MAKERERE UNIVERSITY

COLLEGE OF HEALTH SCIENCES (MakCHS)

DEPARTMENT OF MICROBIOLOGY

NAME COURSE REGISTRATION NO.

MUBIRU SAMUEL EDWARD BMAM 19/U/0823
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.

Procedure for carrying out the catalase test.

 Using a loop or sterile wooden stick, a small amount of a bacterial colony is transferred to a dry
glass slide.
 A drop of 3% hydrogen peroxide is then added to the slide.
Conclusion; production of bubbles of a colorless gas indicates hydrogen peroxide production.

What precautions are taken when carrying out this test?

 The test organisms should not be taken from a blood agar culture.
 The culture used should be 18-24 hours old.
 The hydrogen peroxide used must be fresh as it is very unstable.
 An iron wire loop should not be used.

Staphylococcus species
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.

Procedure for carrying out a slide coagulase test.

 About 10µl of deionized water or physiological saline is added to a slide.
 Several colonies from a fresh culture are collected with an inoculating loop and are emulsified
into the water to obtain a smooth milk coloured suspension.
  A drop of rabbit or human plasma is added to the slide and clumping is observed immediately.

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.

Procedure for carrying out the tube coagulase test.

 Human or rabbit plasma is diluted with physiological saline.
 About 5ml of the diluted plasma is added to a test tube. 5 drops of the test organism culture
are added to the test tube.
 The test tube is then mixed and incubated at 37 degrees Celsius for an hour.
 The tube is finally observed for clot formation. The tube should be examined at 30 minutes
intervals.

What precautions are taken when carrying out this test?

The test cannot be performed from growth on mannitol salt agar. In addition citrated blood should not
be used when performing the test as false positive results can occur.

Deoxyribonuclease (DNAse) test

Differentiates Staphylococcus aureus from Coagulase negative staphylococci
Staphylococcus aureus: DNAse positive
CNS;Coagulase negative staphylococci: 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.

Procedure for carrying out the DNAse test.

 Agar plates are inoculated with the test organism from an 18 hour culture with a sterile
inoculating loop or needle.
 The plates are incubated at 35-37°C for 24 hours.
 The incubated agar plates are flooded with a 1N HCL solution and the excess acid is tipped off.
 Some time is allowed for the reagents to be absorbed into the plates
 The plates are then observed for a clear zone around the colonies within 5 minutes.
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.

Procedure

An inoculum from a pure culture is transferred aseptically to a sterile tube of phenol red mannitol
broth. The inoculated tube is incubated at 35-37°C for 24 hours. A positive test consists of a color
change from red to yellow this indicates a pH change to acidic.

Alternatively, staphylococcus aureus grows on mannitol salt agar to produce yellow colonies.

How does the colony morphology of S. aureus differ from that of S. epidermidis (an example of CNS)
on blood agar?
A clear zone of hemolysis is seen surrounding the colonies of staphylococcus aureus which absent
around the colonies of staphylococcus epidermidis.

List other examples of Coagulase negative staphylococcus

 Staphylococcus saprophyticus
 Staphylococcus lugdunensis

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.

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
Group A Group B Group C Group F Group G Group D
S.pyogenes S. agalactiae S.equi S.anginosus S.dysgalacticae Enterococcus
Subspecies- Subspecies- faecalis
equi equisimilis
S.equi S. milleri S. Bovis
Subspecies-
zooepidemicus
S.constellatus S.intestinalis
Subspecies-
pharyngis

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?
Blood agar is the best medium to demonstrate hemolysis.

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

Β hemolytic Α hemolytic γ hemolytic
S. pyogenes S.pneumoniae Enterococcus faecalis
S. agalactiae Viridans species S. Bovis
NOTE; Enterococcus faecalis can also be an α or β hemolytic and also S.Bovis can also be an α
hemolytic 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.
Procedure
 A beta lysin producing strain of staphylococcus aureus is streaked down the center of a sheep
blood agar plate.
  Streptococcus organisms are then streaked across the plate perpendicular to the aureus within
2mm.
 The plate is incubated at 35-37C in ambient air for 18-24 hours.
Enhanced hemolysis is indicated by an arrow head shaped zone of beta hemolysis at the junction of
the two organisms is a positive result whereas no enhancement of hemolysis is a negative result.
The CAMP test is positive for S.agalactiae.

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 diffusible compound.

Procedure.

 With an inoculating wire or loop, touch two or three morphologically similar streptococcal
colonies and inoculate the slant of the bile esculin medium. Alternatively, streak the surface of
a bile esculin plate with streptococcal colonies.
 Incubate the inoculated tube at 35-37°C for 24 hours

Diffuse blackening of more than half of the slant within 24-48 hours indicates esculin hydrolysis. On
plates, black hoes will be observed around isolated colonies and any blackening is considered positive.
All group D streptococci will be bile esculin positive within 48 hours.

