What is Simple Staining its Principle, procedure, and result?

Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background.

The simple stain can be used as a quick and easy way to determine cell morphology, size, and cell grouping of bacteria. True to its name, the simple stain is a very simple staining procedure because only one stain is used. Any basic dye such as Methylene blue, Crystal violet, or Ziehl’s carbolfuschin can be used to stain the bacterial cells.

Crystal violet produces the best and most consistent results on the variety of microorganisms tested in a laboratory. All three dyes are basic dyes, and therefore work on the same principle; however, crystal violet is the darkest dye and, thus, is most easily viewed through the microscope and gives the best contrast to the unstained background. Another advantage of crystal violet is that most laboratories that work with bacteria will have this stain readily available due to its use in the Gram stain.

Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail

Principle of Simple Staining

The principle of simple staining is based on the principle of producing a marked contrast between the organism and its surrounding, by the use of basic stain. A basic dye consists of a positive chromophore that strongly attracts negative cell components such as peptidoglycan.

Staining with basic (cationic) dyes such as methylene blue, crystal violet, and carbol fuchsin allows interaction with the surface of bacteria because of the presence of negatively charged sugars in the cell wall, which give the bacteria an overall negative charge.

The advantage of simple or direct stains is that the cells are stained with a single cationic dye so it is an easy, fast procedure that allows viewing of cell size, morphology, and cell groupings such as chains or tetrads. Simple staining is effective for vegetative cells but will not easily penetrate spores.

In a simple stain, in contrast to an indirect or negative stain, the cell smears are heat fixed before the staining procedure takes place which, if not done properly, can damage the cell’s integrity.

Simple stains are considered nonspecific stains, which means they stain all microbes in an identical fashion. For a more detailed analysis, specific differential stains such as Gram stain, acid-fast stain, and endospore stains need to be performed.

CAUTION: Dyes used for bacteriological staining are usually aniline dyes so they are potentially carcinogenic and should be handled carefully. Avoid contact, inhalation, or ingestion of dye.

Materials and Requirements

  • Bacterial broth culture or colony
  • Dyes-methylene blue, crystal violet, Ziehl’s carbol fuschin.
  • Immersion Oil
  • Microscope slides (cleaned with 95% ethanol followed by water rinse and dried with laboratory wipes)
  • Inoculating loop
  • Clothespin or clamp
  • Bunsen burner
  • Staining rack
  • Bibulous paper or paper towels
  • Microscope
  • Biohazard container

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps:

Crystal violet stain

  • Solution A: Dissolve 2 g of crystal violet (85% dye) to 20 ml of ethanol.
  • Solution B: Dissolve 0.8 g ammonium oxalate in 80 ml of distilled water.

Combine solution A and solution B for stain working solution. Filter before using. Store up to 1 year at room temperature, away from direct sunlight.

Methylene blue stain

  • Solution A:  Dissolve 0.3 g of methylene blue (90% dye content) in 30 ml of ethanol.
  • Solution B: Dissolve 0.01 g of potassium hydroxide in 100 ml of distilled water.

Combine solution A and solution B for stain working solution. Filter before using. Store indefinitely at room temperature.

Ziehl’s carbolfuchsin stain

  • Solution A: Dissolve 0.3 g of basic fuchsin (90% dye content) in 10 ml of 95% ethanol.
  • Solution B: Dissolve 5.0 g phenol in 95 ml of distilled water.

Mix solution A and solution B together for a stain working solution. Filter before using. Store up to 1 year at room temperature.

Simple staining Procedure

Simple staining procedure is done with Three simple steps 1) Smear preparation, 2)Staining the smear, and  3) Observing the smear.

1) Prepare smear

Starting with a clean grease-free slide, Cleaning of slides is done by the wiping slide with a clean dry cotton cloth and made grease-free by passing it 6 to 12 times through Bunsen flame. Another method of cleaning is to moisten the finger with water, rub it on the surface of soap and then smear the surface of the slide.

