homanc·nko [1021. reprt~sr:nls in wuter, a pcrcentagl' of O\'t'r 6 % of t h(' bacteri al bi oma.,,. production. seems ins tead qui te pnssiblc ll 03].
i trogen r cquirements are also clearly much rcduced. Tht· autoehthv· nous microflora is simultaneously oligonitropbilic. Jannuseh [71J succec('dcd in proving art e, ·idt•n t growth of Flavobacterium aquatile at concentratiort lowcr than l '; of H4- nitrogt'n/L
A Il tlte Lacteria o f tlw antochtlwnous microflora arl' acrobic, and in pari facultati vc anaerobic. Accordiug lo Clark and B urk ll04] PseudomonH'
and Achromobacter t'an grow e,·cn al oxygeu conccntrations lowcr thau 0.5 ~o · M:orcovcr, man y spceit•s an· eapable vf a naerohit' respiration in th t· prescncc of nitratc or nitrit<: , whosc oxygen acts as an c' lcctron acceplor . Owing to tht• eomparativcly low rnetabolis m, oul y small amounts of oxygeu are rcquired. \Vhen bottled, water is sufficiently enrichcd witlt oxygt"n. A further oxygen dilfusion through th t· plastie bottles, from tht• microbio· logica! anglc, should possihl y oeeur only after several weeks.
Fiually, a li thc bac tcria of th e autoch thouous microfi ora are uuquestionably psychrophilic (faeultati ve psychrophilic). Thc mini mal temperature::. for a multiplication a re of O °C. Sueh rnaximum temperalures as 25-30 °C are seldom exceeded. 'l'he optimurn of growth is u <;ually fouud a t + 20 °C. In t he case: of low tempr:raturcs at the spring, multiplication is by far slowt·r. Almost always, higher t empcraturc;s are us(:d aftcr hottling . This i.- E< uff:ìcient to promotc baeterial growth lo a larg(• extcn t .
Of m ajor importau ee for Lite quautitativc dc terminatior• of thn whol 1• auLochthonous microllora, is the corn::cl choict: of tht· culture a nd m t'dia C'onditions. in kecp iu :.: wi th tlll' specific <'11\"Ìrounwntal require nwnl~. Alr<'· ady at the countiug of haetnia in drinking water, t hc differcut eultur!' nu·dia muy cauRc considerahl': dc' i a tions hotlt in tbc; numbcr of coloni es an d in thc rangt' of specie;; ll·l. lO:>. l ()()]. Fnr t he oligoca rbophilic m icroflora d<·· tcrmiuat ion , culture ml"c.lia lo" in nut rif'nt,; must hc used inst('ad. lt is noi ) el ckar tht: extenl Lo whicl1 hacl!~ ri a are: ohliga t c or facultativt· oligoear· hophilie. P.~l'ttelomoltaS an· ccrt ainl y facultati,•e oligoearhophilic for tht· most parl. Tht' nurnlwr of eolonies t•,;-;('ntiall y highcr on nH·Ùia Jo,, in nutril'nts, poiut to thc fac t that a high perct·ntagc~ of flom i;; ohli~a l('. oligocarhopltilit: . Tht>sc· spccit's. in tiH: suhsequc•nt culture in culi\ t•ntiouul media. are vcry un~lahle and can he ;;. till culti,at('d succcssfu lly onl) on aùeqnat~ ·l~
poor med ia. Summing up, we mus t s tate that thc autochtlwnous mieroflora in min erai
watcr, as wdl as in a li other waters very p oor in nutrients, existing in natun·, is mal'kedly oligocarbuphilic and psychrotrophie. \\'e prcsently know ' t'f) liul c of the baet erial species and of tbcir physiol ogical propcrtie.:l. Thc autoehthouous fiora may multiply slowly a nd r enew constautly in the opcu flo" -
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~CHMIOT-LUfiE:'IIZ 105
systems of natura! springs. Aft<' r spring watcr is bottlcd, there occurs in the cuntaitwr a more or lcss rapid bactcrial mtùtiplication, as in s tatic culturcs. This is mainly due to thc increase of the inside surface, and also to the oxygcn eurichment and to thc higher temperatures Juring the storage period.
