{"id":1575,"date":"2016-02-09T07:21:20","date_gmt":"2016-02-09T07:21:20","guid":{"rendered":"http:\/\/www.vtei.cz\/?p=1575"},"modified":"2024-07-16T10:46:04","modified_gmt":"2024-07-16T09:46:04","slug":"detection-of-total-coliforms-and-escherichia-coli-on-chromogenic-medium","status":"publish","type":"post","link":"https:\/\/www.vtei.cz\/en\/2016\/02\/detection-of-total-coliforms-and-escherichia-coli-on-chromogenic-medium\/","title":{"rendered":"Detection of total coliforms and Escherichia coli on chromogenic medium"},"content":{"rendered":"

<\/i> This article is available in Czech only. For translation or more information on this topic, please contact author.<\/h4>\n

Souhrn<\/h2>\n

Tento p\u0159\u00edsp\u011bvek p\u0159edkl\u00e1d\u00e1 praktick\u00e9 zku\u0161enosti s\u00a0chromogenn\u00edm m\u00e9diem CCA, kter\u00e9 se podle novely normy \u010cSN EN ISO\u00a09308-1 pou\u017e\u00edv\u00e1 ke stanoven\u00ed koliformn\u00edch bakteri\u00ed a\u00a0E. coli<\/em> ve vod\u00e1ch s\u00a0n\u00edzk\u00fdm obsahem doprovodn\u00e9 mikrofl\u00f3ry. Jsou uvedeny vybran\u00e9 charakteristiky metody a\u00a0jsou diskutov\u00e1ny hlavn\u00ed probl\u00e9my s\u00a0jeho vyu\u017eit\u00edm ve\u00a0vodohospod\u00e1\u0159sk\u00e9 praxi.<\/p>\n\"baudisova-3\"<\/a>\n

\u00davod<\/h2>\n

V\u00a0dubnu 2015 do\u0161lo v\u00a0souvislosti s\u00a0novelizac\u00ed normy \u010cSN EN ISO\u00a09308-1 [1] k\u00a0v\u00fdznamn\u00e9 zm\u011bn\u011b metody stanoven\u00ed koliformn\u00edch bakteri\u00ed a\u00a0Escherichia coli<\/em>. Jedn\u00e1 se o\u00a0vysoce citlivou metodu, vhodnou pouze pro vody s\u00a0n\u00edzk\u00fdm obsahem doprovodn\u00e9 mikrofl\u00f3ry, p\u0159edev\u0161\u00edm tedy pro dezinfikovan\u00e9 vody (upraven\u00e1 pitn\u00e1 voda, baz\u00e9ny). U\u00a0ostatn\u00edch typ\u016f vod (m\u011blk\u00e9 soukrom\u00e9 studny, povrchov\u00e9 vody, zejm\u00e9na stojat\u00e9\u00a0apod.) je jej\u00ed vyu\u017eit\u00ed sporn\u00e9, p\u0159edev\u0161\u00edm pro vysok\u00fd n\u00e1r\u016fst doprovodn\u00e9 mikrofl\u00f3ry. Tato metoda byla v\u00a0minul\u00fdch letech testov\u00e1na pro p\u0159\u00edpadn\u00e9 pou\u017eit\u00ed v\u00a0p\u0159\u00edrodn\u00edch koupac\u00edch vod\u00e1ch a\u00a0vzhledem k\u00a0n\u00edzk\u00e9 selektivit\u011b (viz\u00a0d\u00e1le) byla vyhodnocena pro tento typ vod jako nevyhovuj\u00edc\u00ed [2].<\/p>\n

