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Unknown Identification Part II

Stock Cultures

Background and Introduction

Once a pure bacterial isolate (single colony) has been obtained, it is important to store the culture under appropriate conditions to maintain it in a viable state for several months.

To set up a reserve stock culture, a deep stab tube is inoculated, incubated, and stored in the refrigerator. To set up a working stock, a slant tube is inoculated, incubated, and refrigerated when not in use. The working stock is used for further tests, and the reserve stock is stored as a source of viable organisms if needed.

The goal of today’s lab is to prepare reserve stocks and working stocks of each of the two unknown bacterial isolates (A# and B#). To avoid accidentally working with a contaminant, both reserve and working stocks will be prepared using a single isolated colony. The instructions below describe how to use only one colony as the source of inoculum for both stocks and as the source of bacteria for a Gram stain.

It is critical to do this work aseptically and not to confuse the A and B cultures. Label all tubes correctly and read the labels before doing anything. Because it has been several days since the cultures were streaked, it is possible that some cells will not stain as expected due to changes in the cell walls. However, the size, shape, and arrangement of the cells should be similar to the first slide.

Procedure

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