Intended learning outcomes (ILOs)
Describe how staining, culture and biochemical
tests are used to identify bacteria.
List the major biochemical tests used to identify
Explain how serological tests can be used to
identify an unknown bacterium.
To identify the importance of animal inoculation in
Intended learning outcomes (ILOs)
To understand the principle and uses of bacteriophage
To know the principle and uses of antibiotic sensitivity
To differentiate between different molecular methods for
To identify phenotypic and genotypic methods used for
The methods used for isolation and identification of organisms
1- Direct microscopic examination of:
✓Fresh unstained film to demonstrate the motility of the
✓Stained film e.g. by Gram stain to study shape (cocci or
bacilli…), size (small or large…), arrangement (group or
chains…) and staining reaction (Gram + or -ve).
2- Cultural Morphology:
This includes the colony morphology;
▪ Size& shape
▪ Mucoid or dry
▪ Opaque or translucent
▪ Pigment production
▪ The effect of the colony on the surrounding medium ( e.g.
haemolysis on blood agar and swarming on nutrient agar).
Pseudomonas culture on nutrient
agar produce greenish exopigment.
Staph culture on nutrient agar
1: Staph. Aureus.
2: Staph. albus
3: Staph. citreus
Culture of E.coli on MacConky
medium (dry rose pink colonies)
Culture of Klebsiella on MacConky
medium (mucoid rose pink colonies))
Proteus on nutrient agar showing
Blood agar plate showing:
1- Complete hemolysis (Beta)
2- Partial hemolysis (Alpha)
3- No hemolysis (gamma))
3- Biochemical reactions:
• Sugar fermentation
• Triple sugar iron test
• Indole test
• Methyl red test
• Citrate utilization test
• Catalase test
• Coagulase test
• Urease test
• Oxidase test
Sugar fermentation test
▪ A set of 5 tubes containing glucose, lactose, maltose, mannite and
sucrose are used.
▪ A positive test gives red reaction with or without gas production.
Triple sugar iron test (TSI)
▪ This test measures sugar fermentation (glucose, lactose and sucrose
and detects H2S production.
▪ Positive H2S gives black coloration of the butt.
▪ This test demonstrate the ability of the bacteria to decompose
the amino acid treptophan present in peptone water and
production of indole which is tested by Kovac's reagent.
▪ Positive reaction gives pink ring while negative one produce
Methyl red test (MR)
This test is done to detect the ability of the bacteria to produce
large amount of acid on fermentation of glucose. Few drops of
MR indicator are used.
Positive reaction gives red colour while negative one gives
Citrate utilization test
The organism that able to utilize the sodium citrate as the
source of carbon, can grow on the media with change of its
colour from the green to blue.
The organism that cannot utilize the citrate, gives no growth
and no change in colour (green).
This test detects the catalase enzyme production by some
baceria e.g. Staphylococci.
It is done by adding a drop of hydrogen peroxide on the test
colony on N. agar.
Rapid effervescence due to oxygen gas production indicates a
Coagulase test is used to detect free coagulase enzyme by
adding few drops of an overnight broth culture of the test
organism to 0.5 ml of human or rabbit plasma diluted 1/10.
A positive result gives a distinct clot.
Some bacteria e.g. Proteus produce urease enzyme which
decomposes urea with the release of amonia. This leads to
alkalinity of medium and change colour of the indicator.
Urease positive bacteria will turn the medium pink after 4-24
Some bacteria produce oxidase enzyme which can reduce
oxidase reagent to a deep purple colour.
The tested colony is picked and smeared on a strip of filter
paper impregnated with oxidase reagent.
4- Bacterial motility on soft agar:
▪ It is tested by stabbing the bacteria in deep column of soft agar.
▪ Non motile organism grows along the stab only.
▪ Motile bacterial growth appears radiating from the stab line
and also on the upper surface of agar.
5- Serological tests:
▪ Slide agglutination test is used for identification of unknown
colony of bacteria using commercially prepared antisera.
6. Animal pathogenicity test
Animal commonly used:
● Importance of animal pathogenicity test;
-Identify certain pathogenic bacteria e.g. T.B
bacilli (characteristic lesion).
-Determine degree of pathogenicity of certain
-Differentiate between related organisms.
-Isolation of organism in pure culture.
-Test ability of toxin production.
-Evaluation of vaccine and antibiotics.
7. Bacteriophages typing
● Lysis of bacteria by one or a series of specific bacteriophages.
● Used to trace the source of infection during epidemics e.g. Staph. aureus.
8. Molecular methods for bacterial identification
● DNA probe:
- Single stranded labeled DNA (probe) hybridize with complementary nucleic
acid sequence in specimen to form double stranded DNA.
● Polymerase chain reaction (PCR):
-Extraction of DNA.
-Amplification of DNA using specific primers in special apparatus.
-The amplified DNA (amplicon) run in gel electrophoresis to it into bands.
● Real-time PCR:
-Automated closed system.
-Rapid and quantitative but expensive.
Plasmid finger printing:
•Separation of plasmid DNA from
•Plasmid DNA is subjected to restriction
•Gel electrophoresis separate the DNA into
a number of bands that are considered as
a fingerprint to each strain.
RFLP Plasmid fingerprint