Biochemical Tests
1- Catalase Test:
Purpose:
It is used to differentiate between Catalase positive-bacteria, such as
staphylococci and Catalase negative-bacteria such as streptococci.
Principle:
Bacterial Catalase Enz.
2 H2O2
2 H2O + O2 (Bubbles)
Methods:
1. Slide Method
2. Tube Method
1- Slide Method:
Take 1-2 drops of 3% H2O2 on a slide.
Mix it with a small quantity of
samplebacteria.bacteria.
2- Tube Method:
Pour 1-2 drops of 3%hydrogen peroxide (H2O2) into a test tube.
Mix it with a small quantity of sample bacteria.
Dr. Refat Al-hakimi
Results:
bubbles production
No bubbles production
positive Catalase bacteria
(Staphylococcus species)
negative Catalase bacteria
(Streptococcus species)
Catalase positive bacteria
Staphylococcus species
Bacillus
Listeria onocytogenes
Gonococcus& Meningococcus
Vibrio cholerae
Campylobacter & shigella
Coagulase Test
Purpose:
It is used to differentiate between Staphylococcus aureus and other
Staphylococcus species .
Principle
Bacterial coagulase
Fibrinogen
fibrin coagulation (clot)
 types of coagulase are produced by Staphylococcus Saureus:
1- Free coagulase
2- Bound coagulase
Free coagulase:
converts fibrinogen to fibrin by
activating a coagualse-reacting
factor present
Oxidase
test in plasma. It is
detected by clotting in
the tube test
Dr. Refat Al-hakimi
Bound coagulase
(clumping factor):
converts fibrinogen directly to
fibrin without requiring a
coagulase-reacting factor. It
can be detected by clumping
of bacterial cells in
the rapid slide test.
Methods:
A- Slide Method
B- Tube Method
A- Slide Method
1- Take one drop of human plasma on a slide.
2- Take part of test colony to the slide by a plastic or wooden
stickacteria.
3- Mix well and look for Clumping (clots )within 10 seconds.
4- acteria.
Results:
Clumping or clots within 10 seconds
No clumping within 10 seconds
coagulase positive
coagulase negative
S. aureus.
other staph.
B- Tube test method (detects free coagulase):
pour 3-4 drops of human plasma into a test tube.
Well Mix it with 12-16 drops of sample bacteria.
— incubate the tubes at 35–370C for 6-12 hours and examine hourly.
— If the test is still negative, leave the tube at room temperature
overnight and examine again.
Dr. Refat Al-hakimi
+
Results:
Clumping or clots in tube
No clumping in tube
Dr. Refat Al-hakimi
positive coagulase
negative coagulase
S. aureus.
other staph.
DNaseTest
Purpose
 It is used to help in the identification of Staph. aureus
 The DNase test is useful when plasma is not available to a coagulase test
or when the results of a coagulase test are difficult to interpret.
Principle
Bacterial DNase enzy.
DNA
DNA hydrolysis (clear zone)
Requirements
 DNA-ase agar.
 Hydrochloric acid solution 1 mol/l (1N).
Method:
Inoculate a plate of DNase agar with a test colony.
Incubate in 37 ˚C overnight.
Cover the surface of the plate with 1 ml/l hydrochloric acid solution.
Look for clearing around the colonies within 5 min of adding the acid.
+
-
Results
Clearing around the colonies
positive DNA-ase
Staphylococcus aureus.
No clearingaround the colonies
Negative DNA-ase
Staphylococcus epidermidis
Dr. Refat Al-hakimi
Bile solubility test
Purpose
It helps to differentiate Streptococcus Pneumonia (soluble in bile and bile
salts), from viridans streptococci (insoluble).
Method
Mix several colonies in 2 ml sterile physiological saline to give suspension.
Divide the suspension between two tubes.
In 1st tube; add 2 drops of the sodium deoxycholate reagent and mix.
In 2nd tube (negative control); add 2 drops of sterile distilled water and mix.
Leave both tubes for 10–15 minutes at 35–37 0C.
Look for a clearing of turbidity in the 2nd tube
Results
Clearing of turbidity
No clearing of turbidity
Dr. Refat Al-hakimi
bile is lysed
bile is not lysed
Strept. Pneumoniae.
viridans streptococci
Oxidase Test
Purpose:
It is used to differentiate between Oxidase and non-oxidase product
bacteria.
