Chemical Bonding

Chemical bond:-  

Chemical bond is the attractive force which holds together the constituents atoms or ions  or molecules present in a substance.

There are three types of chemical bonds: 

Ionic Bond or electrovalent bond

Covalent Bond

Co-ordinate Bond.



Octet Rule: 


Atoms form chemical bonds in order to complete their octet i.e. eight electrons in their valence shell.




Ionic bond or electrovalent bond:

"Ionic bond is formed by the complete transfer of one or more electrons from the outermost energy shell of one atom to the outermost energy shell of another atom". 

Ex:-

When sodium (Na) and chlorine (Cl) are combined, the sodium atoms each lose an electron, forming cations (Na+), and the chlorine atoms each gain an electron to form anions (Cl−). These ions are then attracted to each other in a 1:1 ratio to form sodium chloride (NaCl).






Factors  favouring of Ionic bond:

1)low ionisation energy of the metal atoms.

2)Higher electron affinity of non-metal atoms.

3)Higher value of lattice energy of the resulting Ionic crystals.

4)Large radius of cations and small radius of anions.

5)Large differences of eletronegativity between the combining atoms.




Co-valent Bond:

A covalent bond is a force which binds atoms of same or different elements by mutual sharing of electrons.

Ex:-1:



 2:-







Factors favouring covalent bonds:



1)The combining atoms should have small difference in electronegativity.

2)The combining atoms should obtain octate structure by sharing one or more electrons.

3)High electron affinity.

4)Small atomic size.

5)High ionisation energy.





Bond Length:



"The distance between the nuclei of two atoms bonded together is called bond length".







The lengths of double bonds are less than the lengths of single bonds between the same two  atoms, and triple bonds are even shorter than double bonds.
Single bond > Double bond > Triple bond (decreasing bond length)




HYBRIDIZATION:


The mixing or merging of dissimilar orbitals of similar energies to form new orbitals is known as hybridisation.
               There are many different types of hybridization depending upon the type of orbitals involved in mixing such as sp3, sp2 , sp. sp3d. sp3d2 , etc.


Formation of methane (CH4):
    In methane carbon atom acquires sp3 hybrid states as described below: 
Here, one orbital of 2s-sub-shell and three orbitals of  2p-sub-shell of excited carbon atom undergo hybridization to  form four sp3 hybrid orbitals. 
  The process involving promotion of 2s-electron followed by hybridization.






 
As pointed out earlier the sp3 hybrid orbitals of carbon atom are directed towards the comers of regular tetrahedron.
Each of the sp3 hybrid orbitals overlaps axially with half filled Is-orbital of hydrogen atom constituting a sigma bond.


Because of sp3 hybridization of carbon atom, CH4 molecule has tetrahedral shape.. 


Formation of ethane:


In ethane both the carbon atoms assume sp3 hybrid state. 
One of the hybrid orbitals of carbon atom overlaps axially with similar orbital of the other carbon atoms to form sp3-sp3 sigma bond.
 The other three hybrid orbitals of each carbon atom are used informing sp3-s sigma bonds with hydrogen atoms. 






Each C-H bond in ethane is sp3-s sigma bond with bond length 109 pm. The C-C bond is sp3-sr sigma bond with bond length 154 pm.

Formation of ammonia (NH)3 molecule;


In NH3 molecule the nitrogen atom adopts sp3-hybrid state.
Three of sp3-hybrid orbitals of the N atom are used for forming sp3 (sigma) bonds with H atoms.
The fourth sp3-hybrid orbital carry lone pair of electrons.
The relatively larger lone pair bond pair interactions cause HNH angle to decrease from 109°.281 to 107°. 
The ground state, hybrid state of N atom and orbital overlap in are shown in Fig.










Formation of water (H2O) molecule.


In H2O molecule, oxygen atom adopts sp3 hybrid state.
Two of the sp3-hybrid-orbitals of oxygen contains lone pairs of electrons whereas the other two hybrid orbitals constitutesp3-scr (sigma) bonds with H atoms. 
The lone pair orbitals exert relatively greater repulsive interactions on bond pair-orbitals causing HOH angle to decrease from 109°.28′ to 104.5°. 
The hybridization Of O atom along with orbital overlap in  molecule are shown in Fig.

