Week 4: Laboratory Diagnosis of Bacteria

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Author:
jlyip89
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259546
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Week 4: Laboratory Diagnosis of Bacteria
Updated:
2014-02-01 04:09:40
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Lab diagnosis bacteria
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micro
Description:
Week 4 Micro Lecture
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  1. Procedure for lab diagnosis
    • 1. specimen collection
    • 2. identification/classification of bacteria
  2. Types of specimens collected for pathogen identification (classification)
    • Body Fluids
    • •Blood
    • •Cerebrospinal fluid
    • •Peritoneal (abdominal)
    • •Pleural (chest)
    • •Synovial (joint)
    • •Pericardial (heart)
    • •Urine

    • Others:
    • •Upper respiratory tract
    • •Lower respiratory tract
    • •Wounds, abscesses, and tissues
    • •Genital specimen
    • •Fecal specimen
  3. (6) Polyphasic approach of bacterial identification
    • 1. Direct ID of bacteria
    • 2. morphological characteristics
    • 3. growth characteristics
    • 4. biochemical characteristics
    • 5. serological characteristics
    • 6. genomic characteristics
  4. Direct ID of pathogen from specimen
    Certain human pathogens (e.g.,  the causative agents of tuberculosis, Lyme disease and syphilis) either cannot be cultured or grow extremely poorly in lab.

    Direct detection of bacteria without culture is possible in some cases.
  5. Morphologic Characteristics

    - 3 types
    • - colony morphology
    • - cellular morphology
    • - staining reactions:

    • simple stains
    • - methylene blue
    • - malachite green
    • - basic fuchsin

    • Differential stains
    • - gram stain
    • - acid-fast stain

    • special stains
    • - capsule stain
    • - flagella stain
    • - endospore stain
  6. Growth Characteristics
    - Effects of temperature and pH on bacterial growth

    - Effect of oxygen on bacterial growth

    - Antibiotic sensitivity: in vitro testing of antibiotics against isolated strain (disk diffusion)
  7. Biochemical characteristics: Hemolysis Test

    - what type of test?
    - possible observed patterns?
    • Hemolysis test (hemolysin activity)
    • - plate isolated bacteria on blood agar plate
    • - observe different patterns

    • beta-hemolytic: 
    • complete lysis of red blood cells, resulting in a clear halo around the colony

    • gamma-hemolytic: 
    • non-hemolytic = completely clear

    • alpha-hemolytic: 
    • incomplete lysis of red blood cells, resulting in a greenish halo around the colony
  8. Biochemical Characteristics: Fermentation assay
    Fermentation assay

    –Phenol red (PR) carbohydrate broth

    •Fermentation produces acids from the breakdown of carbohydrate

    •PR indicator of fermentation

    •PR is red at pH > 7 = no fermentation

    •PR is yellow = fermentation
  9. Name all 4 types of biochemical characteristic tests
    • 1 Hemolysis test
    • 2. Fermentation test
    • 3. coagulation test
    • 4. catalase test
  10. Serological characteristics

    - what is it?
    - what types of assays are used?
    - Antigen and antibody reaction

    - Using antibodies to detect antigens (direct evidence for a pathogen) or using antigens to detect specific antibodies in serum (indirect evidence of infection)

    –Enzyme-linked immunosorbent assay (ELISA)

    –Western blot

    –Radioimmunoassay
  11. Genomic Characteristics

    - name 3 methods
    • comparing DNA restriction pattern
    • –Restriction fragment length polymorphism (RFLP)

    • detecting specific bacterial gene(s)
    • –Polymerase chain reaction (PCR)

    –Hybridization with DNA probe

    • determining genomic DNA sequences
    • –16S ribosomal RNA gene sequencing
  12. 16S Ribosomal RNA
    - 30s small subunit of ribosome is composed of one rRNA molecule (16S) and 21 different proteins (S1-S21)

    - This gene is only about 1,550 bp in length in prokaryotes

    - Very conserved DNA sequence among bacterial species

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