The document outlines the essential steps for bacterial smear preparation, which is critical for effective staining in microbiology. It includes requirements, preparation of glass slides, labeling, creating an evenly spread smear, air drying, and fixation methods. Proper technique ensures cell morphology is visible and prevents wash-away during staining.

1. Bacterial Smear Preparation
Anup Muni Bajracharya

2. • The first step in most bacterial staining
procedures is the preparation of a smear.
• A good smear preparation is the key to a good
stain.

4. • Requirements:
• Culture of Bacteria
• Glass slides
• Bunsen burner
• Inoculating loop
• Glassware marking pencil/ Permanent Marker

5. 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.

6. • Step II: Labeling of slides:
• Proper labeling of the slide is essential.
• Every slides should be labeled clearly.
• A lead pencil /permanent CD marker is used to
write on the frosted areas of the glass slide.

7. • 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.

8. i. Broth cultures (liquid medium)
• Re-Suspend 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.
• Allow the smear to air-dry
Different techniques are used for smear preparation depending upon culture media

10. ii. Culture plates (Solid medium)
• Suspension is accomplished by spreading the cells in a circular motion in
the drop of water with the loop. This helps to avoid cell clumping.
• The finished smear should occupy an area about the size of a nickel and
should appear as a translucent, or semitransparent, whitish film
• Place a drop of water into the circle that has been created on the slide.
• Using a sterilized and cooled inoculation loop, obtain a very small sample of
a bacterial colony from the culture palte.
• Gently mix the bacteria into the water drop.

11. • Step IV: Air dry
• Smear should be allowed to dry completely at
room temperature at safe place

12. • Step V: Fixation of smear:
• The purpose of fixation of smear is to preserve and
prevent smear being washed away during staining.
• 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.