What is Sterilization in Place (SIP)

 Most pharmaceutical GMP systems available today incorporate some form of sterilization using steam. In situ sterilization of equipment using a medium called Pure Saturated Steam. The installation comprises of one or more pieces of processing equipment

Example • Fermentor or a centrifugal separator to handle harvests, connected by rigid stainless steel or flexible Teflon-lined piping

Why is there a need for sterilization?

• To increase the level of sterility assurance associated with those products made by aseptic processing.
• The size and configuration of large number of process equipment utilized in the production of Biological/parenteral restricts them to be placed inside an autoclave for sterilization.
• To assure a higher degree of sterility assurance for these items, they should be sterilized in situ rather than sanitized.
• SIP is required as a result of desire for enhanced sterility assurance for aseptically produced materials.
• Steam SIP is in daily use in the Biological & parenteral industry.

What makes SIP a critical process?

• Steam-in-place sterilization enables the entire processing system to be sterilized as a single entity thereby reducing the need for aseptic connections.
• Criticality of system design to achieve sterility with SIP is considered more closely than the steam sterilizer.
• SIP employs the same moist heat mechanism as steam sterilization in autoclaves.

SIP Fundamentals

• SIP differs only slightly from steam sterilization in autoclaves.
• To achieve effective SIP, the elements necessary for process effectiveness inherent in the autoclave design and operation must be provided in the SIP system.

What is a saturated pure steam?

• Saturated steam is a steam-water mixture in which the vapour phase (steam) is in equilibrium with the liquid phase (water or condensate).
• The addition of heat to saturated steam can result in its de-saturation (or superheating).
• The loss of heat from saturated steam will result in its condensation.

SIP Design Considerations

• Complete displacement and elimination of entrapped air.
• Constant bleeds of steam at all low points to eliminate condensate build-up Heating large masses of stainless steel from ambient temperature to 121°C results in the creation of large quantities of condensate.
• Strict adherence to the sterilization procedures.
• Proper maintenance of the sterility after the process.

• SIP system design concepts is achieved through a review of the equipment elements that make up that system.

• The Components of an SIP system:
   Pressure Vessels
   Piping system
   Filters

• The system must be designed in a way that condensate can be readily removed.
• To achieve this objective, system is sterilized in multiple patterns, in which each pattern sterilizes a portion of the larger system.
• Some portions of the system must be sterilized more than once to assure that all portions of the system are fully covered.

Control System Devices in Fermenter

 Fermenter provides defined conditions for the formation of biomass and product in Control temperature, pH, degree of agitation, oxygen concentration, foaming, etc Requires careful monitoring

pH control device

• In batch culture the pH of an actively growing culture will not remain constant for very long.
• The pH controlling device (probe) checks the pH of the fermentation media at specific intervals of time and adjusts the pH to its optimum level by addition of acids or aikalis for maximum yield of the product.
• Maintaining pH to its optimum level is very important for growth of micro-organism to obtain a desired product.
• The control of pH values is ensured with the help of peristaltic pumps, releasing out acid/alkali via silicone tubes.
• For pH, only sterilisable electrodes are used.
• The electrodes used are mainly combined glass reference electrode that can withstand repeated sterilization at 121°C :
• > Silver/silver chloride electrodes with KCI as electrolyte.
• > calomel/ mercury electrodes
• The electrode is connected via leads to a pH meter/ controller.

Temperature control device

•  Temperature control device generaly contains a thermometer or probe, a heating element and cooling coils or jackets around fermenter.
• These are on and off depending on the need for heating or cooling.
• During fermentation process, various reactions take place in a fermenter which leads to the generation of heat in the fermentation media. The leads to increase in temperature which is detrimental for the growth of microorganisms and may slow down the fermentation process.
• Temperature probe is used to measure the temperature of the culture broth in the fermenter vessel like stainless steel Pt100 sensors (platinum resistance electrode).
• A temperature probe is a type of temperature sensor. Some temperature probes can measure temperature by being placed onto the surface. Others will need to be inserted or immersed in liquid to be able to measure the temperature.

Other temperature measurement devices include:

• >Glass thermometers with mercury
• >Pressure bulb thermometers
• >Thermistors (Metal resistant thermometers)
• > Bimetallic thermometers >Thermocouples

Pressure gauge

•  Industrial fermenters are designed to withstand a specific working pressure.
• Pressure measurements are required as a factor of safety.
• It is important to fit the equipment with devices that sense, indicate and control pressure.
• The correct pressure is maintained by regulatory valves controlled by associated pressure gauges.
• Gauges help to ensure there are no leaks or pressure changes that could affect the operating condition of the bioreactor.

Following are some pressure measuring sensors:

• > Bourdon tube pressure gauge
• > Diaphragm gauge
• > Piezoelectric transducer

Dissolved oxygen probe

•  It monitors the dissolved Oxygen in the system. It is measured by DO probe.
• DO electrodes measure partial pressure of dissolved oxygen.
• In case of low oxygen tension in broth, more oxygen is purged in fermenter and/or agitator speed is increased.

The electrodes used to measure DO are:

• > Polarographic electrodes
• > Phase fluorometric oxygen sensor

Rotameter

• It is a device that measures the flow rate of air.
• It indicates rate of air flow into vessel.
• It is attached to air sparger.
• A pressure valve is attached to rotameter for safer operation.

Foam control device

• Foam is produced during the fermentation process as a result of continuous agitation of the fermentation broth.
•  This may lead to spill out of the media out of the fermenter and cause media loss as well as contamination.
• Therefore, it is necessary to remove/break or neutralize this foam with the help of anti-foaming agents.

Foams can be eliminated by two methods:

1. By the use of antifoam agents
2. By mechanical breaking of foam

i]. By the use of antifoam agents

• The set up includes a sensor and a small tank containing anti-foam agent.
• A probe (foam sensing and control unit) is inserted through the top plate.
• It is set at a defined level abuve the broth surface, when the foam rise and touches the probe surface/tip, a current is passed through the circuit which activates the pump and antifoam is released within seconds.

Examples:

• Insaluble oils, polydimethy siloxanes and other silicones, certain alcohols, stearates and glycols.

