Isolation and Identification of

Bacteria

Dr Bereket D. (DVM, MSc,

Asst. Prof.

06/09/2023 1
 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.

06/09/2023 2
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
06/09/2023 3
 1. Isolation in pure
●form
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.
06/09/2023 4
 2.Staining
a. The age ofreaction
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.

06/09/2023 5
 a. Gram
a.
stain
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. [ DECOLOR I
v. Flood the smear with SAFRANIN for 1 minute,
then wash with water. [ COUNTERSTAIN]
vi. Blot the smear, air dry and observe.

06/09/2023 6
 cont
d
● Examine under
microscope

i. Gram positive bacteria‐

violet
ii. Gram negative bacteria‐ pink

06/09/2023 7
 Shape of
Bacteria
● 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).

06/09/2023 8
 i.
Coccus

Staphylococcus
species

Streptococcus
06/09/2023 species 9
 ii.
Bacillus

Clostridium Listeria
spp. spp.

06/09/2023 10
 Ziehl‐Neelsen
Staining
● Acid‐Fast bacilli‐
red

Mycobacterium
06/09/2023 11
tuberculosis
 c. India ink (capsule
stain)
● The capsule stain employs an acidic stain and a
basic stain to detect capsule production.
● Capsules are formed by organisms such as
Klebsiella pneumoniae . Most capsules are
composed of polysaccharides, but some are
composed of polypeptides.

06/09/2023 12
 Staining procedure
● Place a loopful of India ink on the side of a clean slide
● 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.
● Place a clean cover slip over
the preparation avoiding air bubbles.
● Press down, or blot gently with a filter paper strip to
get a thin, even film
● Examine under dry objectives followed by oil
immersion
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Examine under
microscope

Klebsiella
06/09/2023
pneomonae 14
● Spirochetes – brownish
black

Spirochete Treponema Leptospira

s pallidum
06/09/2023 15
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.

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 Shape of the
colony

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 Elevation of the
colony

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 Margins of the
colony

06/09/2023 19
4. Cultural
characteristics
These provide additional information for
the identification of a bacterium.

06/09/2023 20
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.
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 Mac Conkey
Agar

Enterobacter cloacae Eschericia coli on MacConkey

on MacConkey Agar: Agar: growth, with pink colonies
06/09/2023 22
growth with pink
 contd
.

Staphylococcus Streptococcus
06/09/2023 pyogens 23
aureus
 cont
d

06/09/2023Bacillus subtilis Proteus spp. 24

 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.

06/09/2023 25
 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

06/09/2023 26
 Laboratory Objectives

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 Tests To
● Case Study Tests
● Indole
Know
● Methyl Red/Voges Proskauer
● Citrate
● H 2 S production in SIM
● Motility
● Lactose fermentation
● Sucrose fermentation
● Glucose fermentation & gas production
● Triple Sugar Iron Agar (TSI) test
● Staphylococcus identification tests
● MS A

06/09/2023 28
 Indole
Principle: Test
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:
● This test is performed to help differentiate species of the family
Enterobacteriaceae.
Media and Reagents Used:
● Tryptone broth contains tryptophan.
● Kovac’s reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.
06/09/2023 29
 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: Indole Positive:
E.coli
‐Development of bright red
Proteus
color at the interface of the vulgaris
reagent and the broth within Indole Negative:
Salmonella spp.
seconds after adding the
Klebsiella spp.
reagent is indicative of the
06/09/2023 Enterobacter 30
 Methyl Red/Voges‐Proskauer
(MR/VP)
● Properties these test for: Both tests are used to
differentiate species of the family Enterobacteriaceae.
● Media and Reagents Used:
● Glucose Broth
● Methyl Red indicator for M R test
● Voges Proskauer reagents‐ A: 5% Alpha‐Naphthol and
ethanol, B: Potassium Hydroxide; (3:1 ratio) and Deionized
Water.
Principle of M R 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.
06/09/2023 31
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 M R reagent to the broth
Result interpretation:
‐ Positive result is red (indicating pH below 6)
‐ Negative result is yellow (indicating no acid production)

M R Positive:
E. coli

M R Negative:
Enterobacter
aerogenes
Enterobacter cloacae
Klebsiella spp.
06/09/2023 32
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
and add 10.6ml
ml ofofthe broth
VP(A) to a sterile
followed test
by 0.2ml VP: left + and right –
of
tubeVP(B)
Shake the tube gently to expose the medium Positive
to atmospheric oxygen and allow the tube to Klebseilla
remain undisturbed for 10 to 15 mins
pneumoniae
Intrepretation:
Enterobacter
Positive : Pinkish red color at the surface of Negativ
the medium e
Negative : Yellow color at the surface of E.coli
the medium
06/09/2023 33
 Citrate Utilization
●test:
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.
● 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.
● Procedure:
Inoculum is streaked over the slant of Simmon’s
citrate agar in a tube and incubated for 24‐48 hrs.
● Result interpretation:
Growth on the slant and change in colour
to blue of the medium indicates positive result.