Principle:
Bacterial Oxidase Enz.
Phenylenediamine
Deep purple color
Method:
puta piece of filter paper in a clean petri dish.
Add 2 or 3 drops of oxidase reagent on the filter paper.
Take a small quantity of sample bacteriaand Mix it by a wooden stick.
Observe for a color change to a deep blue-purple within 10 seconds.
Results
No color appears within 10 seconds
negative Oxidase.
Deep blue-purple within 10 seconds
positive Oxidase
Examples for Oxidase positive
Neisseria
Pseudomonas
Brucella
Haemophilus
Vibrio
campylobacter
Dr. Refat Al-hakimi
Indole Test
Purpose:
It is important for identification of enterobacteria.
Principle:
Bacterial Enzyme
Tryptophan(amino acid)
Indole production(red ring)
Method:
Prepare peptone water andInoculate samplein peptone water.
Incubate overnight in 37 ˚C.
After incubation period, add drops of Kovac’s reagent to the tube.
Shake gently and then Observe for a red ring on the surface layer within 10
minutes.
-+
Results:
Show as red ring
Remain as yellow ring
Indole positive
Escherichia coli
Proteus vulgaris
Dr. Refat Al-hakimi
positive Indole
negative Indole
Urease Test
Purpose:
It is important for differentiating between enterobacteria that produce the
urease from non- product.
Principle:
Bacterial Urease Enzyme
Urea
Ammonia + Carbon Dioxide (Co2)
Method:
Inoculate a tube of urea agar with a test colony.
Incubate in 37 ˚C overnight.
Observe after 4 hours for a change in color to pink or red.
Results:
Bright pink or bright red color
Yellow color
positive Urease
Proteus
Klebsiella
Dr. Refat Al-hakimi
positive Urease
negative Urease.
Citrate Test
Purpose
It is used to assist in the identification of Enterobacteria.
Principle:
This test is based on ability of bacteriainconsumption of citrate as its
only source of carbon.
Method by using Simmon’s citrate agar (Green color)
Prepare slopes of the medium
First streak the slope with a saline suspension of the test organism and
then stab the butt by using a sterile straight wire
.
Incubate at 350C for 48 hours.
Look for a bright blue color in the medium
Results
Bright blue
No change in colour
Dr. Refat Al-hakimi
Positive citrate (Klebsiella and citrobacter)
Negative citrate
Triple Sugar Iron Test (TSI) or Kligler iron agar (KIA)
Purpose
It is used to differentiate among the members of Enterobacteriaceae
(such as E. coli, Salmonella, Shigella, Klebsiella, Enterobacter etc).
Principle
It differentiates bacteria on their ability to ferment glucose, lactose,
and/or sucrose and on their ability to reduce sulfur to hydrogen sulfide
gas (H2S).
Method
Touch the top of a well isolated by a straight inoculating wire colony.
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.
Incubate the tube for 18‐24 hours at 35oC in an incubator.
Read the result by colour of media
Notes
Red /Red
non-inoculated or negative
Yellow/Yellow
Red /Yellow
Dr. Refat Al-hakimi
Lactose fermentation(coliform bacteria)
Non-lactose fermentation (other enterobacteria)
Red /Yellow
false result (contamination)
SIM agar method (Sulfide-Indole-Motility)
Purpose
It is used for detection H2S production, Indole production and Motility
of the bacteria.
Principle
In Indole
appear red or pink ring
in H2S
appear blackening (due to ferrous sulphate production)
in Motility
appear turbidity (due to motile bacteria)
Method
Inoculate test organism two-thirds into the medium by stabbing.
Incubate at 37°C for 18 –24 hours.
Examine tubes after incubation for motility and H2S production.
After determining motility and H2S production;
add 3-4 drops of Kovac’s Reagent.
