Preparation of bacterial smear

 

PREPARATION OF BACTERIAL SMEAR

Objective: To prepare a bacterial smear suitable for staining and microscopic examination, to observe bacterial morphology and arrangement

Theory:

A bacterial smear is a dried preparation of bacterial cells on a glass slide or it is a thin film of bacterial cells placed on a glass slide. This technique is a crucial step in the microscopic analysis of microorganisms, as it allows the staining and visualization of bacterial cells under different magnifications.

Smear preparation is especially important in simple and differential staining techniques such as Gram staining, acid-fast staining, or spore staining, which help in the identification and classification of bacteria. A well-prepared smear ensures that cells are not too thick (which may hinder observation) or too sparse (making it difficult to find bacteria under the microscope).

In our laboratory, the smear preparation process involves three essential steps:

1. Spreading of bacterial cells on the slide
2. Air drying the smear completely
3. Heat fixing the smear gently over a flame

Heat fixing kills the bacteria and adheres them to the slide. After this, staining is done to enhance visibility under a microscope, allowing the observation of bacterial shape (cocci, bacilli, spirilla), arrangement (chains, clusters, pairs), and basic internal structures.

Materials Required:

• Clean glass microscope slides
• Inoculating loop or sterile pipette
• Bacterial culture (broth or colony from solid media)
• Bunsen burner
• Staining reagents (e.g., crystal violet, Gram stain)
• Distilled water
• Bibulous paper or blotting paper
• Microscope

Procedure:

1. Take a clean grease free slide.
2. If using solid culture: Place a loopful of sterile water on the slide. Pick a small amount of bacterial colony using a sterile loop and mix it in the drop of water to create a uniform suspension.
   If using liquid culture: Take a loopful of culture and transfer it directly onto the slide.
3. Spread the suspension into a thin, even layer using the loop.
4. Allow the smear to air dry completely at room temperature. Do not heat to speed up drying, as it may distort the cells.
5. Once dry, heat fix the smear by quickly passing the slide 2–3 times through the flame of a Bunsen burner, smear side up. Do not overheat.
6. After fixing, apply the chosen stain (e.g., crystal violet for simple staining).
7. Allow the stain to sit on the smear for the recommended time (usually 1 minute).
8. Gently rinse the slide with distilled water to remove excess stain.
9. Carefully blot dry using bibulous paper or allow the slide to air dry.
10. Observe the slide under a compound microscope using 10x (to locate the smear), then switch to 40x and 100x oil immersion lenses for detailed examination.

Results:

• Draw the figure of the prepared bacterial smear as observed under the microscope.
  (Indicate cell shape and arrangement, e.g., cocci in chains, rod-shaped bacilli, etc.)
• During the smear preparation, the bacterial culture was successfully spread into a thin, even layer on the glass slide as shown in the figure.

Observation:

SN	Sample	Stain Used	Colour Observed	Shape	Inference / Result
1	E. coli	Safranin	Red	Rod-shaped (bacilli)	Red-colored bacilli observed

Conclusion:

The preparation of a bacterial smear is a fundamental and essential technique in microbiology. It allows the examination of morphological characteristics, such as shape, size, and arrangement, which are key in identifying and classifying bacterial species.

Precautions:

1. Always use clean, grease-free glass slides to avoid interference with the smear.
2. Use aseptic techniques while transferring bacterial cultures to prevent contamination.
3. Avoid overloading the slide with bacteria; a thin smear gives better results.
4. Allow the smear to air dry completely before heat fixing to avoid distortion of cells.
5. Do not overheat the slide during heat fixing, as it may damage the bacterial structure.
6. Handle the Bunsen burner and slides carefully to prevent burns and breakage.

References:

1. Cappuccino, J. G., & Welsh, C. (2017). Microbiology: A Laboratory Manual (11th ed.). Pearson Education.
2. Pelczar, M. J., Chan, E. C. S., & Krieg, N. R. (2001). Microbiology: Concepts and Applications. Tata McGraw-Hill.
3. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries (Part 2). Cambridge University Press.