We must expressly point out, by way of conclusion, that the increased mtù tiplication of bacteria after bottling of water very low in nutrients, is un ultogcther normal biologica[ proccss. 'fhe indicative or thrcshold valucs of the number of aerobic colonies at a 20 °C incuhation for an untreatcd water, are absolutely meaninglcss from t he biologica[ viewpoint [106). Objectivcly speaking, onJy 2 possibilities are left: c ither the renunciation to auy indicative value as to the numhcr of aerobic colonies st 20 °C, or the need for a nurnher of bacteria equal to O for r.ach bottle. 'fhe latter alternative is possible only through pasteurizatiou or s terilization. But in this (:ase t he product is no longer in its natura} state. From the sanitary angle, of importancc is the constant bactcriological control of the allochthonous polluting flora, and chiefly of the transitory polluting flora, through the 1letermination of t he number of aerobic colonies a t a 37 °C incubation.
Summary. - :\Tatural, non- carbonated minerai water is, like every other natura! water from a spring, ncver s ter ile. Howcver, the microbial level i:; always very low. But aftu its bottling, the level rises rapidly and numhers of more than 10,000 to 100,000/ml can be reached. In principle 2 groups of bacteria of very different origin and properties can be found in t he rnicrobial flora of the bottled, non- carbonatcd minerai water.
Allochthonous bacteria will get into the watcr by contarrùnation from the containers, closures, air or the bottling machines. 'fhey are mostly transitory as they cannot grow in a substrate wi th an extremely low nutrit ive level and die off more or less rapidly.
From the hygicnic point of view the pe.rmanently contaminating flora "ith Pseudomonas aeruginosa as mai n rcprescntative is more serious. These ~ pecial gram- negative bacteria are oligocarbotolerant and can t.hcrefore tnultiply in the minerai walcr of extremely low nutrient lev<'l after a certain adaptation. 'fheir cffective hacteriological contro! is possihle by colony ('ounting with incubation at + 37 °C hut on.ly j ust after bottlin g.
T he autochthonous microbial flora consists of psychrotrophic and of tlist inctly oligocarbophilic, mainly gram- negative bacteria such as .Achro· mobacur, Flavobacleria, Pseudomonas as well as gram- positive A rthrobacter:-pecies. Accorùing to inùirect cxperieuces, this autochtbonous microbial flora rnust be growiug in the open :;ystem uf thc undergrounù source and rt•nf•w itsclf consta n ti y.
The Lottling of the natural spring watcr irnplics a ùrastic environmcutal t' hange from this opcn systcm into a closed one. 'fhen the bactcria start
.Atm. 1•1. Sup<r . .Srmità (19ill) 1%, 93-112
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l Oil BATTE RI(ti.O\.IA OEI.I.F. ACC,Il E )!ll\ERALI NATU RA LI
multiplying morP or l.ess rapidly like in a batch cultu re. Main reasou fur
thi!- is tlw exlension of tlw inner:- s urfacf> of tl11· sy!' tem. T lw multiplical iou of bacteria after bottling of a minerai waler of extn··
mcly low nutri«>nt ) .. ,.-d tht•r~>fnn· Ì!'- an c·nti rely norma! binlogi<'al proct•s,.;. For this reasou , limiti' uf tlw aPrnbic <·olmi\' count at - 20 °C incubati(lu fnr natura) miu•·ral watt·r st•rm noi. tn br justifi•·d.
