Culture Methods

CULTURE METHODS [email protected]  CULTURE METHODS Cultural methods employed depend on the reasons for which they are intended. The purpose of culture is: - To isolate bacteria in pure cultures. - To demonstrate their properties. - To obtain sufficient growth for the preparation of...

CULTURE METHODS [email protected]  CULTURE METHODS Cultural methods employed depend on the reasons for which they are intended. The purpose of culture is: - To isolate bacteria in pure cultures. - To demonstrate their properties. - To obtain sufficient growth for the preparation of antigens and for other tests. - To determine sensitivity to antibiotics. - To estimate viable counts. - Maintain stock cultures.  Cultural methods include: - Streak culture - Lawn culture - Stroke culture - Stab culture - Pour plate method - Liquid culture - Anaerobic culture methods STREAK CULTURE It is used for the isolation of bacteria in pure culture from clinical specimens. One loopful of the specimen is transferred onto the surface of a well-dried plate. The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate. On incubation, separated colonies are obtained over the last series of streaks LAWN CULTURE Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium It provides a uniform surface growth of the bacterium. Uses - For bacteriophage typing. - Antibiotic sensitivity testing. - In the preparation of bacterial antigens and vaccines. STROKE CULTURE Stroke culture is made in tubes containing agar slope/slant. Uses - Provide a pure growth of bacterium for slide - Agglutination and other diagnostic tests. STAB CULTURE Prepared by puncturing a suitable medium (gelatin or glucose agar) with a long, straight wire. Uses - Demonstration of gelatin liquefaction/ gelatin hydrolysis test. - Oxygen requirements of the bacterium under study. - Maintenance of stock cultures. POUR PLATE CULTURE Uses Gives an estimate of the viable bacterial count in a suspension for the quantitative urine cultures. - Agar medium is melted and cooled to 45oC. - 1 ml of the inoculum is added to the molten agar. - Mix well and pour to a sterile petri dish. - Allow it to set. - Incubate at 37oC, colonies will be distributed throughout the depth of the medium. ENZYME DETECTION OXIDASE TEST ✓Detects the presence of an enzyme oxidase produced by certain bacteria which will oxidize phenylenediamine in the reagent to a deep purple colour ✓Positive test is indicated by the development of a purple colour. ✓Oxidase positive – Pseudomonas, Vibrio, Neisseria ✓Oxidase negative – Salmonella, Shigella INDOLE TEST ✓Used to detect indole production from tryptophan present in peptone water. ✓Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4 (p)-dimethylaminobenzaldehyde. ✓Positive test is indicated by a pink ring. ✓Negative indole test – yellow ring ✓Indole positive – E.coli ✓Indole negative – Klebsiella, Salmonella UREASE TEST ✓ Done in Christensen’s urease medium. ✓ Used to detect organisms that produce urease. ✓ Urease produced by the organisms splits urea into ammonia and CO2. ✓ Urease positive – pink colour ✓ Urease negative – yellow colour ✓ Positive – Proteus, Klebsiella ✓ Negative – E.coli, Salmonella CATALASE TEST ✓ Catalase-producing organisms, when exposed to hydrogen peroxide, produce visible bubbles within 30 seconds. ✓ Differentiates staphylococci, which are catalase positive, from streptococci, which are catalase negative. ✓ Other catalase-positive organisms include Listeria, C. diphtheriae, the family Enterobacteriaceae, Pseudomonas, Mycobacterium tuberculosis, etc COAGULASE TEST ✓ Used to differentiate Staphylococcus aureus (positive) from coagulase-negative Staphylococcus (CONS). ✓ Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen in plasma to (insoluble) fibrin  Carbohydrate fermentation TRIPLE SUGAR IRON AGAR (TSI) Composite media is used to study different properties of a bacterium eg., Sugar Fermentation, Gas production, and H2S production ✓Contains 3 sugars – Glucose, Lactose, Sucrose. ✓Ferrous Sulphate ✓Sodium Thiosulfate ✓pH indicator (Phenol Red)  TSI REACTIONS ✓Yellow slant / Yellow butt – Lactose fermenters ✓Pink slant / Yellow butt – Non-lactose fermenters. ✓Pink slant / no colour change – Non fermenters ✓Black colour– H2S production. ✓Gas bubbles or cracks in the medium–gas production. Lactose fermenters – E.coli, Klebsiella Non-lactose fermenters– Salmonella, Shigella H2S – Proteus  METHYL RED TEST ✓To differentiate enterobacteria ✓Organisms ferment glucose, producing sufficient acidity in a buffered medium to give a Brick red colour change. Positive – E. Coli, Proteus Vulgaris Negative – Serratia marcescens, Enterobacter, Aerogenes. STAINING TECHNIQUES STAINING CLASSIFICATION Stains are substances applied to biological or non-biological specimens to enhance contrast, making it easier to visualize and study them under a microscope. The classification helps researchers and scientists choose the most appropriate staining method for their specific objectives. The three primary categories of positive staining are: ✓Simple stains ✓Differential stains ✓Special stains SIMPLE STAINING Involves the use of a single dye to uniformly color all cells or structures in a specimen. Provides basic contrast for easy visualization. Highlights the overall