Enzymes- types and their functions for dmlt and paramedical students

Different types of Enzymes in the human body and their functions:

What are enzymes and what is their function?

1)Enzymes are naturally occurring proteins that are found in the bodies of certain living things, including humans and other animals, and that cause chemical changes such as breaking down food in the stomach.

2)Within the human body, enzymes can be found in bodily fluids, such as blood, saliva, the gastric juices or the stomach and fluids in the intestines.

3)In general, enzymes serve as catalysts for biological functions, including natural, involuntary bodily functions, such as blood clotting.

Enzymes have three main characteristics.

1) They increase  the rate of a natural chemical reaction.
2)They typucally only react with one specific  substrate or reactant,
3) Enzyme activity is regulated and controlled within the cell  through several different means, including regulation by inhibitors and activators.

  There are 6 major classes of enzymes found in the body.

The following are the names of enzymes and their functions.

Ligase:
This enzyme in the body requires ATP  and binds nucleotides together in the nucleic acids.
It also binds simple sugars in polysaccarides.

Lyase:
This enzyme in the body breaks the bonds between carbon atoms or carbon nitrogen bond.

Hydrolase:
This enzyme in the body breaks large molecules into similar molecules by adding a water molecule.

Transferase:
This enzyme in the body cuts a part of one molecule and attaches it to another molecule.

Isomerase:
The atoms in a molecule are rearranged without changing their chemical formula.
This helps in getting carbohydrate molecules for certain enzymatic processes.

Oxido-reductase:
This enzyme removes hydrogen or electrons from one molecule and donates it to another molecule.

This enzyme is mainly involved in mitochondrial energy production.

Kinase:
This enzyme in the body attaches a phosphate group to a high energy  bond.
It is a very important enzyme required for ATP production and activation of certain enzymes.

Structure of Atoms for all paramedical students kaarnataka

Structure of Atoms:
          An atom is the smallest unit of matter that retains all of the chemical properties of an element.

Atoms consist of three basic particles: protons, electrons, and neutrons.

The nucleus (center) of the atom contains the protons (positively charged) and the neutrons (no charge).

Atoms have different properties based on the arrangement and number of their basic particles.

Subatomic Particles:

Protons:

Protons were discovered by Ernest Rutherford in the year 19191.

Protons are having positively charge.

Protons are having mass is- 1.672x10^-24kg

Protons are exist in nucleous.

Electrons:

Electrons were discovered by J.J.Thomson in 1897.

Electrons are having negatively charge. 

Electrons are having mass is- 9.109x10^-28kg

Electrons surround the atomic nucleus in pathways called orbitals.
While electrons are revolving they lose or gain energy.

The distribution of electrons in orbit by using formula 2n^2

Neutrons:

Neutrons are having zero charges.

Neutrons are having mass is 1.6749x10^-27 kg.

Determination of Equivalent Mass Method — I (Hydrogen Displacement Method):

Principle:

The known mass of a metal to react with dilute acids and volume of hydrogen produced in the reaction is measured and the equivalent mass of an element is calculated using formula

o This method is useful for the metals which react, or dissolves in mineral acids and liberates hydrogen gas. e.g. Mg, Zn, Al, Ca, Zn, Sn etc.

1) Clean and weigh accurately a piece of metal (Mg / Zn / Al) whose equivalent mass is to be found and place it in a conical flask containing distilled water.
2) The mouth of the conical flask is fitted with a cork through which side tube (gas carrying tube) and a thistle tube are inserted as shown in the diagram.
3) Using side tube the conical flask is connected to graduated (calibrated) test tube called Eudiometer.
4) Eudiometer tube is completely filled with water and is inverted on the side tube as shown in the diagram.
5) The bottom part of thistle tube is dipped in the water in the flask.
6) Mineral acid like HCI is added to the flask through the thistle funnel. 

7) The reaction between the metal and acid takes place and hydrogen gas is liberated which is collected in eudiometer by downward displacement of water.

8) When the evolution of gas has stopped the mouth of the eudiometer tube is closed with a thumb and carried to a tray of water where the level of water inside and outside the tube is equalised and the volume of hydrogen gas is read at atmospheric pressure.

Observations: 

The weight of metal piece = W g

The volume of hydrogen collected = V dm3. 

Atmospheric pressure = P mm 

Aqueous tension= f mm of Hg

Absolute temperature = T K

Calculations: 

Now the volume of hydrogen at S.T.P. from the above data using following formula is calculated. 

