Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

The overall goal of this procedure is to prepare a bacterial pellet from a positive blood culture that can be used as a sample for various applications. This is accomplished by first mixing a positive blood culture with sterile water Following centrifugation. The pellet is resuspended in ammonium chloride to lice the red blood cells after a second round of centrifugation.

The pellet is resuspended in water. The final step is to use the obtained pellet for bacterial identification by matrix assisted laser desorption ionization, time of flight, gram staining, antibiotic susceptibility testing, and or PCR based tests. Ultimately acquired results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

The main advantage of this technique over existing methods such as bacterial subculture of positive blood cultures, is to reduce the bacterial identification by about six to 24 hours and to reduce the time to antibiotic resistance results by about 24 hours. This method can help to rapidly adapt the antibiotic treatment and to improve the outcome of patients suffering from bloodstream infections. Though this method can be applied to routine identification methods such as OV and to conventional anti-microbial sustainability testing approaches, it can also be applied to new technologies aiming at ification of VUL factors and or resistant mechanism such as ESBL and CARBAPENEMASE activities.

Visual demonstration of this method is important since the bacterial PAL preparation is crucial in order to obtain enough microbial material for downstream applications, demonstrating the procedure will be Christian Durel, Maria Cortesi, Maria ti, and Kim Giza, all of whom are laboratory technicians from my lab To prepare a positive blood culture for subsequent centrifugation. Sterilize the blood culture cap by adding 70%ethanol on the cap of the bottle and burning it. Then move the bottle to a laminar flow hood.

Collect five milliliters of the blood culture with a 20 gauge needle syringe. Next, add the five milliliters of blood culture to a 50 milliliter centrifuge tube containing 45 milliliters of sterile water and mix the sample. Centrifuge the sample at 1000 Gs for 10 minutes.

At room temperature, remove the supernatant with a laboratory vacuum pump. Then resuspend the pellet in one milliliter of homemade ammonium chloride. Lysing solution.

Break down the pellet by scraping and vortexing before centrifusion the sample at 140 gs for 10 minutes At room temperature following centrifugation, discard the supernatant, which mainly contains red blood cell debris. If the pellet remained hemorrhagic Resus, suspend the pellet in two milliliters of sterile and distilled water to further lice residual red blood cells and to wash the pellet after centrifuging the sample as before, discard the supernatant, then resuspend the pellet in 200 microliters of water and vortex the tube for direct identification from the blood pellet. First, transfer one microliter of the pellet to a MALDI target plate and let it dry on a 42 degree Celsius heating platform.

Overlay the sample with one microliter of 70%formic acid and let it dry on the heating platform. Next, overlay the sample with one microliter of Maldi Matrixx and again, let it dry on the heating platform. Proceed to maldi TOF MS analysis with a MALDI TOF MS system as described by the manufacturers.

If the identification score is invalid at the species level, perform a protein extraction of the blood pellet sample by first mixing 20 microliters of the pellet with one milliliter of 70%ethanol. Then centrifuge the sample at 13, 000 Gs for two minutes at room temperature, discard the supernatant and add 25 microliters of 70%formic acid and 25 microliters of 100%aceto nitrile to the pellet vortex vigorously to resuspend the pellet following centrifugation at 13, 000 Gs for two minutes. At room temperature transfer one microliter of the supernatant onto a MALDI target plate and let it dry on a 42 degree Celsius heating platform.

To perform bacterial identification and antibiotic susceptibility testing or a ST first, prepare a plastic tube containing three milliliters of sterile 0.45%sodium chloride solution compatible with the automated microbial system. Add a sufficient volume of the blood culture pellet to the solution and vortex to obtain a bacterial suspension corresponding to a McFarland turbidity of 0.6 to 0.8 as measured using a densitometer machine. Next, inoculate the automated gram-positive or gram-negative cards for identification.

Also inoculate the automated a ST cards for antimicrobial susceptibility testing of gram-positive cocci and gram-negative bacteria as described by the manufacturer. To perform antimicrobial susceptibility testing by disc diffusion assay. First, prepare a glass tube containing three milliliters of sterile 0.9%sodium chloride solution.

Then add a sufficient volume of the blood culture pellet to the solution to obtain a bacterial suspension corresponding to a McFarland turbidity of 0.5 as measured by a densitometer machine. Vortex the bacterial suspension to inoculate the bacterial suspension. First, dip a sterile cotton swab in the bacterial suspension and gently remove excess fluid by turning the swab on the inside wall of the glass tube.

Then spread the bacterial suspension evenly onto the entire surface of Mueller. Hint and auger or Mueller hint and auger for fastidious organisms by swabbing in three directions or by using an automated plate rotator for application of antimicrobial discs on inoculated auger plates. First, closely apply the discs to the surface of the dried inoculated auger plates manually or by using a susceptibility disc dispenser.

Then incubate the plate at the appropriate temperature and atmosphere as described in the antimicrobial susceptibility Testing European committee on antimicrobial susceptibility testing disc diffusion method to perform the automated PCR based diagnostic test. First, use a micro pipette to transfer 50 microliters of the blood culture pellet into the sample reagent vial provided with the MSA automated PCR based diagnostic test kit. Then vortex the vial for 20 seconds using a one milliliter micro pipette.

Dispense the sample into the specimen port of the cartridge. Insert the cartridge into the automated PCR system and start the assay as described by the manufacturer in the laboratory of Lausanne. The result is directly validated and transmitted to the clinicians thanks to the connectivity of the automated PCR system used with a laboratory interface system or LIS.

If a result is invalid, we recommend repeating the assay after tenfold dilution of the bacterial pellet. Since this is often due to PCR inhibitors, the ammonium chloride hemolysis produced a very high quality blood culture pellet with little erythrocyte debris as compared to the negative control without ammonium chloride. Gram staining of positive blood culture of Escher Coli Coagulase negative staphylococcus capita and streptococcus disc AE was performed before and after the preparation of the blood culture.

Bacterial pellet preparation of the pellet using this approach allows concentrating the microorganism by about 100 to 1000 fold. Overall, there are several downstream applications that can be performed starting from the blood culture pellet, which all benefit from a reduced time to results without reduced sensitivity and specificity as compared to assays done on isolated colonies. Once mastered pate, preparation can be done in about 30 minutes if it is properly performed.

Don't forget that working with blood culture can be extremely hazardous and precautions such as working with gloves and under laina flow hood should be taken while performing this procedure.