This experiment tests the ability of staphylococcus aureus to utilize human hemoglobin as a sole iron source. First, isolate red blood cells from fresh human blood. Purify the hemoglobin by high performance liquid chromatography to ensure the integrity of hemoglobin.
Now add hemoglobin into iron depleted medium to supply the iron in the form of hemoglobin Next culture. S reus. In the presence of hemoglobin and measure its ability to utilize hemoglobin as an iron source.
The results show that as reus proliferates in the presence of hemoglobin as a sole iron source. This method can help answer key questions in the field of microbiology, such as whether a pathogen can utilize host proteins as sources of iron, and which mechanisms are required to utilize iron using this process. So this method can provide insight into iron acquisition of staph reus from hemoglobin.
It can also be applied to the studies of other bacteria and how they use iron containing proteins from the host. Demonstrating this technique will be GLA Ani and Catherine Haley from my laboratory. First, obtain freshly isolated human blood, supplemented with an anticoagulant and maintain at four degrees Celsius throughout the purification Pellet the red blood cells by centrifugation for 20 minutes at 15, 000 Gs.Carefully aspirate the snat and gently resuspend the pellet in ice cold, 0.9%sodium chloride solution After three washes, resus, suspend the pellet in one volume of ice cold 10 millimolar triss, HCL to ly the RBCs by osmotic pressure.
Then add toluene to 20%final volume and incubate at four degrees Celsius on a rotator overnight. Next centrifuge. The lysate at 20, 000 Gs per one hour.
Collect the middle hemolysate fraction, leaving the upper toluene layer and the pellet undisturbed. Pass the hemolysate through a 0.44 micrometer syringe filter. If the solution contains particulate matter and cannot be passed through the filter, repeat the high speed centrifugation.
Now purify the hemoglobin with a high performance liquid chromatography anion exchange column. Use a mobile phase A of 10 millimolar tris, HCL, and a mobile phase B of 10 millimolar tris, HCL plus 0.5 molar sodium chloride. Run a 0%to 100%gradient of solvent B over two minutes at two milliliters per minute flow rate.
Monitor the elution based on absorption at 410 nanometers and 280 nanometers. Collect the fraction characterized by a bright red color and a prominent absorption peak. Dialyze the elution against phosphate buffered saline for approximately 16 hours.
To sterilize the sample, pass it through a 0.22 micrometer syringe filter for determining the hemoglobin concentration. Prepare HP standard solutions in PBS. Now mix the standard solutions or the sample solutions with two xra kins reagent at a one-to-one ratio in 96 well plates after a 15 minute incubation measure absorbance at 540 nanometers.
Plot a standard curve and determine the HP concentration of the samples five to 15 milligrams per milliliter. Yields are typical run aliquots of 15 to 20 micrograms purified hemoglobin sample on two 15%STS page gels. Stain one of the gels and transfer the proteins from another gel onto a nitrocellulose membrane to immuno blot for hemoglobin freeze and store one milliliter aliquots of the isolated hemoglobin in liquid nitrogen from a frozen stock streak S aureus on triptych, soy agar, and incubate at 37 degrees Celsius for 20 to 24 hours.
Inoculate single colonies into five milliliters of RPMI containing E-D-D-H-A and culture at 37 degrees Celsius with shaking at 180 RP M overnight. Pellet the bacteria by centrifugation for five minutes at 7, 500 Gs.Then Resus, suspend the bacteria in RPMI containing 0.5 millimolar E-D-D-H-A, and normalize to an OD 600 of about three. Next, prepare N-R-P-M-I containing 2.5 micrograms per milliliter, HB and 0.1 to one millimolar E-D-D-H-A to chelate the free iron.
Now subculture 10 microliters of bacterial suspension into one milliliter of N-R-P-M-I plus E-D-D-H-A plus HP incubate the cultures at 37 degrees Celsius for up to 48 hours with shaking at 180 RRP M every six to 12 hours. Remove 50 microliters of the culture mix with 150 microliters of PBS in 96 well plates and take OD 600 readings. In this HPLC purification of human hemoglobin from hemolysate, the absorbence of eluate at 280 and 410 nanometer wavelengths is measured with fraction five containing the hemoglobin.
Typically, yields of five to 15 milligrams of hemoglobin per milliliter are obtained per preparation. The purified hemoglobin was analyzed by SDS page in duplicate, and the gels were either stand for proteins or transferred onto nitrocellulose and immuno blotted. Next, the purified human hemoglobin was tested for ability to support the growth of wild type asus and ASUS that lacks the hemoglobin receptor required for hemoglobin derived iron acquisition.
The increase in optical density of the cultures over time indicates that in N-R-P-M-I plus E-D-D-H-A plus hb, the wild type ASUS proliferates, but the mutant does not. In contrast, the ability to utilize supplemented free iron is identical in both wild type and mutant strains, neither proliferates in the absence of an source. In this comparison of media supplemented with hemoglobin purified from fresh blood or lyophilized hemoglobin, the hemoglobin purified from blood requires ISDB for growth.
While lyophilized hemoglobin enables proliferation of the ISDB mutant Once mastered, a proper purification of hemoglobin can be completed in a few hours. Remember, the donors are potential carriers of bloodborne pathogens, so treat human blood as a biohazardous material.
