Introduction: The microtitre plate assay (MTPA) is widely used for measuring biofilm formation but has not been validated for non-typeable Haemophilus influenzae (NTHi).
Aim: This study aims to validate the MTPA for NTHi and investigate the effect of frozen storage of cultures on subsequent biofilm formation.
Method: Four NTHi clinical isolates and Pseudomonas aeruginosa (PAO1) as a positive control were grown in broth and standardised inoculum added to quadruplicate wells of a 24 well microtitre plate. Final growth (FG) was determined by absorbance after 20 hrs incubation and biofilm (BF) then measured by absorbance after crystal violet staining. The relative biofilm production (BF/FG) was determined over 13 plates on the same and different days. MTPA was subsequently used on an additional 4 fresh NTHi isolates and repeated after freezing the isolates at -80oC for 2 weeks, 1, 2, 3 and 6 months.
Results: Mean and SD of BF/FG ratio (n=13) and SD of the BF/FG between plates on single days (n=5) are as follows: strain 1 (1.58, 0.73, 0.40), strain 2 (0.58, 0.23, 0.11), strain 3 (0.15, 0.08, 0.03), strain 4 (0.16, 0.17, 0.10) and PAO1 (0.29, 0.07, 0.03). For freezing experiment, the mean/SD of BF/FG ratio (n=4) for each strain, fresh and frozen for 2 weeks, 1,2,3 and 6 months are as follows: strain A (1.35/0.17, 1.99/0.27, 2.27/0.22, 2.31/0.28, 2.61/0.25, 3.04/0.32), strain B (1.96/0.25, 1.92/0.54, 2.44/0.27, 2.59/0.52, 1.83/0.34, 2.80/1.10), strain C (0.69/0.26, 0.94/0.09, 1.10/0.09, 1.07/0.14, 1.03/0.18, 0.38/0.14), strain D (0.12/0.16, 0.12/0.05, 0.10/0.04, 0.14/0.04, 0.24/0.17, 0.22/0.10).
Discussion: MTPA using BF/FG as a measure of the ability of NTHi to form biofilms and standardised inoculum appears reproducible and applicable to strains stored by freezing. Additional studies are required to investigate apparent increased biofilm formation ability in some strains after frozen storage.