microbial staining

• DYES

  A general-purpose

  coloring agent
• STAINS

  A mixture of selected dyes

  to color a particular

  biological specimen
• Chromophore

  molecules in a given material that absorbs particular wavelengths of visible light, and in doing so confer color on the material
• Auxochrome

  color intensifier/helper, a functional group of atoms with one or more lone pairs of electrons when attached to a chromophore, alters both the wavelength and intensity of absorption
• Benzene ring

  stains is an organic compound with ________
• Classification of stain based on charge

  acid, basic, neutral
• acid stain (anionic stain)

  Chromogen is negatively charged, Used to stain the positive component of the microbial cell, e.g Eosin, Nigrosin, India Ink, Congro red
• basic stain (cationic stain)

  Chromogen is positively charged, Used to stain negatively charged

  component of microbial cell, e.g Methylene blue, Safranin, Malachite Green, Basic Fuchsin, Crystal Violet
• neutral stain

  Complex salt of dye acid with dye base, Complex salt of dye acid with dye

  base, e.g Giemsa stain, Leishman stain
• Requirements for Staining

  Stain

  Mordant

  Decolorizer

  Accentuater
• Mordant

  a chemical that serves as a link between the dye and the substrate
• Decolorizer

  a solvent of ethanol and acetone, is used to remove the dye
• Accentuater

  intensify staining
• Slide Preparation


  smear, aseptic transfer, heat fix
• Different Staining Methods used in Microbiology

  simple, differential, special
• Simple Staining

  uses only a single dye that which does not differentiate between different types of organisms
• 2 types of Simple Staining

  Direct or Positive Staining & Indirect or Negative Staining
• Direct or Positive Stain

  steps:

  
• Neagtive stain

  acid dye, (-) chromogen, repelled by (-) cell wall, cells (colorless and seen against dark background)

  
• DIFFERENTIAL STAINING

  distinguishes organisms based on their interactions with multiple stains, Separation into groups: gram stain & acid fast stain, Visualization of Structure: Flagella, Capsule, Endospore
• Hans Christian Gram

  1884: Developed the Gram stain as a means to differentiate pneumococci

  from Klebsiella pneumonia.
• Acid-fast stain

  distinguishes different types of bacteria based on the wax content of their cell wall. e.g Mycobacterium spp.

  
• Paul Ehrlich

  1882 developed the acid-fast stain as a means of staining the tubercle bacillus, Mycobacterium tuberculosis, his original method has undergone modification by Ziehl and Neelsen that are still used today
• Flagella Staining

  technique is used to visualize the presence and arrangement of flagella
• Leifson Flagella Stain

  method uses tannic acid (more solvable in alcohol) and a dye

  
• Ryu-based Wet-Mount Flagella Stain

  simple and is useful when the number and arrangement of flagella arecritical in identifying species of motile bacteria, a reagentthat are stable for longertime periods.

  
• Capsule stain


  to differentiate capsular material from bacterial cells, negative staining, hiss' method, maneval's method
• Negative staining (India Ink)

  background seems darker/black, bacterial cell (violet), capsule (clear halo)

  
• Hiss' Method


  bacterial cell (dark violet), background (brighter color), capsule (light violet)

  
• Maneval’s Method


  A bacterial cell appears bright red-pink. The background appears dark blue in color. The capsule appears as a clear halo.

  
• Endospore Staining



  to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers. Spores are normally impervious to stains. Schaeffer-Fulton Method, Endospores in green, Vegetative cells in red or pink

  
• STAINING


  technique used to enhance contrast in samples, to define cell size, shape, arrangement, to study chemical properties and structures, to demonstrate the purity of culture, to observe certain structures
• Bacterial Identification Problems


  most habitats harbor microbes in complex associations. microbiologist usually need to grow them under artificial conditions. they are not visible to the naked eye. widely distributed.
• Six I's


  
Inoculation, isolation, incubation, identification, inspection, information gathering

  
• Inocuation 


  Cultivate an inoculum into a container of a fresh nutrient medium
• Sterile/Aseptic Technique


  using practices and procedures to prevent contamination from pathogens.
• Isolation Techniques


  Streak plate method, loop dilution/ pour plate technique, spread plate technique
• Streak Plate Method


  sample/inoculum is diluted by streaking it across the surface of the agar plate. streaking in successive areas of the plate, the inoculum is diluted to the point where there is only one bacterial cell deposited every few

  millimeters on the surface of the agar plate.

  
• Loop Dilution or Pour Plate

  the sample is inoculated, also with a loop, into a series of cooled but

  still liquid agar tubes so as to dilute the number of cells in each

  successive tube in the series

  
• Spread Plate Method


  a small volume of liquid, a diluted sample is pipetted onto the surface of

  the medium and spread around evenly by a sterile spreading tool