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Cold shock response in various organisms is induced by an abrupt downshift in temperature and leads to a dramatic increase in production of a homologous class of cold shock proteins. These proteins are essential for low temperature survival of bacteria. To identify CSP from diversified microflora, immunoassay was developed. A small 14 kDa protein from cold tolerant mutant, CRPF8 of Pseudomonas fluorescens was concentrated and fractionated by HPLC and antisera was raised. Specificity of anti-CRPF8 was checked using western blot analysis and further confirmed by Immunoelectron Microscopy. Bacterial strains from various habitats were isolated and their crude protein was purified. CSPs were characterized from crude extract using anti-CRPF8. Expression of CSPs was observed only in bacterial strains isolated from temperate region and negligible or no expression was observed in bacterial strains isolated from arid zones. Therefore this anti-CRPF8 can be used as immunological tool for the identification of CSP from diversified microorganisms.