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.
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 It is messy, complicated, and prone to operator error.
The method also requires a large number of cells although a membrane-filtration technique has been reported; Romero, et al However, it is also central to phenotypic microbial identification techniques. A visible loopful of cells from a single, well-isolated colony is mixed into the drop. If the mixture becomes viscous within 60 seconds of mixing KOH-positive then the colony is considered gram-negative.
The reaction depends on the lysis of the gram-negative cell in the dilute alkali solution releasing cellular DNA to turn the suspension viscous. This method has been shown effective for food microorganisms Powers , and for Bacillus spp Carlone et al , Gregersen , although it may be problematic for some anaerobes Carlone et al , but also see Halebian et al This test has the advantage of simplicity, and it can be performed on older cultures.
False negative results can occur in the test by using too little inoculum or too much KOH DNA-induced viscosity not noticeable. False positive results can occur from too heavy an inoculum the solution will appear to gel, but not string , or inoculation with mucoid colonies.
This can serve as a valuable adjunct to the tradition gram stain method von Graevenitz and Bucher L-alanine aminopeptidase is an enzyme localized in the bacterial cell wall which cleaves the amino acid L-alanine from various peptides. Significant activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microorganisms so far studied display no or very weak activity Cerny , Carlone et al.
To perform the test, the reagent is used to make a suspension with the bacteria. Aminopeptidase activity of the bacteria causes the release of 4-nitroaniline from the reagent, turning the suspension yellow. The test is especially useful for non-fermenters and gram-variable organisms, and is a one step test with several suppliers of kits.
Results of the test are available in 5 minutes. A popular combination of fluorescent stains for use in gram staining particularly for flow-cytometry involves the use of the fluorescent nucleic acid binding dyes hexidium iodide HI and SYTO When the dyes were used together in a single step, gram-negative organisms are green fluorescent by SYTO 13 while gram-positive organisms are red-orange fluorescent by HI which overpowers the green of SYTO 13 Mason et al There are commercial kits available for this procedure, which requires a fluorescent microscope or a flow cytometer.
Sizemore et al developed a different approach to fluorescent labeling of cells. Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin.
This methodology makes use of the same reaction used for the chromogenic LAL test. Gram-negative organisms, with bacterial endotoxin, initiate the LAL coagulase cascade which results in activation of the proclotting enzyme, a protease.
In the LAL test, this enzyme cleaves a peptide from the horseshoe crab coagulen, resulting in a clot. It can also cleave a peptide from a synthetic substrate, yielding a chromophore p-nitroaniline which is yellow and can be measured photometrically at nm Iwanaga Gram-positive organisms, lacking endotoxin, do not trigger the color change in this method, while gram-negative organisms do trigger it.
Results are available within 10 minutes. The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems.
Unfortunately, the gram stain methodology is complex and prone to error. This operator-dependence can be addressed by attention to detail, and by the use of controls on the test. Additional steps might include confirmatory tests, of which several examples were given. As with all microbiology assays, full technician training and competent review of the data are critical quality control steps for good laboratory results. Experts at the Microbiology Network are ready to assist with consulting or training to meet your needs.
Our team of CGMP consultants and trainers stands ready to help you. Call us. The first consultation is free. This basic dye is positively charged and, therefore, adheres to the cell membranes of both gram negative and positive cells. After applying crystal violet and waiting 60 seconds the excess stain is rinsed off with water.
Next, a mordant is used. This binds to the crystal violet making a large complex that adheres to the cell membrane. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.
The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.
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