DIAGNOSTIC MICROBIOLOGY

Definitive microbiologic diagnosis of an infectious disease usually involves one or more of the
following 5 basic lab techniques : direct microscopic visualization of the organism , cultivation and
identification of organism, detection of microbial antigens , detection of microbial DNA or RNA and
detection of an inflammatory or host immune response to microorganisms.
In many infectious disease, pathogenic organisms can often be directly visualized by microscopic
examination of patient specimens such as sputum,urine,and CSF.The organism microscopic morphology
and staining chracteristics can provide the first screening step before doing a specific identification.The
organisms to be examined do not need to be alive or able to multiply.icroscopy yields rapid and
inexpensive results.Some of the microscopic visualization techniques include : Gram-staining, Acid-fast
stain (Ziehl-Neelsen) , India ink preparation , potassium hydroxide prepation.
GRAM STAINING
This technique separates bacteria into 2 classifications according to their cell wall composition.
If a specimen on a microscope slide is treated with a solution of crystal violet and then iodine, the
bacterial cells will stain purple.If the stained cells are then treated with a solvent such as alcohol or
acetone, Gram-positive organisms will retain the purple stain while Gram-negative species will lose the
stain and become colourless. Addition of the counterstain safranin stains the colourless Gram-negative
bacteria pink or red.
The Gram stain is important therapeutically because gram-positive and gram-negative bacteria
differ in their susceptibility to various antibiotics.Gram stain then is used to guide the initial therapy until
a more definitive identification of the microorganisms can be carried out. In addition, the morphology of
the stained bacteria can sometimes be diagnostic.For example, gram-negative diplococci in urethral pus
indicates a presumptive diagnosis of gonorrhea.
However, there are limitations in this technique as it requires a large number of microorganisms
(>10 organisms/ml) .Microorganisms that lack cell walls such as mycoplasma also cannot be identified
using Gram stain.
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ACID-FAST STAIN (Ziehl-Neelsen)

This technique is used to identify organisms that have waxy material (mycolic acid) in their cell
walls.Certain acid-fast bacteria retain the carbolfuchsin stain after being washed with an acidic
solution.The most clinically important acid-fast bacterium is Mycobacterium tuberculosis which appears
pink,beaded and slightly curved. Acid-fast staining is reserved for clinical samples from patients
suspected of having Mycobacterial infection.
Ziehl-Nelson stain: acid-fast organism takes up carbolfuchsin and appear red.
Non acid-fast organism accept the methylene blue counterstain and appear blue

CAPSULE STAIN
In capsules stain, capsules are seen as clear halo around bacterial cell. The capsule stain
employs an acidic stain and a basic stain to detect capsule production.Capsules are formed by organisms
such as Klebsiella pneumoniae . Most capsules are composed of polysaccharides, but some are
composed of polypeptides. The capsule differs from the slime layer that most bacterial cells produce in
that it is a thick, detectable, discrete layer outside the cell wall. Some capsules have well-defined
boundaries, and some have fuzzy, trailing edges. Capsules protect bacteria from the phagocytic action of
leukocytes and allow pathogens to invade the body. If a pathogen loses its ability to form capsules, it
become non-virulent.
Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces.
Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the
cell itself using a basic stain. We use India ink and Gram crystal violet. This leaves the capsule as a clear
halo surrounding a purple cell in a field of black.
It is useful in detecting Cryptococcus neoformans in CSF. One drop of centrifuged CSF is mixed
with one drop of India ink on a slide. Then, 1% CVS applied to unheated slide.Then it is washed off with
20% aq. Copper sulphate solution & dried. Capsules stained pale violet whereas the cells stained deep
violet. Organisms identified using this technique includes Bacillus anthraxis, Streptococcus pneumoniae,
Klebsiella.

SPORE STAIN
The spore wall is relatively impermeable, but dyes can penetrate through heating the
preparation. The same impermeability prevents decolourization of the spore by alcohol treatment which
decolorizes vegetative cells. Spores are stained with malachite green & the vegetative cell
counterstained with safranin.
Examples of organisms stained using this method are Clostridium and Bacillus spp.