The Research Of Single And Mixed Bacterial Biofilm On The Surface Of Porous Titanium Alloy Growth Characteristics

[objective] to establish microbial biofilm on the surface of porous titanium alloy materials in vitro model;Observation material surface epidermis staphylococcus aureus and staphylococcus aureus formation of a mixed microbial biofilms and microbial biofilms in growth characteristics, the in vitro growth dynamics, the differences between the ultrastructural aspects;For related research and provide theoretical basis for clinical work.[method ]porous titanium alloy material chosen as the experimental material.The experimental group:Standard strains by staphylococcus epidermidis ATCC35984 positive (biofilm formation) separate training group (table Portugal positive group), staphylococcus aureus ATCC25923 standard strain (biofilm formation positive) separate training group (s. aureus positive group) and the epidermis staphylococcus standard strains ATCC35984 positive (biofilm formation) and standard strains of staphylococcus aureus ATCC25923 positive (biofilm formation) positive training set of the above three groups of bacteria are mixed with porous titanium alloy material culture, 10 cases in each group, a total of three groups.Porous titanium alloy material (10 cases in each group) are immersed in 400 ul epidermis staphylococcus aureus, staphylococcus aureus, and a mixture of both bacteria liquid,at 350 c respectively in the constant temperature incubator culture 2h,4 h, 6 h, 12 h, 24 h, 48 h, 72 h, porous titanium material surface biofilm in vitro model is set up, respectively in each time point after porous titanium alloy material, staining with crystal violet staining method, and DMSO decoloring after 15 minutes per hole orifice 100 ul decoloring liquid - 100, traces of enzyme label plate measuring instrument is used to test 490 nm absorbance, using multifunctional enzyme standard determination of each hole in 490 nm wavelength optical density (OD) value, OD value of the size of the adhesion ability strong and the weak reaction, indirectly reflect the microbes form biofilm thickness, only to join TSB culture medium as a blank control.Actual OD values for each experiment hole OD value minus the OD value of blank control hole, experiment repeated six times.Test the ability of biofilm formation.Dimethyl benzene sulfonic acid sodium (XTT colorimetric method) by using multifunctional enzyme standard determination of each hole decoloring liquid at wavelength of 450 nm OD value of bacteria cell activity to OD value of the size of the reaction.Actual OD values for each experiment hole OD value minus the OD value of the ck empty, experiment repeated six times, in order to evaluate the biofilm growth kinetics in vitro;Laser confocal microscope vision detecting the thickness of the biofilm in the group, the unit within the bacterial population, construction of biological membrane surface three-dimensional fluorescence image;Biological membrane surface ultrastructure of scanning electron microscope observation.[results] :1. The crystal violet staining method to detect biofilm formation ability results: epidermis staphylococcus aureus, staphylococcus aureus, group and mixed group, the group starts 6 hours with bacterial biofilm formation, and in 8 h on bacterial biofilm obviously thickening, in 12 h, 24 h period, the bacterial biofilm growth trend is relatively stable,thickening of bacterial biofilm in a slow growth period, as the extension of incubation time, and since cultivation of 24 hours,thickening of biofilm growth and thickening speed, begin to cultivate 6 h and continue to develop to the whole process of 72 hours, mixed in positive bacterial biofilm thickness more than epidermis staphylococcus aureus and staphylococcus aureus single group, epidermis staphylococcus group thickness is greater than the staphylococcus aureus.2. XTT colorimetric method to detect biofilm growth kinetics results: since cultivation to 48 hours before the mixed bacteria group OD value below the epidermis staphylococcus single single group, but higher than that of staphylococcus aureus in 48 hours,72 hours time mixed bacteria group OD value higher than the epidermis staphylococcus gradually, in the whole training process of mixed bacteria groups and the epidermis staphylococcus OD value is always higher than that of staphylococcus aureus group;Compared the 12 h difference between groups was statistically significant (P < 0.05).Porous titanium mixed bacteria group, 2 h, 4 h, time OD value lower than that of staphylococcus aureus group has no statistically significant difference (P > 0.05).6 h after each time point, OD value was significantly higher than that of staphylococcus aureus group, the difference was statistically significant (P <0.05).3. The laser confocal microscopy observations:Porous titanium alloy material and mixed bacteria, staphylococcus aureus and epidermis staphylococcus culture began 6 h of biofilm growth, 12 h to start each bacterial biofilm became obvious raised thick until 48 h, mixed group and the epidermis staphylococcus bacteria biofilm thickness at the thickest, to 72 h stable biofilm thickness, the porous titanium mixed bacteria group in 2 h to 72 h the train during the formation of the biofilm is always greater than the thickness of staphylococcus aureus and epidermis staphylococcus single group (P < 0.05), mixed bacteria group of porous titanium alloy material surface colony number 6 h, 12 h, 24 h,48 h, 72 h, respectively, than the epidermis staphylococcus group gold and single yellow staphylococcus bacteria group,and difference has statistics meaning (P <0.05).Mixed bacteria group of fluorescence image and 3 d reconstruction tip: hybrid biofilm hummocky lung surface rendering undulating shape, protuberant part of living bacterium, dead bacteria division in ravines between hummocky uplift;Biofilm tomography tip: set is in 12 h outer layer of the mixed bacteria living bacterium proportion is as high as 90%, living bacterium is still as high as 80%, 48 hours until 72 hours training living bacterium was down to 50% live bacteria in biofilm convex,dead bacteria in hybrid biofilm concave part, with the percentage of the underlying dead bacteria increased, the surface of living bacterium depolymerization release,continue to spread increases the possibility of engraftment.Mature biofilm on the surface of porous titanium alloy materials mixed bacteria group structure more complex than a single bacterium group.Laser confocal microscope to detect hints of staphylococcus aureus and epidermis staphylococcus aureus in a total of 6 h, 12 h, 24 h, 48 h, 72 h, mixed bacteria group of bacterial biofilm thicker, mixed bacteria and the single bacterial biofilm thickness difference to be markedly, with statistical significance (P < 0.05).4. Scanning electron microscopy (sem) results showed that as the training time raised,48 h, 72 h two biofilm formation, the timing of the more mature as research object,the groups of porous titanium alloy material with the extension of incubation time, the surface are gradually formed a crumb,cascading sample stacking the complex three-dimensional structure of the hybrid biofilm, visible epidermis staphylococcus aureus and staphylococcus aureus bacteria is mixed crumb growth, a large number of epidermis staphylococcus aureus and staphylococcus aureus to form multiple layers of complicated block structure.And epidermis staphylococcus aureus and staphylococcus aureus single bacteria group, compared to the same culture group with the time point of porous titanium alloy mixed bacteria biofilm structure more complicated, the three-dimensional hierarchical structure more apparent, the staphylococcus aureus and epidermis staphylococcus and adhesion on the surface of the material more closely,biofilm peripheral staphylococcus aureus and epidermis staphylococcus quantity more.[conclusion]1. Biofilm formation positive epidermis staphylococcus aureus, staphylococcus aureus mixed culture can be formed on the surface of porous titanium alloy material than a single bacterium more thick, more dense, structure more complex mature mixed bacterial biofilm.2. The mixed bacteria group of bacteria in the porous titanium alloy surface adhesion ability is stronger, the surface of the material also more adhesive on the number of bacterial colonies.3. Mixed bacterial biofilm surface uneven, living bacterium ratio is higher, to a convex concave parts dead bacteria ratio is higher, the die/live bacteria gradually reduce the proportion of outside introversion.4. Living bacterium mixed bacterial biofilm surface more easily than other experimental depolymerization release, continue to spread colonize new colony formation, which may be lead to clinical biological materials mixed microbial infection in delay no more, and more single bacterial infection more difficult to control one of the important reasons.