ENVIRONMENTAL FACTORS EFFECTING THE GROWTH OF MICROORGANISMS As we have studied microorganisms must be able to respond to variations in nutrient level, and particularly to nutrient limitations. The growth of the microorganisms as greatly effected by the chemical and physical nature of the surroundings. There are two types of factors effecting the growth of microbes i.e. bio-chemical we will firstly discus the bio-physical factor which are temperature, PH, water and solute, radiation pressure and oxygen connections. 1. Temperature: Environmental temp profoundly affects microorganisms, like all other organisms. Microorganisms are usually unicellular and their temperature varies with that of the external environment. A most important factor external environment. A most important factor influencing the effect of temperature on growth is the temperature sensitivity of enzymes - catalyzed redactions. Every 10oc rise in temperature will double the rate of the bio-chemical reactions of enzymes. Because the rate of each reaction increases, metabolism as a whole is more active at higher temp and the microbes grow faster. Beyond a certain point farther increases actually solve growth, and sufficiently wish temperatures are which comp decith lethal high temp damage microbes by denaturing proteins, enzymes, transport carries microbial members are also disrupted by temperature extremes, the lipid bilayer simply disintegrate. Thus although functional enzyme, operate more rapidly at higher temperature, the microbes may be damaged to such an extent that growth is inhibited because the damage cannot be repaired. At very low temperatures members solidify and enzymes donst work rapidly. Microorganisms can be placed into fire clarets on the bases of temperature ranges for growth. i. Psychrophiles

Crow well at 0oc and have an optimum growth temperature of 15oc or lower, the maximum is around 20oc. they are readily isolated from Arctic and antartic habitats because 90% of the ocean is 5oc or colder. Example:

The pschrophilic alga chlomydomonas nivalis can actually turn a snowfield or glacier pink with its bright read spores. Other examples are psendomonas, vibrio, alealigenes, Bacillus, arthrobactor, moritella, photo bacterium and shewanella. Adaptations: The pschropkyles are adapted to their environment in several ways.i.

Their enzymes, transport systems, and

proteinsynthetic by mechanism function well at low. Temperatures.ii.

The

cell

membranes

of

pschrophillic

microbes have high level of fatty acids and remain seminudewhen cold indeed may pschoophyles being to lack cellular constituents at temperature higher then 20oc because of cell membrane disruption. ii. Pscrotrophs or facultative psgonrophiles Many species can grow at 0o to 7oc even though they have optima between 20 and 30oc, and at about 35oc. these are called facultative psychrophiles. Pschrotrophic bactona and fungs are major factors in the spoilage of refrigerated foods. iii. Mesophiles Mesophiles are microorganisms with growth optima around 20 to 45oc. they often have a temperature minimum of 15 to 20oc. their maximum is about 45oc or lower. Most microorganisms falo within this category most also human pathogens are meosphiles as nigh be expected since their environment is a firstly constant 37oc. iv. Thermophiles They can grow temperatures of 55oc or higher. Their growth minimum is usually around 45oc and they often have option between 55 and 65oc. The voat majority are prokaryotes although a few algeo and ufngi are thermophilic. Thermophiles differ from mesophiles in having much more heat stable enzymes and protein synthesis systems able to motion at high

temperatures. Their membranes lipids are also more saturated than those of mesophiles and have higher melting points. Therefore thermophile membranes remain intent at higher temperatures. v. Hyperthermophiles Thypertheronophiles have optimum growth temperatures between 80oc and 113oc are called hyperthermophiles. Their minimum temperatures is 55oc or above example pyroeoccus abyssi and pyrodietium occultum are examples of marine hyperthemophiles found in hot areas of seafloor.

Picture Temperature (2) Fig(1.3) Temperature ranges for microbial growth. Microorganisms can be placed in different cases based on their temp ranges for growth. 2. Pressure Most microorganisms spend their liver on land or on the surface of water, always subjected to a pressure of 1 atmosphere (atm), and are never affected significantly by pressure. Yet the deep be (ocean if 1,000 m or more in depth) is 75% of the total ocean value. The hydrostatic pressure can reach 600 to 1,100 atm in the deep sea, while the temperature is about 2 to 3 oc despite these extremes, bacteria survive and adept. Their are two types of microbes on the baris of pressure. i. Barotoleront: Increased pressure does adversely effect them but not as much at as it does non tolerant bacteria.ii. Basophilic

They grow more rapidly at high pressures. Some bacteria in the gut of the of the deep sea invertebrates such as amphipods and holothurians are truly basophilic.

These gut bacteria may play and important role in nutrient recycling in the deep sea. One basophilic has been recovered from the Mariana pinch near the Philippines (depth about 10,500m ) that is actually unable to grow at pressure below about 100 to 500 atm when incubated at 20oc. thus far basophilic have been found among serial bacterial genera (e.g. photobacteriam shewanella, colwellia). PH: Ph is the measure of hydrogen ion activity in the solution and is defined as the negative logarithm of the hydrogen in concretion (evpvessed in terms of morality). Ph log[H+] The Ph scale extends from Ph oo (1.0 mH+) to PH 14.0 (1x10-14 MH+) and each unit represents a tenfold change in H+ confutation. It is not surprising that PH dramatically affects microbial growth. Each species has a definite PH growth range and PH-Optional. There are three types of microbes on the basis of PH. 1. Acidophilus Have their growth optimum between Pho and 5.5 Example: the archaic feoroplasma Acidarmanus, Picrophiles, lyanidium ladarium etc. Most fungi profer sligulty acidic sorrow dings, obutpmito 6 alogo also seem to faror slight acidity 2. Newtrophiles Grovatoptium PH between 5.5 to 8.0 Example are Escherichias euglexa, paramecium. 3. Alkalophiles Prefer the PH of range of 8.5 and 11.5. Examples, bacillus alcalophiles, natronsbatemium 4. Extreme alkathophiles Prefer have growth optima to PH 10 or higher. In general micabial groups have different / character PH references. Although microorganisms will often grow over wide ranges of PH and for from their optima, there are limit to their tolerance. Drafting radiations in PH canharm microorganisms by disrupting the plasma membrane or inhibiting the activity of enzymes sulglobus, acontiam,

and membrane transport proteins procaryoles die of the internal PH drops much below 5.0 to 5.5. changes in the external Ph might after thus reduce their availability to the organism. Several mechanisms for the maintances of a neutral cytoplasmic PH have been proposed. Plasma membrane may be relatively impermeable newtrophiles appear to achange potassium for protams using an antiparty transport system. Extreme alkalophile like bacillus alcalopkilus maintain their PH closer to neutrality by changing external Na-ions for external protons. Internal buffering may also contribute to ph homeostasis.

OXYGEN CONCENTRATIONOxygen also effects the growth of microbes on the badis of oxygen CMC we can classify microbes as under. 1. Aerobe: An organism able to grow in the presence of atmosphere oxygen is an aerobe fungi and algal ph. 2. Anaerobe: That grow in the absence of oxygen is anaerobe. Example, clostridium, fisobacterium. 3. Obligate Aerobe Completely dependent on atmosphat oxygen for growth Example, pseudomonas, micrococcus lutes, onycoboterium, most algal, fungi and protozoa. 4. Facallotive Anaerobe Dont required oxygen for growth, but do grow better in its prlocace. Example, Escherichia, enterocoecas, secharonycas, cevisiae, In the presence of oxygen their will be aerobic respiration 5. Aerotolerants Anaerobes Simple ignore oxygen and grow equally well whether in is present or not. Example. Enterococcas facecalis 6. Obligate Anaerobe Dont tolerate oxygen at all and die in its presence . Example: bactericides, Fusobacterium, Clostridium Pasteurianum, Nethanococcus, Aero. Aerotolerant and strict anaccore dont generate energy through respiration but and mug employ fermentation or anaerobic pathways for this purpose. 7. Microaerophiles They are damaged by the normal atmospheric 02 (20%) and require or level below the range of 2 to 10% for growth. Example campylobacter, spirillum volutons, treponema pallidum. The nature of bacterial 02 responses can be readily determinded by growing the bacteria in cultures tubes filled with a solid culture medium a special medium like thioglycollate broth which contains reducing agent to laver 02 levels.

