Microbiology - 003 - Bacterial Smear and Simple Stain

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Because bacteria are, for the most part, transparent, we use stains to give them color for microscopic observation. Making a bacterial smear prepares the bacteria to be stained and is the first step of most staining procedures. A successful smear will have a single layer of bacteria fixed to the slide, ready to be stained and then observed under a microscope. 
Get a clean slide. If you’re staining bacteria from a broth culture, transfer a loopful of the broth directly to the slide. Otherwise, add a drop of water to a sterilized loop and place it on the slide, then sterilize the loop again. Transfer a very small amount of bacteria from a colony on a plate to the drop of water and mix. Spread the water-bacteria mix around to about the size of a dime. Air dry the slide, allowing the water to evaporate. This will deposit the bacteria on the slide’s surface. However, at this point the bacteria are easily rinsed off by most staining protocols. They must be fixed, or stuck, to the slide so that they do not wash away. 
Heat fix the bacteria by using a slide holder or clothespin to pass the slide through the flame of a Bunsen burner two or three times. This kills the bacteria and attaches them to the slide. Take care not to overheat the slide. 

Allow the slide to cool. At this point, you have a bacterial smear that is ready to be stained. 

One basic example of a staining technique is a simple stain. Take your prepared bacterial smear and use a staining bridge to suspend it above a staining trough or sink. Use a dropper to cover the smear completely with stain – in this example, we’re using crystal violet. Allow the stain to sit for 60 seconds, then rinse the slide with water to remove excess stain. Get a piece of bibulous paper and gently blot the slide. Don’t wipe – this will disturb the bacteria. The slide is now ready to be viewed under a microscope. If your staining was successful, the bacteria should stand out from their background.

Because bacteria are, for the most part, transparent, we use stains to give them color for microscopic observation. Making a bacterial smear prepares the bacteria to be stained and a simple stain is a quick and easy way to observe bacteria.

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