Process Bacteriological laboratory Diagnosis
Bacterial Identification Revealing of the organism up to species level, itinvolves the use of methods to obtain pure cultures from single colonies, followed by tests designed to characterize and identify the isolate can be done by: Culture characteristics and Colonial Morphology. Biochemical Reactions. Toxin production. Animal inoculation. Serologic Tests. Molecular Tests.
Colonial Morphology: Appearance • Macroscopic growth features: • Shape. • Margin. • Elevation. • Size. • Texture. • Appearance. • Pigmentation. • Opacity.
Colonial Morphology: Pigment production Endopigment production Exopigment production Pseudomonas aerogenosaproduces green pigment • E.g. golden colonies with staphylococcus aureus
Colonial Morphology: Haemolysis on Blood Agar Beta haemolysis Alpha Haemolysis E.g. streptococcus mutans • E.g. streptococcus pyogenes
Colonial Morphology: Indicator media MacConkey agar Mannitol Salt agar E.g. staphyolcoccusaureusis Mannitol fermenter • E.gE.coli is a lactose fermenter
Biochemical reactions • he ability of the organism to attack various substrates to produce particular metabolic products. Common biochemical reactions involve: • Presence or absence of single enzyme: • Catalase test: • Oxidase test: • Coagulase test: • Urease test: • DNAase test • Indole test: • Presence of metabolic pathways; • Sugar fermentation test. • Citrate utilization. • VP test. • Methyl red test. • Other: • H2S production
Catalase test: Principle: Testing the ability of bacteria to produce the enzyme which is capable to break H2O2 to produce water& oxygen +vecatalase test appear as bubble in the medium e.g. catalase +ve (staphylococci)
Oxidase test: Principle: The cytochrome oxidase enzyme catalyzes the transport of electrons from a donor compound to the final electron acceptor, oxygen. using an artificial electron donor, phenylenediamine, a redox is used to reduce the cytochrome oxidase. If the enzyme is present, the colorless dye will turn a purple to blue color. E.g. Oxidase +ve (Pseudomonas, Neisseria)
Coagulase test Co-agulaseis the enzyme produced by staphylococcus aureuswhich is capable of converting fibrinogen to fibrin (to produce clot). Two types of the test: Tubes coagulase Slide coagulase To detect the free coagulase To detect the bound coagulase +VE -VE +VE -VE 11
Urease test • tests the ability of an organism to produce an enzyme urease. • hydrolyzes urea to ammonia and carbon dioxide. • The broth contains the pH indicator phenol red. • Phenol red turns yellow in an acidic environment and pink in an alkaline environment. • urea is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink. • E.g to urease positive (proteussp and helicobacter pylori)
Indole test • The indole test examines the ability of a bacteria to breakdown the amino acid tryptophan. • The presence of indole is confirmed by the addition of a few drops of Kovac's or Ehlrich reagents. • The red ring at the top indicates a positive test. • E.g. to indole positive E.coli.
Hydrogen sulphide (H2S) production • his test determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism. • agar contains peptone, sodium thiosulfate, and ferrous ammonium sulfate. • When hydrogen sulfide gas is produced, it combines with iron compounds to produce FeS, a black precipitate. • E.g to H2S producer (salmonella)
Sugar fermentation test • Testing the ability of the bacteria to enzymatically converting sugars to acid (lactic acid, acetic acid etc.), neutral (ethyl alcohol etc.), gases (carbon dioxide, hyrogen, etc). • The medium contains: • Broth. • Suger tested (glucose , lactose etc). • pH indicator phénol red (yellow in acidic medium) . • Durham‘stubes for gas collection.
