CHAP4:PREPARATION AND STAINING OF SPECIMEN

• Steps in preparation of the smear
• steps in preparation of smear
• steps in preparation of smears

  1. prepare the glass slide
  2. Label the slide
  3. preparation of smear
  4. heat fixation
• Staining


  -bacterial cells are so small that they must be colored with some dye to be studied more
• Smear


  -usualy begins by spreading a thin film of the specimen over a slide
• fixation process


  -it is allowed to air dry,and then passed through the flame of bunsen burner to heat and drying the smear.This mainly to stick the organisms to the slide
• Basic dyes


  -colored portions of the positively charged colored.Which attracted to the outside of bacterial cells
• basic dyes color are:

  • crystal violet
  • methylene blue
  • safranin
  • carbolfuchsin
  • malachite green
• Acidic dyes


  -dyes that are negatively charged and is not attracted to the bacteria and color the background,leaving the bacteria unstained.
• Acidic dyes color are;

  • nigrosin
  • india ink
  • eosin
• Staining procedure are;

  1. Simple staining
  2. Differential staining
  3. gram-staining method
  4. Acid-fast or Ziehl-Neelsen staining method
  5. acid-fast bacteria
  6. non-acid-fast bacteria
  7. Staining specific microbial structures
  8. Special staining method
  9. negative staining
• Simple staining

  • one stain{basic dye}is applied to a heat-fixation and rinsed off
  • all microbes would take on the color of dye
• widely used simple stains are;

  1. Loeffler's alkaline methylene blue
  2. Carbol fuchsin
  3. Gential violet
  4. Safranin
• capsules and spores are not stained with simple stains
• flagella cannot be demonstrated at all in this way{simple staining}
• Differential staining


  -more complex staining methods

  -divided bacteria into groups,depending on their reaction to the chemicals used
• Gram-Staining method

  • most important staining technique in microbiology
  • developed in 1884 by Dr.Cgristian Gram
  • it is differential stain used to sort bacteria into 2 groups.{gram-positive and gram-negative
• Steps in application of Gram staining method

  1. application of crystal violet
  2. application of iodine
  3. alcohol wash
  4. application of safranin
• Gram-positive


  -bacteria from which the blue color of the stain iodine bacterial cell complex cannot be removed
• Gram-negative


  -those from which it can removed and which are stained with the counterstain
• Gram-positive organism will be purple{blue}

  Gram-negative organism will be red{pink}
• Gram-positive bacteria


  -tend to be killed by penicillin and detergent
• Gram-negative bacteria


  -are more resistant to antibiotics
• Acid-fast or Ziehl-Neelsen staining method is also known as hot method
• Steps in Acid-fast or Ziehl-Neelsen staining method

  • carbol fuchsin is steamed into a heat-fixed smear for 5 minutes{primary stain}
  • slide is cooled and gently but thoroughly rinsed
  • acid-alcohol is applied{decolorization}to remove fushsia color from all bacteria except acid-fast.
  • methylene blue is applied as the counterstain
  • rinse,blotdry and examine
• Acid-fast bacteria


  -are those that retain stain{carbol fuchsin}even when treated with acid-alcohol{red/pink}
• Non-acid-fast bacteria


  -completely decolorized when treated with acid-alcohol and stained with the counterstain.{blue}

  1cell wall do not posesses complex lipids,fatty acids and waxes
• Special staining method

  • important special stains-are those for capsules,spores,flagella and metachromic granules
  • stains primarily designed to demonstrate metachromatic granules and especially valuable in identifying Corynebacterium diphtheriae
• Important special stain;

  1. albert's stains-using toluidine blue and malachite green
  2. neisser's stain-using methylene blue
• Negative{relief}staining

  -microorganism such as treponema pallidum are not stained by ordinary dyes and may be visible by the process known as negative staining

  -in this method the background is stained

  -microbes appear as colorless objects against a gray-black background

  -microbes are mixed with india ink or 10%nigrosin

  -congo red can also be used in negative staining-has been used display spiral organisms