Fig (1.2) oxygen can and bacterial growth. An illustration of the growth of bacteria with varying responses to oxygen, each dont represent its surface. The surface, which is directly eaposel to atmosphenic oxygen, will be convoke. The oxygen cutent of the medium cleareases with the depth until the tube. The presume of could absence of the enzymes superoxide disecutcese and cutlasses for each type are shown. Oxygen accept electors and is readily reduced because its two aiter abital electrons are unpaired fleapoteins, servial other cell constituents and radiation promote oxygen reduction. The result is usually some combination of the reducing products, such as superposed radical. Lydroghin peroxide, and hydrogen radical 02 + e 02 + e + 2Ht H202 + e + Ht 02 (superoxide radical) H202 (hydrogen peroxide) H20 + OH (hydroxyl practical)

The products are extremely tocic because they are powerful oxidizing agents and rapidly cellar constituents. Many microbes posses enzymes that offerd protection against toxic 02 Prodects. Obligate aerobes and facltativ anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase, which eateries the deduction of 02 Radical and H202 respect only peroxides can also be used to desroy hydrogen peroxide. 202 + 2H + 02 + NAD+ Aero tolerant microbes amy lack catolase but almost always have superoxide dismutase all strict anaerobel lack both SOD and catalase and therefore cannot tolerate oxygen. Solutes and Water Activity Because a selective permeable plasma membrane separates microorganism form their environments, they can be affected by changes in the asthmatic concentration of their surroundings. On the basis of solutes there are two types of solutions 1. Hypotonic Solution Hypotonic solution is that solution which contains small am ant of solute be with lower osmotic concentration. When a cells place inside hypotonic solution water will enter the cell and cause it to burst unless small thing is done to prevent the influx. The osmotic can of the cytoplasm can be reduced be inclusion bodies. 2. Hypertonic Solution

A solution with higher amant of salute care is called hyportonic solution it needs mmore soluent (H20) to solurate therefore when a cell is place in a hypertonic environment water will leve the cell and the palmer membrane shriek away from the wall a process know as plasmolysis. This delydrates the cell and may damage to the pasma meubrance, the usually because meteorically inactive and caeases to grow on the basis of solutes microorganisues can be classified as under.

(i) Halophiles Have up adopted completed to hypertonic, solin condition that require high levels of Nasal to grow. Example holobacterium, dunaliella, Egto thiordaspora. (ii) Extreme Halophiles These microbes grow live in the extreme of halopulic condition be which contains way high can dint of Nacl about 0.7m. These when hellhole bacteria contain enormous quantity of potassium in order to remain hypertonic to their environment, the internal potassium cone may read to 4 to 7m. The enzymes ribosomes, and transport proteins of these bacteria high levels of potassium for stability and activity. (iii) (iv) Obligate halophiles Completely dependent on Nucl for their growth . Facultative holophiles They grow in suite environment (can to 2% or 15%). If it is not present they also sustain their lives Growth factors Microorganisms often grow and reproduce when mainendts and sources of energy, certain nitrogen phosphorous and sulphar are supplied. These organism have onzume and pathiways necessary to synthesize all cell compounds required for their well being. Many microbes on the after hand, lack one or more essential enzyme therefore they camont manufactured all indispen side (necessary) constituents but must obtains them or their precursors from the environment. Organic compounds required because they are essential cell components ore pressures of such components and can not be synthesized by the organism are called growth factor there are three major calluses of growth factor.

i. Amino acids Not synthesized by microbes, needed for protein synthesis ii. Purines and Phrimidines Nacleid acid synthesis iii. Vitamins Vitamins are small organic molecules that usually make up all or part of enzyme cofactors, and only very small anoint sustain growth. Some microbes require many timings for example enterococcus facials needs aright different vitamins for growth others growth factors are also see home (from homfgobin or cytrochiomes) is required by haemophilus influnzea nad some mycoplasmuas need cholesterol example of vitamins required y microbes biotin, cyanocobalimib folic acid, lipor acid, patphinic acid, pyridoxine (E6) thiaonine (B1) etc. STERILIZATION The term sterilization is derived from a Latin word sterilis which means unable to produce offsprings or barren. Sterilization is a process by which all living cells, viable spores both pathogenic & non pathogenic, viruses and various are either destroyed or removed from an object or medium. a sterile object is totally free of viable spores & microorganisms, and other microbial infectious agents. Or Sterilization means the freeing of an article or a medium from all living microorganism including viruses bacteria their spores and fungi both pathogenic & non pathogenic Sterility is an absolute form there is no such thing as almost sterile an object or environment is not consider stabile as long as it contain even a single viable microbe used an inanimate object media not on body surface Uses:Sterilization is required for the culture media, suspending fluids reagents containers and equipments used in microbiology it is also required for medical and surgical instruments and material used in procedures that in value penetration into the blood tissues and other normally

sterile parts of the body e.g in surgical operations and diagnostic aspire simply the uses of sterilization as summarize for prevention of culture media form bacteria used for medical +surgical instruments food drags manufacturing for enduring safety from contaminating organisms. PHYSICAL AGENTS STERILIZATION 1. Sterilization by dry feat a. Red hat b. Flaming c. Hot air oven 2. Sterilization by moist heata. Aautodaving (temp above 1000c 12ic) b. Boiling (temp-1000c c. Pasteurization (at temp below 1000c)

3. Low temperature 4. Radiation 5. Filtration 6. Desiccation 7. Osmotic pressure Chemical agents 8. Sterilizing gas a. Ethylene oxideb. Beta propiolactone gas 9. Formaldehyde and glidaraldehyde 1. STERILIZATION BY MOIST HEAT (temp above 1000c)

The most heat kills the microorganisms probably by denaturing and coagulating their enzymes, structural proteins and nucleic acids in the absence of water heat does not coagulate the protein. Most heat sterilization, usually autoclaving is the most frequently used method of sterilization because bacterial end spores are resistant to boiling at (1000c at sea level) they must be exosed to a higher temperature, This cannot be achieve unless the processor is increased, for this purpose an automate chamber is used this instrument sterilizes with saturated steam under procedure. In autodauc the pressure

increases the boiling point of water, thereby increasing the temperature to which water can be heated. Water is boiled to produce steam which is released through jacket and enter into chamber (of autoclave) the air initially present in the chamber is forced out until the chamber is filled with saturated heat and the outlets are closed. Hot saturated steam continues to enter antil the chamber reaches the desired temperature usually 1210C at a pressure of 15 Ib/in20. At this temperature the saturated heat defies all vegetative alls & end spores within 15 minutes (for a small volume of liquid) the higher container of liquid like flask require more time after liquid have been autoclaved, pressure should be returned to normal by exhausting the steam very slowly from the chamber. Materials not suitable for autoclaving include powders heat sensitive comports and water insoluble substances (such as miner oil) 2. PASTEURIZATION:(temp below 1000c)

The pasteurization is a Process in which substance like milk aye heated at temperature well below heating boiling, to destroy the pathogenic microbes the process of pasteurization was first introduced by Louis Pasteur in 1860. Milk can be pasteurized in two ways in the alder method the milk is heated at 63 0C for 30 minutes followed by rapid cooling large quantities of milk are now usually subjected to flash pasteurization or high temperature short term (HTST) pasteurization, which consist of quick heating to about 720C for 15-min then rapid coding the dairy industry also sometimes sues ultrahigh temperature (OHT) sterilization milk & milk products are heated at 140 to 1500C for 1-3 seconds. The pasteurization is sufficient to kill the vegetative calls of milk borne pathogens e.g mycobacterium Boris, salmonella, streptococcus and bracelet (but not to till sterilize the milk flash method. 3. STERILIZATION BY BOILING (Temp 1000c)

Heating increases the temperature of water until it reaches 1000C further beating fails to raise the temperature of water. Boiling at 1000C for 10-15 minutes is sufficient to kill the vegetative cells and eukaryotic spores, but the temperature of boiling water (1000C) is not high enough to destroy the bacterial end spores that may survive hours of boiling.

