MICROBIOLOGY

840-266-MI

OVERVIEW OF PRINCIPLES OF
BIOCHEMICAL TESTS FOR
BACTERIAL IDENTIFICATION

THE MICHENER INSTITUTE for Applied Health Sciences

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 DIVISION OF CONTINUING EDUCATION
MICROBIOLOGY
840-266-MI
OVERVIEW OF PRINCIPLES OF BIOCHEMICAL
TESTS FOR BACTERIAL IDENTIFICATION

Table of Contents

PAGE

OBJECTIVES 1

RESOURCE MATERIAL 5

CARBOHYDRATE-BASED TESTS 6
CARBOHYDRATE FERMENTATION AND GAS PRODUCTION 7
OXIDATIVEIFERMENTATIVE TEST (OFF) 8
METHYL RED TEST 10
VOGES-PROSKAUER TEST 11
LACTOSE FERMENTATION: THE ONPG TEST 11
PROTEIN-BASED TESTS 17
GELATIN LIQUEFACTION TESTS 17
DECARBOXYLASE TESTS 20
PHENYLANANINE DEAMINASE TEST 22
UREASE TEST 23
INDOLE TEST 24
IMViC 25
MISCELLANEOUS TESTS 29
NITRATE TEST 29
DEOXYRIBONUCLEASE TEST 30
TESTS FOR CARBON UTILIZATION 32
CITRATE TEST 32
MALONATE TEST 33
MOTILITY 33
CYTOCHROME OXIDASE TEST 34
CATALASE TEST 36
MULTITEST MEDIA 41
TRIPLE-SUGAR IRON MEDIUM (TSI TEST) 41
SULPHIDE INDOLE MOTILITY MEDIUM (SIM TEST) 46

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OBJECTIVES

The objective indicates what you should know, understand and be prepared to explain upon completion of
this module. The self assessment question and answers will enable you to judge your understanding of the
material.

Upon completion of this module, the student should be able to:

l. Name one example of each of the following substances found in biochemical tests:
a) A monosaccharide
b) A disaccharide
c) A polysaccharide
d) A sugar alcohol.

2. Name two types of enzymes able to attack amino acids and state whether the pH that results would be acid
or alkaline.

3. For each of the tests in this unit;

a) Briefly explain the theory or principle
b) List the substrate(s) present and the enzyme(s) being tested for
c) List the indicator(s) incorporated in the medium (if applicable)
d) List the reagents (s) added after incubation (if applicable)
e) Describe the inoculation procedure used
f) State the incubation time and temperatures
g) Describe the form in which the medium is used, e.g. liquid, semi-solid or solid; tube or plate
h) State if the medium or specific ingredients in the medium were sterilized by pressure steam sterilization
(the autoclave) or by filtration
i) When relevant to the explanation of the test principle, list media constituents and important concentrations
j) Describe a positive and a negative result
k) Describe correct reporting practices, using the correct formats
I) Describe the use of controls.

4. For the following indicators, give the colors in acid and alkaline conditions:
a) Phenol red
b) Neutral red
c) Methyl red
d) Bromcresol purple
e) Bromthymol blue.

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5 Describe the purpose for adding a Durham tube to a fluid medium.

RESOURCE MATERIAL

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MICROBIOLOGY
840-266-MI
OVERVIEW OF PRINCIPLES OF BIOCHEMICAL

TESTS FOR BACTERIAL IDENTIFICATION

INTRODUCTION

Although much microbial Identification is done today using commercial miniaturized or automated systems,
the majesty of these are based on the classical principles outlined in this module. Understanding these
principles is essential for troubleshoot and quality control m most commonly used systems.

Accompanying this module are 35 mm transparencies, which can be obtained from the TIMT library for ease
in studying and comparison, tests that are similar (such as carbohydrate based or protein-based tests) have
been grouped together. All the test results described can be obtained after 18-24 h of incubation at 35°C;
exceptions will be noted.