Positive: Klebsiella
06/09/2023 Left‐ Positive: Right‐ 34
pneumoniae
Negative
 Oxidation‐Fermentation (OF)
test (Hugh and 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
06/09/2023 35
 Result interpretation:
● Oxidising organisms, eg Pseudomonas
species, produce an acid reaction in
the open tube only
● Fermenting organisms, eg
Enterobacteriaceae, produce an acid
reaction throughout the medium in
both tubes
● 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
06/09/2023 36
 Motility
Test
● Property it tests for: This test is done to help
differentiate species of bacteria that are motile from
non‐motile.
● Media and Reagents Used: Motility media
contains tryptose, sodium chloride, agar, and a
color indicator.
● How to Perform Test: Stab motility media with
inoculating needle.
● 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
06/09/2023 37
easier to see.
Motility

From left to
right: +
+ –

06/09/2023 38
 Lactose
● Property itFermentation
tests for: This tests for the bacteria’s ability to
ferment lactose.
● 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.
● How to Perform Test: Inoculate lactose broth with inoculating loop.
● Results
● A positive result is yellow after indicator is added (indicating
lactose fermentation)
● A negative result will have no color change or will be reddish.

06/09/2023 39
 Sucrose
● Property it Fermentation
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
● Media and Reagents Used: Sucrose broth contains beef extract,
gelatin peptone, and sucrose. Phenol red indicator is added to
indicate an acid end‐product.
● How to Perform Test: Inoculate sucrose broth with inoculating loop.
● Results
● A positive result is yellow after indicator is added
(indicating sucrose fermentation)
● A negative result has no color change or is reddish.

06/09/2023 40
 Glucose Fermentation and
Gas Production
● 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
● 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.
● How to Perform Test: Inoculate broth with inoculating loop.
● Results
● A positive result for acid is yellow after indicator is
added (indicating glucose fermentation)
● A positive result for gas is a bubble in the Durham
tube.
● A completely negative result has no color change or reddish
color and no bubble.
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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
06/09/2023 42
Triple Sugar Iron Agar (TSI)
test
● 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.
● 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.
06/09/2023 43
Triple Sugar Iron Agar (TSI) test
(contd…)
● Result a b c d
1. interpretation:
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), H2S
production = Glucose fermentation
only. [figure: 1(c)]
3. Alkaline slant/acid butt (K/A) =
Glucose fermentation only. [figure:
1(b)]
4. Acid slant/acid butt (A/A), with gas
production = Glucose, sucrose,
and/or lactose fermenter. [figure:
 1(a)]
Quality control:
A/A, with gas: E. Figure (1): TSI
coli results
K/A, H2S:
06/09/2023
Salmonella typhi 44
 Mannitol Salt Agar
(MSA)
● 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.
● Media and Reagents: MSA media contains
nutrient agar, mannitol, 7% sodium chloride and
phenol red indicator.
● How to Perform Test: Inoculate an MS A plate
using streak plate method and incubate 24‐48 hours.

06/09/2023 45
 MSA
● Reading Results:
●
Results
If the organism is tolerant to salt it will grow.
● If the organism is not tolerant to salt it will not grow.
● If the salt tolerant organism can ferment mannitol then there will
be yellow zones around the colonies.
● If the salt tolerant organism cannot ferment mannitol then the media
will remain pink.

06/09/2023Growth with no mannitol Growth with + mannitol 46

fermentation. fermentation.
Test for
enzymes
● Catalase test
● Oxidase test
● Urease test
● Coagulase test
● Nitrate
reduction

06/09/2023 47
 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 O2 bubbles occurs. The lack of catalase is
indicated by a lack of bubble production.
catalase
2H2o2 2H2o + o2 (gas
bubbles)

06/09/2023 48
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 H2 o2 to H 2 o and
o2
• Optimal pH for catalase action is 7.
06/09/2023 49
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.

