Bulk transport

BULK TRANSPORT:    cells have also developed mechanism to transport across the membrane, larger molecules cannot pass through the membrane , generally like secretory molecules like protein, hormones .  This transportation  is done by the formation of  carrier vesicles.

ENDOCYTOSIS:  molecules are transported insides the cells by a process called endocytosis.

It is of two type :

1.Pinocytosis: intake of fluid ion through the vesicles, is called pinocytosis and its also called cell drinking.

2.Phagocytosis: intake of food , cell eating , for eg: macrophages, white blood cells , and amoeba etc.

EXOCYTOSIS:  molecules are transported outside the cells by a process called exocytosis. Exocytosis perform secretion , excretion, and ephagy.

Active transport

                       Active transport:

§ Cell has the continuously translocation of K+ ions , into the cell by active transport mechanism at the expense of metabolic energy.

§ Most of the inorganic substances or ions , uncharged particles like, amino acids , and carbohydrates are actively transported across the membrane.

§ Cell always intake the essential nutrients from the outside , many cells equilibrium of diffusion can be adjusted by coupling energy yielding reaction, so the cells are able to translocates solute or metabolite against the concentration gradient.

For eg: movement of Na+  and K+ ions pump.

§ATP DRIVEN ACTIVE TRANSPORT:

§ Na+ and k+ pump : In all cell membrane having a high content of k+ ion at the inner side and high Na+ ions at the outer side of cells, so here these are pumped against their concentration gradient. 

§ Here integral membrane protein is Na+ and k+ ATPase.

§ Na + and k+ ATPase exist in hetero tetramer  form . Its having  two subunits, α ‌and β and it also  exist in dimer form. On Hydrolysis  of ATP, gives ADP+ iP and protein changes its conformation transport 3 Na+ ions outside and 2K+ ions at the inside the cell membrane , against their concentration gradient these are pumped  at the opposite direction , so its required energy.

§ When the hydrolysis of ATP is not done then the transport of molecules is not done so its also called the coupling mechanism.

LOW DRIVEN ATP ACTIVE TRANSPORT: The molecules are transported across the membrane via coupled with the ions[ Na+ and H+ ions] 

Ø For eg: glucose is diffused by simple diffusion or via facilitated mechanism but in intestine and kidney glucose is absorbed against the concentration gradient  by an active transport system .

Ø  glucose molecules is release into the cell against concentration gradient  by the protein conformation change , after  release the glucose , protein pick up the Na+ ion  and pump them out side the cells to balance the ions concentration.

Ø Glucose is also transported into the blood stream by  diffusion.

facilitate transport

FACILIATED TRANSPORT: 

It is a type of transport in which:

the uncharge molecules are passed from the cell membrane with the help of  carrier molecules, that’s means for a specific substance a specific carrier molecules are found. 

Ø These carrier proteins are pick up the molecules and  these protein changes their conformation according to the substance  and release the substance inside the cell.

Ø If the carrier is saturarted with solute molecule, the rate of solute transport is maximum.

MECHANISM OF PASSIVE TRANSPORT

Mechanism of passive transport: passive transport can continue to occurs if the absorbed solute is immobilised . Cations have tendency to move from electropositive to electronegative side while anions have tendency to move from electronegative to electropositive side.

There are two mode of active transport  :

i.Lipid matrix permeability: - lipid soluble substances can pass from the semi permeable membrane according to their solubility and concentration gradient

ii. Hydrophilic membrane channel: - these are the channels which are formed by tunnel proteins, channels makes the membrane semipermeability so water can pass outside and inside according their osmotic gradient , co2and o2 also can diffuse from through these channels as per their conc. Gradient .

TYPES OF TRANSPORT MECHANISM

Transport mechanisms are described as four types:

1)Passive transport

2)Faciliated transport

3)Active transport

4)Bulk transport

PASSIVE  TRANSPORT OR DIFFUSSION:

§Simple diffusion :-

                                molecules are passing through membrane on their concentration gradient from higher to lower concentration due to their own kinetic energy.

§It does not require any metabolic energy.

§[solution = solute particle in less amount + solvent in higher amount],   when the solute particles arranged uniformly then solution is called homogenous solution.