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CARBOHYDRATE-BASED TESTS

Carbohydrates, also known as sugars, are common substrates upon which bacterial enzymes act. There are
four classes of carbohydrates commonly used In biochemical tests:

Monosaccharides, which have one sugar unit; examples include:

-Five-carbon (pentose) sugars: arabinose, xylose
-Six-carbon (hexose) sugars: glucose (dextrose), fructose, galactose.

Disaccharides: two-sugar units; for example:

Polysaccharides (starch)

Sugar alcohols, e.g. mannitol or dulcitol.

When bacterial enzymes attack sugars, the resulting product(s) generally produce an acid pH. in order to
detect this acid (and, therefore, to say that a test result is positive), pH indicators are often included m the
formula of the medium (or are added after incubation). See module 880-028, Introduction to Bacterial
Physiology and Growth, for a discussion of indicators.

The carbohydrate-based tests to be discussed include:

 A simple test for carbohydrate fermentation and gas production
 The Oxidative/fermentative (O/F) test
 The Methyl red test
 The Voges-Proskauer test
 The ONPG test for lactose fermentation.

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CARBOHYDRATE FERMENTATION AND GAS PRODUCTION

Glucose (dextrose) IS the most frequently tested substrate, but other sugars can be substituted.
 The basal broth medium includes:
 Peptone -a nitrogen source
 Beef extract -nutrient source
 Sodium chloride
 Phenol red -a pH indicator
 Water

The base is sterilized in an autoclave. The sugars, which are filter-sterilized, are aseptically added afterwards
to achieve a final concentration of 0.05-0.10 (0.5-1%). In order to ensure that any acid produced is a
breakdown product of the specific sugar, it is critical that the peptones be free of any trace sugars. It is also
important that the sugar concentration be high enough so that enough acid is produced to neutralize alkali
produced through protein degradation.

Some bacteria not only produce acid from carbohydrate breakdown, but also gases such as carbon dioxide
and hydrogen gas. Organisms producing gas are termed aerogenic; those producing no gas are anaerogenic.
Gas can be detected by looking for a gas bubble caught m a small inverted test tube, called a Durham's tube,
Inserted inside the larger tube

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This Durham's tube is added to the culture tube of media before autoclaving. (Lactose, sucrose, salicin and
xylose are usually filter-sterilized, since heat may degrade the carbohydrates.) The steam generated during
sterilization pushes any air out of the Durham's tube, so that there is no air trapped m the tube prior to
inoculation. If a bubble IS seen after sterilization the tube of media should not be used.

An example of the use of gas production to separate two closely related organisms is Escherichia coli
(aerogenic) from Shigella sp. (generally anaerogenic.)

OXlDATIVE/FERMENTATlVE (OFF) TEST

Many pathways are used by bacteria to break down carbohydrates to obtain energy for cell metabolism.
These pathways can be placed into two main groupings:
 Aerobic pathways
 Anaerobic pathways

Aerobic reactions need oxygen to function and generally produce a lot of energy for the cell; these are
oxidative reactions. Anaerobic reactions, occurring in the absence of oxygen and producing much less energy,
are termed fermentative.

As a rule, facultative bacteria (able to grow aerobically and anaerobically) will use oxidative pathways,
reverting to fermentative ones when oxygen is absent. Strict aerobes use only aerobic pathways, whereas
strict anaerobes use only fermentative pathways. The Off test is used to determine whether an organism uses
carbohydrates Oxidative or representatively The most frequently used substrate is glucose, although others
may be used.

THE MEDIUM

The basal medium is semi-solid, containing a reduced peptone content plus an indicator, bromthymol blue.
After the bulk medium has been sterilized, the filter-sterilized sugar is added to the still-molten base and the
entire medium is dispensed into tubes, forming a butt.

THE TEST

Two tubes of off medium are stab-inoculated and one of the tubes is layered With 1 cm of sterile mineral oil.

