Diagnostic Techniques In Bacteriology
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Diagnostic techniques in medical microbiology
Dr. Mejbah Uddin Ahmed
Diagnosis of infectious diseases
Infectious disease is diagnosed by:
Clinically
Radio logically
Laboratory investigation microbiological hematological
and other
Diagnostic microbiology Patient Microbiology Laboratory
Diagnosis
Diagnostic microbiology
Several procedures are involved in the diagnostic microbiology:
Different types of microscopy.
Cultural method.
Serological method.
Molecular method.
Microscopy
Microscopy: Is done for direct or indirect visualization of organism. Different types of microscopy are available:
Light microscopy: -Unstained preparation. -Stined preparation.
Immunoflourescence.
Electronmicroscopy.
Microscopy Simple stain: A single basic dye such as: methylene blue or basic fuchsin is used as simple stain. Negative stain: This technique is useful in the demonstration of bacterial capsule. Impregnation stain: Demonstration of spirochetes and bacterial flagella. Differential staining: They impart different colours to different bacteria (Gram’s stain and acid -fast stain.)
Simple staining
A single staining agent is used such as methylene blue, carbol fuchsin, crystal violet or safranin.
It highlights the entire microorganism.
Cellular shapes and basic structures are visible.
Gram stain
Developed by the Danish bacteriologist Christian Gram in 1884.
Classifies bacteria into two groups based on differences in cell wall structureGram – positive & Gram- negative
The gram stain is a very important early step in some medical situations may provide enough information to select an antibiotic.
Steps of Gram Stain
Ziehl - Neelsen 1.
Primary Stain by heated carbol fuchsin.
2.
Decolorize with 20% sulfuric acid and alcohol separately or with acid alcohol (which contains hydrochloric acid and ethanol).
3.
Wash the slide in water.
4.
Counterstain usually with methylene blue or malachite green.
Immunofluorescence
Direct: detection
of antigen by use of fluorescence labeled antibodies
used
for diagnosis of viral infection, Chlamydia, legionella, Treponema pallidum, Giardia, Pneumocystis
Indirect: detection
of antibody.
Electron-microscopy Important diagnostic technique for many viral infections
Culture Used in bacterial, fungal, viral, parasitic diagnosis Mainstay of bacterial diagnosis Liquid media based culture Solid agar based culture Ensures organism available for susceptibility testing epidemiological studies
Serology
Depend on the detection of either antibody and/or antigen in patients serum.
Antigen detection useful in acute diagnosis
Antibody detection may be delayed
Serology
Types of tests:
Precipitation.
Agglutination.
Complement fixation (CFT).
Radioimmunoassay (RIA).
Enzyme immunoassays (EIA)
Immunochromatographic test (ICT).
Enzymelinked Immunosorbant assay(ELISA)
Molecular techniques
Allows the detection of either RNA or DNA Western
blotting - protein detection
Southern
blotting - DNA detection
Polymerase
chain reaction - DNA or
RNA detection
Dr. Mejbah Uddin Ahmed
Diagnosis of infectious diseases
Infectious disease is diagnosed by:
Clinically
Radio logically
Laboratory investigation microbiological hematological
and other
Diagnostic microbiology Patient Microbiology Laboratory
Diagnosis
Diagnostic microbiology
Several procedures are involved in the diagnostic microbiology:
Different types of microscopy.
Cultural method.
Serological method.
Molecular method.
Microscopy
Microscopy: Is done for direct or indirect visualization of organism. Different types of microscopy are available:
Light microscopy: -Unstained preparation. -Stined preparation.
Immunoflourescence.
Electronmicroscopy.
Microscopy Simple stain: A single basic dye such as: methylene blue or basic fuchsin is used as simple stain. Negative stain: This technique is useful in the demonstration of bacterial capsule. Impregnation stain: Demonstration of spirochetes and bacterial flagella. Differential staining: They impart different colours to different bacteria (Gram’s stain and acid -fast stain.)
Simple staining
A single staining agent is used such as methylene blue, carbol fuchsin, crystal violet or safranin.
It highlights the entire microorganism.
Cellular shapes and basic structures are visible.
Gram stain
Developed by the Danish bacteriologist Christian Gram in 1884.
Classifies bacteria into two groups based on differences in cell wall structureGram – positive & Gram- negative
The gram stain is a very important early step in some medical situations may provide enough information to select an antibiotic.
Steps of Gram Stain
Ziehl - Neelsen 1.
Primary Stain by heated carbol fuchsin.
2.
Decolorize with 20% sulfuric acid and alcohol separately or with acid alcohol (which contains hydrochloric acid and ethanol).
3.
Wash the slide in water.
4.
Counterstain usually with methylene blue or malachite green.
Immunofluorescence
Direct: detection
of antigen by use of fluorescence labeled antibodies
used
for diagnosis of viral infection, Chlamydia, legionella, Treponema pallidum, Giardia, Pneumocystis
Indirect: detection
of antibody.
Electron-microscopy Important diagnostic technique for many viral infections
Culture Used in bacterial, fungal, viral, parasitic diagnosis Mainstay of bacterial diagnosis Liquid media based culture Solid agar based culture Ensures organism available for susceptibility testing epidemiological studies
Serology
Depend on the detection of either antibody and/or antigen in patients serum.
Antigen detection useful in acute diagnosis
Antibody detection may be delayed
Serology
Types of tests:
Precipitation.
Agglutination.
Complement fixation (CFT).
Radioimmunoassay (RIA).
Enzyme immunoassays (EIA)
Immunochromatographic test (ICT).
Enzymelinked Immunosorbant assay(ELISA)
Molecular techniques
Allows the detection of either RNA or DNA Western
blotting - protein detection
Southern
blotting - DNA detection
Polymerase
chain reaction - DNA or
RNA detection
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