§For eg : cells are having higher amount of  k+ ions instead of environment , so cells draw k+ ions from environment.

§Membrane of kidney cells , epithelial cells, red blood cells and muscles cells are impermeable to substance unless their transport is facilitated .All membranes are permeable for o2 , co2 , and water .

Diffusion down concentration gradient: 

Simple diffusion :In a concentration solution , a substance separated by its dilution , so the solute molecule move from higher to lower concentration until equilibrium is reached , it a physical phenomenon and its does not depends on cellular energy.

  Empirical term of diffusion can be explained by the first law of fick:             

                         dm/dt= - DA [dc/dx]

                                                       dm/dt= flow of matter in per moles

                                                      dc/dx = concentration gradient

                                                     D = diffusion gradient. [cm2 s-1]

Diffusion down the electrical gradient:                                        In a solution the solutes are dissociates into ions and having charge. Cell are also having charge particles inside and outside the media, cell membrane maintain their potential difference, this difference is measured by MICROELECTRODE.  Its range from 50mV to 100mV.  The ions which involved to form potential gradient are k+ and Na+ ions .

                              Each individual substance will diffuse independently of others as per gradient of its own concentration, diffusion pressure, or partial pressure.

OSMOSIS:  Diffusion of water from semi permeable membrane that occurs under the influence or osmotically active solution.

                     ^= RTc               c= molar concentration

                                                R= constant

                                                 T= temperature

                                                ^= osmotic pressure

                                                

PUMP CARRIER AND CHANNELS [ MEMBRANE TRANSPORT]

PUMP, CARRIER AND CHENNELS

§ MEMBRANE TRANSPORT:

§  Cell exchanges both matters and energy between themselves and the environment through the plasma membrane.

§In this transport molecules are entered into the cell and metabolic waste expelled via plasma membrane & this traffic meticulosly regulate.

§ plasma membrane is impermeable and it prevents water soluble substances from escaping the cell.

§Plasma membrane has evolved a vectorial transport mechanism to bring in them and out the waste

Ø Molecules can diffuse across the membrane down the concentration gradient , but the rate of diffusion will be depends upons the size of the molecules , types of charges it bears and degree of lipids solubility. 

Ø Permeability of uncharged molecules is very slow. For eg: sugars , amino acids , nucleotides, glucose etc.

Ø these are transported via MTP mechanism[ Membrane transport protein mechanism].

Ø MTP mechanism is specific for a specific substances called UNIPORT .

Ø Some MTP mechanism two molecular species simultaneously in same direction so its called SYMPORT, and if in opposite direction called ANTIPORT……
§  Tranport proteins are called COPORT.
§Membrane protein transfer the molecules by making an aqueous channel called channel protein.
 some molecules are passing through the membrane by attaching with the protein molecule are called , carrier proteins.

Introduction about spectrophotometer

                                                    SPECTROPHOTOMETER

PRINCIPLE: A spectrophotometer is an instrument which is used to measure the amount of light that a sample absorbs. in it a beam of light is passing through the sample and then sample absorb the light and its measure the intensity of light which is reaches to a detector.

INSTRUMENTATION: it is a source of rediation . itscontains both deutrium[D] and hydrogen [H] lamps which emitts the light wavelength range 160-375 nm and produce UV rediations. here a tungsten lamp also employed for as a source of visible light.this lamp use the wavelength range 350-2500 nm . for the selection of wavelength a monochromator is used. monochromators contains the following component parts like , an entrance slit, a collimating lens , a dispersing device,a prism , a focusing lens , an exit slit. in which the sample and the reference sample are placed that must be transparent are called CUVETTES. in UV visible spectroscopy photomultiplier tubes are commonly used as a detector. it consist photoemmisive cathod , several dynode and an anode . photo multiplier are very sensitive to UV and visible rediation.

WORKING:                                       

• This works on the principle of lambert beer's low .
• its works on IR and visible range of light [100-780nm]
• it measure absorbance or transmission of monochromic light by the test sample.                     T=l/b , A= log10 T 
• where T= transmittance , A = absorbs
• switch on the instrument before 10 min. of use 
• now set the wavelength 
• set the absorbance or transmittance with the help of blank.
• now take the absorbance of test sample one by one.

