Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

1. Preparation

1. Before beginning, wash hands thoroughly and put on appropriately sized gloves.
2. Sterilize work surface with 5% sodium hypochlorite (bleach) and dry thoroughly.
3. Place an inoculating loop in an empty 120 mL Erlenmeyer flask so that it does not touch the bench top while working.

2. Growth Media and Cultures

1. Gather four plates of Mannitol Salt Agar (MSA), Eosin Methylene blue agar (EMB), and eight tryptic soy agar (TSA) from the refrigerator (these can be purchased commercially).
2. Place plates onto the cleaned work area.
3. Gather the following broth cultures: Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Proteus vulgaris. Use caution: as these are aerobic organisms, the caps of the tubes should be slightly loose.
4. Place all cultures in a test tube rack on the cleaned work surface.

3. Transferring Cultures and Incubation

1. Stand or sit at the bench with all of the materials in reach.
2. To use aseptic technique to transfer the bacteria to the plate, first flame the loop until it is glowing orange - and then allow it to cool in the air.
3. While the loop cools, take the broth culture of E. coli, and then open the cap with using your pinky finger.
4. Flame the opening of the tube quickly. This prevents contamination.
5. Dip the loop into the tube and streak the organism onto the first quadrant of an EMB plate.
6. After the first streak is performed, flame the loop, and then perform a second streak crossing the first streak only once or twice. This will allow for single colony isolation and to determine if there is any contamination.
7. Repeat this until all four quadrants of the plate have been streaked.
8. Now, transfer each of the rest of the four organisms in the same streak plating manner so that the final product is one MSA plate, one EMB plate, and two separate TSA plates per organism.
9. Once all organisms have been transferred, flame the loop one final time and put it away.
10. Place 1 TSA plate with each organism into the gas packet, and then shake a gas sachet before placing it into the chamber alongside the plates. Seal tightly.
11. Incubate all plates at 37°C overnight.
12. Sterilize the work surface one final time with 5% bleach.

4. Reading and Recording Results

1. Remove the plates from the incubator and place them onto a clean lab bench
2. Take note of the growth as of the organisms on each plate in your lab notebook. In particular, take detailed notes on:
   1. Numbers and size of colonies (if present) on each plate, for each plated strain
   2. Appearance of the agar surrounding any colonies growing on the MSA plates
   3. Appearance of any colonies growing on the EMB plates
   4. Differences in growth between the TSA plates grown outside versus inside the gas package