Lecture Notes on TSI, IMViC, Selective and Differential
Media, Enterobacteriacea, MIO, LIA, Oxidase Test
THE INTESTINAL BACTERIA
Many intestinal bacteria are classified in the tribe Enterobacteriaceae.
These are gram negative rods which are able to ferment glucose.
Enterobacteriacieae which are also capable of fermenting lactose are
called coliforms. Coliforms are generally considered to be opportunistic
pathogens and non-pathogens, however there are many instances of severe,
life-threatening infections caused by coliforms. Coliforms are very important
as INDICATORS OF FECAL CONTAMINATION. Some common coliforms are:
Recently a number of pathogenic strains of E. coli have been
A number of non-coliform Enterobacteriaciae are important pathogens. Since
these non-coliforms are LACTOSE NEGATIVE, lactose fermentation tests are
important in trying to identify them.
Salmonella typhi causes TYPHOID FEVER, a severe systemic
infection which begins several weeks after ingesting the organisms. The
disease is characterized by high fever, headache, a rash on the abdomen
(rose spots), constipation and/or diarrhea and organisms in the stool,
blood and urine.
Other Salmonellae cause a less severe gastroenteritis commonly called
Salmonella "food poisoning" although this is more accurately called a "food
Shigella dysentariae causes bacillary dysentery. In this
disease, frequent bloody, mucoid watery stools are passed. The organism
invades and kills the intesinal epithelium. This causes white cells to
accumulate and these can be seen in the stool upon microscopic examination.
Yersinia enterocolitica causes diarrheal disease similar
to Shigella. This organism is frequently associated with pigs and therefore
undercooked pork is associated with infections.
Other lactose negative Enterobacteriaceae which are not classified as pathogens
include Proteus sp. and Serratia marcescens.
There are a number of non-Enterobacteriaceae which are important intestinal
Vibrio cholerae causes cholera.
Vibrio parahemolyticus is a marine organism which frequntly
contaminates seafood. It causes an acute diarrheal disease 24-48 hours
after ingestion of raw or incompletely cooked shellfish and less frequently,
Partially cooked poultry can be the source of Campylobacter jejuni.
This organism can cause diarrhea, often with blood and pus, 1 to 7 days
Helicobacter pylori, an organism believed to be associated
only with humans, is now known to be the cause of gastric ulcers
LABORATORY METHODS FOR THE INTESTINAL BACTERIA
A number of selective isolation techniques are used for fecal organisms.
SELENITE BROTH is used mainly to enrich for Salmonella
and to some extent Shigella. This broth is called an enrichment
broth because it suppresses the growth of most intestinal organisms while
allowing the growth of the pathogens thereby increasing the chances of
finding the pathogens.
Plating media such as:
Salmonella-Shigella (SS) agar,
Eosin Methylene Blue (EMB) agar
MacConkey (MAC) agar
contain a variety of dyes and bile salts to allow the selective growth
of the gram negative enteric organisms. These media also contain sugars
(such as lactose) and pH indicators to allow one to distinguish between
different organisms based on their metabolic abilities. Hence lactose fermentors
will appear different from other organisms which are unable to ferment
TRIPLE SUGAR IRON (TSI) agar is used to help identify the biochemistry
of the Enterobacteriaceae. This medium contains three sugars and a pH indicator:
On EMB, Lactose (+) organisms appear purple red, sometimes with a metalic
green sheen. Lactose (-) organisms appear light colored and transparent.
On MAC, Lactose (+) organisms are purple-red to brick-red often with a
zone of precipitate surrounding the colonies. Lactose (-) organisms can
be light pink to white and they are often transparent.
SS agar is highly selective for Salmonella and Shigella and these form
colorless colonies often with black centers indicative of H2S production.
Most other organisms don't grow on this medium. If they do they will be
This medium also contains iron salts which will react with any H2S
produced by the growing organism to form a black precipitate.
An organism capable of fermenting only glucose and neither of the other
two sugars would form only a very small amount of acid (why?).
Whereas lactose and / or sucrose fermenters will form relatively large
amounts of acid.
There are three possible sugar reaction interpretations possible on
Alkaline / Alkaline (Red/Red) = No sugars fermented
Alkaline / Acid (Red/Yellow) = Only glucose fermented
Acid / Acid (Yellow/Yellow) = Lactose and/or sucrose fermentation occured
in addition to glucose fermentation.
Notice that an Acid / Alkaline reaction is not an option. (Why?)
The INDOLE test measures the ability of the microorganism to degrade
tryptophan into indole, ammonia and pyruvic acid:
The METHYL RED - VOGUES PROSKAUER test measures the types of products
produced in the fermentation of glucose.
The MR test determines if acid products such as lactic and formic acids
The VP test determines if neutral products such as ethyl alcohol, acetoin
and butanediol are formed.
The MR test
uses methyl red as a pH indicator which turns red at acidic pH, hence red
is a positive MR test.
Acetoin reacts with alpha-napthol under alkaline conditions to form a pink
or red product which takes several minutes to form.
The CITRATE test determines whether an organism is capable of using
citrate as a sole carbon and energy source. Such organisms will produce
alkaline end products in the presence of oxygen and these can be measured
with a pH indicator. Bromthymol blue is green at neutral pH and blue at
alkaline pH, therefore blue represents a positive test.
LYSINE IRON AGAR
This medium allows you to test
The possible color reactions after 24-48 hours of incubation are:
(1) if an organism can decarboxylate lysine;
(2) if an organism can deaminate lysine; and
(3) if an organism can produce H2S.
If the organism is producing hydrogen sulfide a black precipitate will
be visible. H2S production is usually not as strong in this
medium as it is with TSI.
Alkaline /Alkaline (purple/purple) = lysine decarboxylase positive
Alkaline / Acid (purple/yellow) = lysine not broken down = negative test
Red pigment / Acid (red/yellow) = lysine deaminase positive
MOTILITY INDOLE ORNITHINE
This medium allows you to
Hazy growth or growth spreading away from the line of inoculation indicates
(1) determine if the organism is motile;
(2) see if the organism can decarboxylate ornithine; and
(3) see if the organism can break down tryptophan to produce indole.
The decarboxyation of ornithine will cause the medium to become alkaline,
The indole test can be performed on this medium by dripping several drops
of Kovac's reagent onto the top of the medium. A bright red color is positive
purple color throughout the tube = positive for ornithine decarboxylase
yellow color in the bottom or throughout = negative for ornithine decarboxylase
The oxidase test will determine if the organism possesses cytochrome
c, one of the electron carrier proteins found in the electron transport
chain. (Even though many bacteria are capable of respiration and therefore
have an electron transport chain, not all bacteria have the same cytochrome
proteins in the chain. For instance, E. coli and the other Enterobacteriaceae
lack cytochrome c but have cytochrome o and cytochrome d instead. On the
other hand, Pseudomonas aeruginosa, a number of other organisms as well
as our mitochondria use cytochrome c in aerobic respiration.)
The test uses a compound called tetramethyl-p-phenylenediamine which
is clear in the reduced (electron rich) state and purple in the oxidized
(electron poor) state. In the presence of an organism with cytochrome c,
the reduced tetramethyl-p-phenylenediamine will donate its electrons to
the cytochrome c and become purple colored.
©2008 by John M. Berestecky
Wet a small piece of white paper towel or filter paper with the
Use a wooden applicator stick to take some growth from TSA or blood
agar and then smear this onto the wetted paper.
An immediate purple reaction is positive for oxidase
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