CELL BIOLOGY LAB 5 Preparation of Smear Making

CELL BIOLOGY LAB. 5 Preparation of Smear

Making films : A good smear is one that, when dried, appears as a thin whitish layer or film. There are two types of culture from which a smear can be prepared: a. Smear from broth cultures: Two to three loopsful of suspended cells are applied to the slide with a sterile inoculating loop without spreading. Leave the slide to dry.

b. Smear from solid culture: Place a loopful of tap water or saline on the slide in which the cells will then be emulsified by spreading; because bacteria cultured in solid medium produce thick, dense growth and are not amenable to direct transfer to the slide. So they must be diluted. The cells are transferred by a sterile loop, by touching the culture to prevent the transfer of too many cells. Then allow the smear to dry.

2. Heat fixation: This is performed by rapid passage of the air dried smear two to three times over the flame of Bunsen burner. Unless fixed on the slide, the bacterial smear will be washed away during the staining procedure. During fixation the bacterial proteins are coagulated and fixed to the slide.

3 -Staining of films: A- The stains are poured directly or filtered on the slid. B- When staining is complete, the dye is washed off with water. C- The slid is allowed to dry in the vertical position or is placed between two sheets or filter paper. D- The drying of the film is completed over the Bunsen flame, then examined under the microscope (with oil immersion lens).

Types of staining techniques 1. Simple staining (use of single stain): Used for visualization of morphological shape (cocci, bacilli, and spirals) and arrangement (chains, clusters, pairs, and tetrads) 2. Differential staining (use of two contrasting stains): Used for separation into groups e. g. . Gram stain and acid-fast stain. 3. Special staining: Used for visualization of structures e. g. , flagella stain, capsule, and spore stain.

Gram stain : for diagnostic identification of various bacteria consist from : 1. Crystal violet for 60 seconds 2. -Lugol iodine for 60 seconds 3. Absolute alcohol "ethanol" 4. Carbol fuchsine for 45 seconds

Acid-Fast stain (Ziehl-Neelsen stain): A few species of bacteria in the genera Mycobacterium do not readily stain with simple stains so stained with this stain to be visualize. 1. - Strong carbol fuchsine (10%) 2. -Sulphuric acid (20%) 3. Acid alcohol 4. -Loffler's methylene blue.

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