Obviously the
microorganisms
seen with an unaided eye. Therefore, it is almost necesary to visualize them (
microbes) with the help of
a specially and specifically designed device known as
microscope.
Interestingly, quite a few microorganisms are easily visible more willingly in comparison to others
by virtue of either their larger inherent dimension (size) or more rapidly observable characteristic
features. In actual practice, it has been duly observed that there are substantial number of microorganisms
which need to undergo systematic and methodical several
staining techniques whereby their cell
walls
, membranes, and other relevant structural features critically happen to lose their opacity
(opaqueness) or
colourless natural status.
Metric Units of Length :
Both the microorganisms along with their integral component parts
do possess very small physical features ; therefore, they are usually measured in units which are evidently
not-so-common to most of us in daily routine. The
microorganisms are measured in the metric
units of length
(i.e., the ‘Metric System’). Importantly, the standard unit of length in the domain of
the
‘metric system’ is the meter (m), which remarkably has the major advantage of having the ‘units’
that are invariably related to one another by factors of 10, such as : 1 m
≡ 10 decimeters (dm) ≡ 100centimeters (cm) ≡ 1000 millimeters (mm) : as shown below :
Preparation of Bacterial Specimens for Light Microscopy
As a large segment of living microorganisms invariably appear
almost colourless when seen
through a
standard light microscope, one should always subject them to a highly specific treatment for
possible vivid observation.
Staining (or colouring) is regarded to be one of the widely accepted phenomenon
to accomplish the aforesaid objective.
The various aspects of
‘staining’ shall be duly elaborated in the following sequential manner,
namely :
•
Stained preparations
•
Preparation of smears for staining
•
Gram staining
•
Differential staining
•
Miscellaneous staining e.g.,
Capsule staining ; Endospore staining ; Flagellar staining.
1. Stained Preparations
In usual practice a large number of investigative studies related to the specific shapes and cellular
arrangements of various microbes are effectively carried out with the help of
stained preparations. In
other words, different means and ways to colour the microorganisms with a particular and appropriate
dye (
i.e., staining) is performed meticulously so as to emphasize certain structures vividly and explicitely.
It may be worthwhile to state here that before one commences the
‘staining’ of the microbes they should
be duly fixed (or attached) onto the surface of the
microscopic slide ; naturally without proper fixing,
the requisite stain could wash them off the slide instantly.
2. Preparation of Smears for Staining
The
‘fixing’ of specific specimen may be accomplished by first spreading a resonably thin filmof the material onto the surface of the microscopic slide. In fact, this ‘thin film’, is termed as smear,
which is subsequently air dried. The air dried slide is now carefully exposed to a low flame of a Bünsen
burner a number of times, taking special care that the
smear side is always up. The aforesaid most
common
‘staining methodology’ comprising of air-drying followed by flame-heating allows the fixing
of the microorganisms onto the surface of the slide, and invariably
kill them completely. After this, the
‘suitable stain’
is adequately applied, and subsequently washed off with ample slow-running water.
The wet slide is now gently blotted with absorbent paper. The resulting slide having the
stained
microorganisms
are actually ready for detailed microscopic examinations, whatsoever.
3. Gram Staining
Hans Christian Gram (1884) – a Danish bacteriologist first and foremost developed the well
known staining procedure called as
Gram staining. Since, its inception earned a well-deserved recognition
across the globe by virtue of the fact that it categorically divides microorganisms into
two major categories,
namely : (
a) Gram-positive*, and (b) Gram-negative**.
Methodology :
The various steps involved are as follows :
(1) A
heat-fixed bacterial smear is duly covered with the following staining reagents in a
sequential manner, namely : (
a) crystal violet (i.e., a basic purple dye) which eventually imparts its
colour to
all cells ; and hence usually referred to as a primary strain ; (b) iodine solution i.e., clearly
washing off the purple dye after a short while, the smear is covered with iodine solution that serves as a
mordant***
; (c) alcohol**** i.e., the iodine is washed off thereby causing a ‘decolourizing effect’ ;
and (
d) safranin – a basic red due (or other appropriate agent) i.e., to act as a counterstrain.
(2) The resulting
‘smear’ is washed again, blotted dry, and carefully examined microscopically.
(3) In this manner, the purple dye (crystal violet) and the iodine combine with
each bacterium
thereby imparting to it a distinct purple or dark violet colouration.
Gram-positive Bacteria :
The bacteria which ultimately retain the purple or dark violet colouration
even after the alcohol treatment to decolourize them are grouped together as
Gram-positive bacteria.
Besides, it has been duly observed that as these specific class of microorganisms do retain the original
purple stain, they are significantly not affected by the
safranin counterstain at all.
Gram-negative Bacteria :
The bacteria that eventually lose the crystal violet, are duly
counterstained by the
safranin ; and, therefore appear red in colour.
The characteristic features enumerated below for
Gram +ve and Gram –ve bacteria vividly
justifies why the
Gram-staining technique renders some microorganisms purple-violet and others redin appearance.
4. Differential Staining
In
bacteriology, a stain for instance Gram’s stain which evidently enables one to differentiate
distinctly amongst the various kinds of bacteria. It may be emphasized at this material time that unlike
simple stains
, the differential stains very much interact altogether in a different manner with specifically
different types of microorganisms ; and, therefore, this criterion may be exploited to afford a clear cut
distinction amongst them. In actual practice, however, the differential stains largely employed for
microorganisms are (
a) the Gram’s stain ; and (b) the Acid-Fast Stain.
