Transcript
Laboratory diagnosis of bacteria
Prepared bySamira Fattah
Assis. Lec.College of health sciences-HMU
Lab 1
Methods of bacterial identification
• Microscopic examination.• Cultural appearance.• Biochemical reactions.• Serological identification.• Animal inoculation.• Bacteriophage typing.• Molecular methods.
Microscopic examination
• Gram stain: pneumococci, gonococci, staphylococci , meningococci
• Acid fast stain: mycobacterium tuberculosis, leprae
• Fluorescence : -direct -immunoflorescence
Fluorescent viability stain for showing live (green) and dead (red) bacteria
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.
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.
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.
Biochemical reactions
1) Sugar fermentation2) Indol test3) Urease test4) Oxidase test5) Catalase test6) Coagulase test7) DNAse test8) Gelatinase test
- 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
Coagulase test
is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci. fibrinogen fibrin (clot formation)
coagulase
Oxidase Test
• All Enterobacteriaceae are oxidase-negative.
• This test is used to differentiate enterobacteriaceae from Pseudomonas which is oxidase positive.
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.
Analytical profile index (API):
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.
Vitek system
Vitek card
Serological identification• Antigen detection– e.g. latex agglutination
• Antibody detection– e. g. agglutination tests, complement fixation tests, indirect
immunofluorescence
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
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.
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.
•
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.
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