DR . R. MAHENDIRAN RAMASAMY

  REAL TIME PCR   AIM:  Demonstration of Real-Time PCR through experimental samples  INTRODUCTION:  Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation ofnucleic acids. It monitors the amplification of a targeted DNA molecule during the PCR (i.e.in real time), not at its end, as in conventional PCR. Real-time PCR is the technique ofcollecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. Using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a million-fold using sequence specific oligonucleotides, heat stable DNA polymerase, and thermal cycling. The PCR is the cyclic reaction based on the rapid change in temperature during each step. During PCR, our gene of interest is amplified as well as quantified. Real-time quantitative PCR is the reliable detection and measurement of products generated during each cycle of...

  I SOL A T I ON A N D SE P ARA T I ON OF C H R O M OSO MA L D N A FR OM B A C TE R I A   AI M : To iso l a te a nd s e p a r a te t h e c h r omosomal D N A f r o m ba c te r ia   P RI N C I P LE:   G e nom i c ( c h romosomal) D N A f rom e i t h e r b ac t e r ia, plant or a ni m a l ce ll c a n be iso l a ted b y l y sis m e thod. F or pl a nt ce ll or b ac t e ri a l c e l l , f i rst ce ll w a ll is me c h a nic a l l y disrupted. The ce ll w a ll is b r o k e n e n z y ma t ic a l l y using e n z y m e s l i ke l y so z y me. W h e n b a c te r ia a r e l y s e d und e r a lkaline c ondi t ions both D N A a nd p r oteins a re p rec ip i tat e d. A f ter the a ddi t i on of ace tate- c ontaining n e utr a l iz a t i on buf f e r the la r ge a nd le s s supe rc oi l e d c h r omos o mal D N A a nd p rot e ins p rec ip i tat e , but the small b ac te r ial D N A plasmids ca n r ...

  CAPSULE STAINING INTRODUCTION: 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. AIM : To prepare a smear of an encapsulated bacterium and stain it’s capsule and to visualize the capsule and to differentiate the capsule from the cell body. PRINCIPLE: 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, back...

  SPORE STAINING INTRODUCTION: In 1922, Dorner published a method for staining endospores. Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster The endospore stain is a differential stain which selectively stains bacterial endospores. The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers. AIM: To perform spore Staining technique to demonstrate the Spore in a culture. PRINCIPLE: Bacterial endospores are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions. The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state. In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is wate...