Broth dilution method

Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3 (2) 163-175... 

The broth dilution method was used in these studies. Pseudomonas organisms were cultivated in I per cent peptone, 3 per cent NaCi medium, and Dipl, pneumoia. Sir, pyo nes and CoU. Dipktk riae were cultivated on heart infusion broth containing 0 5 per cent glucose. All other bacteria were cultivated on heart infusion broth. F gi were grown in Sabouraud s medium. 

EDso (mg/kg) for mean 2 log reduction compared to vehicle-treated control. Micro-broth dilution method. 

The broth dilution method is a simple procedure for testing a small number of isolates, even a single isolate. It has the added advantage that the same tubes can also be used for minimum bactericidal concentration(s) (MBC) tests. 

The main advantage of the broth dilution method for MIC determination lies in the fact that it can easily be converted to determine the MBC as well. 

MIC values were detennined by the broth dilution method (63). 

Since it has been observed by the authors that modifications of the Rammelkamp broth dilution method for assay (28) enjoy the greatest popularity in laboratories today, a composite of these modifications, as used with satisfaction in the authors laboratory, is presented here. Practically all assay procedures for antibiotics discovered after penicillin are patterned on the penicillin assay itself. Therefore it serves our purpose to outline the assay for newer agents by indication of changes or substitutions which must be made in the basic (penicillin) method. 

The antibacterial activity of the nanocomposites was investigated by modified Kirby-Bauer diffusion plate method against gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus). An equal quantity of the nanocomposites with different Ag-NPs concentrations was pressed into pellets of diameters about 13 mm and thickness of 1-2 mm and incubated with the bacteria at 37 °C. Zone of inhibition was measured after 24 h of incubation. Broth dilution method was employed to determine the values of MIC (Minimum Inhibitory Concentration) of PPy-NTs Ag-NPs nanocomposites against E. coli and 5. aureus. 

The MIC value which is defined as the minimum concentration of an antimicrobial that is able to restrict the growth of microbial after whole night incubation have been determined by broth dilution method. Initial concentration of the nanocomposites for the test is taken as 10 mg/ml and diluted twice in each time up to a concentration of 0.0195 mg/ml. Nanocomposites with different concentrations have been subjected to E. coli and S. aureus to investigate the value of MIC. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoIiiun bromide) is added to the solution after 16 h incubation in Luria Berteni media in 96 well microtiter plate and incubated for 45 min in dark. Live bacterial cells which are capable to produce succinate dehydrogenase (enzyme) which reacts with MTT and forms formazan complexes with purple appearance. So the well having minimum concentration of sample where the complex does not form i.e. which does not appear purple is considered as the MIC value of the compound and is presented in the Table 7. It is observed that the MIC value of the nanocomposites decreases with the increase in Ag-NPs concentration in the nanocomposites and is minimum in the case of nanocomposites with 15 wt% of Ag-NPs. 

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