Microbiology Laboratory

PREPARATION OF BACTERIAL SMEAR

Written by MicroDok

Bacterial smear is defined as a dehydrated or dried preparation of a bacterial suspension (cells) on a clean glass slide. The source of bacteria for the preparation or making of a slide can be from an agar slant, broth (liquid) culture and from a culture plate.

Procedure of making a bacterial smear

  • Using a grease pencil, mark one end of the slide with the name of the bacterial culture.
  • Transfer a speck of the bacteria to a drop of normal saline on a clean glass slide. If the bacteria is from a liquid medium (i.e. broth), place 1-2 drops or loops of bacteria on a clean glass slide.
  • Spread out the bacteria (from a broth or solid culture) mixture over a large area on the glass slide. Always ensure that a thin preparation is made and, that a space is left on each of the four sides of the glass slide.
  • Air dry the glass slide at room temperature (25-28oc) while ensuring that it is free from dust.
  • Heat-fix the smear by passing it through a Bunsen burner flame three times or by flooding the smear with 70% methanol or ethanol and allowed for 2 min. Always ensure that the prepared smear is on top of the glass slide as you pass it through the Bunsen burner flame.
  • After heat-fixing, the fixed smear can be stained on a staining rack using different types of dyes depending on the staining technique used. There are different staining techniques that are available in the bacteriology laboratory. The staining can be simple or Some of the staining techniques used in the bacteriology laboratory include:
  • Gram staining: For dividing bacteria into either Gram positive or Gram negative.
  • Ziehl-Neelsen staining: For detecting acid-fast bacilli e.g. Mycobacterium.
  • Albert staining: For detecting Corynebacterium diphtheria from sputum specimen.
  • Wayson staining: For detecting Yersinia pestis
  • Loeffler methylene blue stain: For detection of Bacillus anthracis.
  • Malachite green staining: For detection of bacterial endospores.
  • Crystal violet staining: For detection of bacterial spores.
  • Robinow’s staining: For detection of bacterial DNA.
  • Capsule staining: For detection of bacterial capsules.
  • Add a drop of immersion oil on the stained slide.
  • Examine the slide under the oil immersion objective lens (x100)

Heat-fixing is defined as the microbiological technique of attaching bacterial cells onto the surface of a glass slide by the use of a Bunsen burner flame or 70% of methanol. A gentle heating of a bacterial cell on a slide when passed through the blue flame of a Bunsen burner actually kills the bacteria but nevertheless, heat-fixing does not distort the cell structure of the organism. The cell structure of the bacteria cell becomes distorted if and only when the slide is over-heated. Heat-fixing of bacterial cells allows the internal and external structures of a microorganism to be intact during staining and microscopy.

Techniques of making good bacterial smear

  • Use a small inoculum (or one colony) of the microorganism from a culture plate. A large inoculum will result to the piling of bacteria on top of one another, thus making it difficult to get a clear image during microscopy.
  • One or two loops of the bacterial cell suspension should be placed on a clean glass slide and spread out over a large area. This technique applies to a liquid medium containing a bacterial cell.
  • Inoculum of bacterial cell from a culture plate should be diluted with one or two drops of physiological (normal) saline and spread out properly.
  • Allow the prepared slide to dry properly before staining and subsequent microscopy.
  • Glass slides containing smears of bacteria should be kept free from dust particles.

Qualities of a well prepared bacterial smear 

  • The shape of the bacterial cell will not be distorted or unclear during microscopy.
  • The bacteria will be properly and evenly separated from one another after spreading out on the slide and subsequent microscopy.
  • During staining of the prepared smear, the bacteria will not be washed off the slide

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