Biochemical testing (gram negative)

Triple Sugar Iron Test (TSI)

Definition

It is a type of test that contains 3 sugars (Lactose, Sucrose, and Glucose) and also iron. It also
contains Agar as solidifying agent (TSI is a semi-solid media having slant and butt).

Composition:

Composition Description
0.1 % glucose If fermented, it produces acid that turns
the butt in test tube a yellow in color.
1% Sucrose If both fermented, it produces a large
1% Lactose amount of acid that turns the butt and
slant both yellow in color.
Iron: ferrous sulfate Indicator of H2S formation
Phenol Red Indicator of acid production (it is yellow in
acidic condition while red in alkalic
condition)
Peptone It acts as a source of nitrogen (for
production of ammonia)
Agar It acts as the solidifying agent

Uses:

 To differentiate among the different groups of Enterobacteriaceae based on their ability

to ferment glucose, lactose and/or sucrose.
 It differentiates between groups capable of reducing sulfur to hydrogen sulfide (Sodium
Thiosulfate -> Hydrogen sulfide)

Materials:
 TSIA slant
 Given sample of bacterial cultures (E.coli, salmonella typhi, shigella sonnei)
 Inoculating wire
 Burner
 Incubator
Procedure:

1. Take 5-7 ml of the autoclaved TSIA media in a sterile test tube and slant was made by tilting
the media till it solidified.
2. Using a flamed inoculating loop, pick a colony from the bacterial culture.
3. Inoculate the organism in TSIA slant by stabbing in the butt and then with the same loop
streaking on the whole slant surface of the medium
4. Incubate at 37°C for 18-24 hours.
5. Observe for the color change in the slant and butt, gas production and H2S production.

Interpretation/results:

Red slant/Red butt = no fermentation (If neither lactose/nor sucrose is not fermented both slant
and butt will be red)

Red slant/Yellow butt = only glucose fermentation (If lactose is not fermented, the butt will be
yellow since it is oxygen-deficient but it contains more glucose than the slant. but on the slant
the acid (less acid as media in slant is very less) will be oxidized to carbon dioxide and water by
the organism and the slant will be red (alkaline or neutral pH).
Yellow slant/yellow butt = lactose and/or sucrose fermentation (large amount of acid is
produced)

Black slant/ black butt = if H2S is produced, the black color of ferrous sulfide is seen.

Yellow slant/yellow butt with cracks/bubbles = it indicates production of gas.

Notes:

-A/A(Acidic slant/acidic butt) -K/AH2S(ferrous sulfide is seen)

-K/A(Alkaline slant/acidic butt) -K/K(Alkaline slant/alkaline butt)

IMVIC Test

Definition:

It is a four type of test which consists of:

I- Indole Test: process that break down the amino acid

M- Methyl Red: Glucose Oxidation occurs

V- Voges-Proskauer: Production of neural end products

C- Citrate: Citrate Fermentation

Types of IMVIC test:

Indole test

Definition

Is a type of test that determines the bacterial species to detect the ability of the organism to
degrade the amino acid (tryptophan) to produce indole. This test is done on sulfide-Indole-
Motility. The bacteria with the tryptophanase will cleave to the tryptophan which produces
indole, pyruvic acid and ammonia.

The production of indole can be identified by Kovac’s or Erhling’s agent which is composed of 4
(p)-dimethylamino benzaldehyde, that reacts with indole that gives a red color compound as a
result.

Uses:

 used as an aid in the identification and differentiation of gram-positive and gram-negative

organisms
 it is used for screening for the ability of an organism to break down the amino acid
tryptophan and produce indole

Procedure:

Two methods are in use:

1. a conventional tube method in which it requires an overnight incubation, It identifies weak

indole producing organisms and
 2. a spot indole test, which detects rapid indole producing organisms

Materials:

Incubator
Inoculating loop
Sterilized tubes containing 4ml of broth culture
Bacterial samples from culture ( E.coli, P.vulgaris, E. aerogenes K. pneumoniae)

Reagent:

Kovács reagent (3, 5):

Ingredient Amount
Amyl or isoamyl alcohol, reagent grade (butyl alcohol may be substituted) 150.0 ml
p-dimethylaminobenzaldehyde (DMAB) 10.0 g
HCl (concentrated) 50.0 ml
Procedure:

Dissolve DMAB in the alcohol. Gentle heating might be required to get the aldehyde into solution.

