CA2240237A1 - Culture medium - Google Patents
Culture medium Download PDFInfo
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- CA2240237A1 CA2240237A1 CA002240237A CA2240237A CA2240237A1 CA 2240237 A1 CA2240237 A1 CA 2240237A1 CA 002240237 A CA002240237 A CA 002240237A CA 2240237 A CA2240237 A CA 2240237A CA 2240237 A1 CA2240237 A1 CA 2240237A1
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- Prior art keywords
- culture medium
- agar
- analysis set
- sugar
- gel
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
Abstract
A culture medium for the detection of streptococcus mutans in saliva and/or in plaque is described, whereby the culture medium comprises at least one sugar in addition to the agar. The mass ratio of the agar to the at least one sugar varies between 1:0.6 and 1:12.
Description
Culture mediu~
~he present invention relate~ to a culture medium for the detection o~ streptococcus mutans in saliva or in plaque ~ith the o charac~eristics of the generic part of patent claim 1 a~ well a~ to an a~alysis ~et with the characteri~ticR of the generic p~rt of patent claim 18.
In the field of p~eventive denti~try it i~
known to ex~ine in the saliva or the plaque of the teeth the pre~ence of streptococcu~ mutan~
and to determin~ eheir quantitati~e concentration. In order to carry o~t ~uch a quali~tive a~d quantitative detection a given amount of ~aliva or an ~mount of plaque having been ~cratched off the teeth iB applied onto a culture medium, where~y in this case the known mitis sali~ariu~ agar iB often used.
For the e~tabli~hment of a diagnosis, particularly for estimating the risk of cariogenicity, it is nece~ary to determine the concentration of lactobacill~ in the ~aliva and/or in the plaque, whereby in thi~ ca~e a culture medium i~ al~o u~ed. A sample of the ~aliva, re~pectively of the plaque, i~ applied onto thi~ culture medium based on a rogo~a agar a~ thi~ i~ al~o de~cribed a~ove for the detection o~ the ~treptoco~u~ mutan~, ~o that the i~cuba~ed culture medium can be visually evaluated on a quantitati~e and qualitative basi~ after a corre~ponding ~ime of incubation.
The culture ~edium used herefore and ba~ed on a o ueual ~ogOsa agar ha~ the following componition:
Agar a~ar 19 g Tryptone 10 g Yea~t extract 5 g Glu~o~e 20 g Pota~ium hydrogenpho~p~ate6 g Ammonium hydrogencitrat~ 2 g Manganese~ ulphate l-hydr~te0.1 g Iron-II-~ulph~te 7-hydr~te 0.03 g Tween ~0 1 g For the manufacture of the ready-to-apply rogo~a culture medium the afore indicated compo~ent~ are added to 1 l of water.
For the establi~hment of an impecca~le dia~no~i~, par~ichl~ly for defining concrete step~ to a~oid the risk of cariogenicity, it i~
moreover nece~ary to mea~ure the buffering capacity o~ the ~aliva. In the analyei~ ~et being available nowaday~ this buffering capacity i~ mea~ured by ~prinkling a tect strip which is ~oaked with an indicator ~ith ~aliva, whe~eby the nece~a~y color change o~ the test st~ip i~ ~hen vi~ally de~er~ined after an exposure time of about two minutes. Hereby the test strip used herefore, however, only allow~
a coar~e mea~uring of the buffering capacity being defined a~ pH-value, so that the measuring of the buffering capacity i6 not ~afe and depend~ o~ the skill o~ the corre~ponding u~er.
The pre~ent in~ention ha~ t~e obje~t of di~posin~ a ~ulture medium of the indicated sort which make~ po~ible a ~afer statement about the concentration of ~treptococcus mutan~.
Thi~ object i~ realized by mean~ of a culture medium with the ~ignlficant characteristic~ of patent claim 1.
The inventive culture medium of the detection of streptococcus mutans in saliva and/or in plaque provides t~at the inventive culture medium contain-~ at least one ~ugar in addition S to one agar, where~y ~he ~a~ ratio of the agar to the at lea~t one sugar varies between l:o.
and 1:12.. In other ~ord~, the thu~ inventive culture ~edium differs from the known culture medium ~n ~hat way tha~ the inve~tive culture lo m~dium compri~e-~ at least one sugar in the afore indicated ma~ ~atio.
It was ~urprisingly notcd that the inventive culture medium show~ a high ~electivity con~erning ehe ~treptococcus mutanB, whereby thie high ~electivity for ~treptococcus mutan~
i~ referred to the ~act that, in contra~t to u~ual culture mediums being available in trade, ~he inventive culture medium additionally comprises a sugar. Compared to u~ual culture mediumfi thi~ impro~ed ~ele~ivity o~ the inventive culture medium is expre~ed in that the inventive culture medium ~hows a ~ignificantly higher germ yield of ~treptococcu~ mutan~, which was con~irmed by 135 Qaliva isolates o~ 55 children. This supplemen~ary sugar additive doe~ not impede the preparing of a ready-to-apply culture medium from the inventive culture medium, ~hereby for the ~imple and rapid preparation the inventive dry culture medium is only taken up into the re~lred a~ount of water.
The inventive culture medium can also be manuf~tured in a particularly ~i~ple ~anner since here~ore it i~ only neces~ary to mix a ~uitable agar with the required ~ugar amount, whereby the mixing of the agar with the ~ugar can be carried out not only during the manufacture o~ a ready-to-apply culture medium, me~ning duri~g the taking up of the agar and of ~he ~u~ar into water, but al~o by mixing the dry agar with the at lea~t one sugar.
A ~ir~t e~h~A~ent of the inventive culture medium provide~ that hereby the ma~ ratio of the agar to the at least one -~ugar lie~ between 1:3.8~ and 1:4 2~. ~ereb~ it wa~ noted that thiY preferred embodiment of the inventive culture ~edium wa~ taken up into water in a particularly ~imple and rapid manner, ~o that a ready-to-apply culture medium can be manufactured corre~pondingly ~imple and rapid.
A ~urther embodiment of the inventive culture medium ~howing an extremely high selecti~ity for the detection o$ ~treptococcu~ mutans provides that hereby the ma~s ratio of the agar ~o ~he at lea~t one ~ugar varie~ between 1:5.1 and 1:10. Althou~h t~ia embodiment of the inventive aulture ~edium has an extremely hig~
Bugar concentrAtion it could ~e aurpriai~gly noted that the ~treptococcu~ mutans extracted o from hu~an ~ali~a or pla~ue grew perfectly and ~e~ectively on a culture medium being mixed ~ith su~ar in such a ~ay.
The term agar i~ ~ed above and ~ubsequently, whereby thi~ term means particularly the mucilage ext~acted and dried ~rom red seaweed a~ well a~ highly molecular galac~o~ides, pa~ticularly ~ B of galaktoae-~ulphate e~ters, a~ far as ~pecia~ agar~ are ~ub~equently not mentioned.
In respec~ to the sugar contained in the inventive culture medium it i~ to be noted that thi~ sugar can be a monoaaccharide and/~r a mono~accharide derivative. Ho~ever the in~entive culture medium preferably compri~ea a~ sugar a di~accharide, particularly aucroae and/or ~ eucro~e dexivative.
Depending on the corre~ponding field of application, the inventive culture ~ediu~
consists in the simplest cafie only of the u~ual agar and the a~ore described sugar in the mentioned mass ratios. If such an embodiment i,Q
then applied by skilled epecial staff a~, for example, in correspondin~ ho~pitals, it i8 only necessary to take up the inventive culture ~edium into a coxre~po~;n~ amoun~ of w~ter, so that then corre~ponding dishes, for example Petri dishes, can be filled with this ready~to-apply, gel-like cu~ture ~edium. In order to ~a~ely and reproducibly distingui~h the streptococc~ m~ea~s ~ulti~a~ed in the ~aliva or in the plaque and the additionally and eventually cultivated streptô~occu~ mutan~, particularly the s~eptococcus para~ang~is and ~o the streptococcus sallvarius, aft¢r incubating with the saliva, re~pe~tively with the plaque, and after the ~ubsequen~ lncubation, it is recl_. n~d to carry out a dilution ~erie~
belng u~ual for ~icrobiological e~Am~n~tions.
Hereby the saliva, respectively the plaq~e, is dilu~ed by a phy~iological salt eolution to a ten~h ~o thousandth part of its original concentration, which cause~ an evident and effective BUppres~ion of th¢ gro~th of the a~ore mentioned strep~ococcus and of lactobacillu~, actinomycete~, ~taphylococcus or gram-negative bacterla. Thi~ again leads to a particularly safe and reproduci~le detection and vicual quantification of the ~treptococcus mutans.
A particularly simple and safe detection of strep~ococcus mutans which can renounce on the afore mentioned dilution ~eries without showing a fal~ification of the re~ult provide~ that hereby an inventive culture ~edium iR used w~ich co~pri~e~ ~t le~t o~e peptide antibiotic co~plex in addition to t~e agar and to the at least one sugar, wh~reby the concentration of this peptide antibiotic complex varies between 0.001 ~ by weight and 0.0005 % by weight relative to the total weight of the agar and the added ~ugar.
The afore de~cribed e~bodiment of the inventive culture medium particularly comprises as peptide antibiotic complex the bacitracin being ava~lable i~ t~de. In order to m~nufacture from thl~ embodiment of the inventive culture me~ium a ready-~o-apply culture medium it is only nece~ary to ta~e up the agar containing the at lea~t one sugar and the bacitracin into the ~equired amo~n~ o~ water. It was S particularly noted that such a rea~y-to-apply, inventivç c~lt~re medi~ then effectively suppre~e~ the growth of a mixed ~lora, particula~ly the ~th of ~ep~ococcu~
salivariuR and/or streptococcu~ para~angui~, lo without reducing the yield of strepeococcus ~utan~ in a ~tati~tically ~ignificant ~a~ner.
In re~pect to such ~aliva ~mp~e~ or plaque samples ~ic~ ~ompri~e~ al~o other ~treptococcus type~ in addition to the ~treptoco~cu~ mutan~, thic e~h~;ment of the inventive culture ~ediu~ makee it thu~ po~sible to easily and effective~y distingui~h ~he streptococcus mutans and the other streptococcus or ~he afore mentioned other microor~ani~m~.
A p~rticularly ~uitable agar being particularly u~ed in the inventive culture medium is the ~l~eady initially mentioned mitiB ~alivariu9 agar.
Hereby a typical miti~ ~alivarius a~ar aj it iY
available in trade and cold by the firm DIFCO, Augeburg, ha~ ehe following compo~ition Bacto trypto~e 8 g to 12 g, particularly 10 g Proteose peptone no. 3 3 g to 7 g, particularly 5 g Proteose peptone 3 g to 7 g, particularly 5 g lo ~acto dextro~e 0.5 g to 1.5 g, particularly 1 g ~acto ~ucrose 30 g to 60 g, particularly 50 g Pota~ium hydrogenpho~phate 3 g to S g, particularly 4 g ~ye, particularly O.oS g to o.09 g, trypan blue particularly 0.075 g Dye, parti~ularly 0.0006 g to o.ooo1 g, bacto cryetal violet particularly 0.008 g ~acto agar 13 g to 17 g, particularly 15 g For manufacturing a ready-to-apply culture medium the afore mentioned components are ~eirred into 1 1 of water.
In order to facilitate particularly the ~pplication of the inventi~e culture medium a further embodiment o~ the inventive culture medium provides that the culture medium iB
applied onto one ~ide of an inert ~upport (dip-~lide; solid c~rrie~). For ~he detection of ~treptococcu~ mu~an~ in ~aliva it i~ only nece~ary to remove the s~erile packed culture medium being appl ied onto an inert ~upport, pareicularly onto a plastic ~upport, from the lo s~erile packing and to add the saliva sample or the plaque ~ample to be ex~ined to thi~
culture medium, ~ that the corre~ponding st~eptococcu~ ~utan~ ~oncen~ration can be detected after a corre~ponding ~terile incubation.
