DEPARTMENT OF ZOOLOGY (Star Science Department, Conferred by UGC)

Transcription

1 DEPARTMENT OF ZOOLOGY (Star Science Department, Conferred by UGC) Hands-on experience via Lab Sessions (State of Art Labs with latest Machinery/ Equipment/ Software) DEV SAMAJ COLLEGE FOR WOMEN Re-accredated A Grade College (2 nd cycle) with 3.75/4 CGPA (Highest in India, ) by NAAC Banglore College of Excellence Status Award by the UGC, New DelhI Ferozpur City, Punjab, India ,

2 Dept. of Zoology Star Science Dept Conferred by UGC New Delhi

3 OBJECTIVE OF THE LAB Enhance Critical Thinking Learning Skills. Provide a Broad Overview and Application of Advanced Technology Develop an understanding of the Physiological and Biochemical Experiments and its relation with Research and development Explore the Physical, Morphological, and Physiological Characteristics of animals in Natural diversity

4 LAB FACILITIES 1. Cell Biology & Histology Lab 2. Biochemistry Lab 3. Molecular Genetic lab 4. Physiology Lab 5. Invertebrate lab 6. Vertebrates lab

5 RESOURCES IN THE LAB Cell Biology & Histology Lab : Computer attached High Magnification Light Microscope, Light Microscopes, Microtome, Hot Plates, Oven, Camera Lucida, Laminar flow. Biochemistry Lab: Double distillation unit of water, UV/VIS Spectrophotometer, Colorimeter, Centrifuge, Homogenizer, Autoclave, three digit Weighing Balance, Paper & Thin Layer Chromatography Molecular Genetic lab: Thermal Cycler, Deep Freezer, Electrophoreses Unit, UV Transilluminator, Cold Centrifuge.

6 Continue Physiology Lab: Animals storage box, Metabolic study cage, Sphygmomanometer, stethoscope, Hb apparatus, water bath Invertebrate lab- Specimen of all Phyla, Staining kits, Insect collection Net, Insect Collection Box, Permanent stained slides Vertebrates lab: Specimen of all Vertebrate classes, Stuffed animals, preserved animals, desiccators, dissection tray

7 MICROTOMY Microtomy is the means by which tissues can be sectioned and attached to the surface so that examination by microscopy can take place. The basic instrument used in microtomy is a microtome into which cutting tool is clamped.

8 General Processes Fixation Staining Dehydration Cutting Clearing Embedding

9 Steps in the processing of tissues Fixation preservation of tissues in its original condition. Dehydration removal of water from tissues. Embedding infiltration of paraffin wax. Microtomy preparing thin slices of tissues. Mounting arranging tissues on slides. Staining colouring of tissues.

10 This is the process by which the constituents of cells and tissue are fixed in a physical and partly also in a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds, call fixatives.

11 Tissues are dehydrated by using increasing strength of alcohol; e.g. 50%, 70%, 90% and 100%. The duration for which tissues are kept in each strength of alcohol depends upon the size of tissue, fixative used and type of tissue; e.g. after fixation in aqueous fixative delicate tissue need to be dehydrated slowly starting in 50% ethyl alcohol directly whereas most tissue specimens may be put into 70% alcohol.

12 Impregnated tissues are placed in a mould with their labels and then fresh melted wax is poured in it and allowed to settle and solidify. Once the block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly. After the block has completely cooled it is cut into individual blocks and each is trimmed.

13 Set the wax block on the Hub of Microtome and cut the section with the help of Microtome. Spread the section on glass slides

14 Staining Without staining, the tissue section would remain translucent and we would still have difficulty identifying the relevant cell types and features. Staining using colored dyes provides a mechanism for introducing contrast.

15 Staining Cell components such as nucleic acids with a net negative charge (anionic) stain more readily with basic dyes and are termed basophilic cationic components, such as proteins with many ionized amino groups, have affinity for acidic dyes and are termed acidophilic. Examples of basic dyes are toluidine blue, alcian blue, and methylene blue. Hematoxylin, staining behaves like a basic dye basophilic tissue components.

16 Tissue Staining Basophilic Basophilic Stain with basic dye [dye+cl-] Toluidine blue, methylene blue, hematoxylin, alcian blue Nucleic acids, some cytoplasmic components (rrna and rer), glycosaminoglycans and acidic glycoproteins Acidophilic Stain with acidic dye [Na+dye-] Orange G, eosin, acid fuschin Mitochondria, cytoplasm, secretory granules, ECM proteins

17 H&E Stain (Eosin is the counterstain to Hematoxylin) Hematoxylin produces a dark blue or purple color, staining DNA in the cell nucleus and other acidic structures (such as RNA-rich portions of the cytoplasm and the matrix of cartilage). In contrast, Eosin stains other cytoplasmic components and collagen. In many staining procedures certain structures such as nuclei become visible, but other parts of cells remain color free.

