Microbiology Notes | Biochemistry,Immunolgy,Cell biology

The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. A  capsule  is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The  capsule  differs from the  slime layer  that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain employs an acidic stain and a basic stain to detect capsule production. Principle of Capsule Staining Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained. Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with  india ink or nigrosin or congo red . India ink is difficult to obtain nowadays; however,

Aim: To isolate the genomic DNA from E .coli DH5α cells. Principle: The isolation and purification of DNA from cells is one of the most common procedures in contemporary molecular biology and embodies a transition from cell biology to the molecular biology  (from in vivo to  in vitro) . The isolation of DNA from bacteria is a relatively simple process. The organism to be used should be grown in a favorable medium at an optimal temperature, and should be harvested in late log to early stationary phase for maximum yield.The genomic DNA isolation needs to separate total DNA from RNA, protein, lipid, etc. Initially the cell membranes must be disrupted in order to release the DNA in the extraction buffer.  SDS (sodium dodecyl sulphate)  is used to disrupt the cell membrane. Once cell is disrupted, the endogenous nucleases tend to cause extensive hydrolysis. Nucleases apparently present on human fingertips are notorious for causing spurious degradation of nucleic acids during purific

Principle: When bacteria are lysed under alkaline conditions both DNA and proteins are  precipitated. After the addition of acetate-containing neutralization buffer the large and  less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA  plasmids can renature and stay in solution. In prokaryotes, plasmid is double stranded, circular, and is found in the cytoplasm. The cell membranes must be disrupted in order to release the plasmid in the extraction buffer. Solution 1 contains glucose, Tris, and EDTA. Glucose provides osmotic shock leading to the disruption of cell membrane, Tris is a buffering agent used to maintain a constant pH8. Plasmid can be protected from endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a necessary cofactor for most nucleases. Solution II contains NaOH and SDS and this alkaline solution is used to disrupt the cell membrane and NaOH also denatures the DNA into single strands. Solution III