Gram staining is one empirical technique of distinguishing bacterial types into two big groups (Gram-positive and also Gram-negative) based on the chemical and also physical nature of your cell walls. The technique is named after the inventor, the Danish scientist Hans Christian Gram (1853-1938), who occurred the an approach in 1884 (Gram 1884). The importance of this determination to correct identification the bacteria can not be overstated together all phenotypic methods begin with this assay.

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The basic Method

First, a loopful the a pure society is smeared on a slide and allowed to waiting dry. The society can come native a special suspension of a liquid society or a pure nest from a plate rely in water ~ above the microscopic lense slide. Necessary considerations: take a small inoculum—don’t do a special smear the cannot be fully decolorized. This could make gram-negative organisms appear to be gram-positive or gram-variable. Take a fresh culture—old societies stain erratically. resolve the cells to the slide by warm or through exposure to methanol. Warmth fix the on slide by passing the (cell next up) with a fire to warmth the glass. Do not let the glass come to be hot to the touch. Crystal violet (a an easy dye) is then included by extending the heat-fixed cells with a all set solution. Enable to stain for around 1 minute. Briefly rinse the slide v water. The heat-fixed cells have to look purple at this stage. Add iodine (Gram’s iodine) systems (1% iodine, 2% potassium iodide in water) for 1 minute. This acts together a mordant and also fixes the dye, making that more difficult to decolorize and reducing few of the variability that the test. Summary rinse v water. Decolorize the sample by using 95% ethanol or a mixture the acetone and alcohol. This can be excellent in a secure stream, or a collection of washes. The important facet is to ensure that all the shade has come the end that will do so easily. This step washes far unbound crystal violet, leaving Gram-positive organisms stained purple v Gram-negative biology colorless. The decolorization the the cell is the most “operator-dependent” step of the process and the one the is most most likely to it is in performed incorrectly. Rinse v water to protect against decolorization. Rinse the slide v a counterstain (safranin or carbol fuchsin) i beg your pardon stains all cells red. The counterstain stains both gram-negative and also gram-positive cells. However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s result is only seen in the previously colorless gram-negative cell which now show up pink/red. Blot tenderness and permit the on slide to dry. Carry out not smear.

What’s walking On?

Bacteria have a cell wall made increase of peptidoglycan. This cell wall provides rigidity come the cell, and protection indigenous osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cabinet wall, gram-negative bacteria have actually a thin cell wall surface and an outer phospholipid double layer membrane. The decision violet stain is little enough to pass through through the procession of the cell wall of both species of cells, however the iodine-dye complicated exits just with challenge (Davies et al. 1983)

The decolorizing mixture dehydrates cell wall, and also serves together a solvent come rinse the end the dye-iodine complex. In Gram-negative bacteria it also dissolves the external membrane that the gram-negative cell wall surface aiding in the release of the dye. It is the thickness the the cell wall that characterizes the an answer of the cells to the staining procedure. In addition to the plainly gram-positive and gram-negative, there space many types that room “gram-variable” through intermediate cell wall surface structure (Beveridge and also Graham 1991). As provided above, the decolorization action is crucial to the success that the procedure.

Gram’s an approach involves staining the sample cell dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining v a red dye. The cells with a thick cell wall surface appear blue (gram positive) as decision violet is preserved within the cells, and also so the red dye cannot be seen. Those cells v a slim cell wall, and therefore decolorized, appear red (gram negative).

It is a prudent exercise to always include a optimistic and an unfavorable control on the staining procedure to confirm the accuracy of the results (Murray et al 1994) and also to perform proficiency testing on the capacity of the technicians to correctly analyze the stains (Andserson, et al. 2005).

