Microbial Staining

• Dyes


  A general-purpose coloring agent
• Stains


  A mixture of selected dyes to color a particular biological specimen
• Composition of stains


  • Organic compound with Benzene ring (benzene derivatives)
  • Chromophore: a group of atoms and electrons forming part of an organic molecule (benzene) emits color as a result
  • Auxochrome: a functional group of atoms with nonbonded electrons which cannot provide color but when attached to a chromophore, alters both the wavelength and intensity of the chromophore for absorption; helps a dye to bind to the object that is to be colored; ionizes chromogen and allows them to form salts
  • Chromogen: chromophore attached to a benzene ring (di pa considered as stain unless may auxochrome)
• Acid stain


  Anionic stain (negative ang chromogen), used to stain the positive component of the microbial cell
• Acid stains examples


  • Eosin – targets the cytoplasm, RBC, collagen, & muscle fibers
  • Nigrosin – commonly used to determine the presence of capsule; also used for sperm vitality
  • India ink – similar to nigrosin
  • Congo red – used for detection of cell walls of plants and fungi; also targets outer membrane of gram-negative bacteria; produces amyloid appendages (used for the visual detection of amyloid in muscle and nerve fresh frozen sections in patients who have amyloidosis)
• Basic stain


  Cationic stain (chromogen is positively-charged), used to stain the negatively-charged component of the microbial cell

  
• Basic stains are attracted to negatively charged molecules in the cell including nucleic acids (DNA and RNA) and some proteins
• Neutral stain


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


  • Giemsa stain
  • Leishman stain
• Requirements for staining


  • Stain
  • Mordant – intensifies stains; fixates or holds a stain down to a molecule or surface, e.g., cell membrane; allows stain to penetrate deeply (e.g., Iodine)
  • Decolorizer – removes excess stain (e.g., ethanol & acetone)
  • Accentuator – increases color intensity (e.g., phenol for pH control); used in histopathological studies to prevent degradation

  
• Smear


  In circle formation
• To prepare a smear mount


  1. Prepare a small sample of your specimen

  2. Aseptic Transfer: transfer the sample onto a slide

  3. Spread the drop into an even smear spread thinly across the length of your slide

  4. Heat fix: Allow the smear to dry completely before undergoing fixation to adhere the smear to the slide via heat fixation or methanol fixation

  5. A cover slip is optional
• Simple staining


  Uses only a single dye that which does not differentiate between different types of organisms, generally makes organisms of the sample the same color
• Direct or Positive staining


  Single color, stained by simple dye solution, mostly basic stains (+ chromogen)
• Indirect or Negative Staining


  Dye is being intensified by mordants or being decolorized or being accentuated, commonly uses acid dyes (- chromogen), repelled by (-) cell wall = repelled by bacteria (di nya nasstain ang bacteria pero causes the formation of deposits), cells appear colorless, are seen against dark background

  
• Differential staining


  Uses multiple stains to classify multiple specimens into groups (e.g., based on chemical composition of their cell walls)
• Gram stain procedure


  1. Stained with basic dye = CRYSTAL VIOLET (both gram-positive and gram-negative will appear purple in this step)

  2. Mordant = IODINE (both would still remain purple)

  3. Decolorizer = ETHYL ALCOHOL (differential step) (yung mga hindi nagstay ang stain mawawash-off)

  4. Counterstain = SAFRANIN (didikit sa gram-negative; why it appears pink)
• Hans Christian Gram (1853-1938) 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, Mycobacterium spp. (acid-fast bacteria = bacteria with the ability to resist decolorization by acids during staining procedures), Mycolic acid (waxes) prevent dye from coloring the cells
• Acid-fast stain procedure


  1. Primary stain = CARBOLFUCHSIN (or carbol fuchsin or carbol-fuchsin) (30s)

  2. Decolorizer = ACID ALCOHOL (15-20s)

  3. Counterstain = METHYLENE BLUE (30s)
• Paul Ehrlich (1854-1915) developed the acid-fast stain as a means of staining the tubercle bacillus, Mycobacterium tuberculosis
• Flagella staining


