Negative Stain

NEGATIVE STAIN (Nigrosin)

What makes a stain a negative stain? It is acidic with its negative anion on the chromophore (color portion of the dye). That means the color portion of the dye has a negative charge. Bacteria cells also have a negative charge, so the bacteria cells repel the chromophore (dye). Therefore, the cell will have no color. Just the background is stained.

Negative Stain Prep

With NO gloves on, clean several slides, rinse, then squirt alcohol on the slides and dry with a paper towel and set them aside on a clean paper towel. Place one SMALL drop of Nigrosin to the edge of a slide and set aside.

We will be using aseptic technique, so we need the Bunsen burner, gloves, and lab coat.

MOUTH BACTERIA

Clean four slides, rinse, and dry.

Place one drop of Nigosin on the edge of a slide.

Scrape the pointed end of a toothpick between your teeth to pick up some plaque.

Wipe the plaque from the toothpick to the edge of the slide.

Mix the plaque with the stain using the toothpick in a tap-and-mix motion.

Throw the toothpick in the biohazard bag.

Take a clean slide (not a coverslip) and place it at a 45 degree angle facing TOWARD the stain drop. Slide it into the drop and wait five seconds so the capillary action draws the stain up and down the edge of the slide.

Maintaining the angle, quickly draw the top slide across the bottom slide to smear the drop. Leave the slide out to air dry.

The bacteria cells will look clear against a gray background.

You do not need to heat fix this slide, and don’t use a coverslip.

Whenever you look at a slide with oil immersion, you can change the objectives back to one of the lower two lenses, but do not ever use the hi-dry lens on a slide that already has oil on it, or it will get oil on the wrong lens.

TAKING PURE COLONY WITH A NEEDLE

Place on drop of Nigrosen in the center of a new slide. Take the sample with an inoculating needle instead of a loop. A loop picks up too much of the colony and it will be hard to see the individual bacteria cells. An inoculation needle picks up a much smaller amount of sample, so it will be easier to see.

Sterilize the needle from the tip to the end of the wire where it attaches to the handle by passing it through the tip of the inner blue cone of the flame. Cool it by dipping the wire tip into the agar in a clear area of the plate. What will happen if we touch a hot needle to a colony? It will be destroyed and change the normal morphology (size, shape, and arrangement of cells). Remember, when you lift the lid of the plate, hold it above the plate as a shield from air contaminants. Touch the inoculating needle to one colony and replace the lid.

Touch the inoculating needle tip into the Nigrosin drop and mix well by stirring in circles with the tip of the needle, but do not enlarge the drop much. Place a spreading slide at 45° to the drop, contact the drop for 5 seconds and spread the stain across the slide. Sterilize the needle again, and because it has sticky Nigrosin on it, rinse in water and wipe with a towel before you return it or use it again. This will remove the Nigrosin that the flame does not remove.

There is no need to heat-fix Nigrosin because the stain makes the bacteria stick to the slide. Just let it air dry. When your slides are all prepared, you can clean up your area and remove your gloves. When viewing the Nigrosin slide, observe under 1000x and oil. Observe the colony morphology (color, margin, elevation) of the pure culture you took a sample from. Also observe what you see under the microscope, including shape of cells (bacilli, cocci, etc) and arrangement (staphylo, tetrads, strepto, etc).