The Bactérie S with negative Gram are highlighted by a technique of coloring called Coloration of Gram. The bacteria with gram negative appear pink then with the Microscope. The technique of coloring rests on the characteristics membrane S and of wall of the bacterium. The Coloration of Gram is a determining factor in the Taxonomie (classification) bacterial.
Principle of coloring of negative GramAfter fixing of the Bacterium S on a blade (two possible methods: fixing with the ethanol with 90° or by passage of the blade in the flame, known as smear ):
- the blade is plunged in a first dye: the purple of gentian . The purple one of gentian is coloring powerful, (Toxique and Cancérigène). It will cross the wall S and membrane S of the bacteria and to fix itself in their Cytoplasme S. Ainsi at this stage all the cells are coloured purple.
- the blade is then treated with the Lugol (diiode) in solution which is used as corrosive : it will reinforce purple gentian contained in the Cytoplasme of the bacteria.
- One drives out then the purple one with a solution of ethanol to 90°. This stage is crucial because makes it possible to discriminate the bacteria negative Gram bacteria positive Gram. The principle is the following: the bacteria negative Gram have a wall low in Peptidoglycane, component which because of its small quantity will make it possible alcohol to carry purple gentian, this one disappearing from the cytoplasm. The bacterium is thus faded (for more information, to see the characteristic of negative the Gram bacteria ).
the blade, after being rinsed, is briefly plunged in a second dye, the fuchsine . This dye crosses all the walls and membranes and will color pink all the bacteria which were faded during the preceding stage; the bacteria positive Gram being coloured purple (darker) will not appear pink (more clearly).
The blade is then recovered, rinsed and dried, then observed with the optical Microscope generally with the objective x100 + oil with immersion. The bacteria negative Gram will appear pink. The observation of their color is generally accompanied by structural descriptions (e.g. bacillus or hull).
Characteristics of negative the Gram bacteria
Description of the envelope
The bacteria negative Gram have a structure which is organized in three great parts: (of outside towards the interior)
- the external membrane = the wall
- space periplasmic and Peptidoglycane
- the plasmic membrane
The external membrane is in direct contact with the external medium. It mainly made up of Phospholipide S is organized into double-layered (part Hydrophile outside and left Lipophile inside) but contains also many intrinsic proteins, in particular the proteins of transport called Porine S. Beaucoup of negative Gram (e.g. Salmonella ) have also a compound nonprotidic called Lipopolysaccharide or LPS; this Immunogenic compound little , whose capacity Pathogène (lipid A) is included in the external membrane, is activated at the time of the lysis of the bacterium. The external membrane is connected to the Peptidoglycane by the Lipoprotéine of Braun.
Space periplasmic is a storage space of Enzyme S, of Nutriment S… It has many of other functions, in particular in certain stages of the Synthèse of proteins and in the Métabolisme. It is located between the external membrane and the plasmic membrane and it contains a layer of Peptidoglycane S. The layer of peptidoglycane (also called muréine) is relatively thin at the negative Gram contrary to the positive Gram.
The plasmic membrane is similar to the external membrane (except the LPS). It has porous proteins leading in space periplasmic (Synthèse of protein). The plasmic membrane contains many other proteinic complexes of vital importance for the bacterium (as ATP synthase ) which have paramount roles in the bacterial metabolism.
Examples of negative Gram bacteria
- Family of the Enterobacteriaceae :
- Family of the Vibrionaceae :
- Family of the Pseudomonadaceae :
- Kind Pseudomonas
- Family of Neisseriaceae
- Species Neisseria meningitidis (responsible for the bacterial Meningitis)
the Mycobactérie S: a noncolourable exception because by the method of Gram bus acido-alcoolo-resistant.
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