Biological Techniques, Biology tutorial

Types of microscopes and their uses content:

Biology is a practical science, and biology students are generally needed to spend some time in laboratory. The kinds of activities the student will carry out differ depending on exact field they are in. Plant geneticist, for instance, at times spends time out in fields gathering plants, whereas molecular biologists may utilize complex equipment like DNA sequencing machines. Even so, there are few basic methods that all beginning lab students must understand. Biological techniques are systems or procedures which are utilized to study living things. They comprise approaches, experimental and computational methods, protocols and tools for biological research.

Class of microscope:

There two types of microscopes which students can recognize in any laboratory. They are simple and compound microscopes magnify the object directly, and conversely, compound microscopes magnify the object, and illustrate objects in the reverse position. Students can observe that what is left in image is right in object when moving slide in the certain direction.

Kinds of microscopes:

There are several kinds of microscopes which you can find in market today. All you require to do is to find out what it is used for. Different kinds of microscopes and their essential functions are as given below.

Complex Compound Microscope: Light or optical kinds of complex compound microscopes combines eyepiece lenses, objective, and light condemner lens and magnify an image of small objects

Simple Compound Microscope: The simple compound microscope is related to complex compound microscope in function. Though, difference is that the simple compound microscope is most utilized to magnifying objects usually. It has no light condenser joined under stage as compared to complex compound microscope.

Simple Magnifying Lens: Simplest light microscope is magnifying lens. It is generally hand held. It magnifies object and generally useful for field work.

Stereo / Dissecting Microscope: The stereo or dissecting microscope, combines two objectives lenses, and 2 eyepieces to view the object. When you utilize this microscope, you will see 3-D images of object on stage.

Light microscopy and functions:

Significant features available in the microscopes. To utilize light microscope resourcefully, you require understanding essential microscopy: dark field, bright field, additionally, phase contrast, and oil immersion. Consider the following; Focal Plane, Contrast, Resolution, and Recognition (CFRR) of sample when you see it.

Bright field microscopy: In bright field microscope, light source is from below stage. Light travels through specimen, by the objective lens to the eye through eyepiece.

Phase contrast microscopy: Detail of transparent living cells is noticeable in phase contrast microscopy. Though, inadequate contrast between structures with comparable transparency may happen. Phase contrast is better than bright field microscopy if specimen is transparent and high magnifications are required.

Dark field microscopy: It is a cheaper substitute to phase contrast microscopy. In dark field, reflected light from particles on slide passes by combined lenses to the eye. To view specimens in liquid sample, dark field is best.

Parts and functions of a modern microscope:

The parts of the typical modern complex compound microscope are given below:

  • Eyepiece (EP): Eyepiece lens is joined to top of tube. Standard lens powers are 5X or 10X of 15X or 16X.
  • Revolving nosepiece (RN): This transferable part house two or more objectives lenses.
  • Nosepiece (NP): There are three or four objective lenses on nosepiece with 4X, 5X, 10X, 40X, 100X powers. Low powers (4X, 5X, 10X, 40X) are known as dry scan objectives.
  • Condenser Lens (CL): CL use to focus light onto specimen on microscope stage. Use it most at the higher powers (40X and 100X) to give you sharper image than those microscopes with no CL.
  • Diaphragm Knob (DN): It handles disc directly above CL. DN is use to differ amount and size of cone light reaching slide from below.
  • Condenser Mover (CM): It is use to move condenser up or down, to get good quality of light.
  • Focus Knobs (FN): It make rough and fine adjustments to be focused. There are 2 knobs on microscope, large knob is for coarse focus, and small one is for fine focus.
  • Rack Stop (RS): It is use to lock stage level after adjusting how close objective lens can get to slide.
  • Stage Holder (SH): Seat on which stage is resting, join it with FK knob that move stage up or down when you turn FK knob.
  • Arm (AR): It joins eyepieces and objectives to base.
  • Stage and Slide Mover: Stage mover knob is utilized to move stage forward or backward to position best quality specimen. While, small lower knob moves slide to either left or right to position the best specimen for view

Operating the microscope:

Proper method to operate microscope is to begin focus with lowest power objective lens (OB) while you look by side and move lens down as close to slide without touching it. Next look through eyepiece lens and focus upward only until image is sharp. After having sharp image at low power lens, turn revolving nosepiece (RN) to click next higher power lens and perform minor adjustments with fine focus knob.

Preparation of microscope slides:

Slides making is significant part of medical, biological, veterinary and forensic sciences. Two major kinds of preparation are utilized:

a. Temporary Preparations:

These preparations may be required for matter of minutes or hours only. They are mounted in water or dilute 1, 2, 3-propanetriol (glycerol) or other fluid of low volatility. After test they are discarded.

Material under test can be fixed and stained or even examined in the living state, for instance protozoa like amoeba. In this situation a harmless aqueous stain like 1% methylene blue can be utilized - it would then be called as vital stain. Preparation of stained specimens for microscopical study either temporary or permanent generally involves three processes.

i) Fixation:

For fresh tissues, major objective of fixation is to kill tissues quickly by precipitating proteins. Reagent utilized is known as fixative. Different fixatives are utilized depending on nature of tissue whether soft of hard. Tissues must be washed well after fixation, using same solvent as stain, to remove all traces of fixative.

ii) Staining:

Object of staining is to accentuate distinction between different components of the tissue or organ.

iii) Mounting:

Mounting media used for temporary preparations have water and 1, 2, 3-propanetriol (glycerol) 30%-50% aqueous solution.

b. Permanent Preparation:

If slide is to be kept for long-term reference, for days or even years, it should be made as the permanent preparation. This is attained by:

1. Dehydrating specimen after staining (usually with the graded series of alcohols);

2. Clearing it - treating it with the solvent which is mutual soluble between alcohol and mounting medium [traditionally clove oil or 1-2 dimethylbenzene (xylene); and

3. Mounting it under a coverglass in preservative that dries hard and has refractive index similar to glass. Making of permanent stained preparation mounted in Canada balsam involves five processes:

(i) Fixation (ii) Staining (iii) Dehydration, (iv) Clearing, and (v) Mounting

Dissection guide:

Dissection is the major part of biology practical. Meaning of dissection is to cut open animal to determine structure of its parts, to describe their boundaries and show clearly their mutual relations. Dissection comprises mostly in removing connective tissue that binds many parts together. Some dissecting guide to help is given below:

Preparation for Dissection:

Materials Required: Dissection trays, Petridishes, Dissection kit, Chloroform, Ethane, Formalin, Urethane, Microscope, Table lamp, Animals.

Process: One has to carry out many tasks before and after dissection.

Procuring Animals: Orders for animals must be placed with animal supplier according to number of students in class. This must be completed two-three days before dissection is to be completed.

Anaesthetizing the Animals: Before dissection, animals are provided anesthesia. Chloroform and ether are utilized as anaesthetizing agents. Rats, frogs and pigeons are usually freshly chloroformed for dissection. Preservatives usually utilized are formalin (5% or 8% or 10%) and 70% alcohol.

Setting up of dissection Trays: Large animals such as rat, frog, pila, fish, leech and other animals are dissected in dissection trays while small animals like cockroach and small insects can be dissected in petriplates.

General Rule of Dissection:

The common anesthesia utilized in biology laboratory and chloroform and ether. For rats, frogs and pigeons, it is better to dissect them immediately after anesthesia.

Formalin (5%, 8% or 10%) and 70% alcohol are common laboratory preservatives. It is generally better to perform dissection after you have completed theoretical studies.

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