Record result of indole
Pink or red
color ring
Dr. Refat Al-hakimi
No color
change
Positive
Negative
Indole
Indole
Escherichia coli in SIM
Medium
Motile, indole positive and
hydrogen sulfide negative
positive indole
(Escherichia coli and
Proteus vulgaris)
negative indole (other
enterobacteria)
Proteus mirabilis in
Motile, indole negative
and hydrogen sulfide
positive
H2S positive
indole positive
Motile positive
Proteus vulgaris
Escherichia coli
All enterobacteria are motile
except:
Proteus mirabilis
Salmonella Typhi
Proteus vulgaris
Morganella
Morganii
Vibrio cholera
Vibrio
parahaemolyticus
Dr. Refat Al-hakimi
Shigella species
non
motile
Klebsiella pneumoniae
Identification of Staphylococcus aureus
LABORATORY FEATURES
Specimens:
1- Pus
2- swabs from infected sites
3- sputum
4- cerebrospinal fluid
5- blood for culture.
6- Faeces when foodpoisoning is suspected.
Microscopic examination
Staphylococci are Gram positive cocci graplike clusters also sometimes
singly and in pairs They are non-motile and non-capsulate.
Culture
- Most Staphylococci grow well aerobically and few strains grow
anaerobically
- Temperature range for growth is 10–42 ºC, with an optimum of 35–37C.
A- Blood agar, chocolate (heated blood)agar:
- S. aureus produces yellow to cream colonies after overnight incubation
- Some strains are beta hemolytic when grown aerobically.
- Colonies are slightly raised.
Dr. Refat Al-hakimi
S.aureus
S.epidermidis
B- MacConkey agar:
- Smaller colonies are produced after overnight incubation at 35–37 ºC.
- Most strains are lactose fermenting.
C- Mannitol salt agar:
- A useful selective medium for isolating S. aureus from faecal specimens
when investigating staphylococcal food-poisoning.
- It can also be used to screen for nasal carriers.
- S. aureus ferments mannitol and is able to grow on agar containing
70–100g/l sodium chloride.
- Mannitol salt agar containing 75 g/l sodium chloride .
Biochemical tests
S. aureus is:
- Catalase positive .
Dr. Refat Al-hakimi
- Coagulase positive.
- DNase positive.
Novobiocin
S.epidermidis -------------------------Susceptible for Novobiocin
S. saprophyticus----------------- Resistant for Novobiocin
Dr. Refat Al-hakimi
Identification of Streptococcus pyogenes
LABORATORY FEATURES
Specimens:
1- throat swab (avoiding saliva contamination)
2- swabs of pus
3- serous fluid depending on the site of infection
4- blood for culture.
Culture media should be inoculated as soon as possible or a swab placed in a
tube of silica gel.
Microscopic examination
- Streptococci are Gram positive cocci arranged in short chains, but also in
pairs and singly
- Long chains are formed in fluid cultures.
- The organisms are non-motile.
Culture
Blood agar:
- S. pyogenes produces betahaemolytic colonies (clear zone of complete
haemolysis ) small, colourless, dry, shiny or mucoid colonies.
Dr. Refat Al-hakimi
- Haemolysis is more marked under anaerobic conditions as seen in colonies
growing below the agar surface .
Note : Do not use human blood because this may contain unwanted substances
such as citrate (e.g. donor blood), antibiotics, or antibodies such as ASO or
anti-M protein that could interfere with the growth of S. pyogenes.
MacConkey agar:
S. pyogenes does not grow on this medium
Biochemical tests
S. pyogenes is:
- Catalase is negative
bacitracin
S. pyogenes -----------------------Sensitive for bacitracin
S.agalactiae----------------------Resistant for bacitracin
Serology
- ASO antibody in serum is helpful in diagnosing rheumatic fever.
Dr. Refat Al-hakimi
Identification of Streptococcus pneumoniae
LABORATORY FEATURES
Specimens:
1234-
Sputum.
exudate.
blood for culture.
cerebrospinal fluid.
Microscopic examination
- S. pneumoniae is a Gram positive elongated (lanceolate) diplococcus also
forms short chains.
- Pneumococci are capsulated and non-motile .
- In Gram stained smears from specimens, the capsule can often be detected as
an unstained empty area around the diplococcus .
Culture
Blood agar:
- S. pneumoniae forms translucent or mucoid colonies, 1–2 mm in diameter.
- In young cultures the colonies are raised but later become flattened with
raised edges, giving them a ringed appearance .