After removing the soapy filling with a clean cloth the surface becomes clean and grease-free. If the slide is perfectly clean drop can spread on its surface in a thin even film, otherwise, the water collects into small drops, and film cannot be made.

Transfer a loopful of the broth culture to the center of the slide. If a colony from an agar plate is being used, mix a loopful of bacteria into a drop of water on the slide to make a suspension. Use a second slide to place a large drop of water on top, and then use your inoculating loop to obtain the amount of water needed for the smear.

Be sure to mix a broth culture before taking a sample because many bacteria are not spread evenly throughout the broth, and you may miss your sample. Too little bacteria will be difficult to locate on the slide.

Too much bacteria from a colony added to the drop of water will make a thick, pasty smear that will not allow good staining and will be too thick for microscopic observation. Usually, a whole colony is too much touching the colony with the loop should be sufficient.

Using the inoculating loop, spread the sample out on the slide to achieve a thin layer and allow it to air dry. “Too much liquid (broth culture or water) will result in a large sample area on the slide and will take longer to air dry”.

Holding the slide with a clothespin or clamp, heat-fix the dry smear by gently passing the slide smear-side-up through a Bunsen burner flame 2 to 3 times. Do not overheat or bacterial cell walls will be damaged and cell morphology will be destroyed. The slide should not become so warm that you cannot touch it.

2) Stain the smear

Place the slide on a staining rack and apply the stain to the slide for 1 min. If the stain is too light upon microscopic viewing, increase the staining time. Increase staining time by 5 to 10 sec if the stain is too light. If stained for too long a period, stain crystals can form on the slide, producing artifacts that distort the view of the bacteria.

Rinse the stain from the slide with water from a wash bottle or slowly running tap water until the runoff is clear usually 5 to 10 sec.

Direct the water flow to the end of the slide and allow the water to run across the stained culture so that the water force does not disrupt the smear. Tap water is often suggested for the rinsing of stains since it is readily available and the type of water used for rinsing does not affect the outcome of staining results. Only rinse until the water is clear as it runs off of the slide.

Blot the slide dry with bibulous paper or paper towels. Do not rub the smear, only blot. Be sure the bottom of the slide is clean.

3) Observe the slide

Observe the smear using oil and the oil immersion lens (a coverslip is not necessary). Viewing is best where there is a single layer of cells on the slide. Staining smears from colonies is also easier and more consistent. After viewing, dispose of all slides in the appropriate biohazard container.

Results

The background will be clear with the bacterial cell stained the color of the dye used. The overall shape and arrangement of the sample bacteria should be observable. The bacterial cells usually stain uniformly and the color of the cell depends on the type of dye used.

If methylene blue is used, some granules in the interior of the cells of some bacteria may appear more deeply stained than the rest of the cell, which is due to the presence of different chemical substances.

Troubleshooting

Common problems that occur throughout the protocol and possible solutions are outlined in Table

ProblemSolution
Cannot bring cells into focusI) Make sure the smear side of the slide is on top.
II) Add immersion oil to the top of the smear when viewing with a 100× immersion lens
Stain is too light or too darkI) Experiment with incubation time the longer the dye is present the darker the stain
No cells are found within the smearI) Broth culture was used and not mixed before the sample was taken some cells float on top or sink as a pellet to the bottom of a tube.
II) Usually broth culture must be turbid for good cell concentration for viewing.
III) Usually, colonies give better results than broth culture
Smear is too thick or pasty to see throughI) Reduce the amount of colony transferred into the water
Difficult to focus because smear not adhering appears that pieces are floatingI) Smear not completely air dried before heat fixing
II) Heat fixing not sufficient pass 3 to 4 times through the flame
Cells look lysed or distortedI) Too much heat reduce heat fixing
Large crystals on a slideI) The stain was not adequately rinsed off of smear make a new slide
II) Stain was not adequately rinsed off of smear make a new slide
Oval purple organisms present larger than bacteriaI) Reagents contaminated with yeast that Gram stain purple

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