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...

  ACID-FAT STAINING INTRODUCTION: It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’scarbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining. AIM: To differentiate the Acid fast and Non -Acid fast bacteria by the acid fast staining technique. PRINCIPLE: When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsi...

                                                          GRAM STAINING INTRODUCTION: Gram Staining is the common, important, and most used differential staining techniques in microbiology,which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms. AIM: To observe the morphology and the arrangement of the given bacterial smear by the gram Staining technique. PRINCIPLE : When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content ...