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Practical Medical Microbiology

Practical Medical Microbiology. Lab # 6 Laboratory Identification of other Gram Negative Bacteria and Fungi. Classification of Gram Negative Bacteria. Gram negative Bacteria: aerobes. Pseudomonas aeruginosa : Microscopic examination. Morphological Description: Type of stain: Gram stain

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Practical Medical Microbiology

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  1. Practical Medical Microbiology Lab # 6 Laboratory Identification of other Gram Negative Bacteria and Fungi

  2. Classification of Gram Negative Bacteria

  3. Gram negative Bacteria: aerobes

  4. Pseudomonas aeruginosa:Microscopic examination Morphological Description: • Type of stain: Gram stain • Gram reaction: Gram -ve • Color: Pink • Shape: Rods • Non-spore forming bacteria • Motile (have one or more polar flagella) • Species of Medical Importance: Pseudomonas aeruginosa

  5. Pseudomonas aeruginosa: Cultural Characteristics and Biochemical tests Culture Media: • Growth on Cetrimide Agar Diagnostic Laboratory Tests: • Gelatin liquefaction as Bacillus sp. • Oxidation/Fermentation(O/F) test • Oxidase test

  6. Culture MEDIA: Cetrimide Agar Composition Cetrimide + MgSO4 + Glycerol Cetrimide: Inhibits all microorganisms except Pseudomonas aeruginosa MgSO4 + Glycerol:Enhances pigment production of Pseudomonas aeruginosa(pyocyanin) SELECTIVE MEDIUM

  7. biochemical tests: Oxidation/Fermentation (O/F) test O2 No O2 Pseudomonas Sugars + O2 Oil to prevent O2 Oxidation Acids Bromocresol purple Obligate aerobe

  8. Biochemical tests:Gelatin Liquefaction Pseudomonas aeruginosaandBacillus Sp. (Positive) • Nutrient broth • + Gelatin (12-15%) • Gelatin isliquified • by m.o. producing • Gelatinase enzyme E. Coli (Negative)

  9. Diagnostic laboratory tests:oxidase test Pseudomonas spp. are oxidase positive, i.e. possess bacterial cytochrome oxidase. b) A colony on a piece of filter paper + oxidase reagent → dark blue color a) A colony + oxidase reagent (dimethyl-p-phenylenediamine hydrochloride) → blue color

  10. Classification of Gram Negative Bacteria

  11. Curved/helical shaped gram negative rods: Vibrio sp. • Microscopic Examination • Type of Stain: Gram Stain • Gram reaction: Gram Negative • Color: Pink • Shape: Curved rods • Motile with polar flagella

  12. Vibrio sp: culture media ThiosulphateCitrate Bile Salt Sucrose Medium (TCBS) Constituents: Na citrate + Na thiosulfate + Bile salt + Sucrose

  13. Vibrio sp: Culture media (TCBs) • Principle: • The high pH 8.5-9.5 inhibits all intestinal flora except Vibrio sp. • Bile salts inhibit the growth of gram positive bacteria SELECTIVE MEDIUM Other Vibrio spp. don’t ferment sucrose but give alkaline metabolic end product (blue color) Sucroseis fermented by Vibrio choleraeproducing acid that turns colour of indicator yellow DIFFERENTIAL MEDIUM Thymol Blue/Bromothymol Blue (BTB) Indicator

  14. Vibrio sp: culture media (TCBS) Vibrio cholera: Sucrose Fermentor Other Vibrio sp.: Non-Sucrose Fermentors

  15. Vibrio sp: culture media (tcbs) Uses of TCBS Medium: • Isolation of Vibrio spp. from clinical specimen • Differentiation between Vibrio choleraeand other species

  16. FUngi

  17. Types of fungi Types of Fungi A. Molds (Filamentous Fungi) B. Yeast (Bacteria-like Fungi)

  18. Classification of Pathogenic Fungi

  19. Diagnostic tools for identification of pathogenic Fungi

  20. Specimen collection Specimens for fungal microscopy and culture may be: • Scrapings of scale, after cleaning with alcohol. • Hair which has been pulled out from the roots. • Skin biopsy. • Moist swab from a mucosal surface (inside the mouth or vagina. • A swab should be taken from pustules.

  21. MorphologyDirect Microscopy Potassium hydroxide (KOH) stained with blue or black ink Unstained wet mount Stained Dried Smear Histopathology of biopsy with special stains

  22. OPPORTUNISTICFUNGal infections 1- Candida albicans (Candidiasis) • Culture Medium: - Sabouraud Dextrose Agar • Used to culture fungi • it has a low pH that inhibits the growth of most bacteria • it may also contains the antibiotic gentamicin to specifically inhibit the growth of Gram's-negative bacteria • Microscopic Shape: - Ovoid cells (yeast) or filamentous (pseudohyphae).

  23. Candida albicans • The ability ofCandida albicansto convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence. Candida albicans showing germ tube

  24. OPPORTUNISTICFUNGal infections 2-Aspergillussp. (Aspergillosis) • Culture Medium: - Sabouraud Dextrose Agar • Microscopic Shape: - Filamentous (mold), carrying conidia (spores)- HP (x400).

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