Characteristics of a perfect antifoam agent :

• Long durability (slower consumption).
• Low viscosity (ease of pumping & dosing)
• Stability of the product as supplied (for storage)
• Stability of the product as dispersed into the foamant (for effectiveness).
• High activity (low dosing)
• It should be Effective at low & high temperatures
• It should be Effective at low & high pH
• It should be Effective in high salt concentrations
• It should be not toxic to culture Microorganisms.

ii]. By mechanical breaking of foam

• Mechanical antifoam devices like discs, propellers, brushes or hollow cones are attached to the impellors shaft above the broth surface.
• Foam is braken down when it is thrown against the walls of the fermenter.

Basic Fermenter Design : External, Agitation & Aeration, Inlets and Outlets

  De Becze and Liebmann (1944) used the first large scale (above 20 litre capacity) fermenter for the production of yeast. But it was during the First World War, a British scientist named Chain Weizmann (1914-1918) developed a fermenter for the production of acetone.

   Since importance of aseptic conditions was recognised, hence steps were taken to design and construct piping, joints and valves in which sterile conditions could be achieved and manufactured when required.
  The first pilot fermenter was erected in India at Hindustan Antibiotic Ltd., Pimpri, Pune in the year 1950.

Basic criteria of the fermenter

• The vessel should be capable of being operated aseptically for a number of days and should be reliable in long-term operation and meet the requirements of containment regulations.
• Adequate aeration and agitation should be provided to meet the metabolic requirements of the microorganism.
• However, the mixing should not cause damage to the organism.
• Power consumption should be as low as possible.
• A system of temperature control should be provided.
• A system of pH control should be provided.
• Sampling facilities should be provided.
• Evaporation losses from the fermenter should not be excessive.
• The vessel should be designed to require the minimal use of labour in operation, harvesting, cleaning and maintenance.
• Ideally the vessel should be suitable for a range of processes, but this may be restricted because of containment regulations.
• The vessel should be constructed to ensure smooth internal surfaces.
• The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant to facilitate scale-up.
• The cheapest materials which enable satisfactory results to be achieved should be used.
• There should be adequate service provisions for individual plants.

Parts of fermenter

1. Materials used for body construction.
2. Seals 
3. Mixing components

•  Impellers
•  Spargers
•  Baffles

4. Sampling point
6. Bottom drainage system (outlet)
7. Air filter.

1. Material used for fermenter

Properties of the material used for fermenter designing :

• Non-corrosive
• Non-toxic
• Tolerable to repeated steam sterilization Cycles
• Withstand high pressure
• Resist pH changes
• Selection of the material also depends on the type of fermentation process to be carried.

Mainly two types of materials are used worldwide for the manufacture of fermenters:

1. Glass
2. Stainless steel

i]. Glass fermenters

• On a small scale (Laboratory scale) (1 to 30 dm³), it is possible to use glass.
• Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of the vessel.
• These can be of two types:

1. > A glass vessel with a round or flat bottom and a top flange carrying plate. All vessels of this type have to be sterilized by autoclaving.
2. > A giass cylinder with stainless-steel top flange carrying and bottom solid plates. Vessels with two stainless steel plates cost approximately 50% more than those with just a top plate.

• These top flange carrying plates provides port for the entrance of media, inoculum, buffers and anti-foam agents.
• At pilot and large scale, when all fermenters are sterilized in situ, any materials used will have to be assessed on their ability to withstand pressure sterilization and corrosion and on their potential toxicity and cost.

ii]. Stainless steel fermenters

• Fermenters are normally constructed of stainiess steel or at least have a stainless-steel cladding (or layering) to limit corrosion.
• The American Iron and Steel Institute (AISI) states that steels containing less than 4% chromium are cliassified as steel alloys and those containing more than 4% are classified as stainiess steels.
• Mild steel coated with glass or phenolic epoxy materials has occasionally been used.
• Stainless steel - 304 and 316 grade coated with epoxy or glass lining are into use.
• The corrosion resistance of stainless steel is thought to depend on the existence of a thin hydrous oxide film on the surface of the metal.
• The composition of this film varies with different steel alloys and different manufacturing process treatments such as rolling, pickling (removal of impurities, such as stains, inorganic contaminants, rust) or heat treatment.
• The film is stabilized by chromium and is considered to be continuous, non-porous, insoluble and self healing.
• If damaged, the film will repair itself when exposed to air or an oxidizing agent.
• The minimum amount of chromium needed to resist corrosion will depend on the corroding agent in a particular environment, such as acids, alkalis, gases, soil, salt or fresh water.
• Increasing the chromium content enhances resistance to corrosion, but only grades of steel containing at least 10 to 13% chromium develop an effective film.
•  The inclusion of nickel in high percent chromium steels enhances their resistance and improves their engineering properties.
•  The presence of molybdenum improves the resistance of stainless steels to solutions of halogen salts and pitting (small holes) by chloride ions in brine or sea water.
• Corrosion resistance can also be improved by tungsten, silicone and other elements.
• AISI grade 316 steels which contain 18% chromium, 10% nickel and 2-2.5% molybdenum are now commoniy used in fermenter construction.
•  Increasing the chromium content enhances resistance to corrosion, but only grades of steel containing at least 10 to 13% chromium develop an effective film.
• The inclusion of nickel in high percent chromium steels enhances their resistance and improves their engineering properties.
• The presence of molybdenum improves the resistance of stainless steels to solutions of halogen salts and pitting (small holes) by chloride ions in brine or sea water.
• Corrosion resistance can also be improved by tungsten, silicone and other elements.
• AISI grade 316 steels which contain 18% chromium, 10% nickel and 2-2.5% molybdenum are now commonly used in fermenter construction.

2. Types of Seals

  It is important to consider the ways in which a reliable aseptic seal is made between glass and glass, glass and metal or metal and metal joints such as between a fermenter vessel and a detachable top or base plate.

• Between a fermenter vessel and a detachable top or base plate.
• Sealing assembly for stirrer shaft.

i]. Between a fermenter vessel and a detachable top or base plate

a) Gasket seals are suitable with glass to glass joints.

• Nitryl or butyl rubbers are normally used for these seals as they will withstand fermentation process conditions.

b) Lip seals are suitable with glass metal joints.

• These are generally made of silicone and are fluorosilicone elastomers.

c) O ring seals are suitable with metal to metal joints.