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.
Procedure.
 Inoculate one or two colonies of streptococcus D species from an 18-24 hour culture into 6.5%
NaCl broth.
 Incubate the tube at 35-37°C in ambient air for 48 hours.
 Examine tubes for turbidity after 24 hours and if negative, test again at 48 and 72 hours.
The test is positive for enterococcus faecalis and hence forth turbidity is observed
in the test tube and the test is negative for other strteptococcus D so the test tubes remain
clear.

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).
Procedure.
  Using an inoculating loop, select three to four well- isolated colonies of the alpha hemolytic
organism to be tested. An 18-24 hour culture of isolated organism can also be used for testing.
 Streak the isolate onto one-half of a TSA-5% sheep agar plate so as to obtain confluent growth.
Use of any other media other than TSA-5% sheep blood agar is not recommended as a false
result may result.
 Using sterile forceps, place an optochin disk onto the surface of the agar.
 Press the disk gently with the sterile forceps or loop so that the disk adheres firmly to the agar
surface.
 Incubate the plate at 35-37°C for 18-24 hours in 5-10% carbon dioxide enriched environment.
 If zone of inhibition is present, measure the diameter with a millimeter ruler or caliper.

A presumptive identification for S.pneumoniae can be made if the alpha hemolytic colony produces a
zone of inhibition of 14mm or greater around the disk. Organisms producing smaller zone sizes (6-
14mm) should be tested for bile solubility and only identified as a pneumonococci only if bile soluble.
The test is negative when zone of inhibition or a zone of inhibition less than 14mm and it indicates
streptococcus pyogenes and S.mitis species.

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.
Procedure.
 A drop of bile reagent is placed near a suspected 18-24hour colony.
 The drop is then gently rolled over several representative colonies by tilting the plate. The
colonies should not be dislodged with the bile reagent.
 The plate is kept right side up and is incubated at 35°C for 15-30 minutes or until the drop has
evaporated. The plate can also be placed on a heat block as a substitute for the use of an
incubator.
 The flattening of the colony is observed. Proper observation should be made that the colony
does not simply float away.
Bile solubility is demonstrated as disintegration or flattening of the colony within 30 minutes
leaving an area of alpha hemolysis where the colonies were located. A positive test indicates the
presence of S.pneumoniae. a negative result is demonstrated when there is no change in integrity
of the colony within 30 minutes and this is observed for viridans species.
What other tests can be used to differentiate among the Streptococcus species?
The AccuProbe-Pneumococcus test is used to aid in the identification of atypical pneumococci and to
help differentiate between viridans Streptococcus strains
PYR test is a test which is used in the identification of group A beta-hemolytic Streptococci and in the
differentiation of Enterococcus from group D Streptococci. 
 
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.

Procedure 
1. Strip of  filter paper are soaked in a freshly prepared 1% solution of tertramethyl-p-phenylene-
diamine dihydrochloride.
2. After draining for about 30 seconds, the strips are freeze dried and stored in a dark bottle tightly
sealed with a screw cap.
3. For use, a strip is removed, laid in a petri dish and moistened with distilled water.
4. The colony to be tested is picked up with a platinum loop and smeared over the moist area.
5. A positive reaction is indicated by an intense deep-purple hue, appearing within 5-10 seconds, a
“delayed positive” reaction by colouration in 10-60 seconds, and a negative reaction by absence of
colouration or by colouration later than 60 seconds.

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 .
Procedure 
1. With a straight inoculation needle, touch the top of a well-isolated colony.
2. Inoculate TSI by first stabbing through the 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 at 35°-37°C in ambient air for 18 to 24 hours.
4. Examine the reaction of medium.

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.
Procedure 
1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated
colony.
2. Incubate aerobically at 35 to 37C for up to 4-7 days.
3. Observe a color change from green to blue along the slant.
 

Give examples of some of the Gram-negative rods that are positive for this test.
Klebsiella pneumoniae and Proteus mirabilis 
Citrobacter 
Enterobacter serratia 
Pseudomonas aeruginosa

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.
Procedure of Urease Test

1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate slant with
1 to 2 drops from an overnight brain-heart infusion broth culture.
2. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 48 hours to 7 days.
3. Examine for the development of a pink color for as long as 7 days.

Give examples of some of the Gram-negative rods that are positive for this test.
Proteus species 
Klebsiella pneumoniae 

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.

Procedure 
1. Touch a straight needle to a colony of a young (18- to 24-hour) culture growing on agar medium.
2. Stab once to a depth of only 1/3 to ½ inch in the middle of the tube. Be sure to keep the needle in
the same line it entered as it is removed from the medium.
3. Incubate at 35°-37°C and examine daily for up to 7 days.
4. Observe for a diffuse zone of growth flaring out from the line of inoculation.

Give examples of Gram-negative rods that produce hydrogen sulphide

Salmonella species eg Salmonella typhi,typhimurium 
Proteins species eg Proteus mirabilis and Proteus vulgaris 
 

Give examples of Gram-negative rods that are indole positive

Escherichia coli 
Proteus vulgaris

Give examples of Gram-negative rods that are motile

Escherichia coli 
Salmonella typhi
How do the colony morphologies of Enterobacteriaceae differ in different media?

