NAME: ……………………………………………………………………. REG. NO.

: ……………………………………………………………

PRACTICAL SESSION TWO

This second session will involve

1. Classification and identification of the different Gram-positive cocci and Gram-
negative rods that are commonly seen clinical settings
2. Learning the key biochemical tests used in the identification of bacteria

Practical Timetable

Date Group
13/12/22 C1
14/12/22 C2
20/12/22 B1
21/12/22 B2
3/1/23 A1
4/1/23 A2
10/1/23 E1
17/1/23 E2
18/1/23 D1
24/1/23 D2

Please ensure that you do read the principles, as these will be examined in both the
progressive exam and the final exam

The overall objective of this practical is to classify and identify the different Gram-
positive cocci and Gram-negative rods that are commonly seen in clinical settings.

The different bacteria that will be demonstrated during this practical session include:
1. Gram-positive cocci: Staphylococcus species, Streptococcus species
2. Gram-negative rods: Lactose-fermenting bacteria, Non-lactose fermenting
bacteria

By the end of the practical session, using the different biochemical tests, you should be
able to:
1. Differentiate between Staphylococcus species and Streptococcus species
2. Differentiate between Staphylococcus aureus and Coagulase negative
staphylococci (CNS)
3. Differentiate among the different Streptococcus species
4. Differentiate between the different Enterobacteriaceae
5. Identify Pseudomonas aeruginosa and differentiate it from Enterobacteriaceae

You are expected to write up what you did during the practical session. Include the
answers to the questions that have been asked in this guide. The deadline for handing in
is two weeks from the time you do the practical.

While you will not have a demonstration on the Gram-positive rods, you are required to revise
them in your free time, and differentiate between them.

Practical session two guide v.4

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During the practical, you will also revise different components of bacteriology that were studied
last semester including morphology, growth and nutrition of bacteria
1. Introduction to the light microscope
2. Gram-staining
3. Culturing microorganisms

Gram-staining
Gram-staining reaction distinguishes most bacteria as Gram-positive and Gram-negative,
according to whether or not they resist decolonization with acetone alcohol after staining with
crystal violet and subsequent treatment with iodine.
a. What feature of bacteria does the Gram-stain depend on?
b. How would you interpret the findings of a Gram-stain?
c. What is the purpose of differentiating bacteria based on their Gram staining
reaction?
d. Revise the differences between Gram-positive bacteria and Gram-negative
bacteria.

Culturing microorganisms
Cultural techniques are used to isolate pathogens in pure culture so that they can be identified,
and if indicated, tested for their susceptibility to antimicrobials. Most bacteria can be cultured
artificially providing the culture medium contains the required nutrients in the correct amounts,
and the osmotic pressure and pH of the medium are also correct; and the microorganisms are
incubated in an atmosphere and temperature most suited to their metabolism.
a. Revise the different types of media used in the microbiology laboratory.
b. How do you describe colonies on a plate?

Practical session two guide v.4

NAME: ……………………………………………………………………. REG. NO.: ……………………………………………………………

For each of the biochemical tests, write up how each was demonstrated in the laboratory

Practical session two guide v.4

NAME: ……………………………………………………………………. REG. NO.: ……………………………………………………………

Gram-positive cocci: Staphylococci and Streptococci

Catalase test
Staphylococcus species are differentiated from Streptococcus species using the CATALASE
TEST.

Staphylococcus species: Catalase positive

Streptococcus species: Catalase negative

Principle: Catalase is an enzyme that decomposes hydrogen peroxide into water and oxygen.
An organism is tested for catalase production by bringing it into contact with hydrogen peroxide.
Bubbles of oxygen are released if the organism is a catalase producer.

What precautions are taken when carrying out this test? HINT: medium on which the organism
is grown and instrument used to pick a colony from the growth on the medium

Staphylococcus speicies

Coagulase test
Staphylococcus aureus is differentiated from Coagulase negative staphylococci using the
COAGULASE TEST.

Staphylococcus aureus: Coagulase positive

Coagulase negative staphylococci: Coagulase negative

Principle: Coagulase causes plasma to clot by converting fibrinogen to fibrin.

