Una

• Stains


  A mixture of selected dyes to color a particular biological specimen

  
• Dyes


  A general-purpose coloring agent
• Classification of stains based on charge

  • Acid
  • Basic
  • Neutral
• Acid stain


  Chromogen is negatively charged, used to stain the positive component of the microbial cell
• Basic stain


  Chromogen is positively charged, used to stain negatively charged component of microbial cell
• Neutral stain


  Complex salt of dye acid with dye base, stains both positive and negative charged components of microbial cell
• Neutral stains


  • Giemsa stain
  • Leishman stain
• Requirements for staining


  • Stain
  • Mordant
  • Decolorizer
  • Accentuater
  • Slide Preparation
  • Smear
  • Aseptic Transfer
  • Heat fix

  
• Staining methods used in microbiology

  • Simple staining
  • Differential staining
  • Special staining

  
• Simple staining


  Uses only a single dye that does not differentiate between different types of organisms

  
• Types of simple staining

  • Direct or positive staining
  • Indirect or negative staining

  
• Direct or positive staining


  One reagent used, basic dye stains bacteria

  
• Negative staining


  Acid dye is repelled by negatively charged cell wall, cells appear colorless against dark background

  
• Differential staining


  Distinguishes organisms based on their interactions with multiple stains
• Types of differential staining

  • Gram staining
  • Acid-fast staining

  
• Gram staining


  Differentiates bacteria into gram-positive and gram-negative groups
• Acid-fast staining


  Distinguishes bacteria based on the wax content of their cell wall, used for Mycobacterium spp.
• Types of special staining

  • Flagella staining
  • Capsule staining
  • Endospore staining

  
• Flagella staining


  Visualizes the presence and arrangement of flagella
• Flagella staining methods


  • Leifson flagella stain
  • Ryu-based wet-mount flagella stain

  
• Capsule staining


  Differentiates capsular material from bacterial cells
• Capsule staining methods


  • Negative staining using India ink
  • Hiss' method
  • Maneval's method

  
• Endospore staining


  Differentiates bacterial spores from vegetative cells and spore formers from non-spore formers
• Endospore staining method


  • Schaeffer-Fulton method

  
• Staining is a technique used to enhance contrast in microscopic samples

  
• Applications of staining in microbiology

  • Define cell size, shape, arrangement
  • Study chemical properties and structures
  • Demonstrate purity of culture
  • Observe certain structures

  
• Bacterial identification is challenging due to microbes being widely distributed, present in complex associations, and not visible to the naked eye
• Six I's of bacterial identification

  • Inoculation
  • Isolation
  • Incubation
  • Identification
  • Inspection
  • Information Gathering
• Inoculation


  Cultivating an inoculum into a container of fresh nutrient medium
• Isolation techniques


  Colony, streak plate, loop dilution/pour plate, spread plate
• Incubation


  Controlling atmospheric gases and temperature to allow microbial growth
• Types of microbial cultures

  • Pure (axenic)
  • Mixed
  • Contaminated
• Identification techniques


  • Morphology
  • Staining
  • Biochemical tests
  • DNA assays

  
• Types of culture media

  • General/non-selective
  • Enriched
  • Selective
  • Differential
  • Reducing
  • Carbohydrate fermentation
  • Transport
  • Assay
• Some microbes can only grow on live cells or animals (obligate parasites)
• Animals are an important source of antibodies, antisera, antitoxins, and other immune products used in therapy or testing
• 3 Types of Cell Work

  • Chemical
  • Transport
  • Mechanical
• Laws of Thermodynamics


  Metabolism is the sum of all chemical reactions within a living organism
• 2 Classes of Metabolism

  • Release energy
  • Require energy

  
• Metabolism


  An energy-balancing act