• These are made of Polytetrafluoroethylene (PTFE) and Neoprene.

ii]. Sealing assembly for stirrer shaft

• Stirrer shaft is a device providing agitation.
• It must be sealed properly ensuring a long term aseptic operation

There are various types of sealing assembly such as:

• Mechanical seal
• Packed gland seal
• Magnetic drive.

3. Mixing components

• Structural components involved in mixing such as :

1. Impellor for agitation
2. Sparger for aeration
3. Baffles for breaking vortex

Importance of Agitation and Aeration

• Important factor in a fermenter. 
• Provides a provision for adequate mixing of the contents of a fermenter.
• Mixing helps to disperse the air bubbles, suspend cells, enhance heat and mass transfer in the medium.

Location of mixing components are

• Impeller : shaft in the centre of the fermenter
• Sparger : below the impeller (at base)
• Baffles : Along the side walls of the fermenter

i]. Impellors or agitators

Agitation should ensure that a uniform suspension of microbial cells is achieved in a homogenous nutrient medium.

• Agitation is done using impellers.
• These are mounted on the drive shaft and into the fermenter through its lid (flange).
• These are made up of impellor biades and the position may be varying according to the need and size of the fermenter.
• These impellors or blades are attached to a motor on lid.
• Impellors may not be required in small scale fermenters.

The major function of an impellor is to aid in proper mixing of the following components uniformly throughout the fermentation vessel :

1. > Suspended microorganisms (microbial cells),
2. > Media components
3. > Oxygen (air bubbles)
4. > Heat transfer.

• Impellor blades reduce the size of the air bubbles by breaking them and distributes them uniformly into the fermentation media.
• Impellors also helps in breaking of foam bubbles in the head space of fermenter.
• This foam formed during fermentation process can cause a major problem of contamination. Therefore, it is important to breakdown these foam so that they don't over flow out of the fermenter and cause contamination.

Impellors classification : Basic impellors

a] Disc turbines or Rushton turbines consists of a disc of a disc with a series of rectangular vanes set in a vertical plate around the circumference.

b] Vaned discs has a series of rectangular vanes attached vertically to the underside. Air from the sparger hits the underside of the disc and is displaced towards the dreamstim vanes where the air bubbles are broken up into smaller bubbles.

c] Open turbines of variable pitch

d] Marine propellerss - The vanes of a variable pitch open propeller are attached directly to a boss on the agitator shaft. In this case the air bubbles do not initially hit any surface before dispersion by the vanes or blades.

Modern Agitator Developments 

Four other modern agitator developments are derived from open turbines. The new turbine designs make it possible to replace Rushton turbines by :

• larger low power agitators which do not lose as much power when aerated,
• Which are able to handle higher air volumes without flooding and give better bulk blending and heat transfer characteristics in more viscous media.
• Good mixing and aeration in high viscosity broths may also be achieved by a dual impeller combination, where the lower impeller acts as the gas disperser and the upper impeller acts primarily as a device for aiding circulation of vessel contents.

ii]. Spargers or aerators

•  A sparger is an aeration system through which sterile air is introduced in the medium of fermentation tank.
•  Spargers are located at the bottom of the fermentation tank.
• Glass wool filters are used in a sparger for sterilization of air and other gases.
• The sparger pipes contain small holes of about 5-10 mm.
• Through these small holes pressurized air is released in the aqueous fermentation media.
• The air released is the form of tiny air bubbles.
• These air bubbles helps in mixing of media.
• Impeller blades disperses air released through sparger into medium.

Types of spargers

1. Porous sparger

• These type of spargers are used mainly in laboratory scale non- agitated fermenter (without impellors).
• These are mainly made up of ceramics.
• Pressure drop occurs across the sparger.
• Fine holes may become blocked due to microbial growth around holes with low pressure.

2. Orifice sparger (a perforated pipe)

• These are used in small or large scale, agitated/non- agitated fermenters.
• Spargers have holes of at least 6mm diameter.

3. Nozzle sparger (an open or partially dosed pipe)

• It is used in most modern mechanically stirred fermenter design from lab to industrial scale.
• It has a single open or partially closed pipe to provide the stream of air bubbles.

iii]. Baffles

Baffles are mounted on the walls of a fermenter.

• 4, 6 or 8 baffles may be used in a fermenter.
• Baffles are metal strips roughly 1/10th of vessel diameter and attached radially to the fermenter wall.
• The major function of baffles is to break the vortex formed during agitation process by the impellors.
• If the vortex, formed during agitation, is not broken, the fermentation media may spill out of fermenter and be a major cause of contamination. Therefore, it is important to break the vortex using a barrier in the form of baffles.

It is recommended that baffles should be installed so that a gap existed between them and the vessel wall, so that there is a scouring action around and behind the baffles thus minimizing microbial growth on the baffles and the fermenter walls.

4. Ports

  Ports are located on the top flange carrying plate of the fermentation vessel.
Different ports are present for the supply of different components via silicone tubes:

• Feeding port: for fermentation media (in case where media is sterilized ex-situ),
• Inoculation port : for seed culture (microorganism).
• Buffer port : for buffers (acid/ alkali)
• Anti-foam port: for anti-foaming agents.

Care should be taken that the port provides aseptic transfer.
The reservoirs for the nutrients and inoculum and associated piping are steam sterilized in situ.
Addition is done using peristaltic pump only after aseptic connection has been established.

5. Sampling point

• Sampling point is used for time to time withdrawal of samples to monitor fermentation process and quality control.
• This sampling point should provide aseptic withdrawal of sample.

6. Bottom drainage system

• It is aseptic outlet present at the bottom of fermenter for removal of entire fermented media and products formed after the fermentation process is completed.
• It is different from the sampling point.

