MLT 415 – Fundamentals of Microbiology Objectives: 1. To prepare smear from broth/plate culture. 2. To understand the technique and principle of Gram stain. 3. To stain the smear using Gram stain. 4. To report the result of the unknown smear. Principles: Most bacteria have no colour, so they generate little contrast in the microscope feld. Therefore, to see bacteria with the microscope, it is necessary to apply colour by using a staining reagent. Once stained, the bacteria may be observed and studied with respect to their shape, size and arrangement. Microbial smear is a very small amount of microbial growth that spread on a clean slide and drying by air. The purpose of making a smear is to fx the bacteria onto the slide and to prevent the sample from being lost during a staining procedure. A smear can be prepared from a solid or broth medium. The preparation of a stained bacterial smear involves several steps. Preparing the smear requires attention to a number of details that help prevent contamination of the culture and ensure safety to the preparer. Each step in this procedure has an important reason and each should be followed carefully as a prerequisite to successful work. First, a loopful of the bacteria is placed on a glass slide. The bacterium was spread on the glass slides with a circle/oval movement. The smear should not too thick as it will cause the arrangement and morphology of the bacteria become difcult to observe. The normal saline is use during the smear preparation to act as emulsion if the bacterium was taken from a solid media. The smear need to be drying frst before fxing with the heat, partly to ensure they remain attached to the slide. There are a few reasons of fxation process is to kill the microbes, fx the microbe cells to the slide and prevent their removal during washing steps. Page | 1