 Where Volume of hydrogen at S.T.P. Vo dm3, 

Pressure at S.T.P. Po mm of Hg = 760 mm = 1.013 x 10^5 N/m2 

The absolute temperature at S.T.P. To K = 273 K 

Microbiology - bacterial Growth

BACTERIAL GROWTH

Growth of Bacteria is the orderly increase of all the chemical constituents of the bacteria. Multiplication is the consequence of growth. Death of bacteria is the irreversible loss of ability to reproduce.

Bacteria are composed of proteins, carbohydrates, lipids, water and trace elements.

Factors Required for Bacterial Growth
The requirements for bacterial growth are:

(A)) Environmental factors affecting growth, and

(B) Sources of metabolic energy.

A. Environmental Factors affecting Growth:
1. Nutrients. Nutrients in growth media must contain all the elements necessary for the synthesis of new organisms. In general the following must be provided : (a) Hydrogen donors and acceptors, 
(b) Carbon source, (c) Nitrogen source, (d) Minerals : sulphur and phosphorus, (e) Growth factors: amino acids, purines, pyrimidines; vitamins, (f) Trace elements: Mg, Fe, Mn.

Growth Factors: 
A growth factor is an organic compound which a cell must contain in order to grow but which it is unable to synthesize. These substances are essential for the organism and are to be supplied as nutrients. Thiamine, nicotinic acid, folic acid and para-aminobenzoic acid are examples of growth factors.

Essential Metabolites: These metabolites are essential for growth of bacterium. These must be synthesized by the bacterium, or be provided in the medium. Mg, Fe and Mn are essential trace elements.

Autotrophs live only on inorganic substances, i.e. do not require organic nutrients for growth. They are not of medical importance.

Heterotrophs require organic materials for growth, e.g. proteins, carbohydrates, lipids as source of energy. All bacteria of medical importance belong to heterotrophs. Parasites may depend on the host for certain foods. Saprophytes grow, on dead organic matter.

2.   pH of the medium. Most pathogenic bacteria grow best in pH 7.2-7.4. Vibno cholerae can grow in pH 8.2-9.0.

3.   Gaseous Requirement
(a) Role of Oxygen. Bacteria may be classified into four groups on oxygen requirement :
(i)    Aerobes. They cannot grow without oxygen, e.g. Mycobacterium tuberculosis.
(ii)   Facultative anaerobes. These grow under both aerobic and anaerobic conditions. Most bacteria are facultative anaerobes, e.g. Enterobacteriaceae.
(iii)Anaerobes. They only grow in absence of free oxygen, e.g. Clostridium, Bacteroides.
(iv)   Microaerophils grow best in oxygen less than that present in the air, e.g. Campylobacter.

Aerobes and facultative anaerobes have the metabolic systems: (1) cytochrome systems for the metabolism of oxygen, (2) Superoxide dismutase, (3) catalase.

Anaerobic bacteria do not grow in the presence of oxygen. They do not use oxygen for growth and metabolism but obtain their energy from fermentation reactions. Anaerobic bacteria are killed by oxygen or toxic oxygen radicals. Multiple mechanisms play role for oxygen toxicity : (1) They do not have cytochrome systems for oxygen metabolism, (2) They may have low levels of superoxide dismutase, and (3) They may or may not have catalase.

(b) Carbon dioxide. All bacteria require CO2 for their growth. Most bacteria produce CO2. N. gonorrhoeae and N. meningitides and Br abortus grow better in presence of 5 per cent CO2.

4.   Temperature. Most bacteria are mesophilic. Mesophilic bacteria grow best at 30-37°C. Optimum temperature for growth of common pathogenic bacteria is 37°C. Bacteria of a species will not grow but may remain alive at a maximum and a minimum temperature.

5.   Ionic strength and osmotic pressure.

6.   Light. Optimum condition for growth is darkness. 

B.Sources of Metabolic Energy
Mainly three mechanisms generate metabolic energy. These are fermentation, respiration and photosynthesis. An organism to grow, at least one of these mechanisms must be used.