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OXIDATIVE REACTION

If the organism inoculated produces acid m the "open" tube (the one without oil) only, then the organism is
said to attack the carbohydrate oxidatively. The indicator, bromthymol blue, turns from a neutral green to all
acid yellow in the top of the open tube. The closed tube (layered with mineral oil) shows no challenge. (The
acid remains at the top of the open tube, since It is prevented from diffusing by the semi-solid medium.) The
two tubes on the left are included as uninoculated controls.

FERMENTATIVE REACTION

If the organism attacks sugars fermentatively, acid will be produced m both open and closed tubes, turning
the indicator yellow

NO ACTION

Report the test as having no action If there is a neutral reaction in both tubes or an alkaline reaction in the
open tube.

Examples
 Oxidative: Pseudomonas aeruginosa
 Fermentative: all enterobacteriaceae
 No action: Alcaligenes sp.

METHYL RED TEST

The methyl red (MR) test is discussed with the VogesProskauer (VP) test (described following), since both
tests are done on a single tube of medium. The glucose substrate is incorporated into a broth containing:
 Peptone
 Buffer
 Glucose
 Water

Some bacteria break down glucose, producing a variety of acid end-products called mixed acids. The
production of these acids results in a very low pH -pH 4.2 or less. To detect this low pH after incubation, a
few drops of methyl red indicator is added to the medium.

The medium is split to perform both the MR and VP tests. To the portion used for the MR test add a few
drops of methyl red. .A positive methyl red (MR) test shows all immediate red colour in the medium; a
negative test, yellow

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VOGES-PROSKAUER TEST

Those organisms negative m the MR tests are usually positive m the VP test. Voges-Proskauer-positive (VP+)
organisms produce a neutral product called acetoin or acetylmethylcarbinol. Acetoin is detected by adding
alpha-naphthol ( a -naphthol) and KOH.The medium is split to perform both the MR and VP tests. To the half
used for the VP test, add about 5 drops of a -naphthol and 2 drops of KOH. Because oxygen speeds up the
reaction, the tube contents are mixed well after the reagents are added. A positive VP reaction may not occur
Immediately. Therefore; the tubes are left for 10 minutes and shaken periodically during that time to ensure
adequate aeration. A VP+ test is red at the top of the tube. A VP-test has no redness in the top layer.

Examples

LACTOSE FERMENTATION TESTS. THE ONPG TEST

In any medium containing both glucose and lactose, bacteria will attack glucose first and then attack the
lactose when the glucose is used up. In order for the cell to degrade lactose into glucose and galactose, the
bacterium must have two enzymes:

 Beta-galactoside permease allows lactose to enter the cell, where

 Beta-galactosidase hydrolyses the lactose into glucose and galactose

All lactose fermenters have both of these enzymes, but a group of bacteria called slow or late lactose
fermenters have, beta-galactosidase (and therefore can split lactose), but lack permease.

The orthomtrophenyl galactopyranoside (ONPG) test detects bacteria that are beta-galactosidase-positive but
permease negative.

The substrate, ONPG, is not only split by beta-galactosidase but It is also able to penetrate the bacterial cell
by passive diffusion because of Its small Size, thus eliminating the need for permease. When the colourless
ONPG is split, It produces two compounds (Figure 2), galactose and the yellow-coloured orthonitrophenol.
Therefore, If an organism is a lactose fomenter, It has beta-galactosidase and will, therefore, split the ONPG
molecule to produce orthonitrophenol.

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Both permease and beta-galactosidase are inducible enzymes, meaning that they are not always continuously
present in the cell. These enzymes must be induced by the presence of the substrate. This means, in order to
get a rapid ONPG test, it is best to inoculate organisms that have been previously grown on a medium
containing lactose such as MacConkey or TSI (triple-sugar Iron) agar.

