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VOL. 75 (2), 217-231, 2009 (CU)II IN VIVO INTERACTION WITH CEFOTAXIME

Group C was used as a control to know the different substances
which belonged to the animals in order to not to be confusing with
the free cefotaxime throughout the study.

Five rats of the groups A and B, were sacrificed after 30, 60, 90,
120, 150, 180 and 210 minutes after the antibiotic administration.
The blood, liver, spleen, kidney, lung and heart of those rats were
extracted and after that, they were measuring their free cefotaxime
concentration by HPLC.

Residual microbicide activity was tested by antibiogram
experiments in agar plates using bacteria strains from the CECT
(Spanish Type Culture Collection, Burjasot, Spain). Those strains
were Escherichia coli CECT 434 = ATCC 25922, described as standard
for sensibility, Escherichia coli CECT 616 = ATCC 8739, described
for assays with liquid with metal, Staphilococus aureus spp aureus
CECT 435, Staphilococus aureus spp aureus CECT 239 = ATCC 6538
and Bacillus subtilis CECT 356 = ATCC 6633 as «a priori» negative
control since the species was not listed among the affectable bacteria
by cefotaxime.

The strains were maintained in TSA media at refrigeration
teemperature and precultured in tubes with 10 mL TSB medium at
37 ºC for 24 hours before the experiments.

2.2. Chemical and reagents

Cefotaxime sodium was supplied by the Teaching Hospital of
Madrid. Copper (II) acetate was obtained from Merck. Trichloracetic
acid was supplied by Scharlau, this substance was used in
concentrations of 10% and 20%.

The mobile phase reagents used were HPLC grade methanol from
Scharlau. Water used was from a Milli-Rho-Milli-Q system
(Millipore, Bedford, MA, USA). Phosphate buffer (0.1 M) was
prepared with phosphate monopotasic anhydride and o phosphoric
acid supplied by Merck.

The agar media used were TSA, a general bacterial growth media,
and blood agar, both cooked as recommended the Merck’s recipes.

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