REPRODUCTION
Bacteria reproduce by binary fission. Multiplication takes place in geometric progression. The nucleus (chromosome) undergoes duplication prior to cell division. When the cell grows about twice its size, the nuclear material divides, and a transverse septum originates from plasma membrane and cell wall and divides the cell into two parts. The two daughter cells receive an identical set of chromosomes. The daughter cells separate and may be arranged singly, in pairs, clumps, or chains.

GROWTH CURVE
The growth curve indicates multiplication and death of bacteria. When a bacterium is inoculated in a medium, it passes through four growth phases which will be evident in a growth curve drawn by plotting the logarithm of the number of bacteria against time.
Number of bacteria in the culture at different periods may be :
 (1) Total count. It includes both living and dead bacteria, or 
(2) Viable count. It includes only the living bacteria. 
Microbial concentration can be measured in terms of cell concentration, i.e. the number of viable cells per unit volume of culture, or of biomass concentration, i.e. dry weight of cells per unit volume of culture.

Growth Phases

1. Lag Phase. In this phase there is increase in cell size but not multiplication.
Time is required for adaptation (synthesis of new enzymes) to new environment. 
During this phase vigorous metabolic activity occurs but cells do not divide.
Enzymes and intermediates are formed and accumulate until they are present in concentration that permits growth to start. 
Antibiotics have little effect at this stage.

2. Exponential Phase or Logarithmic (Log) Phase.

The cells multiply at the maximum rate in this exponential phase, i.e. there is linear relationship between time and logarithm of the number of cells.
Mass increases in an exponential manner. 
This continues until one of two things happens: either one or more nutrients in the medium become exhausted, or toxic metabolic products, accumulate and inhibit growth. 
Nutrient oxygen becomes limited for aerobic organisms. 
In exponential phase, the biomass increases exponentially with respect to time, i.e. the biomass doubles with each doubling time. 
The average time required for the population, or the biomass, to double is known as the generation time or doubling time. 
Linear plots of exponential growth can be produced by plotting the logarithm of biomass concentration as a function of time.
Importance : Antibiotics act better at this phase.

3.  Maximal Stationary Phase.
Due to exhaustion of nutrients or accumulation of toxic products death of bacteria starts and the growth cease completely. 
The count remains stationary due to balance between multiplication and death rate. 
Importance: Production of exotoxins, antibiotics, metachromatic granules, and spore formation takes place in this phase.

4. Decline phase or death phase. In this phase there is progressive death of cells. 
However, some living bacteria use the breakdown products of dead bacteria as nutrient and remain as persister

Microbiology - Cultural media for dmlt students

TYPES OF CULTURE MEDIA:

Media are of different types on consistency and chemical composition.A. On Consistency:
1.   Solid Media. Advantages of solid media: 
(a) Bacteria may be identified by studying the colony character, 
(b) Mixed bacteria can be separated. Solid media is used for the isolation of bacteria as pure culture.

 'Agar' is most commonly used to prepare solid media. 

Agar ispolysaccharide extract obtained from seaweed. 
Agar is an ideal solidifying agent as it is : (a) Bacteriologically inert, i.e. no influence on bacterial growth, 
(b) It remains solid at 37°C, and 
(c) It istransparent.

2.Liquid Media. It is used for profuse growth, 
e.g. blood culture in liquid media. 
Mixed organisms cannot be separated.

B. On Chemical Composition :
1.Routine Laboratory Media
2.Synthetic Media.
These are chemically defined media prepared from pure chemical substances. 
It is used in research work.

ROUTINE LABORATORY MEDIA
These are classified into six types:
(1) Basal media, (2) Enriched media,
(3) Selective media, (4) Indicator media, (5) Transport media, and
(6) Storage media.

1. BASAL MEDIA. Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media.
Examples: Nutrient broth, nutrientagar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media.

2.ENRICHED MEDIA. The media are enriched usually by adding blood, serum or egg.
Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media.

3.SELECTIVE MEDIA. These media favour the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria.
Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media. 
Antibiotic may be added to a medium for inhibition.

4.INDICATOR (DIFFERENTIAL) MEDIA. 

An indicator is included in the medium. A particular organism causes change in the indicator, 
e.g. blood, neutral red, tellurite. 

Examples: Blood agar and MacConkey agar are indicator media.

5.   TRANSPORT MEDIA. 
These media are used when specie-men cannot be cultured soon after collection. 

Examples: Cary-Blair medium, Amies medium, Stuart medium.