Résumé L rs cara.ctéristiq11PS mirrobìologiq11ef' de /'(•att minérale naturrllr.
non ga;eust•s). - L'l'au rninérale non ,zazeusc· n 'est, commi' tout\'s Irs autn·~ caux de snurct·:; nalurt•ll t•s. jamait< toul à fait s téri lt·, mais toujouri' <'xtrf.memcnt pauYrf' en germes. Aprè:- l"emboutl'illag(• un accroissC'ment s ignificati{ dc·.- microorga nis mcs présenti' dans l' eau minérale l's t constaté Pt s'élève à un nombn' atteignant 10.000 à 100.000 gcrmesjml.
E n principe. h·s g roupt•s dr m icroorganismes de la flon· microhielll' dans l' eau minérale non gazeuse p«>uvent etrc d 'origine et de caractère très dinrs.
Lcs microbes a llocbthonPs entrent dans l'eau par contamination par les bouteilles, par Ics capsules, l'air ou Ics installa tioos d'embouteillage. La plupart dcs microbcs Dt' se préseutent là que dc façon transitoire, car ils n t' se multiplicnt pas dans des suhstrats pauvres en subs tances nutritives, et meurent asscz rapidem ent.
Ce qui est plus inquétan t du point de vue hygièniqur est la contaminat ion permanente provoquée par Pseudomona.s aNuginosa. Ces hact éries gramnégatives son t oligocarbotolérantes et peuvcnt se muJtiplier mcm r dans l"eau extrèmement pauvre en substanc«>s nutritives, après adaptation. Un contròlc bactériologique efrectif est possib]._. p ar la détcrmination du nombn· de colonirs à + 37 °C immédiatt·ment après l'embouteillagc.
La flore bactéricll e autochtbonc est composée de bact éries psychrntrophcs et bactérics oligocarbophilc·s et gram- néga tives commc Ics groupt'-" de AchromobactPr. Flat·obacterium ('t Pseudomonas comme éga lemcnt le·,.. groupes dc Arthrobacter. Aprè" le~< expérienccs r cceuillics d<· façon indirccte, la fl ore microbielh- autochthone peut déja se multiplier lcn temcnt dans lf' syst ème ou,·ert des sourccs sou terraines et sc renouYelcr constamrnent. Après r cmbouteillage de l'eau de la soun·e lcl'i conditions du milir u pour l'accroi:-;semcnt dcs bact éries se modifient totalemcnt d'tul système ouvert à un s ystème rcstant. Un accroi;;!'emcnl d t' bactérics plus ou moin;, rapidf• con•· mencc, c'cst - à- dirc comm e dan,.. une culturt' statiquc.
La ruisnn principali' en est l'accrois!wm enl dc!' surface;; int erne.". L 'accroÌSS('Jllent plu:- intt' n;;i, t' aprt•s r r m bou t.t>illage dc 1\·a u llliné-ra)p
pauvrc en substancc·s nutriti' ('~ «>!'t donc un proce!'sus normal du point d t· vuc biolog ique. LeR 'aleur:; limites du nomiJrc d(' colouies a«~robiC's à t- 20 °C ne sont donc pas biologiquemenl signitirati, ·r s pour l"eau minéralc na tur('llt· .
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32: 1-7.
.!1111. 11/ . • Srtptr. Sanil1ì (1Uò6) 12, 93- 112
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22. Rcu; c.- t;NI\.A)Il', K . Il. 1'.170. Auf'kei ll iUllJ! u~i tlo-r \l i···hull!.l , ., ... '' '""'rll 1111 Hohruel7
:-rhrijlo·llr. l n. Il tl»t'r Uod••n LuflftyJl.. 31. l'o-•·hrr. St Ullt!.Jrt.
23. ~~~ l,l, f:n. G. 19~ l. \ orkollltnfll uu tl ut•df"ululll! tt•chui•ch IIIIN\OoUII'!'ht t>r b.oktt'rÌt' ll in
der trillkW(hoen't• r,urJ!Un~. 7.Ml lrabl. lJakll'ri(l/. l iri!. l. 4 bl . Orip.. A . 22i : SU 55.
