Rapid detection of microbial contamination in water samples with bioluminescence method
,1,* Saman hosseinkhani
,2 Maryam monsefshokri
1. Department of Biochemistry, Science and Research branch, Islamic Azad University.
2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University.
3. Department of Biochemistry, Science and Research branch, Islamic Azad University.
In pharmaceutical and food companies, the colony count technique is a common method for evaluating microbial contamination. however, this technique has some disadvantages such as being time-consuming, tedious and need skills. therefore, there is a need for a rapid and sensitive method to measure bacterial communities in samples. bioluminescence is one of the few types of luminescence that light is produced by living microorganisms. atp reacts with luciferin-luciferase enzymatic complex and the light emitted is measured by the device. atp is composed of an adenine ring, a ribose sugar, and three phosphate groups, and work as cellular energy currency in living cells. atp is available in both intracellular and extracellular forms that extracellular atp doesnt need to be extracted, but intracellular atp needs extraction due to its placement in the cell. atp production is heavily controlled in cells, its measurement can indicate the number of live cells in the environment. in this method, atp produced by biological systems is rapidly measured by enzyme measurement in the presence of luciferase. the amount of atp measured can be used to evaluate cell proliferation, apoptosis, cell toxicity and detection of microbial contamination. the simplicity of operation, fast, sensitive, specific are some advantages of this method. therefore, the atp bioluminescence method can be used to detect the concentration of atp by the cell_solver agent to identify contamination in various aquatic environments.
Material and instrumental
all chemicals were of analytical grades obtained from commercial sources. luminometer fb14 (berthold) and a centrifuge mpw260r (med instrument) were used for measuring the emission and pre-concentration process.
expression and purification of luciferase
e. coli strain bl21 was transformed with pet-28a. bacteria containing pet-28a plasmid were initiated by adding 10μl of an overnight culture to 10ml of lb broth with ampicillin. this culture was grown at 37 ºc for four hours, then lactose was added to a medium culture for an overnight for expression of a protein. then the bacteria were centrifuged and bacterial suspension was sonicated. after sonication, the lysed cell was centrifuged and kept the supernatant for purification. in this method, the protein was purified using an affinity column (ni-nta- sepharose). sds-page was used to confirm the presence and purity of the sample.
total bacterial atp measurement procedure
in this method, 5 μl of bacterial sample was added to the cell and first treated with of cell_solver as the extraction solvent, then the extract was neutralized by addition of the diluent. aliquot of 10 μl of luciferin-luciferase reagent was added to the cell and the bioluminescence was measured as total atp. for measuring of extracellular atp, the bacterial sample was added to the cell and mixed with luciferin-luciferase reagent without any extraction solvent and bioluminescence was measured.
Purification of protein
a fraction of 1 to 5 purification luciferase enzyme a bond is about 62kda and its purification is more than 95%. expression enzyme has his-tag binds. histidine tags have an affinity for a nickel. the supernatant was transferred to the column, then washing and elution buffer was used to remove impurities isolate the luciferase protein from the column, respectively.
effect of type and concentration of stabilizer on assay
the effect of type and concentration of stabilizer on the assay were studied while other operating conditions were kept constant. bsa, sucrose, and cyclodextrin were compared instability of the enzyme. the fig1 showed that the emission when used sucrose is more than another component. for the concentration of sucrose, as indicated in fig2, luciferase activity increased up to 3 mm of sucrose and decreased at higher concentrations, at lower volumes.
figures of merit
under to optimum conditions, the detection limit was . the calibration graph was linear in the range .
analysis of water samples
various water samples such as urban, pool, well, seawater were measured in the linear range of the atp calibration curve using cell_solver. in all samples, except for urban water samples, the atp was found with this method.
A sensitive method of atp bioluminescence for detection of microbial contamination in aqueous samples has been described. the simplicity of operation, fast, specific and selective are some advantages of this method. our results show that the bioluminescence system may be employed as a rapid method to detect bacterial in samples without cultivation.
Atp, bioluminscence, luciferase.