How do you prepare a Gram stain?


How do you prepare a Gram stain?

The performance of the Gram Stain on any sample requires four basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Gram’s Iodine), rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, counterstaining with …

What are the reagents needed for the Gram stain procedure?

Gram staining technique requires simultaneous use of chemical reagents for a fixed period followed by washing; Primary stain (crystal violet), Mordant (iodine), Decolorizer (ethanol or acid-alcohol) and Counter stain (safranin or dilute carbol-fuchsin).

What 4 reagents are used in the Gram stain?


  • Crystal violet (primary stain)
  • Iodine solution/Gram’s Iodine (mordant that fixes crystal violet to cell wall)
  • Decolorizer (e.g. ethanol)
  • Safranin (secondary stain)
  • Water (preferably in a squirt bottle)

How do you interpret gram stain results?

A Gram stain is colored purple. When the stain combines with bacteria in a sample, the bacteria will either stay purple or turn pink or red. If the bacteria stays purple, they are Gram-positive. If the bacteria turns pink or red, they are Gram-negative.

What is the importance of Gram staining?

The main benefit of a gram stain is that it helps your doctor learn if you have a bacterial infection, and it determines what type of bacteria are causing it. This can help your doctor determine an effective treatment plan.

What is the principle of Gram staining?

Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as gram-positive bacteria. The stain is retained because the cell wall of a gram-positive bacteria is not as permeable as the cell wall of a gram-negative bacteria.

What is Safranin used for in Gram staining?

Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endospore staining. It can also be used for the detection of cartilage, mucin and mast cell granules.

Is safranin basic or acidic?

Flagella Staining

Table 2. Simple Stains
Stain Type Specific Dyes
Basic stains Methylene blue, crystal violet, malachite green, basic fuschsin, carbolfuschsin, safranin
Acidic stains Eosine, acid fuchsin, rose bengal, Congo red
Negative stains India ink, nigrosine

What is crystal violet used for in Gram staining?

Methyl violet 10B is the active ingredient in a Gram stain. In the Gram staining method, crystal violet is used to differentiate between Gram Positive and Gram Negative bacteria.

How does Gram staining help identify bacteria?

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. They are stained pink or red by the counterstain, commonly safranin or fuchsine.

Which bacteria Cannot be Gram stained?

Atypical bacteria are bacteria that do not color with gram-staining but rather remain colorless: they are neither Gram-positive nor Gram-negative. These include the Chlamydiaceae, Legionella and the Mycoplasmataceae (including mycoplasma and ureaplasma); the Rickettsiaceae are also often considered atypical.

How do you Gram stain bacteria?

There are six basic steps:

  1. Apply a smear of bacteria on to a slide.
  2. Add about 5 drops of Hucker’s Crystal Violet to the culture.
  3. Add about 5 drops of iodine solution to the culture.
  4. Tilt slide and decolorize with solvent (acetone-alcohol solution) until purple color stops running.
  5. Add about 5 drops of Safranine O.

Does Gram stain work for all bacteria?

Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet.

Why Decolorizing is the most critical step in Gram stain?

The Gram stain is the most important staining procedure in microbiology. This layer makes up 60-90% of the gram positive cell wall. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell.

What can go wrong in Gram staining?

Several factors may affect the results of Gram staining: If the smear is too thick, proper decolorizing will not be possible. If the smear is overheated during heat fixing, the cell walls will rupture. Cells from old cultures may stain Gram negative even if the bacteria are Gram positive.

What happens if you forgot the Decolorizer in a Gram stain?

95% ethanol, because it removes the primary stain (crystal violet) from Gram-negative cells. If missed, then the bacteria would remain purple and give a false positive result.

Why is Gram stain differential?

The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process. Gram negative cell walls have an outer membrane (also called the envelope) that dissolves during the alcohol wash.

What does alcohol do to Gram negative bacteria?

In the case of Gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the Gram-negative cell wall (and may also damage the cytoplasmic membrane to which the peptidoglycan is attached).

What kills gram negative?

Fourth-generation cephalosporins such as cefepime, extended-spectrum β-lactamase inhibitor penicillins (piperacillin/tazobactam, ticarcillin/clavulanate) and most importantly the carbapenems (imipenem/cilastatin, meropenem, ertapenem) provide important tools in killing Gram-negative infections.

How does age affect gram reaction?

How does the age of a culture affect the gram stain reaction? a. Old cultures can convert to gram-variable or gram-negative giving erroneous results. If done too long, gram-positive can look gram-negative and if done too short, gram-negative and look gram-positive.

How does ethyl alcohol kill bacteria?

Alcohol kills germs through a simple chemical process known as denaturation. Denaturation occurs when alcohol molecules bond with the fat membrane encasing a virus or bacteria cell. As the fat membrane is broken down, the inside of the cell — including all of its critical components — becomes exposed.

Is denatured alcohol a disinfectant?

Denatured Ethanol (DE) or Isopropanol (IPA)? The effectiveness of alcohol as an anti-bacterial or anti-fungal disinfectant increase as the molecular weight increases. Denatured Ethanol is considered more effective as a virucidal disinfectant, as isopropanol is not effective against non-enveloped viruses.

Which is better isopropyl or ethyl alcohol?

The World Health Organization suggests that 70% ethyl alcohol is superior to isopropyl alcohol against the influenza virus, however, both provide adequate germicidal properties. It’s widely recognized that ethanol in this concentration is effective against many viruses and bacteria.

Why is 70 alcohol preferred to 95 alcohol as an antiseptic?

70% IPA solutions penetrate the cell wall more completely which permeates the entire cell, coagulates all proteins, and therefore the microorganism dies. Extra water content slows evaporation, therefore increasing surface contact time and enhancing effectiveness.

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