06/09/2023 50
Catalase
test….
Methods:

1. Slide method

2. Tube method
06/09/2023 3. Direct plate method
51
Catalase
test….
Positive Negative
• Micrococcus • Streptococcus
• Staphylococcus • Clostridium
• Bacillus
• Listeria monocytogenes
• Enterobacteriacae
• Gonococcus and
Meningococcus
• Vibriocholerae
• Pseudo/Aero/Plesiomonas
06/09/2023 52
Oxidase
Principle
Test
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.

06/09/2023 53
 Oxidase

Test….
Cytochromes are iron containg hemoprotiens and in aerobic respiration
they transfer electrons(H) to oxygen to form water.
 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

Quality controls
Positive control‐
Pseudomonas spp
Negative control – E. coli
06/09/2023 54
Oxidase
Test…..
Positive Negative
• Pseudomonas ● Enterobacteriaceae
spp. ● Acenitobacter spp.
• Aeromonas spp.
• Vibrio spp.
• Alcaligenes spp.
• Neisseria spp.
• Haemophilus sps

06/09/2023 55
 Urea Hydrolysis (Urease
● Property test)
it tests for: This test is done to determine a
bacteria’s ability to hydrolyze urea to make ammonia using
the enzyme urease.
● Media and Reagents Used: Stuarts Urea broth (pH 6.8)
contains a yeast extract, monopotassium phosphate,
disodium phosphate, urea, and phenol red indicator.
● 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
● How to Perform Test: Inoculate Urea broth with
inoculating loop.

06/09/2023 56
 Reading
●Results:
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.

06/09/2023 57
 Coagulase
● Principle:
‐
test
This test is used to differentiate Staphylococcus aureus (positive)
coagulase
fromnegative Staphylococci. S. aureus produces two forms of
coagulase: bound and free.
‐ Bound coagulase or clumping factor, is bound to the bacterial cell wall
reacts
and 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
formation
the of a clot when S. aureus colonies are incubated with
plasma.
● 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
06/09/2023
for clot formation. 58
 Coagulase
● Reading
A. Results:
Slide test:
Results
‐ 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: Clot of any size
Positive:
Negative No clot
Coagulase
: Positive : Staphylococcus aureus a b
Coagulase negative: Staphylococcus epidermidis

06/09/2023 59
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:
● Aid in the species deferentiation of
i) Haemophilus duceryi(‐) and Haemophilus vaginalis(‐)
from other Haemophillus spp.
ii) Neisseria mucosa(+) from other Neisseria spp
● Aid in the identification of Enterobacteriaceae(+)
06/09/2023 60
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
06/09/2023 61
 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.

06/09/2023 62
Nitrate reduction
test….

06/09/2023 63
 Gram positive
flowchart

present

06/09/2023 64
 Gram negative
flowchart

06/09/2023 65
Key identification characteristics
for Enterobacteriaceae
GENUS/SPECIES Fermentation of MR VP Indole Citrate Urease H2
Gas
G L S M
Escherichia coli (+) (+) (+) (+) (+) (+) (‐) (+) (‐) (‐) (‐
Shiegella (+) (‐) (‐) (+) (‐) (+) (‐) (‐/+) (‐) (‐) (‐
Shiegella sonnei (+) (+) (‐) (+) (‐) (+) (‐) (‐) (‐) (‐) (‐
Salmonella (+) (‐) (‐) (+) (+) (+) (‐) (‐) (+) (‐) (+
Klebsiella Pneumo. (+) (+) (‐) (+) (+) (‐) (+) (‐) (+) (+) (‐
Enterobacter (+) (‐) (+) (+) (‐) (+) (‐) (+) (‐) (+) (+
Serratia (+) (+) (‐) (+) (+) (‐/+) (+) (‐) (+) (‐) (‐
Proteus (+) (‐) (‐) (+) (‐/+) (+) (‐) (+) (‐/+) (+) (+
morganella (+) (‐) (‐) (+) (+) (+) (‐) (+) (‐) (+) (+
Yersinia (+) (‐) (‐) (+) (‐) (+) (‐) (‐/+) (‐) (‐/+) (‐

G: Glucose,
06/09/2023 L:Lactose, S:Sucrose, M: Manitol, MR: Methyl Red, VP:
66
Voges Proskauer
 Thank you

06/09/2023 67