Therefore boiling is considered a procedure for disinfection rather then sterilization, and is used for disinfection of drinking water, and objects not harmed by water. Although high temperature of boiling water kills more effectively than pasteurization. It is not routinely used in food industries b/c such high temperatures often damage the product. 4. STERILIZATION BY DRY HEAT: Many objects are best sterilized are placed in an oven at 160-1700C for 2-3 hours. Microbial death results from the oxidation of all constituents and denaturation of proteins. The dry heat is generally used for sterilization in three ways.

Red Heat Inoculating wires, points of forceps and spatulas are sterilized by holding them almost vertical in the flame of banes burner, until they are seen to be red-hot. b. Flaming Scalpel blades, needles, mouth at culture tubes, glass slides and cover slips are sterilized by pasing the article through a gas or spirit flame without allowing them to become red-hot. c. Hot air oven This is the main means of sterilization by dry heat. The oven is used heated with electricity and has a thermostat that maintains the chamber air, the items and sterilized in even at 160 0C- 1700C for 2-3 hrs. The hot air oven is used for sterilization of glassware, such as syringes, test tubes, petridishes, pipettes and flask, metal instruments such as scalpels, forceps, scissors materials such as oils, jellies, powders, fats and greases which are impermeable to moisture. 5. Low temperature Low temperature inhibits the growth and reproduction of the microorganism but not cause complete sterilization, only use as a static, Freezing items at -200C or lower stops microbial growth because of low temperature and absence of liquored water but when temperature increases than again growth starts.

Some microorganisms will be killed by ice crystal due to disruption of call membrane. The remigration greatly slows the microbial growth and reproduction but does not halt it completely most pathogens are hemophilic microbes + do not grow well at temperatures around 40C. 6. Filtration Filtration is the preferred method to reduce the microbial population in solutions. liquids & gases can be sterilized by passing them through filters. The microbes are retained by filters that have process smaller than the size of the microbes there are two main types of filters. Depth Filters The depth filters consist of fibrous materials or granular materials that have been handed into a thick layer filled with twisting channels of small diametric the solution contains microbes is sucked through this layer under vacuum and microbial cells are removed by physical screening or entrapment and also by adsorption to the surface of filter material. The depth filters are made up of diatomaceous earth (Barkefielf filters) asbestos or of other similar materials. Membrane Filters The membrane filters are made up to cellulose acetate, cellulose nitrate, polycarbonate polyvinylidere fluoride or other synthetic materials. These filters are power membranes having 0.1 mm thickness, are used to remove vegetative cells but not viruses, with a pore size of about 0.2um in hamster. The viruses are not trapped b/c they have much smaller size than pores of the filters. These membranous filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics and other heat sensitive solutions. 7. Radiation Energy transmitted through a spur in variety forms, called radiation. The two types of radiation used to kill the microbes are a. Ultraviolet radiation b. Lionizing radiation

a. Ultraviolet radiation The ability of sunlight to kills bacteria & other microbes is mainly due to the ultra-violet rays that it contains the effectiveness of ultra-violet light as a sterilizing agent increases with decrease in wavelength. The greatest antimicrobial activity of VV light occurs at 250-260nm wavelength. The most significant form of lesion caused by uv radiation is the formation of the thymine dimmers (T=T) But addition of hydroid group to the bases also occurs as a result the DNA replication is stop and organism cannot grow.

Cells have repair mechanisms against uv. Induced barrage involutes cleavage dimmers in that either of the

presence of visible light (photo reactivation) or excision of damageable bases which is not dependent upon visible light (can occur in dark). Ultra violet rays from suitable shielded lamps have been used ot reduce the number of bacterial cell in atmosphere but for safety their intersity has to be restricted b. Ionizing radiation (also tram Michal pook for Oetiul)

The lionizing radiation is an excellent sterilizing agent and penetrate deep into objects, destroy bacterial enclospores and vegetative calls. Lionization ionizing radiation include x-rays, gama rays and cosmic rays these have higher energy and penetrating power the uv. Radiation and kill manly by the production of free radials e.g

production of -0H radicals by the hydrolysis & water these highly reactive radials can break covalent bond in DNA there by killing the organism. Sulfhydryle containing compounds such as amino acid cytokine, case protect the DNA from fraradical attack. R-rays, X-rays kill vegetative cells readily but spokes are remark resistant probably because of their lower water content X-rays are used in medicine for sterilization of heat-sensitive items such as surgical gloves, sutures, plastic items like syringes. 8. Ethylene oxide and Beta propiolactone gases The either oxide and betapropro Cactone as used as sterilizing gases. Ethylene oxide (Eto) is both microbicidal and sporicidal and kill by combining with cell proteins sterilization is carned out in a special ethylone oxide sterilizer, very much resembling outoclave in appearance B/C the ethylene oxide is explosive, it is usually supplied in a 10-20% concentration mixed with either C20 or dichlorodifluromethane

extensive aeration of sterilized materials is necessary to remove residual Eto because it is so toxic Betapropiolctone (BPC) is occasirally employed as sterilizing gas . in liquied form it has been used to sterilize vaccines & sera the BPC destroys microber more readily than Eto but does not perietrate materials well & may be carmogerive for this reason BBL is not used extensively as Eto. Formal detiyde and glutoraldehyde H-C19-H Formaldehycle H-C110-CH2-CH2-CH2-C110-H Glutoraldehyle

Both Aldihydes, formldyde & glutural dehyde can be used as chemical sterilants. They are highly reactives compounds that canbine with nulve aciols & proteins and inactivate them by crossling & alkaliting maleculs. The glutaraldelyde which has two reactive groups (-cooh) is 10 times more effective then formaldelyde and is less toxic See other agents from Michaels book of Microbiology

Sanitization Sanitization is closely related to disinfection in sanitization the microbial population is reduced to levels that are considered safe for public health standards. Sanitization is used to clean eating utensils in restaurants the chemical used is called sanitizer, like disinfectant it is only use in inanimate media or object. Disinfection The disinfection is the process of killing, inhibition or removal of microorganisms may remains alive. Like the sterilizing agents the disinfectants jare used only an inanimate (living) objects and media and near on true tissues (body surfaces) Disinfection is generally employed if sterilization is either impossible or unnecessary the purpose of disinfection is to minimize the risk of infection or product spoilage by reducing the number of microbes, especially pathogens in the environment. Antisepsis (Sepsis=Growth)

The antisepsis is the prevention of infection in living tissues and is accomplished with antiseptics which are the chenicals used to prevent or inhibit the growth of pathogens in tissues, they also reduce the total microbial population. The antiseptics are less toxic than disinfectants as they are used in living tissues

CHEMICAL DISINFECTANTS AND ANTISEPTICS Any different chemicals are available for use as disinfectants + antiseptics, each has own aolvantages and disadvantages. In reelecting an agent it is important to keep the characteristics of a closeable an agent it is important to keep the characteristics of a clesirable disinfectant. 1. The disinfecting must be effective against a wised range of infector against C+ve+-ve bacteria acia fast bacteria, high dilution and in presence of organic matter. 2. Although chemical must be toxic for infectious agents but is should not be toxic for the human being. 3. The disinfectant must be stable (upon storage), odorless or with a pleasant odor, soluble in water and lipids for penetration into microorganisms and have a low surface tension. If possible the disinfection should be inexpensive Chemicals which are used as disinfectants are listed as fallows. 1. Phenol and phenolics 2. Halogens 3. Alcohols 4. Detergents 5. Heavy Metals 6. Quaternary Ammonium commands (or detergents) 7. Sterilizing gases (also use as dissentients) 8. Dyes 9. Metalic salts 10. Aldiphyde

1. Phenol and Phenolics The phenol was the first widely used disinfectant and antiseptic. In 1861 joseph used it to reduce the risk to infection during operations. Action the phenol is easily penetrable the organic mather, and denature the protein & disrupting cell membrane the phenol is effective in killing all vegetative cells although not very active against spores & none eloped viruses phenol itself is now used very rarely because of its toxic nature as it valise irritations of skin. But think are a number of derivatives of phenol call phonemics, that have greater antibacterial antibacterialacterial activity & lower toxicity then phoned also. Today the phenol + phenalics such as cresols, xyenals, orthophenylphenal, hexaloro pheneare used as disinfectants in laboratories and hospitals.

