Transcript Red slant with yellow butt

Single media and multiply test
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.
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• 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 one-fourth 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 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. 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.
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• 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 by-products (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.
• 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.
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• The fermentation of the sugars causes the anaerobic butt to turn
yellow and stay yellow. However, if only glucose is used, even
though the butt 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
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. 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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The End
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