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PROTEIN-BASED TESTS

As in carbohydrate testing, the products of protein or amino acid breakdown can be used for organism
identification. Protein-based tests include:

 Gelatin liquefaction tests

 Lysme decarboxylase and orruthme decarboxylase tests
 Phenylalanme deaminase test
 Urease test
 Indole test

Amino acids are degraded by enzymes. .Each amino acid has both an amino and a carboxyl group (Figure 3).

Two types of enzymes can act on each of these parts of the amino acid:

 Decarboxylase enzymes are able to split off the carboxyl group (Figure 4A), to produce an alkaline
end-product.
 Deaminase enzymes cleave off the amino group (Figure 4B), to give an acid end product.

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GELATIN LIQUEFACTION TESTS

Principle: Gelatin is a simple protein substrate, easily digested by "gelatmase"-producing organisms.

Normally having the consistency of Jelly, gelatin turns to liquid after bacterial digestion. This liquefaction of
gelatin provides the basis of a simple test.

TEST-TUBE METHOD

The organism is stab-inoculated into a gelatin butt. If gelatin is degraded a bacteria, the gelatin changes from
a solid to a liquid state (Figure 5).

However, a tube of fluid gelatin does not necessarily indicate a positive test, since gelatin is itself fluid at
30'C or warmer. All supposedly positive gelatin tubes are refrigerated, ill order to check that they remain fluid
at colder temperatures (Figure 6).

Gelatin can be modified by formalin so that It remains solid at incubator temperatures. If charcoal particles
are incorporated in formalin-treated gelatin tablets or pellets, gelatmase-producing bacteria can break down
the charcoal-gelatin pellet, releasing free charcoal particles throughout the medium and producing a black
butt (Figure 7).

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DECARBOXYLASE TESTS

There are two decarboxylase tests commonly used: Iysine and ornithine decarboxylase. The following
principles apply to both, but Iysine will be used as the example. The test medium Consists of:

 A basic growth medium

 Lysine or ornithine (an ammo acid substrate)
 Glucose (a carbohydrate substrate)
 Bromcresol purple (a pH indicator).

The uninoculated medium is coloured purple. You will recall that bromcresol purple is yellow m acid and
purple in alkaline conditions. The question arises: How can a positive test be detected, since the end product
is alkaline and this medium is initially purple?

CONTROL SYSTEM

In order to ensure that an alkaline or positive reaction is due to decarboxylase, a control is incorporated that
includes only the substrate glucose and lacks the amino acid. The organism attacks this carbohydrate
substrate; and since the reaction is acid, the indicator a turns yellow Therefore, by adding this control to the
test system, we are assured that: ..
 The organism is living
 It ferments glucose.

Why it is necessary to include the glucose at all? Glucose is incorporated because the acid conditions
produced from Its breakdown facilitate the decarboxylation reacllon. What If an organism is not able to
ferment glucose? The test is designed for Enterobacteriaceae, all of which ferment glucose; bacteria unable
to ferment glucose should not be tested using this system.

TEST SYSTEM

What happens m the control also takes place m the test system -organisms that do or do not decarboxylate the
ammo acid first attack the glucose. The resulting reaction is again
acid, producing a yellow colour.
 In the negative test, since the organism is unable to decarboxylate the Iysine, no alkaline by-product is
produced; therefore, the indicator remains yellow
 In the positive test, which is cloudy and purple, the alkaline products turn the indicator to purple.

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PREVENTION OF FALSE POSITIVE REACTIONS

False positive reactions, I.e. alkaline reactions, can occur in this test If lysme or ornithine decarboxylase-
negative bacteria are allowed to attack the nonspecific proteins found in media peptones.

This nonspecific protein degradation occurs only in the presence of oxygen. Therefore. to prevent false
positive reactions, It is essential that the medium surface be over layered With sterile mineral oil (Figure 8)
before incubation.

Failure to inoculate the medium with sufficient organism to produce growth is also a form of "false positive";
This is easily detected by a lack of media turbidity

NOTE: This test is Invalid If the control tube is not yellow the test cannot be read If the oil overlay is
Omitted.

