Noe Miyashita

Attomol-level ATP bioluminometer for detecting single bacterium: ATP calibration curve is linear from 0 to 5 amol

We have developed an automated high-sensitive ATP bioluminometer for detecting single bacterium. The apparatus consists of a tube rack for setting reagents and samples, two washing baths for preventing sample carry-over from dispenser nozzle, and x-, y-, z- actuators for moving the dispenser, and an high-sensitive optical system. The reaction tube was selected to reduce the background signal intensities for the ATP bioluminescence measurement. The background signal intensity of the reaction tube was 18 RLU, which is almost the same as the dark counts of the photomultiplier (16 RLU). The ATP calibration curve was linear from 0 to 5 amol (its slope = 22.4 RLU/amol and 3.3 SD of the blank sample signal = 17.9 RLU), and the detection limit of 0.8 amol was obtained. The relationship between intracellular ATP and CFU in Escherichia coli (ATCC25922) was kept linearity from 0 to 20 CFU, and the intracellular ATP (amol) per CFU was calculated to be 3.3 amol/CFU (R2 = 0.9713). Moreover, the relationship between intracellular ATP and CFU in Staphylococcus aureus (ATCC25923) was also kept linearity from 0 to 30 CFU, and the amol/CFU was calculated to be 1.6 amol/CFU (R2 = 0.9847). The automated ATP bioluminometer has ultra-high sensitivity and will be a powerful tool for measuring ATP luminescence derived from small number of bacteria.

New Approach for Setting a Management Criterion in Microbiological Monitoring Using Rapid Microbiological Methods

The application of rapid microbiological methods (RMM) to bacterial monitoring in pharmaceutical manufacturing processes is now a key topic, since timely microbiological data are critical for product release, continuous process improvement and quality control. An automated, highly sensitive detection system has been developed which can measure the amount of ATP in a sample in 2 h with one hundredfold more sensitive than the conventional ATP method. One of the major subjects for adoption and implementation of RMM is how to set the criterion value for practical microbial control. This value was conventionally been set by experimental rule and indicated as the number of colonies counted after incubation in a particular medium. We have adopted a new approach to set a criterion value which enables assessment in whether the status of the object is normal or not. By setting this criterion value, it is possible to conduct the microbiological control with the intended probability of false-positive and false-negative. In this approach the probability distribution model of the measurement value of each object in a normal status has been established by performing repetitive measurement of each object. We have suggested and verified the probability distribution form of the ATP measurement value using measurement data of the standard bacterial solution of Staphylococcus aureus. The theoretical value of the model was in good agreement with the actual measured value. The results suggest it is possible to set an applicable management criterion value using this model and to conduct new microbiological monitoring using RMM.