General Microbiology Laboratory
Biochemical Tests
H2S Production
 In this lab we have chosen two bacteria that are H2S producers
and motile, and one bacterium that is nonmotile and does not
produce H2S.
 Salmonella is a facultatively anaerobic gram-negative rod that
occurs in humans, warm- and cold-blooded animals, food, and
the environment. It is H2S positive and motile.
 Proteus is a gram-negative facultatively anaerobic rod that
occurs in the intestines of both humans and a wide variety of
animals and polluted waters. It is motile and produces H2S.
 Klebsiella ( inflammation of the lungs) is a facultatively
anaerobic gram-negative rod that occurs in human feces,
clinical specimens, soil, water, fruits, and vegetables. It is
nonmotile and does not produce H2S.
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H2S Production in SIM
 The most convenient medium for testing for indole and/or hydrogen
sulfide production is SIM medium (SIM is an acronym for sulfide,
indole, and motility). the SIM medium contains peptones (Cysteine) and
sodium thiosulfate as substrates, and ferrous ammonium sulfate,
Fe(NH4)SO4, as the H2S indicator.
 Hydrogen sulfide is produced when amino acids containing sulfur
(Cysteine ) are metabolized by microorganisms.
 Once H2S is produced, it combines with the ferrous ammonium sulfate,
forming an insoluble, black ferrous sulfide precipitate that can be seen
along the line of the stab inoculation. If the organism is also motile, the
entire tube may turn black. This black line or tube indicates a positive
H2S reaction; absence of a black precipitate indicates a negative
reaction.
 SIM agar may also be used to detect the presence or absence of motility
in bacteria as well as indole production.
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Hydrogen Sulfide Production.
This is a tube semisolid
agar that can also be
used to demonstrate
bacterial motility.
It is inoculated by
stabbing the wire loop
(or preferably a straight
wire inoculating needle)
straight down the middle
of the agar to about onefourth the depth of the
medium and
withdrawing the wire
along the same path.
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H2S Production in SIM
 How to Perform Test: Stab SIM media with inoculating
needle.
 Property it tests for: This test is used to help differentiate
species of the family Enterobacteriaceae. This test is used to
determine the ability to reduce sulfur into H2S.
 Media and Reagents Used: SIM media contains the sulfur
containing amino acid, cysteine, sodium thiosulfate, & peptonized
iron or ferrous sulfate.
 Reading Results: H2S will react with
the iron or ferrous sulfate and produce a black precipitate. A positive
result has a black
or brown precipitate present and a negative
result has no black precipitate.
 Any blackening of the medium is considered
a positive test for H2S.
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Triple sugar Iron Agar (TSIA)
 Triple sugar iron agar tests for 3 things---sugar fermentation
(glucose/lactose/sucrose), CO2, and H2S. It is a good medium for
some bacteria, although you can run all of these tests in other
ways, such as running individual sugar discs, SIM for H2S, and
phenol red glucose for CO2. Gases are easy to spot. The H2S is
identified the same way as in SIM, but be sure that the black is
Inside of the medium.
 Carbon dioxide is identified by cracks and bubbles inside of the
medium, sometimes a few bubbles and sometimes enough to push
the slant up to the top.
 There are 3 possibilities of sugar reactions and different reactions
in different areas (butt vs. slant) of the medium, so the
physiology behind it is pretty complex. The outcome of sugar
use is always acid, so the pH indicator phenol red will turn
yellow---reported as A. No use of the sugar or alkaline byproducts (which is NO sugar use) from the other non-sugar
nutrients in the medium will cause the indicator to stay the same
color red/orange or maybe even change it to a red---reported as a
K.
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 The reactions in TSIA are reported as slant (A or K), butt (A or K), a circle
around the butt for CO2, and + for H2S. For example K/A +H2S = red
slant, yellow butt, with both CO2, and H2S.
 The fermentation of the sugars causes the anaerobic butt to turn yellow
and stay yellow. However, if only glucose is used, even though the slant
turns yellow only after a few hours it will revert to red because the protein
in the medium is broken down to alkaline products when the small amount
of glucose is used up. If lactose and/or sucrose are used, the large amount
of fermentation products neutralizes the basic products and the slant stays
yellow.
 Therefore,
• A/A = glucose and lactose and/or sucrose are used
• K/A = glucose alone
• K/K = no sugars used
 There is no way to get a A/K reaction when using a G-rod on this medium.
IF YOU do, it means that
 1) you did not inoculate correctly with a stab and streak
 2) you inoculated something other than a G- rod.
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Principle
The triple sugar iron (TSI) agar test is generally
used for the identification of enteric bacteria
(Enterobacteriaceae).
It is also used to distinguish the Enterobacteriaceae
from other gram-negative intestinal bacilli by their
ability to catabolized glucose, lactose, or sucrose, and
to liberate sulfides from ferrous ammonium sulfate or
sodium thiosulfate.
TSI agar slants contain a 1% concentration of lactose
and sucrose, and a 0.1% glucose concentration. The
pH indicator, phenol red, is also incorporated into the
medium to detect acid production from carbohydrate
fermentation
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SOME COMMON SUGAR REACTIONS IN TSIA
 NOTE: This is a stab and streak inoculation.
THIS MEDIUM IS RUN ON GRAM - RODS ONLY!
A/A
E. coli, Yersinia, Aeromonas, Vibrio
K/A
Salmonella, Edwardsiella, Shigella
K/K
nonfermenters such as
Pseudomonas and others
A/K
erroneously inoculated a Gram + or
some other weird thing
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 How to Perform Test: :
 1-Inoculate the medium using an inoculating NEEDLE. Stab the inoculum
down through the butt, then pull the needle out and streak up the slant ( Do
NOT take another inoculum to do the slant).
2-Incubate at 30 or 37 degrees C.
 Property it tests for:
 Identify the various sugar reactions in TSIA.
 Identify the presence of carbon dioxide and hydrogen sulfide gases.






Media and Reagents Used:
TSIA per unknown
Reading Results:
A/A = yellow throughout
K/A = red slant, yellow butt
K/K = red or red/orange throughout


carbon dioxide = bubbles or breaks in medium
black precipitate = hydrogen sulfide
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From left to right:
Uninoculated control
Red slant and red butt, no black color= no
fermentation of glucose, sucrose or lactose. No
Hydrogen sulfide produced
Red slant and black butt= no lactose or sucrose
fermentation, H2S has been produced
Red slant with yellow butt= no lactose or sucrose
fermentation, lactose is fermented, no H2S has
been produced
Yellow slant, yellow butt and black coloration=
Lactose, sucrose and glucose fermented, and H2S
has been produced
Yellow slant, yellow butt and lifting and/or cracking of
media, no black coloration= Lactose, sucrose and
glucose fermented, H2S has not been produced
but gas has been produced
Yellow slant, yellow butt and no lifting and/or cracking
of media, no black coloration= Lactose, sucrose
and glucose fermented, H2S has not been
produced nor has gas been produced
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Limitation of the procedure
(1) If screw-cap tubes are used, leave the caps loose after
inoculating the tubes to prevent excessive disruption of the
agar should large amounts of gas be produced during
incubation.
(2) Record the butt as acid production if the black color of FeS
masks the color in the butt.
(3) Hydrogen sulfide production may be evident on Kligler Iron
Agar
(4) Do not use an inoculating loop to inoculate a tube of Triple
Sugar Iron Agar. While stabbing the butt, mechanical splitting
of the medium occurs, causing a false positive result for gas
production
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End of lecture
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