The Inhibition And Destructive Of Tuoguan San On Staphylococcus Aureus Bacterial Biofilm In Vitro

ObjectiveTo explore an appropriate method for establishing vitro standing model of Staphylococcus aureus bacterial biofilm so as to provide model for research of Tuoguansan’s intervention on S.aureus BF.And to discuss Tuoguansan’s suppressive and destructive effects on S. aureus BF. MethodsThe Staphylococcus aureus bacterial biofilm(ATCC25933) should be revived, inoculated, made a passage and cultivated in order to make the standard bacterium solution whose concentration is 1x109cfu/ml. Make the preliminary characterization of S. aureus BF’s formation using the 24 pore plate cultivation safranine dye means. Use the cell climbing slice of 8 mm diameter as the carrier; use LB broth for cultivating and change the fluid every day; after 4 days, use the scanning electron microscope to observe in order to further confirm that the bacterial strain can form BF; observe the S. aureus BF’s shape under different magnifications.Use the cell climbing slice of 8 mm diameter as the carrier; use 4 different types of Tuoguansan concentration on ripe BBF; after 4 days, confirm the best concentration in Tuoguansan’s effects on S. aureus BF. it is divided into the blank group and Tuoguansan-with-different-concentration group. Apply Tuoguansan 3 da ys after modeling; use continuous viable count to measure quantity of viable organism 4 days after using the medicine. Use the cell climbing slice of 8 mm diameter as the carrier. The scanning electron microscope is used to observe the six-day dynamic growth trend of S. aureus BF and the influence on BF exerted by the daily use of vancomycin(VAN). There are the blank group and the vancomycin group separately. The 96 pore plate is used for the cultivation; use vancomycin for the vancomycin group every day. Starting from the second day, samples are taken every day. The crystallization is dyed violet. The enzyme labeling instrument is used to measure the optical density value and draw the growth curve of the S. aureus BF and the growth curve after being affected by VAN. There are the blank group, vancomycin preventive group and Tuoguansan preventive group separately. The cell climbing slice is used as the carrier. The 24 pore plate is used for the cultivation. Samples are taken on the 4th day and the 7th day. After being processed, they are observed through the scanning electron microscope and compared with each other in regard to the preventive effects of VAN and Tuoguansan on the growth of S. aureus BF. There are the blank group, the vancomycin group and the Tuoguansan group(The medicine is used with the vancomycin group and the Tuoguansan group 48 hours later) separately. The cell climbing slice is used as the carrier. The 24 pore plate is used for the cultivation. Samples are taken on the 4th day and the 7t h day. After being processed, they are observed through the scanning electron microscope and compared with each other in regard to the destructive effects of VAN and Tuoguansan on the mature S. aureus BF. Results1. Use 24 pore plate to culture S. aureus for 4 days, then perform safranin staining to form flaky red matter that is visible with naked eyes. This is preliminarily classified as the formation of biofilm. 2. The cell climbing slice is used as the carrier to culture for 4 days, then conduct such treatments as removing airborne viable particles, gradient elution using ethanol, isoamyl acetate replacement, carbon dioxide critical point drying by using the carbon dioxide critical point drying instrument, adhesion, and metal spraying by using HCP-2 sputter coater. After these treatments, the formation of membranous BBF can be seen through the conduct scanning electron microscopy. And at the same time, we can watch the shape of biofilm. 3. The most suitable Tuoguansan concentration on S. aureus BF: use Tuoguansan on the mature S. aureus BF for 4 days, the bacteria number of 0.1g/ml group is smaller and there is no biofilm formed. 4.Comparison of continuous viable count after Tuoguansan’s effects on S. aureus BF: after 4 days use of Tuoguansan with different concentrations, during the continuous viable count, the number of live bacteria from the different concentration groups of Tuoguansan is significantly less than that of the blank group(p<0.05). 5.The growth trend of S. aureus BF and the influence on it e xerted by the functions of vancomycin(VAN): through dynamic watching of the blank group by SEM and OD measurement with ELIASA, it is observed that, S. aureus BF starts to increase since the first day, and reaches the peak at the fourth day, then again starts to decrease. For the VAN group, BBF quickly increases to the peak on the first day of using vancomycin, then gradually declines, and bacterial biofilm gradually decreases. 6. The preventive effect of Tuoguansan on S. aureus BF: whether culturing for 4 days or 7 days, there is biofilm in the vancomycin prevention group, but the amount of biofilm is small when culturing for 7 days; there is no biofilm in the Tuoguansan prevention group, and the number of bacteria is very small. 7. The destructive effect o f Tuoguansan on S. aureus BF: as for the mature S. aureus BF, there is biofilm in the combination group of Western and Chinese medicines on the 4th day after medicine intake, but the amount decreases on the 7th day; however, there is no biofilm in the Tuoguansan group on the 4th day or on the 7th day after medicine intake, and the number of bacteria is very small. ConclusionTuoguansan can restrain the formation of Staphylococcus aureus bacterial biofilm, S.a BF and destroy the formed mature Staphylococcus aureus bacterial biofilm, S.a BF in vitro.