The overall goal of this procedure is to rapidly identify enterococcus species from positive blood culture bottles using peptide nucleic acid fluorescent and C two hybridization. Once blood cultures have been prepared and gram tested to confirm the presence of Enterococci culture, samples are fixed onto PNA fish microscope slides. The slides are then incubated with PNA probes specific to the organisms of interest.
Once the probes have hybridized, a stringent washes performed to remove excess non hybridized probe. Mounting media is added to the slides, which are then sealed with cover slips. The enterococcus species present in the sample can be identified by fluorescence microscopy.
This PNA Fish identification protocol is unique in providing a rapid and specific method for the identification of enterococcus from palsy. Patient blood culture samples Incubate two blood culture bottles, one aerobic and one anaerobic containing venous blood from a patient suspected of sepsis in an automated blood culture instrument at 35 degrees Celsius. Once the device indicates that it has detected microbial growth, remove the bottle, gently swirl the blood culture bottles to evenly resuspend the bacteria.
Then using a sterile syringe and needle aseptically transfer approximately five milliliters of blood from each bottle into a separate screw cap tube. Once all the specimens have been allotted, use a sterile pipette to transfer a drop of blood from each tube onto a separate glass microscope slide. Allow the slide to air dry for approximately 15 minutes.
Then after the slide is completely dry, immerse the slide in methanol to fix the sample. After the slides have been fixed, wash them and perform a standard gram stain to determine the morphology of the organisms present in each blood culture. Examine the stain slides using a microscope with an oil immersion objective.
Enter a caucus species will appear as gram-positive cocci typically assembled in pairs or short chains. Place one drop of fixation solution in the well of a p and a fish microscope slide. Gently mix the tube of blood culture to be tested.
Then transfer 10 microliters of culture onto the drop of fixation solution and pipe it up and down to emulsify the sample. Place the slides on a heating plate at 55 degrees Celsius for 20 minutes to fix the smears in each test. Be sure to include positive and negative control slides for assay quality assessment.
If e callous control slides can be purchased from a Van DX or prepared using reference cultures. Once the smear is ready, place one drop of EFI callous O-E-P-N-A on the well containing the blood culture sample, and also on the positive and negative control wells. To avoid contamination of the probe stock solution, be sure not to allow the dropper bottle tip to touch the blood smear.
Place a cover slip on top of the sample, taking care not to create air bubbles. Then incubate the slide on a heating block at 55 degrees Celsius for 30 minutes. To allow the PNA to hybridize following the incubation place the slides in a slide carrier.
Immerse the loaded slide carrier into a staining dish containing prewarm 55 degrees Celsius Wash Solution. To remove the excess PNA off the sample and gently slip off the cover slip by agitating the slide in the buffer or by using forceps. Incubate the slide at 55 degrees Celsius in wash solution for 30 minutes.
Then remove the slide from the staining dish and allow it to air dry. Once the slide is dry, add one drop of room temperature mounting medium to the slide and carefully place a cover slip on the sample. Examine the slides on a fluorescent microscope using a dual band filter within two hours after preparation.
Using this method, iffy, callous, shown in in green on the left can be distinguished from eum in red. In the middle panel on the far right is a negative result. Three things to remember for achieving successful results are instrument temperatures must be held at the set temperatures.
You need to follow the stain procedure very closely as outlined in the package insert. And finally, you need to have a reader that is experienced and does a thorough reading of the slide. This protocol is useful in making therapy decisions for the treatment of sepsis caused by enterococcus.
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