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Bls 206 lecture 1
 

Bls 206 lecture 1

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    Bls 206 lecture 1 Bls 206 lecture 1 Presentation Transcript

    • BLS 206: DIAGNOSTIC MICROBIOLOGY HOZA, A.S (2010) ROAD MAP …………….
      • WHAT IS EXPECTED OUT OF THIS COURSE….
      • AT THE END OF THE COURSE, YOU WILL BE ABLE TO APPLY: MOLECULAR/CONVENTIONAL DIAGNOSTIC MICROBIOLOGY TECHNIQUE IN D’SE Dx!
      • NB: 1. THERE SHALL BE @ LST 3 EXAMS.
      • 2 . NO FORMAL LECTURE NOTES WILL BE GIVEN
      • Qn1: Highlight the major steps involved during Gram’s stain in a diagnostic lab
      • Qn2: Briefly explain why Gram –ve bacteria don’t retain basic stain after decolourization. (Less than 2 lines)
      Getting 2 know @other in BLS 206!
    • Laboratory Diagnosis of Infection Ask the lab for a diagnosis, expecting a yes or no, but often end up with just a maybe…
    • Basic Principles of Lab diagnosis
      • Clinical assessment
      • Collecting and transporting specimens
      • Microscopy
      • Culture
      • Sensitivity
      • Non-cultural diagnostic methods
      • Virological diagnosis
    • Diagnosis of Bacterial Infection Patient Clinical diagnosis Haematology Biochemistry Non-microbiological investigations Radiology Sample Take the correct specimen Take the specimen correctly Label & package the specimen up correctly Appropriate transport & storage of specimen
    • A proper clinical assessment is essential for optimal use of laboratory services!
    •  
    • Collecting the correct specimen
      • Pernasal swabs for pertussis
      • Sputum , not saliva
      • Blood culture bottles, not clotted blood
      • Correctly timed Gentamicin assays
      • Pus, not swabs
    • Getting the specimen to the lab
      • Problems in delay or inappropriate storage
        • delay in diagnosis & treatment
        • pathogens die
        • contaminants overgrow
      • Blood cultures directly into incubator
        • not refrigerator!
      • CSF straight to lab
      • Don't put an entire surgical specimen into formalin!
        • Send a portion to microbiology in a sterile container
    • Collecting the specimen correctly
      • Take an mid-stream urine
        • avoids contamination with perineal flora
      • CSF
        • Avoid contamination
        • Avoid bloody tap
      • Throat swab
        • Make the patient gag!
      • Blood cultures
        • Avoid contamination with skin organisms
    • Specimens & Infection Control
      • Please be considerate to lab staff!!
        • Label hazardous specimens
      • Don't send specimens to the lab without proper packing
        • Leaking or blood-stained specimens are not acceptable!!!
    • Factors limiting usefulness of bacteriological investigations
      • wrong sample
        • e.g. saliva instead of sputum
      • delay in transport / inappropriate storage
        • e.g. CSF
      • overgrowth by contaminants
        • e.g. blood cultures
      • insufficient sample / sampling error
        • e.g.in mycobacterial disease
      • Animal/patient has received antibiotics
    • Diagnosis of Bacterial Infection culture on plates or in broth identification by biochemical or serological tests on pure growth from single colony microscopy Decolorise Counterstain Stain unstained or stained with e.g. Gram stain sensitivities Serodiagnosis DNA technologies by disc diffusion methods, breakpoints or MICs
    • Microscopy Unstained preparations
      • “ Wet prep”
      • Dark-ground illumination for syphilis
    • Microscopy Stained preparations
      • Gram-stain
      • Acid-fast stain
        • Ziehl-Neelsen
      • Fluorescence
        • Direct, e.g. auramine
        • Immunofluorescence
    • Culture of Bacteria
      • Solid media
        • Agar plates
          • For Identification
          • For Enumeration
        • Slopes
        • For safe long-term culture, e.g. Lowenstein-Jensen media for TB
      • Liquid media (broth)
        • For enrichment or maximum sensitivity
    • Advantages of Solid Media
      • isolation of single clonal colonies
        • get bacterium in pure culture
      • identify by colonial morphology
      • quantification by colony-forming units
    • Identification of Bacteria
      • Morphology
      • Growth requirements
      • Biochemistry
      • Enzymes
      • Antigens
    • Non-cultural diagnostic methods
