Reaktion #164446

Reaktionsgleichung

NaOH

X-100

EDTA

→

dopamine

Reaktionsbedingungen

Aufarbeitung

1. 1
   FiltrationThis mixture was then filtered through a 0.2 micron syringe
2. 2
   Filtrationfilter (typically polyethersulfone), which traps the bacteria
3. 3
   WaschenA small volume of alkaline rinse solution (50 mM NaOH)
4. 4
   Sonstigewas flushed through the filter
5. 5
   Waschento rinse away any artifactually reactive metabolites
6. 6
   ExtraktionAlkaline extraction solution (75 mM NaOH)
7. 7
   Sonstigewas back-flushed through the filter
8. 8
   Wascheneluting the bacteria
9. 9
   workup.ADDITIONA small volume (typically 0.3 ml) of this neutralized sample was added directly to an assay tube
10. 10
    workup.ADDITIONcontaining the lyophilized BacTx™ reagents (
11. 11
    workup.ADDITIONcontaining MBTH, trehalose, and Ficoll)
12. 12
    workup.ADDITIONcontaining MBTH, trehalose, and Ficoll

Vorschrift

In the first method (Experiment 1; FIG. 26), several milliliters of urine were combined with an alkaline detergent solution (1% Triton X-100, 50 mM EDTA, 10 mM NaOH). This mixture was then filtered through a 0.2 micron syringe filter (typically polyethersulfone), which traps the bacteria. A small volume of alkaline rinse solution (50 mM NaOH) was flushed through the filter, to rinse away any artifactually reactive metabolites. Alkaline extraction solution (75 mM NaOH) was back-flushed through the filter, eluting the bacteria trapped on the surface of the filter. The eluted bacteria were heated at 80° C. for several minutes and then combined with an equal volume of neutralization solution (150 mM MOPS). A small volume (typically 0.3 ml) of this neutralized sample was added directly to an assay tube containing the lyophilized BacTx™ reagents (lyophilized hemolymph from Manduca sexta, dopamine, lysostaphin and a separate lyophilized pellet containing MBTH, trehalose, and Ficoll). The BacTx™ reagents consist of lyophilized hemolymph from Manduca sexta, dopamine, lysostaphin and a separate lyophilized pellet containing MBTH, trehalose, and Ficoll. In the presence of bacterial peptidoglycan, the hemolymph proteolytic enzyme cascade is triggered, resulting in the oxidation of dopamine to quinones which form a bright red adduct with MBTH. An increase in absorbance at approximately 500 nm over time, e.g. in 1-90 minutes, is thus an indication of bacteria in the urine sample. In this experiment, a urine sample which had been spiked with 2E6 CFU/ml of E. coli prior to the assay yielded a readily observed bright red color which intensifed over a 90 minute period, while a normal, unspiked urine sample yielded no color development (see Results below).

Quelle