A~ already repeatedly mentioned above, a ready-to-apply cult~re medium can be eaoily and rapidly manufactured from the inventive culture medium, ~he~eby ~uch a ready-to-apply culture medium preferably ha~ gel-like con~i~tency. , whereby water can be added to the culture medium in order to produce the gel-like con~i~tency. For generating the gel-like consi~tency t~e dry inventive culture ~edium comprising ~he agar, preferably the miti~
salivariu~ agar, ~hich contain~ the at lea,~t on~ ~ugar and, if necee~ry, the peptide anti~iotio ~o~plex, pre~erably the bacitracin, ie only taken up into water, whereby ~he de~ired gel-like con~i~tency can then be ad~usted ~y varying ehe amount of the added water.
Shch a gel-like development of the ready-to-apply culture ~ediu~ preferably co~pri~es between 70 g to 150 g of the agar, peptide antibiotic the miti~ ~alivariuB agar, between 42 g and 1.8000 g of the at lea~t one BUgar and a~ much water as i~ required, prefe~ably between 888 g and 100 g, for ~an~acturiny the gel-like con~i~tency. If, in addition to the gel-like and ready-to-apply culture medium, the afore ~e~cribed peptide antibiotic complex, preferably the bacitra~in, i~ to be added, the amount of this peptide antibio~ic complex, preferably the amount of the bacitracin, then variee ~etween ~.~ mg and 12 mg, e~ch xelati~e to the total amount of the at lea~t one ~u~ar and of the agar, paxticularly the mitis ~alivarius agar.
In order to fa~ ate the determination preci~ne~ of the cultivated ~treptococcu~
mutan~ in re~pect to the afore de~cri~ed and ~ub~equently described e~odiments of ~he inve~tive culture medium in which the culture medium iB applied on~o an iner~ ~upport, a further development o~ the inventive culture ~edium provides that hereby the inert ~upport is dy~d with dark color, preferably dyed black, or that it con~ist~ o~ a corresponding dark plastic ~terial.
Another ~m~o~iment of the inventive cultu~e me~iu~ being at the ~ame time B~ table for the de~ection of streptococ~us mueans and of lac~obacillu~ or yeast fungus provides that the in~enti~e ~ulture ~edium whi~h comprises the at lea~t one ~4gar ~nd, if ~eceu~ary, the peptide antibiotic co~plex in addition ~o the agar, particularly the mitis ~alivarius agar, i~
applied onto one side of the i~ert support, preferably a plastic ~upport, wherea~ on the other side of the inert support a further culture ~ediu~ for the detection of laceobacillus in the saliva and/or in the plaque or a further culture medium for the deteation of yea~t ~ungus in the ~aliva and/or ~he plaque i~ depo~ited. Hereby such an embodiment of the in~entive ~ulture medium allows that the concentration ~f ~treptococ~us mutan~ and la~tobacillus or of str~ptococcus mutan~ and ye~et fungus ~n the ~aliva and/or in the plaque bei~g required for the Carie~
prophylaxi~ can ~e concurrently determined with one sing~e te~t, 50 that ~uch a development of the inventive cultuze ~edium can ~e applied al~o particularly for the rapid and safe diagno~i~ in the denti~t'~ practice.
In the afore de~cribed development of the in~entive cult~re medium by which lactobacillu.
can be determined conc~rrent~y with ~treptococcus mutan~ a culture medium ~a~ed on a rogo~a agar i~ 6elected a~ further culture medium, whereby ~uch a culture mediu~ then ha~
a chemical conetruction a~ initially described.
In a particularly advantageou~ development o~
the ~fore de~ribed embodi~ent of the inventive culture mediu~ the further culture medium comprises at least one sugar in addition to the u~ual rogoca agar or to ~he cul~ure medium for the detection of yea~t fung~, whereby ~hi~ a~ leaet one ~ugar preferably i8 gluco~e, ~ucro~e and/or a gluco~e derivative, re~pectively a ~ucrose derivative. Particularly if the further culture medium contalns ~he rogo~a agar or the culture medium for ~he detection of yea~t fungu~ and the at lea~t one ~ugar in a ma~s ratio of between l:0.~ and 1:6, S then su~h modi~ied further culture medium ~how~
a h~gh selecti~ity and yield of lactobacillus, recpectively of yea~t fung~, ~o that ~hi~
e~bodiment allow~ a preci~e and reproducible, qualitative ~nd qu~ntitA~ive de~ermination o~
lactobacillu~, re~pectively yea~t fung~, in the ~al iva a~d/or i~ the plaq~e.
If in the a~ore de~cribed embodimen~ one ~ide of the inert ~pport i~ coated with the culture medium for the de~ection of the streptococcuq mutans and the other ~ide of the inert ~upport i~ coated with the culture mediu~ for the detection of the yea~t funguB in the ~aliva and/or in the plaque, thi~ e~bodiment of the inventive culture medium then allow~
particularly for children b~ing under ~our ~ears old, preferably howev~r for the a~e of le~ ~han three year~, a particularly easy and concurrent deter~ination of an eventual pre~ence of yeajt fungu~ in the ~aliva or in the pla~ue of the~e children in addition to ~treptococcu~ mutans. Especially in the ca~e of CA 02240237 l998-06-lO
children infected ma~ively with carie~, partic~larly with the nur~ing bo~tle ~yndrome, it wa~ noted that yea~t fungu~ are important indlaator germ~. Hereby a culture medium for the detec~ion of yea~e fungu~ with the following compo~ition is preferably u~ed:
Chromopeptone 8 g to 12 g Glucose 15 g to 25 g lo Chloramphenicol 0.3 g to 0.7 g Dye mixture (chro~ogen mix) 1.5 g to 2.S g Agar 10 g to 20 g The agar 601d under the trade name "BL
CHROMagar Candida~ ~Manufacturer: ~ecton ~ickin~on 6~b~, Heidel~erg~ i~ particularly ~uitable for the detection of yea~t ~ungu~.
In ord~r to manufactu~e ~ re~dy-to-apply culture medium for the detection of yeast fungu~ the afore indi~ated component,s are taken up into one liter of deionized water, so that gel-like culture medium i~ produced to which, if de~ired, further amounts of ~ugar of the described concentration range~ can be added.
In order to facilitate the application of the inert ~uppor~ ~oated on one side with the inventi~e ~ulture medium and coated on the other ~ide with the further c~lture medium, a further develop~ent of the inve~tivo cult~re medium provides that the fuxther cult~re medium ha~ a ge~-like con~i~ten~y, whereby the gel coneain~ between 70 g and 250 g of the further c~lture medium snd between 930 g and 750 g of water.
pa~ticularl~ ~uitable and advantageous development of the afore de6crib~d embodime~t of ~he inven~ive culkure medium ~hich can be applied in each denti~t'~ practice provide~
that the inert eupport which i~ co~ted on one ~ide with the gel-l~ke culture mediu~ on the baei~ of the agar, preferably the miti~
~all~riu~ ag~r, containing at lea~t one ~ugar and the peptide antibiotic complex, particularly bacit~acin, and which is ~oated on the other ~ide with the gel-like further culture medium on the basis of the afore described rogosa agar or the culture medi~m for the detection of yea~t fungu~ i~ to be arranged ~ithln a ga~tight lockable ~torage ~e~el.
Hereby the ga~tight lockable storage ve~el ~erve~ for ~he ~terile ~torage and for the CA 02240237 lgss-06-lo transport of thi~ e~bodiment of the ~nventive culture medium and guaranties at the same time that, after the adding of the corre~ponding ~aliva ~ample~, respecti~ely plaque ~a~ples to ~oth culture mediums, the thus treated culture ~ediume can be incubated in the stora~e vessel, where~y a C02 relea~ing ~able~, preferably an alkali carbonate tablet, re~pectively an alkali hydrogencarbonate tablet, can be arranged ~ithin ~he etor~e ve~el particularly during the incubation. Hereby thi~ C02 reloa~ing tablet cau~e~ the fact that a corre~ponding C02 atmo~phere i~ adju~ted ~ithin the gastight lockable ~torage veesel during the incubation time, whereby, on one hand, the selectivity of the inventive culture medium for the streptoco~cus mutan~ and the lactobacillus, respectively the yeast ~ungu~ increa~ed and, on the other hand, unde~ired side reaction~ are excluded. Such ah em~odi~ent is perfe~tly ~uitable for the concurrent detection of ~treptococcus mutanB and lactobacillu~, re~pectively yea~t ~ungus, in the dentist ' B
practices, whereby this development of the Z5 inventive culture ~edium then allow~ a reproducible and ~uantified determination r~garding these ~icroorgani~m~.
lg The present invention rela~e~ further~ore to a analy~i~ ee~ for the ~imple e~tablishment of a diagnosis, particularly for evaluating the ri~k S of cariogenicity.
Thi~ object i~ reali~ed according to the inven~ion by an analysis set havin~ the significant ~aracte~ietic~ of patent claim 18.
The inventive analysis set comprises a culture medium being arranged o~ one side of ~n inert support and being of the afore de~çribed ~ort for the det~ction of streptococcus mutans, whereas the other side of the inert support is co~ted ~ith the fu~ther culture mediu~ baeed on the afore de~cribed rogosa agar for the detec~ion of lactobacillus or with a culture - mediu~ of the detection of yea~t fungus.
ln a development of the in~entive analysis set the analysis #et can prefera~ly comprise ~t leaet one carpule filled with an acid, pref~ral~ly with ~ O . 005 n hydrochloric aoid, a8 ~ell as at least one cannula.
The inventive analy~is 8et ~hows a range of ad~antage~. Fir~t of all it i~ to be noted that the afore deecribed in~en~ive analy~i~ set allow~ a preci~e qualit~ti~e and quantitative a~ well as concurrent determination of ~treptococcu~ mutan~ and lactoba~lllus, respectively yeast fungue, in ~aliva an~/or in pla~u~. In addition to that the afore mentioned development of the in~entl~e analyRi~ Bet can make possible a determination o~ the buffering lo c~p~city of the ~ali~a. Thi~ is achieved by taking up a defined amount of saliva into the carpule bein~ filled with an acid, prefe~ably with a O.OOS n hydrochloric acid, ~o that after mlxing the ~aliva with the acid the pH-value of thi~ liquid mixture can be easily determined, whereby the pH-v~lue i~ a quantitative indication o~ the bu~fering capacity o~ the ple.
In order ~o ~ke ~p ~he s~liva into the carpule filled with ~he a~id the carpule of the inventive analy~i~ aet i8 ~it into a usual ~edi~al ~yring~. The cannul~ contai~ed i~ the analysi6 ~et is mounted onto the ~yringe, 50 that then the carpule i~ filled wlth a given amount of the ~aliva sample, xe~pectively the pl~que ~uapen~ion, ~y mean~ of the ~yringe by pulling back the ~yri~ge pi~ton to a given mark or to a gi~en stopper. After ~ixin~ the ~ample with the acid the evaluation can be realized by interpreting the color change. After ~he determination o~ the concentration of streptococcus mutan~, lactobacillu~ or yeast fungus and even~ally o~ ~he buffe~ing capacity the inventi~e analy~ln set can be easily ~asted.
A ~urther development of the a~ore de~cribed inventive analy~i~ set p~ovides that the analynis set co~pri~eC at lea~t one c02 ~eleasing tablet. Hereby the C02 relea~ing tablet e~enti~lly c~use~ that, after incubating the cultu~e ~edium ~o~ the detection of streptococcus mutans and the furthe~ c~lture mediu~ ~or the detection of lactob~eillus, respec~ively for the detection o~ yea~t fungu~, the i~ert ~upport coated with those two cult~re medium iB ~tored in a ga~tight, lockable ~torage vessel, whereby the C02 relea~ing tab~et i~ a~anged within this gastight ~torage ve~el during ~he incubation. Thi~ C02 relea~ing tab~et cau~es the ga~tight storage ve~el i~ ~et under a corresponding C02 atmosphere durin~ the incu~atlon, ~o that, on one hand, the selectivity for the detection of the ~treptococcu~ mu~an~ and lactob~cillu~ i~
~o~re~pondingly increased and, on the oeher hand, unde~ired ~ide reactions are e~cluded becau~e of the CO2 atmo~phere.
