This document outlines various methods used to identify unknown bacterial species, including microscopic examination, culture-based techniques, biochemical tests, serological identification, and molecular methods. Microscopic examination involves staining bacterial smears to determine cell morphology and arrangement. Cultural characteristics like colony morphology, pigmentation, and hemolysis are also examined. Biochemical tests identify metabolic pathways and enzyme production. Serological tests use antibodies to identify species. Molecular methods like PCR and DNA probes allow for rapid and accurate identification compared to older phenotypic techniques. A combination of these methods is usually required to definitively identify an unknown bacterial species.

1. Prepared By
DR. Hanan Fathy
DR. Sabah Al-Khawagah
Diagnosis of Bacterial
Diseases

2. Intended learning outcomes (ILOs)
 Describe how staining, culture and biochemical
tests are used to identify bacteria.
 List the major biochemical tests used to identify
bacteria.
 Explain how serological tests can be used to
identify an unknown bacterium.
 To identify the importance of animal inoculation in
bacterial identification.

3. Intended learning outcomes (ILOs)
 To understand the principle and uses of bacteriophage
typing.
 To know the principle and uses of antibiotic sensitivity
test.
 To differentiate between different molecular methods for
bacterial identification.
 To identify phenotypic and genotypic methods used for
bacterial typing.

4. Bacterial identification
 The methods used for isolation and identification of organisms
are:
1- Direct microscopic examination of:
✓Fresh unstained film to demonstrate the motility of the
organism.
✓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).

5. Gram negative cocci (pink in
colour)

6. Gram positive bacilli (violet in
colour)

7. Spiral shape bacteria (spirochetes)
Gram-negative curved bacilli (V. cholera)

8. Gram positive cocci arranged in
clusters

9. 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).

10. Pseudomonas culture on nutrient
agar produce greenish exopigment.
Staph culture on nutrient agar
produce endopigment.
1: Staph. Aureus.
2: Staph. albus
3: Staph. citreus
1
2
3

11. Culture of E.coli on MacConky
medium (dry rose pink colonies)
Culture of Klebsiella on MacConky
medium (mucoid rose pink colonies))

12. 1
3
2
Proteus on nutrient agar showing
swarming growth
Blood agar plate showing:
1- Complete hemolysis (Beta)
2- Partial hemolysis (Alpha)
3- No hemolysis (gamma))

13. 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

14. 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.

15. 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.
Un-inoculated

16. Indole test
▪ 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
yellow ring.

17. 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
yellow colour.

18. 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).

19. Catalase test
 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
positive test.

20. Coagulase test
 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.

21. Urease test
 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
hr.

22. Oxidase test
 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.

23. 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.

24. 5- Serological tests:
▪ Slide agglutination test is used for identification of unknown
colony of bacteria using commercially prepared antisera.

25. 6. Animal pathogenicity test
●Animal inoculation:
Animal commonly used:
-Guinea pigs.
-Mice.
-Rabbits.
● Importance of animal pathogenicity test;
-Identify certain pathogenic bacteria e.g. T.B
bacilli (characteristic lesion).
-Determine degree of pathogenicity of certain
bacteria.
-Differentiate between related organisms.
-Isolation of organism in pure culture.
-Test ability of toxin production.
-Evaluation of vaccine and antibiotics.

26. 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.

27. Bacteriophage typing
A number of specific phages leads to lysis of the bacteria.

28. 8. Antibiotic susceptibility test
●Used for selection of the proper antimicrobial agent to be used for treatment.
●Disc diffusion method.

29. Antibiotic sensitivity test (Disc diffusion method)

30. 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.

32. Dot blot hybridization

33. Conventional PCR

35. Agarose gel electrophoresis

36. Polymerase chain Reaction (PCR)

37. 9. Bacterial typing
I- Phenotypic methods:
● Biotyping.
● Antibiotic susceptibility.
● Bacteriophage typing.
● Serotyping.
II- Genotypic methods:
● Plasmid finger printing.
● Restriction fragment length polymorphism (RFLP).

38. Genotyping
Plasmid finger printing:
•Separation of plasmid DNA from
chromosomal DNA.
•Plasmid DNA is subjected to restriction
enzyme digestion.
•Gel electrophoresis separate the DNA into
a number of bands that are considered as
a fingerprint to each strain.
RFLP Plasmid fingerprint

39. Plasmid Finger Printing