Gram Staining Technique

Background

Gram staining is the most essential and universally used staining technique in bacteriology laboratory, was firstly introduced by Danish bacteriologist Hans Cristian Gram in 1884. The purpose of this experiment is to determine the shape and Gram stain of the bacteria under a microscope, which have consistent differences in their cell wall. The reason for staining bacteria is because of very fact that almost all bacteria are transparent and cannot be seen through the microscope. The Gram stain is a type of differential stain that allows a (micro) biologist to spot the variations between organisms and/or differences among identical organism. Gram staining, bacteria requires the employment of aseptic technique to ensure the sterility of the experiment. To be better understand and recognize, the technique used to differentiate two large group of bacteria; gram positive (purple-colored) and gram negative (pink-colored). Gram-positive bacteria is characterized by having a thick layer of peptidoglycan. It makes up 90% of the cell wall of the bacteria. When stained, this type of bacteria will become purple. And another Gram-negative bacteria has thin layer of peptidoglycan, and is called a slime layer. In contrast to Gram-positive, it only makes up 10% of a cell wall, but has a high lipid content. When stained with counter stain safranin, gram-negative bacteria will be stained pink.

"Details of the chemical mechanism of the Gram stain, In aqueous solutions crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both gram-positive and gram negative cells. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple. When added, iodine interacts with CV+ to form large CVI complexes within the cytoplasm and outer layers of the cell. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells .With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell. After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added. At the completion of the Gram stain the gram-positive cell is purple and the gram-negative cell is pink to red." (;asm)

 

 

Methods:

Aseptic technique was used throughout the experiment- make general smears of a slide containing sample (unknown bacteria) and let slide completely dry then heat fix by (holding with side holder) running it over the Bunsen burner flame approximately 3 times and let cool.

Step 1: once the slide has cooled, apply crystal violet dye; primary stain for approximately 30 seconds the rinse with H₂O.

Step 2: apply the Gram’s iodine; mordant for one minute and rinse with H₂O again.

Step 3: Decolorize the smear with alcohol (ethyl alcohol or acetone or 1:1 ethanol-acetone); decolorizer wash for no more than 15 seconds.

Step 4: Counterstain with safranin; secondary stain for 45 seconds to 1.5 minutes then rinse with H₂O.

Step 5: Blot dry with bibulous paper.

Step 6: Examine with microscope including oil immersion.