Chapter 2_microbes identification tests.pptx
- 2. 2 • At the end of this lecture, you will be able to apply………… - Phenotypic diagnostic methods for microbes identification - Some basic principles of microbiology testing => Task of the methods to make the microbes visible and measurable. Out lines
- 3. 3 • the analysis of sample, the synthesis of results (of several samples), and the clinical consultation • Together these form the basis for diagnosis, therapy and infection control. The basic principle is…. - Clinical diagnosis or assessment - Collecting and transporting specimens - Microscopy/morphology - Cultural diagnostic methods - Immunological methods Component of diagnostic microbiology
- 4. 4 Major groups of the microbial world: • Bacteria • Fungi • Algae • Parasite • Viruses Major features of microbes: • Small and diverse (appearance, genetics).
- 5. 5 I. Phenotypic (conventional) II. Genotypic: Genetic and Molecular techniques (to be covered in Chapter 4) Methods used to identify microbes - Over the past century, the identification and differentiation of microorganisms has principally relied on microbial morphology and growth variables (Phenotypic). - Advances in molecular biology over the past 10 years have opened new avenues for microbial identification and characterization (Genotypic). • The method microbiologist use to identify microbes fall into two categories:
- 6. 6 ‘Old fashioned’ methods via biochemical, serological and morphological are still used to identify many microorganisms. • Examining specimens to detect, isolate and identify pathogens Phenotypic characteristics: a) Morphology : Microscopic (wet preparation – Staining preparation) • Macroscopic (Cultural Characteristics) are traits that can be accessed with the naked eye e. g appearance of colony including shape, size, color of the colony (pigment), speed of growth and growth pattern in broth- nutrient requirements for the growth of the organism Growth on different laboratory conditions and media- Can determine whether the microbe is a bacteria, fungus, or protozoa) b) Biochemical (Metabolic differences) characteristic are traditional for microbes identification • include enzymes (catalase, oxidase, decarboxylase), fermentation of sugars, capacity to digest or metabolize complex and sensitivity to drugs can be used in identification c) Immunological tests: serological tests are great value in the diagnosis of many bacterial, fungal and viral infections. I. Phenotypic methods to identify microbes
- 7. 7 Disadvantages Poor discriminatory power Difficulties in typing Not provide enough information about microorganisms for today's needs. Phenotypic Methods
- 8. 8 1- Microscopy: Microorganisms can be examined microscopically for…. a) Morphology • Size and shape of cells • Identification of bacteria • Prokaryote arrangement of cells • Gram staining • Microscopic structures and characteristics (such as flagella)
- 9. 9 1-Simple stain: one dye is used e.g methylene blue stain – reveals shape- size- cell arrangement The most commonly used stain in diagnostic microbiology is the Gram stain 2- Differential stain: Gram stain: differentiation between Gm+ve and Gm–ve bacteria. Some microbes have unique characteristics that can be detected by special staining procedures 3- Acid fast reaction (Ziehl-Neelsen stain) staining acid fast bacilli 4- (spore stain): bacterial endospores 5-Structural stains –reveal certain special structures of cell parts not revealed by conventional methods such as: capsule (capsule stain - flagellar stains- granules (volutin) Morphology and staining reactions of bacteria:
- 10. 10 Different bacterial morphologies. 2- Differential stain: Gram stain: differentiation between Gm+ve and Gm–ve bacteria • Primary stain (Crystal violet) • Mordant (Gram’s Iodine mixture) • Decolorization (ethyl alcohol) • Secondary stain (Saffranin red)
- 11. 11 Gram staining procedure and cell wall structure > Gram-positive organisms will appear purple, whereas gram-negative organisms will appear pink. Procedure
- 12. 12 • Wet mounts and hanging drop mounts - allow examination of characteristics of live cells: size, motility, shape, and arrangement • Fixed mounts - are made by drying and heating a film of specimen. - This smear is stained using dyes to permit visualization of cells or cell parts Specimen Preparation for Optical Microscopes
- 13. 13 Dyes create contrast by imparting a color to cells or cell parts • Basic dyes –cationic, positively charged chromophore • Acidic dyes –anionic, negatively charged chromophore • Positive staining –surfaces of microbes are negatively charged and attract basic dyes • Negative staining–microbe repels dye, the dye stains the background Staining Staining reactions of dyes
- 14. 14 1- Light Microscope (digital) 2- Stereo microscope 3- Dark field Microscope 4- Electron Microscope (transmission- Scanning) Microscope Types 1
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- 17. 17 - Some microbes have unique characteristics that can be detected by special staining procedures Special Strains - (a) Acid-fast-stained smear showing pink colored bacilli of M. tuberculosis (Tiwari et al., 2005). - (b) Haematoxylin and eosin stain lymph node biopsy showing many large, encapsulated yeast cells of C. neoformans (Khan et al., 2003).