Slide coagulase test: most strains of S. aureus have a bound coagulase or “clumping factor” on
the surface of the cell wall. This factor reacts directly with fibrinogen in plasma, causing rapid
cell agglutination. Any strain that is negative with slide coagulase test must be confirmed with a
tube coagulase test.
Tube coagulase test: the coagulase detected by this method is secreted extracellularly and
reacts with a substance in the plasma called “coagulase reacting factor” to form a complex
which, in turn, reacts with fibrinogen to form fibrin

What precautions are taken when carrying out this test? HINT: Plasma used

Deoxyribonuclease (DNAse) test

Differentiates Staphylococcus aureus from Coagulase negative staphylococci

Staphylococcus aureus: DNAse positive

CNS: DNAse negative

Principle: Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA). The test organism is

cultured on a medium which contains DNA. After overnight incubation, the colonies are tested
for DNAse production by flooding the plate with a weak hydrochloric acid solution. The acid
precipitates unhydrolyzed DNA. DNAse producing colonies are therefore surrounded by clear
areas indicating DNA hydrolysis.

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Mannitol fermentation
Differentiates Staphylococcus aureus from CNS

S. aureus: positive
CNS: negative

Principle: mannitol salt agar has a high salt concentration (7.5% NaCl) which favors growth of S.
aureus and discourages the growth of other organisms. Additionally, S. aureus ferments
mannitol. S. aureus can be detected by the presence of a yellow zone around isolated colonies,
indicating production of acid from mannitol.

Test S. aureus CNS

Catalase test Positive Positive
Coagulase test
-Slide coagulase Positive Negative
-Tube coagulase Positive Negative
DNAse Positive Negative
Mannitol salt Positive Negative

To conclude that an organism is S. aureus (or not), all the above tests should be carried out.

How does the colony morphology of S. aureus differ from that of S. epidermidis (an
example of CNS) on blood agar?

List other examples of Coagulase negative staphylococcus

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NAME: ……………………………………………………………………. REG. NO.: ……………………………………………………………

Streptococcus species

Catalase test
Staphylococcus species are differentiated from Streptococcus species using the CATALASE
TEST.

Staphylococcus species: Catalase positive

Streptococcus species: Catalase negative

Principle: Catalase is an enzyme that decomposes hydrogen peroxide into water and oxygen.
An organism is tested for catalase production by bringing it into contact with hydrogen peroxide.
Bubbles of oxygen are released if the organism is a catalase producer.

What precautions are taken when carrying out this test? HINT: medium on which the organism
is grown and instrument used to pick a colony from the growth on the medium

Grouping of streptococci
Streptococci are grouped according to the Lancefield grouping system by Rebecca Lancefield
-based on antigens detected in these organisms
-cell wall polysaccharides (A, B, C, F, G streptococci)
-cell wall lipoteichoic acids (Group D streptococci, Enterococcus species)
Give examples of Streptococci that fall into each of the groups mentioned above

Haemolysis
Streptococci may first be divided into those that produce a soluble hemolysin and those that do
not.

Beta (β) hemolysis: cause a clear zone of hemolysis on the medium

Alpha (α) hemolysis: cause a green pigmentation with a narrow zone of partial hemolysis
Gamma (γ) hemolysis: have no effect (no hemolysis) on the medium

What medium best demonstrates hemolysis characteristics of Streptococcus species?

Give examples of β-hemolytic, α-hemolytic and γ-hemolytic Streptococcus species.

Antibiotic discs
Bacitracin and Co-trimoxazole (SXT) antibiotics are used to differentiate among the different
Streptococci.

CAMP test
This test is for the presumptive identification of group B beta-hemolytic streptococci. It was first
described in 1944 by Christie, Atkins, and Munch-Petersen (CAMP).The hemolytic activity of the
beta-hemolysin produced by most strains of S. aureus is enhanced by an extracellular protein
produced by group B streptococci. Interaction of the beta-hemolysin with this factor causes
“synergistic hemolysis”, which is easily observed on a blood agar plate.

Bile –Esculin test

This test differentiates Group D streptococci from other Streptococci species. It is based on the
ability of certain bacteria to hydrolyze esculin in the presence of bile. These bacteria grow in the
presence of bile salts. Hydrolysis of the esculin in the medium results in the formation of glucose
and a compound called esculetin, which react with ferric ions to form a black diffusable
compound.

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Salt tolerance test

This test is useful for the presumptive identification of the enterococcal group D organisms,
which have the ability to grow in the presence of 6.5% NaCl incorporated into broth medium.
This test is used to distinguish Enterococcus species from the other Group D streptococci, e.g.
S. bovis and S. equinus.

Optochin susceptibility test

This test differentiates Viridans streptococci from S. pneumoniae. Ethylhydrocupreine
hydrochloride (optochin), a quinine derivative, selectively inhibits the growth of Streptococcus
pneumoniae at very low concentrations (5 ug/mL).

