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6.

Techniques in microbiology
Microscopy
1. Types of light Microscope
a) Bright field (components, oil immersion lens, resolving
power( ability of a lens to separate distinguish between
small objects that are close together))
b) Darkfield : to observe living and unstained cells. The field
surrounding a specimen appears black, while the object
itself is brightly illuminated, b’cos the background is dark.
c) Phase-contrast : to observe living cells, permits detailed
examination of internal structures in living organisms.
Uses a ring shaped diaphragm.
d) Fluorescence : expose a specimen to UV (short WL) and
forms an image of the object with the resulting
fluorescent light. Light passes through an exciter filter and
allows desired WL.
2. Electron microscope ; a beam of
electron is used instead of light, to
examine objects smaller than 0.2 m.
Use electromagnetic lenses instead of
glass lens.
Techniques for observing
microorganisms
Direct observation (wet mount, hanging drop) –
unstained, examine with ordinary bright field
illumination for observation of motility.
Staining
1. simple staining
2. differential staining - divided into 3 types
gram staining
acid fast staining(Ziehl-Neelsen method): to
identify Mycobacterium tuberculosis, M.
leprae. They do not bind stain easily
1. heat mixture of fuchsin and phenol
2. wash with acid-alcohol, not decolourized. Hence
remain red. Non acid fast bac. –decolourized
and stained with methylene blue for
counterstain = appear blue.
Endospore staining
(Schaeffer-Fulton)
stained by heating bacteria with
malachite green
wash cell free of dye with water
counterstain with safranin
the technique yield a green endospore
resting in a pink to red cell.