6.   STORAGE MEDIA. 
Media used for storing the bacteria for a long period of time.
Examples: Egg saline medium, chalk cooked meat broth.

COMMON MEDIA IN ROUTINE USE

Nutrient Broth. 500 g meat, e.g. ox heart is minced and mixed with 1 litre water. 10 g peptone and 5 g sodium chloride are added, pH is adjusted to 7.3. Uses: (1) As a basal media for the preparation of other media, (2) To study soluble products of bacteria.

Nutrient Agar. It is solid at 37°C. 2.5% agar is added in nutrient broth. It is heated at 100°C to melt the agar and then cooled.

Peptone Water. Peptone 1% and sodium chloride 0.5%. It is used as base for sugar media and to test indole formation.

Blood Agar. Most commonly used medium. 5-10% defibrinated sheep or horse blood is added to melted agar at 45-50°C. 
Blood acts as an enrichment material and also as an indicator.
Certain bacteria when grown in blood agar produce haemolysis around their colonies. 
Certain bacteria produce no haemolysis.

 Types of changes : (a) beta  (b)haemolysis. 
The colony is surrounded by a clear zone of complete haemolysis,
e.g. Streptococcus pyogenes is a beta haemolytic streptococci, 

(b) haemolysis.
The colony is surrounded by a zone of greenish discolouration due to formation of biliverdin, 
e.g. Viridans streptococci, (c) Gamma (y) haemolysis, or, No haemolysis.
There is no change in the medium surrounding the colony,

Chocolate Agar or Heated Blood agar.

Prepared by heating blood agar.
It is used for culture of pneumococcus, gonococcus, meningococcus and Haemophilus.
Heating the blood inactivates inhibitor of growths.

MacConkey Agar. 

Most commonly used for enterobac­teriaceae.
It contains agar, peptone, sodium chloride, bile salt, lactose and neutral red. 
It is a selective and indicator medium :

(1)   Selective as bile salt does not inhibit the growth of enterobactericeae but inhibits growth of many other bacteria.

(2)   Indicator medium as the colonies of bacteria that ferment lactose take a pink colour due to production of acid. Acid turns the indicator neutral red to pink. These bacteria are called 'lactose fermenter', 
e.g. Escherichia coll. 

Colourless colony indicates that lactose is not fermented, i.e. the bacterium is non-lactose fermenter, 
e.g. Salmonella. Shigella, Vibrio.

Mueller Hinton Agar. Disc diffusion sensitivity tests for antimicrobial drugs should be carried out on this media as per WHO recommendation to promote reproducibility and comparability of results.

Hiss's Serum Water Medium.

This medium is used to study the fermentation reactions of bacteria which can not grow in peptone water sugar media, 
e.g. pneumococcus, Neisseria, Corynebacterium.

Lowenstein-Jensen Medium.

 It is used to culture tubercle bacilli. It contains egg, malachite green and glycerol. 
(1) Egg is an enrichment material which stimulates the growth of tubercle bacilli, (2) Malachite green inhibits growth of organisms other than mycobacteria,
(3) Glycerol promotes the growth of Mycobacterium tuberculosis but not Mycobacterium bovis.

Dubos Medium. 
This liquid mediumis used for tubercle bacilli. In thismedium drug sensitivity of tubercle bacilli can be carried out.
It contains 'tween 80', bovine serum albumin, casein hydrolysate, asparagin and salts.
Tween 80 causes dispersed growth and bovine albumin causes rapid growth.

Loeffler Serum. 
Serum is used for enrichment. 
Diphtheria bacilli grow in this medium in 6 hours when the secondary bacteria do not grow.
It isused for rapid diagnosis of diphtheria and to demonstrate volutin granules.
It contains sheep, ox or horse serum.

Tellurite Blood Agar. 
It is used as a selective medium for isolation of Cotynebacterium diphtheriae.
Tellurite inhibits the growth of most secondary bacteria without an inhibitory effect on diphtheria bacilli.
It is also an indicator medium as the diphtheria bacilli produce black colonies. Tellurite metabolized to tellbrism, which has black colour.

EMB (Eosin-methylene blue) Agar.

A selective and differential medium for enteric Gram-negative rods.
Lactose-fermenting colonies are coloured and non lactose-fermenting colonies are non pigmented. 
Selects against gram positive bacteria.

XLD (Xylose Lysine Deoxychoiate).