2 1. GA l '\, .l. 19611. As~u.r ÌHf! a(•ct•ptahle lt•wl• in propri••tur) <1ruf!'· ]Jru,t: ( .a;mr•lic J ru/
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poli~:rilirhr r.,l!!P fll lll!l'll A.rclt. Jfy~. 142: 62t• 63 1.
:!6. Z lbUII: R'IANC\, \\ • 195U. Z ur h ) ~t il' l H' dt•r mim·rnlwa,•crfuhrol.. :ll ivu. l. 1-.r~dmi-'t' 'ou
b~skhti~un~:t•·n uuol unll"r.urhw•~en • .. 4rrh. lly~. 1-'2: (JIJ'.I -62.;.
2i. <~.:11 111 N«,. A. l IJ.'i9. LdJer 'di!' e•·gcbni~·~· :i-jillll'Ìj!t'r loal..tc•riolo~i·l'iu r l.. un t roU1·n in nuncrulbmnut"nhctrirlll'u , Arch. llyg. 14.3: 4% -l72.
:.!il. \ tA«,oò\, A. & D. ( .R.\UVI'\. J 97 1. ~: lUÙ<' dc <toci<IUt' phfnom\-111'• pnu' aut inllucnrcr
la chnrg•· n1icrobit'unt dt,. cnux mino~raJ.·,. t'Il hnul <•ill••• p lustirpw• l'l \l'In·. l nd. Alimrnt.
''"'· 91. 531 539.
2\1. \ O•S, 1.. l'lì l. J•orl .. c hrillt' iu der a~cp l !!>Chen niJltillun;: \1111 lt·lwH•IIIÌIIelu. l.mlralbl .
Jlulaniol. III J!.- f . Abt. Ori[; .. B. 159: 335-351.
311. IIA'i,.l '\, t;_ 1967. R ut ionelll' fl liss igkcitsl..onft·kt iuoir·rnu!! 111 tl<·r ph.~rm.<tit• anil tler « bott le paci.. ::1111 >>. Phurm. fnrl. 29: 1173-!175.
Jl. Scuom 111., r . & \1. L Ldii};N i l:C" '· 1.!173. Uri(•utie rcutl•· untt·r .. uchuu!!t'll itlll'r tl.t• \ t•rltaltcn
\o n U.llill rno;porl'll ".ILU'C'IIII drr polyiithy h·u\ crarhei lUOj:. r erJIOf J.. Uuflfioch. 24:
l90 I'Jl .
:1:!. LI nn. '- IU:KI """ S<:ut:l.llll lt .'- , 'J. 19ì::l. B•·tleullllll( rh-s 1'-l'ÌIIl!(•·hnhf·· \oli l'ut'l..,toflo·u
lu". P.u·J..millo•ln fu r nichl-·ll'rih· lt·lll'll•ttiÌ th'l. l ffJWd •. Rwad.•rft. 24: i7 Ili,
;B. \\ ~LUI<\l "LII. h. . I l. J<Jill. ~~~n li•.Jtion nud dc•ÌJifd .. tiou iu d o•r phar11wz•·nll•du·n nnd
iirztlil•lwll l' r."i•. l. /'. 1·. l nfiJrrmz/wnsd,.•nsr l : 3\1 55.
:s 1. \ o:-.,., 1· •. & B. '\l ot.T/.1,1\ , 19i3. Llltt•r<uf• h ouo~l'll u lwr tlit• o lwrfliil-ht·nkeiullahl ,.,lnul içrto·r
l.un•htollo· lur dit· ldtt'lhmalleh•·rpo,·J..""I! Jfrlrftll l '.~· 28: l 7'.1 1111>.
:l:i. DAVI.~, M. S. &. J. ll . BA'l't.:M '-"' 11)(,11. H•·lalÌH' huani<lit~ .tuoi t h~ killin,.: o l J..rcl• 1ia. l. Olo~rn a l iou• "" l•.,drrrit·ltia rolt an d M icrncu•·•·u • h •nd,. il..l i•·u• . . l . IJu C'I• riul 80 .;:: :;;o .