In a further e~odi~ent of the inventive a~ly~i~ set ehe analysi~ ~et comprise~ ~t least one paraffin piece and~or a pH-value i~dicator. Hereby thix paraffin piece ~erves for the ~illing of ~he corre~po~ding patient whose saliva, respectively plaque i~ to be ex~m;n~d in res~ect to the presence of ~treptococcus mu~an~ and lacto~cillus, re~pectively yea~t fungu~, ¢hew~ thi~ paraffin piece for a given time before the execution of the co~resp~d~ng ~ ion, in order to thus animate the salivation, whereas the pH-value indicator i~ employed to determine the pH-valu~
of the saliva, a~ thi~ iB de~ribed later on.
In order to furtherly ~implify the determination of the buffering capacity of the saliva in the afore described embodimen~ of the ~5 inventive analy~ e~, a further development of the inventive analysis set provides that hereby the carp~le ¢ompri~es also an indicator, particularly a liquid indicator, in addition to the acid being pareicularly contained in ~he carpule in a given amount and concentration.
Hereby it is rendered po~ible to directly def ine the buf f ering capacity of the saliva sample by interpreting the color change after the filling o~ a 8ali~a ~ample, respectively a pla~ue ~uBpen~ion, intb the carpule, whereby ~he safety and reproducibility of the determination of the bu~fering capa~i~y iB
fu~therly i~proved.
It is ~l~o pos~i~le to provide the analysis set with a pH-value indicator a~, for example, a liquid i~dicator, with a ~olid indicator and~or with a indicator strip, 80 that hereby t~e buffered ~aliva can be removed from the carpule in order to then deter~ine its pH-value.
The afore described indicator preferably i~ a liquid ~niver~al indic~tor, whereby thiP liquid universal indicator has a pH-value indicating range of between pH 4 and pH 10.
In order to ~acilitate the vi-~ual defini~ion of the buffering capacity of the ealiva, re~pectively of the plaque suspeneion, , it is particularly advantageou~ that the analy~is ~et containe color ~cale, eo that the bu~ering capacity of the corre~pon~in~ ~ample can be de~ermined in a particularly ea~y and rapid s manner by vi~ually compaxing the color ~c~le with the obta$ned color yield of the çarpule which i~ filled with the ~aliva and which ~ontain~ the univer~al indicator a~ ~ell as the afore described acid.
Another advantageou~ development of the inventive analy~i~ set provide~ ~hat the analy~is ~et comprises at lea~t on~ ma~k which can be arranged on the inert support, so that by thi~ ~a~k the cul~ure mediu~ which is arranged on on~ side of ~he inert ~uppor~ and which i~ based on the modified agar, pa~ticulaxly the mitiu ~alivarius ~gar, and/or the ~urther culture ~edium which i~ arranged on the other ~ide of ~he inert ~upport and which is ~a~ed on the rogo~a agar, re~pectively for ~he detec~ion of ye~t fu~gu~, can be ~ubdivided into single ~ection~. This ~mbodiment of the inventive analy~i~ see makes it thus poosibl~ to optionally subdivide one c~lture ~edium or both culture medium~ in single 8ections, 80 that single mouth areas or CA 02240237 l998-06-lO
particularly dental area~ can be specifically examined fo~ streptococcuR mutane and l~ctobacillu~ or yea~ fungu~ with one single te~t. Herefore it is only ~ecessary to apply S thi~ 6ample removed from the mo~h area, re~pectively from the ~pecific tooth, onto the section of the co~re~rond; ngly ~ubdivided c~lture ~edlum, ~o that after the inc~bation the microbiological ~ondition6 can easily be defined in re~pect to their concentration of streptococcu~ mutan~ and/or lac~oba~illus or of ~t~e~ococcu~ mutan~ and~or yeast fungus. T~i~
maQk i~ preferably provided wit~ an in~c~iption part, ~o that an a~ign~en~ of the obtained valuee to ~pe~ial mo~th ~reaB, re~pe~tively dental area~, can ~afely be carried o~t.
In another em~odiment of the inventive anal~sis set this analy~is eet compri~e~ moreover a calibrated ve~el for taking up the saliva Here~y it i~ ~ade po~sible to measure a further parameter during the ~aliva e~-nAtion, whereby thic para~eter ig the ealiva volume generated by the patient by chewing the afore de~cribed paraf~in piece for a ~iven time.
A further advantageoue develop~ent of the inventive analy~ et provide~ that the a~aly~i~ qet compri~e~ ~oreover a di~po~able ~yringe. Hereby thi~ ~ispoeable syring~ ~erve~
to inject saliva or a plaque ~u~pen~ion into ~he carpule filled ~ith the acid, preferably ~lth the 0.005 n hydrochloric acid, and wi~h the univercal indlcator, whereby the amount o~
the injected ~aliva can be determined optionally in the disposable ~yringe or in a particularly advantageou~ manner by a mark located on the carpule up to which mark the gliding plug i~ di~placed in the axially oppo~ite direction o~ the piercable membrane.
Thi8 embodiment o~ the analy8i~ -~et guaranties that each u~er of the inventive analy~ et find~ ~ll in~tru~en~ required for the dete~mina~ion of the buf~e~in~ capacity of saliva in thi~ analysi~ set, ~o that ~he corre~ponding analyei~ can be carried out in a p~r~iclllarl~ ~imple ~nd ~apid and thul3 ~ery economical manner.
The afore described embodiments o~ the inventive cult~re medium in which the culture ~edium, re~pectively the afore de~cribed fur~her culture medium, i~ applied each onto one ~ide of an inert ~upport can be ufied in a particularly ~imple, rapid and reproducible manner for the dete~tion of ~treptococcus mutan~ on one hand and for the detection of lactobacillu~ or yea~t fungu~ on the other hand. In thi~ detection method firat of all a saliva ~a~ple or a pla~ue ~ample i~ taken from th~ patient and applied onto the culture medium for the detection of otreptococcue ~utans a~
well as onto the further culture medium for the detection of lactobacillu~, respectively ye~t fungus. The culture medium~ thus loaded with the ~ali~a sample are then transmiteed p~eferably i~ a ~a~tight, lockable ve~sel having been event~ally ~llled before with a CO2 atmo~phere into a suitable incubator, so that the corre~po~ding ~torage ve88el i8 then inçuba~ed for a given incubation time, preferably between 24 and ~8 hours at a temperature o~ 37 nc + 4 ~C.
After e~pi~ation of the incubation time a vi~ual evaluation and ~uantification of the streptococcu~ mutan~ cultivated on ehe first culture m~dium arranged on one side of the inert ~upport as well as of the lactobacillus, re~pectively the yeast fungus, cultivated on the further culture medium arranged on the other ~ide of the inert support. By a vi~u~l comparison with the corresponding ~tandard~ the con~ent~ation o~ strepto~oc~u~ mu~ans and lactobacillu~, re~pectively of ~treptococcu~
mutans and yeast fungu~ ~an then be quantified, as thi~ i~ u~ual in microbiology.
Advantageou~ development~ of the inventive culture medium a~ well as of the analysis set lo are indi~ated in the ~ubclai~s.
The inventive culture medium and the inventive analy~i,q ~et are .qubsequently explained by an example in connection with the drawings.
Fi~ure 1 show~ a ~chematical, partly per~pective embodiment of the ana~yui~ ~et;
Figure 2 Bhow~ the cul~ure medium in a se~tional view along the line A-B in figure 1, whereby the culture medium is contained in the analy~i~ Ret according to figure 1 and arranged on an inert ~upport;
Fi~ure 3 show~ an enlarged vie~ of the ma~k ~hown in ~igure 1 in a top view;
~ig~re 4 ~how~ a view of the culture medium b~ing arranged on a ~upport, whereby this view can be compared with figure 2. The culture medium ha~ the m~k ~ho~n in figure 3; and Figure 5 shows a sectional view of a carpule .
In the figures 1 ~o 5 the same part~ are xeferred to the corre~ponding number~.
A~ ~hown in fi~ure 1 ~he analy~ et 1 fir~t of all compri~es a corre~ponding amount of cannula~ 2, ~hereby the embodiment ~hown in ~igure 1 ex~mpl~rily indi¢~te~ three ca~ula~
The analy~ et 1 moreover compxi~e~ an amount of carpules 8 which corresponds to the amount of the cannula~ z, ~he~e~y in ~ igure 2 one single ca~pule 8 i~ only examplarily ~hown. Th~
- more detailed con~txuction of thi~ carpule 8 i~
subse~ùently explained by figure 5.
In addition to the carpule B and the cannulas 2 the analy~is set co~prise~ furthermore a corre~ponding amount of inert plastic supports 3 a coated wi~h culture mediums, whereby these plactic Bupport~ 3 a co~ted with culture mediums 3 are explained more detailed in the figuree 2 and 4. The analysis ~et al~o compri~e~ a mask 4 being schematically ~hown in figu~e 1, where~y the ~a~k 4 i6 de~cribed more detailed by the figure 3. The analysis set 1 furthermore co~pri~ number of paraffin blocks 5 which corre~pond~ to the number of cannula~ 2 and a number of C02 releasing tablet 7 which corre~pond~ to the number of pl~etic support~ 3 a, whereby in ~igure 1 on~y one paraf~in block 5 and one C02 relea~ing tablet 7 i~ examplarily ~hown. Moreover the analy~i~ set 1 compri~e'~ a ~lor ~cale 6.
As shown be~t in ~he sectional view~ of the figures 2 and ~, each inert pla~tic 6upport 3 a compri~e~ a grip ~ection 13, whereby a dieh-~h~ped area 14 iB directly adjoined to both sides of the grip section 13. Hereby the di~h-shaped area 1~ co~p~i~e~ a ridge-like ~ide wall sec~ion 15, where~y only ~he front-~ide ridge sections 15 are shown in the figure~ 2 and 4, The di~h- ~haped ~rea 14 ~erve to take ~p the culture medium 3 which i~ nu~bered ~ith 16, re~pectively with 17, in the ~igure~ 2 and 4, ~hereby one of the cultu~e mediums 16, re~pectively 17, i8 the afore described miti~
sallvarius agar and the other culture medium 17, re~pectively 16, i~ th~ al~o a~ore de~c~ibed rogo~a a~ar or i~ a culture medium for the detection of the yeast ~ungu~. The inert plastic suppor~ 3 a is arranged within a gastight lockable storage ve~xel 18, whereby the ~torage ven~el 18 i~ clo~ed up with the grip ~ec~ion 13.
It surely i~ po~ible to form the inert pla~tic ~upport 3 a in such a way that an in~erior ~rip section 13 is ~onnected with the di~h-shaped area 14, eo th~ ~he ~hole pla~tic ~upport 3 a i~ arranged within the ~torage ve~çl 18 and corre~pondingly ~lo~ed up ~ith a ~crew cap.
However thi~ embodiment i~ not 6hown in the figure~ 2 and 4.
The culture medium being arranged on the di~h-shaped area 14 and bein~ ~ased on a ~odi~ied miti~ salivariu6 agar i~ s~ch a oulture medi~m 16 a8 it i~ manu~actured as ~ollows.
~ir~t of all a purcha~able mitis salivarius agar a~, for example, from DI~C0 i~ ~hosen, whereby the pu~ha~able mitis salivariu~ agar contains the following compo~ents:
Bacto tryptose 20 g Proteose peptone no. 310 g Proteo~e peptone 10 g Bacto dextro~e 2 g Bacto ~ucro~e 100 g Potas~ium hy~ e~pho~phate 8 g Trypan blue 0.15 g Bacto crystal violet0.0016 g ~acto agar 30 g 360 g sucrose (Merck 1.07651) and 3 mg bacitracin (Sigmar ~-0125) are added to the fir~t half o~ this dry culture medium bein~
sub8equently named culture medium I, and 600 g sucro~e (Merck 1.07651) and 5 mg bacitracin (Sigmar b-0125) are added to the eecond half of the dry culture medium being ~ub~equently named culeure medium II, whereafter ~he culture medium~ I a~d II are mixed. The culture mediums I and II are ~hen ~olved in deionized water under the ~ormation of one liter of ready-to-apply culture ~ediums I and II ~ereafter the water i~ boiled. Then the culture mediums are ~terilized at lZl ~~ for lS ~inute,~. After having aooled down, the gel-like culture medium~ are applied onto the inert plastic support 3 a whi~h i~ a black p~a~ic aupport.
