How to Perform a Gram Stain

IntroductionChristian Gram was a bacteriologist who developed the gram

stain. He invented the gram stain while working in a morgue in Berlin in 1884 with Carl Friedlander. His original purpose was to find a technique that would make bacteria more visible in the stained areas of the lung tissue he was studying. During his work, he found that Typhus bacillus, the bacteria that causes typhoid fever, did not retain this particular stain. This led to the discovery that different types of bacteria have different structural components. This laboratory technique is used today to differentiate between the two large groups of bacterial species, gram-negative and gram-positive bacteria, based on their cell wall structure.

Gram-positive bacteria are identified by their thick, multilayered peptidoglycan layer. They do not contain a periplasmic space or barely any lipopolysaccharide (LPS) content. Also, gram-positive bacteria do not contain lipids or an outer membrane. When a gram stain is performed, gram-positive bacteria will retain the crystal violet dye and stain dark violet or purple. This is because the outer layer of peptidoglycan absorbs the stain.

Gram-negative bacteria have a single layered peptidoglycan layer. They do contain a periplasmic space and an outer membrane. They have a high lipid and LPS content as well. When a gram stain is performed, gram-negative bacteria will stain red or pink. This is because alcohol in the decolorization step interacts with the lipids of the cell membrane and the outer lipopolysaccharide membrane is washed away. Then the peptidoglycan layer is exposed, which will absorb the red safranin stain.

Below are a list of materials and step-by-step instructions with images on how to perform a gram stain. There is also a glossary at the end of the instructions for those who may be unfamiliar with microbiology and gram staining terms.

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Materials

o Bunsen Burnero Strikero Glass slideo Sterile loopo Microscopeo Distilled watero Crystal Violeto Iodineo Safranino Distilled Watero Bacteria sample

Steps

1. Light the Bunsen burner with the striker and hold the sterile loop over top for 30 seconds. Use this loop to obtain a small sample of bacteria.

2. Drop 1-2 drops of distilled wateronto the glass slide. Add the bacteria sample to the water and create a thin film. Let the slide sit to completely air-dry.

It is important to hold the loop over the Bunsen burner to make sure the loop is sterile without any viable organisms on it. This is to avoid contamination.

It is important to let the slide completely dry so the water molecules do not interfere with the stains and the appearance of the cells under the microscope.

3. Heat fix the slide by slowly passing it 3-4 times through the top of the flame of the Bunsen burner.

4. Add drops of the crystal violet stain to cover the whole bacteria sample on the slide. Let the stain sit for 30 seconds.

5. Rinse of the stain with distilled water.

6. Add drops of the Iodine stain to cover the

WARNING: Do not keep the slide over the flame for too long. If the bacteria are exposed to too much heat they will become denatured and will not be able to properly absorb the stains for accurate identification.

The crystal violet stain will dissociate into two ions CV+ and Cl-. The ions penetrate through the cell wall of both types of bacteria. The CV+ ion will interact with the negatively charged components of the bacteria staining it purple.

WARNING: Do not over rinse because the bacteria could be removed from the slide.

Iodine is in the form of negatively charged ions, which will interact with the CV+ ions. A complex of

whole bacteria sample on the slide. Let the stain sit for 30 seconds then gently rinse the stain off the slide with distilled water.

7. Decolorize the sample by holding the slide at an angle and gently running the alcohol across it. Stop immediately when the run off becomes clear or after 20 seconds then rinse with distilled water.

8. Add drops of Safranin, the counterstain, to cover the whole bacteria sample on the slide. Let the stain sit for 30 seconds.

Iodine is in the form of negatively charged ions, which will interact with the CV+ ions. A complex of

WARNING: Do not over decolorize because this will remove the stain from both types of bacteria. Alcohol interacts with the lipids on the bacteria’s cell membrane. Therefore, gram-negative bacteria will loose their outer cell membrane, which will cause the CV-I complex to wash off. Gram-positive bacteria will become dehydrated and trap the CV-I complex its cell wall.

Safranin is used to stain gram-negative bacteria, a pink-red color, to be viewed under the microscope and be differentiated from gram-positive bacteria.

9. Rinse off the stain with distilled water and let the slide

air-dry.

10. Examine the slide under a microscope to identify the type of bacteria present.

ConclusionOverall, gram staining is a very

simple and efficient way to differentiate between gram-positive and gram-negative bacteria. This is a widely used technique in the medical field performed on body fluids or on biopsies of individuals who are suspected of having a bacterial infection. A diagnosis can be made more quickly from a gram stain than by creating a cell culture. Also, the gram stain results are helpful when choosing the correct antibiotic for treatment because certain antibiotics only work against certain types of bacteria.

Glossary

o Cell membrane: A biological barrier that separates the outside environment from the inside of the cells. It controls the movement of certain molecules, ions, and substances in and out of the cell.

o Cell wall: A rigid and tough layer that surrounds the cell membrane and provides the cell with protection and structural support.

o Denature: A change in a molecule’s structure because of extreme environmental conditions.

o Distilled water: Water in which all impurities are removed by boiling the water and condensing the steam into a clean container.

o Ions: A difference in the number of electrons and protons give an atom a net positive or net negative charge.

Gram-postive bacteria stain purple. Gram-negative bacteria stain a pink-red color.

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o Lipids: Biological molecules which store energy and are components of a cell membrane. Examples are fats, sterols, wax and vitamins.

o Lipopolysaccharide: A lipid and polysaccharide (carbohydrate) linked together found on the outer membrane of gram-negative bacteria.

o Peptidoglycan: The cell wall of bacteria composed of sugars and amino acids.

o Periplasmic space: The space in between two selectively permeable membranes.

o Viable: A living organism or an organism that is functional.

Gram -negative bacteria under a microscope.

Gram -positive bacteria under a microscope.

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