7. Air Filter

• Oxygen-enriched air is used for sparging fermenters, to achieve the desired broth aeration and dissolved oxygen levels.
• Fermenter air should be free of unwanted airborne microorganisms and bacteriophage contamination.
• It is important to prevent the entry of unwanted microorganisms from the environment, that could interfere with the growth and multiplication of the selected fermentation organisms; contamination would impact fermentation yield and compromise product quality.
• Depending on the nature of the fermenter microbial cultures in use, it may also be important to remove undesirable microorganisms from fermenter air exhaust before releasing it into the environment.
  Fermenter air filtration involves filtration of both inlet and exhaust air with pre-filtration and final filtration procedure.
• Fermenter air filtration involves filtration of both inlet and exhaust air with pre-filtration and final filtration procedure.

i]. Inlet air filter

It is used to filter the air that is coming inside the fermenter to prevent any contamination of the fermentation broth.
It consists of following components:

• > Compressor : A compressor is a mechanical device that increases the pressure of a gas by reducing its volume.
• > Conditioner : a system for controlling the humidity and temperature.
• > Pre-filters : Fermenter air pre-filtration of compressed air protects downstream final air filters. It is composed of fibrous materials which removes solid particulars such as dust pollen, mould and bacteria from air.
• > Final sterile filters : Final fermenter air filtration provides a sterile barrier between compressed inlet gas supply to fermenters and fermenter broth contents via sparger.

II). Exhaust air filter

  It is used to filter the air that is going out of the fermenter to prevent the environment from being contaminated by any biohazards.

• >Pre-filters : Removes undesirable microorganisms to avoid their escaping out of the fermenter.
• > Final sterile filters : Final fermenter air filtration provides a sterile barrier between fermenter air exhaust and the surrounding environment.

Also read

Control System Devices in Fermenter

Major Differences between Antigens (Ags) & Antibodies (Abs)

  An antigen is any foreign molecule that interact with cells of immune system and could induce an immune response.
  Antibodies are globular proteins or immunoglobulins that react specifically with antigen that stimulated their production.
These are the basic difference between Antigens and Antibodies.

Major differences between Antigen and Antibody

Difference No.1: Definition

Antigen

• Antigens are any substance or molecules that interact with antibodies or by T cell receptor when complexed with MHC are called antigens.
• It include components of cell walls, capsules, flagella, toxins, viral proteins, bacteria and other microorganisms.
• Theoretically any foreign body that is capable of inducing an immune response can be called as an antigen.

Antibodies

• Antibodies are globular proteins or immunoglobulins synthesized by plasma cells of B cells that react specifically with antigen that stimulated antibody production.
• There are millions of antibodies inside our body that is specifically designed against different antigens in the surrounding.

Difference No.2 Chemical Nature

Antigen

• Antigens are generally proteins but can be carbohydrates, lipids or even nucleic acids.
• In the case of virus carbohydrate polysaccharide can be antigen.

Antibody

• Antibodies are glycoproteins made up of amino acids and small amount of carbohydrates.
• Antibodies also called immunoglobulins.

Difference No.3 : Basic Structure

Antigen

• Antigenic structure is highly complex in structure and composition.
• Antigens can be Simple to complex.
• Each antigen will interact with a specific antibody, this interactions is highly specific.

Antibody

• Antibody's 'Y' shaped structure consisting of 4 polypeptide chains, 2 heavy chains (H-chains) and 2 light chains (L-chains) joined by disulphide bonds.
• Each antibody contains two antigen binding site at the Fab region (variable region).

Difference No.4 : Interactions

Antigen

• The region of the antigen that interacts with the antibodies is called epitopes.
• A pathogen has many epitopes.
• This epitope interaction specifically with different antibodies.

Antibody

• The variable region of the antibody that specifically binds to an epitope of antigen is called paratope.
• Generally, a Y shaped antibody like IgG has 2 identical paratopes.

Difference No.5 : Role in Immune System

Antigens

• Antigens causes disease or allergic reaction.
• E.g. Cholera, Malaria, Polio, HIV etc.

Antibody

• Antibodies are involved in our defence system.
• Protects the system by immobilisation or lysis, agglutination, precipitation of antigenic material.
• Antibodies are the master molecules that is protecting us from diseases.

Alkaline Phosphates (ALP) : Level, Quantification principle, Isoenzyme & Clinical Symptoms

 Alkaline phosphates enzyme plays an important role in the growth and development of bones and teeth. It is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules.

  It is produced by osteoblasts of the bone and is associated with calcification process. It is localized in cell membranes and hence an ecto-enzyme.

  Alkaline phosphatase hydrolase enzyme catalyzes hydrolytic removal of phosphate group from mono-phosphoric esters including glycerophosphates, phenyl-phosphates, nucleotides, and proteins at alkaline pH (9 and 10) as its name indicates.
It is activated by magnesium (Mg++) and manganese(Mn++).

  Alkaline phosphatase (ALP) is an enzyme present in the canalicular and sinusoidal membranes of the liver and also active in many other tissues, particularly in bone, kidney, intestine and placenta. It is useful diagnostic test in bone and liver disorders.

Principle of Quantitative Analysis of ALP

  The principle for quantitative analysis of ALP in lab is Alkaline phosphatase in serum acts on the substrate paranitrophenyl phosphate buffered at pH 10 at 37°C to liberate paranitrophenol which gives yellow colour complex and this is measured at 405 nm.

Isoenzymes of Alkaline Phosphates

  It exists in six forms of isoenzymes. The isoenzymes are due to variation in carbohydrate content (sialic acid content), so they can also be called as isoforms. Affinity electrophoresis using polyacrylamide gel is used in separation and identification of fraction of ALP isoenzymes to differentiate liver or bone disorders.

The six isoenzymes of ALP are:

1. Alpha-1ALP:

•  Alpha-1 ALP is 10% of total ALP.
•  It moves in alpha 1 position in electrophoresis.
• It is synthesized by epithelial cells of biliary canaliculi and increased in obstructive jaundice, biliary cirrhosis and to some extent in metastatic carcinoma of liver.

2. Alpha-2, heat labile ALP:

• It is 25% of total ALP. 
• It is alpha 2 heat labile, stable at 56°C but loses its activity when kept at 65°C for 30 min.
• It is synthesized by hepatic cells and increased in hepatitis.

3. Alpha-2, heat stable ALP:

• It is only 1% of total ALP. It is also alpha 2 but heat stable isoenzyme.
• It is not destroyed at 65°C but inhibited by phenylalanine.
• It is of placental origin and found in blood in normal pregnancy.
• It increases in 2nd and 3rd trimester of normal pregnancy and its decrease indicates placental insufficiency and foetal death.

4. Pre-Beta ALP:

• It constitutes 50% of total ALP.
• It is pre-beta ALP and is most heat labile, loses its activity at 56°C within 10 min.
• It originates from the bone and is increased in rickets and Paget's disease.

