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
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
- Expectations: be able to obtain results and interpretations if
given a sample and this table.
|yellow slant/yellow butt
||aerobic and anaerobic fermentation of sucrose and/or lactose (and glucose)
|red slant/yellow butt
||aerobic: glucose fermented till it ran out then peptones
anaerobic fermentation of glucose (butt
ferments glucose slower)
|red slant/red butt
||aerobic and anaerobic: no fermentation, peptones used (not
|red slant/butt unchanged
||aerobic: no fermentation, peptones used
anaerobic: little or no growth (not an enteric)
|no change in slant & butt
||aerobic and anaerobic: no fermentation, bacteria is growing slowly if at all
|black precipitate, esp. in butt
||sulfur reduction has occurred (producing H2S)
|cracks, breaks or lifting in agar/font>
||gas production during fermentation
- 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
- Observe what the media in the tube and record its color and appearance in your
- Label the tube, include your name, the date, and either the bacteria
sample or the test's name.
- DDo NOT Use your Wire loop, as this will soon ruin it. Use your
inoculating needle for this experiment.
- 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.
- 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.
- Remind the instructor to set up the control tubes (uninoculated
controls) which are essential for observing and comparing the results.
- Place your tubes in the rack for this experiment.
- 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).
- Look at what is happening aerobically (on the slant) and anaerobically (in
the butt). Growth may occur in both locations.
- 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.
- 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
- Check your results against the table, the results are complex. Make
your interpretations. Use pictures in the Atlas as an aid.
- 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.)