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

DEV SAMAJ COLLEGE FOR WOMENRe-accredated ‘A’ Grade College (2nd cycle) with 3.75/4 CGPA (Highest in

India,2013-14) by NAAC Banglore

College of Excellence Status Award by the UGC, New DelhIFerozpur City, Punjab, India-152002,

Hands-on experience via Lab Sessions (State of Art Labs with latest Machinery/ Equipment/ Software)

Dept. of ZoologyStar Science Dept Conferred by UGC New Delhi

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

1. Cell Biology & Histology Lab

2. Biochemistry Lab

3. Molecular Genetic lab

4. Physiology Lab

5. Invertebrate lab

6. Vertebrates lab

LAB FACILITIES

Cell Biology & Histology Lab : Computer attached HighMagnification Light Microscope, Light Microscopes,Microtome, Hot Plates, Oven, Camera Lucida, Laminar flow.

Biochemistry Lab: Double distillation unit of water, UV/VISSpectrophotometer, Colorimeter, Centrifuge, Homogenizer,Autoclave, three digit Weighing Balance, Paper & Thin LayerChromatography

Molecular Genetic lab: Thermal Cycler, Deep Freezer,Electrophoreses Unit, UV Transilluminator, Cold Centrifuge.

RESOURCES IN THE LAB

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Physiology Lab: Animals storage box, Metabolic study cage,Sphygmomanometer, stethoscope, Hb apparatus, waterbath

Invertebrate lab- Specimen of all Phyla, Staining kits, Insectcollection Net, Insect Collection Box, Permanent stainedslides

Vertebrates lab: Specimen of all Vertebrate classes, Stuffedanimals, preserved animals, desiccators, dissection tray

MICROTOMY

Microtomy is the means bywhich tissues can be sectionedand attached to the surface sothat examination bymicroscopy can take place.

The basic instrument used inmicrotomy is a microtome intowhich cutting tool is clamped.

General Processes

Fixation

Dehydration

Clearing

Embedding

Cutting

Staining

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.

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

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

Impregnated tissues are placed in a mould withtheir labels and then fresh melted wax is poured init and allowed to settle and solidify.

Once the block has cooled sufficiently to form asurface skin it should be immersed in cold water tocool it rapidly.

After the block has completely cooled it is cutinto individual blocks and each is trimmed.

Set the wax block on the Hub of Microtome and cut the section with the help of Microtome.

Spread the section on glass slides

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.

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.

Tissue Staining

Basophilic Acidophilic

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

Stain with acidic dye [Na+dye-]

Orange G, eosin, acid fuschin

Mitochondria, cytoplasm, secretory granules, ECM proteins

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 acidicstructures (such as RNA-rich portions of the cytoplasmand the matrix of cartilage).

In contrast, Eosin stains other cytoplasmiccomponents and collagen. In many stainingprocedures certain structures such as nuclei becomevisible, but other parts of cells remain color free.

Study of Histology

Study through computer associated light microscope

Students Electronic light microscope

Polymerase Chain ReactionPCR, polymerase chainreaction, is an in-vitrotechnique for amplificationof a region of DNA whosesequence is known orwhich lies between tworegions of known sequence

Primer Selection

Target Selection

Sample Preparation

Critical Steps in PCR

DNA template

Primers

Enzyme

dNTPs

Mg2+

buffers

Reaction Components

Anneal at 55⁰ C

Extension at 72⁰ C

Denature at 94⁰ C

Step Cycle ProgrammeDenaturationTrade off between denaturing DNA and

not denaturing Taq Polymerase

Taq half-life 40min at 95 °, 10min at 97.5°

94° C

AnnealingTrade off between efficient annealing and

specificity

2-5 ° below Tm

ExtensionTemperature optimum for Taq Polymerase

72 °

Sample

DNA Extraction

Addition of premix reagent

Automated Amplification

PCR Protocol

DetectionMutation Detection

SNP Quantification Genotyping

Merits of PCR Generates and Modifies

DNA

Much faster

Specificity

Simplicity

ApplicationsGenome mapping and gene function

determination

Detection of drug resistance

genes

Forensic (DNA fingerprinting)

Biodiversity studies ( e.g.

evolution studies)

Quantitative Proteins Estimation

Specificity and Sensitivity of the Method

Specificity of an assay relates to how good the assay is in discriminating between the requested analyte and interfering substances

Sensitivity of an assay is a measure of how little of the analyte the method can detect

There are many Spectrophotometric method for determining the protein concentration

Bicinchoninicacid method

biuret Lowry BradfordSpectrometric (A280/A260)

There are a wide variety of protein assays available. but each assay has its own advantages and limitationsThe factors that you should consider in choosing a method:

• Sensitivity• The presence of interfering substance• Time available of the assay

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.

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 ).

Concentration of unknown sample

Standard curve are most commonly used to determine

the concentration of a substance, using serial dilution of

solutions of known concentrations(standard solutions)

Tube Casein Standard Unknown

A

B

C

D

E

F

Set up 7 tubes as follows:

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.

Glimpse of Biochemistry lab

Blood Group

Detection

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

Basic Fundamental

Common Lab Method- Slide Method

Principle:When red cells are mixed with various

reagent anti-seras (soluble antibody),agglutination will occur on the slides containingcells positive for (possessing the antigen) thecorresponding antigen.

No agglutination will occur when the red cellsdo not contain the corresponding antigen.

Reagents

Anti-A antibodiesAnti-B antibodiesAnti-D

SlidesSterilized NeedlesAlcohol Wooden applicator.

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 D4. 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.

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

VERTEBRATE LAB SPECMEN

PHYLA/ CLASS

UROCHORDATAPISCES AMPHIBIAREPTILIAAVESMAMMALIA

INVERTEBRATE LAB SPECIMENPHYLUM

PROTOZOAPORIFERACOELENTRATAPLATYHELMINTHESASCHELMINTHESANNELIDAARTHROPODAMOLLUSCAECHINODERMATA

RULE OF THE ZOOLOGY LABAlways 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.

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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.

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

Gratitude From the Dept. of Zoology