The first stage in the majority of staining procedures is often smear preparation, which is a basic skill in the microbiology lab. The ensuing staining process will be directly impacted by the quality of the smear. Depending on the material or culture media, there are slight variations in the smear preparation.
Principle of Bacterial Smear?
Glass microscope slide preparation:
Clean slides are necessary for the creation of microbiological smears. Slides must be washed with soap and water or scouring powders, followed by a water rinse and a rinse of 95% alcohol, to remove grease or oil from the fingers. Dry the slides after washing them, then store them on paper towels in the lab until you’re ready to utilize them. Keep in mind to grasp the polished slides by their edges.
Labeling slides:
A slide must be properly labeled. With a glassware marking pencil, the organism’s initials can be inscribed on either end of the slide on the surface where the smear will be applied. It is important to take precautions to prevent staining substances from coming into touch with the label.
Smear preparation:
It’s essential to stay away from smears that are too thick or dense. When excessive amounts of the culture are utilized to prepare the sample, a lot of cells are concentrated on the slide, resulting in a thick or dense smear. With this kind of preparation, it is more difficult to see the shape of individual cells and less light can flow through.
A minimal quantity of bacterial culture is all that is needed for smears. Any smear that dries to a thin, whitish layer or film is considered to be desirable. You should be able to read your textbook’s print despite the stain. Depending on whether the smear was created from a solid media culture or a broth, different methods are employed.
Cultures in broth:
Re-suspend the culture by tapping your finger on the tube.
Using a sterile inoculating loop, apply one or two loops to the center of the slide and distribute them equally over an area roughly the size of a dime, depending on the size of the loop. The smears should be placed on the lab table and left to air dry.
Cultures from solid media:
Organisms grown in a solid medium create thick, dense surface growth and cannot be transferred directly to a glass slide. By adding one or two loops of water to the center of the slide where the cells will be emulsified, these cultures must be diluted.
Using a loop or needle, disseminate the cells in the drop of water in a circular motion to achieve suspension. Cell clumping is less likely as a result. The final smear should be about the size of a nickel and have the appearance of a confluent, translucent, or semitransparent, white film. The smear needs to be given time to finish drying off at this stage. Don’t blow on or wave the slide around in the air.
Heat fixation:
The bacterial smear will wash away during the staining process if it is not fixed to the glass slide. Heat fixation, in which the bacterial proteins are coagulated and fixed to the glass surface, prevents this from happening. The air-dried smear is quickly passed over the flame of the Bunsen burner two or three times to fixate heat.
What are the materials required to prepare a bacterial smear?
- Tidying up microscope slides.
- Trays for staining and newspaper.
- Bottle of water (for rinsing).
- Bacterial cultures:
- Escherichia coli
- Staphylococcus aureus
- Micrococcus luteus
- Pseudomonas aeruginosa
What is the protocol for Bacterial Smear Preparation?
Smears from Broth Medium:
Label the first three clean slides with the organism’s initials and assign them the numbers 1, 2, and 3. By tapping the culture tube with your finger, you can resuspend the sedimented cells in the broth culture.
1. Place one loopful of culture on Slide 1, two loops on Slide 2, and three loops on Slide 3, all with sterile loops.
2. Spread the cell suspension into an area roughly the size of a dime by moving the loop in a circular motion.
3. Permit the slide to completely dry by air.
4. Fix the preparation with heat.
Smear from a Solid Medium:
Put the organism’s initials on four clean slides. Slides 1 and 2 should be marked with a loop (L), while Slides 3 and 4 should be marked with a needle (N).
1. Add one to two loops of water to each slide using a loop.
2. On Slide 1, emulsify the cells in water by touching the entire loop to the culture. On Slide 2, simply touch the tip of a sterile loop to the culture before emulsifying it in water. On Slides 3 and 4, repeat Steps 1 and 2 using a sterile inoculating needle.
3. Enable complete air drying of all slides.
4. Fix the preparation with heat
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