Stanoven potu vybranch indiktorovch mikroorganism vpotravinch pomoc automatizovan metody TEMPOMikrobiologie potravin, IV. ronkstav hygieny a technologie mlka, FVHE, VFU BrnoListopad 2011

TEMPOAutomatizovan metoda stanoven potu MOZaloena na principu MPN (miniaturizace metody)

MPN - Sriov edn & inkubaceInkubace

Stanoven potu3/2/2MPN statistick tabulka= Most probable Numberx.10y cfu/g

TEMPO 3 ady zkumavek 16 zkumavek v jedn ad

Acidifikace media zpsoben rozkladem cukr 4MU fluorescencepH 74MU bez fluorescencepH 6TEMPO TC: acidifikaceNegativn : fluorescencePozitivn : nen fluorescenceTotal Coliforms (TC) Stanoven celkovho potu koliformnch MOfluorescenn pH indiktor :4-Methylumbeliferon (4MU)

Stanoven potu E. coliMUGb - D glukuronidza(E. coli)Glukuront4-methylumbeliferon+TEMPO ECspecifick enzymatick substrt pro detekci E. coli : 4-methylumbeliferyl-D-glukuronid (MUG)Pozitivn: fluorescence

- - - + + - - - - - + - + - - -- + + + + + + + + + + + + + + ++ + + + + + + ++ + + + + + + +Detekce

MPN kombinace16 / 15 / 4cfu/gMPN tabulka+edn- - - + + - - - - - + - + - - -- + + + + + + + + + + + + + + ++ + + + + + + ++ + + + + + + +

TEMPO SystemReader teka (interpretace vsledk)InkubacePprava vzorkuFiller - Inokulace

TEMPO System

TEMPOPrep

TEMPORead

TEMPOFiller

TEMPOReader

WIFI

PreparationStation

ReadingStation

Plnc stanice Preparation stationTempo kitFiller PCteka rovch kdStojnkyDispenzoryVortex

rov kd Barevn rozlien

TEMPO KIT - kartaTransfer tubeCitliv oblast, riziko kontaminace

TEMPO KIT -Vialky s mdiemDehydratovan mdium

rov kdBarevn rozlien+Vzorek potraviny + PPV (10-1)

edc roztokSekundrn:

Destilovan voda

STERILN

Re-hydratace media ve vialce

EDNedc roztok(sekundrn)

Vzorek(1/10)

Celkov objem

Finaln edn3 ml

+

1 ml

=4 ml

1/403,9 ml

+

0,1 ml

=4 ml

1/400

Stanoven potu - rozptedn 1/40:10 to 49 000 cfu/gedn 1/400:100 to 490 000 cfu/gedn 1/4000:1 000 to 4 900 000 cfu/g

Pprava -FillerInkubaceteka rovch kdPipraven vzorky do filleruFiller

Inkubace

TEMPO C / 24 - 27 h30C +/- 1C37C +/- 1C

Oznaen vzorku

Naten rovho kdu vialky

Po naskenovn kartyNastaven edn

tec stanice

Kontola doby inkubace

5.3 E4 CFU/gTEMPO - teka

Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*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*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 distributionIn 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*From that most probable number, refer it to an MPN statistic table, and determine your concentration.Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*3 times 16 wells, mimicking a MPN 16 tubes method.16 wells of 2.25 L16 wells of 22.5 L16 wells of 225 L

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.Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*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*16/15/4 is the Most Probable Number, that will be converted into a concentration through a statistic table.

Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*Automated Microbial Enumeration SystemFull range of specific enumeration mediumUnique disposable enumeration CardAutomated inoculation/sealingAutomated reading and interpretationTracebility from sample to resultTempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*The TEMPO system has two workstations:The preparation station with its computer science and the TEMPO Filler-SealerThe 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*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.

Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*The transfer tube is already inserted in the card.Handle the card without touching the tip of the transfer tube.Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*For single use.

Contain, for certain tests, irritant or toxic components (see package inserts)

Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*Sterile diluent water or equivalent purified water validated by the user.Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*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*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*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.Tempo Specialist Team Tempo Training Tool Version 2 September, 01, 2005*Temperature tolerances: +/- 1C

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