Etiket Arşivleri: Staining

Staining ( Chapter 6 )


The sections, as they are prepared, are colourless and different components cannot be appreciated. Staining them by different coloured dyes, having affinities of specific components of tissues, makes identification and study of their morphology possible. Certain terminologies used in the following account are given below.


Substances stained with basic dyes


Substances stained by acid dyes

Vital staining

Staining of structures in living cells, either in the body (in vivo) or in a laboratory preparation (in vitro). e.g. Janus green is taken up by living cells and stains the mitochondria.

Metachromatic staining

There are certain basic dyes belonging to aniline group that will
differentiate particular tissue components by staining them a different color to that of original dye. The phenomenon is known as metachromasia. The tissue element reacting in this manner are said to be exhibiting metachromasia. The generally accepted explanation of this phenomenon is that change in color is due to polymerization.

Sulfated substances are highly metachromatic e.g. Mast cell granules. These contain Heparin which is highly sulfated.
Some of the common metachromatic dyes are :
Methylene blue Methyl violent
Thionin Crystal violent
Toluidine blue

Thionin and toluidine blue dyes are commonly used for quick staining of frozen selection using their metachromatic property to stain nucleus and cytoplasm differently. Tissue components often demonstrated by metachromatic stains :
Anyloid material, Mast cell granules
Mucin Cartilage

Direct staining

Application of simple dye to stain the tissue in varying shades of colours.

Indirect staining

It means use of mordant of facilitate a particular staining method or the use of accentuator to improve either the selectivity or the intensity of stain.

Progressive staining

Stain applied to the tissue in strict sequence and for specific times. The stain is not washed out r decolorised because there is no overstaining of tissue constituents. Staining is controlled by frequent observation under microscope.

Regressive staining

Tissue is first overstained and then the excess stain is removed from all but the structures to be demonstrated. This process is called differentiation and should always be controlled under microscope.


Partial or complete removal of stain from tissue sections. When the colour is removed selectively (usually with microscopic control) it is called differentiation. In case decolourization is to restain the selection with some other stain, acid alcohol treatment is the method of choice.


In regressive staining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies.


Bacterial Staining Endospores

Bacterial Staining
Endospore Location
Simple Stain
Endospore Stain
Differential stain
Endospore vs. Spore Stain
1 = MG with Heat
Decolorize = H2O
Counterstain = Safranin
Endospores = Green
Vegetative = Pink

Laboratory‎ > The Gram Stain and Simple Stain Smear

FE204 Microbiology

Lab report

The Gram stain and simple stain Smear 


       Preparation of smear and application of simple stain to observe size,shape, arrangement, color of the microorganisms using oil immersion objective.And application of differential staining method and classify microorganism as Gram positive, Gram negative.




•                    Crystal violet

•                    Safranine

•                    Distilled Water

•                    Iodine

•                    Alcohol

•                    Bibulous paper

•                    Microscope

•                    E.coli in Nutrient broth culture

•                    Sterile Glass slide

•                    İnoculating loop

•                    Forcep

•                    İmmersion oil

•                    Bunsen burner

•                    Xylol

•                    Methylene blue


       To preparation of smear, firstly in aseptic conditions a sterile microscopic slide was taken.It was flamed through a bunsen burner.After that, a few loops of E.coli broth culture was taken. The inoculum was putted on the slide.Then was waited for air drying.Next, heat fixation was applied and smear was prepared.

      In Gram staining process, firstly the ready smear was placed to staining pool.Then, a few drops of Crytal violet was added and was waited for 1 minute.After waiting, it was washed with water. Next, a few drops of Iodine in order to form CV-I complex was added and was waited for 1 minute.Then,it was washed with water again. After the this step, the smear was decolorized by using alcohol and it was washed with water.After that, 1 drop of Safranine was added and was waited for 1 minute.Then, it was washed with water and was dried with a paper.Finally, before it was examined under microscope at 100x, a few drops of oil added on the slide.And was decided the bacterial smear is G(+) or G(-).



       Bacteria are too small to see without the aid of a microscobe. Even with a microscope, bacteria cannot be seen easily. Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope.There are many different ways to stain bacteria so that they can be more easily visualized under the microscope. Some stains can also be used to identify and classify bacteria. The Gram stain is a differential stain that allows you to classify bacteria as either Gram-positive or Gram-negative.

      Using a single stain to color a bacterial cell is commonly referred to as a simple stain.  The most common dyes for this type of stain are methylene blue, fuchsin, and crystal violet.    Staining times for most simple stains are relatively short, usually from 30 seconds to 2 minutes, depending on the affinity of the dye.  After a smear has been stained for the required time, it is washed off gently, blotted dry, and examined directly under oil immersion.  This type of slide is useful in determining basic morphology and the presence or absence of certain kinds of granules.

      Bacteria that decolorize easily are called Gram-negative (red) and those that retain the primary stain are called Gram-positive (purple). Bacteria stain differently because of differences in their cell walls. Gram-positive cell walls consist of many layers of peptidoglycan. The crystal violet-iodine complex is larger than either the crystal violet or iodine molecules that entered the cell and the complex cannot pass through this thick cell wall. Gram-negative bacteria have a thin layer of peptidoglycan and an outer lipopolysaccharide layer. The alcohol dissolves the lipopolysaccharides so that the crystal violet-iodine complex can wash out of the cell.

      Before bacteria can be stained, a smear of bacteria must be made on a slide and heat fixed. A smear is made by spreading a bacterial suspension on a clean slide and letting it air dry. The dry smear is heated on a hot plate or passed through a flame several times to heat fix it. Heat fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break, a process called autolysis. The heat also enhances the adherence of bacterial cells to the slide. Finally, it is very important to prepare thin smears.  If a smear is too thick one will not be able to see individual cells, their arrangement, or the details of their microstructures.  Thick smears with large clumps of cells can entrap the stain preventing it from being washed.  The first step in preparing a smear depends on  whether the organism has been growing in liquid or solid media.  Two or more loopfuls of liquid media containing the organism can be placed on the slide.  One can use an inoculating loop to disperse organisms from solid media into a drop of water on the slide.