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PHENYLALANINE DEAMINASE TEST

Another ammo acid, phenylalanine, is broken down by the enzyme phenylalanine deaminase. The medium is
a basal agar medium containing the substrate phenylalanine. Organism is inoculated using a zig-zag
streaking, and incubated. After 2-24 h, the product phenylpyruvic acid (PPA) is detected by running ferric
chloride (FeC13) down the surface of the slant.
A positive test is indicated by a green colour, where the FeCl3 was added to the tube.
The negative test is buff-yellowish colour, due to the unreacted FeCl3 added.

Very few organisms are positive for phenylalanine deaminase, so it is a useful test for screening purposes.
Providencia and Morganella species will give a positive reaction after only a few hours of incubation 35°C.

UREASE TEST

The substrate urea is also incorporated into a basal agar medium and slanted in tubes. Unlike the procedure to
test phenylalanine, an indicator, phenol red, is included. Urea is sensitive to heat and so must be filter-
sterilized before being added to the sterile basal medium. The slant surface is inoculated by "zig-zag"
streaking.

Urea is attacked by organism that makes urease, producing an alkaline end-product: ammoina (Figure 9). The
alkaline end-product turns the indicator from a buff colour to a bright pink, which is a positive test. A
negative test appears buff or yellow

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INDOLE TEST

An ammo acid substrate, tryptophane, is attacked by the enzyme tryptophanase to produce the by-products
indole and pyruvic acid. To detect indole, a reagent called Kovac's is used. Kovac's reagent contains:

 Amyl alcohol -extracts indole to the surface of the medium, where It can be detected

 HCL -provides acid conditions for the reaction to occur

 P-dimethylammobenzaldehyde -the reactive ingredient; indole plus p dimethylammobenzaldehyde
produces a pink-red dye.

A positive test result shows a rd ring at the top of the tube where Kovac's reagent was added. A negative test
result shows a yellow ring.

IMViC

Indole is the main component of the IMViC (indole, MR, VP, citrate) reaction. Some organisms have a
typical IMViC as illustrated in Table 2.

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NUSCELLANEOUSTESTS

The following group of tests are not easily classified into a particular group and so are placed here for
convenience. Included m This section are:

 Nitrate test
 Deoxyribonuclease test
 Tests for carbon utilization
-Citrate test
-Malonate test
 Motility tests
 Cytochrome oxidase test
 Catalase test.

NITRATE TEST

This test determines whether an organism is able to reduce nitrate to nitrite or (even further) to nitrous oxide
or nitrogen gas. The broth medium contains:

 Potassium nitrate
 Beef extract
 Peptone.

A Durham's tube is placed in the broth prior to autoclaving.

One of three reactions may be reported from this test:

Nitrates are reduced to nitrites (NO) ~ N02)

Nitrates are reduced to nitrates and beyond, to gases (NO) ~ N02 ~ N20 or N2)
Nitrates (NO) are not reduced.

In order to detect if nitrates are reduced to nitrite, two reagents are added to each incubated test:

Sulfanilic acid
α -naphthylamine (since α -naphthylarmine is carcinogenic, dimethyl α-naphthylamine is preferred).

The reagents react with nitrites to produce a red dye if no red colour results, two possibilities exist:

Nitrates have not been reduced

Nitrates have been reduced beyond nirites, to gases.

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HOW CAN WE DETECT WHICH IS ACTUALLY OCCURRING?

It may be helpful to determine whether any unreduced nitrate still remains In the tube, by adding Zinc dust.
Zinc is able to reduce nitrates to nitrites. If zinc is added to a tube of uninoculated nitrate broth plus the two
reagents (sulfanilic acid and a -naphthylamine), a red colour appears.

Therefore, if a red colour appears in a "negative" test after the addition of Zinc dust, then it can be assumed
that the nitrate had not been reduced by bacteria but by the zinc instead.

