BIOCHEMICAL TESTS FOR BACTERIAL TRIPLE SUGAR IRON AGAR (TSI)

IDENTIFICATION •orange red medium with a slant and a butt

Stab = butt
BIOCHEMICAL TESTS Streak = slant
 Provide additional information for the •a composite media used to study different
identification of the bacterium properties of a bacterium:
 Primary involved – enzyme ✓sugar fermentation
Produced by certain bacteria ✓gas production
 BAP and CAP = culture media for ✓H2S production
biochemical tests •Differential medium
 Include: •In addition to peptone, yeast extract &
•Oxidase test agar, it contains 3 sugars: Glucose, Lactose,
•Triple sugar iron agar (TSI) Sucrose
•Methyl Red •Ferrous ammonium sulfate = indicator for
•Vogues Proskauer H2S production
•Indole test •Indicator: Phenol red (acid=yellow;
•Citrate utilization alkaline=red
•Urease test
TSI REACTIONS
OXIDASE TEST ❑CHO Fermentation
•detects the presence of enzyme “oxidase” acid (A)= yellow
produced by certain bacteria which will alkaline (K) = red
reduce the dye (tetramethyl-p-phenylene
diamine dihydrochloride) •A/A = 3 sugars were fermented (Glucose,
Lactose, Sucrose) ALL YELLOW
Positive rxn= purple color •K/A = only glucose was fermented (red
Oxidase (+) - Pseudomonas, Vibrio, slant, yellow butt)
Neisseria •A/K = only lactose and sucrose were
Oxidase (-) - Salmonella, Shigella fermented
•K/K = no sugar was fermented (red slant,
•Enzyme is expressed and contained by red butt)
microorganisms
•Sub straights for reaction = culture media ❑H2S production
•Gram negative bacilli w purple reaction = •(+) Black color
pseudomonas, vibrio Blackening or black precipitate
•Differentiate pseudomonas from vibrio =
morphology = green metallic sheen ❑Gas production
(aeruginosa and e coli) •Gas bubbles or crack in the medium
•Single out most likely present
•Red slant, red butt = no gas, no h2s
•Red slant, yellow butt = no gas, no h2s K/A
•Yellow slant, yellow butt + space =gas + no
h2s
•Yellow slant, yellow butt (blackening)=no
gas + h2s
•Red slant, yellow butt (blackening +
space)=gas + h2s
INDOLE TEST
•Used to detect indole production by the
organism
•Tryptophanase produced by organisms
-Break down tryptophan to indole
•After overnight incubation, 5 drops of
indole reagent (Kovac’s reagent) is added
•Positive result is indicated by a pink ring
Indole positive –E.coli
Indole negative –Klebsiella, Salmonella

Urine sample >> Check on gram stain –

gram-negative bacilli
•Culture on BAP and MAC
•MacConkey – pink colonies
Eliminate salmonella – non lactose
fermenting

METHYL RED
•Detects the production of stable acids
(lactic acid, acetic acid or formic acid)
during fermentation of glucose

