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isolation and identification of bacteria

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 R.KAVITHA, M.PHARM
LECTURER
DEPARTMENT OF PHARMACEUTIC
SRM COLLEGE OF PHARMACY
KATTANKULATUR
 IDENTIFICATION METHOD
The most important task of a bacteriology is to
identify the pathogens from the clinical sample so
that appropriate treatment can be instituted.
There are several methods to identified the different
type of bacteria.

1. Isolation in pure form

2. Staining reaction
3. Morphology of bacterial colony
4. Cultural characteristics
5. Metabolism
6. Biochemical properties
1. Isolation in pure form
y Studies on the biochemical, antigenic and other characters
of bacteria can be done only if the organism available in the
pure form.
Technique:
a. Plating on solid culture media‐ clinical sample is streaked
onto a solid medium (like: MacConkey agar, nutrient agar
or blood agar) in such a way so as to ensure isolated
discrete colonies.
b. Use of selective growth condition‐most important
example of this is the growth of anaerobic bacteria which
will not take place in an environment having oxygen.
 2.Staining reaction
a. The age of the culture is important. In older cultures ,
staining characteristics either vary or are not brought out
well. Simple stains bring out the best morphology.
Differential and special stains are necessary to bring out
characteristics like: gram negative and gram positive
bacteria, Acid fast and non acid fast , spirochetes, capsule
and Flagella, etc.
 a. Gram stain
a. Gram stain divides the bacteria into Gram positive & Gram
negative.

The basic procedure goes like this:

i. Take a heat fixed bacterial smear.
ii. Flood the smear with CRYSTAL VIOLET for 1 minute, then wash
with water. [PRIMARY STAIN]
iii. Flood the smear with IODINE for 1 minute, then wash with
water.
iv. Flood the smear with ETHANOL‐ACETONE, quickly, then wash
with water. [DECOLORI
v. Flood the smear with SAFRANIN for 1 minute, then wash
with water. [COUNTERSTAIN]
vi. Blot the smear, air dry and observe.
 contd
y Examine under microscope

i. Gram positive bacteria‐ violet

ii. Gram negative bacteria‐ pink
 Shape of Bacteria

y Bacteria display three basic shapes:

i. round‐ cocci, (from the Greek kokkos ‐ a berry),
ii. rod shaped – bacilli (from the Latin bacillus ‐ a stick
or rod),
iii. spiral (quelled).
 i. Coccus

Staphylococcus species

Streptococcus species
 ii. Bacillus

Clostridium spp. Listeria spp.

 Ziehl‐Neelsen Staining
y Acid‐Fast bacilli‐ red

Mycobacterium tuberculosis
 c. India ink (capsule stain)
y The capsule stain employs an acidic stain and a basic
stain to detect capsule production.
y Capsules are formed by organisms such as Klebsiella
pneumoniae . Most capsules are composed of
polysaccharides, but some are composed of
polypeptides.
 Staining procedure
y Place a loopful of India ink on the side of a clean slide
y A small portion of the solid culture is suspended
in saline on the slide near the ink and then emulsified in
the drop of ink, or else, mix a loopful
of liquid culture of specimens like CSF with the ink.
y Place a clean cover slip over
the preparation avoiding air bubbles.
y Press down, or blot gently with a filter paper strip to get a
thin, even film
y Examine under dry objectives followed by oil immersion
Examine under microscope

Klebsiella pneomonae
y Spirochetes – brownish black

Spirochetes Treponema pallidum Leptospira

3. Morphology of the bacterial
colony
i. Shape: circular, irregular, radiate or rhizoid.
ii. Size: diameter in mm
iii. Elevation: flat, raised, low convex, dome shaped
iv. Margin: Entire, wavy, lobate, filiform
v. Surface: smooth, wavy, rough, granular, papillate,
glistening etc.
Shape of the colony
Elevation of the colony
Margins of the colony
4. Cultural characteristics
These provide additional information for the
identification of a bacterium.
A. On solid medium the following characters
are observed
i. Shape: circular, irregular, radiate or rhizoid.
ii. Size: The size of the colony can be a useful characteristic
for identification. The diameter of a representative
colony may be measured.
iii. Elevation:
iv. Margin: Entire, wavy, lobate, filiform
v. Surface: smooth, wavy, rough, granular, papillate,
glistening etc.
vi. Size in mm
vii. Texture : dry, moist, mucoid, brittle, viscous, butyrous
(buttery).
viii. Color : colorless, pink, black, red, bluish‐green.
 Mac Conkey Agar

Enterobacter cloacae on Eschericia coli on MacConkey Agar:

MacConkey Agar: growth, with pink colonies
growth with pink colonies
 contd.