If, however, no change occurs after addition of the Zinc, It can be assumed that the nitrates were reduced
beyond nitrites, to gas. You may notice the presence of gas in the Durham's tube; this constitutes added proof
of gas production.

DEOXYRIBONUCLEASE TEST

The substrate deoxyribonucleic acid (DNA) is used to detect bacteria that have the enzyme
deoxyribonuclease (DNase). This enzyme is able to break down the large DNA macromolecule into smaller
subunits. The test system used is an agar-based medium containing DNA. Bacteria are spot-inoculated onto
the medium; as the organism grows (and If It produces the enzyme), the enzyme will diffuse out from the
point of inoculation in a uniform radius (Figure 10).

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This zone containing the DNA fragments can be detected m two ways:

 By addition of HCI to the plate surface

 By incorporation of toluidine into the medium.

HYDROCHLORIC ACID METHOD

After incubation, the plate can be flooded Within HCL. Hydrochloric acid reacts with intact DNA to produce
an opaque precipitate; but wherever the DNA has been split into smaller molecules, a clear area appears.
Therefore, If the organism produces DNase, a clear zone will be visible around the DNase-producing colony
(Figure 11), With the rest of the plate remaining opaque or cloudy

TOLUIDINE BLUE METHOD

If toluidine blue, a metachromatic dye, has been incorporated m a DNA test plate, It complexes with intact
DNA and gives the plate a blue colour. However, if the DNA has been split into smaller molecules by a
DNase-producing between, the dye shifts to its metachromatic pink colour. Therefore, a positive test will be
detected by the pink colour around the growth

TESTS FOR CARBON UTILIZATION

Both the citrate and the malonate tests use the same indicator system; both are tests designed to determine If
an organism is able to use the substrate (either citrate or malonate) as the sole source of carbon for growth.

CITRATE TEST

The medium contains no other carbon but that m sodium citrate. If the organism can utilize the Citrate, It will
show growth on the medium, and the indicator bromthymol blue will turn from a green to a blue colour.
A negative test shows no growth and the indicator remains the original green colour. The positive test is
reported as growth plus a colour change. The medium must be closely

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Examined to see whether there is growth. Contamination from dirty tubes may cause a false positive reaction;
If only a color change occurs, the test must be repeated. It is Important to use a very small amount of
inoculums (the zigzag streaked medium is stabbed to get rid of excess inoculums), so that carryover from
other tests is not used by the organism for growth.

MALONATE TEST

The inalonate test is another carbon utilization test. As With the citrate test, a blue color plus growth is a
positive test and a green color plus no growth is a negative test. Malonate is usually a fluid medium.

MOTILITY

The presence of flagella on bacteria is frequently useful for Identification. Since stained microscopic methods
tend technically to be difficult to do, other indirect techniques have been developed to detect flagella.

USE OF SEMI-SOLID MEDIA

The most common method involves the stab-inoculation of a butt of semi-solid medium. If the organism has
no flagella (and is therefore not motile), it will fail to migrate out from the centre stab-line. If it is motile, the
whole tube will become cloudy due to movement of the organisms toward the perimeter of the tube (Figure
12).

It is important when inoculating to hold the tube steady while it enters and leaves the tube, so that only one
line is made. The agar concentration must be less than that used for plate media.

TETRAZOLIUM METHOD

Tetrazolium salts are frequently added to motility media to detect bacterial growth. Wherever a living
bacterium is growing, it will convert colorless tetrazolium to a red formazan dye. If the medium appears red
throughout the tube, this indicates that the organism is motile and has grown out to the tube's periphery. If the
organism is

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nonmotile, it will form only a single red line along the inoculation stab. The motility of strict aerobes cannot,
however, be determined using a stab media technique, for they grow on the medium surface and not in its
depths. Wet preparations for detection of motility, flagella stains, or microscopic methods such as electron
microscopy must be used, instead.

