8.4: Lab Procedures- Enterotube inoculation

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Learning Outcome

Learn how to inoculate an enterotube

Enterotube Inoculation Procedure:

1. Take one enterotube, record your initials, and unknown number on the label.

2. Remove both caps. The tip of the inoculating wire is under the white cap, it is already sterile. Do not heat sterilize this wire. Using your NA plate of your unknown culture, pick a well isolated colony directly with the tip of the enterotube inoculating wire (Figure 1). A visible amount of inoculum should be seen at the tip and side of the wire. Avoid touching agar with wire.

3. Inoculate enterotube by first twisting wire, then withdrawing wire through all 12 compartments applying a turning motion (Figure 2). Reinsert wire (without sterilizing) into enterotube, using a turning motion through all 12 compartments, until the notch of the wire is aligned with the opening of the tube (Figure 3). The tip of the wire should be seen in the citrate compartment. Break wire at the notch by bending. The portion of the wire remaining in the tube maintains anaerobic conditions necessary for the true fermentation of glucose, production of gas, and decarboxylation of lysine and ornithine.

4. With the broken off part of the wire, punch holes through the foil covering the air inlets of the last 8 compartments (adonitol, lactose, arabinose, sorbitol, Voges-Proskauer, dulcitol/PA, urea, and citrate) in order to support aerobic growth in these compartments (Figure 4). Replace both caps.

5. Incubate at 30ºC for 18-24hrs with enterotube lying on its flat surface or in an upright position. Allow air circulation between incubated tubes.

6. Interpret and record all reactions with exception of Voges-Proskauer. All other tests must be read before the Voges-Proskauer test is performed as the reagents added for these tests may alter the remainder of the enterotube reactions. The Dulcitol/PA and H₂S/Indole compartment results will not be used since they are not reliable if read after 2 days. After all reactions are recorded, follow directions below for completion of VP test.

Voges-Proskauer Test Reagent Addition:

1. Place enterotube horizontally on the bench with glucose compartment pointing downward. By using a toothpick, punch a hole about 3-4mm in diameter in the plastic film above the VP chamber.

2. Add two drops of 40% KOH solution and three drops of 5% alpha-naphthol. A positive test is indicated by the development of a red color within 20 minutes. Do not wait longer than 20 minutes before reading the results!

Watch this video 1 on Enterotube Inoculation

Watch Video 1: Enterotube inoculation performed in our Microbiology laboratories at NC State. URL:https://youtu.be/3pmaDdZPLJg