I have aliquoted soluble media of bacteria. Just want to know what is the best method to analyse the sample - might contain small/large antimicrobial peptides?
Test the culture medium for inhibition of bacterial growth, either in a broth microdilution assay or by a disc diffusion assay.
Dear Raymond,
Attached please find a recent review on antimicrobial peptides that contains references for the methods to identify this kind of entities.
Pharmaceuticals 2013, 6, 1543-1575; doi:10.3390/ph6121543
Review
Antimicrobial Peptides
Ali Adem Bahar 1,2 and Dacheng Ren 1,2,
Received: 22 October 2013; in revised form: 21 November 2013 / Accepted: 25 November 2013 /
Published: 28 November 2013
Abstract: The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics).
Keywords: antimicrobial peptide; biofilm; persister
Hoping this will be helpful,
Rafik
I would have go with Adam Shapiro suggestion - disk diffusion assay. Centrifuge the culture to remove cells, filter sterilize the soup to remove left over cells and place the liquid on paper disks (you can make your own disks - punch holes a Whatman 3 paper and autoclave the disks with a little water). Spread your subject bacteria on a plate, let it dry for 1- minutes and place the soaked disks.
For higher volumes you can spread the subject bacteria as above, and make a hole in the agar (I use sterile 200ul tip, upside down). Fill the hole with the tested liquid.
Yoram
Respected Researchers,
I would like to introduce a rapid colorimetric assay for detection of Antimicrobial Peptides (AMP). One might use Dimyristoylphosphatidylcholine (DMPC) and diacetylene based polymer for the rapid detection of AMP.
Supramolecular assemblies constructed from polymerized polydiacetylene (PDA) lipids have been shown to exhibit colorimetric transitions, which are induced by AMPs. The colorimetric transitions occurring in the PDA-based aggregates are associated with structural perturbations within the conjugated backbone of the polymer.
You might find supporting literature all over the web. For reference, I am also attaching a manuscript.
Regards
Pallavi Thakur,
Researcher, DRDO
All above answers are accurate will give you antimicrobial activity from the culture broth.The simplest will be agar well or disc diffusion method. The other method is turbidometric method where you can determine the antimicrobial activity by measuring optical density(OD). For the identification of peptide antibiotic you need to go for further for theanalysis by FTIR, GCMS/LCMS, NMR, elemental carbon nitrogen hydrogen ratio etc.which should be the later part of your project.
The best and simple methods are broth microdillution assay and agar well diffusion method. For better result you can follow the colorometric method mentioned by Pallavi Thakur.
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