      • Antigen detection
        • e.g. latex agglutination
      • Antibody detection
        • e. g. agglutination tests, complement fixation tests, indirect immunofluorescence
      • Molecular methods
        • Polymerase Chain Reaction
    • Sensitivity tests
      • on solid media
        • disc diffusion technique
      • in liquid media
        • minimum inhibitory concentration (MIC) test
      • Breakpoint methods
      • E-test
    • I think it’s time for a short Break!! Oooh yeah!!!
    • Bacterial cultivation Part 1a Selective /differential media
    • Lab activities:
      • Form 8 groups of about 8 students
      • Each group will have 1 type of specimen
        • Exercise
      • Demo selective and differentiation plates
      • Streaking bacteria on differentiation plates
      • One group report after each practical MUST be submitted
    • Cultivation
      • The process of growing microorganisms in culture by taking bacteria from the infection site (in vivo or environment) and grow them in artificial environment in the laboratory (in vitro).
    • Growth needs
      • Fastidious bacteria – relatively complex growth needs
      • Non-fastidious bacteria- relatively basic and straightforward growth needs
    • Phases of growth media
      • Broth:
        • Growth of bacteria will change the liquid from clear to turbid (cloudy).
      • Solid:
        • Agar plates
        • Slants
          • Bacterial cells inoculated on solid media will multiply enough to be seen by naked eye.
    • Colony- (clone)
      • Colony - A bacterial population derived from one bacterial cell.
        • The cells within the colony have identical, genus, species, genetic and phenotypic characteristics.
      • Pure bacteria - derived from a single colony.
      • Selection of a pure colony -most important for bacterial identification
    • Media classification and function
      • Enrichment – Used to enhance growth of a particular pathogen
      • Supportive - support growth of most non fastidious bacteria
    • Media classification and function
      • Selective - Contain inhibitory agents that are inhibitory to all organisms except those sought
      • Differential - Contain factors that allow bacterial species to manifest certain metabolic characteristics that distinguish them from other species.
      • Media can be both selective and differential based on the ingredients of the medium.
    • Blood agar plate (BA)
      • Nutrient agar with 5% sheep blood
      • Cultivation of fastidious and non fastidious bacteria.
      • Differential – Identify hemolysis - Some bacteria secrete enzymes that lyse red blood cells (hemolysins) such that a clearing around the colony appears.
        •  hemolysis- complete clearing (white hemolysis)
        •  hemolysis – incomplete clearing (green hemolysis)
        •  hemolysis- no hemolysis
    •  
    •  
    • Mannitol Salt Agar (MSA)
      • Both selective and differential medium.
      • High salt concentration - inhibits most bacteria.
      • Selective for Staphylococcus sp.
      • Differentiate between Staphylococcus sp. by the sugar mannitol fermentation .
      • Mannitol fermention produce acids that change the pH of medium.
      • Peach color- neutral- no fermentation
      • Bright yellow- Acidic – mannitol fermentation ( Staph. coag. pos.- Staph. aureus)
    •  
    • MacConkey Agar (MAC)
      • Selective and differential medium.
      • Selective - Gram positive bacteria are inhibited by the presence of bile salts and crystal violet inhibitors in the medium
        • Most of gram negative bacteria will grow.
      • Differentiate - Between Gram negative bacteria by their ability to ferment lactose .
      • Pink colonies- Bacteria that ferment lactose (precipitation of some salts in media by acid production).
      • Pale colonies- Non fermenters
    •  
    • Eosine Methylene blue (EMB)
      • Differentiatial between lactose fermenting and non fermenting enteric bacteria
    • Tellurite Glycine Agar (TGA)
      • Selective- Tellurite glycine and lithium inhibit most bacteria
      • Preferential growth of Staphyloccoci coagulase positive (Staphyloccocus aureus)
    • Bacteria streaked in lab
      • Staphylococcus aureus
      • Staphylococcus epidermidis
      • Salmonella pullorum,
      • E.coli
      EMB and TGA S.aureus S.pullurum S.epidermidis E.coli
    • OK guys, it’s the end of presentation, C U next time...!!