It is used to isolate Salmonella and Shigella species from stool specimens. This is a selective media.

SS (Salmonella-Shigella) Agar. 

It isa selective medium used to isolate Salmonella and Shigella species. 

SSAgar with additional bile salt is used if Yersinia enterocolitica issuspected.

DCA (Desoxycholate Citrate Agar).

It is used for isolation of Salmonella and Shigella.
The other enteric bacteria are mostly inhibited (a selective medium).
It is also a differential (indicator) medium due to presence of lactose and neutral red.

Tetrathionate Broth. This medium isused for isolating Salmonella from stool. It acts as a selective medium. It inhibits normal intestinal bacteria and permits multiplication of Salmonella.

Selenite F Broth. Uses and functions are same as that of tetrathionate broth.

Thiosulphate-Citrate-Bile-Sucrose (TCBS) Agar. TCBS agar is a selective medium used to isolate Vibrio cholerae and other Vibrio species from stool.

Charcoal-yeast agar. Used for Legionella pneumophila. Increased concentration of iron and cysteine allows growth.

Tellurite-Gelatin Agar Medium (TGAM). It may be used as transport, selective and indicator medium.

Campylobacter Medium. This selective medium is used to isolate Campylobacter jejuni and Campylobacter coli from stool.

Cary-Blair Medium. It is used as a transport medium for faeces that may contain Salmonella, Shigella, Vibrio or Cam­pylobacter species.

Amies medium is used for gonococci and other pathogens.

Peptone Water Sugar Media. These indicator media are used to study 'Sugar fermentation'. 1 % solution of a sugar (lactose, glucose, mannitol etc) is added to peptone water containing Andrade's indicator in a test tube. 
A small inverted Durham tube is placed in the medium. The media are colourless. 
After culture, change of a medium to red colour indicates acid production. 
Gas, if produced collects in Durham tube.

Motility Indole Urea (MIU) Medium.
This is used to differentiate enterobacteria species by their motility, urease, and indole reactions.

KIA (Kligler Iron Agar).
This is a differential slope medium used in the identification of enteric bacteria.
The reactions are based on the fermentation of lactose and glucose and the production of hydrogen sulphide.

Christensen's Urea Medium. This is used to identify urea splitting organisms,
e.g. Proteus. A purple pink colour indicates urea splitting.

Bordet-Gengou Medium. This medium is used for culture of Bordetella pertussis.

 Increased concentration of blood allows growth.
It contains agar, potato, sodium chloride, glycerol, peptone and 50% horse blood. Penicillin may be added to it.

Microbiology - Sterilization and Disinfectants fot dmlt,dotat,ddt students

• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 
  
• 

Sterilization

Sterilization is the killing or removal of all microorganisms, including bacterial spores which are highly resistant. Sterilization is an absolute term, i.e. the article must be sterile meaning the absence of all microorganisms.

Disinfection is the killing of many, but not all microorganisms. It is a process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e. pathogens must be killed. Some organisms and bacterial spores may survive.

Disinfectants are chemicals that are used for disinfection. Disinfectants should be used only on inanimate objects.

Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill microorganisms, e.g. on the surface of skin and mucous membranes.
Uses of Sterilization
1.   Sterilization for Surgical Procedures: Gloves, aprons, surgical instruments, syringes etc. are to be sterilized.

2.   Sterilization in Microbiological works like preparation of culture media, reagents and equipments where a sterile condition is to be maintained.
CLASSIFICATION OF METHODS 
Sterilization and disinfection are done by :
(A). Physical Agents
     1.   Heat
     2.   Radiation
     3.   Filtration

(B). Chemical Agents
In practice, certain methods are placed under sterilization which in fact do not fulfill the definition of sterilization such as boiling for 1/2 hr and pasteurization which will not kill spores.

STERILIZATION BY HEAT
Heat is most effective and a rapid method of sterilization and disinfection. Excessive heat acts by coagulation of cell proteins. Less heat interferes metabolic reactions. Sterilization occurs by heating above 100°C which ensure lolling of bacterial spores. Sterilization by hot air in hot air oven and sterilization by autoclaving are the two most common method used in the laboratory.