Staphylococcus aureus Streptococcus pyogens

contd

Bacillus subtilis Proteus spp.

 B. IN A FLUID MEDIUM FOLLOWING
CHARACTERS ARE OBSERVED

i. Degree of growth‐ Absence, scanty, moderate,

abundant etc.
ii. Present of turbidity and its nature.
iii. Presence of deposit and its character.
iv. Nature of surface growth.
v. Ease and disintegration and odor.
 5.METABOLISM

To classify the differentiate species following aspects are

studied
i. Requirement of oxygen
ii. The need of co2
iii. Capacity to form pigments
iv. Power of hemolysis
Laboratory Objectives
 Tests To Know
y Case Study Tests
y Indole
y Methyl Red/Voges Proskauer
y Citrate
y H2S production in SIM
y Motility
y Lactose fermentation
y Sucrose fermentation
y Glucose fermentation & gas production
y Triple Sugar Iron Agar (TSI) test
y Staphylococcus identification tests
y MSA
 Indole Test
Principle:
Indole test is performed to determine the ability of the organism to split
tryptophan molecule into Indole. Indole is one of the metabolic degradation
product of the amino acid tryptophan
Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing
and deaminating tryptophan with the production of Indole, Pyruvic acid and
ammonia.
Property it tests for:
y This test is performed to help differentiate species of the family
Enterobacteriaceae.
Media and Reagents Used:
y Tryptone broth contains tryptophan.
y Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde,
and amyl alcohol—yellow in color.
Indole test
• Procedure:
‐Inoculate Tryptone broth with
the test organism and incubate
for 18 to 24 hrs at 37 c
‐Add 15 drops of Kovac’s reagent
down the inner wall of the tube
• Interpretation:
‐Development of bright red color
at the interface of the reagent Indole Positive:
E.coli
and the broth within seconds
Proteus vulgaris
after adding the reagent is
indicative of the presence of Indole Negative:
Salmonella spp.
Indole and is a positive test
Klebsiella spp.
Enterobacter aerogens
 Methyl Red/Voges‐Proskauer (MR/VP)
y Properties these test for: Both tests are used to differentiate
species of the family Enterobacteriaceae.
y Media and Reagents Used:
y Glucose Broth
y Methyl Red indicator for MR test
y Voges Proskauer reagents‐ A: 5% Alpha‐Naphthol & ethanol,
B: Potassium Hydroxide; (3:1 ratio) & Deionized Water.
Principle of MR test:
To test the ability of the organism to produce and maintain
stable acid end products from glucose fermentation and to
overcome the buffering capacity of the system
This is a qualitative test for acid production.
MR test (contd…)
Procedure:
‐ Inoculate the MR/VP broth with a pure culture of the test organism and
incubate at 35° for 48 to 72 hrs.
Add 5 drops of MR reagent to the broth
Result interpretation:
‐ Positive result is red (indicating pH below 6)
‐ Negative result is yellow (indicating no acid production)

MR Positive:
E. coli

MR Negative:
Enterobacter aerogenes
Enterobacter cloacae
Klebsiella spp.