CYTOCHROME OXIDASE TEST

In bacteria that possess it, the cytochrome oxidase enzyme is found at the end of an electron transport system
whereby it transfers hydrogen atoms (protons) to molecular oxygen to form water (Figure 13). Both aerobic
and facultative bacteria have cytochrome oxudase; however, anaerobes usually use compounds other than
oxygen as final hydrogen acceptors.

The reagent used to detect cytochrome Oxidase is one of:

 Tetramethyl-p-phenylenendiamine dihydrochloride, or
 Dimethyl-p-phenylenediamine oxalate.

These compounds are redox dyes. In their reduced state, they are colorless; but if they are oxidized by the
cytochrome oxidase enzyme, they turn purple (dimethyl) or dark pink (tetramethyl).

Either of two methods may be used:

 The reagent can be dropped directly on the colonies on the plate; or

 The organism can be rubbed onto a Strip of filter paper saturated With the reagent For the filter paper
test, use either a platinum loop or a very clean chromium loop since the reagent auto-oxidizes and my
turn color spontaneously

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Also, the reagent m solution should be prepared fresh each day to maintain the reagent in a reduced state
(prevent Oxidation), some workers add a reducing agent such as 0.1 % ascorbic acid. When the reagent is
added to the filter paper strips, the strips can be maintained indefinitely in a desiccant. Positive and negative
controls must be incorporated; each tune the test is done.

THE CATALASE TEST

The catalase enzyme also functions at the end of the respiratory chain. When the hydrogen atoms from the
respiratory system are passed to oxygen, water is most often formed. However, sometimes hydrogen peroxide
(H202), which is highly toxic to bacteria, is produced Instead (Figure 14).

Catalase is able to split

the H202 Into water and oxygen, to render it harmless and protect the organism. Most facultative and aerobic
organisms have catalase; however, anaerobes and streptococci lack It. (This is useful, since the catalase test is
used to distinguish streptococci from staphylococci.) It is thought that one reason anaerobes die in air is that
they lack catalase, and are therefore unable to and their environment ofH202.

Method: The presence of catalase enzyme is detected by exposing a culture to 3% hydrogen peroxide. If the
organism has the enzyme, bubbles of oxygen are produced.

One of two methods will be used:

 If the medium is not blood agar, place a drop ofH202 on a bacterial culture on the agar surface. A
positive test is indicated by the production of bubbles (02)

 As an alternate method a small amount of culture from the plate can be placed onto a glass slide. Add
a drop of H20 2 on top of the organism on the slide; if bubbles are produced (02), the test result is
positive. This method must be used if the organism is grown on a blood agar plate because the blood
itself has catalase and will provide a false positive reaction.

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TRIPLE-SUGAR IRON MEDIUM (TSI TEST)

The TSI test is a classic example of a multitest system. It provides a lot of information on a single tube and
gives the experienced technologist a very good idea of winch direction to go, for a final species
Identification. With the increasing use of miniaturized Identification systems, the TSI will be used less and
less, but at the present it is still considered a pivotal test.

The TSI does three main things:

 It detects the fermentation of one or more carbohydrates. It indicates gas production

 It indicates H2S production.

Before discussing the test's principle, it is useful to look at the medium composition and the functions of its
constituents (Table 3). Note especially those in boldface type.

More than any other medium, TSI must be prepared m a particular fashion. Note that the
0.012 (1.2%) agar concentrations will give a solid slant. Sufficient molten media it’s poured into tubes to
produce a medium having a slant of 3.7 cm and a bun of2.5 cm (Figure 15). These specifications are critical
for the accurate working of the test.

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To inoculate the medium, a straight wire with a small amount of organism is stabbed through the centre of the
medium almost to the bottom, stopping 0.5 cm from hitting the glass. The Wire is then zig-zagged across the
surface. (If small tubes are used, a single streak-line is acceptable.)

CARBOHYDRATE UTILIZATION

If the organism ferments lactose and/or sucrose, it will use glucose first; glucose, being a monosaccharide,
will always be attacked before disaccharides such as lactose and sucrose. If lactose and/or sucrose are
degraded, the TSI will have a yellow slant and a yellow butt.

