Practical Microbiology Lab Manual PDF

Practical Microbiology Safety Precautions in the Microbiology Laboratory Biohazard sign Use: labelling of infectious material Biohazardous labelled waste disposal red bags Use: Disposal of biological waste Safety box...

Practical Microbiology Safety Precautions in the Microbiology Laboratory Biohazard sign Use: labelling of infectious material Biohazardous labelled waste disposal red bags Use: Disposal of biological waste Safety box Use: Disposal of needles and sharp objects 1  Laboratory diagnosis of bacteriological infections 1. Specimen collection. 2. Microscopic examination. 3. Cultivation. 4. Identification by: Culture morphology. Gram-stained film. Biochemical reactions. 5. Typing. 6. Serological identification. 7. Molecular methods. 8. Animal pathogenicity Microscopic examination Stained Smears 1-Simple staining: A single stain is used Show morphology of organism and cellular elements in exudates 2-Differential staining (Gram, Ziehl Neelsen) 3-Special staining (capsule, spore) 2  Differential staining Gram Stain Ziehl-Neelsen Use It differentiates bacteria into Used to stain Mycobacterium. Gram positive and Gram The cell wall of the Mycobacterium negative based on differences contains high lipid (mycolic acids) in cell wall structure content making them unable to be stained by Gram stain Primary stain Crystal violet Conc. Carbol fuchsin in phenol Fixative Iodine (mordant) Decolorizing 95% alcohol 25 % H2SO4 in 95%alcohol or agent 3% HCL in 95% alcohol Counter stain Diluted carbol fuchsin or Methylene Blue (secondary Safranin stain) Gram positive cocci Stain: gram stain 3 Gram negative bacilli Stain: gram stain Acid fast bacilli appear red bacilli against blue background. Stain: (Ziehl Neelsen stain) 4  Cultivation of Bacteria I- Simple Media Contain the essential growth requirements. Nutrient broth Type: Simple fluid media Use: 1-Support growth of many microorganisms. 2- Base for other solid media Nutrient agar Type: Simple solid media Use: 1-Support growth of many microorganisms. 2- Base for other solid media 5 II) Enriched media Contain highly nutritive substances such as blood, serum or egg for fastidious organisms. Blood agar Type: Enriched &Differential Use: 1- Cultivation of fastidious bacteria 2-Differentiate between bacteria according to type of hemolysis (α,β,γ).(Test hemolytic activity) Chocolate agar Type: Enriched Use: Cultivation of fastidious bacteria Loffler’s serum Type: Enriched Use: Cultivation of C. diphtheria 6 III) Selective media They contain inhibitory substances that inhibit growth of certain bacteria and allow growth of others. Lowenstien Jensen media (LJ) Type: Selective &Enriched Use: Selective for Mycobacterium tuberculosis Quiz ?? What is the name of this media? Its type is………… It is used in………………….. 7 IV- Indicator media They contain an indicator that changes its color according to pH due to metabolic activities of particular organisms MacConkey's agar Type: Indicator, Selective &Differential. Use: Isolation of Enterobacteriaeceae & differentiate between lactose fermenter & lactose non fermenters Triple sugar iron agar Type: Indicator Use: Differentiation of Enterobacteriaeceae by testing their fermentative activity and H2S production 8  Anaerobic cultivation Anaerobiosis can be achieved by: 1. Anaerobic culture media (containing reducing substances used to grow anaerobic bacteria) as Robertson's cooked meat 2. Anaerobic GasPack system (anaerobic incubation) Robertson's cooked meat Use: cultivation of anerobic bacteria Anaerobic GasPack system Use: cultivation of anerobic bacteria 9  Identification of the bacteria Conventional methods Non-conventional methods Biochemical reactions Molecular methods *PCR *DNA probe Biochemical reactions 1-Indol test Principle: Indole production from amino acid tryptophan. Interpretation: Positive: Pink ring Negative: Yellow ring 2-Oxidase test Principle: Test ability of bacteria to produce Oxidase enzyme. Interpretation: Positive test: Immediate purple colour Negative test: No purple colour develop. 10 3-Urease test Principle: Test ability of bacteria to produce Urease enzyme Interpretation: Positive test: Deep pink Negative test: Yellow 4-Catalase test Principle: Test ability of bacteria to produce Catalase enzyme Interpretation: Positive test: Gas bubbles (O2 production) Negative test: No bubble 5-Coagulase test Principle: Test ability of bacteria to produce coagulase enzyme 11 6-DNase production test Principle: Test ability of bacteria to produce DNase enzyme that hydrolyze DNA into smaller molecules Interpretation: Positive: colonies surrounded by clear zones Negative: Opaque white 7-Citrate test Principle: It is the ability of an organism to utilize citrate as the sole source of carbon for its growth. Interpretation: Positive: blue color on the slant. Negative: green Analytic Profile index (API) Used for identification of microorganism 12  Antibiotic susceptibility tests It is an in-vitro laboratory method which determines the susceptibility of bacteria to antimicrobials. Disc diffusion method Use: Determine Antibiotic sensitivity Epsilometer (E) Test Use: Determine Antibiotic sensitivity 13  Immunoassays Immunoassays include: I. Immunofluorescence (IF) II. Enzyme-linked immunosorbent assay (ELISA) III. Radioimmunoassay (RIA) IV. Western blot (Immunoblot) V. Flow cytometry VI. Immunochromatography (lateral flow assay) I. Immunofluorescent Techniques (If) Principle: To identify unknown antigens (Direct IF) or unknown antibodies (Indirect IF), a known antibody labelled with a fluorochrome is utilized. UV light is used by a fluorescent microscope to visualize the reaction. Types: i. Direct Immunofluorescence ii. Indirect Immunofluorescence 14 Ii. Enzyme-Linked Immunosorbent Assay (ELISA) A sensitive assay for detection of unknown Ag or Ab using enzyme labeled antibodies. The enzyme in positive samples reacts with a colorless substrate to produce a colored product. Spectrophotometers assess color intensity, which is correlated with the unknown's concentration. TYPES i. Indirect ELISA: - To identify and quantify serum antibodies ii. Sandwich ELISA: - To detect and measure concentration of an antigen (protein) in a sample. 15 III-Immunochromatography A rapid test used for antigen or antibody detection. Typically, these tests use single-use, disposable cartridges or strips that produce observable colored end products interpreted as: 1. Positive (two pink lines) 2. Negative (one line) 3. Invalid (no lines). 16