ENVE 301: Environmental Microbiology Laboratory

Simple Stains: Direct and Indirect Staining


If you cannot saw with a file or file with a saw, then you will be no good as an experimentalist.- Augustin Jean Fresnel, 1788-1827


Supplemetal Reading/Web Sites

Direct and Indirect Staining

Slide Preparation and Troubleshooting


Stains

As you've observed viewing wet mounts in the previous laboratory, most bacteria are difficult to see under the bright field microscope. Bacteria are almost colorless (remember, all cells are composed primarily of water) and therefore show little contrast with the broth in which they are suspended. To visualize bacteria, either dyes or stains, or an alternative source of illumination (phase contrast or differential interferrence constrast) are used. Since staining of bacterial cells is relatively fast, inexpensive, and simple, it is the most commonly used technique to visualize bacterial cells. Staining not only makes bacteria more easily seen, but it allows their morphology (e.g. size and shape) to be visualized more easily. In some cases, specific stains can be used to visualize certain structures (flagella, capsules, endospores, etc). of bacterial cells.

There are several staining methods that are used routinely with bacteria. These methods may be classified as 1) simple (nonspecific) and 2) differential (specific). Simple stains will react with all microbes in an identical fashion. They are useful solely for increasing contrast so that morphology, size, and arrangement of organisms can be determined. Differential stains give varying results depending on the organism being treated. These results are often helpful in identifying the microbe.

Stains (dyes) are chemicals containing chromophores, groups that impart color. Their specificity is determined by their chemical structure. Stains are generally salts in which one of the ions is colored. (A salt is a compound composed of a positively charged ion and a negatively charged ion.) For example, the dye methylene blue is actually the salt methylene blue chloride which will dissociate in water into a positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless. Commonly used microbiological stains generally fall into one of two categories - basic stains or acidic stains ( although there are a few stains such as India Ink) which are neutral). A basic dye is a stain that is cationic (positively charged) and will therefore react with material that is negatively charged. The cytoplasm of all bacterial cells have a slight negative charge whengrowing in a medium of near neutral pH and will therefore attract and bind with basic dyes. Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue. Acid dyes have negatively charged chromophores and are repelled by the bacterial surface forming a deposit aroung the organism. They stain the background and leave the microbe transparent. Nigrosine and congo red are examples of acid dyes.

Note: The dyes used for bacteriological staining are generally aniline dyes, derived from coal tar, which means they are POTENTIALLY CARCINOGENIC and should be handled carefully. Avoid contact with them by keeping them off skin, clothing and benches.

At first glance, the easiest way to stain bacterial cells would appear to be simply mixing the bacterial suspension with the dye and making a wet mount of this mixture. Unfortunately, if you were to try staining bacterial cells in this manner you would find that there was too much background (unbound dye) to allow for visualization of the cells. Therefore, you need to remove the unbound dye. Simply washing off the dye would result in removal of the cells along with the excess dye. Therefore, you need a mechanism to fix the cells to the slide before staining to allow for removal of excess dye while keeping the cells on the slide. A simple method is that of air drying and heat fixing. The organisms are heat fixed by passing an air-dried smear of the organisms through the flame of a gas burner. The heat coagulates the organisms' proteins causing the bacteria to stick to the slide. Be very careful not to over heat the organisms when fixing them to a slide. This distorts the sample of the organisms. Keep in mind the analogy of a fried egg. When you drop a raw egg onto a cold frying pan, it has a certain shape. Start heating it and the proteins (albumin) on the lower surface of the egg precipitate and fix the egg to the pan. At this point there has been a minimal distortion in the shape of the egg as a whole; only a small percent of the proteins have been precipitated. If you keep applying heat, the shape of the entire egg will change and eventually it will be reduced to charred remains. When you heat fix a slide, you want to apply enough heat to precipitate the proteins to allow the cells to stick to the slide but not to drastically change the shape of the cells (or reduce them to charred remains).