This test would be reported as acid over acid or A/A, the upper “A: being the slant reaction and the lower
one, the butt. Some people use the terminology +/+ A/A (or +/+) = glucose, lactose and/or sucrose fermented.

If the organism tested ferments glucose only, then after incubation you will see a red (alkaline) slant and a
yellow (acid) butt. Can you explain why there is acid m the butt only?

If you had examined the inoculated medium after 2-3 hours of incubation, you would have seen a yellow
slant and butt. You will recall that the sucrose and lactose have a

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concentration of 0.01 (1%); however, the glucose has only 1/10 tills amount, 0.001 (0.1%) concentration.
This means that an organism able to use only glucose will consume it relatively rapidly. Once the glucose has
been used up, the organism then attacks the nonspecific proteins found in peptones.

Visualize a tube that is positive for fermentation of glucose only; the alkaline reaction occurs only in the
slant; the butt remains yellow This is termed reversion, because the acid slant turned (reverted) to alkaline
early in the Incubation period due to nonspecific protein breakdown in air. This reversion therefore aids in
detection of glucose-only fermentation.

You would report this test as alkaline (no action) over acid, or NA/A, or +/-NA/A (or +/-) = glucose only
remented.

EFFECT OF SLANT PREPARATION

As you can see, proper slant preparation of TSI medium is critical for reading glucose only fermentation. If
tubes were prepared with no butt (Figure 17), the test for glucose only fermentation would appear as an
NA/NA reaction.

On the other hand, If there was little slant and mostly butt to the medium (Figure 18), the test would be read
as A/A.

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EFFECT OF INCUBATION TIME AND TEMPERATURE

It is critical that the test, be read between 18 and 24 hours after incubation at 35°C. If read before or after
three times, the results will be Incorrect.

GAS PRODUCTION

When some organism ferments carbohydrates, they produce carbon dioxide and hydrogen gas. As these gases
escape, they produce bubbles in the medium or even lift the whole agar medium, often right out of the tube.
Other organism produce cracks in the agar. The medium should be carefully examined for these features. If
gas occurs in the medium, It is reported as: A/A gas or A/Ag or +/ +

H2O PRODUCTION

Some organisms can produce hydrogen sulphide from sulfhydryl compounds. Sodium thiosulphate is
included in TSI to provide these -SH groups. Once the H2S is produced, it reacts with an indicator, ferrous
sulphate, to produce a black precipitate, ferrous sulphide:

If an organism produces H2S, It is reported as:

For example:

The typical reaction of most Salmonella species is NA/AH2S; Salmonella typhi is NA/A with a characteristic
tiny amount of H2S.

SULPHIDE INDOLE MOTILITY MEDIUM (SIM TEST)

The SIM test is an example of a multitask system that provides three tests in one medium:

 Indole production
 Motility
 H2S

SIM medium contains:

Trypticase

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I peptones -contain tryptophane

 Tiuotone
 Ferrous ammonium sulphate -H2S indicator
 Sodium thiosulphate -provides -SH groups
 Tetrazolium
 Agar

The medium is formed as a semi-solid butt and is inoculated by stabbing with a Straight wire to the bottom of
the tube. The wire is withdrawn carefully along the same line. Indole production has been discussed
previously; the principle arid test method is the same. Paradimethylaminobenzaldehyde in Kovac's reagent
reacts with indole to produce a red color at the medium's surface.

Motility has also been discussed previously the tetrazolium has been included to detect microbial growth.
Where blackening due to H2S production fills the tube, it can be assumed that the organism is motile.

Sodium thosulphate provides the sulfhydryl groups for H2S production. Once the hydrogen sulphide is
produced, it can be detected by the H2S indicator ferrous ammonium sulphate, which produces a black
precipitate throughout the medium. (If the organism is nonmotile, H2S will first form only on the line of
inoculation, rather than be spread throughout the tube.)

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