Preparation of Slide - Microbiology for Lab Technicians

Microbiology for Lab Technicians - one year diploma

1. by, Miss Areesha Ahmad Lecturer B.Sc (H), M.Sc , M.Phil and Ph.D scholar (Microbiology) Preparation of Slide

2. Preparation of Microscopic slide

3. P reparation of Microscopic slide Preparing a slide for viewing microorganisms, especially for techniques like acid-fast staining, involves a few careful steps to ensure clear observation. Here’s a general step-by-step guide for preparing a slide for microscopic examination:

4. Procedure 1. Clean the Slide: Start with a clean, dry glass slide. If necessary, wipe it with ethanol and a lint-free tissue to remove any dust, oils, or fingerprints. 2. Prepare the Specimen Smear: Bacterial Specimen : Use an inoculating loop to pick a small sample of bacteria (from a culture plate or liquid culture) and spread it thinly on the slide. If using a liquid sample like sputum, place a drop on the slide and spread it evenly. Tissue or Sample : For solid tissues or larger samples, use a scalpel or needle to obtain a very thin section. Place a small piece on the slide and press it down gently.

5. 3. Air Dry: Allow the smear to air dry completely. Avoid using heat at this stage, as it can cause artifacts in the sample. 4. Heat Fixation: Once dry, pass the slide quickly through the flame of a Bunsen burner (3–5 times) with the smear side facing up. This step: Kills the organisms Fixes them to the slide to prevent washing off during staining Preserves the shape of cells Note : Be careful not to overheat, as this can distort the cells. 5. Staining (Optional): After fixation, apply the desired stain to the slide based on the type of observation you need: Simple Stain : For a basic view, use stains like methylene blue. Gram Stain : For bacterial classification, use crystal violet, iodine, alcohol ( decolorizer ), and safranin . Acid-Fast Stain ( Ziehl-Neelsen ) : Use carbol fuchsin , acid-alcohol as a decolorizer , and methylene blue as a counterstain to view acid-fast bacilli. Fluorescent Stain : For more sensitive detection, fluorochrome stains like auramine-rhodamine can be used.

6. 6. Rinse and Dry: Gently rinse the slide with distilled water to remove excess stain. Blot dry with absorbent paper, avoiding smudging or disturbing the smear. 7. Microscopic Examination: Place the slide on the microscope stage, start with a low magnification to locate the specimen, and switch to higher magnifications as needed. For oil immersion (usually at 1000x), add a drop of immersion oil to the slide and carefully adjust the focus.