Culture medium I then ha~ a raeio of miti~
~alivariu~ agar to ~he added ~ucroee of 1:~.996, wherea~ cultur~ medium II has a ratio of mitis salivarius agar to the a~ded sucrose lo of 1:6.661.
For the manufacture of the culture medium 17 the u~ual rogosa agar i~ used as starting ~ateri~l, whereby the corre~ponding ro~o~a agar lS ha~ the already abovc indicated fo~lowi~g compo~ition:
Agar agar 19 g Tryptone lO g Yeast eXtract S g Gluco~e 20 g Pota6sium hydrogenphosphate6 g Ammonium hydrogencitrate 2 g Mangane~e~ sulphatc l-hydrate0.1 g Iron-II-~ulpha~e 7-hydrate0 03 g Tween 80 l g This rogo~a agar i~ ~olved in 1 1 of water after h~vlng added 1.32 ml of glacial acetic acid. After ~ol~ing the rogo~a agar one liter of a gel-like to pa~ty composition is produced which is applied onto the dish-~haped area 1 of the inert plastic support 3 a whereon it adhere fi .
The figure 3 shows the enlarged and schematical lo view of a mask 4, whereby the ~a~k 4 is formed in ~uch a way that rect~ngular free spaces 25 between the ridges are formed, whereby four of such free spaces 25 are examplarily -~hown in figure 3.
on their bottom side the ridge~ ~hich extend ~ro~s~i~e to the longitudinal ridge~ 2~a have wedge-e~aped c~te~ 28 ~figure 4), ~hereby the~e wedge-~haped cutters 28 cause the division of the culture medium into fields 26, a~ this i~ s~own in figure 4 for the culture medium 16. In orde~ to fix ~he ~k ~ on the ine~t pla~tic support the mask 4 ha s corre~ponding ~la~p section~ 29 which can ~ear in with the ridge-like side wall sections 15.
In figure 5 a carpule 8 is ~chematically ~hown, whereby the carpule 8 ha~ a piercable mem~ane 31 at it~ top Ride and a piston 3~ at itB
boteom side, whereby the piston 32 can be ~xially ~hifted i~ ~he ~i~e~tion of the arrow 33 and vice-ver~ igure 5). A 0.005 n hydrochloric acid and a ~olution of the univer~al indicator are located in the inner space 30 of the carpule 8, whereby the volume of ~oth BolutionB ha~ the t~tal value of 1.27 ml. A limiting element 34 iB arranged at the bottom ~ide of the carpule 8 in ~uch a way that the a~ial ~hif~in~ of the pi~ton 32 in the direation of the arrow 33 is limited by mean~
of thi~ limiting ele~ent 34. Hereby it i~
gu~ra~teed that a maximum total v~l~me 4~ 1, 7 ml of liquid i~ taken up into the inner space ~0 of the carpule 8 during the shifting of the pi~ton 32 in the direction o~ the arrow 33.
If the u~er want~ to employ the afore described analyei~ ~et ~hown in figu~es 1 to 5 the corre8~n~; ng patien~ has to chew firstly the paraffin block 5 taken from the analy~ib ~et 1 for a gi~en time pe~iod (generally for five minutes). Hereby the ~ali~ation of the pa~ient i9 animated, ~o that a corre~ponding measuring cylinder can detect and then measure the spit Oue ~aliva amount.
For ~he ~etermination of the buffering capacity of the ~ali~a, the u~er of the analysis ~et l take~ a carpule 8 from the analy~ et 1, whereby the carpule 8 is filled with the mixture of hydrochloric acid and univer~al indica~or (measuring range pH 4 to pH lo). This c~rpule B i~ then flt into a usual ~yringe, whereby a ca~ula 2 taken from ~he analysi~ set 1 i~ mounted onto the syrlnge. The carpule ~it inco the syrlnge then takes up a given amount of ~liva by a correeponding movement of the ~yrin~e piston which gear8 in with the piston 32 ~figure 5) arranged within the carpule B, whereby the volume of the 6aliva e~ual,~ ~
quarter of the volume of the carpule content.
The axial ~hifting of the ~hiftabl~ pi~ton 32 i~ then limited by the limiting element 34, so that it is guaranteed that always the same amount of ~aliva i~ taken up into the carpule 8.
The carpule 8 being addltionally filled with ~aliva ie then removed from the syringe and ~he dispo~able cann~la belng mounted onto the ~yringe and having a length o~ 3 2 mm and diameter of o.9 mm is removed and wasted.
The carpule 8 removed from the syringe, the carpule 8 being a glacs carpule, then S immediate~y allows a vi~ual evaluation of the colo~ change of ~he carpule content, whereby thi~ color change i9 quantified by the vi~ual comparlson with the color scale 6 of the analysi~ set 1. Herewith the corresponding u.Rer lo then define~ the bu~ering capacity of the saliva.
A furth~r sali~a sample is then applied onto c~lture medium 16 and another saliva sample onto cul~ure ~ediu~ 17 (figure 2), where~y the pla~tic ~upport 3 a coated with the culture mediums 16 and 17 is introduced into the storage ve~el 18 after applying the saliva ~amplç~. Befor~ that a C02 releaRing tablet 7 ~0 was removed from the analy~is eet 1 and introduced into the ~torage ve~el 18.
Afte~ ga~tightly clo~ing the storage vessel 18, ehe storage ve~eel 18 is then transmitted into 2S an incubato~ together with ~he culture medium~
16 and 17 being incubated wi~h ~aliva and being arranged within the ~torage ve~el 18, whereby 3~ .
the corre~ponding storage ve~el i~ then incu~ated, for example, for 24 or ~8 hour~ a~ a temperature of 37 ~C.
After expiration of ~he inaubation time the vi~ual e~aluation and quantification of the cultur~ mediums l~, re~pectively 17, whereby the cul~i~ated streptocoacus mutan~ ~e well a~
the lacto~a~ill~ cultiv~ted on the further lo culture medium are clearly vi~ible Bince they ~tand out again~t the ground in re~pect to the color. The concent~ation of ~trep~ococcu~
m~tan~ and lactobacillu~ can then be detected by ~ vi~ual compari~on with the ~t~n~ard, as lS thic i~ u~ually done in microbiolo~y.
If, in contra~t to that, the ~er wants to examine ~pecial mouth areaR or ~ingle teeth in re~pect to the pre~ent microorgani~m~, he 2~ arrangc~ two or three mask~3 4 of the analy~3i8 Be~ 1 on the culture medium 16 and/or on the culture medium 17, a~ thi~ is shown in f igure 4 ~or the culture medium 16. Hereby aingle fielda 26 are generated on ~he corre~pondi~g c~lture medium a~, for example, o~ the culture medium 16 in figure 4, ~here~y the~e fields 26 are ~traightway incubated with the locally removed :~9 ~aliva qample~, respectively ~he plaque su~pensions. Hereafter the further proce~sing i~ carried out a~ it i~ described above.
~he e~ential advantag~ of this way of proce~ing 1~ the fact that all locally removed ~ample~ are examined at the ~ame time in the ~ame analysi~, BO that corre~ponding to that a ~e~hodical fault can be excluded. ~his lo e~eentially contribut~ to increa~ing the preci~enes~
In order to proo~ that the afore described miti5 salivarius agar (culture mediums I and II) which additionally contains the sugar in dif~erent concentrations i~ clearly superior to the u~ual miti8 ~alivariu~ agar, a large, clinical ~tudy with ~5 children ~nd 13~ ~aliva isolate~ was carried out. Hereby the usual miti8 Balivarius agar wa~ compared to the culture ~edium~ I and II containing the ~ugar, whereby the ~iti~ salivariu~ agar containing the sugar ha~ the compo~ition as it is de~cribed abo~e for th~ culture mediums I and Z5 II.
In the ~cope of ehi~ ~tudy it wa~ noted that the germ yield of ~treptococcus mutans wa~
clearly and ~ignificantly higher for ~he culture m~dium I and II containing ~ugar than the ge~m yield of ~ereptococcu~ mutans for ~he ueual mitis sallvarius agar. In comparieon with the cult~re medi~m I the culture medium I I had a higher germ yield of ~trep~ococcu~ mutan~.
~he present invention relate~ to a culture medium for the detection o~ streptococcus mutans in saliva or in plaque ~ith the o charac~eristics of the generic part of patent claim 1 a~ well a~ to an a~alysis ~et with the characteri~ticR of the generic p~rt of patent claim 18.
In the field of p~eventive denti~try it i~
known to ex~ine in the saliva or the plaque of the teeth the pre~ence of streptococcu~ mutan~
and to determin~ eheir quantitati~e concentration. In order to carry o~t ~uch a quali~tive a~d quantitative detection a given amount of ~aliva or an ~mount of plaque having been ~cratched off the teeth iB applied onto a culture medium, where~y in this case the known mitis sali~ariu~ agar iB often used.
For the e~tabli~hment of a diagnosis, particularly for estimating the risk of cariogenicity, it is nece~ary to determine the concentration of lactobacill~ in the ~aliva and/or in the plaque, whereby in thi~ ca~e a culture medium i~ al~o u~ed. A sample of the ~aliva, re~pectively of the plaque, i~ applied onto thi~ culture medium based on a rogo~a agar a~ thi~ i~ al~o de~cribed a~ove for the detection o~ the ~treptoco~u~ mutan~, ~o that the i~cuba~ed culture medium can be visually evaluated on a quantitati~e and qualitative basi~ after a corre~ponding ~ime of incubation.
The culture ~edium used herefore and ba~ed on a o ueual ~ogOsa agar ha~ the following componition:
Agar a~ar 19 g Tryptone 10 g Yea~t extract 5 g Glu~o~e 20 g Pota~ium hydrogenpho~p~ate6 g Ammonium hydrogencitrat~ 2 g Manganese~ ulphate l-hydr~te0.1 g Iron-II-~ulph~te 7-hydr~te 0.03 g Tween ~0 1 g For the manufacture of the ready-to-apply rogo~a culture medium the afore indicated compo~ent~ are added to 1 l of water.
For the establi~hment of an impecca~le dia~no~i~, par~ichl~ly for defining concrete step~ to a~oid the risk of cariogenicity, it i~
moreover nece~ary to mea~ure the buffering capacity o~ the ~aliva. In the analyei~ ~et being available nowaday~ this buffering capacity i~ mea~ured by ~prinkling a tect strip which is ~oaked with an indicator ~ith ~aliva, whe~eby the nece~a~y color change o~ the test st~ip i~ ~hen vi~ally de~er~ined after an exposure time of about two minutes. Hereby the test strip used herefore, however, only allow~
a coar~e mea~uring of the buffering capacity being defined a~ pH-value, so that the measuring of the buffering capacity i6 not ~afe and depend~ o~ the skill o~ the corre~ponding u~er.
The pre~ent in~ention ha~ t~e obje~t of di~posin~ a ~ulture medium of the indicated sort which make~ po~ible a ~afer statement about the concentration of ~treptococcus mutan~.
Thi~ object i~ realized by mean~ of a culture medium with the ~ignlficant characteristic~ of patent claim 1.
The inventive culture medium of the detection of streptococcus mutans in saliva and/or in plaque provides t~at the inventive culture medium contain-~ at least one ~ugar in addition S to one agar, where~y ~he ~a~ ratio of the agar to the at lea~t one sugar varies between l:o.
and 1:12.. In other ~ord~, the thu~ inventive culture ~edium differs from the known culture medium ~n ~hat way tha~ the inve~tive culture lo m~dium compri~e-~ at least one sugar in the afore indicated ma~ ~atio.