5. Gamma ALP:

• It is 10% of total ALP.
• It is gamma ALP and is inhibited by phenylalanine.
• It is synthesized by intestinal cells and is increased in ulcerative colitis and after gastrectomy surgery.

6. Leukocyte ALP:

• It constitutes 4% of total ALP.
• It originates from leukocytes.
• It is increased in lymphomas and significantly decreased in chronic myeloid leukaemia.

Abnormal ALP Isoenzymes

Regan isoenzyme (carcino-placental isoenzyme):

• It is named after the first patient in whom it was detected.
• It is an abnormal ALP isoenzyme closely resembling placental ALP isoenzyme.
• It is seen in about 15% cases of carcinoma of lung, liver and gut. Chronic heavy smoking also increases regan isoenzyme in blood.

Nagao isoenzyme:

• It is an abnormal ALP isoenzyme present in carcinomas and metastasis.

Normal Levels of ALP

• Normal level of ALP in Adult is 40-125 IU/L.
• Normal level of ALP in Children is 42-362 IU/L.
• In children, ALP levels are more because of the increased osteoblastic activity in children.

Clinical Significance of Alkaline Phosphates

• Moderate (2-3 times) increase in ALP level is seen in hepatic diseases such as infective hepatitis, alcoholic hepatitis or hepatocellular carcinoma.
• Very high levels of ALP (10-12 times of upper limit) may be noticed in extrahepatic obstruction (obstructive jaundice) caused by gallstones or by pressure on bile duct by carcinoma of head of pancreas.
• Intrahepatic cholestasis may be due to virus (infective hepatitis) or by drugs (chlorpromazine).
• ALP is produced by epithelial cells of biliary canaliculi and obstruction of bile with consequent irritation of epithelial cells leads to secretion of ALP into serum.
• Drastically high levels of ALP (10-25 times of upper limit) are seen in bone diseases where osteoblastic activity is enhanced such as Paget's disease (osteitis deformans), rickets, osteomalacia, osteoblastoma, metastatic carcinoma of bone and hyperparathyroidism.

What is Major Histocompatibility Complex ?

 Antigens induce an immune response inside the body. Most of these antigens are large protein molecules some are polysaccharides and a few are glycoproteins or nucleoproteins.

Self antigens are the antigens which are found on the membranes of almost all the cells of human body and other vertebrate animals. These self antigens are known as major histocompatibility antigens. Major histocompatibility antigens are glycoproteins in chemical nature. major histocompatibility antigens are also known as histocompatibility antigens.

Discovery of Major Histocompatibility Antigens

These antigens were discovered when scientists were doing transplantation experiments on mice. Scientists found that sometimes the transplant tissue from the donor mouse was accepted by the recipient mouse but at other times it was rejected by the recipient Mouse.

  Now the question was why the tissues from one individual of a species were destroyed when introduced into member of the same species.
  The answer was given by an American Mouse geneticist George Snell. He gave the reason that this rejection was because the tissues of the donor and the recipient were incompatible. 

  In other words it was discovered that if the animals were not closely related the recipients rejected the grafts. So if the donor Mouse in the recipient Mouse are of the same genetic background or same inbred strain the transplanted tissue is accepted by the recipient. However if the donor and recipient Mouse are of genetically different backgrounds or different inbred mouse strains the transplanted tissue is rejected by the recipient.

A group of closely related genes was the cause of the rejection. This cluster of gene was named the Major Histocompatibility Complex or MHC. The major histocompatibility complex got its name from the fact that, The genes in this region encode proteins which determine whether a tissue transplanted between two individuals will be accepted or rejected.

Major Histocompatibility Complex Genes

It was discovered that these genes encode proteins which act as self antigens on the surface of the cells. These proteins are known as major histocompatibility antigens.
  These antigens gives the answer why rejection of the transplanted tissue happened when a tissue was transplanted from donor mouse to recipient Mouse.
  The Hajor Histocompatibility Antigens of donor Mouse were perceived as foreign or non-self by the recipient Mouse. As a result the immune system of recipient Mouse mount an immune response and result in the destruction of the transplanted tissue.

Major Histocompatibility Complex

In the term histocompatibility "histo" means tissue and "compatibility" means getting along or agreeable. The MHC got its name from the fact that the genes in this region encode proteins that determine whether a tissue transplanted between two individuals will be accepted or rejected.

  The term complex represents that these genes are localised to a large genetic region containing multiple loci. Since the molecules encoded by these genes were found to have main effects on histocompatibility to distinguish them from other molecules encoded by genome having minor effects on histocompatibility.  
They were called major histocompatibility molecules and the genes encoding these molecules were called major histocompatibility complex.

Major histocompatibility complex is defined as - A tightly linked cluster of genes whose products play important role in intercellular recognition and in discrimination between self and non-self.

Major histocompatibility complex genes  are present on chromosome 6 in humans and they are known as human leukocyte antigens (HLA) complex. In case of mice they are present on chromosome 17 and are known as H-2 complex in mice.

MHC molecules are found on all nucleated cells in the body and they play an important role in the development of both humoral and cellular immunity. B cells react with antigen on their own but T cells recognize antigens only in peptide form and that too when combined with an MHC molecule.

The main function of MHC molecules is to bring antigen to the cell surface for recognition by T cells in humans. The genes coding for MHC molecules are found on short arm of chromosome 6.

The MHC molecules are divided into three classes namely
  MHC Class 1
  MHC Class 2 
  MHC Class 3

MHC class 1 molecules are coded at three different locations or low sigh termed a b and c. These glycoproteins are expressed on all nucleated cells.

MHC class 2 genes are situated in the D region and there are several different loci known as DR DQ and DP. These glycoproteins appear only on cells that can process non-self materials and present antigen to other cells.

The area between the class MHC 1 and MHC class 2 regions on chromosome 6 is of MHC class 3 genes. These genes code for complement proteins and cytokines which take part in immune response but these molecules are not expressed on cell surface.

Bacterial Cell Wall Staining by Chance's Method

  Bacterial cell is consisting of various structural components. Cell wall is one of the most important component. Cell wall present outside of the bacterial cell membrane. It gives rigidity, protection and shape to the bacterial cell.

  Based on the structure of cell wall, all bacteria are divided into two groups as

• Gram positive and
• Gram negative.

