Its purpose is to bind the specimen to the slide so that it does not wash off during staining. This process is called heat fixing the specimen to the slide. Put the sample side facing up and label with your initials on one end of the slide. Next, heat fix the slide by placing it on the slide warmer for 5 minutes. Micrococcus luteus, and Saccharomyces cerevisiae.ģ. Prepare three separate smears, one each of Bacillus megaterium. Spread the drop over a small portion of the slide to make a thin film with lightly visible turbidity.Ģ. Next, with a sterile loop transfer a SMALL AMOUNT of the growth to the drop of water and rub the loop around until the material is as evenly distributed as possible to form a just visibly turbid suspension. Preparations of bacteria for staining can be made from growth on an agar plate or from a broth culture.ġ. To prepare a slide from cells grown on an agar medium, first place a SMALL drop, a loopful works well, of water on a clean grease-free slide. Preparation of a Bacterial Smear for Staining Review Lab procedures for operating a Brightfield Light microscopeī. Image 1: Heat Fixing over a bunsen burnerĪ. Be careful not to overheat the slides in this procedureĪfter heat-fixing is complete, you are ready to simple or gram stain your slide.Cells will be rinsed off the slides if they are not heat fixed properly.Heat fixing kills cells, and adheres them to the slide.When slides are completely air-dry, heat fix the bacterial specimen by passing the slide slowly over the flame twice (your instructor will demonstrate this). Air dry the bacterial specimen on the slide (slide warmers may also be used).ĥ. Be careful not to have the two smears run into each other.Ĥ.Mix it well with the saline and spread the mixture over a wider area of the slide. With an inoculation loop or needle, pick up a small amount of bacteria. If you use the saline dropper directly on the slide, do not release a full drop.ģ. This can be done by placing a drop of saline onto your inoculation loop and then transferring it to the slide. Add a small drop of saline to the slide (you will usually put two bacteria on one microscope slide- Follow your instructor’s specific instructions). Note that it is important to recognize the side of the glass slide that you put your bacterial sample on.Ģ. Label a clean glass slide using a red wax marker. Staining procedures - UK Standards for Microbiology Investigations.A Oktari - The Bacterial Endospore Stain on Schaefferįulton using Variation of Methylene Blue Solution. Young cultures (less than or a day old) may have only vegetative cells, whereas older cultures (5 to 7 days old) are excellent for good sporulation.ģ- Heat fixing should be done with minimal flaming as excess heat will destroy the integrity of the cells, causing them to shrink and to aggregate together on the slide.Ĥ- It should be noted that any debris on the slide can also take up and hold the malachite green stain and so caution should be taken when interpreting slides. The smear is therefore ready to be observed under the microscope at the x100 objective with immersion oil.ġ- Some labs do not use paper towels as described in the Schaefer-Fulton procedure (an ill-fitting piece of paper may burn or leak)Ģ- The age of the culture will affect sporulation. Then cover the slide with a mercurochrome solution for two minutes. Wash it with water, wipe it with filter paper. tetani, Bacillus anthracis, Bacillus cereus, Desulfotomaculum spp, Sporolactobacillus spp, Sporosarcina spp,Ĭover the smear with malachite green for a minimum of 45 min. □ Examples of positive endospore staining : Clostridium perfringens, C. □ Vegetative cells : Vegetative cells are brownish-red to pink. Endospore staining (source : Endospores : Endospores are light green.
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