It was ~urprisingly notcd that the inventive culture medium show~ a high ~electivity con~erning ehe ~treptococcus mutanB, whereby thie high ~electivity for ~treptococcus mutan~
i~ referred to the ~act that, in contra~t to u~ual culture mediums being available in trade, ~he inventive culture medium additionally comprises a sugar. Compared to u~ual culture mediumfi thi~ impro~ed ~ele~ivity o~ the inventive culture medium is expre~ed in that the inventive culture medium ~hows a ~ignificantly higher germ yield of ~treptococcu~ mutan~, which was con~irmed by 135 Qaliva isolates o~ 55 children. This supplemen~ary sugar additive doe~ not impede the preparing of a ready-to-apply culture medium from the inventive culture medium, ~hereby for the ~imple and rapid preparation the inventive dry culture medium is only taken up into the re~lred a~ount of water.
The inventive culture medium can also be manuf~tured in a particularly ~i~ple ~anner since here~ore it i~ only neces~ary to mix a ~uitable agar with the required ~ugar amount, whereby the mixing of the agar with the ~ugar can be carried out not only during the manufacture o~ a ready-to-apply culture medium, me~ning duri~g the taking up of the agar and of ~he ~u~ar into water, but al~o by mixing the dry agar with the at lea~t one sugar.
A ~ir~t e~h~A~ent of the inventive culture medium provide~ that hereby the ma~ ratio of the agar to the at least one -~ugar lie~ between 1:3.8~ and 1:4 2~. ~ereb~ it wa~ noted that thiY preferred embodiment of the inventive culture ~edium wa~ taken up into water in a particularly ~imple and rapid manner, ~o that a ready-to-apply culture medium can be manufactured corre~pondingly ~imple and rapid.
A ~urther embodiment of the inventive culture medium ~howing an extremely high selecti~ity for the detection o$ ~treptococcu~ mutans provides that hereby the ma~s ratio of the agar ~o ~he at lea~t one ~ugar varie~ between 1:5.1 and 1:10. Althou~h t~ia embodiment of the inventive aulture ~edium has an extremely hig~
Bugar concentrAtion it could ~e aurpriai~gly noted that the ~treptococcu~ mutans extracted o from hu~an ~ali~a or pla~ue grew perfectly and ~e~ectively on a culture medium being mixed ~ith su~ar in such a ~ay.
The term agar i~ ~ed above and ~ubsequently, whereby thi~ term means particularly the mucilage ext~acted and dried ~rom red seaweed a~ well a~ highly molecular galac~o~ides, pa~ticularly ~ B of galaktoae-~ulphate e~ters, a~ far as ~pecia~ agar~ are ~ub~equently not mentioned.
In respec~ to the sugar contained in the inventive culture medium it i~ to be noted that thi~ sugar can be a monoaaccharide and/~r a mono~accharide derivative. Ho~ever the in~entive culture medium preferably compri~ea a~ sugar a di~accharide, particularly aucroae and/or ~ eucro~e dexivative.
Depending on the corre~ponding field of application, the inventive culture ~ediu~
consists in the simplest cafie only of the u~ual agar and the a~ore described sugar in the mentioned mass ratios. If such an embodiment i,Q
then applied by skilled epecial staff a~, for example, in correspondin~ ho~pitals, it i8 only necessary to take up the inventive culture ~edium into a coxre~po~;n~ amoun~ of w~ter, so that then corre~ponding dishes, for example Petri dishes, can be filled with this ready~to-apply, gel-like cu~ture ~edium. In order to ~a~ely and reproducibly distingui~h the streptococc~ m~ea~s ~ulti~a~ed in the ~aliva or in the plaque and the additionally and eventually cultivated streptô~occu~ mutan~, particularly the s~eptococcus para~ang~is and ~o the streptococcus sallvarius, aft¢r incubating with the saliva, re~pe~tively with the plaque, and after the ~ubsequen~ lncubation, it is recl_. n~d to carry out a dilution ~erie~
belng u~ual for ~icrobiological e~Am~n~tions.
Hereby the saliva, respectively the plaq~e, is dilu~ed by a phy~iological salt eolution to a ten~h ~o thousandth part of its original concentration, which cause~ an evident and effective BUppres~ion of th¢ gro~th of the a~ore mentioned strep~ococcus and of lactobacillu~, actinomycete~, ~taphylococcus or gram-negative bacterla. Thi~ again leads to a particularly safe and reproduci~le detection and vicual quantification of the ~treptococcus mutans.
A particularly simple and safe detection of strep~ococcus mutans which can renounce on the afore mentioned dilution ~eries without showing a fal~ification of the re~ult provide~ that hereby an inventive culture ~edium iR used w~ich co~pri~e~ ~t le~t o~e peptide antibiotic co~plex in addition to t~e agar and to the at least one sugar, wh~reby the concentration of this peptide antibiotic complex varies between 0.001 ~ by weight and 0.0005 % by weight relative to the total weight of the agar and the added ~ugar.
The afore de~cribed e~bodiment of the inventive culture medium particularly comprises as peptide antibiotic complex the bacitracin being ava~lable i~ t~de. In order to m~nufacture from thl~ embodiment of the inventive culture me~ium a ready-~o-apply culture medium it is only nece~ary to ta~e up the agar containing the at lea~t one sugar and the bacitracin into the ~equired amo~n~ o~ water. It was S particularly noted that such a rea~y-to-apply, inventivç c~lt~re medi~ then effectively suppre~e~ the growth of a mixed ~lora, particula~ly the ~th of ~ep~ococcu~
salivariuR and/or streptococcu~ para~angui~, lo without reducing the yield of strepeococcus ~utan~ in a ~tati~tically ~ignificant ~a~ner.
In re~pect to such ~aliva ~mp~e~ or plaque samples ~ic~ ~ompri~e~ al~o other ~treptococcus type~ in addition to the ~treptoco~cu~ mutan~, thic e~h~;ment of the inventive culture ~ediu~ makee it thu~ po~sible to easily and effective~y distingui~h ~he streptococcus mutans and the other streptococcus or ~he afore mentioned other microor~ani~m~.
A p~rticularly ~uitable agar being particularly u~ed in the inventive culture medium is the ~l~eady initially mentioned mitiB ~alivariu9 agar.
Hereby a typical miti~ ~alivarius a~ar aj it iY
available in trade and cold by the firm DIFCO, Augeburg, ha~ ehe following compo~ition Bacto trypto~e 8 g to 12 g, particularly 10 g Proteose peptone no. 3 3 g to 7 g, particularly 5 g Proteose peptone 3 g to 7 g, particularly 5 g lo ~acto dextro~e 0.5 g to 1.5 g, particularly 1 g ~acto ~ucrose 30 g to 60 g, particularly 50 g Pota~ium hydrogenpho~phate 3 g to S g, particularly 4 g ~ye, particularly O.oS g to o.09 g, trypan blue particularly 0.075 g Dye, parti~ularly 0.0006 g to o.ooo1 g, bacto cryetal violet particularly 0.008 g ~acto agar 13 g to 17 g, particularly 15 g For manufacturing a ready-to-apply culture medium the afore mentioned components are ~eirred into 1 1 of water.
In order to facilitate particularly the ~pplication of the inventi~e culture medium a further embodiment o~ the inventive culture medium provides that the culture medium iB
applied onto one ~ide of an inert ~upport (dip-~lide; solid c~rrie~). For ~he detection of ~treptococcu~ mu~an~ in ~aliva it i~ only nece~ary to remove the s~erile packed culture medium being appl ied onto an inert ~upport, pareicularly onto a plastic ~upport, from the lo s~erile packing and to add the saliva sample or the plaque ~ample to be ex~ined to thi~
culture medium, ~ that the corre~ponding st~eptococcu~ ~utan~ ~oncen~ration can be detected after a corre~ponding ~terile incubation.
A~ already repeatedly mentioned above, a ready-to-apply cult~re medium can be eaoily and rapidly manufactured from the inventive culture medium, ~he~eby ~uch a ready-to-apply culture medium preferably ha~ gel-like con~i~tency. , whereby water can be added to the culture medium in order to produce the gel-like con~i~tency. For generating the gel-like consi~tency t~e dry inventive culture ~edium comprising ~he agar, preferably the miti~
salivariu~ agar, ~hich contain~ the at lea,~t on~ ~ugar and, if necee~ry, the peptide anti~iotio ~o~plex, pre~erably the bacitracin, ie only taken up into water, whereby ~he de~ired gel-like con~i~tency can then be ad~usted ~y varying ehe amount of the added water.
Shch a gel-like development of the ready-to-apply culture ~ediu~ preferably co~pri~es between 70 g to 150 g of the agar, peptide antibiotic the miti~ ~alivariuB agar, between 42 g and 1.8000 g of the at lea~t one BUgar and a~ much water as i~ required, prefe~ably between 888 g and 100 g, for ~an~acturiny the gel-like con~i~tency. If, in addition to the gel-like and ready-to-apply culture medium, the afore ~e~cribed peptide antibiotic complex, preferably the bacitra~in, i~ to be added, the amount of this peptide antibio~ic complex, preferably the amount of the bacitracin, then variee ~etween ~.~ mg and 12 mg, e~ch xelati~e to the total amount of the at lea~t one ~u~ar and of the agar, paxticularly the mitis ~alivarius agar.
In order to fa~ ate the determination preci~ne~ of the cultivated ~treptococcu~
mutan~ in re~pect to the afore de~cri~ed and ~ub~equently described e~odiments of ~he inve~tive culture medium in which the culture medium iB applied on~o an iner~ ~upport, a further development o~ the inventive culture ~edium provides that hereby the inert ~upport is dy~d with dark color, preferably dyed black, or that it con~ist~ o~ a corresponding dark plastic ~terial.
Another ~m~o~iment of the inventive cultu~e me~iu~ being at the ~ame time B~ table for the de~ection of streptococ~us mueans and of lac~obacillu~ or yeast fungus provides that the in~enti~e ~ulture ~edium whi~h comprises the at lea~t one ~4gar ~nd, if ~eceu~ary, the peptide antibiotic co~plex in addition ~o the agar, particularly the mitis ~alivarius agar, i~
applied onto one side of the i~ert support, preferably a plastic ~upport, wherea~ on the other side of the inert support a further culture ~ediu~ for the detection of laceobacillus in the saliva and/or in the plaque or a further culture medium for the deteation of yea~t ~ungus in the ~aliva and/or ~he plaque i~ depo~ited. Hereby such an embodiment of the in~entive ~ulture medium allows that the concentration ~f ~treptococ~us mutan~ and la~tobacillus or of str~ptococcus mutan~ and ye~et fungus ~n the ~aliva and/or in the plaque bei~g required for the Carie~
prophylaxi~ can ~e concurrently determined with one sing~e te~t, 50 that ~uch a development of the inventive cultuze ~edium can ~e applied al~o particularly for the rapid and safe diagno~i~ in the denti~t'~ practice.
In the afore de~cribed development of the in~entive cult~re medium by which lactobacillu.
can be determined conc~rrent~y with ~treptococcus mutan~ a culture medium ~a~ed on a rogo~a agar i~ 6elected a~ further culture medium, whereby ~uch a culture mediu~ then ha~
a chemical conetruction a~ initially described.
In a particularly advantageou~ development o~
the ~fore de~ribed embodi~ent of the inventive culture mediu~ the further culture medium comprises at least one sugar in addition to the u~ual rogoca agar or to ~he cul~ure medium for the detection of yea~t fung~, whereby ~hi~ a~ leaet one ~ugar preferably i8 gluco~e, ~ucro~e and/or a gluco~e derivative, re~pectively a ~ucrose derivative. Particularly if the further culture medium contalns ~he rogo~a agar or the culture medium for ~he detection of yea~t fungu~ and the at lea~t one ~ugar in a ma~s ratio of between l:0.~ and 1:6, S then su~h modi~ied further culture medium ~how~
a h~gh selecti~ity and yield of lactobacillus, recpectively of yea~t fung~, ~o that ~hi~
e~bodiment allow~ a preci~e and reproducible, qualitative ~nd qu~ntitA~ive de~ermination o~
lactobacillu~, re~pectively yea~t fung~, in the ~al iva a~d/or i~ the plaq~e.
