Question

Different kinetics methods for antibiotic production

Answer 1

Samples and reference standard
Samples of the commercial product ApralanTM So-
luble (powder for oral solution) supplied by Eli Lilly do
Brasil Ltda (SP, Brazil) were tested. The apramycin sulfate
reference standard (lot RS0192, potency 873 mg mg-1) used
in the tests was kindly provided by Eli Lilly of Brasil Ltda
(SP, Brazil).
Test Microorganism
The test microorganism used in the assay was Esche-
richia coli (ATCC 8739), provided by the Instituto Adolfo
Lutz (SP, Brazil). Cultures of Escherichia coli (ATCC
8739) were maintained in slants of tryptone-soy agar.
Determination of experimental conditions
An aliquot of a fresh culture of Escherichia coli
(ATCC 8739) was transferred to a flask containing 100 mL
of tryptone soy broth and incubated at 32.5±2.5ºC for no
longer than 24 hours. The suspension obtained from this
growth, containing 107
-108
CFU mL-1, was used as the
inoculum. Appropriate amounts of tryptone-soy broth
were inoculated at 1.0, 3.0 and 5.0%.
Solutions of reference standard were prepared con-
taining various concentrations (1, 2, 4, 8, 10, 20, 30, 40,
50, 60, 70, 80, 90 and 100 mg mL-1) of apramycin sulfate,
employing tryptone-soy broth as the diluent. Aliquots of
100 mL of each apramycin sulfate solution were transferred
to a microplate. Also, aliquots of 100 ml of tryptone-soy
broth inoculated with Escherichia coli (ATCC 8739) at 1.0,
3.0 and 5.0% were transferred to the microplate. Negative
controls (NC) and positive controls (CP) were also included
in the microplate. A scheme of the distribution of apramycin
sulfate solutions in the microplate is shown in Scheme 1.
A microplate spectrophotometer (Biotek Elx808,
WI, USA) was used for incubation and reading of mi-
croplates. Incubation was performed at a temperature of
37.0±1.0 ºC for a period of 8 hours. Absorbances were
measured at 630 nm at intervals of 5 minutes.
SCHEME 1 - Scheme of distribution of apramycin sulfate solutions at various concentrations (1, 2, 4, 8, 10, 20, 30, 40, 50, 60, 80, 90
and 100 mg mL-1), negative control (NC) and positive control (PC) and distribution of tryptone soy broth inoculated at 1, 3 and 5%.