SIDDAGANGA MEDICAL COLLEGE AND RESEARCH INSTITUTE, TUMKUR
DEPARTMENT OF MICROBIOLOGY
1B. Microscopy
1) Enumerate different types of microscopes used in diagnostic microbiology laboratory
1. Light microscope
2. Dark field microscope
3. Phase contrast microscope
4. Fluorescence microscope
5. Electron microscope
2. Describe the principle and uses of various microscopes
a) Dark-field microscope
Principle:
Dark field microscope, the object appears bright against a dark background, the dark field condenser
has a central opaque that blocks light from entering the objective lens directly and has peripheral
annular hallow area which allows the light to pass through and focus on the specimen obliquely.
Only the light which is reflected by the specimen enters the objective lens whereas the unreflected
light does not enter the objective, as a result the specimen is brightly illuminated, but
thebackground appears dark.
Uses: 1. visualize living unstained cells
2. thin bacteria like spirochete
b) Phase contrast microscope
Principle:Condenser is similar to dark field [Link] the cone of light pass through the a cell,
some light rays are bent due to variation in density and refractive index within the specimen and are
retarded by about 1/4th of a wavelength the Undeviated light rays strike a phase ring, Deviated rays
miss the ring and pass through the rest of the plate the phase ring is constructed in such a way that
the undeviated light passing through is advanced by 1/4th of a wavelength ,the deviated and
undeviated waves will be about half wavelength out of the phase and will cancel each other when
they come together to form an image ,Background is bright, while the unstained object appears dark
Uses:
1. microbial motility, 2. determining the shape of living cells, detecting bacterial components
,such as endospores and inclusion bodies
c) Fluorescent microscope
Principle:
Fluorescent dyes - exposed to UV rays, become excited and fluorescei.e. they convert short
wavelength rays into longer wavelengthsMercury lamp emits rays that pass through an excitation
filterExcitation filter allows only short wavelength UV light to pass throughExciting rays get reflected
by a dichromatic mirror - fall on the specimen stained by fluorescent [Link] filter – removes
remaining UV light or blue and violet light
Uses: 1. Auto fluorescence (eg. Cyclospora) 2. Microbes coated with fluorescent dye (eg. Acridine
orange dye is used for detection of malaria parasite by method called quantitative buffy coat
examination, auramine phenol is used for detection of tubercle bacilli) 3. Immunofluorescence- it
uses florescent dye tagged immunoglobulins to detect cell surface antigens or antibodies bound to
the cell surface antigens
d) Electron microscope
Principle:
Electrons pass through a magnetic condenser and then bombard on the thin sliced specimen
mounted on the copper slide . The specimen scatters electrons passing through it, and then the
electron beam is focused by magnetic lenses. (objective lens followed by projector lenses) to form
an enlarged, visible image of the specimen on a fluorescent screen . A denser region in the specimen
scatters more electrons and therefore appears darker in the image since fewer electrons strike that
area of the screen. In contrast, electron-transparent regions are brighter
Uses:The most important use of EM in diagnostic microbiology is detection of viruses. It can detect
viruses either—1. Directly from the clinical specimens (e.g. rotavirus from stool) 2. From tissue
cultures 3. after adding specific antiviral antibody to the specimen (known as immune-EM)
3. Label the parts of microscope in the given picture
4. Suggest the type of microscope, desired magnification and method used in the
following case scenarios
Case scenario Microscope Magnification Method
Stool examination for Light 10x, 40x Wet mount (saline &
parasitic eggs Iodine mount)
Urine examination for pus Light 10x, 40x Wet mount
cells in a case of Urinary tract
infection
Bacterial examination of CSF Light 100x Gram stain
sample in case of pyogenic
meningitis
Bacterial examination of Light 10x, 40x Wet mount
tissue fluid from gummatous
ulcer in a case of sexually Light 100x Silver impregnation
transmitted disease method (Levaditi stain,
Fontana stain)
Fluorescent 10x, 40x Direct fluorescent
antibody staining
5. Write the differences between Electron microscope and the Light/Compound
microscope
Features Light/Compound microscope Electron microscope
Highest practical magnification 1,000-1,500 Over 100,000
Best resolution 0.2µm 0.5nm
Radiation source Visible light Electron beam
Medium of travel Air High vacuum
Specimen mount Glass slide Metal grid
Types of lens glass Electromagnet