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INTRODUCTIONOF CULTURE MEDIA Most bacteria can be cultured artificially on culture media containing required nutrients, pH and osmotic pressure. The microorganisms grow in an atmosphere and temperature most suited to their metabolic reactions. The pathogens are isolated in pure culture so that they can be identified and tested for their sensitivity to antimicrobials. The specimens are cultured in known volumes, and the number of bacterial colonies appearing after incubation can be counted. Operation room requirements and blood from blood bank are frequently checked for sterility by using pure culture methods. Vaccines and antitoxins require the growing of bacteria under controlled conditions. Stock cultures are also useful for the teaching institutes for practical training purposes. COMPOSITION OF CULTURE MEDIA The basic ingredients, which are common to the many of the frequently used media are as follows: 1.  Water: It allows fluids to enter and leave cells more readily and to enhance the chemical reactions. 2.Sodium chloride: Presence of sodium chloride maintains the isotonicity  of bacterial cells. 3.Peptones: It is a source of readily available nitrogen. 4.Buffers: They maintain a constant pH in culture media. 5.Indicators:  In culture media the indicators are useful in the detection of acid or alkali production by microorganisms. Indicators such as phenol red, methyl red and Bromocresol purple are used to adjust pH of the culture media. 6.  Solidifying agents: Agar, gelatin, egg yolks and serum are used as solidifying agents of the culture media. 7.Selective agents: These are special chemicals introduced into the culture media for inhibiting some types of bacteria, while allowing other bacteria to grow. Examples: (a) Crystal violet inhibits Staphylococci but not tubercle bacilli (b) 6.5% (w/ v) sodium chloride is inhibitory to most Streptococci but not S. faecalis. 8.   Additive for enrichment: The substances such as sheep blood, horse blood, rabbit serum or calf’s ground hearts allow fastidious organisms to grow since these organisms, may not survive in ordinary culture media. 9.Reducing substances: The substances such as thioglycollate are used to remove free oxygen from the medium for the growth of anaerobic bacteria. THE DIFFERENT TYPES OF CULTURE MEDIA Basic Media These support the growth of microorganisms that do not have special nutritional requirements. They are often used (a) To maintain stock cultures of control strains of bacteria and (b) For subculturing pathogens from selective media prior to performing biochemical and serological dentification tests. Examples: (1) Nutrient agar (2) Nutrient broth. Enriched Media These are enriched with (a) Whole blood (b) Lysed blood (c) Serum (d) Extra peptones and (e) Vitamins to support the growth of particular pathogens such as Hemophilus influenzae, Neisseria and Streptococcus species. Examples: (I) Blood agar (II) Tryptone soya media. Selective Media These media contain substances that accelerate the growth of required pathogens only and prevent or slow down the growth of other microorganisms. Example: XLD agar: It is used for the growth of Salmonellae and Shigellae. The bile salts present in this media inhibit the growth of many fecal commensals Differential (Indicator) Media These contain indicators, dyes or other substances which help to differentiate microorganisms. Example: TCBS agar contains the indicator bromothymol blue which differentiates sucrose fermenting from non-sucrose fermenting vibrio species. Transport Media When specimens are not cultured soon after collection, to prevent overgrowth and also to ensure survival of pathogens, transport media are used. These are mainly used to transport microbiological specimens from health centers to the district pathological laboratories. Example: (1) Amies transport medium (2) Cary Blair medium. DIFFERENT FORMS OF CULTURE MEDIA:- 1.      Solid Culture Media This is used in petri dishes and in test tubes (slope cultures) and prepared by adding Solidifying agent  (1.0 – 1.5%) (w/v). Microorganisms grow on this and form colonies after multiplication. This helps to identify the organism. 2.    Semisolid Culture Media This is prepared by adding Solidifying agent (0.4-0.5%) (w/v) to a fluid medium. These are used mainly as transport media and for the testing of motility of the organisms. 3.   Fluid Culture Media These media are mainly used as biochemical testing media, blood culture media or the enrichment media. PREPARATION OF CULTURE MEDIA Most culture media are available commercially in readymade dehydrated form. It is less costly to use readymade media, since the ingredients are often required in small amounts but available in large quantities if purchased. Some of the chemicals are also difficult to obtain. To ensure good performance and reproducibility in the results the following must be  performed correctly- 1.     