PRECAUTION:

1. handle the cuvette carefully.
2. in each time equal amount of sample is added .
3. adjust the abosorbance or transmission with adjust button.
4. wash the cuvettes each time before and after use because a few part of earlier sample is lefted so wavelength measured not in perfect way its disturbs the measurement .

ADVANTAGES : Its works on high accuracy and reliability , 340-960 nm range, % T , absorbance, concentration, measuring ways , 10 nm bandwidth, 

Introduction about microtome

                         MICROTOME

INSTRUMENTATION:

                                       Microtome use steel , glass and diamond blades depending upon the the specimen being sliced and the desired thickness of the section being cut . steel blades are used to prepare sections of animals or plant tissues for light microscopy histology. Glass knives are used to slice sections of light microscopy and to slice very thin sections for electron microscopy. Industrial grade diamond knives are used to slice hard materials such as bones , teeth and plant matter for both light microscopy and for electron microscopy gem quality diamond knives are used for slicing thin sections of electron microscopy.

PRINCIPLE:

                        microtome is a sectioning instrument that allows the cutting of extremely thin slices of a material known as section . microtome are used in microscopy , allowing for the preparation of sample for observation under transmitted light or electrons radiation . it is a method for the preparation of thin section for materials such as bones, minerals, and teeth.

                                            

                                           

WORKING :

                             Microtome is a common instrument . this device operates with a staged rotary action such that the cutting is part of the rotary motion . in a rotary microtome ,blade is fixed in horizontal position . through the motion of the sample holder, the sample is cut by the knife position , at which point the fresh section remains on the knifes , at the highest point of the rotary motion , the sample holders is advanced by the same thickness as the section that is to be made , allowing for the next section to be made.

The flywheel is many microtomes can be operated by hands . this has the advantages that clean cut be made , as the  relatively large mass of the fly wheel prevents the sample from being stopped during the sample cut. It cuts thickness between 1 and 60 micron meter. For hard material , its cits a semi thin section with a thickness of as low as .5 micron meter.

USES:

1.   Setting up the rotary microtome .

2.   Put the prepared blocks of wax having tissue or organ .

3.   Adjust the sample and rotate the rotor.

4.   The thickness of slides can be adjusted.

UV transilluminator

              UV TRANS ILLUMINATOR

PRINCIPLE: 

                          uv. Transilluminator are used in molecular biology labs to view DNA[oRNA]  that are separated by electrphoresis through an agarose gel. Then added a flouroscent dye which binds with nucleic acid . that’s dye stained  gel to a UVB light source to became visible and fluoresce the DNA segments.

INSTRUMENTATION :                                                                                                                              at the time of electrophoresis , a flourescent dye is used to stained agarose gel which binds with nucleic acids . exposing the stained gel to a UVB light source which causes DNA segments to became visible and flouresce.

PRECAUTIONS: 

1. because EtBr [ ethydium bromide ] isa toxic chemical substance so it has been used safely and handled very carefully.
2. when we want to avoid UVB  as a light source , we can recommended the blue light LED transilluminator such as the one descrides here.
3. a safety lid for viewing the gel which comes in transilluminator , however , when the lid is not in place, safety glasses must be worn when operating the UVB bulb
4. we recomanded thet gloves to be worn bcz EtBr is a toxic substance which is added into the staining gel.

USES :

·       Place sample on the filter area . it is recommanded to place the gel on a gel- tray to protect the filter surface from cut and scratches.

·       Press On\OFF switch on . the uv tbes within the unit should be glowing.

·       Now switch on the main power.

·       When using a transilluminator with multiple UV wavelength dial the knob to the appropriate wavelength setting.

ADVANTAGES:

provision of UV protecting shield.

Best suitable for viewing fluorescent sationed gel.

Gel viewing filter provides high quality performance by facilitating sharpened gel view.

 

Compact in size and light in weight.

Provision of working in both high and low intenties with an aid of simple awitc system.                 

HOT AIR OVEN

                         HOT AIR OVEN

   

PRINCIPLE :

                            It is an electronic device which used sor the sterilization [ sterilization is a technique for the removal of all tyoe of microorganism]. , Hot air oven used dry heat to sterized the articles . when electricity is passed through the heating coil, electrical energy is converted into heating energy and its used for  dry sterilization.