- Pneumococci show alpha-haemolysis, i.e. colonies are surrounded by an
area of partial haemolysis or green colour in the medium (reduced Hb).
Note: When cultured anaerobically on blood agar, some strains of S.
pneumoniae show betahaemolysis
Dr. Refat Al-hakimi
Chocolate and lyzed blood agar:
- S. pneumonia grows well on chocolate agar and lyzed blood agar.
- Growth is enhanced when incubated in a CO2 enriched atmosphere
(candle jar).
Biochemical tests
S. pneumoniae is:
- Catalase negative.
- Sensitive to optochin .
- Bile soluble.
Rapid latex and coagglutination tests to detect capsular pneumococcal antigen
Optochin
Pneumococci--------------Sensitive to optochin
Dr. Refat Al-hakimi
Identification of Neisseria meningitidis
LABORATORY FEATURES
Specimens
123-
CSF.
blood for culture
swabs from haemorrhagic skin lesions .
Microscopic examination
-N. meningitides is a non-motile gram negative diplococcus arranged in groups.
-In smears from specimens, meningococci are found intracellular .
Culture
- N. meningitides is an aerobe with primary cultures growing best in a moist
CO2 enriched atmosphere .
- The temperature range of growth is 25–42 ºC with an optimum of 35–37 ºC.
- Enriched media are required for isolation.
-Specimens should be cultured as soon as possible after collection.
- recommend inoculating C.S.F. into cooked meat medium .
Blood cultures
- Meningococci grow well in Columbia diphasic medium .
- Because sodium polyanetholsulphonate (SPS)may be inhibitory to
meningococci, may be add sterile 1%gelatinto neutralize the effect of SPS.
- Subculture a positive blood culture onto chocolate agar and incubate in CO2.
Dr. Refat Al-hakimi
Chocolate (heated blood) agar
- N. meningitides produces transparent or grey, shiny, 1–2mm colonies .
- The colonies of group B meningococci often appear grey-yellow.
Note: N. meningitidis also grows on Mueller Hinton agar without add of blood.
Biochemical tests
N. meningitidis is
oxidase --positive
Serology
- Direct latex agglutination and coagglutination slide antigen tests
are available to detect antigens to the main groups of meningococci .
Dr. Refat Al-hakimi
Identification of Neisseria gonorrhoeae
LABORATORY FEATURES
Specimens
1- Urethral and cervical exudates.
2- urine (centrifuged) from male patients
3- rectal swab.
4- An eye swab (gonococcal conjunctivitis in newborn is suspected).
Microscopic examination
- N. gonorrhoeae is a non-motile, non-capsulate gram negative diplococcus,
typically seen in pus cells (intracellular) but also extracellularly.
- Morphologically gonococci look the same as meningococci.
Making smears: a swab should be gently rolled on a slide when making a
smear and the preparation methanol-fixed rather than heat-fixed.
Culture
- N. gonorrhoeae is an aerobe or facultative anaerobe.
- It is a fastidious organism Which requires culturing with the minimum of
delay.
Dr. Refat Al-hakimi
- Some strains are unable to survive drying or temperatures below body
temperature.
- An enriched selective medium such as modified New York City medium or
Thayer
Martin medium is required to isolate N. gonorrhoeae from urogenital
specimens.
- Gonococci grow best in a moist carbon-dioxide enriched atmosphere .
- Optimum temperature for growth is 35–36 ºC.
MNYC medium and Thayer Martinmedium
- N. gonorrhoeae grows rapidly producing small, raised, grey or translucent
colonies after overnight CO2 incubation .
- Examine a gram stained smear of the colonies and perform an oxidase test .
Chocolate agar only is available for isolating N. gonorrhoeae.
Biochemical tests
- N. gonorrhoeae is oxidase positive.
Note: Neisseria colonies are strongly oxidase positive and immediately turn
deep Purple the reagent of oxidase is rapidly bactericidal (see above).
Serology
Coagglutination tests for identifying N. gonorrhoeae isolates have been shown
to be specific and highly sensitive.
Dr. Refat Al-hakimi
Identification of Escherichia coli
LABORATORY FEATURES
Specimens
1- Urine
2- Pus
3- Faeces
4- Cerebrospinal fluid(infants)
5- Blood.
