Natural defect is a new type of positive sample that is the nearest to the actual glass crack prepared by artificial way. It is suitable for the packaging materials of conventional glass materials such as cillin bottles and ampoule bottles. The aperture of natural defect can be less than 1 micron, and the minimum can be nano level, which is very suitable for the MALL value study of rigid packaging. Including microbial challenge sensitivity test and head-space protection studies. Thus, natural defects can be used for method validation of ultra-sensitive equipment.