Koliformn\u00ed bakterie jsou podle t\u00e9to normy skupina gramnegativn\u00edch ty\u010dinek netvo\u0159\u00edc\u00edch spory s\u00a0negativn\u00edm cytochromoxid\u00e1zov\u00fdm testem, kter\u00e9 tvo\u0159\u00ed za aerobn\u00edch podm\u00ednek kolonie b\u011bhem 24\u00a0hodin na\u00a0selektivn\u00edm diferencia\u010dn\u00edm m\u00e9diu p\u0159i\u00a0kultiva\u010dn\u00ed teplot\u011b (36 \u00b1 2) \u00b0C. Koliformn\u00ed bakterie vykazuj\u00ed aktivitu \u03b2-D-galaktosid\u00e1zy a\u00a0E. coli<\/em> je ta koliformn\u00ed bakterie, kter\u00e1 z\u00e1rove\u0148 vykazuje aktivitu \u03b2-D-glukuronid\u00e1zy. V\u00fdznamn\u00e1 je zm\u011bna \u201edetek\u010dn\u00edho syst\u00e9mu\u201c koliformn\u00edch bakteri\u00ed a\u00a0E. col<\/em>i oproti d\u0159\u00edv\u011bj\u0161\u00ed metod\u011b zalo\u017een\u00e9 na fermentaci lakt\u00f3zy (koliformn\u00ed bakterie) a\u00a0tvorb\u011b indolu (E. col<\/em>i). Aktivitu \u03b2-D-galaktosid\u00e1zy vykazuje mnohem \u0161ir\u0161\u00ed spektrum bakteri\u00ed (tento enzym \u0161t\u011bp\u00ed lakt\u00f3zu na galakt\u00f3zu a\u00a0gluk\u00f3zu, kter\u00e1 d\u00e1le m\u016f\u017ee a\u00a0nemus\u00ed b\u00fdt fermentov\u00e1na na kysel\u00e9 produkty); podrobn\u011b jsou biochemick\u00e9 vlastnosti jednotliv\u00fdch druh\u016f bakteri\u00ed z\u00a0\u010deledi Enterobacteriaceae<\/em> uvedeny v\u00a0Bergey\u2019s\u00a0Manual of Determinative Microbiology [3]. Po\u010det koliformn\u00edch bakteri\u00ed detekovan\u00fdch na chromogenn\u00edm m\u00e9diu tak b\u00fdv\u00e1 v\u00fdznamn\u011b vy\u0161\u0161\u00ed, ne\u017e byl jejich z\u00e1chyt na d\u0159\u00edve pou\u017e\u00edvan\u00e9m lakt\u00f3zov\u00e9m TTC agaru s\u00a0tergitolem, a\u00a0v\u00fdsledky proto nekoreluj\u00ed. Naproti tomu stanoven\u00ed\u00a0E. coli<\/em> pomoc\u00ed enzymu \u03b2-D-glukuronid\u00e1zy je mnohem specifi\u010dt\u011bj\u0161\u00ed ne\u017e d\u0159\u00edve pou\u017e\u00edvan\u00fd indol test (test na \u03b2-D-glukuronid\u00e1zu byl v\u00a0d\u0159\u00edv\u011bj\u0161\u00ed verzi normy pouze nepovinn\u00fd a\u00a0prakticky se nepou\u017e\u00edval), tak\u017ee v\u00fdsledky stanoven\u00ed E. coli<\/em> na chromogenn\u00edm m\u00e9diu budou v\u00fdrazn\u011b spolehliv\u011bj\u0161\u00ed.<\/p>\n\"baudisova-1\"<\/a>\n

Obr. 1. Koliformn\u00ed bakterie, E. coli<\/em> a doprovodn\u00e1 mikrofl\u00f3ra narostl\u00e1 na CCA
\nFig. 1. Total coliforms, E. coli<\/em> and background microflora grown on CCA<\/h6>\n