Cell wall of Gram positive cell is monolayered while cell wall of gram negative bacteria is bilayered.
Bacterial cell wall can be demonstrated by various special staining methods like Chance's method, Ringer's method & Dyers method. The most common cell wall staining method is Chance's method.

Requirements :

• Clean grease free slide
• Nichrome wireloop
• 24 hrs old culture of bacteria
• 0.5% New fuchsin/Basic fuchsin solution (Basic Stain)
• 0.5 % Condo red solution (Acidic Stain)

Procedure & Steps

• Prepare the smear on the slide under aseptic conditions with the help of wire loop.
• Air dry the smear, but do not heat fix (Because the heat fixation changes the structure of capsule. therefore heat fixation is avoided here).
• Apply 0.5% New Fuchsin for 3 minutes.
• Remove the excess stain (but do not water wash)
• Apply 0.5 % Congo Red for 4 minutes.
• Gentle wash with water
• Air dry and observe under oil immersion objective lens.

Machanism and Principle of chance's cell wall staining method

  New Fuchsin stain is a basic stain. Therefore it is stained cell wall as well as cytoplasm. Cell wall and cytoplasm both are acidic in nature and they are having negative charge on their surface. However here the strong staining of cell wall take place. Because cell wall is more acidic than cytoplasm due to the presence of free carboxylic groups on its surface.

  Congo red is a acidic stain and this acidic stain bind with basic stain which is already present on the cell and removes that basic stain. That means here Congo red acts as a decolorizer. but the  removal of basic stain(New Fuchsin) takes place only from the cytoplasm and not from the cell wall, because it is strongly bound to the cell wall.

  Therefore after water wash cytoplasm becomes colourless while cell wall becomes pink coloured. that means here the role of Congo red is to carry out the decolorization, But the decolorization of only cytoplasm takes place and decolorization of cell wall does not take place.

Microscopic Observation

Spherical and rod shaped bacterial call wall Pink in colour.

 Under the microscope you will observed pink colour cell wall and colourless cytoplasm.

Gram Staining : Principle & Machanism, Steps, Disadvantages and Factors affecting gram Staining

Gram staining is a technique used to differentiate two major groups of bacteria based on their cell wall composition called gram positive and gram negative bacteria.

The technique was named after Hans Christian Gram who developed this method in 1884.
Gram staining is a preliminary test in the bacterial identification process. It plays an important role in clinical microbiology. It helps the medical professionals in the diagnosis of infectious diseases directly from the clinical sample.

The cell wall of gram positive bacteria is completely different from the gram negative bacteria. It is important for them to understand the Gram nature to provide the appropriate treatment for the infection.

Requirements of Gram Staining

• Clean grease free glass slide
• Nichrome wire loop
• Bacterial cell suspension
• 0.5% Crystal violet
• Gram's lodine
• 95% Ethanol
• 0.5% Safranine/basic fuchsin

Steps of Gram Staining

Imagine our sample has a mixed culture with gram positive cocci and gram negative rods

Step-1 (Smear Preparation)

• Prepare a thin smear of culture on a clean glass slide (oil free)
• Allow to air-dry.

Step-2 (Heat Fixation)

• Heat fix by exposing the smear to indirect flame
• Heat fix slides above burner by moving slides through the peak of the flame.
• Never heat fix wet slides !
• Purpose of heat fixing is to kill and immobilize bacteria on slide.

Step-3 (Primary stain)

• Add a few drops of Crystal violet on the smear.
• Wait for a minute.
• Wash the Crystal violet with water.
• At this stage, all cells appear in purple colour.

Step-4 (Mordant)

• Add plenty of Gram's lodine to the smear.
• Wait for 1 minute.
• Rinse with water.
• Cells appear to be purple yet.

Step-5 (Decolourising)

• Decolourise with 95% Ethyl Alcohol for not more than 30 seconds.
• Wash with water.
• At this stage, gram positive cells appear in purple colour And gram negative appear as colourless.

Step-6 (Counter stain)

• Add counter stain Safranin to the smear.
• Wait for 1 minute.
• Wash with water.

Step-7 (Microscopic Observation)

• Dry the slide and put a drop of immersion oil on the smear & Observe under 100X lens.

• Gram positive cells retain the purple colour
• Gram negative cells appear in pink colour.

Machanism of Gram Staining (Principle) :

The widely accepted theory is based on the differences in the cell wall composition between the gram-positive and gram-negative bacteria.

When crystal violet and iodine are added to the smear both will penetrate through the cell wall. And form a large crystal violet iodine complex (CVI complex) within the inner and outer layers of gram positive and gram negative bacteria.

The cell wall of gram-negative bacteria is thin and made of one or two layers of peptidoglycan. In addition to this it has got an outer lipopolysaccharide layer surrounding the cell wall.

  When a decolorizer like alcohol or acetone is added. The outer lipopolysaccharide layer will be completely dissolved leaving the thin peptidoglycan layer exposed.
The effective alcohol makes the peptidoglycan layer become perforated.

  In the decolorization step the gram-negative bacteria failed to retain the CVI complex and become colourless as the complex is washed away.

On contrary to this thick and multi-layered peptidoglycan in gram positive bacteria will be dehydrated by the addition of alcohol. Because of the thick peptidoglycan layer and dehydration by the alcohol treatment the CVI complex gets trapped in the cell.

  Therefore after the decolorization step the gram positive bacteria appear purple in color and the gram negative bacteria become colorless in the last step.

  When a counter stain likes a safranine is added the gram negative bacteria easily absorbed the stain and gives pink color to the cells. The gram positive bacteria remain purple in color due to the retention of CVI complex which is darker than Safranine.

Factors affecting Gram staining

• Age of culture.
• Excessive heat fixation.
• Thick smear or overcrowding of cells.
• Old staining reagents.
• Air drying.
• Over decolourization.

Disadvantages of Gram Staining

Athough the gram staining is used as primary test in the identification process this method will not be able to identify the bacteria to the species level. It will be used in combination of other modern and traditional identification tests
  The CVI complex may get lost from the gram positive bacteria due to over decolorization, which might lead to misinterpretation.
  There were evidences where some old gram positive cultures were not able to retain purple color and therefore observe as gram negative. 