morphology and size of cells. COMMONLY USED SIMPLE STAINS ✓Crystal Violet: Widely used for general bacterial staining. ✓Methylene Blue: Stains a variety of cells and tissues. ✓Safranin: Used in microbiology for staining Gram- negative bacteria. ✓Advantages: - Quick and easy procedure. - Provides a general overview of cell morphology. - Requires minimal equipment and expertise. ✓Limitations: - Lacks specificity for different cell components. - May not differentiate between different cell types. - Limited information compared to more advanced staining techniques. DIFFERENTIAL STAINING Utilizes multiple dyes (primary dye and counter stain) to distinguish between different types of cells or structures based on their chemical and physical properties. Enables the differentiation and classification of microorganisms. Highlights specific structural features for identification. Types of differential staining include: ✓Gram Staining ✓Acid Fast Staining GRAM STAINING In 1884, Hans Christian Gram, a Danish bacteriologist, developed the Gram staining technique as a differential staining method. Gram staining became one of the most widely used and fundamental techniques in microbiology. The primary purpose of Gram staining is to classify bacteria based on the characteristics of their cell walls. Gram-positive bacteria have a thick peptidoglycan layer in their cell wall, which retains the crystal violet dye. Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane, causing them to lose the crystal violet stain during the decolorization step. GRAM STAINING Gram-positive bacteria retain the crystal violet stain and appear purple under the microscope. Gram-negative bacteria do not retain the crystal violet stain and take up the counterstain (safranin), appearing pink. -Primary stain: Crystal Violet. - Mordant: Iodine - Decolorization: Alcohol. - Counterstain: Safranin. ACID FAST STAINING Acid-fast staining is used to identify bacteria that resist decolorization by acid-alcohol. Acid-fast bacteria retain the primary stain despite the decolorization step, appearing red/purple under the microscope. Acid-fast staining is particularly important for detecting Mycobacterium species, including the causative agent of tuberculosis, Mycobacterium tuberculosis. Mycobacteria have a unique lipid-rich cell wall that makes them resistant to standard staining methods. - Primary stain: Carbol fuchsin. - Decolorization: Acid-alcohol. - Counterstain: Methylene blue. ACID FAST STAINING The Ziehl-Neelsen method is a widely used acid-fast staining technique. In this method, carbolfuchsin (a red dye) is used as the primary stain, and acid-alcohol is used for decolorization. - Primary stain: Carbol fuchsin. - Decolorization: Acid-alcohol. - Counterstain: Methylene blue. SPECIAL STAINS ✓Target and highlight specific structures or components in a specimen. ✓Provide detailed examination and visualization of particular elements. ✓Enable the identification and analysis of specific biomolecules or cellular structures. Examples of Special Stains Immunohistochemistry (IHC): Involves using antibodies to detect specific antigens in tissues. Visualize the distribution and localization of proteins within cells or tissues. Used in cancer research, pathology, and neuroscience. ✓ Fluorescence Staining: Utilizes fluorophores to label specific structures in specimens. Enables visualization of fluorescence-labeled components under a microscope. Applied in cell biology, genetics, and live-cell imaging. ✓ Giemsa Stain: Combines eosin and methylene blue to stain blood cells and microorganisms. Used in cytology and histology for detailed cellular examination. Commonly used in diagnosing malaria and studying blood cells. Advantages of Special Staining: Provides high specificity for targeted structures. Enables visualization of specific molecules or cellular components. Allows for multiplexing, studying multiple targets simultaneously. Factors Influencing Staining Techniques ✓ pH: pH level affects the ionization and binding properties of dyes. Different stains may require specific pH conditions for optimal results. ✓ Temperature: Staining at higher temperatures can enhance the rate of dye penetration. Temperature variations can affect the overall staining process. ✓ Duration of Staining: The length of time a specimen is exposed to a stain affects the intensity of staining. Overstaining or understaining may impact the quality of results. ✓ Concentration of Stain: The concentration of the staining solution influences the saturation of color in the specimen. Too high or too low concentrations can lead to suboptimal results. TIPS FOR OPTIMIZING STAINING TECHNIQUES ✓ Standardization: Establish standardized protocols for staining procedures. Ensure consistent staining conditions for reproducibility. ✓ pH Control: Adjust and maintain the pH of staining solutions as per stain requirements. Use buffer systems to stabilize pH. ✓ Temperature Regulation: Monitor and control the temperature during staining processes. Optimize temperature conditions for specific stains. ✓ Quality of Reagents: Use high-quality stains and reagents for accurate and reliable results. Check expiration dates and storage conditions. ✓ Sample Preparation: Properly prepare specimens to ensure uniform staining.