Bile solubility test

Bile salts, specifically sodium deoxycholate and sodium taurocholate, have the capability to
selectively lyse Streptococcus pneumoniae when added to actively growing bacteria in agar or
in broth media. S. pneumoniae produces autolytic enzymes (autolysins) that account for the
central depression or umbilication characteristic of older pneumococcal colonies on agar media.
The addition of bile salts activates the autolysins and accelerates the natural lytic reaction
observed with cultures

Organism Hemolysis Bacitracin SXT CAMP Bile Growth Optochin Bile

test Esculin in solubility
6.5%
NaCl

Group A β S R - - - R -

Group B Β, none R R + - V R -

Group C, F, G β V S - - - R -

Group D
Enterococci Β, α, none R R - + + R -
Nonenterococci α, none R S - + - R -
(e.g S. bovis)

Viridans α, none V S - V - R -
streptococci

Streptococcus α v S - - - S +
pneumoniae

What other tests can be used to differentiate among the Streptococcus species?

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Gram-negative rods:

They are classified according to the colonies on selective/differential media (MacConkey). With
reference to Gram-negative rods, how does MacConkey agar act as a selective and a
differential medium?

Oxidase test
This test is used in the identification of Pseudomonas aeruginosa. (Some enteric bacteria
such as Aeromonas are also oxidase positive).
A piece of filter paper is soaked with a few drops of oxidase reagent. A colony of the test
organism is then smeared on the filter paper. If the organism is oxidase-producing, the
phenylenediamine in the reagent will be oxidized to a deep purple color.

Triple sugar iron (TSI)

From this test we are able to demonstrate acid production from glucose/lactose fermentation at
the slant and the butt (deep) of the tube, Gas production and hydrogen sulphide production.
Fermentation is demonstrated by yellowing of the slant and/or the butt (deep). This indicates
acid production.
• Alkaline slant/Alkaline deep (Red slant/Red butt)
No carbohydrate fermentation. Nonfermentative bacteria, e.g., Pseudomonas aeruginosa

• Alkaline slant/Acid deep (Red slant/Yellow butt)

Glucose fermented; lactose (or sucrose for TSI) not fermented. Non-lactose fermenting bacteria,
e.g., Shigella species

• Alkaline slant/Acid (black) deep (Red slant/Yellow butt/Hydrogen sulphide

production)
Glucose fermented; lactose not fermented, hydrogen sulphide produced. Salmonella species,
Citrobacter species, Proteus species

• Acid slant/acid deep (Yellow slant/Yellow butt)

Glucose and lactose fermented. Lactose fermenting colonies. Escherichia coli, Klebsiella,
Enterobacter species

Citrate utilization
Some organisms utilize citrate as the sole carbon source and ammonia as the only source of
nitrogen.
The organism is cultured in a medium which contains sodium citrate, an ammonium salt, and
the indicator bromo-thymol blue. For a positive result, the medium turns from green to blue due
to the alkaline reaction following citrate utilization. However, growth on the medium (without the
color change) also indicates a positive test as this also indicates utilization.
Give examples of some of the Gram-negative rods that are positive for this test.

Urease production
Some organisms produce urease which breaks down urea producing ammonia, making the
medium to become alkaline. The indicator is phenol red.
For a positive result, the medium changes color and turns to pink.
Give examples of some of the Gram-negative rods that are positive for this test.

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Sulphur indole motility (SIM)

This is a semisolid medium that demonstrates three characterisitics found within the organism.
• Decomposition of Sulphur-containing amino acids leads to hydrogen-sulphide
production. The medium will blacken
• Indole production from tryptophan found in the medium. Indole production is detected by
Kovac’s reagent which contains 4-(p)-dimethylaminobenzaldehyde. This reacts with
indole to produce a red colored compound
• Motility is demonstrated by growth of the organism away from the original stab point
filling the semisolid medium.
Give examples of Gram-negative rods that produce hydrogen sulphide
Give examples of Gram-negative rods that are indole positive
Give examples of Gram-negative rods that are motile

How do the colony morphologies of Enterobacteriaceae differ in different media?

Which bacteria have the characteristic fishy odor? What other characteristic of this
organism on blood agar is specific to this organism?

Describe the colony morphology of Pseudomonas aeruginosa

Which steps would you take to identify colonies of non-lactose fermenting bacteria?

Fastidious bacteria: Haemophilus spp.

How do you differentiate among the different Haemophilus spp. in the lab?
What is satellitetism? How is this test carried out?

Gram-negative cocci
What are some of the examples of Gram-negative cocci isolated from clinical specimen?
Suppose you isolated Gram-negative cocci from a clinical specimen. How would you
conclude which Gram-negative coccus it is?

In summary,

When identifying bacteria:

1. Gram stain result

2. Culture morphology result

3. Identification using biochemical tests

4. Antimicrobial susceptibility test results (if applicable)

Practical session two guide v.4