We maintain individual isolators for varying periods of time. Small experimental isolators are kept running for 1-6 months or so, depending on the experiment. They are completely emptied and resterilized between uses. Breeding isolators can be kept going for 3 years or more. However, prolonged use requires consistent monitoring for damage and replacement of isolator components, such as filters and gloves. This chapter describes our practices for maintaining isolators for long periods. We also describe our methods for working in the isolators to maintain sterility.