Laboratory diagnosis of bacteria

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.