Left: negative/Right: positive

 Voges Proskauer Test (acetoin production)
Principle:
To determine the ability of the organisms to
produce neutral end product acetyl methyl
carbinol (acetoin) from glucose fermentation
Procedure:
Innoculate pure culture of the test organism
into MR/VP broth and incubate for 24 hrs at
37°c
Aliquot 1 ml of the broth to a sterile test tube
and add 0.6ml of VP(A) followed by 0.2ml of VP: left + and right –
VP(B)
Shake the tube gently to expose the medium to Positive
atmospheric oxygen and allow the tube to Klebseilla pneumoniae
remain undisturbed for 10 to 15 mins
Enterobacter
Intrepretation:
Positive : Pinkish red color at the surface of the Negative
medium E.coli
Negative : Yellow color at the surface of the
medium
Citrate Utilization test:
y This test is one of several technique used to assist in the identification of
enterobacteria. The test is based on the ability of an organism to use citrate
as its only sole source of carbon and ammonia as its only source of
nitrogen.
y Principle:
The test organism is cultured in a medium which contains sodium citrate, an
ammonium salt and the indicator bromothymol blue. Growth in the medium is
shown by turbidity and a change in colour of the indicator from light green to blue,
due to alkaline reaction following citrate utilization.
y Procedure:
Inoculum is streaked over the slant of Simmon’s citrate
agar in a tube and incubated for 24‐48 hrs.
y Result interpretation:
Growth on the slant and change in colour
to blue of the medium indicates positive result.

Positive: Klebsiella pneumoniae

Left‐ Positive: Right‐ Negative
Negative: E. coli
 Oxidation‐Fermentation (OF) test
(Hugh & Leifson)
Principle:
Oxidation‐Fermentation test is used to determine the
oxidative or fermentative metabolism of a carbohydrate or its
non utilization.
Fermentation is a anaerobic process and bacterial fermenters
of carbohydrates are usually facultative anaerobes. Oxidation
is a aerobic process and bacterial oxidisers are usually strict
aerobes
Procedures:
‐The method described, sometimes referred to as the Hugh
and Leifson test employs a semi‐solid medium in tubes
containing the carbohydrate under test (usually glucose) and a
pH indicator
‐Two tubes are inoculated and one is immediately sealed with
paraffin oil to produce anaerobic conditions
 Result interpretation:
y Oxidising organisms, eg Pseudomonas
species, produce an acid reaction in the
open tube only
y Fermenting organisms, eg
Enterobacteriaceae, produce an acid
reaction throughout the medium in both
tubes
y Organisms that cannot break down the
carbohydrate aerobically or
anaerobically, eg., Alcaligenes faecalis,
produce an alkaline reaction in the open
tube and no change in the covered tube
 Motility Test
y Property it tests for: This test is done to help differentiate
species of bacteria that are motile from non‐motile.
y Media and Reagents Used: Motility media contains
tryptose, sodium chloride, agar, and a color indicator.
y How to Perform Test: Stab motility media with inoculating
needle.
y Reading Results: If bacteria is motile, there will be growth
going out away from the stab line, and test is positive. If
bacteria is not motile, there will only be growth along the
stab line. A colored indicator can be used to make the
results easier to see.
Motility

From left to right:

+ – +
 Lactose Fermentation
y Property it tests for: This tests for the bacteria’s ability to ferment
lactose.
y Media and Reagents Used: Lactose broth contains beef extract, gelatin
peptone, and lactose. A phenol red indicator is added to indicate acid
production from fermentation.
y How to Perform Test: Inoculate lactose broth with inoculating loop.
y Results
y A positive result is yellow after indicator is added (indicating lactose
fermentation)
y A negative result will have no color change or will be reddish.
 Sucrose Fermentation
y Property it tests for: This test is done to help differentiate species of the
family Enterobacteriaceae. This tests the bacteria’s ability to ferment
sucrose and production of acid end‐product
y Media and Reagents Used: Sucrose broth contains beef extract, gelatin
peptone, and sucrose. Phenol red indicator is added to indicate an acid
end‐product.
y How to Perform Test: Inoculate sucrose broth with inoculating loop.
y Results
y A positive result is yellow after indicator is added (indicating
sucrose fermentation)
y A negative result has no color change or is reddish.
 Glucose Fermentation & Gas
Production
y Property it tests for: This test is done to help differentiate species of
the family Enterobacteriaceae. This tests for the bacteria’s ability to
ferment glucose and produce gas and/or an acid end‐product..
y Media and Reagents Used: Glucose broth contains beef extract,
gelatin peptone, and glucose. A phenol red indicator is added to
indicate an acid end‐product. A Durham tube is added to indicate gas
production.
y How to Perform Test: Inoculate broth with inoculating loop.
y Results
y A positive result for acid is yellow after indicator is added
(indicating glucose fermentation)
y A positive result for gas is a bubble in the Durham tube.
y A completely negative result has no color change or reddish color
and no bubble.
Sugar Fermentation Tests