The phonemics have a disagreeable odor and can cause skin irritation. Hexachlorophene is one of the most popular bacteriostatic against it acts as antiseptic b/c if persists on the skin once applied and reduces skin bacteria for long periods. However it cane casue brain

damage and is now used in hospital nurseries. The soap which is used for bathing by inforts contain 3% luxachlerphone . The pregnant women are advised not to tuse hexachlorphone b/c its absoiption thorugh skin of mother may cause damage to the factor. 2. Alcohols Alcohols are among the most widely used disinfectants and antiseptics. They are bactericidal, fungicidal but not sporcidal, some lipid containing viruses are also destroyed. Alcohols disrupt the plasma memberane and lipid envelope the two most popular alcohol germicides are ethanol and ispropanol usually used in 70-80% concentration.

They and by detaining proteins and dissolving lipids & membrane. Ethanal requires water for maximum activity i.e it is for more effective at 70% then at 100%. The Alcohol water mixture (70%-50%) rapidly inactivates most vegetative be any many viruses but end spores are unaffected. Alcohols are used only for a short form disinfection or antisepsis. 3. Halogens Among the halogens the iodine & chlorine are important anti microbial agents. They are bactericidal and spermicidal. Iodine is used as a skin antiskid and kills by oxidizing all constituents and iodinating cell protons Iodine often used as a tincture of iodine (20% armiger iodine in water ethanol sold of potassium iodide) In dialup solutions of water or ethanol (tincture of iodine) the iodine rapidly inactivates microbes by irreversibly combining with proteins iodine is useful for disinfecting thermometers, for reducing microbes on skin sites selected for surgery or needle puncture, and for treating cuts & wounds. Iodine may chemically combined with large carrier molecule to form iodophores.

Iodophores are stable, no stainable and water soluble, and dissolved, they release free iodine slowly to minimize skin burns & irritation. Commercially available iodophores such as betadine, isodine and surgidine are used as as skin disinfectants. Chlorine is the usual disinfectant for municipal water supply and swimming pools and is also employed in dairy and fod industries. It also oxidizer cellular constituent & protein contents chlorine may be applied as chlorine gas, sodium hypochlorous acid Na (Hclo) or ca (Hclo), all of which yield Hclo and then atomic oxygen the result is oxidation of cellular materials & destruction of vegetative bacteria + fungi but not spores chlorine combine with water form Hclo

Chlorine is an excellent disinfectant of individual use b/c it is effective inexpensive and easy to employ small quantities of drinking water can be disinfected by calzone tablets, which slowly releases the chlorine when added to water and disinfect it in about half hour

4. Heavy metals + their salts The tons of the heavy metals such mercury, siluer, arsenic and zine + copper are used as germicides. Many heavy metals are more bacterio static than bactericidal. These metals have react with acidic group (-COOH) of protein and distingrade the cell protein. A 1% solution of silver nitrate is used to irrigate infected urinary bachelors and to prevents eye infections in infants. The copper sulfates is an effective algicidal in lakes and swimming pools heavy metals combine with proems often their acidic or sulthydryl group and inactiviate them they many also precipitate cell proteins.

5. Detergents Detergents are organic molecules that serve as we thing agents and emulsifiers because they have both polar hydrophilic and nonpalar hydrophobic ends. Although anionic detergents are effective disinfectants but have very less antimicrobial properties. Only the cationic detergents are effective disinfectants the most popular of these cationic detergents are quaternary ammonium compounds characterized by the charged voter nary nitrogen and a long hyarphilc chain they disrupt the microbes & may also denature proclaims cationic detergents like banalkoriam chloride and cetylpyridiniam chloride kill most bacteria but endospores cationic detergents are used as s disinfectants for food utensils and small instruments and as skin antiseptics.

6. Hydrogen Peroxide

(H2O2)

Hydrogen peroxide is used as antiseptic to clean wounds and to disinfect contact lenses. The H2O2 is an oxidizing agent + nontoxic antiseptic when sued in 3% solutions it acts as oxidizing agent but has a very short life in tissues b/c of the presence of enzyme catalase which convert H2O2 to water and oxygen. The release of oxygen and free radicals may be part of the basis for its antibacterial action, especially in deep wounds where the anacrobic bacteria are a problem. Sterilizing gases already discussed Aldehydes from D.R arora book

MICROBIAL GROWTH Mathematics of Growth During the exponential phage each microorganism is dividing at contant intervals. Thus the population doubles in number during a specific length of time called as generation time or doubling time. Suppose that a culture tube is inoculated with one cell that divides every 20 minutes and so on. Because the population is doubling every generation, the increase in population is always 2 where is the number of generation. If no is the initial population number Nt is the population of time t and n is the number of generation in time t a relationship b/w these com be expressed as Nt = No x2 _________1 log No Nlog2 log no Taking loy of both sides of equations Log Nt = N= N= Or Nlog 2 = log Nt log Nt log no/log2 = logNt-log No/0.301________2

The rate of growth during the exponential phase in a batch culture can be expressed in terms of the mean growth rate contant (k( this is the number o generations per unit time, often expressed as generation per unit time or per hour. Dividing the above equation no: 2 by time t gives mean growth rate constant K K= 1/t = logNt-logNo/0.301t __________3 The time taken by a population to double its size is called the mean generation tiem or mean doubling time, and is represented by g After time g the pop. Is doubled (t=g) i.e then Nt=2No By substituting Nt=2No into equation#3 as me get

Hence the mean generation time is the reciprocal of the mean growth rate constant, the mean generation time (g) can be calculated directly from a somilogrithim plot of the growth data and growth rate constant calculated from the g value. For example, suppose that a bacterial population increases from 103 cell to 109 cell in 10 hrs. The generation times vary with species of microbes and environmental conditions. They range from less than 10 minutes (0.17hrs) for a few bacteria to several hrs for some eukaryotic microbes generation times in mature are usually much larger then in culture. Growth curve already prepared Measurement of microbial growth In order to study various favorable and unfavorable effects on the growth bacterial growth is essential. Growth in the laboratory can be measuring by on the following methods. Measurement of cell number Measurement of cell mass MEASUREMENT OF CELL NUMBERS There are many ways be which the microbial number is determined. 1. Direct Microscopic Counts

In direct microscopic count method, the cells are counted directly by microscope. Since in direct count method no differentiation b/w living and dead cells, so both viable and nonviable cells are counted by this method. Petrify Hauser counting chamber is used for counting bacterial all (Prokaryotesthe chamber consists of 25 squars considered as slides. The area of the chamber is 1 rquar metor and valume of the each is 0.02mm3

By microscope we can count the number of cells in each squar. The number of the cells should be 107/ml of the sample. We can calculate the average cells the squar as if 2 cells per squar then 2x25/0.02=___________ cell per squar. The # of cells in per mills meter culbein that sample as 2. Electronic counter By this method only larger microorganisms such as algae, protozoa, and nontilamentous yeas to can be control directly with the help of electronic counters such as coulter center this method is not applicable to bacteria (and fangi) in this case too much cell are there in the form of colony while coulter counter can count cell one by one

In this method the microbial suspension is forced through a small tube, hale or orifice an electrical current flows the hole, every time when a single cell passes through this orifice or tube it gives electrical resistance (or conductivity drops) which goes to counter & cell is counted result with large cells and is used in hospital laboratories for to want white and RBCs. This method is quick, inexpensive, but problem is that it counted both viable + non viable cell, and only app cable to large size microbar. The most accurate measurement of number of living microbar is casually viable count serial dilution of the sample by spread plate technique we can isolate the culture be of cells by this method. But this method is apply only to count viable cells e.g # of cells in food sample or water sample/ml.

