Isolation of L-form Bacteria from Urine using Filtration Method

Transition of bacteria to the L-form state is thought to play a possible role in immune evasion and bacterial persistence during treatment with cell wall-targeting antibiotics. However, isolation and handling of L-form bacteria is challenging, mainly due to their high sensitivity to changes in osmolarity. Here, we describe detailed protocols for the preparation of L-form medium, isolation of L-forms from urine using a filtration method, detection of L-forms in urine samples by phase contrast microscopy and induction of L-forms in vitro. The exact requirements for survival and growth of L-forms may vary from strain to strain. Therefore, the methods presented here are intended to act as basic guidelines for establishing L-form protocols within individual laboratories, rather than as precise instructions. The filtration method can lead to a reduction in the number of L-forms in a sample and should not be used for quantification. However, it is the only method used so far for effective separation of cell wall-deficient variants from their walled counterparts and for identification of bacterial strains, which are capable of L-form switching in patients with urinary tract infections. The filtration method has the potential to be adapted for the isolation of L-forms from patients with other categories of bacterial infections and from environmental samples.