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. Microbiology Network on Twitter Counter. Toggle navigation. Gram Staining Scott Sutton, Ph. The Basic Method First, a loopful of a pure culture is smeared on a slide and allowed to air dry.
Set the slide on the benchtop, and allow it to fully air dry. Once dried, light a Bunsen burner to heat-fix the bacteria. Using tongs, pass the slide through the burner flame several times, with the bacteria side up, taking care not to hold the slide in the flame too long, which may distort the cells.
Now, working over the sink, hold the slide level and apply several drops of Gram's crystal violet to completely cover the bacterial smear and then place the slide onto the bench to stand for 45 seconds. Next, hold the slide at an angle and gently squirt a stream of water onto the top of the slide, taking care not to squirt the bacterial smear directly. Now, holding the slide level again, apply Gram's iodine solution to completely cover the stained bacteria and then allow it to stand for another 45 seconds.
Next, carefully rinse the iodine from the slide, as shown previously. While holding the slide at an angle, add a few drops of Gram's decolorizer to the slide, allowing it to run down over the stained bacteria, just until the run-off is clear, for approximately 5 seconds. Immediately, rinse with water as shown previously. This will limit over-decolorizing the smear. Next, holding the slide level again, apply Gram's safranin counterstain to completely cover the stained bacteria.
After 45 seconds, gently rinse the Safranin from the slide with water, as shown previously, and then blot dry with paper towels. Finally, add a drop of immersion oil directly to the slide, and then examine the slide using a light microscope with a X oil objective lens. To begin this staining protocol, first put on the correct personal protective equipment and then ensure that the glass slides that will be used are clean. Next, prepare the solutions. Now, pipette 10 microliters of the Congo red solution onto the slide.
Using a clean, sterile pipette tip, select a single bacterial colony from the LB agar plate. Then, smear the bacterial colony into the dye to produce a thin, even layer.
Completely air dry the bacterial slide for minutes. Now, hold the slide at an angle and gently squirt a stream of water onto the top of the slide, taking care not to squirt the bacteria directly. Continue holding the slide at a degree angle until completely air-dried. Finally, add a drop of immersion oil directly to the slide, and then examine the slide using a light microscope with a X oil objective.
To perform endospore staining, first, prepare a 0. Next, pipette 10 microliters of 1X PBS onto the center of the slide. Smear the bacteria into the liquid to produce a thin, even layer. Now, set the slide on the benchtop, and allow it to fully air dry. Pass the slide through the blue burner flame several times, with the bacteria side facing up. Then, once the slide has cooled, place a piece of precut lens paper over the heat-fixed smear.
Next, turn on a hotplate to the highest setting, and bring a beaker of water to a boil. Saturate the lens paper with the malachite green solution and, using tongs, place the slide on top of the beaker of boiling water to steam for 5 minutes.
Keep the lens paper moist by adding more dye, one drop at a time, as needed. Next, again using tongs, pick up the slide from the beaker and remove and discard the lens paper.
Allow the slide to cool for 2 minutes. Working over the sink, hold the slide at an angle, and gently squirt a stream of water onto the top of the slide.
Now, hold the slide level and apply Safranin to completely cover the slide. Then, allow it to stand for 1 minute. Next, hold the slide at an angle and rinse as previously shown. Allow the slide to air dry on the benchtop. Finally, add a drop of immersion oil directly to the slide, and then examine the slide with a light microscope, with a X oil objective. In the Gram staining protocol, two different colored stains can result.
Dark purple staining indicates that the bacteria are Gram-positive and that they have retained the crystal violet stain. In contrast, reddish-pink staining is a characteristic of Gram-negative bacteria, which instead will be colored by the Safranin counterstain.
Additionally, different shapes and arrangements of bacteria can be visualized after Gram staining. For example, it is possible to differentiate Cocci, or round bacteria, from rod-shaped Bacillus, or identify bacteria, which forms strands, compared to those which typically aggregate as clumps or occur singly.
In a capsule stained microscope image, the bacterial cells will typically be stained purple, and the background of the slide should be darkly stained. Against this dark background, the capsules of the bacteria, if present, will appear as a clear halo around the cells.
Lastly, in endospore staining, Vegetative cells will be stained red by the Safranin counterstain. If endospores are present in the sample, these will retain the malachite green stain and appear bluish-green in color. Subscription Required. Please recommend JoVE to your librarian. Bacteria have distinguishing characteristics that can aid in their identification. Some of these characteristics can be observed by staining and light microscopy. Three staining techniques useful for observing these characteristics are Gram staining, Capsule staining, and Endospore staining.
Each technique identifies different characteristics of bacteria and can be used to help physicians recommend treatments for patients, identify potential contaminants in samples or food products, and verify sample sterility. Bacteria are microscopic living organisms that have many distinguishing characteristics such as shape, arrangement of cells, whether or not they produce capsules, and if they form spores.
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Gram Staining Set-up Wear gloves and a non-flammable lab coat, as dyes will stain hands and clothing. A Bunsen burner is used to heat-fix the bacteria. Use care when working with flame; tie back long hair.
Commercially available Gram stain reagents will be used. Clean microscope slides with laboratory wipes. Smear a bacterial colony into the liquid to produce a thin, even layer.
Note: Do not use cultures older than 24 hours, as bacteria too old could have changes in their cell wall, which will affect the Gram stain results 1, 4.
Completely air-dry slide. Once dried, heat-fix bacteria by passing slide through the flame bacteria side up times. Note: Do not hold the slide in the flame too long or you might distort the bacterial cells 1. Working over the sink, hold the slide level and apply Gram's Crystal Violet to completely cover the heat-fixed bacteria, allow to stand 45 seconds. Rinse excess Crystal Violet by holding the slide at an angle and squirting a gentle, indirect stream of water onto the slide and letting it run down over the stained bacteria.
Do not squirt water directly onto the bacteria. Thin walled bacteria cannot keep the first stain purple so when the second stain red is placed on the organisms they become red or Gram negative. For a video of the Gram stain procedure click here. Gram negative organisms are Red. Hint; Keep your P's together; Purple is Positive. Gram stains are never pink they are red or purple so you don't destroy the rule; keep your P's together.
In microbiology bacteria have been grouped based on their shape and Gram stain reaction. This leads to lots and lots of memorization.
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