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- 19. 19 b) Metabolic differences - Culture characteristics - Biochemical characteristics • The identification of most prokaryotes relies on analyzing their metabolic capabilities such as the types of sugars utilized or the end products produced. • In some cases these characteristics are revealed by the growth and colony morphology on the cultivation media, but most often they are demonstrated using biochemical tests.
- 20. 20 - Culture characteristics The use of selective and differential media in the isolation process can provide additional information that helps to identify an organism. • E.g., if a soil sample is plated onto a medium that lacks a nitrogen source and is then incubated aerobically, any resulting colonies are likely members of the genus Azotobacter. The ability to fix nitrogen under aerobic condition is an identifying characteristic of these bacteria. In clinical laboratories, where rapid but accurate diagnosis is essential, specimens are plated onto media, specially designed to provide important clues as to identify the disease causing organism. • E.g., Urine sample collected from a patient suspected of having a urinary tract infection is plated onto MacConkey agar, which is both selective and differential. • MacConkey agar has bile salts, which inhibit the growth of most nonintestinal organisms, and lactose along with a pH indicator, which differentiates lactose fermenting organisms.
- 21. 21 - (a) Isolation of Azotobacter spp. from soil sample. - (b) Escherichia coli produces red colonies on MacConkey agar. (a) (b) - Culture characteristics (cont…
- 22. 22 • Biochemical test-based identification systems are range from strip cards for specific groups of bacteria (e.g., coryneforms, bacillus, and enterics) to large plate arrays that may be automatically scanned for changes due to pH shifts or redox reactions. Examples of biochemical tests are Catalase test, oxidase test, etc. - Biochemical characteristics Catalase Test - It is a test to detect the presence of the catalase enzyme. - Most organisms possess this enzyme, which is capable of breaking down hydrogen peroxide. - Organisms containing the catalase enzyme will form oxygen bubbles when exposed to hydrogen peroxide. Catalase test
- 23. 23 Oxidase Test - It is a test to detect the presence of the enzyme cytochrome oxidase. - In the presence of this enzyme, the oxidase reagent (N,N,N ,N -tetramethylp- ′ ′ phenylenediamine dihydrochloride) is oxidized and turns from colorless to purple. Oxidase test specimen - Biochemical characteristics (cont…
- 24. 24 C- Serology - The protein and polysaccharides that make up a bacterium are sometimes characteristic enough to be considered identifying markers. - The most useful of these are the molecules that make up surface structures including the cell wall, glycocalyx, and flagella. - E.g., some species of Streptococcus contain a unique carbohydrate molecule as part of their cell wall, which can be used to distinguish them from other species. - These carbohydrates can be detected using techniques that rely on the specificity of interaction between antibodies and antigens. - Methods that exploit such interactions are called serology.
- 25. 25 Serology (cont… - The cell wall of gram-negative bacteria consists of several layers of various polysaccharides. - The periplasm contains peptidoglycan, a copolymer of polysaccharide and short peptides, and a class of β- glucans. - In gram-negative bacilli, the carbohydrate antigens within the wall of the organism are called somatic (associated with the soma, that is, the body of the cell) or O antigens. - The interaction of an antibody with an antigen may be demonstrated in several ways (e.g. enzyme-linked assays). Enzyme-linked fluorescent dye assay.
- 26. 26 Structure of the cell wall of E. coli.
- 27. 27 Today we covered: - Methods Used to Characterize Phenotypic Characteristics of microbes Summary