Tube 1: Negative acid /Negative gas

Tube 2A: Must incubate longer (ambiguous result)
Tube 2B: Positive acid /Negative gas
Tube 3A: Positive acid/ Positive gas
Triple Sugar Iron Agar (TSI) test
y Principle:
TSI agar is used to determine whether a gram negative rod utilizes glucose
and lactose or sucrose fermentatively and forms hydrogen sulphide (H2S).
TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone.
Phenol red and ferrous sulphate serves as indicators of acidification and
H2S formation, respectively. The formation of CO2 and H2 is indicated by
the presence of bubbles or cracks in the agar or by separation of the agar
from the sides or bottom of the tube. The production of H2S requires an
acidic environment and is indicated by blackening of the butt of the
medium in the tube.
y Method:
1. With a straight inoculating wire, touch the top of a well isolated colony.
2. Inoculate TSI by first stabbing through center of the medium to the
bottom of the tube and then streaking the surface of the agar slant.
3. Leave the cap on loosely and incubate the tube for 18‐24 hours at 35oC in
an incubator.
Triple Sugar Iron Agar (TSI) test (contd…)
y Result interpretation: a b c d
1. Alkaline slant/no change in the butt
(K/NC) = Glucose, lactose and sucrose
non‐utilizer (alkaline slant/alkaline butt)
[figure: 1(d)]
2. Alkaline slant/acid butt (K/A) = Glucose
fermentation only. [figure: 1(b)]
3. Acid slant/acid butt (A/A), with gas
production = Glucose, sucrose, and/or
lactose fermenter. [figure: 1(a)]
4. Alkaline slant/acid butt (K/A), H2S
production = Glucose fermentation only.
[figure: 1(c)]

ƒ Quality control:
A/A, with gas: E. coli Figure (1): TSI results
K/A, H2S: Salmonella typhi
K/NC: Pseudomonas aeruginosa
 Mannitol Salt Agar (MSA)
y Property it tests for: This tests for the bacteria’s ability
to tolerate 7% salt concentration and ferment mannitol.
The media is selective because it selects for salt tolerant
bacteria.
y Media and Reagents: MSA media contains nutrient
agar, mannitol, 7% sodium chloride and phenol red
indicator.
y How to Perform Test: Inoculate an MSA plate using
streak plate method and incubate 24‐48 hours.
 MSA Results
y Reading Results:
y If the organism is tolerant to salt it will grow.
y If the organism is not tolerant to salt it will not grow.
y If the salt tolerant organism can ferment mannitol then there will be
yellow zones around the colonies.
y If the salt tolerant organism cannot ferment mannitol then the media will
remain pink.

Growth with no mannitol fermentation. Growth with + mannitol fermentation.

Test for enzymes
y Catalase test
y Oxidase test
y Urease test
y Coagulase test
y Nitrate reduction
Catalase test
Principle:
This test demonstrates the presence of enzyme catalase in
the organism. The enzyme catalase mediates the
breakdown of hydrogen peroxide (H2O2) into oxygen and
water. The presence of the enzyme in a bacterial isolate is
evident when a small inoculum is introduced into
hydrogen peroxide (30% for slide test), and the rapid
effervescence of O2 bubbles occurs. The lack of catalase is
indicated by a lack of bubble production.lase
catalase
2H2o2 2H2o + o2 (gas bubbles)
Catalase test….
• Catalase is present in most cytochrome containing
aerobic and facultative anaerobic bacteria (except
streptococcus spp).
• Hydrogen peroxide forms as one of the oxidative end
product of aerobic carbohydrate metabolism. If this is
allowed to acculmulate in the bacterial cells it becomes
lethal to the bacteria
• Catalase thus helps in converting H2o2 to H2o and o2
• Optimal pH for catalase action is 7.
Catalase test….