Steps Involved in Transcription

 Transcription is a heterocatalytic function of DNA which involves transfer of coded information from DNA through the synthesis of RNA over the template of DNA. Only one of the strand of DNA called sense strand (master strand) is involved.
  The segment of DNA involved in transcription is cistron. It has a promoter region where initiation begins and a terminator region where transcription ends.

Enzyme involved in transcription is RNA polymerase. It is single in prokaryotes. There are three types of RNA polymerase in eukaryotes,
I for 28s, 18s & 5.8s RNA.
II for mRNA and SnRNA and
III for tRNA, 5s RNA & Sc RNA.

RNA polymerase has five polypeptides — σ, α, β, β' and ω).

σ or sigma factor recognises the promoter region while the remaining or core enzyme takes part in transcription.
A rho or ρ factor is needed for termination of transcription.

Steps involved in transcription

a] Activation of Ribonucleotides :

With the help of phosphorylase, energy and phosphoric acid, ribo- nucleoside monophosphates are changed into triphosphates ATP, GTP, CTP & UTP.

b] Cistron :

It has a promoter region where transcription begins and a terminator region where transcription ends.
Promoter region possesses RNA polymerase recognition as well as RNA-polymerase binding sites.
Sigma factor or RNA polymerase gets attached to promoter region of cistron. The two DNA strands separate with the help of unwindase and helix destabilising proteins.
One of them function as sense strand and takes part is transcription. Transcription proceeds in 5' to 3' direction.

c] Base Pairing :

Ribonucleoside triphosphate come to lie opposite complementary nitrogen bases of sense strand (A opposite T, C Copposite G, U opposite A and G opposite C).
Pyrophosphatase hydrolyses two phosphates from cach activated nucleotide. This releases energy.

d] Strand Formation : 

RNA polymerase establishes phosphodiester bonds between adjacent ribonucleotides.
Sigma factor σ recognises.
Core enzymes moves along the sense strand till it reaches terminator region and separates from DNA template in the presense of rho (ρ) factor.
Terminator region contains a stop signal made of 4-8 A (poly A tail) nucleotides.

e] Separation of RNA :

rho (ρ) factor has ATP ase activity. It helps in separating the newly formed RNA or primary transcript from the sense strand of DNA.

f] Duplex Formation :

With the release of primary transcript the sense and antisense strands of DNA re-establish their hydrogen linkages and form duplex. Unwindase and helix destablising proteins are released.

g] Post-Transcriptional Processing :

Primary transcript is often called heterogenous or hn RNA as it is generally bigger than the functional RNA.

1. Cleavage : hn RNA is broken to form smaller pieces. Ribonuclease-P (RNA enzyme) separates 5-7 t RNA precursors from primary transcript.
2. Splicing : Introns or intervening sequences of nonessential nature are removed by nucleases. Ribozyme (RNA enzyme) is one such enzyme.
3. Terminal Additions : Nucleotides are added at the ends of RNA for specific functions, e.g. CCA at 3'ends of tRNA, cap nucleotides at 5'ends of m RNA or poly A nucleotides at 3'end of m RNA.
4. Nucleotide Modifications : Certain nucleotides are methylated, ethylated, deaminated, etc. to produce different chemicals like inosine, methyl cytosine, dihydrouracil, pseudouracil, etc.

The promoters of prokaryotic genes are simple. They generally consist of about 40 bp of DNA that contain two elements :

• a TATA box, located 10 bp upstream from the transcription initiation site and
• a second element, located 35 bp in the 5' direction upstream.

Eukaryotic promoters are more complex and usually consist of several elements. One set of elements ensures that transcription initiates at the correct site. The elements in this set, which of ten include a TATA box, which are usually found within 100-200 bp of transcription start site. Another set of clements, usually located further 5' from the transcription initiation site, can enhance or silence transcription. These work with, or serve as tissue specific or signal response elements.

Ribosomal RNA is also synthesized as a large 45s precursor molecule that is processed into the mature 28s, 18s and 5.8s fomms.

Metabolism: Definition, Terminology, Feaction & Function

The metabolism applies to the assembly of biochemical reactions which are employed by the organisms for the synthesis of cell materials and for the utilization of energy from their environments.

  Metabolism may be defined as 'the sum total of all the enzyme-catalyzed reactions that occur in an organism'.

  The large number of reactions in a cell are organized into a relatively small number of sequences or pathways. It is a highly coordinated and purposeful cell activity, in which multienzyme systems co-operate. This obviously points out to the fact that the metabolism of even a simple unicellular organism is time variant.

Metabolism in microorganisms and the human beings:

a] Microorganisms like bacteria (e.g., Escherichia coli) can double in number every 40 minutes in a culture medium containing only glucose and inorganic salts, or in 20 minutes in a rich broth. 

  The components of the medium are depleted and very little is added to the medium by the cells. Each cell contains hundreds to thousands of molecules of each of about 2,500 different proteins, about 1,000 types of organic compounds and a variety of nucleic acids. It is, thus, apparent that the bacterial cells participate in a variety of metabolic activities.

b] Human adults maintain a constant weight for about 40 years, during which period a total-~1! about 60 quintals of solid food and 45,000 litres of water are metabolised. And yet boll body weight and body composition remain almost constant.

TERMINOLOGY OF METABOLISM

The various processes constituting metabolism may be divided, somewhat arbitrarily, into catabolism and anabolism. Those processes, whose major function is the generation of chemical energy in forms suitable for the mechanical and chemical processes of the cells, are termed as catabolism; whereas those processes, which utilize the energy generated by catabolism for the biosynthesis of cell components, are termed as anabolism.

The various activities powered by catabolism include mechanical movement, growth, reproduction, accumulation of foods, elimination of waste, generation of electricity, maintenance of temperature etc. The various anabolic activities may be exemplified by food manufacture etc.

Some processes can be either catabolic or anabolic, depending on the energy conditions in the cell. These are referred to as amphibolism.

Catabolism reaction
Fuels(carbohydrates, fats) ➞ CO₂+ H₂O + Useful energy
Anabolic reaction
Useful reaction + Small molecules ➞ Complex molecules

FUNCTION OF METABOLISM

In all cells, metabolism enables the cell to perform its vital functions. Metabolism performs following 4 specific functions:

1. to obtain chemical energy from the degradation of energy-rich nutrients or from captured solar energy.
2. to convert nutrient molecules into precursors of cell-macromolecules.
3. to assemble these precursors into proteins, lipids, polysaccharides, nucleic acids and other cell components.
4. to form and degrade biomolecules required in specialized functions of cells.