• lh l)~\ L•. \ l. ~. & .J . ll . B\lt.:~J \:-o.., , I'ICJU. Ho•lal Ì\t' luuuitiÌI\ .u11l tl11· l.. illi11;: of b:wlt·ria.
Jl. S <·l••ct i\1· dt.w~•·• in 0.\.)< l.tll\t' IH'II\11 ) Ub•uriut<·d '"Ilo tlealh . . /. Huri('TrOI. 80: :illtl 5U I .
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SCRliiDT-T.ORENZ 109
37. WJNSLOW, C. E. A. & O. R. BnoOKE. 1927. The vinbility of vnrious species of bactt·ria
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. l nn. 1/(t, ,,·,iptr . ."an itri ( IU76) 12. O~ ll2
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l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l
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11 0 BATTERIOLOC:tA DEI.Ll' ACQn' ~11:-i ERA I.I ~ATURALI
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59. MOFFET, II. L. , D. ALLA~ & T. WILLIA)IS. 196ì . Survival and disseminotion of bacterio in ncbulizers and incubators. Arner . ./. Dis. Chi/d. 114: 13-20.
60. FAVERO, M. S., L. A. CARSON, W. W. BONO & . J, PETERSE'I. 1971. Pscudomonas acroginosa. Growth in dis tilled water from hospitols. Scie11ce. 173: 836-838.
61. BoTZENHARDT, K. & S. RoP:KE. 1976. Lebensfiibigkeit und vermchrung von Pscudornonas oeruginosa in anorganischen salzlosungcn. Arch. Hyg. 154: 509- 516.
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63 . .MITCIIELL, H. C. & A. C. HAYWARD. 1966. Postoperatc urinary-tract infections caused by cont.aminated irrigoting fiuid. Lancet 1: 793- 795.
64. BASSETT, D. C. j., K. J, STOKES & W. R. G. TIIOMAS. 1970. Wound infection with P seudomonas multivorans: a water- borne contaminant of disinfectant solutions. Lancet. J: 1188- 1191.
65. PmLLIPS, 1., S. EvKYN & M. LAKER. 1972. Outbreak of hospital infection coused b y contuminatcd autoclaved fluids. Lancet. 1: 12511- 1260.
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67. GARDNEK, A. 1970. ll"ou.nd infection with Pseudonomas nmltivorans : " rvater- borne cotUaminant. of disinfectant solutioru.
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A mf. /~t. ' fl l,rt. SnHitti (1976) 12, 03- 112
l l
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ion t'truip'Jis. Chi/d.
•Jf o rganic
r bacteria
•nas aeru-
-rudomo-
ent covi-
•s caused
th PseuLanut.
a.sed by
hemical ,lficro-
bor"e
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n f iir ·obiol.
Hev.
'. 75:
H 12
:'CII~IIOT-LURENZ 111
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90. E rsMANN, P. C., F. C. K ULL & R. L . .\1EYER. 19·~9. The bacteriolog ical a•pec ts of d e ionized water. j. A.m. Phorm. A ssoc. Sci. Ed. 38: 88- 91.
'JI. FowLF.R, E. B., C. W. CuRJSTE:>~So:>~, E. T. J UR :'IIEY & W. D. SCHAFER. 1960. Bacterial « infect.ion » of the Omcga W est R1•actor. Nurleoni•·s. 18: 102- 105.
•n. LEtrSON. E. 1962. T he bacterial flora of tli still~dt and , torrd wut er. I. (;eJwral observations , techniq11es und ccology. J,~t. Buti. Burteriol . .Vommclnture Tflxonomy . 12: 133-170.
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l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l
11 2 IIATTEIIIOLOGlA DE f, LE J\ CQUE Ml 'iF; RALI NA1'Ufi AU
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..