Exercise 49: Bacteria of the Gastrointestinal Tract

This exercise deals with how to distinguish between normal flora and bacterial pathogens in the GI tract. Most bacterial normal flora (coliforms) are lactose positive; most pathogens are lactose negative. Major GI tract (enteric) pathogens are found in the genera Proteus, Salmonella and Shigella. These can be separated from coliforms by using various selective and differential media.

Each student will receive an unknown culture containing E. coli and a nonpathogenic species of Proteus, Salmonella or Shigella. By comparing results for the unknown organisms with results for known organisms on demonstration media, students will be able to identify the lactose negative organism after they isolate it.

The selective and/or differential media available are:

1. Hektoen agar plates (selective and differential)
• Inhibitory agent: bile salts (inhibit gram positive organisms)
• Differentiate on the basis of lactose fermentation and H2S production
• pH indicator: bromthymol blue (green when basic, yellow when acidic)
• Lactose +: yellow/orange colonies with orange precipitate in medium
• Lactose -: green/greenish blue colonies (w/ black centers if H2S +)
• H2S indicator: ferric citrate
2. MacConkey’s agar plates (selective and differential)
• Inhibitory agent: bile salts (inhibit gram positive organisms)
• Differentiate on the basis of lactose fermentation
• pH indicator: neutral red (brownish/grayish when basic, hot pink when acidic)
• Lactose +: hot pink colonies with hot pink precipitate in medium
• Lactose -: grayish colonies
3. Simmon’s citrate slants (differential)
• Differentiates on the basis of utilization of citrate as sole carbon source (also, ammonium salts are used as sole nitrogen source)
• Ammonia produced when ammonium salts are metabolized causes pH change to alkaline
• pH indicator: bromthymol blue
• Citrate +: bright royal blue slant
• Citrate –: no change in green color of slant
4. Urea slant (differential) (see previous lab notes)
• Urea is hydrolyzed by urease to produce ammonia (alkaline) and carbon dioxide
• urease +: hot pink throughout at 24 hours (Proteus is positive)
• urease - : yellow to orange (Salmonella and Shigella are negative)
5. TSI (triple sugar iron) agar
• pH indicator: phenol red K = alkaline (red); A= acid (yellow), G = gas
• slant/butt possibilities: K/A, A/A, K/AG, A/AG, K/K with or without H2S
• K/A or K/AG indicates lac-, suc-, glu+
• A/A or A/AG indicates lac+, suc+, glu+
• K/K indicates lac-, suc-, glu-
• (The small amount of glucose in the medium is oxidized at the surface of the slant so that an organism that only ferments glucose turns the butt acid, but the slant reverts to alkaline. When all three sugars are fermented, there is enough acid produced to overcome this effect, so that the slant remains acidic.)
• H2S production: positive reaction is indicated by blackening in the butt
• gas production: bubbles or cracks in the agar indicated gas production

Procedure:

• Day 1: Each student takes one unknown culture. Streak one of each plated medium for isolation. Incubate 24 hours at 37^oC.
• Day 2: Identify lactose negative colonies. Using a well-isolated colony, streak a Hektoen plate for isolation and to make sure you have a pure culture of your unknown. Incubate 24 hours at 37^oC. If there are no well-isolated lactose negative colonies, streak another plate for isolation and consult your TA.
• Day 3: Find a well isolated colony on the second Hektoen plate and use it to inoculate urea, citrate and TSI slants.
• Day 4: Record results. Compare results to those of known cultures. Complete your worksheet.