A bacterial smear is a preparation that includes a small amount of culture spread in a very thin film on the surface of the slide.

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.