  Technique is used to visualize the presence and arrangement of flagella, requires use of mordant to let the dye stay, flagella is a virulence factor (presence of H antigens na inaattack ng antibodies)
• Leifson Flagella Stain


  Uses tannic acid + dye, the tannic acid-dye complex is more solvable in alcohol, alcohol concentration is enough to maintain the solubility of the ingredients, needs 16-24-hour live culture, NO HEAT FIX because madedegrade ang flagella, pag inabsorb ang tannic acid, nagfoform ito ng colloidal precipitate that colorize the flagella. Also, this process reduces pH.
• Ryu-based Wet-Mount Flagella Stain


  Simple and is useful when the number and arrangement of flagella are critical in identifying species of motile bacteria, uses Ryu stain (reagent that is stable for longer time periods), does not require preparation of smear, self-suspension, needs 18-24-hour sample, 2 components/solutions: SOLUTION 1: mordant – tannic acid, aluminum potassium phosphate, phenol, SOLUTION 2: stain – saturated ethanolic soln of crystal violet
• Capsule staining


  Done to differentiate capsular material from bacterial cells, capsule is also a virulence factor since it prevents phagocytosis, positive staining methods = capsule lang, negative staining methods = di nasstaina ng capsule pero nagiging visible by being colorless

  
• Negative Staining using India Ink


  Cell: Violet, Background: Black
• Pag inabsorb ang tannic acid during leifson


  1. Nagfoform ito ng colloidal precipitate that colorize the flagella

  2. This process reduces pH
• Ryu-based Wet-Mount Flagella Stain


  • Simple and is useful when the number and arrangement of flagella are critical in identifying species of motile bacteria
  • Uses Ryu stain (reagent that is stable for longer time periods)
  • Does not require preparation of smear
  • Self-suspension
  • Needs 18-24-hour sample
• Capsule Staining


  • Done to differentiate capsular material from bacterial cells
  • Capsule is also a virulence factor since it prevents phagocytosis
• Capsule staining methods


  • Positive staining methods (capsule lang)
  • Negative staining methods (di nasstaina ng capsule pero nagiging visible by being colorless)

  
• Negative Staining using India Ink


  • Cell: Violet
  • Background: Darker or black
  • Capsule: clear halo
• Hiss' Method


  • Cell: Dark violet
  • Background: Brighter
  • Capsule: Light violet
• Maneval's Method


  • Cell: Bright red-pink
  • Background: Dark blue
  • Capsule: clear halo
• Maneval's stain


  • Basic dye = ACID FUCHSIN
  • Mordant = 10% PERCHLORIDE
  • Accentuator = PHENOL
  • Acetic acid decreases pH of smear
  • Indicator = CONGO RED (red at neutral, blue at acidic)
• Endospore Staining


  • Needs at least a week-old culture para depleted of nutrition
  • Done to differentiate bacterial spore from other vegetative cells and to differentiate spore-formers from non-spore-formers
  • Spores are normally impervious to stains (endospores are not easily penetrated)
• SCHAEFFER-FULTON Method


  • Endospores: green
  • Vegetative cells: pink
  • Steamed for at least 5 minutes para maforce ang malachite green
  • Spores can be found at any region of the cell and they vary in size (spherical, elliptical)
  • Can be used in diagnosis
• Examples of endospore-forming bacteria


  • Bacillus and Clostridium spp.
  • Bacillus anthracis - used in biological warfare
  • Clostridium tetani
  • Bacillus cereus
  • Clostridium perfringens
• Staining is the first step for bacterial identification
• Problems in bacterial identification


  • Most habitats harbor microbes in complex associations
  • Microbiologists usually will need to grow them under artificial conditions
  • They are not visible to the naked eye
  • Widely distributed (ubiquitous)
• Inoculation


  1. Placing a sample into a container of medium that supplies nutrients for growth and is the first stage of culturing

  2. Purpose and outcome: to increase visibility; makes it possible to handle and manage microbes in an artificial environment and begin to analyze what the sample may contain