Standard Inoculum Protocol (SIP) for Bacterial Identification Using an Automated Microbial Identification and Antimicrobial Susceptibility Testing System (aMIAST)

To begin, swab the septum of an automated blood culture bottle containing monomorphic gram-negative organisms, with 70%isopropyl alcohol, inside a Class II A biosafety cabinet. Then, draw one milliliter of blood broth mixture into a sterile syringe equipped with a 21-gauge needle. Dispense a big drop of the mixture onto the surface of a plated media.

After streaking the plates, incubate the cultures at 37 degrees Celsius for 18 to 24 hours. Examine the plates for isolated colonies inside the biosafety cabinet. Next, dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle.

Now pick up three to five morphologically similar colonies with a sterile straight inoculation wire. Transfer the inoculum into the first tube. Use saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63 McFarland.

Then, place a capillary attachment to the gram-negative aMIAST identification card in the first tube. Position the selected cards on the cassette. Now, load the cassette into its position in the filler chamber with the sample barcode facing inward.

Close the door and press Fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now, transfer the cassette from the filling chamber into the loading chamber.

Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The standard inoculum protocol identified 105 out of 204 tested isolates as RAST reportable gram-negatives, with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex being the primary organisms identified.