Title 9. IDENTIFICATION OF BACTERIA WITH EMPHASIS TO DIFFERENT STAINING TECHNIQUES AND BIOCHEMICAL REACTIONS.pdf ✅

PHARMD GURU Page 1 INTRODUCTION:  Microorganisms in nature are never found in pure populations.  Different types of mixed microorganisms are normally present in soil, water, food, air and various parts of the human or animal body.  To study the role played by a specific microorganisms in these environments, it is necessary to isolate the microorganisms in pure culture.  It is extremely important to maintain isolated pure cultures for extended periods in a viable condition.  Most microbiological laboratories usually maintain a large collection of pure cultures as well as subcultures of authentic species purchased from various culture collection centres. Ex: American Type Culture Collection (ATCC), U.S.A; National Collection of Industrial Bacteria (NCIB), Scotland; National Collection of Yeast Cultures (NCYC), England; National Collection of Type Cultures (NCTC), England; National Chemical Laboratory (NCL), India etc.  Identification of unknown bacterial cultures is one of the major responsibilities of a microbiologist.  Samples of blood, tissue, water, food and cosmetics are examined daily in laboratories throughout the world for the presence of contaminants and pathogenic microorganisms.  Pharmaceutical industries and research institutes are constantly screening soil, water, marine samples to isolate new antibiotic, enzyme and vitamin producing microorganisms.  Once these unknown important microorganisms are isolated they must be identified and classified. IDENTIFICATION OF BACTERIA WITH EMPHASIS TO DIFFERENT STAINING TECHNIQUES AND BIOCHEMICAL REACTIONS
PHARMD GURU Page 2 The science of classification is called taxonomy. Bergey's Manual of Systemic Bacteriology has been the official, internationally accepted reference for bacterial classification. In the current edition of Bergey's manual, bacteria is classified into 33 groups called sections rather than into the classical taxonomic groupings of phylum, class, order and family. The relationship of microorganisms in each section is based on characteristics such as morphology, staining reactions, nutritional requirements, physiological properties, cultural characteristics, biochemical tests, serological properties, pathogenicity and genetic characterisation. MORPHOLOGY:  The morphology of microorganisms depends on a number of factors such as the stain studied, nature of culture medium, temperature and duration of incubation.  The size, shape and arrangement of cells is determined by microscopic examination of stained smears. The shape may be spherical, rod, filamentous, comma like or spiral.  They may be arranged singly, pairs, tetrads or in packets of eight or in chains. Unstained wet film or hanging drop preparations are examined under a light microscope for observation of motility.  They may be non-motile (absence of flagella) or motile with monotrichate, lophotrichate, amphitrichate or peritrichate flagella.  The spores may be oval, spherical or ellipsoidal if present. STAINING REACTIONS: To study size, shape, arrangement and properties and differentiate specific groups of microorganisms, biological stains are used. Stain is an organic compound containing a benzene ring with chromophore and auxochrome group. Different staining techniques are used for visualisation, differentiation and separation of bacteria in terms of morphological characteristics and cellular structures. 1) SIMPLE STAINING: In simple staining, the bacterial smear is stained with a single stain e.g. methylene blue, crystal violet, carbol fuchsin, safranin, etc. Basic stains with a positively
PHARMD GURU Page 3 charged chromogen are used. Bacterial nucleic acids and certain cell wall components carry a negative charge that strongly binds to the cationic chromogen. The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells (Fig. 4.1). The surface of a bacterial cell has acidic characteristic because of a large amount of carboxyl groups located on the cell surface due to acidic amino acids. Therefore, when ionisation of carboxyl groups takes place, it imparts negative charge to the cell surface. In nature, H+ is replaced by another positive charged ion. Ex: Na+ or K+ and H+ bonds with oxygen to form water. Thus, surface of an unstained bacterial cell is represented as shown below. Basic dyes are commonly used for the monochrome staining. These dyes are available as a salt of acids. Ex: methylene blue chloride. When methylene blue rehydrates, it ionizes to form methylene blue and chloride ions. The positively charged ions have the colouring property.
PHARMD GURU Page 4 On addition of methylene blue for staining, exchange of MB+ with Na+ on the bacterial cell surface takes place, resulting into ionic bond formation between MB+ and cell surface. Thus, when colouring agent forms ionic bond with cell or cell components, it results into the staining of cell. The most commonly used basic stains are methelyne blue (2 to 3 minutes), crystal violet (1 to 2 minutes) and carbol fuchsin (15 to 30 seconds). 2) NEGATIVE STAINING: In negative staining, bacteria are mixed with acidic stain (eosin or nigrosin) and a smear is prepared. The acidic stain (negative chromogen) does not penetrate the cells because of the negative charge on the surface of bacteria. Hence, unstained cells are easily observed against the coloured background (Fig. 4.2). This technique is also useful in demonstration of bacterial capsule. Negative (indirect) staining is a technique by which bacterial cells are not stained but are made visible against dark background. The acidic stain is used in this staining e.g. eosin, nigrosin, congo red, Rose Bengal stain etc. Acidic stain has negative charge; therefore, it does not combine with negatively charged of bacterial cell surface. On the other hand, it forms a deposit around the cell, resulting in appearance of bacterial cell colourless against the dark background.