Tryptone broth :

Ingredients
Tryptone=10gm
Sodium chloride = 5gm

Procedure:

a. Dissolve the ingredients in 1 liter of sterile water.

b. Dispense 4 ml per tube.
c. Cap tube and autoclave at 121°C under 15 psi pressure for 15 minutes.
d. Store the tubes in the refrigerator at 4 to 10°C.

I. Procedure for conventional tube method

a. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test
organism in tryptophan broth.
 b. Incubate at 37°C for 24-28 hours in ambient air.
c. Add 0.5 ml of Kovac’s reagent to the broth culture.
d. Observe for the presence or absence of ring

Interpretation/Results:

Positive result= A cherry band forms on the surface of media upon the adding of kovac’s reagent.

Example: Escherichia coli

Negative result= No any change of color occurred upon adding a kovac’s reagent

Example: Klebsiella pneumoniae

II. Procedure for spot indole test

a. Place several drops of Indole Spot Reagent on a piece of filter paper.
b. With an inoculating loop or wooden applicator stick, pick a portion of an 18-24-hour
isolated colony from a non-selective media and rub it onto the reagent saturated area of
the filter paper.
c. Examine immediately
Interpretation/Results:
Positive reaction: a blue color can be seen within 3 mins.

Negative reaction: a pink color can be seen within 3 minutes.

Lysine iron agar

Definition:

It is a type of test for the ability to deaminate lysine or decarboxylate lysine. Lysine iron agar
contains the ff: lysine, peptones, a small amount of glucose, ferric ammonium citrate, and sodium
thiosulfate. Deamination (reddish color) or decarboxylation (remains color) of lysine is the
primary function of the test.

Composition:

A liter of lysine iron agar contains 13.5g of the gelling agent agar, as well as the
nutrients lysine (10g), pancreatic digest of gelatin (5g), yeast extract (3g), glucose (1g), ferric
ammonium citrate (0.5g), and sodium thiosulfate pentahydrate (40mg). Additionally, 20mg of
the indicator bromcresol purple is added.

Uses:

 The test is particularly useful for distinguishing different Gram-negative bacilli—

especially among the Enterobacteriaceae.
  It is very useful for the rapid differentiation of Salmonella arizonae from Citrobacter and
Proteus spp.
 Can detect H2S production

Materials:

Inoculating needle incubator

Tube with culture media (lysine iron agar)

1. The medium is tubed, sterilized and slanted so that a short slant and deep butt are
formed.
2. With a straight inoculating needle, inoculate LIA by twice stabbing through the center of
the medium to the bottom of the tube and then streaking the slant.
3. Cap the tube tightly and incubate at 35°-37°C in ambient air for 18 to 24 hours.
4. Examine at 18 – 24 and 40 – 48 hours for growth and color changes in tube butt and slant,
and for blackening at the apex of slant.

Results:

Left side to right:

K/K- positive decarboxylation without H2S R/Y- negative decarboxylation, positive
deamination without H2S K/AH2S- negative decarboxylation with H2S

K/KH2S- positive decarboxylation with H2S

Lysine deamination (detected on slant)

Positive test: purple slant/purple butt (alkaline), the butt reaction maybe masked by H2S
production.

Negative test: purple slant/purple yellow (acid), fermentation of glucose occurs only

Lysine decarboxylation (detected on butt)

 Positive Test: Red slant

 Negative Test: Slant remains purple
H2S Production:

 Positive Test: Black precipitate

 Negative Test: No black color development
 Gas production: the presence of bubbles or cracks in the medium
Biochemical Test

These are the test used for identification of bacteria species which is based on difference in the
biochemical activities of different bacteria. These differences are in carbohydrate metabolism,
protein metabolism, fat metabolism, production of certain enzymes, ability to utilize a particular
compound etc.

References:

http://microbesinfo.com/2013/05/triple-sugar-iron-agar-tsi-test/

https://microbiologyinfo.com/lysine-iron-agar-slants-test/

https://microbeonline.com/triple-sugar-iron-agar-tsi-principle-procedure-and-interpretation/
https://microbiologyinfo.com/indole-test-principle-reagents-procedure-result-interpretation-
and-limitations/

http://www.yourarticlelibrary.com/experiments/importance-of-biochemical-tests-of-
bacteria/26624