ASSAY FOR DETERMINING ANTIBIOTICS IN WASTE
The present invention relates to a simple and easy-to-use method for the rapid determination of the presence of an antibiotic in a waste such as e.g. liquid or solid waste streams from plants. The present invention also relates to a kit comprising an as say and a manual for the rapid determination of the presence of an antibiotic in a waste.
1 . A method for determining the presence or absence of an antibiotic in a lactose-free sample that is essentially lactose-free and/or contains less than 0.01% (w/w) of lactose comprising the steps of:
(a) contacting said lactose-free sample with an assay medium which comprises a microorganism, a gelling agent and an indicator capable of detecting growth or inhibition of the microorganism to obtain an assembly of the lactose-free sample and the assay medium;
(b) incubating the assembly obtained in step (a) for a period of time sufficient to grow the microorganism in case no antibiotic is present in said lactose-free sample; and
(c) detecting growth or inhibition of growth of the microorganism with the indicator,
wherein milk and/or powdered milk is added in step (a).
2 . The method according to claim 1 wherein said assay medium further comprises nutrients.
3 . The method according to claim 1 wherein said indicator is Bromocresol Purple or Bromothymol Blue.
4 . The method according to claim 1 wherein said microorganism is thermophilic.
5 . The method according to claim 4 wherein said microorganism is Bacillus stearothermophilus.
6 . The method according to claim 1 wherein said gelling agent is agar.
7 . The method according to claim 1 wherein solids are removed from said lactose-free sample prior to step (a).
8 . The method according to claim 1 wherein the amount of lactose in said lactose-free sample plus said added milk or powdered milk is from 2 to 5%(w/w).
9 . The method according to claim 1 further comprising a control test selected from:
(i) a known amount of an antibiotic is added in step (a), or
(ii) a known amount of a β-lactam degrading compound is added in step (a), or
(iii) a combination of (i) and (ii).
10 . A method for determining the concentration of an antibiotic in a lactose-free sample that is essentially lactose-free and/or contains less than 0.01% (w/w) of lactose comprising the steps of:
(a) preparing a dilution range of said lactose-free sample by the addition of milk;
(b) contacting each dilution obtained in step (a) with an assay medium comprising a microorganism, a nutrient and an indicator capable of detecting growth or inhibition of the microorganism to obtain a mixture;
(c) incubating each mixture obtained in step (b) for a period of time sufficient to grow the microorganism in case no antibiotic is present in the sample;
(d) detecting growth or inhibition of growth of the microorganism with the indicator;
(e) determining the dilution with the highest dilution factor capable of inhibiting the growth of the microorganism;
(f) determining the concentration of the antibiotic in the dilution determined in step (e), and
(g) determining the concentration of the antibiotic in said lactose-free sample by multiplying the concentration determined in step (f) with the dilution factor determined in step (e) used to make the dilution.
11 - 19 . (canceled)
20 . A kit comprising:
(a) a container with assay medium which comprises a microorganism, a gelling agent and an indicator capable of detecting growth or inhibition of the microorganism to obtain a mixture;
(b) a manual comprising instructions for determining the concentration of an antibiotic in a lactose-free sample that is essentially lactose-free and/or contains less than 0.01% (w/w) of lactose but which contains milk or powdered milk.
21 . A composition comprising a lactose-free sample containing less than 0.01% w/w lactose, a microorganism, a gelling agent, an indicator and one of milk and milkpowder.