The Gram stainThe Gram stain

Thin smear/heat fix

Gram stain:a. Flood slide with crystal violet and

let stain for 1 minute. 

b. Drain off crystal violet and rinse off with distilled water; flood slide

with Gram's iodine for 1 minute. 

c. Rinse off Gram's iodine with distilled water.

 

d. Hold the slide on an angle (preferably with

a clothes pin) and drop 95% ethyl alcohol onto it until the alcohol leaving the slide no

longer has a purple tint; be sure to drop the

alcohol onto the upper portion of the slide so that the smears are subjected to uniform

decolorization. Be careful not to "decolorize" dye from the clothes

pin!! 

e. Rinse with distilled water and flood the

slide with safranin and let stain for 2-3 minutes. 

f. Rinse with distilled water and blot dry with

bibulous paper.

Gram positive

Gramnegative

• Thick peptidoglycan

• 90% peptidoglycan• Teichoic acids• 1 layer• Not many

polysaccharides• In acid-fast cells,

contains mycolic acid

Gram-positive cell walls Gram-negative cell walls

• Thin peptidoglycan• 5-10% peptidoglycan• No teichoic acids• 3 layers• Outer membrane has

lipids, polysaccharides• No acid- fast cells

(mycolic acid)

The characteristic compound found in all true bacterial cell walls is peptidoglycan. The amount of PPG is among one of the differences between the GP and GN cell walls.

Examples:

• Examples of gram-negative bacteria:Spirochetes (spiral-shaped) - causes syphilis, lyme disease Neisseria (cocci) - causes meningococcus, gonorrhea – Our Favorite: E. coli!!

• Six common gram-positive bacteria that infect humans and their shapes follow:Streptococcus (cocci)Staphylococcus (cocci) Bacillus (bacilli, protective spore) - causes anthrax and gastroenteritis Clostridium (bacilli, protective spore) - causes botulism, tetanus, gas gangrene, and pseudomembranous colitis Corynebacterium (bacilli, no protective spore) - causes diphtheria Listeria (bacilli, no protective spore) - causes meningitis