Now this diluted sample on social agar plates

Now in bate these plates for 24hrs after that each microorganism develops into a distinct colony. If calories are 30 or 25 it is not true it is rejected the original number of calls in the sample can be calculated by the number of calories formed only 30-300 calories included in count. If calories are too much then they are rejected, (not counted) e.g if 1.ml of a 1x10-6 dilution yield 150 column then original sample contain 1.5x108 cell/ml 150x1/10(column of samplex1x10-6=1.5x108 cell/ml Usually a special digital colony counter is required to count 300 denies. In this case plate is attached to counter by a wire, when we touch to one colony it is counted, then second, 3rd and so on. DRAWBACKS OF THIS METHOD 1. This method is used for counting only viable cells not non-viable cells. 2. We count one sample in a dish at one temperature total # of cell are not cannot 3. While spreading some cells can not spread, so we cannot say that a single colony come from a single cell. 4. The hot agar used in pour plate techniques may injure or kill sensitive cells, thus we cannot count all the cell b/c only viable cells are counted in this technique 4. MEMBRANE FILTER METHOD In this method membrane filter is used to count the number of all only those filter are used to count the number of cell which have pores size less then bacterial size i.e 0..45 mm.

In membrane filter technique the sample is poured or drawn passed through a membrane filter, the bacteria being large in size then filters pore so are trapped on the membrane surface. The filter obtained is then transfer on the agar plat and incubate it for 24 hrs, so each all form a separate colony. A colony count gives the number of microbes in filtered sample.

2. MEASUREMENT OF CELL MASS It is an indirect method for growth determination in which total population of both living + dead cells is measured for this two methods are comply employed. 1. Turbid metric methods This technique depends on the fact that microbial calls scatter the light which striking then the amount of scattered is directly proportional to the biomass of the call + indirectly related to cell number or turbidity, It cloudy appearance of the medium due to the concentration of bacteria is called turbidity of the culture increases but the optical density decreases as the light strike the bacterial cell and reflects back. If there is no bacterial cell in medium then light pass easily and 100 absorbance occur but there will be no turbidity. But if bacteria cell increase in number then no light will absorbed optical density is measured at the time of culture formation + cell # on not day after incubation.

It a tube of bacterial suspension is place in a spectrophotometer, having two scales on top scale display the parent transition and bottom scale show absorbance of light the greater the turbidity lesser will be the reading of top scale, and the reading of the bottom rule will be fighter. 2. Dry weight determination Is the direct approach weight cells growing in liquid medium are collected by centrifugation,t hen washed and dried. If weighed at this time then this will be weight of water with bacterial cell b/c cell contain 70% water. For the determination of dry cell weight, wrap the cells in tissue paper and dried in a oven at 100 0C and then incubated, until the bacterial weight become constant. This is the dry weight of the bacterial cell. This method is useful for fungi but not very sensitive for bacteria due to their very little weight. 4. By use of sulfuric acid By the use of the HGo4 we can detect the# of cells in culture. It we add H2so4 in culture NH is produced from which we determined that how much NH3 is produced how much is the no of cell in culture by examine that how much NH3 is produced. CONTINUOUS CULTURE There are two system is which microorganisms are grown. One is closed system while other is open one. The close system consist of batch culture while open system consists of continuous culture. Batch culture A medium in which nutrient supplies are not renewed not the waste are removed. In this medium exponential growth remains for only a few generations and then enter into stationary phase due to nutrients limitation and toxic waste accumulation. In industry the batch culture is used for specific time. Continuous future An open system in which constant environmental conditions are maintained through continual supply of nutrients and removal of wastes, called as continuous culture medium.

In this medium or system the microbial population can be kept in exponential growth phase for a long periods. Types of continuous culture system There are two major types of continuous culture system which are The chemostats The tubidostats The Chemo stats Chemo state means to maintain the growth of the cell chemically. A chemostate is constructed so that sterile or fresh medium is fed into the culture vessel at the same rate as the media containing microorganisms is removed. The culture medium for a chemo state possesses an essential nutrient (e.g amino acid) in limiting quantities. This nutrient is known as growth limiting factor. Because of this limited nutmeat the growth rate is determined by the rate at which new

medium is fed into growth chamber and the final cell density depends. On the concentration of limiting nutrient the rate of the nutrient exchange is expressed as the dilution rate d, the rate at which medium (fresh) flows, through the culture vessel relative to vessel volume. Dilution rate = Flow rate (ml/hr)/vessel volume (ml) D=f/v

The cells in the fresh medium are grow until the growth limiting factor is exhausted this fresh medium is then allowed to flew in and similar volume of old culture containing dead cells + toxic substances are allowed to flow out from the bottom of the vessel. The growth rate is directly proportional to the flow rate. This nutmeat exchange is expressed as dilation rate D. if flow rate is 30ml/hr and vessel volume 100ml then dilution rate is D=f/v=30/100=0.30hr As the dilution rate increase the generation time decreases (i.e growth rate rises) the limiting nutrient will be completely depleted under these balance condition. When the dilution rate rises too high then bacterial cell (microbes) flow or washed out with media from the culture used on the other side, before reproduction. At this situation the limiting nutrient concentration will be high b/c few microbes (bacterial cells) are present to use it. The maintain the exponential growth the level of limiting nutrient should be nantain constant. 2. Turbidstat The second type of the continuous culture is the turbidstat turbidostate has a photocell which measures the turbidity of the culture and automatically controls the supply of nutrients to maintain the predetermined turbidity or cell density. The turbidostate differs from chemo state by several ways. The dilution rate in tubidostate is not constant it varies b/c it not depend on chemical Its culture medium lacks a limiting nutrient The turbidostate operate best at high dilution rates while chemo state is stable & effective at lower dilution rates. IMPORTANCE OF CONTINUOUS CULTURE 1. The continuous culture are very useful because they provide a constant supply of cells in the exponential phase and growing at a known rate. 2. They make possible the study of microbial growth at very low nutrient levels (concentration close to those present in natural environment. 3. These systems are essential for the research in many areas 4. Continuous cultures are also used in food and industrial microbiology. Nutritional requirements

Any substance that cane metabolized to provide energy and building material, is called nutrient or simply food. The process of taking up the nutrient is called nutrition The nutrients into the basis of their need are divided into two major groups. 1. Macronutrients 2. Micronutrients Macronutrients Analysis of microbial cell composition shows that 95% fo cell dry weight is made up of a few major elements such as carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous, potassium, calcium, magnesium and iron, these are called micro element b/c they are required by microelement in relatively large amount . the first six (C,O,H,N,S And P) are components of carbohydrates, lipids, proteins + relic acid, while remaining for (ca, Mg, K, Fe) exist as a action in cell + perform variety of role. Carbon hydrogen and oxygen The carbon, hydrogen and oxygen are present in the carbohydrates if carbohydrates available then these elements are available to cell, carbohydrate also provide energy to the cell carbohydrates and other organic molecules are always in reduced form and have electrons, provide more energy to the cell. Carbohydrates and other organic molecules are always in reduced form and have electrons, produce more energy by transfer of electrons to other molecules. e.g lipids are in more reduced state than carbohydrate so provide more energy. One important carbon source that does not supply hydrogen or energy is C2O B/c C2Ois oxidized and lack hydrogen the microbes fix the environmental C2O (i.e reduce it) and use it as cobra source + sunlight as energy source some organism cannot use C2O as carbon source, but they rely on more reduced, complex molecule e.g glucose for carbon supply such are called heterotrophy. Nitrogen Nitrogen is needed for the synthesis of amino acids, pureness, phyrimidines, emerge cofactors and other substances. Being the backbone of protein and N. Acids, the nitrogen is required by all living organism in high concentration. Nitrogen is obtained from both organic + inorganic sources but it is found in cell always in organic form. The principal inorganic compounds providing (N) are

