Gram positive bacteria have cell walls composed of thick layers of peptidoglycan. Gram positive cells stain purple when subjected to a Gram stain procedure. Gram negative bacteria have cell walls with a thin layer of peptidoglycan.

Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives.

The staining method uses crystal violet dye, which is retained by the thick peptidoglycan cell wall found in gram-positive organisms. This reaction gives gram-positive organisms a blue color when viewed under a microscope.

The cell walls of gram-positive bacteria are made up of a thick, mesh-like layer of peptidoglycan. Gram-negative bacteria have only a thin layer of peptidoglycan in their cell walls, but they also have an outer membrane containing lipopolysaccharides.

Because of this difference, gram-negative bacteria are harder to kill. This means gram-positive and gram-negative bacteria require different treatments. Though gram-negative bacteria are harder to destroy, gram-positive bacteria can still cause problems. Many species result in disease and require specific antibiotics.

Escherichia coli (E. coli) is a Gram-negative, rod-shaped, facultative anaerobic bacterium.

E. coli stains Gram-negative because its cell wall is composed of a thin peptidoglycan layer and an outer membrane. During the staining process, E. coli picks up the color of the counterstain safranin and stains pink.

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.

Various methods exist to detect E. coli, amongst them are PCR, gold nanoparticles for a visual colour change confirmation and fluorescent labelled enzymes.

E. coli are facultative anaerobic, Gram-negative bacilli that will ferment lactose to produce hydrogen sulfide.

Glucose Broth with Durham Tubes This is a test commonly used when trying to identify Gram-negative enteric bacteria, all of which are glucose fermenters but only some of which produce gas. Escherichia coli is capable of fermenting glucose as are Proteus mirabilis (far right) and Shigella dysenteriae (far left).

Traditionally, the colony morphology of Escherichia coli is identified as either a rough or a smooth form. The two forms are readily distinguished, as the colonies of the former are rough, flat, and irregular and colonies of the latter are smooth, high, and circular.

Choose your type of bacterial growth media:

LB Broth and LB Agar	Most referenced bacterial media for growth and maintenance of recombinant E. coli strains
M9 Minimal	Commonly used bacterial media for cultivation and maintenance of E. coli strains.

Escherichia coli are typically Gram-negative, rod shaped (2.0–6.0 μm in length and 1.1–1.5 μm wide bacilli) bacteria with rounded ends. The actual shape of these bacteria does, however, vary from spherical (cocci) cells through to elongated or filamentous rods.

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.

Exposing gram negative cells to the decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. This allows the cells to subsequently be stained with safranin.

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 …

The thickness of the smear used in the Gram stain will affect the result of the stain. The step that is most crucial in effecting the outcome of the stain is the decolorizing step.

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. Therefore, when the decolorizer is applied to the smear, the crystal violet will be retained if the bacteria is gram-positive and the bacteria will appear blue or purple.

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.

Disadvantages: Some bacteria are Gram stain variable (positive or negative results) Some bacteria are resistant to Gram stain (i.e. acid-fast bacteria) False results may occur if over-decolorized.

The safranin is also used as a counter-stain in Gram’s staining. In Gram’s staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, the gram-negative bacteria can be easily distinguished from gram-positive bacteria.

Safranin, another positively charged basic dye, adheres to the cell membrane.

A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells.

Often the first test performed, gram staining involves the use of crystal violet or methylene blue as the primary color. With the dissolution of the lipid layer, gram negatives lose the primary stain.

There are three broad categories of biological stains:

• General or Routine Stains: Used to differentiate between the nucleus and the cytoplasm.
• Special Stains: These are used to demonstrate specific elements within the tissue, such as connective tissue, muscle, carbohydrates, lipids, pigments, and nerve tissue.

Pairs of cocci are called diplococci; rows or chains of such cells are called streptococci; grapelike clusters of cells, staphylococci; packets of eight or more cells, sarcinae; and groups of four cells in a square arrangement, tetrads.

METHYLENE BLUE (METH uh leen bloo) is used to treat methemoglobinemia. This is a condition in which the blood loses its ability to carry oxygen through the body.

SIDE EFFECTS: Nausea, stomach upset, diarrhea, vomiting, or bladder irritation may occur. If any of these effects continue or become bothersome, tell your doctor. This medication may cause your urine or stool to turn green-blue. This effect is harmless and will disappear when the medication is stopped.