Metodika<\/h2>\n

Koliformn\u00ed bakterie a\u00a0E. coli<\/em> se stanovuj\u00ed membr\u00e1novou filtrac\u00ed (v\u011bt\u0161inou se filtruje objem 100\u2008ml vzorku), membr\u00e1nov\u00e9 filtry maj\u00ed porozitu 0,45\u2008\u03bcm. Filtr se um\u00edst\u00ed na chromogenn\u00ed m\u00e9dium (Chromogenic-Coliform Agar) a\u00a0kultivuje se (21 \u00b1 3) hodiny p\u0159i (36 \u00b1 2) \u00b0C. Slo\u017een\u00ed Chromogenic-Coliform Agaru (d\u00e1le CCA) je uvedeno v\u00a0tabulce 1<\/em>. CCA je b\u011b\u017en\u011b komer\u010dn\u011b dostupn\u00fd. P\u0159\u00edprava CCA je velmi jednoduch\u00e1, nav\u00e1\u017e\u00ed se p\u0159\u00edslu\u0161n\u00e9 mno\u017estv\u00ed pr\u00e1\u0161ku, resp. granul\u00ed, a\u00a0rozpust\u00ed se v\u00a0destilovan\u00e9 vod\u011b. Nep\u0159id\u00e1vaj\u00ed se \u017e\u00e1dn\u00e9 dal\u0161\u00ed p\u0159\u00eddavky. Nesm\u00ed se autokl\u00e1vovat. Po zchlazen\u00ed na 45\u201350\u2008\u00b0C se agarov\u00e9 m\u00e9dium steriln\u011b rozleje do Petriho misek. PH m\u00e1 b\u00fdt 6,8 \u00b1 0,2 p\u0159i 25\u2008\u00b0C (podle na\u0161ich zku\u0161enost\u00ed deklarovan\u00e9 pH v\u017edy sed\u011blo, p\u0159esto je nutn\u00e9 u\u00a0ka\u017ed\u00e9 \u0161ar\u017ee prov\u00e9st kontrolu). M\u00e9dium je velmi st\u00e1l\u00e9, uva\u0159en\u00e9 vydr\u017e\u00ed minim\u00e1ln\u011b m\u011bs\u00edc, jen je nutn\u00e1 ochrana p\u0159ed jeho vyschnut\u00edm. Kvalita m\u00e9dia se testuje podle normy \u010cSN EN ISO\u00a011133 [4].<\/p>\n

Tabulka 1. P\u0159edepsan\u00e9 slo\u017een\u00ed CCA
\nTable 1. Prescribed composition of CCA<\/h5>\n\"baudisova-tabulka-1\"<\/a>\n

Koliformn\u00ed bakterie rostou jako r\u016f\u017eov\u00e9 a\u017e \u010derven\u00e9 kolonie, E. coli<\/em> jako tmav\u011b modr\u00e9 a\u017e fialov\u00e9 kolonie (viz\u00a0obr.\u00a01<\/em>). Doprovodn\u00e1 mikrofl\u00f3ra roste ve form\u011b bezbarv\u00fdch koloni\u00ed. Koliformn\u00ed bakterie je nutn\u00e9 potvrdit pomoc\u00ed oxid\u00e1zov\u00e9ho testu (koliformn\u00ed bakterie jsou oxid\u00e1za negativn\u00ed), kter\u00fd norma p\u0159ipou\u0161t\u00ed prov\u00e1d\u011bt p\u0159\u00edmo na membr\u00e1nov\u00e9m filtru. P\u0159i p\u0159\u00edpadn\u00e9m p\u0159eo\u010dkov\u00e1n\u00ed koloni\u00ed na neselektivn\u00ed agar (pro dal\u0161\u00ed konfirmaci) je nutn\u00e9 kolonie pe\u010dliv\u011b p\u0159e\u010distit, aby se v\u017edy pracovalo s\u00a0\u010distou kulturou. Pro p\u0159\u00edpadnou dal\u0161\u00ed konfirmaci, resp. pro potvrzen\u00ed aktivity enzym\u016f \u03b2-D-galaktosid\u00e1zy a\u00a0\u03b2-D-glukuronid\u00e1zy, lze vyu\u017e\u00edt nap\u0159.\u00a0ONP test a\u00a0GLR test (Erba, Lachema). Tyto testy lze vyu\u017e\u00edt i\u00a0pro stanoven\u00ed citlivosti a\u00a0specifi\u010dnosti p\u0159\u00edslu\u0161n\u00fdch reakc\u00ed na CCA. P\u0159i v\u00fdpo\u010dtu v\u00fdsledk\u016f je nutn\u00e9 m\u00edt na pam\u011bti d\u016fle\u017eit\u00fd fakt a\u00a0to, \u017ee i\u00a0E. coli<\/em> pat\u0159\u00ed mezi koliformn\u00ed bakterie, a\u00a0mus\u00ed se tud\u00ed\u017e do jejich po\u010dtu p\u0159i\u010d\u00edst:<\/p>\n