Excessive Decolorization

It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The details concerns in this step are provided below (reviewed in McClelland 2001)

Excessive heat throughout fixation: warm fixing the cells, as soon as done come excess, alters the cabinet morphology and makes the cells much more easily decolorized. Low concentration of crystal violet: concentration of crystal violet up to 2% can be used successfully, however low concentrations an outcome in stained cells the are quickly decolorized. The conventional 0.3% equipment is good, if decolorization walk not generally exceed 10 seconds. Excessive washing in between steps: The crystal violet stain is at risk to wash-out with water (but not the decision violet-iodine complex). Do not use much more than a 5 second water wash at any kind of stage that the procedure. Insufficient iodine exposure: The lot of the mordant easily accessible is essential to the development of the crystal violet – iodine complex. The reduced the concentration, the simpler to decolorize (0.33% – 1% typically used). Also, QC of the reagent is vital as exposure to air and also elevated temperature hasten the ns of Gram’s iodine indigenous solution. A closed party (0.33% beginning concentration) at room temperature will shed >50% of easily accessible iodine in 30 days, an open up bottle >90%. Ns of 60% iodine results in erratic results. Prolonged decolorization: 95% ethanol decolorizes much more slowly, and may be recommended because that inexperienced technicians while competent workers deserve to use the acetone-alcohol mix. Ability is necessary to gauge as soon as decolorization is complete. Excessive counterstaining: together the counterstain is also a an easy dye, the is feasible to replace the decision violet—iodine complex in gram- hopeful cells with an over-exposure to the counterstain. The counterstain have to not be left top top the on slide for more than 30 seconds.

Alternatives come the Gram Stain

Gram’s staining technique is plainly no without its problems. It is messy, complicated, and also prone to operator error. The method also calls for a huge number of cells (although a membrane-filtration technique has to be reported; Romero, et al 1988). However, it is also central to phenotypic microbial identification techniques.

This method, and also it’s liabilities, room of instant interest come those associated in eco-friendly monitoring program as one of the most usual isolates in an EM program, Bacillus spp., will frequently stain gram change or gram negative despite being a gram-positive stick (this is specifically true with older cultures). The problems with Gram’s an approach have result in a find for various other tests the correlate v the cell wall surface structure of the gram-positive and also the gram-negative cells. Number of improvements/alternatives come the classical gram stain have showed up in the literature.

KOH cable Test

The KOH String check is done making use of a autumn of 3% potassium hydroxide on a glass slide. A clearly shows loopful of cells from a single, well-isolated colony is mixed into the drop. If the mixture i do not care viscous within 60 secs of mixing (KOH-positive) climate the swarm is thought about gram-negative. The reaction relies on the lysis that the gram-negative cabinet in the dilute alkali solution releasing cellular DNA to turn the suspension viscous. This an approach has been presented effective because that food microorganisms (Powers 1995), and also for Bacillus spp (Carlone et al 1983, Gregersen 1978), back it may be problematic for part anaerobes (Carlone et al 1983, but also see Halebian et al 1981).

This test has actually the benefit of simplicity, and it have the right to be carry out on larger cultures. False negative results can take place in the test by utilizing too tiny inoculum or too much KOH (DNA-induced viscosity no noticeable). False optimistic results can happen from too heavy an inoculum (the solution will show up to gel, but not string), or inoculation with mucoid colonies. This have the right to serve as a valuable adjunct come the heritage gram stain method (von Graevenitz and also Bucher 1983).

Aminopeptidase Test

L-alanine aminopeptidase is an enzyme localized in the bacter cell wall surface which cleaves the amino acid L-alanine from miscellaneous peptides. Far-ranging activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microbe so far studied display no or really weak task (Cerny 1976, Carlone et al. 1983). To carry out the test, the reagent is provided to make a suspension (with the bacteria). Aminopeptidase activity of the bacteria reasons the relax of 4-nitroaniline native the reagent, transforming the suspension yellow. The check is especially beneficial for non-fermenters and also gram-variable organisms, and is a one step test with several carriers of kits. Results of the test are available in 5 minutes.

Fluorescent Stains

A popular mix of fluorescent stains for use in gram staining (particularly because that flow-cytometry) entails the use of the fluorescent nucleic acid binding dyes hexidium iodide (HI) and also SYTO 13. Hi penetrates gram-positive yet not gram-negative organisms, however SYTO 13 penetrates both. Once the water were supplied together in a solitary step, gram-negative biology are green fluorescent by SYTO 13 if gram-positive organisms room red-orange fluorescent by hi which overpowers the green of SYTO 13 (Mason et al 1998). There are commercial kits accessible for this procedure, which requires a fluorescent microscope or a circulation cytometer.

Sizemore et al (1990) arisen a different technique to fluorescent labeling that cells. Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the external peptidoglycan class of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled through the lectin. A different of this technique has additionally been used to “gram stain” microbe in milk for straight measurement by circulation cytometry.