 


Preparation of a Bacterial Smear and Heat Fixation

Source: http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab3.html

If the culture is taken from solid (agar) medium.

  1. Clean the slide using Bon Ami or other suitable cleaning material. Gently dry the slide with a lint free cloth. (alternatively, it is possible to purchase pre-cleaned slides)
  2. Place a very small drop of distilled water on the surface of the slide.
  3. Aseptically remove a small amount of the culture from the agar surface and just touch it several times to the drop of water until it just turns cloudy.
  4. Burn the remaining bacteria off of the loop. (If too much culture is added to the water, you will not see clearly stained individual bacteria.).
  5. Using the loop, spread the suspension over the entire slide to form a thin film.
  6. Allow this thin suspension to completely air dry.
  7. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix. Caution: Too much heat might distort the organism and, in the case of the gram stain, may cause gram-positive organisms to stain gram-negatively. The slide should feel very warm but not too hot to hold.

If the organism is taken from a broth culture:

  1. Clean the slide using Bon Ami or other suitable cleaning material. Gently dry the slide with a lint free cloth. (alternatively, it is possible to purchase pre-cleaned slides)
  2. Aseptically place 2 or 3 loops of the culture on a clean slide. Do not use water.
  3. Using the loop, spread the suspension over the entire slide to form a thin film.
  4. Allow this thin suspension to completely air dry.
  5. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix.


Simple (Direct) Staining with Methylene Blue

 

  1. Prepare a heat fixed smear of the culture you wish to examine
  2. Cover the smear with methylene blue
  3. Allow the dye to remain on the smear for approximately 1 minute. (Note staining time is not critical. Somewhere between 30 seconds and 2 minutes should give you an acceptable stain. The longer you leave the dye on, in general, the darker the stain.)
  4. Wash the excess stain off the slide Pick up the slide by one end and hold it at an angle over the staining tray. Using the distilled water wash bottle, gently wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide.. Wash off any stain that got on the bottom of the slide as well.
  5. Blot off excess stain using bibulous paper. DO NOT rub the slide, rather place the slide between two sheets of bibulous paper and press down gently. Paper will absorb excess dye.
  6. Examine the slide under the brightfield microscope.
  7. Record the shape, arrangement, and approximate size of the organisms. You should have data on two cultures which you have stained.

 


Negative (Indirect) Stains

Source: http://www.wsu.edu/~hurlbert/pages/101lab4.html

  1. Clean the slide using Bon Ami or other suitable cleaning material. Gently dry the slide with a lint free cloth. (alternatively, it is possible to purchase pre-cleaned slides).
  2. Place a small drop (one-two loopfuls) of the negative stain (India ink) near the end of the slide.
  3. Transfer one loop=full of the bacterial sample to the india ink and mix the two togetner.
  4. Hold a clean slide at about a 20o angle to the first slide. Touch the edge of the clean slide to the bacteria/stain mixture so that the mixture spreads across the edge.
  5. Spread the suspension across the surface of the slide by drawing the clean slide away from the mixture. Essentially, you will be using the clean slide to push the mixture across the surface of the slide. When you have finished spreading the slide, place the "clean" slide in a jar of disinfectant.
  6. Air dry the slide. DO NOT HEAT FIX.
  7. Examine under the bright field microscope.
  8. Record the shape, arrangement, and approximate size of the organisms. You should have data on two specimens which you have stained


Assignment: Upon completion of the laboratory you should

  1. Be able to describe the procedure for heat fixation of a bacterial smear and discuss why fixation is necessary.
  2. Define the following: acidic dye, basic dye, simple (direct) stain, negative (indirect) stain.
  3. State the mechanism of and differences between simple and negative staining.
  4. Have accurate labeled sketches of all of the specimens you examined (minimum = 4). Included with the sketches should be a brief description of the specimen, the magnification under which it was viewed, and an approximation of the size.


 

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