Types of Heat :
 A.  Sterilization by moist heat
 B.  Sterilization by dry heat

A. Sterilization by Moist Heat
Moist heat acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell membrane.

(I). Sterilization at 100°C
1.   Boiling. Boiling at 100°C for 30 minutes is done in a water bath. Syringes, rubber goods and surgical instruments may be sterilized by this method. All bacteria and certain spores are killed. It leads to disinfection.
2.   Steaming. Steam (100°C) is more effective than dry heat at the same temperature as: (a) Bacteria are more susceptible to moist heat, (b) Steam has more penetrating power, and (c) Steam has more sterilizing power as more heat is given up during condensation.

Steam Sterilizer. It works at 100°C under normal atmospheric pressure i.e. without extra pressure. It is ideally suitable for sterilizing media which may be damaged at a temperature higher than 100°C.

It is a metallic vessel having 2 perforated diaphragms (Shelves), one above boiling water, and the other about 4" above the floor. Water is boiled by electricity, gas or stove. Steam passes up. There is a small opening on the roof of the instrument for the escape of steam. Sterilization is done by two methods :

(a) Single Exposure for 11/2 hours. It leads to disinfection.
(b) Tyndallization (Fractional Sterilization). Heat labile media like those containing sugar, milk, gelatin can be sterilized by this method. Steaming at 100°C is done in steam sterilizer for 20 minutes followed by incubation at 37°C overnight. This procedure is repeated for another 2 successive days. That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria during incubation and are destroyed during steaming on second and third day. It leads to sterilization.

II. Sterilization above 100°C: Autoclaving
Autoclaving is one of the most common methods of sterilization. Principle: In this method sterilization is done by steam under pressure. Steaming at temperature higher than 100°C is used in autoclaving. The temperature of boiling depends on the surrounding atmospheric pressure. A higher temperature of steaming is obtained by employing a higher pressure. When the autoclave is closed and made air-tight, and water starts boiling, the inside pressures increases and now the water boils above 100°C. At 15 ib per sq. inch pressure, 121°C temperatures is obtained. This is kept for 15 minutes for sterilization to kill spores. It works like a pressure cooker.

'Sterilization holding time' is the time for which the entire load in the autoclave requires to be exposed.

Autoclave is a metallic cylindrical vessel. On the lid, there are : (1) A gauge for indicating the pressure, (2) A safety valve, which can be set to blow off at any desired pressure, and (3) A stopcock to release the pressure. It is provided with a perforated diaphragm. Water is placed below the diaphragm and heated from below by electricity, gas or stove. Working of Autoclave. (a) Place materials inside, (b) Close the lid. Leave stopcock open, (c) Set the safety valve at the desired pressure, (d) Heat the autoclave. Air is forced out and eventually steam ensures out through the tap, (e) close the tap. The inside pressure now rises until it reaches the set level (i.e. 15 Win), when the safety valve opens and the excess steam escapes, (f) Keep it for 15 minutes (holding time), (g) Stop heating, (h) Cool the autoclave below 100°C, (i) Open the stopcock slowly to allow air to enter the autoclave.

Checking of Autoclave for Efficiency. Methods :
(i)  Spores of Bacillus stearothermophilus are used. Spores withstand 121°C heat for up to 12 min. Strips containing this bacteria are included with the material being autoclaved. Strips are cultured between 50°C and 60°C for surviving spores. If the spores are killed the autoclave is functioning properly.
(ii)Automatic Monitoring System.

III. Sterilization below 100°C
1. Pasteurization. Pasteurization is heating of milk to such temperature and for such a period of time so as to kill pathogenic bacteria that may be present in milk without changing colour, flavour and nutritive value of the milk. Mycobacterium bovis, Salmonella species, Escherichia coli and Brucella species may be present in milk. It does not sterilize the milk as many living organisms including spores are not destroyed..

Methods of Pasteurization
(i)  Flash Method. It is "high temperature- short time method". Heating is done at 72°C for 15 seconds.
(ii) Holding Method. Heating is done between 63°C and 66°C for 30 minutes.

2. Inspissation. Inspissation is done between 75°C to 80°C. Inspissation means stiffening of protein without coagulation as the temperature is below coagulation temperature. Media containing serum or egg is sterilized by heating for 3 successive days. It is done in 'Serum Inspissator'.
B. Sterilization by Dry Heat
Mechanisms. (1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte (in absence of water).