4.6.1.4.1. Gram’s Stain
It has already been discussed at length in the Section 4.6.1.3.
4.6.1.4.2. Acid-Fast Stain
Acid-fast stain
is used invariably in bacteriology, especially for staining Mycobacterium
tuberculosis
, and Mycobacterium leprae. This acid-fast stain possesses an inherent ability to get bound
intimately only to such microbes that have a waxy material in their cells (
e.g., all bacteria in the genus
Mycobacterium
). Besides, this particular stain is also employed to identify precisely the disease-producing
stains belonging to the genus
Nocardia.
Methodology :
The various steps involved in the acid-fast stain are as enumerated under :
(1) A specially prepared solution of the red dye
carbolfuschin is generously applied onto the
exposed surface of a heat-fixed bacterial smear ; and the treated slide is warmed* gently for several
minutes.
(2) The slide is brought to the room temperature (cooled) and washed duly with water.
(3) The, resulting smear is now treated with
acidic-alcohol (i.e., a decolourizer) that removes
critically the red stain from microorganisms which are not acid-fast.
(4) Thus, the
acid-fast microbes do retain the red colour (due to carbolfuschin) by virtue of the
fact that the red dye shows far greater solubility in the
waxes present in the cell wall rather than the acidalcohol.
(5) In
non-acid-fast microorganisms, whose cell walls are devoid of specific waxy components,
the dye
carbolfuschin gets readily removed in the course of decolourization thereby rendering
the cells almost colourless.
(6) Finally, the resulting smear is duly stained with
methylene blue counterstain whereby the
non-acid-fast cells
appear blue distinctly and the acid-fast cells as red.
Ziehl-Neelsen Method
(for staining M. tuberculosis) : This method was developed by two noted
scientists, namely : (
a) Franz Ziehl – a German Bacteriologist (1857-1926), and (b) Fried rich Karl
Adolf Neelsen
– a German Pathologist (1854-1894), whereby the causative organism M. tuberculosis
could be stained effectively. A solution of
carbolfuschin is applied duly, which the organism retains
after usual rinsing with
acid-alcohol admixture.
Capsule Staining (or Negative Staining for Capsules)
Capsule :
The bacterial capsule refers to the membrane that particularly surrounds certain
bacterial cells
, thereby offering adequate protection against the phagocytosis* and allowing evasion of
host-defense mechanisms**
.
It has been duly observed that a host of microorganisms essentially comprise of a gelatinous
covering (
i.e., capsule). However, in the domain of medical microbiology the very presence of a capsule
specifically establishes the
virulence*** of the said organism, the extent to which a pathogen may be
able to cause disease.
In general, the
capsule staining is rather more complicated and difficult in comparison to other
kinds of
staining techniques due to the fact that the particular capsular materials are not only water
soluble but also removable during the thorough washing procedure.
Methodology :
The various steps involved during the capsule staining are as stated under :
(1) First of all the microorganisms are carefully mixed in a solution comprising of a
fine colloidal
suspension
of some distinct coloured particles (one may invariably make use of either nigrosin or
India ink
) to afford a dark background.
(2) The bacteria may now be stained duly with a
simple stain, for instance : safranin.
(3) By virtue of the fact that
capsules do have a highly peculiar chemical composition fail to
accept a plethora of
‘biological dyes’ e.g., safranin ; and, therefore, they mostly appear as haloes****
just surrounding every
stained microbial cell.
(4) Importantly, the application of
India ink duly demonstrates a negative-staining procedure
so as to give rise to a distinct contrast between the
capsule and the adjoining dark medium.
Endospore (Spore) Staining
Endospore
refers to a thick-walled spore produced by a bacterium to enable it to survive
unfavourable environmental conditions. In actual practice, the occurrence of
endospores are
comparatively not-so-common in the microbial cells ; however, they may be adequately generated by
several genera of microorganisms. It is pertinent to mention here that the
endospores cannot be stained
by such ordinary techniques as : (
a) simple staining ; and (b) Gram staining, due to the fact the
biological dyes
are incapable of penetration through the wall of the endospore.
Schaeffer-Fulton Endospore Stain (or Schaeffer-Fulton Procedure) :
In the Schaeffer-Fulton
procedure
the endospores are first and foremost stained by heating together the respective
microorganisms with
malachite green, that happens to be a very strong stain which is capable of
penetrating the
endospores. Once the malachite green treatment is duly carried out, the rest of the cell
is washed rigorously free of dye with water, and finally counterstained with
safranin. Interestingly, thisspecific technique gives rise to a green endospore clearly resting in a pink to red cell
As the
endospores are highly refractive in nature, they may be visualized explicitely (i.e.,
detected) under the
light microscope when unstained*.
Flagella Staining
Flagella
(Pl. of Flagellum) usually refers to a threadlike structure that essentially provides motility
for certain
microorganisms and protozoa (one, few, or many per cell), and for spermatozoa (one per
cell).
It has been well established that the
bacterial flagella do represent various structures of locomotion
that happen to be exceptionally small to be visualized with the help of
light microscopes without staining.
Methodology :
The staining technique consists of a tedious and a quite delicate stepwise procedure
that makes use of a stain
carbolfuschin and a mordant so as to build up the desired requisite diameters
of the respective flagella unless and until they are rendered quite reasonably visible under the
light
microscope. Clinical microbiologists
usually exploit the arrangement and the specific number of flagellacritically as diagnostic aids.