日本| Sanitization of the Centricon Plus-70 and Centricon Plus-20 | Tech Note - TN0125EN00 > Product Catalogue | |
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Introduction
Centrifugal concentrator devices are used in a wide variety of applications, including concentration of macromolecules, purification and desalting and buffer exchange by diafiltration. The Centricon® Plus-70 centrifugal filter is a disposable, single-use device designed for rapid processing of aqueous biological solutions in volumes ranging from 15 to 70 mL. It is compatible with swinging-bucket centrifuges only. The Centricon Plus-70 device can concentrate most 70 mL solutions down to 350 µL in just 25 minutes (some solutions may take slightly longer). The information presented in this technical note provides details on the sanitization process used and its effectiveness by comparing colony forming units in the sanitized and non-sanitized devices. The performance of the Centricon Plus-70 was also evaluated by comparing the recovery efficiency of the sanitized device versus a non-sanitized control.
Sanitization Procedure
1. Add 70 mL of 70% ethanol to the devices. Incubate the devices for 5 minutes at room temperature.
2. Separately treat the recovery cups with 70% ethanol.
3. Spin the devices at 3500 x g for 25 minutes at 250 C.
4. Rinse the devices and the retentate cups with sterile Milli-Q water until traces of ethanol are removed. Two to three rinses are sufficient.
Notes
- It is critical that the membrane remains wet after sanitization and before usage. Sterile water can be used to keep the membrane wet.
- The procedure described herein is applicable to all Centricon Plus-20 and Centricon Plus-70 devices containing Biomax and Ultracel membranes. The experimental data detailed in this tech note was generated using Centricon Plus-70 devices containing 5K (Biomax PES) and 100K (Ultracel regenerated cellulose) membranes to serve as an example of the utility of the recommended procedure.
- The Centricon Plus-70 is not autoclavable.
Efficacy of the Sanitization Procedure
Table.1 shows the effect of 70% ethanol treatment of the Centricon Plus-70 on the number of colony forming units (CFU). The data demonstrate a significant decrease of the number of bacterial colonies observed in the sanitized units versus controls. The number of CFUs was determined by adding 70 mL of sterile PBS to sanitized and control Centricon Plus-70 units,centrifuging at 3500 x g for 25 minutes at 250 C, recovering by a reverse spin, and platting the resulting concentrate on agar plates. The number of colonies observed on each plate was determined after a 48 hour incubation at 370 C.
Performance Evaluation of Sanitizated Centricon Plus-70 Devices
The protein recovery of the Centricon Plus-70 was evaluated using two concentrations of test proteins in control (untreated) versus sanitized devices.
Method
Test Devices
Centricon Plus-70.
a- Negative control: Untreated Centricon Plus-70 Devices (5K and 100K)
b- Centricon Plus-70 Devices (5K and 100K membrane) after 70% ethanol sanitization.
Protocol
- Test solutions: Bovine serum albumin (1mg/mL and 0.1mg/mL) for 5K devices and bovine gamma globulin (1 mg/ml and 0.1mg/ml) for 100K devices
- Test solution added to each device.
- Devices placed into the centrifuge with the appropriate rotor.
- Guidelines of the user guide were followed.
Results
Conclusions
The data presented in this tech note clearly demonstrate that the sanitization method described significantly reduced the bacterial load of the Centricon Plus devices. The method also shows that the sanitization procedure does not impact negatively the performance of Centricon Plus. For users of Centricon Plus requiring reduced bacterial loads, sanitization with 70% ethanol is an appropriate option.