Reagents:
3% hydrogen peroxide stored in dark brown bottle
under refrigeration
18 to 24 hrs culture of the organism to be tested
Control organisms used:
Positive control ‐ Staphylococcus aureus
Negative control – Streptococcus spp.
Catalase test….
Methods:

1. Slide method

2. Tube method 3. Direct plate method

Catalase test….
Precautions
y Avoid colonies from blood agar
y 18 to 24 hrs colony only should be used
y Reagent to be fairly fresh as it is very unstable and
easily breaks down on exposure to light
y Reagent to be stored in brown or amber colored bottle
in refrigerator at 4°c
Catalase test….
Positive Negative
• Micrococcus • Streptococcus
• Staphylococcus • Clostridium
• Bacillus
• Listeria monocytogenes
• Enterobacteriacae
• Gonococcus &
Meningococcus
• Vibriocholerae
• Pseudo/Aero/Plesiomonas
Oxidase Test
Principle
Oxidase test is used to determine the presence of
bacterial cytochrome oxidase enzyme using the
oxidization of the substrate “tetramethyl‐p‐
phenylenediamine dihydrochloride” to indophenol a
dark purple colored end product. A positive test
(presence of oxidase) indicated by the development of a
dark purple colour. No colour development indicates a
negative test and the absence of the enzyme.
 Oxidase Test….
ƒ Cytochromes are iron containg hemoprotiens and in aerobic respiration they
transfer electrons(H) to oxygen to form water.
ƒ The reagent acts as an artificial electron acceptor substituting the oxygen. In the
reduced stage dye is colorless , but in the presence of enzyme cytochrome
oxidase dye is oxidised to indophenol blue
Methods

1. Moist filter paper 2. Direct plate method

method
Quality controls
Positive control‐ Pseudomonas spp
Negative control – E. coli
Oxidase Test…..
Positive Negative
• Pseudomonas spp. y Enterobacteriaceae
• Aeromonas spp. y Acenitobacter spp.
• Vibrio spp.
• Alcaligenes spp.
• Neisseria spp.
• Haemophilus sps
Oxidase Test…..
Precautions
• Use glass rod or wooden stick
• Result should be read within 10 secs
• Do not perform the test from colonies growing in
medium having glucose as its fermentation will inhibit
the oxidase enzyme activity
• Reagent should be freshly prepared
• To be stored in dark bottle
• Do not use pigmented colonies or colonies fromMac‐
conkey agar
 Urea Hydrolysis (Urease test)
y Property it tests for: This test is done to determine a bacteria’s
ability to hydrolyze urea to make ammonia using the enzyme
urease.
y Media and Reagents Used: Stuarts Urea broth (pH 6.8)
contains a yeast extract, monopotassium phosphate, disodium
phosphate, urea, and phenol red indicator.
y Principle
To determine the ability of the organism to split urea forming 2
molecules of ammonia by the action of the enzyme Urease with
resulting alkalinity
y How to Perform Test: Inoculate Urea broth with inoculating
loop.
 Reading Results:
y Urea broth is a yellow‐orange color.
The enzyme urease will be used to
hydrolyze urea to make ammonia. If
ammonia is made, the broth turns a
bright pink color, and is positive. If test
is negative, broth has no color change
and no ammonia is made.

y Figure in the right shows negative (left) and

Positive (right) results.
 Coagulase test
y Principle:
‐ This test is used to differentiate Staphylococcus aureus (positive) from
coagulase negative Staphylococci. S. aureus produces two forms of coagulase:
bound and free.
‐ Bound coagulase or clumping factor, is bound to the bacterial cell wall and
reacts directly with fibrinogen. When a bacterial suspension is mixed with plasma,
this enzyme causes alteration in fibrinogen of the plasma to precipitate on the
staphylococcal cells, causing the cells to clump.
‐ Free coagulase is produced extra‐cellularly by the bacteria that causes the
formation of a clot when S. aureus colonies are incubated with plasma.