If in the a~ore de~cribed embodimen~ one ~ide of the inert ~pport i~ coated with the culture medium for the de~ection of the streptococcuq mutans and the other ~ide of the inert ~upport i~ coated with the culture mediu~ for the detection of the yea~t funguB in the ~aliva and/or in the plaque, thi~ e~bodiment of the inventive culture medium then allow~
particularly for children b~ing under ~our ~ears old, preferably howev~r for the a~e of le~ ~han three year~, a particularly easy and concurrent deter~ination of an eventual pre~ence of yeajt fungu~ in the ~aliva or in the pla~ue of the~e children in addition to ~treptococcu~ mutans. Especially in the ca~e of CA 02240237 l998-06-lO
children infected ma~ively with carie~, partic~larly with the nur~ing bo~tle ~yndrome, it wa~ noted that yea~t fungu~ are important indlaator germ~. Hereby a culture medium for the detec~ion of yea~e fungu~ with the following compo~ition is preferably u~ed:
Chromopeptone 8 g to 12 g Glucose 15 g to 25 g lo Chloramphenicol 0.3 g to 0.7 g Dye mixture (chro~ogen mix) 1.5 g to 2.S g Agar 10 g to 20 g The agar 601d under the trade name "BL
CHROMagar Candida~ ~Manufacturer: ~ecton ~ickin~on 6~b~, Heidel~erg~ i~ particularly ~uitable for the detection of yea~t ~ungu~.
In ord~r to manufactu~e ~ re~dy-to-apply culture medium for the detection of yeast fungu~ the afore indi~ated component,s are taken up into one liter of deionized water, so that gel-like culture medium i~ produced to which, if de~ired, further amounts of ~ugar of the described concentration range~ can be added.
In order to facilitate the application of the inert ~uppor~ ~oated on one side with the inventi~e ~ulture medium and coated on the other ~ide with the further c~lture medium, a further develop~ent of the inve~tivo cult~re medium provides that the fuxther cult~re medium ha~ a ge~-like con~i~ten~y, whereby the gel coneain~ between 70 g and 250 g of the further c~lture medium snd between 930 g and 750 g of water.
pa~ticularl~ ~uitable and advantageous development of the afore de6crib~d embodime~t of ~he inven~ive culkure medium ~hich can be applied in each denti~t'~ practice provide~
that the inert eupport which i~ co~ted on one ~ide with the gel-l~ke culture mediu~ on the baei~ of the agar, preferably the miti~
~all~riu~ ag~r, containing at lea~t one ~ugar and the peptide antibiotic complex, particularly bacit~acin, and which is ~oated on the other ~ide with the gel-like further culture medium on the basis of the afore described rogosa agar or the culture medi~m for the detection of yea~t fungu~ i~ to be arranged ~ithln a ga~tight lockable ~torage ~e~el.
Hereby the ga~tight lockable storage ve~el ~erve~ for ~he ~terile ~torage and for the CA 02240237 lgss-06-lo transport of thi~ e~bodiment of the ~nventive culture medium and guaranties at the same time that, after the adding of the corre~ponding ~aliva ~ample~, respecti~ely plaque ~a~ples to ~oth culture mediums, the thus treated culture ~ediume can be incubated in the stora~e vessel, where~y a C02 relea~ing ~able~, preferably an alkali carbonate tablet, re~pectively an alkali hydrogencarbonate tablet, can be arranged ~ithin ~he etor~e ve~el particularly during the incubation. Hereby thi~ C02 reloa~ing tablet cau~e~ the fact that a corre~ponding C02 atmo~phere i~ adju~ted ~ithin the gastight lockable ~torage veesel during the incubation time, whereby, on one hand, the selectivity of the inventive culture medium for the streptoco~cus mutan~ and the lactobacillus, respectively the yeast ~ungu~ increa~ed and, on the other hand, unde~ired side reaction~ are excluded. Such ah em~odi~ent is perfe~tly ~uitable for the concurrent detection of ~treptococcus mutanB and lactobacillu~, re~pectively yea~t ~ungus, in the dentist ' B
practices, whereby this development of the Z5 inventive culture ~edium then allow~ a reproducible and ~uantified determination r~garding these ~icroorgani~m~.
lg The present invention rela~e~ further~ore to a analy~i~ ee~ for the ~imple e~tablishment of a diagnosis, particularly for evaluating the ri~k S of cariogenicity.
Thi~ object i~ reali~ed according to the inven~ion by an analysis set havin~ the significant ~aracte~ietic~ of patent claim 18.
The inventive analysis set comprises a culture medium being arranged o~ one side of ~n inert support and being of the afore de~çribed ~ort for the det~ction of streptococcus mutans, whereas the other side of the inert support is co~ted ~ith the fu~ther culture mediu~ baeed on the afore de~cribed rogosa agar for the detec~ion of lactobacillus or with a culture - mediu~ of the detection of yea~t fungus.
ln a development of the in~entive analysis set the analysis #et can prefera~ly comprise ~t leaet one carpule filled with an acid, pref~ral~ly with ~ O . 005 n hydrochloric aoid, a8 ~ell as at least one cannula.
The inventive analy~is 8et ~hows a range of ad~antage~. Fir~t of all it i~ to be noted that the afore deecribed in~en~ive analy~i~ set allow~ a preci~e qualit~ti~e and quantitative a~ well as concurrent determination of ~treptococcu~ mutan~ and lactoba~lllus, respectively yeast fungue, in ~aliva an~/or in pla~u~. In addition to that the afore mentioned development of the in~entl~e analyRi~ Bet can make possible a determination o~ the buffering lo c~p~city of the ~ali~a. Thi~ is achieved by taking up a defined amount of saliva into the carpule bein~ filled with an acid, prefe~ably with a O.OOS n hydrochloric acid, ~o that after mlxing the ~aliva with the acid the pH-value of thi~ liquid mixture can be easily determined, whereby the pH-v~lue i~ a quantitative indication o~ the bu~fering capacity o~ the ple.
In order ~o ~ke ~p ~he s~liva into the carpule filled with ~he a~id the carpule of the inventive analy~i~ aet i8 ~it into a usual ~edi~al ~yring~. The cannul~ contai~ed i~ the analysi6 ~et is mounted onto the ~yringe, 50 that then the carpule i~ filled wlth a given amount of the ~aliva sample, xe~pectively the pl~que ~uapen~ion, ~y mean~ of the ~yringe by pulling back the ~yri~ge pi~ton to a given mark or to a gi~en stopper. After ~ixin~ the ~ample with the acid the evaluation can be realized by interpreting the color change. After ~he determination o~ the concentration of streptococcus mutan~, lactobacillu~ or yeast fungus and even~ally o~ ~he buffe~ing capacity the inventi~e analy~ln set can be easily ~asted.
A ~urther development of the a~ore de~cribed inventive analy~i~ set p~ovides that the analynis set co~pri~eC at lea~t one c02 ~eleasing tablet. Hereby the C02 relea~ing tablet e~enti~lly c~use~ that, after incubating the cultu~e ~edium ~o~ the detection of streptococcus mutans and the furthe~ c~lture mediu~ ~or the detection of lactob~eillus, respec~ively for the detection o~ yea~t fungu~, the i~ert ~upport coated with those two cult~re medium iB ~tored in a ga~tight, lockable ~torage vessel, whereby the C02 relea~ing tab~et i~ a~anged within this gastight ~torage ve~el during ~he incubation. Thi~ C02 relea~ing tab~et cau~es the ga~tight storage ve~el i~ ~et under a corresponding C02 atmosphere durin~ the incu~atlon, ~o that, on one hand, the selectivity for the detection of the ~treptococcu~ mu~an~ and lactob~cillu~ i~
~o~re~pondingly increased and, on the oeher hand, unde~ired ~ide reactions are e~cluded becau~e of the CO2 atmo~phere.
In a further e~odi~ent of the inventive a~ly~i~ set ehe analysi~ ~et comprise~ ~t least one paraffin piece and~or a pH-value i~dicator. Hereby thix paraffin piece ~erves for the ~illing of ~he corre~po~ding patient whose saliva, respectively plaque i~ to be ex~m;n~d in res~ect to the presence of ~treptococcus mu~an~ and lacto~cillus, re~pectively yea~t fungu~, ¢hew~ thi~ paraffin piece for a given time before the execution of the co~resp~d~ng ~ ion, in order to thus animate the salivation, whereas the pH-value indicator i~ employed to determine the pH-valu~
of the saliva, a~ thi~ iB de~ribed later on.
In order to furtherly ~implify the determination of the buffering capacity of the saliva in the afore described embodimen~ of the ~5 inventive analy~ e~, a further development of the inventive analysis set provides that hereby the carp~le ¢ompri~es also an indicator, particularly a liquid indicator, in addition to the acid being pareicularly contained in ~he carpule in a given amount and concentration.
Hereby it is rendered po~ible to directly def ine the buf f ering capacity of the saliva sample by interpreting the color change after the filling o~ a 8ali~a ~ample, respectively a pla~ue ~uBpen~ion, intb the carpule, whereby ~he safety and reproducibility of the determination of the bu~fering capa~i~y iB
fu~therly i~proved.
It is ~l~o pos~i~le to provide the analysis set with a pH-value indicator a~, for example, a liquid i~dicator, with a ~olid indicator and~or with a indicator strip, 80 that hereby t~e buffered ~aliva can be removed from the carpule in order to then deter~ine its pH-value.
The afore described indicator preferably i~ a liquid ~niver~al indic~tor, whereby thiP liquid universal indicator has a pH-value indicating range of between pH 4 and pH 10.
In order to ~acilitate the vi-~ual defini~ion of the buffering capacity of the ealiva, re~pectively of the plaque suspeneion, , it is particularly advantageou~ that the analy~is ~et containe color ~cale, eo that the bu~ering capacity of the corre~pon~in~ ~ample can be de~ermined in a particularly ea~y and rapid s manner by vi~ually compaxing the color ~c~le with the obta$ned color yield of the çarpule which i~ filled with the ~aliva and which ~ontain~ the univer~al indicator a~ ~ell as the afore described acid.
Another advantageou~ development of the inventive analy~i~ set provide~ ~hat the analy~is ~et comprises at lea~t on~ ma~k which can be arranged on the inert support, so that by thi~ ~a~k the cul~ure mediu~ which is arranged on on~ side of ~he inert ~uppor~ and which i~ based on the modified agar, pa~ticulaxly the mitiu ~alivarius ~gar, and/or the ~urther culture ~edium which i~ arranged on the other ~ide of ~he inert ~upport and which is ~a~ed on the rogo~a agar, re~pectively for ~he detec~ion of ye~t fu~gu~, can be ~ubdivided into single ~ection~. This ~mbodiment of the inventive analy~i~ see makes it thus poosibl~ to optionally subdivide one c~lture ~edium or both culture medium~ in single 8ections, 80 that single mouth areas or CA 02240237 l998-06-lO
particularly dental area~ can be specifically examined fo~ streptococcuR mutane and l~ctobacillu~ or yea~ fungu~ with one single te~t. Herefore it is only ~ecessary to apply S thi~ 6ample removed from the mo~h area, re~pectively from the ~pecific tooth, onto the section of the co~re~rond; ngly ~ubdivided c~lture ~edlum, ~o that after the inc~bation the microbiological ~ondition6 can easily be defined in re~pect to their concentration of streptococcu~ mutan~ and/or lac~oba~illus or of ~t~e~ococcu~ mutan~ and~or yeast fungus. T~i~
maQk i~ preferably provided wit~ an in~c~iption part, ~o that an a~ign~en~ of the obtained valuee to ~pe~ial mo~th ~reaB, re~pe~tively dental area~, can ~afely be carried o~t.
In another em~odiment of the inventive anal~sis set this analy~is eet compri~e~ moreover a calibrated ve~el for taking up the saliva Here~y it i~ ~ade po~sible to measure a further parameter during the ~aliva e~-nAtion, whereby thic para~eter ig the ealiva volume generated by the patient by chewing the afore de~cribed paraf~in piece for a ~iven time.