Media: MRVP broth (pH 6.9), Clark and

Lub’sbroth
•buffered peptone
•glucose
IMViC TEST •dipotassium phosphate
•Indole
•Methyl Red •Positive Reaction: distinct red color
•Voges Proskauer eg.E. coli, Yersinia
•Citrate Utilization
•Negative Reaction: yellow color
eg.Enterobacter aerogenes, Klebsiella
pneumoniae
❑Procedure:
1. Prior to inoculation, allow medium to
equilibrate to room temperature.
2. Using organisms taken from an 18-24
hour pure culture, lightly inoculate the
medium.
3. Incubate aerobically at 37 degrees C. for
24 hours.
a.Place inoculated media in incubator
b.Aerobically – outside candle jar
4. Following 24 hours of incubation,
aliquot 1ml of the broth to a clean test
tube.
5. Reincubatethe remaining broth for an
additional 24 hours.
6. Add 2 to 3 drops of methyl red indicator
to aliquot.
7. Observe for red color immediately
VOGES-PROSKAUER (VP) TEST
•used to determine if an organism
produces acetylmethyl carbinol (acetoin)
from glucose fermentation
•Principle:
Positive Reaction:pink-red color
eg.Viridansgroup streptococci (except
Streptococcus vestibularis),Listeria,
Enterobacter, Klebsiella, Serratia
marcescens, Hafnia alvei, Vibrio eltor,Vibrio
alginolyticus
•Negative Reaction:lack of a pink-red color
eg.Streptococcus mitis, Citrobacter sp.,
Shigella, Yersinia, Edwardsiella, Salmonella,
Vibrio furnissii, Vibrio fluvialis, Vibrio
vulnificus, and Vibrio parahaemolyticus
❑Procedure:
1. Prior to inoculation, allow medium to
equilibrate to room temperature.
2. Using organisms taken from an 18-24
hour pure culture, lightly inoculate the
medium.
3. Incubate aerobically at 37 degrees C. for
24 hours
Place inoculated media in incubator
Aerobically – outside candle jar
4. Following 24 hours of incubation, aliquot
2ml of the broth to a clean test tube.
5. Re-incubate the remaining broth for an
additional 24 hours.
6. Add 6 drops of 5% alpha-naphthol, and
mix well to aerate.
7. Add 2 drops of 40% KOH, and mix well to
aerate.
8. Observe for a pink-red color at the
surface within 30 min. Shake the tube
vigorously during the 30-min period.
CITRATE UTILIZATION
•Utilizes Simmon’sCitrate medium
•detects the ability of certain bacteria to
utilize citrate as the sole source of carbon
•Indicators: Sodium citrate and
bromothymol blue
•If citrate is utilized, alkali is produced
which turns the medium to blue.
Citrate positive –blue colour
Citrate negative –green colour
•Positive –Klebsiella
•Negative –E.coli
UREASE TEST
•Utilizes Christensen’s urease medium
•used to detect organisms that produce
urease
•Urease splits urea into ammonia and CO2
•Reactions:
Urease positive –pink color
Urease negative –yellow color
•Positive –Proteus, Klebsiella
•Negative –E.coli, Salmonella
Gram negative bacilli, MacConkey pink
colonies, yellow urease = e. coli
Uninoculated urea agar = orange color
LYSINE IRON AGAR (LIA) SLANT
•Contains lysine, glucose, ferric ammonium
citrate, and sodium thiosulfate,
bromcresolpurple (pH indicator)
•Primary Function:
-detect lysine deamination (aerobic; slant)
-decarboxylation (anaerobic; butt)
-H2S production = (+) black ppt in the butt
•Principles:
Bacteria + Lysine undergo decarboxylation
(BUTT) = Amine (Alkali) + Bromcresol Purple
= (+) purple butt, (-) yellow butt
Bacteria + Lysine undergo deamination
(SLANT) = Ammonia + Ferric Ammonium
Citrate = (+) dark red slant, (-) purple slant
•Purple slant, purple butt = (-) deamination,
(+) decarboxylation (-) h2s
•Purple slant, purple butt, blackening = (-)
deamination, (+) decarboxylation (+) h2s
•Purple slant, yellow butt = (-) deamination,
(-) decarboxylation (-) h2s
•Purple slant, yellow butt = (+)
deamination, (-) decarboxylation (-) h2s
PHENYLALANINE DEAMINASE (PAD) TEST
•Amino acids metabolized by deaminases
that remove an amine (NH2 ) group
•Deamination produces phenylpyruvicacid
•Add 10% ferric chloride
•Positive reaction = Green color
•Useful in differentiation of:
-Proteus
-Morganella
-Providencia
•Detects deaminase enzyme
MOTILITY TEST
•Observing growth in a semisolid medium
•Agar of 0.4% or less
•Stab single line into media
•Examine movement away from stab line
•Move from point of stab to other areas of
medium
NITRATE AND NITRITE REDUCTION TEST
 Test verification: Add zinc
(-) Red color = nitrate was not reduced
(+) No change = nitrate was reduced

A red color is observed as Nitrate Reagents

A and B react with nitrite produced from
nitrate produced from nitrate by nitrate
reductase or No change in color is when
Nitrate Reagents A and B are added
because nitrite produced from nitrate has
been further reduced.