Dry heat at 160°C (holding temperature for one hour is required to kill the most resistant spores). The articles remain dry. It is unsuitable for clothing which may be spoiled.

1.   Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to 'red' in bunsen burner or spirit lamp flame. Temperature is above 100°C. It leads to sterilization.

2.   Flaming. The article is passed through flame without allowing it to become red hot, e.g. scalpel. Temperature is not high to cause sterilization.

3.   Sterilization by Hot Air

Hot Air Oven (Sterilizer). It Is one of the most common method used for sterilization. Glass wares, swab sticks, all-glass syringes, powder and oily substances are sterilized in hot air oven. For sterilization, a temperature of 160°C is maintained (holding) for one hour. Spores are killed at this temperature. It leads to sterilization.

Hot Air Oven is an apparatus with double metallic walls and a door. There is an air space between these walls. The apparatus is heated by electricity or gas at the bottom. On heating, the air at the bottom becomes hot and passes between the two walls from below upwards, and then passes in the inner chamber through the holes on Me top of the apparatus. A thermostat is fitted to maintain a constant temperature of 160°C.

Preparation of plaster of paris, Bleaching powder and bones in kannada for paramedical and dmlt students

Preparation Bleaching Powder:
     

Ca0C12 (calcium oxychloride) is bleaching powder.
  Bleaching powder is produced by the action of chlorine on the dry slaked lime. for a long time.

Ca(OH)2(s) + Cl2(g) — Ca0C12(s) + H20(1)
    
This consists of a vertical cast iron tower fitted with a hopper at the top through which slaked lime is fed and hot air and chlorine enters near the base in opposite direction.

The reaction takes place in the different shelves which contains rotating rakes.

Bleaching powder is collected in the drum kept at the base.

Physical Properties:
1. It is a pale yellow powder.
2.It has strong smell of chlorine.
3.It is soluble in water.
4.It Melting point is 100°C

Chemical Properties:

1. Reaction with dilute hydrochloric acid:- When bleaching powder is reacted with dilute hydrochloric acid then all the chlorine gas present in it is liberated:
   
   CaOCl2+2HCL--->CaCl2+H2O+Cl2
2. Reaction with dilute sulphuric acid:- When bleaching powder is reacted with dilute sulphuric acid then all the chlorine gas present in it is liberated: CaOCl2+H2SO4---->CaSO4+Cl2+H2O

1. Uses of the Bleaching powder :

1) Bleaching powder is also used in the paper industry .

2) Bleaching powder is commonly used for bleaching clothes.

3) Bleaching powder is used to disinfect drinking water.

4) Bleaching powder is used in the manufacture of chloroform (CHCl3), an anaesthetic.

5) Bleaching powder is used as an oxidising agent.

6) Bleaching powder is used to shrink wool.

PLASTER OF PARIS

Preparation of Plaster of Paris

       Plaster of Paris is prepared by heating gypsum (CaSO₄.2H₂O) to a temperature of 373 K in a kiln. Actually the chemical name of gypsum is calcium sulphate dehydrated. 

 when gypsum is heated then it loses one and half molecules of water of crystallization leaving only half molecule of water of crystallization remaining attached with calcium sulphate.

During this process care should be taken not to heat the gypsum above 373K because if gypsum is heated beyond the temperature 373 K then all the water of crystallization is removed from it which results in anhydrous calcium sulphate which is also called as dead burnt plaster.

Uses/Applications:

1. It is used for making toys, cheap ornaments, cosmetics, black-board chalk, decorative materials and casts for statues. 
2. It is used by dentists for making casts of denture.


3. It is used in chemistry laboratories for sealing air-gaps in apparatus where air tight arrangement is required.


4. It is used for making walls of homes smooth before painting them and for making beautiful designs on the ceilings of houses and other buildings.


5. It is also used as a fire proofing materials.

Bone: 

composition and applications:

Made mostly of collagen, bone is living, growing tissue.

Collagen is a protein that provides a soft framework, and calcium phosphate is a mineral that adds strength and hardens the framework. 

This combination of collagen and calcium makes bone strong and flexible enough to withstand stress.

The composition of the mineral component can be approximated as hydroxyapatite (HA), with the chemical formula Ca10(PO4)6(OH)2. However, whereas HA as has a Ca:P ratio of 5:3 (1.67), bone mineral itself has Ca:P ratios ranging from 1.37 - 1.87.