Microorganism Staining

Staining Bacteria

We most often use basic stains to examine bacteria. Basic stains, due to their positive charge will bind electrostatically to negatively charged molecules such as many polysaccharides, proteins and nucleic acids. Acid stains bind to positively charged molecules which are much less common, meaning acidic stains are used only for special purposes. Some commonly encountered basic stains are crystal violet, safranin (a red dye) and methylene blue. Basic stains may be used alone (a simple stain) or in combination (differential stain) depending on the experiment involved.

Slightly different techniques must be used for preparing bacteria for staining depending on whether the bacteria are growing on agar or in broth. The concentration of bacteria in a colony on an agar plate is extremely high, thus the major problem is having so many bacteria that they form a dense mass in which individual cells stain poorly and whose morphology is hard to distinguish. In broth, the bacteria are relatively dispersed so we have to ensure that we get a sufficient number onto the microscope slide so they're not too hard to find.

Simple Staining

Step 1. Bacteria from colonies. Clean a glass slide and place a small mark slightly off center using a grease pencil. Using your loop, transfer one small drop of water to the center the slide, being careful to be close to but not overlapping the grease pencil mark. Do not transfer too much water because these drops will have to air dry. Sterilize your loop and touch a single colony and transfer the bacteria to the water droplet on the slide and mix well. DO NOT scoop up a whole colony; you'll have vastly too much bacteria. If while mixing you see an opaque slurry of bacteria on the slide you have too many bacteria for effective staining.

Step 1. Bacteria from broth. Clean a glass slide and place a small mark slightly off center using a grease pencil. Mix the broth containing the bacteria well because the bacteria may...