Metabolism are closely interrelated since the synthesis of the molecules, that are a component of cell, requires an input of energy, while at the same time it is obvious that the cell components are needed to provide the energy supply and to control intracellular solute concentrations.

The specialized functions also require biosynthetic processes as well as a supply of energy. Terms catabolism and dissimilation are synonyins and refer to the pathways or routes breaking down food materials into simpler compounds and resulting in the release of energy contained in them.

The processes of anabolism or assimilation utilize food materials and energy to synthesize cell components.

The energy relations of the biological processes, the term excrgonic is used to denote a chemical reaction which liberates chemical-free energy. The term exothermic refers to the total energy liberated including heat.

  As the magnitude of heat energy is small and also that it cannot drive biological reactions, the biochemists are more interested in free energy changes and often use drive term exergonic.

The corresponding energy-consuming term endergonic refers to the processes which require an input of free energy while the term endothermic denotes a total energy requirement (including heat).

The Five Kingdom Classification by Robert Whittaker & It's Limitations

 Robert Whittaker (1969), proposed the first popular classification system called five-kingdom system, which was accepted widely. The classification system is based on three criteria.

1. Cell types

1. Prokaryotie (cells with primitive nucleus and lacking membrane enclosed organelles) and
2. Eukaryotic (cells with well-developed nucleus and membrane-enclosed organelles.

2. Level of cellular organization 

1. Unicellular or
2. Multicellular.

3. Mode of nutrition

1. Photoautotrophic nutrition, which is concerned with use of sunlight as energy source and CO2 as source of carbon.
2. Heterotrophic nutrition, which is concerned with use of organie compounds as source of energy and carbon. It may be absorptive (which means absorption of nutrients by body wall) and
3. Ingestive (which means intake of solid food particles ).

  This system consists of one prokaryotic kingdom of Monera (which are prokaryotes) and four eukaryotic kingdoms - Protista, Fungi, Plantae and Animalia.

The Five Kingdom Classification  by Robert Whittaker

I] Kingdom Monera (Prokaryotes)

• It includes bacteria, cyanobacteria and archaebacteria.
• Unicellular, microscopic, solitary or colonial forms.
• Can respire aerobically or anaerobically and reproduce by asexual, sexual or vegetative methods.
• Act as decomposers and mineralisers and some may be concerned with nitrogen fixation also.
• No membrane bound organelles like mitochondria and Golgi complex. No nuclear membrane.
• Mode of nutrition: Nutritionally. these organisms exhibit great diversity from autotroph to heterotroph, phototroph to chemotroph and organotroph to lithotroph.

II] Kingdom Protista (Unicellular Eukaryotes)

• Usually (phytoplanktons or zooplanktons).
• Organisation ranges from unicellular to multicellular colonial forms. aquatic pue planktonic
• Some are commensals and some are parasites (heterotrophic or absorptive mode of nutrition)
• Some coloured algae contain various types of accessory pigments.
• Reproduction is by asexual as well as sexual modes and both haploid as well as diploid forms exist.
• Locomotion pseudopodial, ciliary or flagellar depending on the type of locomotary appendages present.
• The phytoplanktons are producers whereas the zooplanktons are consumers (holozoic nutrition).

III] Kingdom Fungi (Multicellular Decomposers)

• Include non-green plants that are the important decomposers and mineralizers.
• Hyphae constitute an entangled cottony mass of filaments termed mycelium that may be aseptate coenocytic or septate multicellular.
• Possess a cell wall made up of chitin or fungal cellulose and membrane bound organelles.
• May be solitary unicellular types or multicellular filamentous types called hyphae.
• Plastids are absent.
• Mode of nutrition heterotrophic (absorption and extracellular digestion), saprophytic or parasitic.
• Non-motile in forms but some may produce motile zoospores.
• Embryo formation does not occur but various fruiting bodies are formed.
• Most members pathogenic on plants as well as animals and cause various diseases as rust, smut and mildew (in higher plants).
• Useful fungi include yeast, Penicillium etc. used in bakery, brewery and antibiotic industries.
• Intercellular mycelia give out haustoria that absorb the nutrients from host cells.
• Asexual and sexual reproduction observed: members with no sexual reproduction known fungi imperfecti.
• Edible fungi: mushrooms and morels.

IV] Kingdom Plantae (Multicellular Producers)

• Members consist of muiticellular, green, photosynthetic, primary producers of the biosphere.
• Aquatic as well as terrestrial, large-bodied and non-motile (except bryophytes which produce motile zoospores).
• Cells bound by cellulosic cell wall and contain the photosynthetic pigments or chloroplast.
• Higher plants have complex cellular organization and vascular tissues (tracheophytes).
• Reproduction: by vegetative and sexual methods.
• All plants produce seeds except bryophytes and pteridophytes.
• Nutrition: autotrophic, absorptive (insectivorous plants) or parasitic (totally parasitic e.g. Cuscuta or partial parasite, e.g. Santalum).
• Lower plants possess simple thalloid organization and lack vascular tissue (bryophytes).

V] Kingdom Animalia (Multicellular Consumers)

• Consist of multicellular, eukaryotic organisms, lacking cell wall, plastids and photosynthetic pigments.
• Nutrition: by ingestion and subsequent digestion within the gastric cavity some possess absorption type of nutrition (parasites).
• Reproduction: asexual or sexual in lower forms while predominantly sexual in higher forms.
• Animals: main consumers in the food chain and may be primary consumers (herbivores) or secondary consumers (carnivores).
• Bear locomotory appendages and are, therefore, motile.
• Characterized by well developed muscular and nervous systems.

Limitations of Five Kingdom System

Most microbiologists do not accept the five-kingdom system for following reasons.

1. It includes prokaryotes in Monera, but it does not distinguish between Bacteria and Archaea.
2. There is a great diversity in kingdom Protista. Protozoa are heterotrophs with animal like cell organization. Algae are photoautotrophs with plant like cell organization.
3. Boundaries between Protista, Plantae and Fungi are poorly defined. For example, the red algae is not closely related to plants, still it is included in Plantae.
4. It does not explain about evolution and phylogenetic relationship.