Preparation of Bacterial Specimens for Light Microscopy

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Chapter: Pharmaceutical Microbiology : Identification of Microorganisms

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.

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 phe-nomenon 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.


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.


Preparation of Smears for Staining


The ‘fixing’ of specific specimen may be accomplished by first spreading a resonably thin film of 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.


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 red in appearance.


(1) A heat-fixed bacterial smear of cocci and rods is first duly covered with a basic purple dye (primary stain) e.g., crystal violet, and the dye is washed off subsequently.


(2) Resulting smear is covered with iodine (a mordant), and washed off. At this particular stage both Gram +ve and Gram –ve bacteria are purple in appearance.


(3) The treated slide is washed with ethanol or an alcohol-acetone solution (a decolourizer), and washed with water subsequently. At this stage Gram +ve cells are purple, and Gram –ve cells are colourless.


(4) Final step, safranin, is added as a counterstain, and the slide is washed, dried, and examined micro-scopically. Gram +ve bacteria retain the purple dye, whereas the Gram –ve bateria appear as pink.

[Adapted from : Tortora et. al. Microbiology an Introduction, The Benjamin/Cummings Publishing Co. Inc., New York, 5th edn, 1995].


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.


Gram’s Stain


It has already been discussed at length in the later Section.


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 acid-alcohol.


(5) In non-acid-fast microorganisms, whose cell walls are devoid of specific waxy compo-nents, 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.


Miscellaneous Staining


There are centain equally important staining procedures which do not fall within the techniques discussed under Sections through Hence, these special staining procedures shall be treated individually in the sections that follows :


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 colloi-dal 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, this specific technique gives rise to a green endospore clearly resting in a pink to red cell as depicted in Fig. 4.2.


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 flagella critically as diagnostic aids.


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