How to do gram staining

Cell Staining in Microscopy Types, Techniques, Preparations and Procedures Microscopy refers to the practice that involves the use of a microscope for the purposes of observing small scale structures that cannot be viewed using the naked eye and often cell staining is necessary as structures are difficult to discern due to insufficient contrast. Cell staining is a technique used for the main purpose of increasing contrast through changing the color of some of the parts of the structure being observed thus allowing for a clearer view. There are a variety of microscopic stains that can be used in microscopy.

How to do gram staining

It appears here with permission. Gram staining is an empirical method of differentiating bacterial species into two large groups Gram-positive and Gram-negative based on the chemical and physical properties of their cell walls. The method is named after its inventor, the Danish scientist Hans Christian Gramwho developed the technique in Gram The importance of this determination to correct identification of bacteria cannot be overstated as all phenotypic methods begin with this assay.

The Basic Method First, a loopful of a pure culture is smeared on a slide and allowed to air dry. The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide.

This could make gram-negative organisms appear to be gram-positive or gram-variable. Take a fresh culture—old cultures stain erratically. Fix the cells to the slide by heat or by exposure to methanol.

Ziehl Neelsen Staining -Principle, Procedure and Interpretations – howMed

Heat fix the slide by passing it cell side How to do gram staining through a flame to warm the glass. Do not let the glass become hot to the touch. Crystal violet a basic dye is then added by covering the heat-fixed cells with a prepared solution.

Allow to stain for approximately 1 minute. Briefly rinse the slide with water. The heat-fixed cells should look purple at this stage.

The Basic Method

This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test. Briefly rinse with water. This can be done in a steady stream, or a series of washes. The important aspect is to ensure that all the color has come out that will do so easily.

This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. Rinse with water to stop decolorization.

Rinse the slide with a counterstain safranin or carbol fuchsin which stains all cells red. The counterstain stains both gram-negative and gram-positive cells.

Blot gently and allow the slide to dry. Bacteria have a cell wall made up of peptidoglycan. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane.

The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty Davies et al.

In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure.

As noted above, the decolorization step is critical to the success of the procedure. The cells with a thick cell wall appear blue gram positive as crystal violet is retained within the cells, and so the red dye cannot be seen.

Those cells with a thin cell wall, and therefore decolorized, appear red gram negative. It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results Murray et al and to perform proficiency testing on the ability of the technicians to correctly interpret the stains Andserson, et al.

Excessive Decolorization It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The particular concerns in this step are listed below reviewed in McClelland Excessive heat during fixation: Heat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.

Low concentration of crystal violet: Excessive washing between steps:Here is a simple explanation of what it means to be a gram-positive bacteria. Gram staining involves the application of a series of dyes that leaves some bacteria purple (Gram +) and others pink (Gram -).

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Gram staining is used to determine gram status to classify bacteria broadly.

Gram stain - Wikipedia

It is based on the composition of their cell staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria.

Gram status is important in medicine; the presence or absence of a cell wall changes the bacterium's susceptibility to some antibiotics.

Gram stain or Gram staining, also called Gram's method, is a method of staining used to distinguish and classify bacterial species into two large groups (gram-positive and gram-negative).The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique..

How to do gram staining

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting.

Cell Staining in Microscopy - Types, Techniques, Preparations and Procedures