ammonium (NH4+)and nitrates (N3o) some bacteria such as series of rhizobium can even convert atmospheric inorganic nitrogen to cellular organic nitrogen by a process known as nitrogen fixation. PHOSPHOROUS Phosphorus is the constituent of the nucleic acids, phospholipids nudities like ATP, several cofactors some proteins and other cell components. All microorganisms need phosphorus in the form of inorganic phosphate for the synthesis of the DNA, FNA, ATP and phospholipids for the cell membrane. It is obtained by micro-organisms in its inactive form i.e phosphate ion (P4o-3) from salts of phosphoric acid. SULFUR The sulfur is needed by the microorganisms for the synthesis of substances like a acids synthesis of substances like a acids costume and methionine, some carbohydrates, biotin and thiamine most microbes use sulfate as a source of sulfur and reduce it. Potassium, calcium, magnesium + iron the calcium, potassium, magnesium and iron are also considered as microelement and present in cell as station e.g is required for the activity of some enzymes ca+ contributes to the heat resistant of bacterial end spores. My+2 ion is required for the activity of some enzymes Ca+contributes to the heat resistante of bacterial endospores. My+2 serus a confactor for many enzymes iron Fe+2, Fe+3is s part of cytochromes and a cofactor for enzymes Growth Factors Growth factors are organic compounds which are required by microorganism and cannot synthesize by own self, e.g Amino acids vitamins and urine, pyrimidines are best example of Growth Factors, Amino acids are required for protein synthesis, purine, pyrimidires are required for nuclic acid synthesis while vitamens are essential for growth, form cofactor of enzyme. Not all bacteria require growth factors, but the ones that cannot grown, some need heme (from cytahrome or haemoglobin) as heamophilus influenza and some mycolplasmas need cholesterol as growth factors.

Those microbes which need growth factor are called fastidious microbes e.g E.cali, while microbes which do not require growth factors are called non fastidious. 2. MICRONUTRIENTS All organisms, in eluding micro organisms require several micronutrients or trace elements besides the macronutrients these micronutrients are manganese, cobalt, zine, mickel and copper however these are resent in such small amount that it is difficult to demonstrate a micronutrient requirement. Micronutrient are normally a part of enzymes and co-enzymes as they help in catalysis of reactions & maintenance of protein structure e.g Zn+2 is present in the active site of some enzymes catalyzing the transfer of phosphate groups malydenum is required for nitrogen fixation and cobalt Co2+ is a component of the vitamin B12 NUTRITIONAL TYPES OF MICROORGANISMS Based on nutrition the microbes are divided into different groups. One the basis of carbon, energy and electrons sources the microorganisms are divided into different groups as Energy Source One the basis of energy source the bacteria (microbes) are divided into two groups

1. Photographs These are the microorganism which derive their energy from sunlight by photosynthesis, without C2O production. Instead chlorophyll they contain pigment bearing body called chromatophore. 2. Chemotrophs These microbes live in darkness and obtain energy from the oxidataion of chemical compounds (other organic or inorganic) Carbon sorrce Carbon source the On the basis of carbon source the microorganisms are of two types. 1. Autofrophs:These are microorganisms which use C2O as principal carbon source, also inorganic compounds. 2. Heterotrophs These microbe use organtic molecules as carbon source Election Source On the basis of electron source the microorganisms are divided into two types. i. Lithographs The lithotripsy use inorganic substances as their electron source i. Organotrophs The organutrophs extract electrons from organic compounds. So on the basis of sources of carbon, energy + electrons the microorganisms are divided into four major mutritional groups which are as follows. 1. Photo lithoautotrophs 2. Photo organo heterotrphs 3. Chemolithoautotrophs 4. Chemoarganoheterotrophs 1. Photolithoautotrophs The photoautotrophs use sunlight as energy source C2O or inorganic compounds as carbon source and inorganic substances as electron source Examples Algae, purple and green, sulfur bacteria, cyan bacteria etc are included in this category of microorganism

2. Photoorganohelterotrophs These microorganism use light energy as the source of enrgy, organic compounds as carbon source, and also electron source Examples Purple non sulfur bacteria and green non suffer bacteria etc. Chemolithooutotrophs These microorganisms use C2O C2O or inorganic compounds as carbon source inorganic compounds as energy source and inorganic substances as electrons source Examples Purple non sulfur bacteria and green non sulfur bacteria etc. 3. Chemolithoutotropbs These microorganisms use C2O or inorganic compounds as carbon source, inorganic compounds as energy source and inorganic substances as electron source Examples Sulfur oxidizing bacteria; Hydrogen bacteria, Nitrifying bacteria, iron oxidizing bacteria etc. 4. Chemo orgono heterotrophs These are bacteria (microbes) which use organic compound as carbon source, energy source and also electron source Examples (E.col), Algae fungi, Protozoa, nonphotocynthetic bacteria Mixotrophs Some microbes, in the absence of oxygen use inorganic substances as energy source white in presence of oxygen use organic substances as energy source such microbes are called mixotrophs b/c they depends on environmental factors

COMPARISON OF NUTRITIONAL REQUIREMENTS OF FOUR MAJOR GROUPS OF MICROORGANISMS Energy source Light energy Carbon source C2O or Inorganic substances Organic compounds C2O or inorganic Organic compounds Inorganic substances Electron source Example Algae, blue green algae purple + green sulfur bacteria Purple non sulfur + inorganic substaince

Photolithoautotroph

Photo organoheterotroph

Light energy

green-non-sulfur bacteria Sulfur oxidizing bacteria hydrogen bacteria NonNitrifying bacteria Protozoa, fungi Photosynthetic (E-coli)

Chemolithoautotrophs

Inorganic substances

substances Organic compounds

Chemoriganohetertroph

Organic compounds

Organic compounds

bacteria

CULTURE MEDIA A culture medium is a solid or liquid preparation used to grow, transport and store microorganism in the laboratory. This culture medium contains all the nutrients , which are required for an organisms growth. The nutrients include the source of carbon, hydrogen, oxygen, sulfur phosphorus, nitrogen & growth factors, in culture media physical requirements like temperature, PH, & osmotic presage. Specialized media are essential in the isolation and identification of microorganisms, the testing of antibiotic sensitivities, water and food analysis, indiustrial microbiology and other activities. The culture media may be in the form of liquid (Fluid) or solid media. Fluid mediam is used to grow langer quantities of a culture of bacterium that has already been isolated. Solid medium is usually prepare by heating agar or gelatin to appropriate liquid or broth of some substance. Agar solidify the medium (liquid medium contain no agar). Solid medium is used to isolate a single beacterial species from a mixture of different bacteria. The discovery of agar as a solid tying agent: Earliest culture media were liquid which made the isolation of bacteria to prepare pure cultures extremely difficult Before the discovery of agar the silica gel was used as for solidifying media. In 1881 Robert Koch boiled the potato and sliced it with knife, and then stick the bacteria on slices, slices were unabated beneath bell jar, isolated cells developed into pure colonies but some bacterial cell can not grow well on the potato slices. An assistant of Robert Koch prepare a peptone mix with gelatin ( a protein), bacteria showed growth, but difficulty was that bacteria destroy gelatin. Gelatin can not be inabated it b/c 37c it melted. In 1882 someone thrown the exta soup of seaweed (which contain agar) in next season it became gel. The Eilstemius (one of kochs assistant) called it agar and used it b/c bacteria con not utilize (or destroy) the agar, so it is used in laboratory. Agar iis a polysaccharide obtained from green algal. Iits long chain of polysaccharide is mainly composed of D-galaito pyranose Units.37

TYPES OF MEDIA

1)

Synthetic or defined media

A medium in which composition and amount of all the components is known, is called as synthetic or chemically defined medium. In defined media it is known that which one is carbon source, nitrogen source, sulfur, phosphorus source, etc. e.g photo litho atrophic bacteria such as cyanobacteria and eukaryotic algal can be grown on relatively simple media contusing co2 as a nitrogen source sulfate, phosphate and a variety of minerals. Many chimoorganotrophic microbes can also be grown in defined media eg e6 e.g. The composition of defined media for E.Coli is. Glucose ___________________ 1 g/liter Na HPo4 ____________________ 16.4 g/liter KH2 PO4 ___________________________ 1.5 g/l (N2H4 )2 50 ________________ 2.0 g/l My so4 7HO _______________ 200.0mg/l Feso, 7H2O ________________ 0.5 mg/l Final4 PH 6.8-7.0 The Defined media are used widely in research, as it is often desirable of know what the experimental microorganism is metabolizing.

