Flood the smear with crystal violet, 1 minute. (As the instructor will demonstrate.) Allow the smear to air dry. Use another slide to spread the smear like a blood smear. Use a loop to mix a drop of water, a drop of india ink and a small amount of Klebsiella pneumoniae together at the end of a slide.ī. As a generalphenomenon, organisms with capsules tend to be more virulentpresumably because of their resistance to phagocytosis and killing.Streptococcus pneumoniae exists in a smooth form (encapsulated) and arough form (non-encapsulated). When this material begins to accumulate on the teethit is referred to as dental plaque. For instance, the oralstreptococci produce a glucan based EPS which helps them to attach tothe teeth. This structure helps the bacterial cell to ATTACH TO SURFACES andto AVOID BEING PHAGOCYTOSED. Our procedure, the Gin's Method, uses india ink to colorthe background and crystal violet to stain the bacterial cell'body' That is, we use a stain whichstains the background against which the uncolored capsule can beseen. For this reason we use aNEGATIVE STAIN to visualize them. The polymers which make up the capsule tend to be uncharged and assuch they are not easily stained. Also, heatingmay cause the bacterial cell to shrink resulting in a clear zonearound the cell - which may cause cells which don't have capsules toappear as if they do. Capsular material is very moist (slimy)and any heating will cause it to shrink - it is for this reason thatwe will not heat fix the slide before staining. Most bacterialcapsules are composed of polysaccharide however some genera producepolypeptide capsules. This viscous surfacelayer is also known as the SLIME LAYER, the GLYCOCALYX or theEXTRACELLULAR POLYMERIC SUBSTANCE (EPS). Gram (-) = the coliforms: ( Escherichia, Klebsiella, Serratia), the enteric pathogens: Salmonella, Shigella, Campylobacter The gram morphology of some commonbacteria: Gram (+) = Staphylococcus, Streptococcus, Bacillus, Clostridium Gram (+) cells are more sensitive to lysozyme - a peptidoglycan digesting enzyme. Gram (+) cells are generally more sensitive to those antibiotics which interfere with cell wall biochemistry like penicillin, vancomycin and cephalosporin.Ĥ. Gram (-) cells have Lipopolysaccharide (Endotoxin) in the outer membrane of their cell walls.ģ. Gram (+) cells have thicker cell walls - more peptidoglycan and teichoic acid.Ģ. al.) and I'd like to stress the following points: 1. These are summarized in Table4.1(Tortora et. There are fundamental biological differencesbetween gram (+) and gram (-) cells. On the other hand, gram (-) cells never become gram (+) if the stain procedure is done correctly. Therefore any time the cell wall is damaged the gram stain characteristics of gram positive cells will change. Impure or mixed culture It is believed that the gram staining characteristics of an organism is a function of its cell wall. However this is not anabsolute phenomenon! Sometimes gram (+) cultures will appear gram(-) or GRAM VARIABLE (a mixture of gram (+) and gram (-) cells). GRAM (+) organisms hold onto the crystal violet-iodine complexmore tightly than the GRAM (-) bacteria do. Heat fix and perform the gram stain on this sample.ģ.) Observe all of your specimens under oil immersion and record cell morphology and arrangement and gram reaction. Mix the sample with a loopful of water on a microscope slide and allow the specimen to air dry.

) Use a sterile applicator stick to obtain a sample of the 'crud' on your teeth at the gum line. 1.)Perform the gram stain on smears made form the following organisms:Ģ.

Flood the smear with SAFRANIN, 1 minute, then wash with water. Flood the smear with ETHANOL-ACETONE, quickly, then wash with water. Flood the smear with IODINE, 1 minute, then wash with water. Flood the smear with CRYSTAL VIOLET, 1 minute, then wash with water. It divides most of the EUBACTERIA into two largegroups: GRAM POSITIVE bacteria and GRAM NEGATIVE bacteria. It requires a PRIMARY STAIN and aCOUNTERSTAIN. Also look up all of these stains in your Leboffe and Pierce book.) al., Chapter 3 and about gram positive and negative cells and their cell walls in Chapter 4, especially Table 4.1. (Read about staining techniques in Tortora et. Gram Stain, Capsule Stain and Endospore Stain

Gram positive vs gram negative colors