A further advantageoue develop~ent of the inventive analy~ et provide~ that the a~aly~i~ qet compri~e~ ~oreover a di~po~able ~yringe. Hereby thi~ ~ispoeable syring~ ~erve~
to inject saliva or a plaque ~u~pen~ion into ~he carpule filled ~ith the acid, preferably ~lth the 0.005 n hydrochloric acid, and wi~h the univercal indlcator, whereby the amount o~
the injected ~aliva can be determined optionally in the disposable ~yringe or in a particularly advantageou~ manner by a mark located on the carpule up to which mark the gliding plug i~ di~placed in the axially oppo~ite direction o~ the piercable membrane.
Thi8 embodiment o~ the analy8i~ -~et guaranties that each u~er of the inventive analy~ et find~ ~ll in~tru~en~ required for the dete~mina~ion of the buf~e~in~ capacity of saliva in thi~ analysi~ set, ~o that ~he corre~ponding analyei~ can be carried out in a p~r~iclllarl~ ~imple ~nd ~apid and thul3 ~ery economical manner.
The afore described embodiments o~ the inventive cult~re medium in which the culture ~edium, re~pectively the afore de~cribed fur~her culture medium, i~ applied each onto one ~ide of an inert ~upport can be ufied in a particularly ~imple, rapid and reproducible manner for the dete~tion of ~treptococcus mutan~ on one hand and for the detection of lactobacillu~ or yea~t fungu~ on the other hand. In thi~ detection method firat of all a saliva ~a~ple or a pla~ue ~ample i~ taken from th~ patient and applied onto the culture medium for the detection of otreptococcue ~utans a~
well as onto the further culture medium for the detection of lactobacillu~, respectively ye~t fungus. The culture medium~ thus loaded with the ~ali~a sample are then transmiteed p~eferably i~ a ~a~tight, lockable ve~sel having been event~ally ~llled before with a CO2 atmo~phere into a suitable incubator, so that the corre~po~ding ~torage ve88el i8 then inçuba~ed for a given incubation time, preferably between 24 and ~8 hours at a temperature o~ 37 nc + 4 ~C.
After e~pi~ation of the incubation time a vi~ual evaluation and ~uantification of the streptococcu~ mutan~ cultivated on ehe first culture m~dium arranged on one side of the inert ~upport as well as of the lactobacillus, re~pectively the yeast fungus, cultivated on the further culture medium arranged on the other ~ide of the inert support. By a vi~u~l comparison with the corresponding ~tandard~ the con~ent~ation o~ strepto~oc~u~ mu~ans and lactobacillu~, re~pectively of ~treptococcu~
mutans and yeast fungu~ ~an then be quantified, as thi~ i~ u~ual in microbiology.
Advantageou~ development~ of the inventive culture medium a~ well as of the analysis set lo are indi~ated in the ~ubclai~s.
The inventive culture medium and the inventive analy~i,q ~et are .qubsequently explained by an example in connection with the drawings.
Fi~ure 1 show~ a ~chematical, partly per~pective embodiment of the ana~yui~ ~et;
Figure 2 Bhow~ the cul~ure medium in a se~tional view along the line A-B in figure 1, whereby the culture medium is contained in the analy~i~ Ret according to figure 1 and arranged on an inert ~upport;
Fi~ure 3 show~ an enlarged vie~ of the ma~k ~hown in ~igure 1 in a top view;
~ig~re 4 ~how~ a view of the culture medium b~ing arranged on a ~upport, whereby this view can be compared with figure 2. The culture medium ha~ the m~k ~ho~n in figure 3; and Figure 5 shows a sectional view of a carpule .
In the figures 1 ~o 5 the same part~ are xeferred to the corre~ponding number~.
A~ ~hown in fi~ure 1 ~he analy~ et 1 fir~t of all compri~es a corre~ponding amount of cannula~ 2, ~hereby the embodiment ~hown in ~igure 1 ex~mpl~rily indi¢~te~ three ca~ula~
The analy~ et 1 moreover compxi~e~ an amount of carpules 8 which corresponds to the amount of the cannula~ z, ~he~e~y in ~ igure 2 one single ca~pule 8 i~ only examplarily ~hown. Th~
- more detailed con~txuction of thi~ carpule 8 i~
subse~ùently explained by figure 5.
In addition to the carpule B and the cannulas 2 the analy~is set co~prise~ furthermore a corre~ponding amount of inert plastic supports 3 a coated wi~h culture mediums, whereby these plactic Bupport~ 3 a co~ted with culture mediums 3 are explained more detailed in the figuree 2 and 4. The analysis ~et al~o compri~e~ a mask 4 being schematically ~hown in figu~e 1, where~y the ~a~k 4 i6 de~cribed more detailed by the figure 3. The analysis set 1 furthermore co~pri~ number of paraffin blocks 5 which corre~pond~ to the number of cannula~ 2 and a number of C02 releasing tablet 7 which corre~pond~ to the number of pl~etic support~ 3 a, whereby in ~igure 1 on~y one paraf~in block 5 and one C02 relea~ing tablet 7 i~ examplarily ~hown. Moreover the analy~i~ set 1 compri~e'~ a ~lor ~cale 6.
As shown be~t in ~he sectional view~ of the figures 2 and ~, each inert pla~tic 6upport 3 a compri~e~ a grip ~ection 13, whereby a dieh-~h~ped area 14 iB directly adjoined to both sides of the grip section 13. Hereby the di~h-shaped area 1~ co~p~i~e~ a ridge-like ~ide wall sec~ion 15, where~y only ~he front-~ide ridge sections 15 are shown in the figure~ 2 and 4, The di~h- ~haped ~rea 14 ~erve to take ~p the culture medium 3 which i~ nu~bered ~ith 16, re~pectively with 17, in the ~igure~ 2 and 4, ~hereby one of the cultu~e mediums 16, re~pectively 17, i8 the afore described miti~
sallvarius agar and the other culture medium 17, re~pectively 16, i~ th~ al~o a~ore de~c~ibed rogo~a a~ar or i~ a culture medium for the detection of the yeast ~ungu~. The inert plastic suppor~ 3 a is arranged within a gastight lockable storage ve~xel 18, whereby the ~torage ven~el 18 i~ clo~ed up with the grip ~ec~ion 13.
It surely i~ po~ible to form the inert pla~tic ~upport 3 a in such a way that an in~erior ~rip section 13 is ~onnected with the di~h-shaped area 14, eo th~ ~he ~hole pla~tic ~upport 3 a i~ arranged within the ~torage ve~çl 18 and corre~pondingly ~lo~ed up ~ith a ~crew cap.
However thi~ embodiment i~ not 6hown in the figure~ 2 and 4.
The culture medium being arranged on the di~h-shaped area 14 and bein~ ~ased on a ~odi~ied miti~ salivariu6 agar i~ s~ch a oulture medi~m 16 a8 it i~ manu~actured as ~ollows.
~ir~t of all a purcha~able mitis salivarius agar a~, for example, from DI~C0 i~ ~hosen, whereby the pu~ha~able mitis salivariu~ agar contains the following compo~ents:
Bacto tryptose 20 g Proteose peptone no. 310 g Proteo~e peptone 10 g Bacto dextro~e 2 g Bacto ~ucro~e 100 g Potas~ium hy~ e~pho~phate 8 g Trypan blue 0.15 g Bacto crystal violet0.0016 g ~acto agar 30 g 360 g sucrose (Merck 1.07651) and 3 mg bacitracin (Sigmar ~-0125) are added to the fir~t half o~ this dry culture medium bein~
sub8equently named culture medium I, and 600 g sucro~e (Merck 1.07651) and 5 mg bacitracin (Sigmar b-0125) are added to the eecond half of the dry culture medium being ~ub~equently named culeure medium II, whereafter ~he culture medium~ I a~d II are mixed. The culture mediums I and II are ~hen ~olved in deionized water under the ~ormation of one liter of ready-to-apply culture ~ediums I and II ~ereafter the water i~ boiled. Then the culture mediums are ~terilized at lZl ~~ for lS ~inute,~. After having aooled down, the gel-like culture medium~ are applied onto the inert plastic support 3 a whi~h i~ a black p~a~ic aupport.
Culture medium I then ha~ a raeio of miti~
~alivariu~ agar to ~he added ~ucroee of 1:~.996, wherea~ cultur~ medium II has a ratio of mitis salivarius agar to the a~ded sucrose lo of 1:6.661.
For the manufacture of the culture medium 17 the u~ual rogosa agar i~ used as starting ~ateri~l, whereby the corre~ponding ro~o~a agar lS ha~ the already abovc indicated fo~lowi~g compo~ition:
Agar agar 19 g Tryptone lO g Yeast eXtract S g Gluco~e 20 g Pota6sium hydrogenphosphate6 g Ammonium hydrogencitrate 2 g Mangane~e~ sulphatc l-hydrate0.1 g Iron-II-~ulpha~e 7-hydrate0 03 g Tween 80 l g This rogo~a agar i~ ~olved in 1 1 of water after h~vlng added 1.32 ml of glacial acetic acid. After ~ol~ing the rogo~a agar one liter of a gel-like to pa~ty composition is produced which is applied onto the dish-~haped area 1 of the inert plastic support 3 a whereon it adhere fi .
The figure 3 shows the enlarged and schematical lo view of a mask 4, whereby the ~a~k 4 is formed in ~uch a way that rect~ngular free spaces 25 between the ridges are formed, whereby four of such free spaces 25 are examplarily -~hown in figure 3.
on their bottom side the ridge~ ~hich extend ~ro~s~i~e to the longitudinal ridge~ 2~a have wedge-e~aped c~te~ 28 ~figure 4), ~hereby the~e wedge-~haped cutters 28 cause the division of the culture medium into fields 26, a~ this i~ s~own in figure 4 for the culture medium 16. In orde~ to fix ~he ~k ~ on the ine~t pla~tic support the mask 4 ha s corre~ponding ~la~p section~ 29 which can ~ear in with the ridge-like side wall sections 15.
In figure 5 a carpule 8 is ~chematically ~hown, whereby the carpule 8 ha~ a piercable mem~ane 31 at it~ top Ride and a piston 3~ at itB
boteom side, whereby the piston 32 can be ~xially ~hifted i~ ~he ~i~e~tion of the arrow 33 and vice-ver~ igure 5). A 0.005 n hydrochloric acid and a ~olution of the univer~al indicator are located in the inner space 30 of the carpule 8, whereby the volume of ~oth BolutionB ha~ the t~tal value of 1.27 ml. A limiting element 34 iB arranged at the bottom ~ide of the carpule 8 in ~uch a way that the a~ial ~hif~in~ of the pi~ton 32 in the direation of the arrow 33 is limited by mean~
of thi~ limiting ele~ent 34. Hereby it i~
gu~ra~teed that a maximum total v~l~me 4~ 1, 7 ml of liquid i~ taken up into the inner space ~0 of the carpule 8 during the shifting of the pi~ton 32 in the direction o~ the arrow 33.
If the u~er want~ to employ the afore described analyei~ ~et ~hown in figu~es 1 to 5 the corre8~n~; ng patien~ has to chew firstly the paraffin block 5 taken from the analy~ib ~et 1 for a gi~en time pe~iod (generally for five minutes). Hereby the ~ali~ation of the pa~ient i9 animated, ~o that a corre~ponding measuring cylinder can detect and then measure the spit Oue ~aliva amount.
For ~he ~etermination of the buffering capacity of the ~ali~a, the u~er of the analysis ~et l take~ a carpule 8 from the analy~ et 1, whereby the carpule 8 is filled with the mixture of hydrochloric acid and univer~al indica~or (measuring range pH 4 to pH lo). This c~rpule B i~ then flt into a usual ~yringe, whereby a ca~ula 2 taken from ~he analysi~ set 1 i~ mounted onto the syrlnge. The carpule ~it inco the syrlnge then takes up a given amount of ~liva by a correeponding movement of the ~yrin~e piston which gear8 in with the piston 32 ~figure 5) arranged within the carpule B, whereby the volume of the 6aliva e~ual,~ ~
quarter of the volume of the carpule content.