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COMPLEX MEDIA Media that contain (Ingredients) components of unknown chemical composition are called complex media usually compiled media are used because a single complex medium meet the nutritional requirements of many different microorganisms. In addition in a complex medium the composition of nutrients is not fixed, her each type of microbe can easily grow. Complex media contain undefined components like 1. Peptones 2. Beef extract (or meat extract) and yeast extract 1. Peptone is proteolyses, digestion product of meat casein, soya meal gelatin and other protein source. It services as soruce of carbon, energy and nitrogen. Beef extract contains a acids, peptides nucleotides, organic acids, vitamins and minerals. Beef extract or yeast extract is an excellent source of b vitamins as well as nitrogen and carbon compounds. If a solid medium is needed for surface cultivation of microbes liquid media can be solidified with addition of % agar, most commonly 1.5% is used agar is a sulfated polymer composed mainly of D-galaxies, 3,6 anhydro-1-galactose and D-glucuranic acid, it is usually extracted from red-algae agar is well suited as solidifying agent b/c after melting it can be coaled at 0-402 before hardening (Melt at 80-90OC)/ Three commonly used complex media are 1. Alutrient broth, 2. Tryptic soy broth 2. Nutrient broth Peotone __________5gk Beef extract 3gK 2. Tryptic soy broth Tryptone ________17gk Reptone _________3gk Glucose _________2.5gk Nacl_____________ 5gk K2S4O ___________2.5gl The nutrient agar media is also a complex media having composition reptone_____5gk, Beef extract____3gk, agar ____is___2kg 3. Enriched media The enriched media contain the nutrients required to support the growth of many (a wide variety) microorganisms, including some of the more fastidious pathogens.

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These media are simple, defined to which many substances such as blood, serum, glucose, yeast extract are added to enrich the media or to enhance the bacterial growth the enriched media are commonly used to isolated as many microbes as possible. Examples of the enriched media are cryptic say agar, blood agar, chocolate agar etc. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar medium is enriched with heat-treated blood which turns brown and gives the medium brown calour so it is called as chocolate medium. Enrichment media Enrichment media contain specific composition which encourage the growth of a particular microbe in a sample with complex mixture of microbes whin a substance is added to the medium then desired microbe becomes the dominant specses because it is better adapted than its competitors to grow in medium Example 1. Serenity broth for salmonella 2. Gram negative broth for salmovalla + shigella 3. Alkaline peptone water for vibrato cholera Selective media The media which support or allow the growth of particular support or allow the growth of particular microorganisms while inhibiting the growth of others, are called selective media. The media are used to suppress the growth of anwanted microbes Many clinical specimens contain a mixture of two or more microorganisms the isolation + identification of any desired organism may be carried out by using selective media which permits the growth of that desired microbes inhibiting the growth of others. Selective media may be simple or enriched media to which selective factors such as dyes, high salt concentration has been added selective media may contain substainces such as antibiotics, potassium fell rite, bilesalfsete which selecfiuelely inhibif the growth of some bacteria of allow the growth of others e,g

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Maiconkey agar media and eosin methyline bluc (EMB)are commonly used selective media which contain bale salts and crysfal villef dye which favour the grom negative bacteria by inhibiting the growth of gram positive bacteria. Differential media:- these media differentiale b/w the different groups of bacteria (microbes) and even permits fenfatire identify action of microorganisms based an their biological charotferistics . These media contain indicators that distinguish b/w organisms on the basis of their appearance on the mediam. These media allow cerfain organisms to produce macroscopically distinct colonies (or characteristic,zones around colonies)which are helpful in differentiafing these organisms. From others in the souple Blood agar is both a differential medium of and an enriched one. It distinguishes b/o hemolytic and non-hemolytic bacteria. Hemolytic bacteria (e.g streptococci staphylococci) produce clear zones around. Their colonies b/c of red blood cell destruction. Macconlcey agar is also both differential and reflective medium. Since it contains lactose and red dye, (lactose fermenting colonies are appear pink to red in calour and are easily distinguished from colonies of non-fomenters. ISOLATION OF PURE CULTURES The pure culture is one that contains a single species. The pure culture is a population of cells arising from a single cell to characterize an individual species. IMPORTANCE:Pure cultures are is important that development of pure culture techniques by Robert Koch transformed microbiology. For the adequate study of microbe a pure culture is needed. Pure cultures can be used to determine biochemical immunological properties pure cultures are also needed for other microbial applications suchy as harvesting useful compounds produced by microbes simply. A culture that arising form a single type of cell is called as pure culture. After the development of pure culture techniques, most pathogens responsible for major human disease have been discovered e.g Laruen discovered plasmodium which cause malaria, Friend discovered streptococcus peneumonia which cause pneumonia, Bassi discovered silkworm disease is caused by fungus. THE SPREAD PLATE TECHNIQUE: The spred plate technique iis one of the method to isolate the pure culture. For this method we need frees nutrient agar in a Petri dish and a broth culture41

In this method first we take an agar plate and then a small volume (Iml of dilute microbial mixture (or broth culture) containing about 30-300 cell is transferred to the centre cuenly over the surface with as sterile bent glass rod, and then put the agar plate in wbation the dispersed cells develop into isolated colonies. So we can study the characteristics of an individual conlony by this techniques. As the number of colonies are equal to the number of viable organisms in the sample, so spread plates can be used to count the microbial population. STEAD PLATE TECHNIQUE: Pure colonies can also be abstained first we will form nutrient agar media (peptone sg, beef extract 3g, and agar 1.5g). this nutrient agar media is pured into petri dish and allowed to solidified in a incubator. After that a mixture of microbes is from sferred to the edge of this agar plate with an inoculating wire loop or swab, and then streaked out over the surface in the form of four quadrant. After that incubate the plate at 37oC for 24 hrs. After incubation the reparate colonies are obtained. PURE PLATE TECHNIQUE This method is used for counting the number of living bacteria or groups of bacteria in a liquid culture suspension Prepare serial dilution of original sample to reduce the microbial population to oblain repartee colonies Then starting with the greatest dilution put about iml of each dilution into each of pcm petri cish by pipette. Then pure into each dish about 10ml of clear nutrient agar (melted coaled jat 45oC) Mix well by rapidly moving the plate for 10 seconds. Allow the agar to set and incubate at 37oC for 24 hrs. after incubation colonies will be seen well distributed throughout the depth of medium and on be enumerated using colony counters. The plates containing 30-300 colonies are counted. The total number of colonies equals the number of viable microbes in the diluted sample. Q: 7 what is Microbiology? Microbiology is the study of lining rogainisms of microscopic size, which include bacteria fungi, algae, protozoa, and infectious agents that are called viruses. It is concerned with their form, structure reproduction. Physiology, metabolism, and classification. It includes the study of their contribution in nature, their relationship to each after and to other lining organisnts, their effects on human beins and onto their42

animals and plants, their abilities to make physical and cynical changes in our environment. On the positive side microbiology has its share in contributing as a model in studies of genetics, molecular biology in addition to suggestiong diagnostic methods, precention and control of carious diseases of microbes origin. Generic engineering is another magnificent gift of microbes with other technique of generic manipulations. On of the attractive features of microbiology is the amount of investigation and work remaining to be done. Many decisions affection the future of the world may depend upon and involve the activities of micro organisms in areas like food precaution pollution control, energy production and the control and treatment of diseases. In short microbiology has assumed a position of great importance in madder society. Microorganisms are closely associated with the health and welfare of human beings. Some microorganisms are beneficial and others are detrimental. For example, microorganisms are involved in the making of yogurt, chasse and wine, in the production of penicillin, interferon, and alcohol and in the processing of domestic and inclustrial wastes. Microorganisms can cause disease, spoil food, and deteriorate materials. Like, iron pipes, glass liens and wood pilings. Many miroorgansisms or microbes occur as single cell i.e. unicellular. In unicellular. Organisms all the life processes ore performed by a single all. Some are malticellulor and still other such as viruses, don not have a true cellular appearance. Some organisms called anaerobes are capable of caring out their essential functions in the absence of free oxygen where other organisms can manufacture the essential compounds for their needs from amphora sours of nitrogen and C02 other microorganisms such as viruses and certain bacteria are totally dependent for their existence. on the cell of higher forms of life. Microorganisms grow in tissues. Q: 8 The Discovery of Microorganisms

