Principle

Smear preparation technique consists of spreading small volume of sample on a slide and air drying the film before staining and microscopy. Bacterial smears must be prepared prior to any of the staining techniques.

Step I: Preparation of the glass slide:

• Clean, grease free slides are needed for smear preparation.
• Grease or oil from the fingers on slides must be removed by washing the slides with soap and water
• Finally rinse the slide with 95% alcohol and dry it.
• Hold the slide by their edge.

Step II: Labeling of slides:

• Proper labelling of the slide is essential.
• Every slides should be labelled clearly.
• A lead pencil is used to write on the frosted areas of the glass slide.

Step III: Preparation of smear:

• An evenly spread smear should be prepared covering area of 15-20mm diameter.
• Avoid thick and dense smear because thick smear prevent light penetration to visualize the morphology of cell.
• A good smear is one that, when dried, appears as a thin whitish layer or film. The print of textbook should be legible through the smear.
• Different techniques are used for smear preparation depending upon culture media

i. Broth cultures (liquid medium):

• Resuspend the culture by tapping the tube with your finger.
• Depending on the size of the loop, one or two loopfuls should be applied to the center of the slide with a sterile inoculating loop and spread evenly over an area about the size of a dime.
• Set the smears on the laboratory table and allow to air-dry

ii. Culture plates (Solid medium):

• Organisms cultured in a solid medium produce thick, dense surface growth and are not amenable to direct transfer to the glass slide.
• These cultures must be diluted by placing one or two loopfuls of water on the center of the slide in which the cells will be emulsified.
• Transfer of the cells requires the use of a sterile inoculating loop or a needle.
• Only the tip of the loop or needle should touch the culture to prevent the transfer of too many cells.
• Suspension is accomplished by spreading the cells in a circular motion in the drop of water with the loop or needle. This helps to avoid cell clumping.
• The finished smearshould occupy an area about the size of a nickel and should appear as a translucent, or semitransparent, confluent whitish film

Step IV: Air dry

• Smear should be allowed to dry completely at room temperature at safe place

Step V: Fixation of smear:

• The purpose of fixation of smear is to preserve and prevent smear being washed away during staining.
• Smears are fixed by heat, alcohol and occasionally by other chemical.

i. Heat fixation

• After smear is air dried completely, rapidly pass the 3-4 times through flame of Bunsen burner or sprit lamp.
• Avoid too much heating.
• After heat fix, allow the smear to cool before staining.

ii. Alcohol fixation:

• Allow smear to air dry completely
• Fix the smear with one or two drops of 70% alcohol, and leave it for 2 minutes until the alcohol dries up.

Requirements:

1. 24 hours culture of Bacillus cereus or Staphylococcus aureus
2. Glass slides
3. Bunsen burner
4. inoculating loop
5. needle, and glassware marking pencil

Procedure

Smears preparation from a Broth Medium

• Label a clean slides with the initials of the organism
• Resuspend the sedimented cells in the broth culture by tapping the culture tube with your finger.
• With a sterile loop, place one loopful of culture on Slide
• With a circular movement of the loop, spread the cell suspension into an area approximately
  of 15-20mm diameter.
• Allow the slide to air-dry completely. This may be done by placing the slide on a drying tray
  attached to a micro incinerator or by placing the slide on the bench.
• Heat fix the smear over flame of Bunsen burner by rapidly passing slide 3-4 times over flame.
• Examine each slide for the confluent, whitish film or haze and record your results in the Lab
  Report.

Smears preparation from a Solid Medium

• Label a clean slide with the initials of the organism
• Using a loop, place one to two loops of water on center of slide.
• With a sterile loop, touch the entire loop to the culture and emulsify the cells in water on Slide
• Allow all slides to air-dry completely
• Heat fix the smear over flame of Bunsen burner by rapidly passing slide 3-4 times over flame.
• Examine each slide for the confluent, whitish film or haze and record your results in the Lab Report.

Preparation of bacterial smear

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