Staining of Microorganism: Negative Staining, Simple Staining, Differential Staining, Simple Staining, Endospore Staining, Lactophenol cotton blue mounting


The microbial cells are very small and transparent in nature, so it is very difficult to observe them under microscope. Staining is uses in order to understand the structure and chemical nature of microorganism and make visible. Thus, staining is the method of artificially coloring microorganism to allow it to visualize under the microscope.

 Dyes or stain are synthetic chemicals with respect to their color property in the anionic and cationic form, they can be divided into acid dyes and basic dyes. An acid dye is the salt of a colored acid while basic dye is the salt of a colored base. Acid fuchsin and erythrosine are acid dyes while methylene blue and crystal violet are the basic dyes. Basic dyes have greater affinity for the nuclear region of the cell whereas acid dyes have stronger tendency to combine with the cytoplasm. In the bacteriological studies, basic dyes are generally used.

Depending upon the purpose and type of organism to be studied, the various techniques of staining is available, they are as follow:

Simple Staining

The simple staining techniques contain only one dye that allow visualization of gross, morphological characteristics of cells like shape, size, cell arrangement, etc.

Materials required: 24 hours old bacterial cultures, methylene blue stain, staining tray, glass slides, inoculation loop, spirit lamp, tissue paper, microscope, etc.


  • Take a clean, dry glass slide.
  • Take a loopful of water on the center of slide.
  •  Aseptically transfer small amount of bacterial suspension in to water droplet with inoculation loop.
  • Spread the drop over the center of the slide to about 1 cm diameter and allow the film to air dry.
  • Fix the smear by passing the slides rapidly over the flame.
  • Flood the smear with the methylene blue dyes for 3 minutes.
  • In order to remove the excess stain, wash the slide in a gentle stream of tap.
  • Blot excess water by using tissue paper, and allow to air dry.
  • Observe under microscope accordingly.

Negative Staining

Negative staining is the simple staining technique. In negative staining, instead of basic dyes, acidic dyes are used, for example nigrosine, India ink. As bacterial cells are negatively charged, the acidic dyes do not stain the microbes but stains only the background. Thus, after staining under microscope, the microbes appear transparent in a dark background. The main advantages of negative staining are that there is less morphological distortion since cells are not subjected to vigorous physical or chemical treatment.

 Materials required:

 24 hours old bacterial culture, Nigrosine stain (Dissolve 10gm Nigrosine in 100ml of boiling distilled water, add 10 drops of formalin as preservatives) slides, inoculation loop, spirit lamp, microscope, etc.


  • Using a sterile wire loop, transfer a loopful of bacterial suspension to the center of a clean slide.
  • Add a small drop of nigrosine solution on the drop of the suspension on the slide.
  • Mix and spread thoroughly to form a thin film.
  • Air dry; do not heat fix.
  • Observe under microscope.

Differential Staining

Differential staining for example gram staining is employed to visualize and differentiate between organism. It is useful in presumptive identification of organism before carrying out several other tests such as serological and biochemical tests.

The gram staining was developed in 1884 by a Danish physician, Dr. Hens Christian Gram. It is a very useful staining method for classifying and identifying bacteria into two major groups i.e., gram negative and gram positive. In this process, the fixed bacterial smear is subjected to four different reagents called crystal violet (primary stain), Iodine solution (mordant), alcohol (decolorization agent) and safranin (counter stain). In grams staining, the bacteria which retain the primary stain i.e., organisms that appear dark blue or violet are called gram positive, while the bacteria that lose the primary stain and counter stained by safranin i.e., appear red are called a gram negative.

Gram Stain Protocol (Differential Staining Techniques)

Fig: Gram Stain Protocol (Differential Staining Techniques)

The difference is staining responses to the gram stain can be related to chemical and physical differences in their cell walls. The gram-negative bacterial cell wall is thin, complex, and multilayered, with relatively significant lipid content in addition to protein and mucopeptides. The higher amount of lipid is readily dissolved by alcohol, resulting in the formation of larges pores in the cell wall which facilitate the leakage of crystal violet- iodine complex and resulting in the decolorization of the bacterium which later takes the counter stain and appears red.

In contrast, the gram-positive cells walls are thick and chemically simple, composed mainly of protein and cross linked mucopeptides. When treated with alcohol, it causes dehydration, and closure of cell walls pores, thus not allowing the loss of crystal violet iodine complex and cells remain blue.

Materials required:

24-hour old Staphylococcus and Escherichia cultures, crystal violet, grams iodine solution, 95% ethanol, safranin, staining tray, droppers, inoculation loop, glass slides, blotting paper, microscope, etc.


  • On separate glass slides, apply thin smears of Staphylococcus and Escherichia.
  • Let the smears air dry and fix the smear by heat.
  • For 30 seconds, apply crystal violet stain.
  • Gently, wash the slide with distilled water for a few seconds.
  • Apply iodine solution for 1 minute.
  •  Decolorize with 95% ethanol by adding it drop by drop on the titled slide until no more colors flows from the smear.
  • Wash it with distilled water.
  •  Flood the slide with safranin (counter stain) for 30 seconds.
  •  Slightly, wash with distilled water and air dry.
  • Observe under microscope.

Endospore staining

Under unfavorable conditioning for growth, specialized structures called spores or endospores are formed within certain gram-positive cells such as Bacilli, clostridia, Sporosarcinae, etc. These spores lack metabolic activity and are resistant to heat, freezing, poisonous chemicals, and radiation. Spores are heat resistant due to their high calcium and dipicolinic acid content. An endospore develops in a characteristics position within a cell i.e., central, subterminal or terminal. In favorable conditions, endospores return to its vegetative state.

The endospore has low affinity for stain but once stained they revisit decolorization. For staining, it is required to soak endospore in stain for longer period in the steam.

Materials required: 48 hours old cultures of bacillus, Malachite green, safranin, staining tray, glass slides, inoculation loop, blotting paper, microscope, etc.


  •  Prepare a smear of Bacillus on a clean slide. Air dry and fix with heat.
  • Flood the smear with malachite green.
  •  Heat the slides to steaming for 5 minutes and add more stain to the smear for the time and time.
  •  Gently, wash the slide gently with running tap water until all excess stain is removed.
  •  Counter stain with safranin for 30 seconds.
  •  Wash smear with water. Blot it and air dry.
  •  Observe the slide under microscope using immersion objective.

Lactophenol cotton blue mounting of fungi

Lactophenol cotton blue stains the fungal cytoplasm and provides a light blue stain the fungal cytoplasm and provides a light blue background, against which the walls of hyphae can readily be seen. Lactophenol cotton blue is made up phenol, which serves as a fungicide; lactic acid which acts as a clearing agent; cotton blue, which stains the cytoplasm of fungus and glycerine, which gives a semi- permanent preparation.

 For rapid and routine examination of almost all types of fungi, spores and spore bearing structure are teased out on a clean slide on a drop of lactophenol cotton blue and a cover glass placed over the preparation which is then ready for microscopic examination.

 Materials required: Fingal culture, Lactophenol cotton blue, needless glass slides, cover slips, 70% alcohol, microscope, etc.


  •  Please a drop of lactophenol cotton blue on a clean slide.
  •  Transfer a small tuft on the fungus into the drop of stain using a needle.
  • Gently tease the material using the needles.
  •  Gently, place a cover slip without trapping air bubbles in the stain.
  •  Observe under microscope using low power and high-power objective.

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