Exercise 13: Differential Media Tubes

Introduction

The tube media used in this exercise do not contain any selective agents to inhibit certain types of organisms. They do contain indicators that will change colors based on the biochemical activity of the organism inoculated. Some of these test require that addition of a chemical reagent to produce the differential result. The media used in this exercise are: Triple Sugar Iron Agar (TSI), Sulfur Indole Motility Medium (SIM), Simmons Citrate Agar (CIT), and Phenylalanine Agar (PDT).

Triple Sugar Iron Agar: TSI is used to differentiate bacteria on the basis of their fermentation characteristics for glucose, lactose, and sucrose. Both the slant and butt sections of the media need to be inoculated for this test to work properly. Organisms that ferment either lactose or sucrose will produce acid (A, yellow) in both the slant and the butt. Those that ferment only glucose will produce an acid (A, yellow) butt while the slant is alkaline (K, pink). Those that do NOT ferment any carbohydrates will produce an alkaline (K, pink) slant and butt. Some organisms also produce CO2 gas as a fermentation product. This is detected as bubbles or cracks in the medium. The medium also contains ferrous sulfate for sulfur reduction as well. As before a black precipitate indicates a positive result for sulfur reduction.

SIM Medium: This medium allows for the determination of three bacterial characteristics in a single tube: sulfur reduction, indole production from tryptophan, and motility. Determining motility in SIM medium is dependent on the proper method of inoculation. Students must inoculate in a straight line (stab) using a needle. Motile organisms can move away from the inoculation line and are detected by the radiating growth extending outward in all directions from the central stab line. If the medium turns black, it is an indication of sulfur reduction and a positive test. The lack of a black precipitant in the medium indicates no sulfur reduction and a negative reaction. The presence of indole may be detected by adding a few drops of Kovac’s reagent to the tube. The reagent will immediately turn red if the reaction is indole positive. If the reagent retains its amber color, it is indole negative.

Citrate Utilization Test: The citrate test is a minimal medium used to differentiate members of the Enterobacteriaceae, all of which are facultative anaerobes. They have the ability to ferment carbohydrates in addition to aerobic respiration. Some can use citrate as their sole carbon source and perform citrate fermentation. Bacteria that can utilize the citrate also produce ammonia (NH₃) and ammonium hydroxide (NH₄OH), increasing the pH of the medium. Bromothymol blue dye changes from green to blue when the pH is >7.6. Conversion of the medium from green to blue is a positive citrate test result.

Phenylalanine Deaminase Test: Phenylalanine deaminase is an enzyme that can remove the amine group (NH₂) from the amino acid phenylalanine. A reagent containing ferric chloride (FeCl₃) must be added to the phenylalanine medium after incubation. Formation of dark green/black color indicates the presence of phenylalanine deaminase, while orange is negative.

Course Intended Outcomes

Describe different types of media according to their physical state, chemical composition and their function and list some of their applications; interpret microbial growth patterns.

Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Inoculation needle
• Test tube rack
• Additional materials and cultures will be assigned by your instructor

Procedure, Day 1

Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Use an inoculation needle for the differential tubes. Sterilize the needle the same as you would the loop. Make sure the entire length of the needle is sterile.
3. Roll the sterile inoculation needle across the surface of a culture to pick up the sample. DO NOT stab the media of the prepared cultures.
4. Proper inoculation technique is critical to producing good results. Each medium should be inoculated exactly as described. Also see figure 13.1.
5. For TSI: Stab the butt section of the tube, then use the side of the needle to streak the slant.
6. For SIM: Stab the semisolid medium from top to bottom in a straight line. Use a rapid motion and be careful to avoid any stirring motion with needle.
7. For CIT: Stab the butt section of the tube, then use the side of the needle to streak the slant.
8. For PDT: Use the side of the needle to streak the slant only.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Clearly label tubes using label tape with your group name or initials, organism name, and section day/time.
4. Place inoculated tubes in the rack labeled “to be incubated” on the back bench.

Procedure, Day 2

1. Collect your group’s tubes from the back bench area labeled “last week’s lab”.
2. Examine your tubes to determine results for each organism inoculated. Remember that you should have 4 tubes for each organism – one of each type.
3. If the organism grew, what was its reaction with the indicator in the media?
4. Remember to add the required reagents to observe the results in the SIM and PDT tubes. Add 3 drops of indole (Kovac’s) reagent to the SIM tube. Add 3 drops of ferric chloride reagent to the PDT tube.
5. Record your results in your lab notebook.
6. Examine tubes from other groups so that you can see results for all the organisms and representative positive and negative results for each media type.
7. Students can also observe prepared tubes at the front bench for representative positive and negative results.

Lab Clean-up

Return bacteria cultures to the front bench after inoculations are complete.

Remove labels from tubes and place in the tube rack ABOVE the biohazard container.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Triple Sugar Iron Agar (TSI)

Sulfur Indole Motility (SIM)

Citrate Utilization (CIT)

Phenylalanine Deaminase (PDT)

Critical Thinking Questions

Many organisms that can use citrate in metabolism still produce a negative result in the citrate test. Why?

If an organism can ONLY ferment glucose to produce acid, what will result should it produce in a TSI tube?

What type of molecule is phenylalanine and what cell products is it found in?

If a bacteria is positive for motility, what specialized cell structure(s) must it possess?