Mathods Of Detecting Bacillus Atrophaeus

Microbial spores are usually considered to be much more resistant to sterilization processed than most other types of microorganisms. Bacillus atrophaeus is a suitable reference organism for testing the effectiveness of dry-heat, ethylene oxide, microwaves and electrothermal deactivation sterilization processes. Due to their resistance characteristics, B.atrophaeus spores are used for producing biological indicators for sterilization.So far, application of B.atrophaeus spore is the main object in most studies; however, few studies on detection of B.atrophaeus have been reported. In order to develop methods of detecting B.atrophaeus, a series of studies about physiological, biochemical and molecular characteristics were carried on to help lay a solid foundation for application of it.Ten kinds of bacillus species were selected in which B.atrophaeus ATCC9372is considered as type culture strain to develop methods of detecting B.atrophaeus based on these.First, morphology and growth characteristics of B.atrophaeus ATCC9372were observed. A medium containing tyrosine was created in which pigment formation was observed by B.atrophaeus ATCC9372, while not by other tested species. This special medium, therefore, is selected as a means for identification of B.atrophaeus. API50CHB experiment was also conducted, but it failed to identify B.atrophaeus. Strains were tested by using the following biochemical characteristics:growth with7%NaCl; oxidase activity; nitrate reduction to nitrite; starch degradation; glycerin utilization; gelatin liquefaction; mannose, mannitol, arabinose and amygdalin utilization. By comparing these biochemical differences, a kind of biochemical standard for identifying B.atrophaeus ATCC9372was developed. All in all, A method for identification of B.atrophaeus through physiological and biochemical characteristics is drawn up.Second, segments of B.atrophaeus RecA and16S rRNA genes were amplified by PCR with universal and RecA primers. Therefore, RecA primers were selected as special primers of PCR which was used for identification of B.atrophaeus. This paper presented two kinds of DNA extraction methods. One is water-boiling method, the other is kit extract method. In addition, a pair of control group was set up for better identification method which makes results more accurate and efficient.Third, quality test to detection method of B.atrophaeus was then carried out. On the one hand, four kinds of bacteria tablets, which were used as biological indicators for sterilization, were tested under detection method made before. The results showed that bacteria contained in the tablets are all B.atrophaeus. It also presents evident to the feasibility of method of identification of B.atrophaeus. On the other hand, water samples from natural environment were tested. The results showed that the concentration of B.atrophaeus is the same as well as accuracy through two kinds of methods for detecting B.atrophaeus. To sum up, two kinds of method for detecting B.atrophaeus are successfully made up.Fourth, In order to evaluate methods, a preliminary study about quality control parameters was carried out. These parameters included the recovery of standard addition and precision. The result showed that accuracy and precision of the method are practical.In summary, this paper successfully developed two kinds of methods for detecting B.atrophaeus.