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Unknown Identification Part III

Background & Introduction

Today you will be performing several tests on your unknown by using your working stock from last lab. These will help you start narrowing down what your unknowns might be. Next lab we will start performing the definitive tests. Remember that the goal is to use tests that eliminate broad categories of organisms as possibilities first and then narrow down between similar groups using presumptive or definitive tests. This method prevents you from doing too many unnecessary tests while still being confident in your identification.

Gram-positive unknown: Culture A 

_____ • Perform a Gram stain using growth from the working slant. This should be done first! If there is any sign of contamination it will be necessary to inoculate a fresh stock before proceeding. 
_____ • Inoculate one tube of TSB using an inoculating loop.  
_____ • Inoculate one tube of Nitrate broth using an inoculating loop. 

_____ • Inoculate motility agar using a needle to make one stab down the center to the bottom of the tube. 

_____ • Perform the catalase test
_____ • If your unknown is a Gram-positive coccus, streak one plate of Blood Agar. T-streak using the entire plate; use the inoculating loop to make several stabs in a streak-free region of the agar. 
_____ • If your unknown is a Gram-positive rod, inoculate one NSM plate for a future spore stain T-streak the plate for isolation .  
_____ • If your unknown is a Gram-positive rod, perform an acid-fast stain 

Gram-negative Unknown B

________ Perform a Gram stain using growth from the working slant. This should be done first.

________ Inoculate 1 tube of TSB using an inoculating loop.

________ Inoculate one tube of nitrate broth using an inoculating loop.

________ Inoculate motility agar using a needle to make one stab down the center to the bottom of the tube.

________ Inoculate 1 mannitol, 1 glucose, and 1 lactose broth tube. Use an inoculating loop.

________ Inoculate one tube of MR-VP medium with an inoculating loop. This will need to be incubated for 2-5 days. Place rack labeled MR-VP.

________ Perform an Oxidase test.

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