The axial ~hifting of the ~hiftabl~ pi~ton 32 i~ then limited by the limiting element 34, so that it is guaranteed that always the same amount of ~aliva i~ taken up into the carpule 8.
The carpule 8 being addltionally filled with ~aliva ie then removed from the syringe and ~he dispo~able cann~la belng mounted onto the ~yringe and having a length o~ 3 2 mm and diameter of o.9 mm is removed and wasted.
The carpule 8 removed from the syringe, the carpule 8 being a glacs carpule, then S immediate~y allows a vi~ual evaluation of the colo~ change of ~he carpule content, whereby thi~ color change i9 quantified by the vi~ual comparlson with the color scale 6 of the analysi~ set 1. Herewith the corresponding u.Rer lo then define~ the bu~ering capacity of the saliva.
A furth~r sali~a sample is then applied onto c~lture medium 16 and another saliva sample onto cul~ure ~ediu~ 17 (figure 2), where~y the pla~tic ~upport 3 a coated with the culture mediums 16 and 17 is introduced into the storage ve~el 18 after applying the saliva ~amplç~. Befor~ that a C02 releaRing tablet 7 ~0 was removed from the analy~is eet 1 and introduced into the ~torage ve~el 18.
Afte~ ga~tightly clo~ing the storage vessel 18, ehe storage ve~eel 18 is then transmitted into 2S an incubato~ together with ~he culture medium~
16 and 17 being incubated wi~h ~aliva and being arranged within the ~torage ve~el 18, whereby 3~ .
the corre~ponding storage ve~el i~ then incu~ated, for example, for 24 or ~8 hour~ a~ a temperature of 37 ~C.
After expiration of ~he inaubation time the vi~ual e~aluation and quantification of the cultur~ mediums l~, re~pectively 17, whereby the cul~i~ated streptocoacus mutan~ ~e well a~
the lacto~a~ill~ cultiv~ted on the further lo culture medium are clearly vi~ible Bince they ~tand out again~t the ground in re~pect to the color. The concent~ation of ~trep~ococcu~
m~tan~ and lactobacillu~ can then be detected by ~ vi~ual compari~on with the ~t~n~ard, as lS thic i~ u~ually done in microbiolo~y.
If, in contra~t to that, the ~er wants to examine ~pecial mouth areaR or ~ingle teeth in re~pect to the pre~ent microorgani~m~, he 2~ arrangc~ two or three mask~3 4 of the analy~3i8 Be~ 1 on the culture medium 16 and/or on the culture medium 17, a~ thi~ is shown in f igure 4 ~or the culture medium 16. Hereby aingle fielda 26 are generated on ~he corre~pondi~g c~lture medium a~, for example, o~ the culture medium 16 in figure 4, ~here~y the~e fields 26 are ~traightway incubated with the locally removed :~9 ~aliva qample~, respectively ~he plaque su~pensions. Hereafter the further proce~sing i~ carried out a~ it i~ described above.
~he e~ential advantag~ of this way of proce~ing 1~ the fact that all locally removed ~ample~ are examined at the ~ame time in the ~ame analysi~, BO that corre~ponding to that a ~e~hodical fault can be excluded. ~his lo e~eentially contribut~ to increa~ing the preci~enes~
In order to proo~ that the afore described miti5 salivarius agar (culture mediums I and II) which additionally contains the sugar in dif~erent concentrations i~ clearly superior to the u~ual miti8 ~alivariu~ agar, a large, clinical ~tudy with ~5 children ~nd 13~ ~aliva isolate~ was carried out. Hereby the usual miti8 Balivarius agar wa~ compared to the culture ~edium~ I and II containing the ~ugar, whereby the ~iti~ salivariu~ agar containing the sugar ha~ the compo~ition as it is de~cribed abo~e for th~ culture mediums I and Z5 II.
In the ~cope of ehi~ ~tudy it wa~ noted that the germ yield of ~treptococcus mutans wa~
clearly and ~ignificantly higher for ~he culture m~dium I and II containing ~ugar than the ge~m yield of ~ereptococcu~ mutans for ~he ueual mitis sallvarius agar. In comparieon with the cult~re medi~m I the culture medium I I had a higher germ yield of ~trep~ococcu~ mutan~.
Claims (26)
1. A culture medium for the detection of streptococcus mutans in the saliva and/or in the plaque, characterized in that the culture medium contains at least one sugar in addition to an agar, whereby the mass ratio of said agar to the at least one sugar varies between 1:0.6 and 1:12.
2. The culture medium according to claim 1, characterized in that the culture medium has a mass ratio of said agar to said at least one sugar of between 1:3.89 and 1:4.25.
3. The culture medium according to claim 1, characterized in that the culture medium has a mass ratio of said agar to said at least one sugar of between 1:5.1 and 1:10.
4. The culture medium according to one of the preceding claims, characterized in that the culture medium comprises as sugar at least one disaccharide, preferably sucrose and/or a sucrose derivative.
5. The culture medium according to one of the preceding claims, characterized in that the culture medium contains between 0.001 % by weight and 0.0005 % by weight of a peptide antibiotic complex.
6. The culture medium according to claim 5, characterized in that said peptide antibiotic complex is bacitracin.
7. The culture medium according to one of the preceding claims, characterized in that said agar is a mitis salivarius agar.
8. The culture medium according to one of the preceding claims, characterized in that the culture medium is applied on one side of an inert support.
9. The culture medium according to one of the preceding claims, characterized in that the culture medium has gel-like consistency, whereby water is added to the culture medium in order to produce the gel-like consistency.
10. The culture medium according to claim 9, characterized in that the gel-like culture medium comprises between 70 g and 150 g of said agar, particularly of a mitis salivarius agar, between 42 g and 1.800 g of said at least one sugar and between 888 g and 100 g of water.
11. The culture medium according to claim 10, characterized in that the gel-like culture medium comprises between 1.4 mg and 12 mg of said peptide antibiotic complex, preferably bacitracin.
12. The culture medium according to one of the preceding claims, characterized in that the culture medium is applied on one side of said inert support, preferably a plastic support, and that on the other side of said inert support a further culture medium for the detection of lactobacillus in the saliva and/or in the plaque or a further culture medium for the detection of yeast fungus in the saliva and/or the plaque is deposited.
13. The culture medium according to claim 12, characterized in that said further culture medium is based on a rogosa agar for the detection of lactobacillus.
14. The culture medium according to claim 13, characterized in that said further culture medium moreover comprises at least one sugar.
15. The culture medium according to claim 14, characterized in that said further culture medium contains the agar, preferably the rogosa agar, and the sugar in mass ratio of between 1:0.2 and 1:6.
16. The culture medium according to one of the claims 12 to 15, characterized in that said further culture medium has a gel-like consistency, whereby the gel contains between 70 g and 250 g of the further culture medium and between 930 g and 750 g of water.
17. The culture medium according to one of the preceding claims, characterized in that the inert support being coated on one side with said gel-like culture medium and on the other side with said further gel-like culture medium is to be arranged within a gastight lockable storage vessel.
18. An analysis set with a culture medium arranged on an inert support according to one of the preceding claims, characterized in that the analysis set (1) comprises at least one inert support (3 a) being coated on one side with said culture medium (3, 16, 17) and on the other side with said further culture medium (17, 16).
19. The analysis set according to claim 18, characterized in that the analysis set comprises, in addition to said inert support (3 a), at least one carpule (8) filled with an acid, preferably with a 0.005 n hydrochloric acid, and at least one cannula (2).
20. The analysis set according to claim 18 or 19, characterized in that the analysis set comprises at least one tablet (7) releasing CO2.
21. The analysis set according to one of the claims 18 to 20, characterized in that the analysis set (1) comprises at least one paraffin piece (5) and/or a pH-value indicator.
22. The analysis set according to one of the claims 18 to 21, characterized in that the carpule (8) is filled with an indicator, preferably with a liquid indicator, in addition to the acid.
23. The analysis set according to claim 22, characterized in that the indicator is a liquid universal indicator with a pH-indication range between pH 4 and pH 10.
24, The analysis set according to one of the claims 18 to 23, characterized in that the analysis set (1) comprises a color scale (6) to measure the pH-value of the carpule (8) being filled with the indicator.
25. The analysis set according to claim 18 to 24, characterized in that the analysis set comprises at least one mask (4) which can be fixed onto the inert support (3 a), so that said culture medium (16; 17) and/or said further culture medium (17; 16) can be subdivided into single sections (26).
26. The analysis set according to one of the claims 18 to 25, characterized in that said analysis set (1) comprises moreover a calibrated vessel for taking up the saliva.
21. The analysis set according to one of the claims 18 to 26, characterized in that said analysis set (1) comprises at least one disposable syringe.
21. The analysis set according to one of the claims 18 to 26, characterized in that said analysis set (1) comprises at least one disposable syringe.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19724970 | 1997-06-13 | ||
| DE19724970.1 | 1997-06-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2240237A1 true CA2240237A1 (en) | 1998-12-13 |
Family
ID=7832344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002240237A Abandoned CA2240237A1 (en) | 1997-06-13 | 1998-06-10 | Culture medium |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0885969B1 (en) |
| JP (1) | JP3459877B2 (en) |
| AT (1) | ATE204908T1 (en) |
| CA (1) | CA2240237A1 (en) |
| DE (2) | DE19825933A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6309835B1 (en) * | 1999-05-27 | 2001-10-30 | Koninkiijke Philips Electronics N.V. | Methods for quantitating the efficacy of oral care products |
| JP2002027975A (en) * | 2000-07-17 | 2002-01-29 | Bml Inc | Culture medium for selecting mutans streptococcus |
| KR100460605B1 (en) * | 2001-07-11 | 2004-12-08 | 박윤중 | Production of the flavor-enhancer by fermentation |
| JP4091912B2 (en) | 2001-07-27 | 2008-05-28 | 学校法人日本大学 | Streptococcus sobrinus isolation method and selective medium |
| EP1869469B1 (en) * | 2005-03-17 | 2011-12-07 | Phigenics, LLC | Rapidly detecting and quantifying viable legionella |
| US20070042454A1 (en) * | 2005-08-17 | 2007-02-22 | Princeton Separations, Inc. | Device and method of detecting streptococcal mutans |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3890200A (en) * | 1973-06-14 | 1975-06-17 | Forsyth Dental Infirmary | Selective medium for streptococcus mutans |
| US4468456A (en) * | 1982-03-29 | 1984-08-28 | Warner-Lambert Company | Medium for differentiating Streptococcus mutans |
| JPS6027389A (en) * | 1983-07-25 | 1985-02-12 | Yakult Honsha Co Ltd | Fusion and regeneration of protoplast of bacterium belonging to genus lactobacillus |
| US4692407A (en) * | 1985-01-31 | 1987-09-08 | Forsyth Dental Infirmary For Children | Method for the determination of Streptococcus mutans |
-
1998
- 1998-06-10 EP EP98110716A patent/EP0885969B1/en not_active Expired - Lifetime
- 1998-06-10 CA CA002240237A patent/CA2240237A1/en not_active Abandoned
- 1998-06-10 AT AT98110716T patent/ATE204908T1/en active
- 1998-06-10 DE DE19825933A patent/DE19825933A1/en not_active Ceased
- 1998-06-10 DE DE59801288T patent/DE59801288D1/en not_active Expired - Lifetime
- 1998-06-12 JP JP16532198A patent/JP3459877B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH1169970A (en) | 1999-03-16 |
| EP0885969A1 (en) | 1998-12-23 |
| DE59801288D1 (en) | 2001-10-04 |
| ATE204908T1 (en) | 2001-09-15 |
| JP3459877B2 (en) | 2003-10-27 |
| DE19825933A1 (en) | 1999-01-07 |
| EP0885969B1 (en) | 2001-08-29 |
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Legal Events
| Date | Code | Title | Description |
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| EEER | Examination request | ||
| FZDE | Discontinued |