Staining Techniques



Microorganism is the scientific term used to describe a living organism of a very small magnitude that can be studied only by the usage of microscopes. Microbiology is the field of science that studies microorganisms, such as : bacteria, virus, fungi and protozoa. The process of staining includes the preparation of the wet mount or smear preparation which is a culture of microorganisms over the cover slip [1]. 

Bacteria are small prokaryotic cells whose length ranges from 0.2 to 1 micrometer and that are found in different environments. They are characterized by the ability to move and to reproduce each 20 minutes. Bacteria have a crucial role about the environment and all the other living organisms, because they have the ability to recycle dead plants and animals, fixate the nitrogen the plants need to function regularly, contribute to the foods by adding by giving them their characteristic scent, making the environment cleaner by converting the dangerous materials into neutral ones, by a process called bioremediation [2]. 

Bacterial staining is the procedure that enables the researchers to have better images of the microorganisms unders the microscope by coloring of the bacteria by specific dyes. There are 4 types of staining, respectively: 

  • Negative staining is the term used to describe the method that only stains the bacteria’s background and exhibiting the bacteria colorlessly. It is a very beneficial method for the determination of the size and the arrangement of the cells by the usage of the nigrosin that is an acid stain that becomes negative by providing H+ion and is repelled by the positively charged of the most bacterial cells [3]. 
  • Simple staining is the term used to descibe the method that enables a view of the cells’s shape, size and arrangement by the usage of simple basic dyes (ex:crystal violet,basic fuchsin and methylene blue). This strain realizes the colorization of the bacteria because the basicity it gains when donating OH ions or accepting H+ ions, allows it to adhere to the negative cell membrane [4]. 
  • Gram staining is the method developed by Hans Christian Gram that enables discerning between Gram-positive bacteria and Gram-negative bacteria due to the different colors they exhibit after reacting with crystal violet. This difference in reaction is caused by the presence of the peptidoglycan that is a polymer composed of several chains of polysaccharides and peptides.  The gram-negative bacteria exhibit the red color of the counterstain (safranin), because of the inability of the thin layer of the peptidoglycan to keep the crystal violet’s color bound, while the gram-positive bacteria that contain a thick layer of the peptidoglycan exhibit the purple color of the crystal violet stain [5]. 
  • Endospore staining is a differential method, because of the usage of more than one stains developed to observe the spores, that are keratin structures produced each 6-8 hours, able to survive in extreme conditions, consisting of a passive metabolism, de-hydrative, exhibit resistance to the desiccation, different chemicals used in the laboratory, radiation and the heat. The first stain used is malachite, a water-soluble stain that is encountered in the cell by the help of the heat and enables the cell exhibit a green color. The second stain is the safranin that enables the other part of the cell except the spores to develop a red color [6]. 


Negative staining 

  • Microscopic slides                                      
  • Nigrosine solution 
  • Bacillus subtilis 

This experiment is performed near the Bunsen burner. Initially, 30 µl nigrosine dye is positioned over the microscopic slide by using a micropipette and afterwards 20 µl Bacillus subtilis is adjoined over it and mixed with it by the help of the pipetting. Finally, this preparation is scattered over the slide by using another slide and observed with a light microscope. 

Simple staining 

  • E.coli`s slant culture  
  • Methylene blue 

Initially, 20 µl E.coli is positioned over the slide by the help of a micropipette and this mixture is stirred over the slide by a heated inoculation loop. Afterwards, the smear preparation is positioned in order to get dried in the room temperature and later it is slipped over the flame in order to fix the microorganism`s sample. Later, 300µl Methylene blue is positioned over the microscopic slide for 1 minute. After that the microscopic slide is washed with water and dried so that all the drops are eliminated from the slide`s surface. At the end of the process, the slide is observed by the help of the microscope. 

Gram staining 

  • Crystal violet 
  • Safranin 
  • Iodine dye 
  • Bacterial mixture 

Initially, 20 µl bacterial mixture is positioned over the slide by using a micropipette and this sample is scattered over the slide by using a heated inoculating loop. This smear preparation is placed over the bench under the room temperature in order to get dried. Afterwards, the slide is slipped over the flame of the Bunsen burner. 300 µl Crystal Violet is positioned over the slide for one minute. Later, the stain is removed after being washed with water and 300 µl Iodine dye is positioned over the slide for 1 minute. After that, 300 µl Ethanol alcohol (95M) is used to wash the slide for 20 seconds and then water is used to wash the slide for approximately 2 seconds. 300 µl Safranin is added for 1 minute and after that washed with water for 2 seconds. The slide is dried by using a blotting paper and later a lamella is positioned over it. Finally, the sample is observed by using a light microscope.  

Endospore staining 

  • Malachite green 
  • Safranin 
  • Bacillus subtilis 
  • Spore staining kit  
  • Electric hot plate 
  • Small beaker 

Initially, 20 µl Bacillus subtilis is positioned with a micropipette over the slide and the sample is scattered around the microscopic slide by using a heated inoculating loop. Preparation smear is dried under room temperature and later, slipped across the flame of the Bunsen Burner 3 times. A toweling paper is placed over the microscopic slide and this slide is positioned over the electric hot plate. 100 µl Malachite green is placed over the toweling paper and dried for a short period of time. This step is repeated 4 times. After the last drying process, the preparation smear is washed with water for 30 seconds. Afterwards, 300 µl of Safranin is positioned over the slide for 20 seconds and the washing process with water is repeated again. After the water is removed from the slide by using blotting paper, lamella is positioned over the slide and observed under the light microscope.  


In the negative staining, a dark background with light particles showing the presence of Bacillus was expected, but the image of our sample reflects the Bacillus bacteria in a green light color and the background as whitish. A possible reason why this has occurred can be, because of contamination of the sample during the smear preparation, such as: preparing a thick layer of smear instead of a thin one, burning of the cells or because of visualization of the sample while the sample is still wet.  

Simple staining method was successful and due to our image the E.coli‘s cells shape, size and arrangement could be visualized. The ability of E.coli cells to appear as blue is because of the ability of the positively charged dye to adjoin to the cytoplasm of the E.coli that carry a negative charge. 

Gram staining method also led to satisfactory results and a classification of bacteria as positive and negative was made. In this method, Crystal Violet contributed to give the cells a purple color, while iodine helped as a co-agent of crystal Violet to create an insoluble mass of stain. Alcohol contributed to decolorize the purple color from the Gram negative bacteria, because of their thin layer of peptidoglycan that is unable to keep the purple color bound to itself. Decolorizing does not occur in Gram positive cells, because of their thick layer of peptidoglycan to keep the purple color bound to its structure. Safranin contributes to give the pink color to the decolorized Gram negative. The samples were observed with compound light microscope assisted by the oil immersion to contribute to a clearer view.  

Endospore staining’s results were satisfactory, because our group’s image showed that the endospores reflected the green color they had gained by the Malachite green and the other cells reflected the red color of the Safranin stain.  


1. retrieved 09.03.2016 from 

2. retrieved 09.03.2016 from 

3. retrieved 10.03.2016 from 

4. retrieved 10.03.2016 from 

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6. retrieved 11.03.2016 from 











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