Microscopic examination
E. coli is a gram negative usually motile rod.
Culture
- E. coli is a facultative anaerobe.
-Optimum temperature for growth is 36–37 ºC with most strains growing
over the range18–44 ºC.
MacConkey agar and CLED agar:
- E. coli is ferments lactose, producing smooth pink colonies on MacConkey
agar and yellow colonies on CLED agar.
Dr. Refat Al-hakimi
CLED
XLD and DCA agar:
- Yellow colonies are produced on XLD agar.
- Growth of E. coli is usually inhibited on DCA agar.
XLD
Blood agar:
Colonies of E. coli may appear mucoid and Some strains are haemolytic.
Dr. Refat Al-hakimi
Biochemical tests
- KIA:----- acid /acid(Y / Y )with gas production and no H2S production.
- Indole----------------positive
- urine nitrite test------positive.
- Citrate and H2S-----negative.
- IMViC test------ (+ + _ _)
Dr. Refat Al-hakimi
Identification of Klebsiella species
LABORATORY FEATURES
Specimens:
1- Urine.
2- Pus.
3- Sputum .
4- Infected tissue.
Microscopic examination
Klebsiellae are Gram negative, non-motile, usually capsulated rods.
Culture
Klebsiellae are facultative anaerobes.
Blood agar:
Klebsiellae produce large , grey white usually mucoid colonies.
Dr. Refat Al-hakimi
MacConkey agar and CLED medium:
- Most klebsiellae are lactose-fermenting, producing mucoid pink colonies
on MacConkey agar and yellow mucoid colonies on CLED medium .
Biochemical tests
- KIA:--------acid /acid(Y / Y )with gas production& no H2S production.
- Citrate-----positive.
- Urea -------positive.
-Indole------negative.
Dr. Refat Al-hakimi
Identification of Salmonella species
LABORATORY FEATURES
Specimens
ENTERIC FEVER
1- Blood---- detected during the first ten days of infection.
2- Faeces---detected during the third week of infection
3- Urine ----- isolated from about 25% of patients after the second week
of Infection especially from those with urinary schistosomiasis.
DIARRHOEAL DISEASE
1- Faeces
2- Blood (during times of fever)
BACTERAEMIA
- Blood
Microscopic examination
Salmonella are gram negative rods, actively motile, non-sporing and
non-capsulate (exception of S. Typhi that is a capsulated).
Culture
- Salmonella are facultative anaerobes.
- They grow at 37 ºC(an optimum temperature).
XLD agar:
- H2S producing salmonellae form pink-red colonies with black centres.
- Non H2S producing salmonellae form pink-red colonies without black centres
Dr. Refat Al-hakimi
DCA and MacConkey agar:
- Salmonellae produce non-lactose fermenting pale coloured colonies which
on DCA, have black centres(H2S-producing salmonellae).
Blood agar (subculture):
Salmonella produce grey-white ,non haemolytic colonies.
Biochemical tests
- KIA –---------alkaline/ acid (pink/ yellow),gas production
(exception of S. Typhi)and H2S production with S. Typhi.
- Urease, lactose and indole ------negative
Serological tests
Widal test
- Ig M antibody immunoassays.
-
Dr. Refat Al-hakimi
Identification of Shigella species
LABORATORY FEATURES
Specimens:
- A fresh faecal specimens may be watery with little blood, mucus,
and pus cells also have an alkaline pH unlike amoebic dysentery have an
acid PH.
Microscopic examination
- Shigellae are Gram negative, non-sporing, and non-capsulate rods
also non-motile.
Culture
- Shigellae are facultative anaerobes.
- They grow at 37 ºC (an optimum temperature).
- A selective medium is required to isolate Shigella species from faeces.
XLD agar:
- Shigellae produce red-pink colonies without black centres.
DCA and MacConkey agar:
- Shigellae produce non-lactose fermenting pale coloured colonies.
- On prolonged incubation, S. sonnei forms pink colonies.
Salmonella-Shigella (SS) agar
- Despite its name, this medium is not suitable for isolating shigellae as it is
inhibitory to most strains.
Biochemical tests
- KIA –---------alkaline/ acid(pink/yellow) .