Citrate utilization test Testing ability of the bacteria to use Citrate as carbon source. The Simmon'sCitrate Agar Slant contains: Sodium Citrate as the carbon source. Ammonium Phosphate as the nitrogen source. Bromthymolblue as the pH indicator (greenat acidic blue at alkaline). bacteria use the enzyme Citrase to degrade the citrate. bacteria convert Ammonium Phosphate to Ammonia and Ammonium Hydroxide. Both of these molecules will cause the agar to become alkaline and produce a blue color. e.g. Klebsiellasp
Voge’sProskauer test Testing the ability of the bacteria to utilize glucose in butyline glycol pathway and produces Acetylemethylecarbinol (AMC) or Aacetoin. Contain pepton, glocose and buffer. the end products will react with alpha naphthol and KOH (Barrit's reagents) produce red maroon color.
Methyl red test Testing the ability of the bacteria to utilize glucose in mixed acid fermentation pathway and produces stable acidic end-products (lactate, acetate). Contain pepton, glocose and buffer. the acids will overcome the buffers in the medium and produce an acidic environment. methyl red is added, if acidic end products are present, the methyl red will stay red.
Biochemical reactions • Analytical Profile Index (API): • Fast identification system combines biochemical reactions and computor database for group of bacteria. • Examples API20E, API Staph 6 7 0 6 5 1 3 = Staphylococcus sp
Toxin production Elek's test (corynebacteriumdiphteriae) Nagler’s reaction. (clostridium perfringens • Testing the ability of the bacteria to produce certain toxin (invitro) or observe its effect (invivo).
Animal inoculation ( animal Pathogenicity test) Animals commonly used: guinea pigs. rabbits. mice. Importance of pathogenicity test: • Differentiate pathogenic and non pathogenic bacteria. • Isolation organism in pure form. • To test ability of toxin production. • Evaluation of vaccines and antibiotics.
Serologic tests • immunologic methods taking advantage of the specificity of antigen antibody binding to certain bacterial components: • Cell wall antigens (O Ag). • Capsular antigen (K Ag). • Flagellar antigen (H Ag). • Example for Serological test; • Latex agglutination test. • Haemagglutination. • Complement fixation. • Counterimmuno-electrophoresis (CIE). • Enzyme linked immunosorbent assay (ELISA). • Fluorescent antibody test.
Molecular tests • highly sensitive and specific method for pathogen detection by identification of its nucleic acid in patient specimen. • detect a short nucleotide sequence of DNA or RNA (target sequence) that is unique to the pathogen. • Examples of Molecular tests used: • Polymerase Chain reaction (PCR). • Ligase Chain Reaction. • Plasmid profile analysis.
Bacterial typing • Revealing of the organism beyondto species level. • It is the ability to discriminate between similar strains (population of bacteria from single cell). • Used for: • investigation and control of epidemics. • Identification of pathogen reservoir and source of transmission. • Surveillance. • Study Pathogenesis of the particular bacteria. • Two categories: • Phenotypic methods. • Genotypic methods.
Phenotypic typing • Bacterial Typing based on expressed phenotypic characteristics. • Biotyping. • Antibiograms. • Serotyping. • Bacteriophage typing.
Phenotypic typing: Biotyping • Based on biochemical & cultural characteristics like colonial morphology, growth requirements, carbon source utilisation and toxin production. • Example: Vibrio Choleraebiotypes.
Phenotypic typing: Antibiogram typing • Testing the clinical isolates (pathogen) for antibiotic resistance or sensitivity.
Phenotypic typing: Serotyping • Using species specific Antibodies to screen for antigens expressed on bacterial cells. • Examples: • Agglutination. • Immunofluorescence.
Phenotypic typing: Bacteriophage typing • Bacteriophages are viruses that infect and lyse bacteria. • Bacteriophage typing is based on sensitivity of bacterial isolates to type-specific bacteriophages. • Example: • Staphylococcus aureusbacteriophage typing.
Genotypic typing • Based on analysis of genetic (DNA-based) relatedness of bacterial strains. • Plasmid analysis. • Restriction Endonuclease analysis. • PCR based (RAPD). • Pulse-field gel electrophoresis. • Ribotyping. • DNA sequencing: • MLST (Multilocus Sequence Typing). • SNP (Single Nucleotide Polymorphism).