The overall goal of this procedure is to examine platelet rich plasma or PRPs in vitro antimicrobial properties. First, isolate platelet rich plasma from a donor rabbit. Next, prepare a bacterial inoculum, then activate the PRP using thrombin and mix with the bacterial inoculum.
Incubate the mixture, sample, and measure the resulting colony forming units. The antimicrobial properties of PRP are demonstrated by the reduction of CFU in PRP treated samples compared to the control samples. Without PRP treatment, Platelet reach plasma is less likely to induce BOL resistance compared to conventional BOL treatments.
It also has properties that promote wound healing, which may have a synergistic effect on infection prevention. In the current protocol, a twice ification approach was performed and the platelet concentration of PRP was standardized to two times 10 to the six platelets per approximately 10 times above the baseline in blood, leukocyte reached PRP was used. Since leukocytes are also involved in direct bacterial kidney and antigen specific immune response, which may contribute to the antimicrobial properties of PRP First anesthetize a rabbit by inhalation of isof fluorine.
Draw two milliliters of 0.129 moles per liter. Tri sodium citrate into a 20 milliliter syringe. Sterilize the rabbit ear using 70%ethanol.
Using a 25 gauge butterfly needle. Draw blood from the rabbit ear vein and mix the blood with the trisodium citrate solution by gentle agitation. Transfer the anticoagulated blood to a 50 milliliter plastic centrifuge tube.
Take an Eloqua of 10 microliters of blood to determine the baseline platelet count using hemo cytometry in a swing out rotor. Set the acceleration and break velocity to low and centrifuge the blood at 300 Gs for 10 minutes. At room temperature, carefully transport the centrifuge tube to a cell culture hood without disturbing the layers.
Transfer all of the plasma buffy coat and two to three millimeter thick red blood cell layer into a 15 milliliter plastic tube. Centrifuge the transferred sample at 3000 Gs for 15 minutes. At room temperature, transfer the supernatant of platelet poor plasma to a new tube.
Obtain platelet rich plasma by adjusting the platelet concentration in the remaining blood sample with PPP to obtain two times 10 of the six platelets per microliter as determined by hemo cytometry from an overnight streaked blood agar plate of S reus. Inoculate five colonies into five milliliters of Mueller Hinton broth vortex. Loosen the cap and incubate for two hours at 37 degrees Celsius.
Read the optical density using a spectrophotometer. Adjust the inoculum to an optical density based on the predetermined standard curve. Make a 100 fold dilution in PBS to obtain one times 10 to the six CF Use per milliliter and place the inoculum on ice.
Set up and label sterile disposable five milliliter round bottom polystyrene tubes. For sample groups as indicated, first add P-R-P-P-P-P or PBS. Then add the thrombin solution for activation.
Next, add Mueller hinton broth, then the ESUs inoculums to obtain a final concentration of one times 10 of the fifth CFUs per milliliter. Incubate the tubes at 37 degrees Celsius with orbital agitation at 150 RP M at predetermined time points. Mix the solutions in each tube to ensure that the bacteria are not trapped inside the PRP gel.
Take 10 microliters of each sample. Dilute serially with sterile 0.9%saline and pipette a 100 microliter Eloqua of each dilution onto a triptych soy agri plate. For CFU counting culture, the agri plates overnight at 37 degrees Celsius.
Then count and record the plate colonies. Plot data on a logarithmic scale with time on the x axis and CFU per milliliter. On the Y axis, platelet-rich plasma is reproducibly prepared using a twice centrifugation approach.
PRP has strong antimicrobial properties against several bacteria, including methicillin resistant and sensitive aureus, group-based streptococcus and RIA gonorrhea at one and two hour time points. MRSA bacteria are commonly found in hospitals worldwide. This killing assay shows.
PRP has strong in vitro antimicrobial properties against MRSA at one and two hour time points. Quantitative analysis indicates an up to 100 fold reduction in CFUs is obtained using PRP compared to PPP and PBS controls at one hour time. Point blood smears of PRP prepared from the twice centrifugation approach has about 10 times the number of platelets compared to whole blood disadvantage facilitates consistent reproducible results.
The number and the quality of platelets are the key factors influencing the success of this experiment. Following this procedure, other bacterial strings can be evaluated and the PRP can be examined in vivo as well to explore its anti-microbial properties in animal infection models after its development. This technique paved the way for researchers in the field of PRP to explore the potential use of PRP in preventing infection and promoting wound healing in animal and clinical settings.