- Lactose ------negative (S. sonnei is a late lactose and sucrose fermenter).
- H2S----------negative.
- Urease -------negative.
- Oxidase -----negative.
- Citrate -------negative.
Dr. Refat Al-hakimi
Identification of Proteus
LABORATORY FEATURES
Specimens
1- Urine
2- Pus
3- sputum.
Microscopic examination
- They are gram negative pleomorphic rods, actively motile, non-capsulate.
- Motility is not as easily observed at 35–37 ºC .
Culture
Blood agar:
When cultured aerobically, most Proteus cultures have a characteristic
‘fishy’odour. Also produce of Swarming on surface of media.
Bile salt, alcohol, dying, chemicals and increasing of agar inhibit of swarming.
MacConkey, CLED, XLD media:
- Proteus produces individual non-lactose fermenting colonies .
- Swarming is prevented on MacConkey agar and XLD agarbecause these
media contain bile salts.
- Swarming is inhibited on CLED agar because it has an electrolyte deficient.
Dr. Refat Al-hakimi
Biochemical tests
- KIA-----alkaline/ acid (pink/ yellow) gas production and H2S production.
- Urea----positive rapidly (differentiate it from salmonellae and shigellae).
- Phenylalanine deaminase-------positive.
- Beta-galactosidase (ONPG) negative.
- Indole negative (P. vulgaris is indole positive).
Dr. Refat Al-hakimi
Identification of Pseudomonas aeruginosa
LABORATORY FEATURES
Specimens
12345-
Pus
Urine
Sputum
Effusions
Blood for culture.
Microscopic examination
- P. aeruginosa is a Gram negative, non-sporing motile rod.
- Some strains are capsulate.
Culture
- P. aeruginosa is an obligatory aerobe.
- It is usually recognized by the pigments it produces including pyocyanin a
blue-green pigment and pyoverdin (fluorescein) a yellow-green fluorescent.
- Cultures have a distinctive smell due to production of 2-minoacetophenone.
- P. aeruginosa grows at 35–37 ºC (an optimum temperature).
Blood agar:
- P. aeruginosa produces large, flat ,spreading colonies which are often
Haemolytic and usually (90% of strains) pigment-producing.
- The pigments diffuse into the medium giving it a dark greenish-blue colour.
- When the culture is left at room temperature ,pigment colour is more intense.
Dr. Refat Al-hakimi
MacConkey agar and CLED medium:
- P. aeruginosa produces pale coloured colonies on MacConkey agar and
green colonies on CLED medium.
- Compared with blood agar ,pigment production is less marked.
Biochemical reactions
- KIA-----------pink/ pink (alkaline/ alkaline),no gas and no H2S production.
- Oxidase------positive.
KIA
Dr. Refat Al-hakimi
Identification of Haemophilus influenzae
LABORATORY FEATURES
Specimens
1234-
cerebrospinal fluid (CSF.)
nasopharyngeal specimens
pus
blood
 Specimens must be cultured as soon as possible and not refrigerated.
Dr. Refat Al-hakimi
Microscopic examination
- H. influenzae is a small non-motile Gram negative coccobacillus or short rod.
- Long thread-like and pleomorphic forms may be seen in CSF (with pus cells)
or following culture.
- It is best stained using dilute carbol fuchsin as the counterstain.
- The capsule can be demonstrated by using specific antiserum.
Culture
- H. influenzae grows poorly anaerobically.
- Growth is best achieved in a moist CO2 enriched atmosphere.
- They grow at 37 ºC (an optimum temperature).
- Media must contain haemin or other iron-containing porphyrin and
nicotinamide adenine dinucleotide (NAD) or its phosphate (NADP).
- The porphyrin requirement is referred to as growth factor X and the NAD
or NADP requirement as growth factor V(from s.aureus).
- H. influenzae grows on chocolate agar because it contains factors X and V.
- the media must be prepared from horse or rabbit blood but no sheep blood.
- H. influenzae produces very small colonies on horse or rabbit blood agar
(colonies may appear betahaemolytic).
Biochemical reactions
- They are not used routinely to identify Haemophilus species.
Serological tests
They are commercially available for the identification of H. influenzae in
cultures and specimens by agglutination techniques.
Dr. Refat Al-hakimi