Enterobacteriac Medium in which it is Colony appearance

eae grown
E. coli nutrient agar large, thick, grayish white, moist, smooth opaque or
partially translucent disks
Klebsiella MacConkey agar pink mucoid colonies
Proteus Nutrient agar Swarming
Shigella MacConkey or EMB Colorless (lactose-negative) colonies
agar
Morganella Solid media Non swarming appearance
Providencia MacConkey agar colourless
 

Which bacteria have the characteristic fishy odor?

Proteus mirabilis

What other characteristic of this organism on blood agar is specific to this organism?
These species swarm over the blood agar plate, obscuring the colonies of other organisms.
 

Describe the colony morphology of Pseudomonas aeruginosa

Pseudomonas aeruginosa appear blue-greencolonies.Pseudomonas produces large, opaque,
flat colonies with irregular margins. The blue green colonies are due to production of two
pigments pyocyanin which is blue-green in color, pyoverdine which is yellow-green in color .
Pseudomonas aeruginosa grown in media is also characterized by grape-like smell. Pseudomonas isn’t
lactose fermenting Bt it will still grow on MacConkey using the peptone in the agar. In MacConkey
agar, Pseudomonas aeruginosa forms flat and smooth colonies that are between 2 and 3mm in
diameter. Generally, these colonies have regular margins and have an alligator skin-like appearance
when viewed from above.

Which steps would you take to identify colonies of non-lactose fermenting bacteria?
The bacteria are inoculated on MacConkey agar .
1.Incubate at 35°-37°C 
2.Observe for the result 

Results 
Organisms that ferment lactose and thereby produce an acidic environment will appear pink because
of the neutral red turning red. Bile salts may also precipitate out of the media surrounding the growth
of fermenters because of the change in pH. Non-fermenters will produce normally-colored or colorless
colonies.

Fastidious bacteria: Haemophilus spp.

How do you differentiate among the different Haemophilus spp. in the lab?
The organism is inoculated on nutrient agar . 
Discs of factor X, V, and XV are placed on to the agar 
Growth is observed around disc for factor XY , because Haemophilis is fastidious and requires these
nutrients factor X and Y for growth . 
 
What is satellitetism?
Satellitetism  is the phenomenon in which certain bacterial species grow more vigorously in the
immediate vicinity of colonies of other unrelated species, owing to the production of an essential
metabolite by the latter species.

Principle 
Haemophilus spp. typically grows on chocolate agar as smooth, flat or convex buff, or slightly yellow
colonies. Chocolate agar provides hemin (X factor) and NAD (V factor), necessary for the growth of
Haemophilus spp. Haemophilus influenzae needs both factors, hemin and NAD, for its growth. Blood
agar contains hemin (factor X) but lacks NAD. NAD is also termed as V factor. Although H.
influenzae requires both hemin and NAD, other Haemophilus species require only NAD for growth.
Species that need NAD will not grow on Blood agar unless they are haemolytic and can release the
NAD from the RBCs. However, they will grow well on Blood agar in vicinity to colonies of Staphylococci
which are capable of providing NAD. Staphylococcus aureus, produce NAD as a metabolic by product
which diffuses into the surrounding medium and enhances growth of Haemophilus in the proximity of
the Staphylococcus colony.

How is this test carried out?

Procedure:
1. Culture the sample containing H. influenzae in chocolate agar.
2.Isolate a typical colony and perform Gram staining (H. influenzae is gram negative)
3.Sub-culture the isolated colony to Blood agar and Chocolate agar.
4.When using a sterile inoculating loop place a single streak of Staphylococcus aureus on a blood agar
plate that has been inoculated with a suspected Haemophilus influenzae
5.Incubate plates for 24 h at 35 to 37°C in 5% CO2.
6.Examine for the presence of colonies that satellite around staphylococcus streak on Blood agar.
7.Use colonies from sub cultured chocolate agar plate to perform further tests for identification to the
species level or for susceptibility testing. 
Results interpretation:
 Positive test:
A positive test is indicated by the growth of tiny colonies of Haemophilus influenzae surrounding
the Staphylococcus streak.
Staphylococcus lyses the red blood cells, releasing hemin (x factor) and NAD (v factor) for growth of
Haemophilus spp. Therefore, Haemophilus spp. grow adjacent to the streak line where the nutrients
are available.
A positive result for a tiny gram-negative rod or coccobacillus suggests that the organism is of the
genus Haemophilus.

Gram-negative cocci
What are some of the examples of Gram-negative cocci isolated from clinical specimen?
Neisseria gonorrhoeae
Moraxella catarrhalis 
Neisseria meningitidis

Suppose you isolated Gram-negative cocci from a clinical specimen. How would you conclude which
Gram-negative coccus it is?

Moraxella catarrhalis hydrolyses tributyrin whereas Neisseria species do not.