H₂S Production, or Triple Sugar Iron (TSI) Agar (Atlas pgs.78-79)

TSI is a differential media that can detect fermentation and hydrogen sulfide production.    It is a rich medium containing a pH indicator, peptones, several sugars (testing for fermentation) and sulfur (testing for sulfur reduction that produces hydrogen sulfide gas).  It is poured to have both a slant (on the top) and a butt (on the bottom).  The slant is to allow for aerobic growth, the butt to allow anaerobic growth (or at least reduced oxygen).  It is a fairly complex test, having a number of combination results that are possible.  When fermentation occurs, acid products are made which will change the color of the media from orange to yellow.  If fermentation occurs with the production of gas, there will be cracks, breaks, or lifting of the agar in the tube.  If fermentation of the sugars does not occur, the bacteria may digest the peptones, releasing alkaline end products.  This will lower the pH and turn the medium red.  If sulfur is reduced, there will be a black precipitate, which especially is visible in the butt.  If the tube becomes largely black, it may not be possible to read the tube for fermentation.  If nothing happens (no change) the medium will stay orange.

One complication is that three sugars are present, and that glucose is present in limiting amounts.  For this reason, it is usually not possible to tell which sugar has been fermented, unless only glucose is fermented.  If glucose is the only sugar that is fermented, then the result may be a red slant/yellow butt because when the glucose runs out peptones may be digested which will turn the slant red, but the butt ferments glucose slower, so it remains yellow.

Its purpose is aid in the identification and differentiation of members of Enterobacteria (enterics) from other Gram- bacilli.

• Many combinations of results are possible with this test.
• Expectations:  be able to obtain results and interpretations if given a sample and this table.

1. Obtain a tube containing a slant with a butt.  A butt is where the agar goes straight across the bottom of the tube, when there is a slant above it.  Make sure that the butt is greater than a half an inch, otherwise there may not be anaerobic growth.
2. Observe what the media in the tube and record its color and appearance in your notebook.
3. Label the tube, include your name, the date, and either the bacteria sample or the test's name.
4. DDo NOT Use your Wire loop, as this will soon ruin it. Use your inoculating needle for this experiment.
5. Aseptically transfer bacteria to the tube.  This is done by flaming an inoculating needle (not the loop), cooling it well, picking up bacteria on the needle by rolling the tip in a colony, transferring some to the slant by streaking the slant in an S-like pattern.
6. Keep the needle uncontaminated and pick up some more bacteria and then stab the needle straight down into the butt, going nearly to the tube bottom. Pull the needle straight out the way you came in.  Do not push the needle against the bottom of the tube as this may distort it.  Make sure that the needle is cooled well as it is thin and heat can easily move down the wire.
7. Remind the instructor to set up the control tubes (uninoculated controls) which are essential for observing and comparing the results. 
8. Place your tubes in the rack for this experiment. 
9. Read the tubes for fermentation at 24 hours, read them for Hydrogen sulfide production at 48 hours (if the tubes are black, 24 hours will work).  For slow growing or slow fermenting organisms, up to twice the time may be needed to make observations.  (If your results are unclear, continue incubating them for up to twice the above times).

Results:

1. Look at what is happening aerobically (on the slant) and anaerobically (in the butt). Growth may occur in both locations.
2. Often holding the tubes up to a light will aid your observations.  Also roll the tube, because sometimes a color change is only near the surface and has to be seen at the correct angle. 
3. Look for the occurrence of fermentation (a yellow color), peptone digestion (red), no change (orange), H2S production (black), gas production (cracks, lifting) in both the slant and the butt, compare your tube to an uninoculated control.  
4. Check your results against the table, the results are complex.  Make your interpretations.  Use pictures in the Atlas as an aid.
5. Use the thioglycollate test to establish true anaerobic growth, but use the TSI test to confirm the oxygen requirements found in the thioglycollate test.  (For example, if you had growth only in the top of a thioglycollate tube, you should only see growth in the slant of a TSI tube.)