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 structure-
Gram – 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