Abstract:

Abstract:Objective To investigate the biofilm(BF) formation rule of nontypeable Haemophilusinfluenzae (NTHi) in vitro,and to observe the internal structure of BF by scanning electron microscope (SEM).Methods NTHi ATCC49247 was investigated in the present study，Pseudomonas aeruginosa (PA) PAO1 was cultured as positive control,at the same time blank control group was set up.The BF of the  bacteria were cultured and then collected on day 1,2,3,4,5,6, and 7.The BF formation was detected by crystal violet staining and plate counting and the structure of BF formed by ATCC49247 was observed under SEM on day 3.Results The plate colony counting of biofilm BF  by ATCC49247 and PAO1 raised during first 3 d,and then declined to (0.823 6±0.007 5)×10⁷ cfu•mL^-1 and(0.942 6±0.019 9)×10⁷cfu•mL^-1 respectively on day 7. The differences between two groups were statistically significant on day 3,4,5, and 6(P<0.05).The differences between different time points in the same bacteria group were statistically significant(P<0.05).The densities of  BF formed by ATCC49247 and PAO1 raised during the first 3 d.The absorbances on 570 nm wavelength(A570) in two groups were 2.717 4±0.017 2 and 2.885 3±0.039 0 ,respectively;and then the A570 values in two  groups declined to 0.151 7±0.074 5 and 1.196 9±1.108 5, respectively on day 7;the differences between bacteria groups and blank control were statistically significant(P<0.05);the differences between two bacteria  groups were  statistically significant on day 3,4,5, and 6(P<0.05);the differences between different time points in the same bacteria group were statistically significant (P<0.05).On day 3,the obvious BF formed by ATCC49247 were observed under SEM.Conclusion BF could be formed by NTHi in vitro；crystal violet staining,plate colony counting and SEM could be taken as conventional methods to detect BF.

Key words: nontypeable Haemophilus influenzae, biofilm, crystal violet staining, plate colony counting, scanning electron