Step 1: crystal violet (CV) dissociates in aqueous solutions to form CV + and chloride (Cl −) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative bacteria. The CV + ion interacts with negatively charged components of bacterial cells and stains the cells purple;
How does crystal violet work in Gram staining?
The gram stain utilizes crystal violet as the primary stain. 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.
Does crystal violet stain all bacteria?
Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram negatives lose the primary stain.
How does crystal violet interact with the bacterial cell wall?
The crystal violet dye adheres to the thick peptidoglycan layer of the cell wall present in gram-positive bacteria, staining them violet or purple when visualized under a light microscope.
What does crystal violet stick to on a bacteria?
The primary stain (crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet.
Why is crystal violet used in simple staining?
Basic stains, such as methylene blue, Gram safranin, or Gram crystal violet are useful for staining most bacteria. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged.
How does crystal violet bind to a cell?
One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture.
Why do gram-positive bacteria stain purple and not pink?
gram stain test Gram-positive bacteria remain purple because they have a single thick cell wall that is not easily penetrated by the solvent; gram-negative bacteria, however, are decolorized because they have cell walls with much thinner layers that allow removal of the dye by the solvent.
Why do gram-positive bacteria sometimes appear purple and sometimes pink?
Under a microscope, gram-positive bacteria appear purple-blue because their thick peptidoglycan membrane can hold the dye. The bacteria is called gram-positive due to the positive result. Gram-negative bacteria stain pink-red. Their peptidoglycan layer is thinner, so it doesn't retain the blue color.
Why do Gram negative bacteria do not retain crystal violet stain?
Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost.
Does crystal violet stain live or dead cells?
Crystal violet will in fact stain living cells (though it is toxic) as well as dead cells. The viability assay is only to be used on adherent cells. It does not differentiate live and dead cells on the plate, the dead cells are simply washed off because they are no longer adherent.
Which type of bacteria stain pink during Gram staining?
Gram-negative bacteriaGram-negative bacteria have cell walls with thin layers of peptidoglycan (10% of the cell wall) and high lipid (fatty acid) content. This causes them to appear red to pink under a Gram stain.
What is crystal violet used for in chemistry?
Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria.
How to stain a gram with crystal violet?
Crystal violet staining solution is prepared in the same way as Liquid A used in Gram stain. Take a small quantity of culture and mix with physiological saline to prepare a smear. Stain the smear with crystal violet solution. Observe under oil immersion lens (Figure 2.9 (A) and (B) ).
Why is CV-I trapped in a Gram-positive cell?
The large CV–I complexes become trapped within the Gram-positive cell due to the multilayered nature of the peptidoglycan;
What is the color of the germinal tube and blastospore?
Figure 2.9. (A) Germinal tube and blastospore of Candida albicans (crystal violet stain). The germinal tube and blastospore appear purple when C. albicans is incubated in 0.5–1 ml human serum or in sheep serum for 2–4 h at 37 °C and stained by the crystal violet staining method. (B) Pseudohypha chlamydospore of C. albicans (crystal violet stain). Pseudohypha chlamydospores of C. albicans appear purple when stained with crystal violet. The chlamydospores are big, spherical, thick-walled, and they are located at the tip or side wall of unevenly stained pseudohypha.
What color is the blastospore?
Germinal tube and blastospores are stained bright blue after C. albicans are incubated in 0.5–1 ml human serum or in sheep serum for 2–4 h at 37 °C and stained with lactophenol cotton blue stain.
What color are pseudohypha chlamydospores?
Pseudohypha chlamydospores of C. albicans appear purple when stained with crystal violet. The chlamydospores are big, spherical, thick-walled, and they are located at the tip or side wall of unevenly stained pseudohypha.
How to stain a slide with lactophenol?
Staining protocol: Place a drop of lactophenol cotton blue staining solution onto a clean slide, and mix the fungal culture or clinical sample with the staining solution. Place a coverslip on top and heat gently. Press the coverslip gently to remove any bubbles. Observe the slide under an oil immersion lens ( Figure 2.10 (A) and (B)).
What is Gram stain used for?
