4.5: Review of Staining Procedures - Biology

To help you review the staining procedures in Labs 3 and 4, fill out the table below with information about these staining procedures. This information should include (but not be limited to) the following:

  • What does the staining procedure tell you about bacterial cell structure, or the types of structures produced by bacteria?
  • Is the staining procedure used to detect specific types of cells? If so, what are they?
  • What do the positive and negative results look like at the end of the procedure?
  • Is there any clinical relevance to the results of the staining procedure?
  • Any other information that would help you to understand what the staining procedure detects and how it works.

Exercise (PageIndex{1})

Staining ProcedureInformation
Silver Nitrate
Negative Staining

Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy

Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations, including high inter-observer variability and the capacity to label only one marker per tissue section. This review details various highly multiplexed techniques that have emerged to circumvent these constraints, allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition, cellular functional and cell-cell interactions. Among these techniques, multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) has emerged to be particularly promising. mIHC/IF provides high-throughput multiplex staining and standardized quantitative analysis for highly reproducible, efficient and cost-effective tissue studies. This technique has immediate potential for translational research and clinical practice, particularly in the era of cancer immunotherapy.

Keywords: immunofluorescence immunohistochemistry immunotherapy multiplex overview.

© 2020 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.

Gram Stain under Microscope

Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram positive bacteria or gram negative bacteria.

Gram positive bacteria will typically have a stronger affinity for crystal violet on applying gram's iodine than the gram negative cell wall.

Being a mordant, gram's iodine forms a complex with crystal violet in the stain that has attached more tightly to the cell wall of gram positive bacteria than that of the gram negative bacteria.

Whereas the gram positive bacteria stain violet as a result of the presence of a thick peptidoglycan layer in the walls of their cell , the gram negative bacteria stain red, due to the thinner peptidoglycan layer in their cell wall (a thicker peptidoglycan layer allows for the retention of the stain, but a thinner layer does not).


The staining involves 3 major steps/processes that include:

o Staining with crystal violet (a water soluble dye)

o De-colorization (using ethanol/acetone)

o Counterstaining (using Safranin )

Due to the differences in the thickness of the peptidoglycan layer on the cell walls of these bacteria, gram positive bacteria will retain the crystal violet stain after the de-colorization process using ethyl alcohol/acetone.

After staining the sample with crystal violet, ethyl alcohol is used to decolorize the sample. It achieves its purpose by dehydrating the peptidoglycan layer by tightening and shrinking it. In doing so, large crystal violet cannot penetrate the tightened layer of peptidoglycan, and hence it is trapped in the cell wall of gram positive bacteria.

On the other hand, the outer membrane of the gram negative cells cannot retain the crystal violet iodine complex and hence the color is lost.

Safranin is a lighter stain as compared to crystal violet and thus it does disrupt the purple coloration in the gram positive cells.


In an aqueous solution, crystal violet dissociates into ions of CV+ and CV-. These ions penetrate the walls and membranes of both gram positive and negative cells.

CV+ will interact with the negatively charged components of the bacterial cells, and take up the purple coloration. On adding iodine, iodine cations (I - or I 3 - ) interact with CV+, which results in the formation of larger complexes of CVI within the cytoplasm and the outer layers of the cell.

On adding the decolorizing agent (ethanol), it interacts with the membrane lipids of both the gram positive and gram negative positive and gram negative.

This results in the loss of the outer membrane, which in turn leaves the peptidoglycan layer exposed. For the gram negative cells, ethanol causes the walls to be leaky and hence they cannot hold the large complexes of CV-L during de-colorization.

In some of the staining processes using gram stain, a pattern of gram-variables are obtained, which is a mix of pink and purple.

Some generas , such as Arthrobacter , Actinomyces and Corynebacterium have a cell wall that is particularly sensitive to breakage during cell division.

This results in gram negative staining of the gram positive cells.

On the other hand in cultures of Clostridium and Bacillus, the reduced thickness of peptidoglycan during growth coincides with an increased number of cells that in turn stain gram negative.


1. The primary stain (crystal violet reagents for staining)

o 2 grams of crystal violet (certified 90 percent of the dye content)

o 20ml of ethanol (95percent vol / vol )

o 0.8 grams of ammonium oxalate,

o 80ml of distilled water,

Mix A and B so as to obtain crystal violet staining reagent and store for 24 hours.

2. M ordant (grams iodine)

o grams of potassium iodide,

o 300ml of distilled water,

Using a mortar, iodine and potassium iodide are ground, while slowly adding water with continued grinding until all the iodine has completely dissolved. (Store this in an amber bottle)

3. D ecolorizing agent

o Ethanol, 95 percent ( vol / vol )

However, acetone or 1:1 acetone with ethanol,

4. Counterstain ( safranin )

o 10ml of the stock solution (2.5 grams Safranin O and 100ml of 95 percent ethanol)

o 90ml of distilled water


It is important to note that the thickness of the sample smear on the slide is an important consideration during the preparation of the sample. The smear should not be too think or too thin.

Bacteria - smear the sample on the slide using an inoculating needle. This can also be done by introducing a drop of saline on the slide followed by the sample and then mixing.

This should then be left to air dry before heat fixing by carefully passing the slide through the Bunsen burner (avoid burning the sample).

Actinomycetes - same as bacteria, but by trying to get a portion of the colony on the slide while it is still intact, this can be achieved by using a scalpel.

Staining Procedure

o Flood the slide with crystal violet staining reagent for about 1 minute ,

o Wash the slide using a gentle, indirect stream of tap water for about 2 seconds, flood the slide with a mordant (Gram’s iodine) then wait for a minute,

o Wash the slide for the again in a gentle, indirect stream of tap water for about 2 seconds,

o Flood the slide with the decolorizing agent then wait for 15 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear,

o Flood the slide using counterstain safranin ( and wait for about a minute (30 seconds to 1 minute)

o Wash the slide using a gentle and indirect stream of tap water to a point where the color appears in the effluent and then blot dry the absorbent paper,

o Add a drop of immersion oil on the stained sample and observe under the microscope

Gram staining helps to characterize bacteria as gram positive or gram negative allowing microscopist enthusiasts/professionals to verify a bacterial cell's wall and membrane which in turn influences various facets of its pathogenicity and level of virulence.

Note : Before running the gel make sure that the gel, gel apparatus and samples are ready.

  1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus.( Make sure that the short plate always faces inside and if you have got only one gel to run use the dummy plate that is available to balance).
  2. When the plates are secured, place them in the cassette and then lock it.
  3. Place them in the gel running tank.
  4. Fill the inner chamber of the tank with buffer.(Now it is easy to remove the comb, since it is lubricated).
  5. Remove the comb CAREFULLY(without breaking the well).
    [Now the gel is ready to load the samples]
  6. Rinse the loading tip a few times with distilled water. (Make sure that all the water is poured out before loading the samples.)
  7. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe with distilled water after loading for a few times .
  8. Attach the power supply by putting the lid (Make sure that the connection is in correct way ie., black - black and red - red). Set the voltage upto 180 V and run for 1 hour.(Don't allow the dye front to go out of the gel).

General Staining and Segmentation Procedures for High Content Imaging and Analysis

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

Keywords: CellMask CellTracker High content imaging High content screening Nuclear segmentation Segmentation.

Watch the video: Gram Staining Procedure Animation Microbiology - Principle, Procedure, Interpretation (January 2022).