steps of bacterial diagnosis in lab

1. Laboratory diagnosis of
bacteria
Prepared by
Samira Fattah
Assis. Lec.
College of health sciences-HMU
Lab 1

2. Methods of bacterial identification
• Microscopic examination.
• Cultural appearance.
• Biochemical reactions.
• Serological identification.
• Animal inoculation.
• Bacteriophage typing.
• Molecular methods.

3. Microscopic examination
• Gram stain:
pneumococci, gonococci, staphylococci ,
meningococci
• Acid fast stain:
mycobacterium tuberculosis, leprae
• Fluorescence :
-direct
-immunoflorescence

4. Fluorescent viability stain for showing live
(green) and dead (red) bacteria

5. Cultural characters
• Pigment production:
o Endopigment (restricted to the colonies):
• Golden yellow with
Staphylococcus aureus.
• White with Staph. epidermidis.
o Exopigment (the color diffuses in the
surrounding medium):
• Green exopigment with
Pseudomonas aeruginosa.

6. Hemolysis on blood agar:
o Complete (beta) hemolysis:
• Staphylococcus aureus and
Streptococcus pyogenes.
o Partial (alpha) hemolysis:
• Streptococcus viridans and
pneumococci.
o No (gamma) hemolysis:
• Enterococci.

7. Effect on lactose of MacConkey’s agar:
o Lactose fermenters:
• Appear as rose pink colonies.
• Example: E. coli & klebsiella.
o Non Lactose fermenters:
• Appear as pale colonies.
• Example: salmonella & shigella.

8. Biochemical reactions
1) Sugar fermentation
2) Indol test
3) Urease test
4) Oxidase test
5) Catalase test
6) Coagulase test
7) DNAse test
8) Gelatinase test

9. - Catalase test:
• Is used to differentiate between staphylococci(catalase +ve)
and streptococci(catalase –ve).
• Principle:
Catalase
enzyme
2 H2O2 2 H2O + O2
• Procedure
– Smear a colony of the organism to a slide
– Drop H2O2 onto smear
– Observe

10. Coagulase test
is used to differentiate Staphylococcus aureus from
coagulase-negative staphylococci.
fibrinogen fibrin
(clot formation)
coagulase

11. Oxidase Test
• All Enterobacteriaceae are oxidase-negative.
• This test is used to differentiate enterobacteriaceae
from Pseudomonas which is oxidase positive.

12. Motility test
• to determine whether a bacterium is motile.
• Non-motile organisms which lack flagella, are usually going to
form a single line of growth that does not spread into the
surrounding area. While a motile bacterium will grow and
make a hazy zone around the stab line.

13. Analytical profile index (API):

14. Automated bacterial identification systems:
o Principle:
- Examples: Vitek system
- These systems identify the organism and its antibiotic
sensitivity by detecting color changes or turbidity in special
plastic cards inoculated with the organism.
- Such cards are composed of tiny wells that contain substrates
for detection of biochemical reactions and antibiotic sensitivity.
- Once the card has been inoculated and placed in the
instrument, it will automatically perform all readings.
- Results are available within 4-6 hours.

15. Vitek system
Vitek card

16. Serological identification
• Antigen detection
– e.g. latex agglutination
• Antibody detection
– e. g. agglutination tests, complement fixation tests, indirect
immunofluorescence

17. Animal inoculation
• The use of laboratory animals (mice, guinea pigs, rabbits) is now
limited due to the advancement in medical microbiological
techniques.
Guinea pig Mouse

18. But they could be used :
1. For growing the organisms that do not grow
on culture such as lepra bacilli.
2. To determine the virulence factor of an
organism. For example if injection of diphtheria
in a guinea pig caused its death, this means
that the organism is toxigenic.

19. Bacteriophage typing
• Bacteriophages are viruses which infect the bacterial
cells and cause their lysis.
• Different types of a certain bacteria are lysed by
different phage groups.
• If a phage is added to a plate inoculated with
susceptible bacteria, a zone of lysis will appear around
the phage drop.
•

21. Molecular methods
Nucleic acid probes:
o Principle
The probe consists of sequence of single stranded DNA or RNA
that forms a covalently bonded hybrid with the specific
complementary strand of the nucleic acid of the organism.
The probe is labeled either to radioactive substance as iodine 131
or enzyme.