不可分型流感嗜血杆菌生物膜体外形成规律及其扫描电镜形态学

目的:探讨不可分型流感嗜血杆菌（NTHi）生物膜（BF）体外形成规律，采用扫描电子显微镜（SEM）观察BF内部结构。方法:以NTHi标准菌株ATCC49247为研究菌株，以铜绿假单胞菌（PA）标准菌株PAO1为阳性对照菌株，同时设空白对照组，分别培养1、2、3、4、5、6和7 d时，使用平板计数法和结晶紫染色法检测BF形成情况，并于3 d时采用SEM检测ATCC49247的 BF结构。结果:平板计数法检测BF内菌落数，ATCC49247和PAO1培养3 d时活菌数逐渐上升至最高，而后逐渐下降，至7　d时分别降至（0.829 4±0.007 5）×10⁷cfu•mL^-1和（0.942 6±0.019 9）×10⁷cfu•mL^-1，培养3、4、5和6　d时2组间比较差异均有统计学意义（P<0.05），同种细菌各不同时间点间比较差异有统计学意义（P<0.05）。结晶紫染色检测BF致密程度，ATCC49247和PAO1培养3 d时 BF致密度逐渐上升至最高，波长570 nm吸光度（A570）值分别为2.717　4±0.017　2和2.885　3±0.039　0，而后逐渐下降，至7　d时分别降至0.151 7±0.074 5和1.196 9±1.108 5，各时间点2组细菌与空白组比较差异均有统计学意义（P<0.05），培养3、4和5 d时2组细菌间比较差异均有统计学意义（P<0.05），同种细菌不同时间点比较差异有统计学意义（P<0.05）。SEM观察，培养3 d时ATCC49247形成了典型的BF结构。结论:ATCC49247可以在体外形成BF，结晶紫染色、平板计数和扫描电镜可作为检测BF的常规手段。

Biofilm formation of nontypeable Haemophilus influenzae in vitro and morphology of biofilm under scanning electron microscope

Objective To investigate the biofilm(BF)formation rule of nontypeable Haemophilus influenzae(NTHi)in vitro,and to observe the internal structure of BF by scanning electron microscope(SEM).Methods NTHi ATCC49247was investigated in the present study,Pseudomonas aeruginosa(PA)PAO1was 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 ATCC49247was observed under SEM on day 3.Results The plate colony counting of biofilm BF by ATCC49247and PAO1raised during first 3d,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 ATCC49247and PAO1raised during the first 3d.The absorbances on 570nm wavelength(A570)in two groups were 2.717 4±0.017 2and 2.885 3±0.039 0,respectively;and then the A570values in two groups declined to 0.151 7±0.074 5and 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 ATCC49247were 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.