LAL-based Assay

Charles flow Laboratories has just exit a product come be supplied with their PTS tool – the PTS Gram identifier (Farmer 2005). This methodology makes use that the exact same reaction used for the chromogenic LAL test. Gram-negative organisms, v bacterial endotoxin, initiate the LAL coagulase cascade which results in activation the the proclotting enzyme, a protease. In the LAL test, this enzyme cleaves a peptide from the horseshoe crab coagulen, resulting in a clot. That can also cleave a peptide indigenous a artificial substrate, yielding a chromophore (p-nitroaniline) which is yellow and can it is in measured photometrically in ~ 385 nm (Iwanaga 1987). Gram-positive organisms, lacking endotoxin, do not create the color readjust in this method, while gram-negative organisms do trigger it. Results are available within 10 minutes.


The differentiation the bacteria right into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems. This job is usually completed through the usage of Gram’s Staining Method. Unfortunately, the gram stain methodology is complex and prone to error. This operator-dependence can be handle by attention to detail, and by the usage of controls ~ above the test. Additional steps could include confirmatory tests, of which several examples were given. As with all microbiology assays, full technician training and competent evaluation of the data are crucial quality regulate steps for great laboratory results.


Anderson, N L, et al. 2005. Cumitech 3B, high quality Systems in the Clinical Microbiology Laboratory. Coordinating ed., D. L. Sewell. ASM Press, Washington, D.C. Beveridge, TJ and also LL Graham. 1991. Surface Layers the Bacteria. Microbiol Rev. 55(4):684-705. Carlone, GM et al. 1983. Methods for differentiating Gram-Positive native Gram-Negative Bacteria. J Clin Microbiol. 16(6):1157-1159. Cerny G. 1976 an approach for difference of gram an unfavorable from gram confident bacteria. Eur J Appl Microbiol. 3:223-225. Davies, J. A., et al. 1983. Chemical mechanism of the Gram Stain and also Synthesis of a brand-new Electron-opaque Marker for Electron Microscopy which Replaces the Iodine Mordant that the Stain. J Bacteriol. 156(2):837-845. Farmer, T. 2005. A Definitive, Rapid alternate to the Gram Stain Assay. Quick Microbiology Newsletter. 3(8):2-3 Gram, C. 1884. The Differential Staining the Schizomycetes in tissue Sections and in Dried Preparations. Fortschitte der Medicin. 2:185-189. Gregersen, T. 1978. Rapid technique for Disitinction of Gram-negative from Gram-positive Bacteria. Appl Microbiol Biotech 5(2):123-127. Halebian, S et al. 1981. Rapid technique That Aids in separating Gram-Positive native Gram-Negative Anaerobic Bacteria. J Clin Microbiol. 13(3):444-448. Holm, C. And also L. Jespersen. 2003. A Flow-Cytometric Gram-Staining methods for Milk-Associated Bacteria. Appl Environ Microbiol. 69(5):2857 – 2863. Iwanaga, S. 1987. Chromogenic Substrate for Horseshoe Crab clotting Enzyme: Its application of the Assay of bacter Endotoxins. Haemostasis. 7:183. McClelland, R. 2001. Gram’s Stain: the vital to Microbiology. Med rap Observer. April pp. 20-31. Murray, RGE et al. 1994. Determinative and also Cytological irradiate Microscopy. IN: approaches for General and Molecular Bacteriology P. Gerhardt, editor-in-chief. ASM Press, Washington, DC. Pp21-41. Powers, EM. 1995. Efficacy of the Ryu Nonstaining KOH technique for promptly Determining Gram reaction of Food-Borne and also Waterborne Bacteria and also Yeasts. Appl Environ Microbiol. 61(10):3756-3758. Romero, S et al. 1988. Rapid an approach for the Differentiation of Gram-Positive and also Gram-Negative Bacteria ~ above Membrane Filters. J Clin Microbiol. 26(7):1378-1382. Sizemore, RK et al. 1990. Alternating Gram Staining method Using a Fluorescent Lectin. Appl Environ Microbiol. 56(7):2245-2247 von Gravenitz, A and also C Bucher. 1983. Accuracy of the KOH and also Vancomycin test in determining the Gram Reaction that Non-Enterobacterial Rods. J Clin Microbiol. 16(4):983-985.

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