Weighing and dissolving of the ingredients 2.     Addition of heat sensitive material 3.     pH testing 4.     Dispensing and sterilization 5.     Sterility testing and quality control 6. Storage Note 1.      The heat sensitive ingredients such as blood or serum should be brought to room temperature and added when the medium has cooled to about 50°C. 2.      A fluid medium should be tested for accurate pH by using a narrow, range pH paper. 3.      For sterilizing culture media, it is necessary to use manufacturer’s instructions. The commonly used methods for sterilization are- a) Autoclaving (b) Steaming at 100°C and (c) Filtration. It is necessary to use correct temperature and correct length of time. Precautions:- 1.  Autoclaving is used to sterilize most agar and fluid media. 2. Steaming at 100°C: Media such as Cary Blair transport medium contain ingredients that would break down above 100°C. Steaming can be performed in an autoclave with a loose lid. 3.    Filtration: Serum and solutions containing carbohydrates, urea, etc. are heat sensitive and hence cannot be autoclaved. Hence, the media containing such substances are filtered to remove bacteria. 4. Sterility testing: Media in tubes and bottles: Incubate the entire batch at 37°C overnight. Contamination is indicated by appearance of turbidity in a fluid medium and growth on a solid medium. 5.  Control of media: Appropriate control species are used to inoculate slants or plates of the medium (quarter part). After overnight incubation, the cultures are examined for (a) Degree of growth (b) Size of colonies and (c) Other characteristics. 6.Storage of culture media: Dehydrated culture media and dry ingredients (agars, peptones, bile salts, etc.) can be stored at room temperature (25°C ± 5°C) in a cool and dry place, away from

Bacteria:- Bacteria are single-celled microorganisms that have a relatively simple structure compared to other living organisms. They are prokaryotic organisms, which means they lack a true nucleus and membrane-bound organelles. Bacteria are some of the most numerous and diverse organisms on Earth, and they can be found in various environments, including soil, water, air, and inside the bodies of other organisms. Here is an overview of the basic structure of bacteria: 1. Cell wall bacterial cell wall is a rigid structure that surrounds the cell membrane of most bacteria. It provides structural support and protection to the cell, helping it maintain its shape and resist changes in the surrounding environment. The composition and structure of bacterial cell walls can vary widely among different types of bacteria, and these differences are used to classify bacteria into two main groups: Gram-positive and Gram-negative. Gram-Positive Bacteria cell wall:-  Gram-positive bacteria have a thick layer of peptidoglycan, a polymer made up of sugars and amino acid, in their cell walls. This layer is located outside the cell membrane and provides strength and rigidity to the cell wall. In addition to peptidoglycan, Gram-positive cell walls often contain teichoic acids, which are polymers of glycerol or ribitol phosphate. These acids contribute to the overall negative charge of the cell wall. Gram-Negative Bacteria cell wall:  Gram-negative bacteria have a thinner layer of peptidoglycan in their cell walls, located between the inner and outer membranes. Outside the peptidoglycan layer, there is an outer membrane made up of lipopolysaccharides (LPS) and proteins. LPS molecules are composed of lipid A (which anchors the LPS molecule in the outer membrane), a core polysaccharide, and an O antigen (which varies among different bacterial species). The outer membrane acts as a barrier against certain chemicals, including antibiotics, making Gram-negative bacteria often more resistant to these substances than Gram-positive bacteria. Function of bacterial cell wall:- Structural Support:  The cell wall maintains the shape of the bacterium and prevents it from bursting or collapsing due to changes in osmotic pressure. Protection:  The cell wall provides protection against physical damage and helps the bacterium resist the effects of harmful substances in the environment. Barrier:  In Gram-negative bacteria, the outer membrane acts as a barrier that can exclude or allow the passage of specific molecules into the cell. Virulence Factor:  Some components of the cell wall, such as lipopolysaccharides in Gram-negative bacteria, can trigger immune responses in the host organism, contributing to the bacterium’s virulence. 2.    Cell Membrane (Plasma Membrane):  The bacterial cell membrane, also known as the plasma membrane or cytoplasmic membrane, is a vital structure that surrounds the bacterial cell. It serves as a selectively permeable barrier, separating the interior of the cell from its external environment. The cell membrane is a phospholipid bilayer embedded with proteins and other molecules, and it performs several essential Functions of cell membrane in bacterial cells: 1.      