    

  WORKING :

v Set the temprature.

v Wait for temperature maintainance.

v Keep the paper wrapped glasswares , media etc. in the hot air oven.

   INSTRUMENTATION:

1.   Double walled chamber made of stainless steel or aluminium .

2.   A fan is set at the bottom of the oven which works to circulates the steam of hot dry through chamber resulting in rise temperature to sterilized the materials .

3.   There is a thermostates controlling the temperature .

4.   A thermometer is fitted for recording the temperature of the oven.

      USES:

v DRY sterilisation.

v Drying of glassware’s [ petriplates , tubes , flasks, pipettes].

v Heating of chemicals used for the preparation of primary standard .

v Maintain the sterility of already sterilized glass wares.                 

PRECAUTIONS:

v Do not keep open the door for long time.

v Keep the materials only after temp reach the settled point.

                                

GEL ROCKER

             GEL ROCKER

PRINCIPLE: 

                     A gel rocker is a device which used in labotories for moleculer and biological mixing applications . rockers are often used in a place of shakers when less aggressive mixing is required . rockers are commoly used for staining and de- staining of gels after electrophoresis, hybridization, washing , blotting , cell culture, and gentle mixing.

WORKING: 

                       Two dimensional rockers use a plateform to remove in a seasaw motion to create waves in liquid laboratory samples. Three dimensional rocjers have aplateform in a three dimensional gyratory motion to create  a gentle swrilng of sample.

                                                Typical features of laboratory rockers includes variable speeds , and till angles. Rockers are often designed to accept stackable plantform or may be outfitted with multiple tiers to increase capacity without increasing the footprint . plateform surfaces are usually covered with rubber pads to prevent objects from slipping during operation.     

FEATURES: 

·      

            Attractive design .

·        Compact in size.

·        Control by DC motor current.

·        Speed 0-100 oscillations per min.

·        Timer 0-99mins.

·        Movement in see saw .

·        Approx. wt. 25kg.

·        Power consumption 200watt.

·        Applications may includes blotting gel staining and culture aeration.

  

APPARATUS WHICH ARE USED IN BIOTECHNOLOGICAL LABORATORY

ELECTRONIC COLONY COUNTER

INSTRUMENTATION AND WORKING : 

                                                                          A device which used to count the colonies of bacterial cell that growing in a culture medium. It is usually consist of an illuminating transparent sheet or plate dividing into sections of known area . petridishes those containing the culture colonies of bacteria are placed over it and then counting the colonies according to the number within the area viewed.

It contains – auto mark pen, wolffhuegel grid glass plate, 110mm magnifying lens , net weight- 3.5kg, supply – 230v AC 50 HZ .

PRINCIPLE :

                           Electronic colony counter is designed to count the accurate colonies of bacterial and mould cells in a petridish within a few time. The purpose of plate colonies counting is to estimate the number of cells present based on their ability to give rise to colonies under some specific conditions of nutritient medium, temprature and time.

USES:

o   Place the petriplate on the center.

o   Use any pen or just point it to count correctly.

o   For more accuracy adjust the senctivity , lighting , beep.

PRECAUTION:

       I.            While the majority of microrganisms are not pathogenic to human and have never been shown to cause illness , under unusual circumstances a few microorganism that are normally pathogenic can act as pathogenic .

     II.         

   All                                                   material , media , tubes , plates, loops, needles, pipettes and other item used for culturing microorganisms should be sterilized by autoclaving .

  III.                                                            use a disinfectant , bleach or 70% ethanol solution, tp wipe down branches and work areas both before and after working with cultures.

  IV.                                                                                                                           Use a disinfectant to clean your hands before and after working with microrganism . nondisinfectant soap will remove surface bacteria and can be used if disinfectant soapin not availav[ble gloves may be use be as protectant.

                                                                                                                                            Use pipette bulbs or pipetting device for the aspiration and disapearing of liquid medium .

  VI.                                                                                                                               Do not eat and drink in the lab because there are many bacterias which are not beneficial for our health.

VII.                                                                                                                              Label everything clearly.