Gram dyeing method

staining Principle

Gram staining all bacteria can be divided into gram-positive bacteria (G + ) and Gram-negative bacteria ( G - ) two categories, is the most commonly used bacteriological staining identification. The reason for staining bacteria can be divided into G + bacteria and G - bacteria, is generally believed that because the Gram stain is based on special chemical components of bacterial cell walls were stained of.

and by mordant after initial transfection, the cells form a water-insoluble macromolecular crystal violet-iodine complex. Because of gram-positive bacteria cell wall thick peptidoglycan content and higher degree of crosslinking molecules which more closely, so that when eluted with ethanol, peptidoglycan cell dehydration due to significant contraction, coupled with its substantially containing lipid, it can not be dissolved in an ethanol slot walls, thus, crystal violet and iodine complex remains firmly retention in the cell wall, it presents purple. Gram-negative bacteria because of the thin wall, and the low content of cross-linked peptidoglycan loose, so that after the case of ethanol, peptidoglycan mesh hard to shrink and, coupled with its high lipid content, so after when the lipid was dissolved in ethanol , the large gap will appear in the cell wall, cell wall complex was easily eluted, thus by ethanol after bleaching, and colorless cells. Then again the red dye-stained, Gram-negative bacteria will get a new layer of color - red, while gram-positive bacteria is still on purple.

operation flow

Gram staining method generally comprises four steps First dye, mordant, bleaching, stained, and specific methods of operation are:

( 1) producers.

conventional smears taken seed culture was dried, fixed.

use of active growth of young cultures as Gram stain; smear should not be too thick, to avoid a false positive result of incomplete bleaching; fixing flame not overheating (hot not suitable to slide).

(2) First dye.

crystal violet was added dropwise (to just cover the appropriate pellicle film) staining 1-2min, washed with water.

(3) mordant.

to residues using iodine water washed, and treated with iodine cover about 1min, washing with water.

(4) bleaching.

residual water on a filter paper to suck the slide, inclined slides, on white background, with a dropper ethanol stream bleaching plus 95% ethanol until no purple effluent, immediately washed with water.

ethanol bleaching is the key to Gram staining operations: lack of bleaching, false-negative bacteria are stained positive bacteria; excessive bleaching, false-positive bacteria are dyed-negative bacteria. Thus bleaching time is generally 20-30s.

(5) counterstaining.

were stained with Safranin about 2min, washed with water.

(6) microscopic examination.

After drying, oil microscope. Cells were stained blue-purple is gram-positive bacteria, dyed red for gram-negative bacteria.

Common species

common gram-positive bacteria Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), Streptococcus pneumoniae, Bacillus anthracis, diphtheria, tetanus, etc. .

common gram-negative bacterium Shigella, Salmonella typhi, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Bordetella pertussis and Vibrio cholera and meningococcus.

main role

Gram staining significance lies in the identification of bacteria, many of the bacteria are divided into two categories, Gram-positive and Gram-negative bacteria.

in the treatment of most gram-positive bacteria are susceptible to penicillin (penicillin-insensitive Mycobacterium tuberculosis): Most pyogenic bacteria belong to gram-positive bacteria, so that they can produce exotoxin pathogenic to humans, you can choose penicillin family, such as ceftriaxone.

Gram-negative bacteria, most of the intestinal bacteria are mostly gram-negative bacteria, which produce endotoxins, rely on endotoxin make people sick. Gram-negative bacteria and is not sensitive to penicillin (but Neisseria meningitidis is meningococcal meningitis and gonorrhea sensitive to penicillin), may be selected fluoroquinolones such as norfloxacin, levofloxacin, also you can choose to macrolides such as clarithromycin, roxithromycin. So, the first to distinguish between pathogenic bacteria are gram-positive bacteria or negative bacteria, the antibiotic of choice in terms of great significance.

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