y Method:
A. Slide test: (for bound coagulase)
‐ Place a drop of coagulase plasma on a clean, dry glass slide.
‐ Place a drop of distilled water or saline next to the drop of plasma as a
control.
‐ With a loop or wooden stick, emulsify a portion of the isolated colony being
tested in each drop.
‐ Mix well and rock the slide gently for 5 to 10 seconds.
B. Tube test: (for free coagulase)
‐ Emulsify several colonies in 0.5 ml of rabbit plasma (with EDTA) to give a
milky suspension.
‐ Incubate tubes at 35oC in ambient air for 4 hrs. Check for clot formation.
‐ If negative at 4 hrs, incubate at room temperature overnight and check again
for clot formation.
 Coagulase Results
y Reading Results:
A. Slide test:
‐ Positive: Macroscopic clumping in 10 seconds
or less in coagulated plasma drop and no
clumping in saline or water drop.
‐ Negative: No clumping in either drop.
‐ Note: All negative slide tests must be
confirmed using the tube test.
B. Tube test:
‐ Positive: Clot of any size (a)
‐ Negative: No clot (b)

a b
Coagulase Positive : Staphylococcus aureus
Coagulase negative: Staphylococcus epidermidis
Nitrate reduction test
Principle:
This test is used to determine the ability of the organism to
reduce nitrate to nitrites or fee nitrogen gas. The reduction of
nitrate to nitrite is detected by adding sulphanilic acid and
alpha‐naphthylamine. The sulphanilic acid and nitrite reacts
to form a diazonium salt which then reacts with alpha‐
naphthylamine to produce a red, water soluble azo‐dye.
Purpose:
y Aid in the species deferentiation of
i) Haemophilus duceryi(‐) and Haemophilus vaginalis(‐) from
other Haemophillus spp.
ii) Neisseria mucosa(+) from other Neisseria spp
y Aid in the identification of Enterobacteriaceae(+)
Nitrate reduction test….
Procedure:
• In order to determine if a bacteria can reduce nitrate, the
test organism is inoculated into nitrate broth [an
undefined medium that contains large amounts of nitrate
(KNO3)]. After incubation, alpha‐naphthylamine and
sulfanilic acid are added . These two compounds react with
nitrite and turn red in color, indicating a positive nitrate
reduction test
• If there is no color change at this step, nitrite is absent. If
the nitrate is unreduced and still in its original form, this
would be a negative nitrate reduction result. However, it is
possible that the nitrate was reduced to nitrite but has been
further reduced to ammonia or nitrogen gas (which evolved
out). This would be recorded as a positive nitrate reduction
result
Nitrate reduction….
• To distinguish between these two reactions, zinc dust
must be added. Zinc reduces nitrate to nitrite. If the test
organism did not reduce the nitrate to nitrite, the zinc
will change the nitrate to nitrite. The tube will turn red
because alpha‐naphthylamine and sulfanilic acid are
already present in the tube
• Thus a red color after the zinc is added indicates the
negative nitrate reduction test.
Nitrate reduction test….

Negative

Negative Posiitive Positive

Nitrate reduced to Nitrate not

Nitrate not Nitrate reduced to NH3 or N2 gas, reduced
reduced nitrite nitrite absent
Nitrate reduction test….

Addition of Zn dust or
Gram positive flowchart

present
Gram negative flowchart
View publication stats

Key identification characteristics for

Enterobacteriaceae
GENUS/SPECIES Fermentation of Gas MR VP Indole Citrate Urease H2
G L S M
Escherichia coli (+) (+) (+) (+) (+) (+) (‐) (+) (‐) (‐) (‐)
Shiegella (+) (‐) (‐) (+) (‐) (+) (‐) (‐/+) (‐) (‐) (‐)
Shiegella sonnei (+) (+) (‐) (+) (‐) (+) (‐) (‐) (‐) (‐) (‐)
Salmonella (+) (‐) (‐) (+) (+) (+) (‐) (‐) (+) (‐) (+
Klebsiella Pneumo. (+) (+) (‐) (+) (+) (‐) (+) (‐) (+) (+) (‐)
Enterobacter (+) (‐) (+) (+) (‐) (+) (‐) (+) (‐) (+) (+
Serratia (+) (+) (‐) (+) (+) (‐/+) (+) (‐) (+) (‐) (‐)
Proteus (+) (‐) (‐) (+) (‐/+) (+) (‐) (+) (‐/+) (+) (+
morganella (+) (‐) (‐) (+) (+) (+) (‐) (+) (‐) (+) (+
Yersinia (+) (‐) (‐) (+) (‐) (+) (‐) (‐/+) (‐) (‐/+) (‐)

G: Glucose, L:Lactose, S:Sucrose, M: Manitol, MR: Methyl Red, VP: Voges Proskauer