Gram stain and lactophenol cotton blue stain are used to visualize certain fungal structures that are significant for identification. These include the germinal tube, hyphae, and spores and are especially important in clinical analyses of oral mucosal diseases. For example, in the identification of thrush, angular stomatitis is closely related ...
Why do Gram positive bacteria stain 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
Why do bacteria stain?
The purpose of staining bacteria is to see, for example, how thick of a layer of peptidoglycan their cell wall has. In the Gram stain, a gram-negative bacteria will stain red or pink because the rinse took out the primary dye and the Safrinin (secondary dye) took over the coloring as the coucter-stain. In a gram-positive bacteria, since it has a thick-layer of peptidoglycan, not all of the Crystal violet color will be rinsed out of the cell wall, so it will be blue or purple. Basically, it’s for helping classifying and determining what the bacteria is composed of. There are also stains for determining if a bacteria has endospores, negative stains (where the background is stained but not the cell), etc. It’s very useful tool to help identify bacteria without necessarily killing the cell.
Why do we use Gram stain?
That is the basis of Gram staining because Gram negative bacteria which stain pink with Gram stain are the most dangerous bacteria to humans and the bacteria which we have developed a good deal of our antibiotics to target. The reason we use the Gram stain is its rapidity. It is still used despite the fact that more modern approaches to ‘bar-code’ bacteria based on their DNA sequence will identify the bacterial species
What color is Gram positive?
Gram positive bacteria can be stained with saffranin. If you are doing a simple stain and saffranin is the only dye you are using, all of your cells will appear pink/red regardless of cell type.
Why is staining bacteria important?
Thanks for A2A. Staining the bacteria is not just to make it visible under the microscope. It has other important uses too. Usually bacteria are subjected to the gram staining technique before viewing under the microscope. This helps us to distinguish between gram negative and gram positive bacteria depending on the color retained after staining. I will not get into the details of gram staining as several better explanatory materials are available on the subject. In essence, staining bacteria is not just to increase their visibility under the microscope, but also to classify them, which is very important if one wishes to narrow down the species that a given bacterium belongs to.
What is Gram staining?
Bruckner What is Gram Staining? 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 ...
What is the difference between Gram positive and Gram negative?
That said, the Gram-positive/Gram-negative difference is very important in bacterial physiology. Gram-positive organisms have an inner membrane and then a thick outer cell wall of peptidoglycan (which reta ins crystal violet, making it nice and purple under the microscope!). Gram-negative organisms, on the other hand, have an inner membrane, a thin cell wall of peptidoglycan outside that (which loses its crystal v
What is the process of staining cells with crystal violet?
The process involves three steps: Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water.
Why do Gram positive bacteria stain 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.
How Does Gram Staining Work?
Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves three steps:
How to stain a cell?
How To- Staining Protocol and Concerns: 1 Make a slide of cell sample to be stained. Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times. 2 Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet. 3 Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. 4 Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. 5 Add the secondary stain, safranin, to the slide and incubate for 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope.
How long to incubate crystal violet?
Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet.
Does alcohol decolorize a cell?
However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. Add the secondary stain, safranin, to the slide and incubate for 1 minute.
Why do bacteria stain?
If methyene blue is used, some granules in the interior of the cells of some bacteria may appear more deeply stained than the rest of the cell, which is due to presence of different chemical substances.
How long does it take for a smear to stain?
Staining. Cover the smear with methylene blue and allow the dye to remain in the smear for approximately one minute (Staining time is not critical here; somewhere between 30 seconds to 2 minutes should give you an acceptable stain, the longer you leave the dye in it, the darker will be the stain).
What is a simple stain?
The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria. True to its name, the simple stain is a very simple staining procedure involving a single solution of stain.
How to clean a slide that has been stained?
Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.
How to get blue off of a slide?
Using distilled water wash bottle, gently wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide.
How to transfer bacteria to a slide?
Using a sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to a slide (clean grease free microscopic slide) or transfer an isolated colony from a culture plate to a slide with a water drop.
Does microbiology use simple staining?
Diagnostic microbiology laboratory generally does not perform simple staining methods. Differential staining such as Gram Staining and AFB Staining are commonly used to identify and differentiate the bacterial isolates. Simple staining can be useful in the following circumstances.
