Objectives: During this laboratory, you will:

1. Complete identification of Unknown No. 2.
   
2. Perform the initial steps to identify the bacteria in Unknown No. 3.

1. Laboratory 6 Continuation and Completion of Unknown No. 2

• Record the results from biochemical tests set up in Lab 6
• Inoculate additional media and/or perform additional tests to complete your identification of your Unknown No. 2.
• When identified, turn in identification sheet (page 6-8).

B. Unknown No. 3

• Gram stain isolated colonies from plates to verify the gram stain recorded in Lab 6
• Use a commercial identification kit for identification of gram-negative organisms
• Use appropriate media and reagents for the identification of gram-positive organisms

Student work: Day 2 of Unknown No. 3

1. Observe your plate(s) and confirm that growth on selective media reflects the results recorded for the Gram stain of material from your unknown in Laboratory #6. If not, consult an instructor.
2. Perform tests appropriate for identification of each organism based on your Gram stain and culture characteristics observed on the CNA and Mac plates streaked from Laboratory 6. Note: Tests require isolated colonies; therefore, if your plates do not contain isolated colonies, restreak for isolation and perform the indicated tests in the next laboratory.
3. Gram-positive organisms should be identified using the information learned in Lab2.
4. Gram-negative organisms can be identified using a commercial identification kit.

The Crystal Enteric Identification System is a miniaturized system of tests for identification of enteric organisms. These tests are modifications of classical methods that measure metabolic endproducts and enzyme activities. These tests include tests for fermentation, oxidation, degradation, and hydrolysis of various substrates. There are also chromogen-linked substrates to detect enzymes that microbes use to metabolize various substrates.

After inoculation with an isolated colony from a clinical specimen (you will use an isolated colony from your Unknown No. 3), the system is incubated for 18-20 hours at 37^o C. The system is then examined for color changes for each substrate included. Color changes result from metabolic activities of the microbe. The resulting pattern of the 30 reactions in the system is converted into a ten digit profile number that is used as the basis for identification using a code book of profiles. Proceed as follows using Unknown No. 3:

1. Remove Crystal lids from pouch, and discard the dust cover and desiccant.

2. Place one (no more) loop-full of Unknown No. 3 into a tube of Enteric/Stool ID Inoculum Fluid; then recap tube and mix well for 15-20 seconds to evenly suspend bacterial cells.

3. Mark Crystal Base with your name, date, and the code of your Unknown No. 3.

4. Pour the entire content of inoculum into the target area (reservoir) of the base.

5. Distribute the inoculum by holding the base in both hands; tilt the base to roll the inoculum gently along the tracks until all of the wells are filled. Roll back any excess fluid to the target area and place the base on the bench. Place cotton over target to absorb fluid.

6. Align the Crystal lid so that the labeled end of the lid is on top of the target area of the base.

7. Push the lid down until a slight resistance is felt; then, place thumbs on edges of the lid towards the middle of the panel and push downwards until the lid snaps into place (Listen—2 clicks).

8. Place your Crystal Enteric Identification System in the container as per instructor's direction.