The fixation procedure is the same regardless of smear source, plate or broth. Place your needle/loop in the center of the drop and with a spiraling circular motion spread the bacteria on the slide.Prick it with your sterile needle, or slightly scoop the edge of the colony with your sterile loop.This serves to both dilute your bacteria and give you something to spread around.Aseptically transfer a loop-full of sterile water to the center of the slide. Regular tap water or the de-ionized water in your rinse bottles are often contaminated with bacteria. Be sure to use sterile water to dilute your samples. You may want to use an inoculating needle to transfer your organism to the slide. You can scoop a lot of organisms off with your loop. Because the broth is full of protein, the smear will usually stay spread out and not bead up on the surface of the slide. Use a spiraling, circular motion to spread out the drop. Use the flat part of the loop to smear the broth drop around the slide.Aseptically transfer a loop-full of organism onto the center of your slide. Be sure to carefully mix the culture tube to suspend the bacteria in the broth. Smear from Brothīroth cultures are usually easier to work with because the cells are already diluted in the broth. Smears that are too thick will most likely wash off the slide regardless of the fixation method. If your slide is wet and fix it in methanol, it will most likely wash off the slide. If your slide is wet and you heat fix it, the bacteria will boil and the cellular morphology will be lost. Do this consistently on the same end of the slide to help orient your slide.īe patient and take the time to let your slide air dry before proceeding with adhering it to the slide. Be sure to label the far edge of the slide. It is very easy to get confused which side of the slide your smear is on. You have lots of room on your slide use it! It helps to initially draw a circle on the bottom of the slide so you know where to look for your smear. You are striving for a light suspension of cells that will leave a faint cloudy deposit on your slide. Dispose of your completed slides in the disinfectant bucket at your bench. Heat or methanol fixation is not guaranteed to kill the organism. The loop is very flexible and it is easy to zing off a loop-full of organisms. Materialsīe careful of aerosols when transferring bacteria from your loop to the slide. It will undoubtedly take you several tries before you are successful. While the goals are the same for both, evenly and lightly dispersed cells firmly adhered to the slide surface, the techniques are slightly different. You will be preparing slides for staining from both broth and agar surfaces. The cells typically shrink in size and will exhibit some changes in shape and extra-cellular matrixes. All procedures that attach the bacteria to the slide result in some morphological changes. The bacteria need to be firmly attached to the slide so they are not washed off during the staining procedures.Large blobs of cells also do not stain properly and could yield erroneous results from the improper staining. If there are too many bacteria on the slide they will form a big glob and you will not be able to see the morphology of the individual cells. The bacteria must be evenly and lightly dispersed.There are two important things to consider when preparing a slide for staining: ![]() You must firmly attach your bacteria to a glass slide before you can stain them. Not only are most bacteria very small, they are also very clear and difficult to view under a microscope without first staining.
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