Stanovení počtu vybraných indikátorových mikroorganismů v potravinách pomocí automatizované metody TEMPO® Mikrobiologie potravin, IV. ročník Ústav hygieny a technologie mléka, FVHE, VFU Brno Listopad 2011

TEMPO Automatizovaná metoda stanovení počtu MO Založena na principu MPN (miniaturizace metody) Preparation Station: Samples 1:10 diluted are brought to the prep. station. A determined amount of that sample will be placed into a vial containing dehydrated media. The mixture (sample + growth media) will be transferred into the card through the TEMPO Filler Instrument. After the filling process, the card will be transferred in incubation (regular lab incubators) Reading Station: After incubation, the card is read by the TEMPO Reader and the result of that test is displayed on the computer. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

MPN - Sériové ředění & inkubace 10-2 1 ml 10-3 10-4 Inkubace 3 + 2 + 3/2/2 Součet pozitivních zkumavek More than one tube is performed for each dilution (here: MPN – 3 tubes) The MPN method requirements are: Tube independence (every tube is unique) Homogeneous and random distribution In other words, every tube should have the same probability of bacterial presence. Once the incubation time is over, determine the number of positive tubes in the same dilution, and thus determine your most probable number. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Stanovení počtu x.10y cfu/g 3/2/2 MPN statistická tabulka = Most probable Number MPN statistická tabulka From that most probable number, refer it to an MPN statistic table, and determine your concentration. x.10y cfu/g Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

TEMPO Patentovaná karta pro stanovení počtu (miniaturizace metody) 2.25 µl 22.5 µl 225 µl Patentovaná karta pro stanovení počtu (miniaturizace metody) 3 times 16 wells, mimicking a MPN 16 tubes method. 16 wells of 2.25 µL 16 wells of 22.5 µL 16 wells of 225 µL The card is being filled with the sample mixture + dehydrated medium in the tempo Filler Instrument. During incubation, microorganisms will grow inside the 48 wells. 3 řady zkumavek – 16 zkumavek v jedné řadě Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Acidifikace media způsobená rozkladem cukrů TEMPO TC: acidifikace Total Coliforms (TC) – Stanovení celkového počtu koliformních MO fluorescenční pH indikátor :4-Methylumbeliferon (4MU) Acidifikace media způsobená rozkladem cukrů 4MU bez fluorescence pH £ 6 4MU fluorescence pH ³ 7 Pozitivní : není fluorescence Negativní : fluorescence

Stanovení počtu E. coli TEMPO EC specifický enzymatický substrát pro detekci E. coli : 4-methylumbeliferyl-D-glukuronid (MUG) + b - D glukuronidáza (E. coli) MUG Glukuronát 4-methylumbeliferon Pozitivní: fluorescence

- - - + + - - - - - + - + - - - - + + + + + + + + + + + + + + + Detekce - - - + + - - - - - + - + - - - - + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + Depending on the number and size of the positive wells, the tempo system deduces the number of targeted bacteria present in the original sample according to a calculation based on the MPN Method. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

- - - + + - - - - - + - + - - - + + + + + + + ++ + + + + + + + MPN kombinace - - - + + - - - - - + - + - - - - + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + MPN tabulka + ředění cfu/g 16/15/4 is the Most Probable Number, that will be converted into a concentration through a statistic table. 16 / 15 / 4 Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

TEMPO System Příprava vzorku Filler - Inokulace Automated Microbial Enumeration System Full range of specific enumeration medium Unique disposable enumeration Card Automated inoculation/sealing Automated reading and interpretation Tracebility from sample to result Reader – čtečka (interpretace výsledků) Inkubace Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

TEMPO System The TEMPO system has two workstations: The preparation station with its computer science and the TEMPO Filler-Sealer The reading station with its computer science as well and the TEMPO-Reader. Both workstations are transmitting data through the WIFI (Wireless Fidelity) Both software applications (tempo_prep and tempo_read) are under control (parameters setting) through a dedicated software (TEMPO_Admin). Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Plnící stanice Preparation station Tempo kit PC Filler Čtečka čárových kódů Dispenzory Overview of the preparation station: Ergonomic tests have been performed worldwide to allow the optimal configuration of the workstation. The user can perform 144 tests (6 boxes of 24 tests) without standing up. Stojánky Vortex Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

TEMPO KIT - karta Čárový kód Barevné rozlišení Transfer tube The transfer tube is already inserted in the card. Handle the card without touching the tip of the transfer tube. Citlivá oblast, riziko kontaminace Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

TEMPO KIT -Vialky s médiem Dehydratované médium Čárový kód Barevné rozlišení + For single use. Contain, for certain tests, irritant or toxic components (see package inserts) Vzorek potraviny + PPV (10-1) Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Ředící roztok Re-hydratace media ve vialce Sekundární: Destilovaná voda STERILNÍ Sterile diluent water or equivalent purified water validated by the user. Re-hydratace media ve vialce Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

ŘEDĚNÍ Ředící roztok (sekundární) Vzorek (1/10) Celkový objem Finalní ředění 3 ml + 1 ml = 4 ml 1/40 3,9 ml + 0,1 ml = 4 ml 1/400 The sample is already 1/10 diluted in the tempo bag with the primary diluent. Do not allow the sample to come into direct contact with the culture medium (in powder) before the medium has been reconstituted. In others words, first add the secondary diluent and then add the sample. This is to prevent from sample contaminations on the nose of the dispensers. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Stanovení počtu - rozpětí Ředění 1/40: 10 to 49 000 cfu/g Ředění 1/400: 100 to 490 000 cfu/g Ředění 1/4000: 1 000 to 4 900 000 cfu/g The dilutions (1/400 for TVC test and 1/40 for the other tests) can be set in the administration software. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Příprava -Filler Čtečka čárových kódů Filler The filling tray is put in the automat, the cards are filled and sealed. Then, the cards are removed and placed in the incubators. The filling process is Vitek-like. Just the sealing is internalized. Připravené vzorky do filleru Inkubace Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Inkubace TEMPO °C / 24 - 27 h 30°C +/- 1°C 37°C +/- 1°C Temperature tolerances: +/- 1°C Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Označení vzorku Meat 12354-55 By pressing on F8, you can identify your sample, either with the keyboard or by screening its barcode. Then, press OK to validate. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Načtení čárového kódu vialky Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Po naskenování karty Nastavení ředění After selecting the row of the desired sample, scan the card you will associate with the vial and vortex that vial in parallel. Then press OK You can modify the default dilution, if it has been done previously in the vial. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

Čtecí stanice

Kontola doby inkubace TEMPO_Read software is dedicated to the reading station. Tempo Specialist Team – Tempo Training Tool – Version 2 – September, 01, 2005

TEMPO - čtečka 5.3 E4 CFU/g