18 Study of Histology Students Electronic light microscope Study through computer associated light microscope

19 Polymerase Chain Reaction PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence

20 Critical Steps in PCR Sample Preparation Target Selection Primer Selection

21 Reaction Components DNA template Primers Enzyme dntps Mg 2+ buffers

22 Step Cycle Programme Denaturation Denature at 94⁰ C Extension at 72⁰ C Anneal at 55⁰ C Trade off between denaturing DNA and not denaturing Taq Polymerase Taq half-life 40min at 95, 10min at C Annealing Trade off between efficient annealing and specificity 2-5 below Tm Extension Temperature optimum for Taq Polymerase 72

23 PCR Protocol Sample DNA Extraction Addition of premix reagent Automated Amplification Detection Mutation Detection SNP Quantification Genotyping

24 Merits of PCR Generates and Modifies DNA Much faster Specificity Detection of drug resistance genes Simplicity Biodiversity studies ( e.g. evolution studies) Genome mapping and gene function determination Forensic (DNA fingerprinting) Applications

25 Quantitative Proteins Estimation

26 Specificity and Sensitivity of the Method Sensitivity of an assay is a measure of how little of the analyte the method can detect Specificity of an assay relates to how good the assay is in discriminating between the requested analyte and interfering substances

27 There are many Spectrophotometric method for determining the protein concentration Bicinchoninic acid method biuret Lowry Bradford Spectrometric (A280/A260) There are a wide variety of protein assays available. but each assay has its own advantages and limitations The factors that you should consider in choosing a method: Sensitivity The presence of interfering substance Time available of the assay

28 Lowry Method Is based on two chemical reactions: The first reaction is the reduction of copper ions under alkaline conditions, which forms a complex with peptide bonds. The second is the reduction of Folin-Ciocalteu reagent by the copper-peptide bond complex, which subsequently causes a color change of the solution into blue with an absorption in the range of 650 to 750 nm detectable with a spectrophotometer.

29 Standard Curve There is a linear relationship between absorbance and concentration. The amount of protein in the sample can be estimated using a standard curve of a selected standard protein solution such as (casein ). Standard curve are most commonly used to determine the concentration of a substance, using serial dilution of solutions of known concentrations(standard solutions) Concentration of unknown sample

30 Set up 7 tubes as follows: Tube Casein Standard Unknown A B C D E F Add 3ml reagent C to all tubes. Mix and let stand at room temperature for 15 min. Add 0.5 ml of Folin-Ciocalteu reagent. (Add this reagent to one tube at a time and immediately after adding it mix well). Let the tubes stand at room temperature for 45 min. 5. Read absorbance at 660 nm against the blank.

31 Glimpse of Biochemistry lab

32 Blood Group Detection

33 Basic Fundamental ABO blood group antigen present on red blood cells (Natural Antigens) IgM antibodies present in the serum Clumping take place with antigen antibody reaction

34 Common Lab Method- Slide Method Principle: When red cells are mixed with various reagent anti-seras (soluble antibody), agglutination will occur on the slides containing cells positive for (possessing the antigen) the corresponding antigen. No agglutination will occur when the red cells do not contain the corresponding antigen.

35 Reagents Anti-A antibodies Anti-B antibodies Anti-D Slides Sterilized Needles Alcohol Wooden applicator.

36 Procedure: 1. On one side of slide labeled anti-a place one drop of antibody A. 2. On the middle of slide labeled anti-b place one drop of antibody B. 3. On last side labeled anti-d place one drop of antibody D 4. Place one drop of cells in each antibody containing circle. 5. Carefully mix each solution with a separate applicator stick. 6. Tilt slowly for one minute, then observe for the agglutination.

37 Interpretation of the results Strong agglutination of RBCs in the presence of any ABO grouping reagent constitutes a positive result. A smooth suspension of RBCs at the end of 2 minutes is a negative result. Samples that give weak or doubtful reactions should be retested by Tube test ABO grouping

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40 VERTEBRATE LAB SPECMEN PHYLA/ CLASS UROCHORDATA PISCES AMPHIBIA REPTILIA AVES MAMMALIA

41 INVERTEBRATE LAB SPECIMEN PHYLUM PROTOZOA PORIFERA COELENTRATA PLATYHELMINTHES ASCHELMINTHES ANNELIDA ARTHROPODA MOLLUSCA ECHINODERMATA

42 RULE OF THE ZOOLOGY LAB Always wear overcoat while working in laboratory. it should be washed at least once a week. Keep the hair and loose garments under check. Always mop the bench with disinfectant such as 2% phenol before and after use. Be systematic and logical keep a faithful record of all the experiments and observations. Update it regularly and submit it for evacuation at the end of each exercise. Always wear gloves if there are cuts on hand or working with hazardous chemicals. Be familiar about the working of instrument prior to handling it independently keep all the laboratory equipments at their respective place after use. Don t remove any culture ir any other article from the laboratory.

43 Continue Dispose of infectious material, cultures and contaminated material carefully in container of disinfectant and check its infecting activity regularly. Dispose of all cultures after autoclaving and pipettes should be placed in disinfectant after use. Work either using laminar flow chamber or light the burner at least five minutes prior to making any inoculations and work near the burner. Always discard the disposable and use cotton, match s stick, paper pieces etc to trash can and never into the sink.

44 Output of the laboratory Work Familiar with Modern Tool & Technology Expert in Handling the Instrument Trained in Lab Experiment Able to Perform an Independent Research Project

45 Gratitude From the Dept. of Zoology