Selective Permeability:   The phospholipid bilayer of the cell membrane acts as a barrier that prevents the passage of most substances, including ions and large molecules, into and out of the cell. Only specific molecules, such as gases (like oxygen and carbon dioxide) and small hydrophobic molecules, can freely diffuse through the lipid bilayer. Other substances, such as nutrients and waste products, require specialized transport proteins to facilitate their movement across the membrane. 2.      Nutrient Uptake:  Bacteria need various nutrients to survive, including sugars, amino acids, and ions. Specialized membrane proteins, such as transporters and permeases, help actively or passively transport these nutrients into the cell, allowing the bacterium to obtain the necessary building blocks and energy for its metabolic processes. 3.      Waste Elimination:  Similarly, the cell membrane aids in the removal of waste products and metabolic byproducts from the bacterial cell. These waste products are expelled from the cell through specific transport mechanisms 4.      Energy Production:    Bacterial cell membranes are also the sites of electron transport chains and ATP synthesis in many bacterial species. These processes are essential for the generation of energy in the form of ATP (adenosine triphosphate), which powers various cellular activities. 5.      Maintaining Cell Shape and Integrity:       The cell membrane plays a role imaintainingthe shape and integrity of the bacterial cell. It provides structural support and helps the cell maintain its specific shape, especially in the absence of a rigid cell wall. 1.      Sensory Functions: The cell membrane contains proteins and receptors that allow bacteria to sense changes in their environment. These proteins can detect environmental signals, such as changes in temperature, pH, or the presence of specific molecules, triggering appropriate cellular responses. 3. Cytoplasm:  The bacterial cytoplasm is the semi-fluid substance inside a bacterial cell, enclosed by the cell membrane. A complex, gel-like matrix contains various cellular components and is the site of numerous biochemical reactions essential for the bacterium’s survival and growth. Here are some key aspects of bacterial cytoplasm:   Composition: The cytoplasm is primarily composed of water, making up the majority of its volume. In addition to water, the cytoplasm contains ions, enzymes, nucleotides, amino acids, sugars, and other small molecules that are crucial for the bacterium’s metabolism. Genetic Material: The bacterial chromosome, a single circular DNA molecule, is located in the cytoplasm. Unlike eukaryotic cells, bacteria do not have a membrane-bound nucleus, so their genetic material is dispersed in the nucleoid region within the cytoplasm. The nucleoid lacks a membrane and is simply an area where the genetic material is concentrated. Ribosomes: Bacterial cytoplasm contains ribosomes, which are the cellular structures responsible for protein synthesis. These ribosomes read the genetic information encoded in mRNA (messenger RNA) and use it to assemble amino acids into proteins. Metabolic Reactions: Various metabolic pathways occur within the bacterial cytoplasm. These include processes such as glycolysis (the breakdown of glucose), the citric acid cycle (Krebs cycle), and the synthesis of essential molecules like nucleotides, amino acids, and fatty acids. Enzymes catalyzing these reactions are found in the cytoplasm. Storage Granules: Some bacteria store reserve materials such as glycogen, polyhydroxyalkanoates (PHAs), or sulfur granules in the cytoplasm. These storage compounds serve as a source of energy

ACID FAST STAIN:- •       A few types of bacteria, such as the mycobacteria and Nocardia species, do not stain using common staining techniques or, if stained, they produce a variable reaction because their walls are not permeable to the rosaniline dyes in common staining regimens. •       The cell walls of the mycobacteria contain mycolic acids giving the cell walls a high lipid content because of which these bacteria are difficult to stain. •       Visualization of these cells in samples require higher concentrations of the dye solution and/or a heating period. •       The expression “acid fast” is derived from the observation that even with the addition of hydrochloric acid to the alcohol decolorizer, some of the stained cells retain the primary stain (carbolfuchsin) •       Cells that release the primary stain (carbolfuchsin) with decolorizing will be visible after the counterstaining step is complete.  •       Bacteria described as acid fast will appear red when examining specimens using bright-field microscopy.  •       Non-acid-fast cells and field debris will appear blue. •       Acid fastness is a characteristic that is shared by just a few organisms, so staining to determine if organisms possess this trait is useful in microbial identification schemes. ACID-FAST STAINING: HIDTORICAL BACKGROUND:- •       In 1882, Robert Koch discovered the tubercle bacillus and described the appearance by using a complex staining procedure.  •       Franz Ziehl was the first to use carbolic acid (phenol) as the mordant. •       The acid-fast stain is performed on samples to demonstrate the characteristic of acid fastness in certain bacteria and the cysts of Cryptosporidium and Isospora. •       Clinically, the most important application is to detect Mycobacterium tuberculosis in sputum samples to confirm or rule out a diagnosis of tuberculosis in patients. •       Friedrich Neelsen kept Ziehl’s mordant, but changed the primary stain to the basic fuchsin (first used by Ehrlich in 1882). This method is known as the Ziehl-Neelsen method. •       In this method heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells because of which the technique is called the “hot staining” method. •       In 1915, Kinyoun published a method that has become known as the “cold staining” method because the heating step was removed in favor of using a higher concentration of the carbolfuchsin primary stain. COMMON ACID-FAST STAINING METHOD:- •      Three methods of acid fast staining:            –     Ziehl-Neelsen (hot),          –     Kinyoun (cold), and           –     Auramine-Rhodamine Fluorochrome (Truant method).  •      The slides produced by Ziehl- Neelsen and the Kinyoun methods can be visualized using a standard bright-field microscope.  .The fluorochrome method is used by large laboratories that have a fluorescent (ultraviolet) microscope. Ziehl-Neelsen or (Hot) method for acid-fast staining:- •      Requirements:-              –     Carbolfuchsin stain:                •      Basic fuchsin,            0.3 g                •      Ethanol,                      95% (vol/vol),                 •      10 ml Phenol, heat-melted crystals,                                                                    5 ml                 •      Distilled water,           95 ml Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water. Mix and let stand for several days. Filter before use. –     Decolorizing solvent:    •      20% H2SO4 Solution •     Counterstain:                           –     Methylene blue           Procedure:- • Heat fix an air dried smear at 80ͦͦC for at least 15 minutes or for 2 hours on an electric hot plate at 65ᶱC-70ᶱC.  •     Place a slide with an air-dried and heat-fixed smear on suitable staining device. Cut a piece of absorbent paper to fit the slide and saturate the paper with the carbolfuchsin stain. Or cover the smear with carbolfuchsin. •     Keep the preparation moist with stain and steaming for 5 minutes. •     Wash the film in a gentle and indirect stream of tap water until no color appears in the effluent. •     Repeat the decolorizing and the washing until the stained smear appears faintly pink and the fluid washing off the slide runs clear. •     Flood the smear with the methylene blue counterstain for 20 to 30 seconds, and wash with tap water. •      After air drying, examine under oil immersion. Acid-fast bacteria appear red, and non-acid-fast bacteria appear blue. 2.Kinyoun (cold) method for acid-fast staining •     Kinyoun carbolfuchsin solution:  –     Solution A: Dissolve 4 g of basic fuchsin in 20 ml of ethyl alcohol. –     Solution B: Dissolve 8 g of phenol (melted) in 100 ml of distilled water. –     Mix solutions A and B together and allow to stand for a few days. •     Acid-alcohol decolorizing agent:  –     Ethanol, 95% (vol/vol), –     97 ml Hydrochloric acid (concentrated), 3 ml •     Methylene blue counterstain: –     Methylene blue chloride, 0.3 g Distilled water, 100 ml Dissolve by shaking.       Procedure:- • Flood slides with Kinyoun’s carbolfuchsin reagent and allow to stain for 5 minutes at room temperature.  •     Rinse with deionized water and tilt slide to drain. •     Decolorize with acid-alcohol for 3 minutes and rinse again with deionized water. •     Redecolorize with acid-alcohol for 1-2 minutes or until no more red color runs from the smear. •     Rinse with deionized water and drain standing water from the slide surface by tipping the slide. •     Flood slide with methylene blue counterstain and allow to stain for 4 minutes.  •     Rinse with distilled water and allow to air dry. •      Examine under high dry (400X) magnification, and confirm acid-fast structures under oil immersion (1000X) Auramine-Rhodamine Fluorochrome  or Truant method for acid-fast staining • Fluorescent staining reagent:  –     Auramine O,        CI 41000, 1.50 g –     Rhodamine B,     CI 749, 0.75 g Glycerol, –     75 ml Phenol (heat melted crystals), –     10 ml Distilled