The organisms which are not visible to the unaided eye are called micro organisms, long before the discovery of microorganisms certain process caused by their life activities, such as fermentation of wnin juice milk, yeast etc were known to man. Even before microorganisms were seen, some investigators suspected their existence and responsibility for causing diseases. The roman philosopher laeretius and the

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physician Girolama Fracastore (1473-1553) were first who suggested that diseases were caused by invisible organisms inholed or ingested. The earlies miroscopic observation appear to have been made between 1625-1630 on bees and weevils be the Italian Frances stellutiv using a microscope probabley supplied by Galileo. However the firs person to observe and describe microorganisms accurately was the amateur microscopes antonym van leeuwenhock, of halland 1683. Robert Hooke aacortemparay of leeuwenhock developed compounded miroscopea in 1665(06-1672) and confirmed lecawenhocks observation. (jasem (1590) was first fortance person who sucdessfully magnified object using hand lens. Later on van plaeciz in 1762 proposed that each disease was coused by a separate agents i.e. microorganisms. Pasteur in 1857 established that frrmentaiotn was the result of microbaila activity. Different types of fermentation were assocated with different kind o mifoorgaanisms. He intrchedeed techniques of strrilization and diculped steam ssterilizer, het air oven and autoclave. Robert koch (Father of bacteriology) perfected bacterialogycal techniques chiring his studies on the culture bacillus (1876). He introchied staining techniques and also method of obtaining bacteria in pure culture using solie media. He discovered bacillus tuberculosis 1882 vibrato cholera 1883 and also demonstrated kochs phenomena which s the expression of hypersensitivity phenomena of mycobaderium tuberculosis. He also suggested criteria. Before blaining the organism reponsibel for disease, according to which (last paragraph of first question) 1. 2. 3. 4. Q: 9 The microorganism must be present in every casse of the disease but absent form health organisms The suspected microorganism must be isolated and grown in apure culture. The some disease must result whine the isolated microorganisms in injunted into a health host The same microorganism must be isolated again from the diseased host. Define Microbiology. When also you know about the branches of microbiology? Write a note on the application of microbiology?44

Microbiology It is the specie branch of biologyic science which deals with the study of mcroorganismes. The organism less then about I millimeter in diameter can not be seen. With the unaided eye and must be examin3d with the microscope are called miroorganisme. These include bacterial, fungi, algae, protozoa, and infectious agents called virus . Microbiology deal with the form, structured reproduction, physiology m metabolism and classification of microorganisms. It includes the study of their distribution in nature, their relationship to each other and to other living organisms, their effects on human beings and pants & animals and their ability to make changes in our environments. The microorganisms (To small unto 0.2 mm) need magnification to increase the size for this purpose instrument used in microscope. Light microsope magnified the microorganism upto 1000-1500mm, more magnification can be sun bey electorn microscope which mangifyses objects bout 10000mm. To small components (cell components ) also need further magnification to increase their size viruses 0.01mm to 0.02mm. Bacteria 0.1mm to 750mm. Two bacteria that are seen with out a microscope are thiomargarta and epulopiscium. Branches OR applied & main area of microbiology? We can divide the branches of microbiology into two types. (a) (b) Branches of microbiology on the basis of classification of microorganism. Branches on the basis of characteristic of microbiology

The branches on the basis of classification of microbiology are as follows. 1. Bacteriology Bacteriology is that branches of microbiology which deals with the study of bacteria. It includes their infection, isolation, morphological and physiological characteristics and their effect on environments. 2. Parasitology The branch of microbiology which deals with the study of microorganisms, which cause the infection n living organisms is called parasiatlogy, it deals with their isolation,

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pmicophalogy, reprochuciton, and their effect, on orgaisms or mode of infection protozea, theelminths. 3. Mycology The branch of microbiology which deals with study of fungi is called mycology. It deals with their morphological and physiological characteristics, reprochuciton nutrituion, and their effect on the environment (yest, Mildest) 4. Phycology The branch of microbiology which deals with the study of the algae is called Phycalogy. 5. Virology The branch of microbiology which deals with the study of the infectious agents called (microorganism due to smallest size) viruses. The vurses are obligate intracellular parasites which are totally dependent on the host cell. Virology deal with nature, morphology reproduction, and mechanism of the action of viruses on the hosts. The main areas or braches of microbial as a result o characteristics of microorgansimss are as follows. 1. medical microbiology 2. aquatic microbiology 3. environmental microbiology 4. soil microbiology 5. industrial microbiology 6. Genetic engineering microbiology 7. food microbiology 8. immunology 9. clinical microbiology 10. community microbiology 11. diagnostic microbiology 12. microbial ecology 13. agricultural microbiology 1. Medical Microbiology The branch of microbiology which deals with the study of microorganisms (pathegenci microorganism) which cause diseases in the animal, plants human being and other46

living organisms, is called medical microbiology. It includes their islation, and diagnosis the medical microbialogists study the ways in which microorganisms cause diseases. The medical microbiology also include the prevention of diseases by using the antimicrobial substance. 2. Aquatic microbiology OR (marine microbiology) Aquatic microbiology deals with the study of microorganisms and their activities in fresh, estuarine and marine waters, including lakes, rivers, springs, bays and seas. The aquatic microorganisms inclueed viruses, bactera, algae, protozoa, and microscoic fungi which inhabit these natural waters. Some are purely water living while others enter into water from air or soil or from industrial and domestic waster. The aquatic microorganisms are of great importance, they can produce they different material in the water, clean the environment in the water, and decrease pollution in the aquatic environment. They may affect the health of humans and other animal life. They occupy a key role or positon in the foood chain by providing rich nurish ment for the next higher level of aquatic life. They are instrumental in the chin of biochemical reaction which accomplish recyding of element e.g. in mineralization 3. Environmental Microbiology The environmental microbiology deals with the study of microbiology live in the air and cause the different diseases in the living organisms. Indoor disease are very much concreted and dangerous, while outdoor disease in whole environmental) are less concentrated and hence lesed dangerous. But it depends on the type of organisms cousing diseases. Some enerinomebntsla mccroogansims. Are useful, they produce oxygen. Some fix the atmospheric nitrogen to other complex compound, which are used in nucleic acid so microorgansismsm are important for life and with out them on life can exit. 4. Food Microbiology Food microbiology deals with the microorganisms present in the food infection and food intoxification microorganisms ore association in a variety of ways, with all of the food we eat. They may influence the quality availability and quantity of oufood. Naturally accruing foods such as fruits and vegetables normaly contain some microorganisms and may bey contataminated with additional rganisms during handling food can reserve as a medium of growth for microorganisms an this growth may couase47

the food to undergo decomposition and spoilage. Food amy also carry pathogenic microorganisms as a result transmit disease. It they allowed to grow in certain food products they produce toxic substance yogert, chees, sauerkrautek. 5. Soil Microbiology Soil microbiology is that branch of microbiology wihc deals with the study of soil microorganisms. The wastes of the humans and other animals their bodies and tissues of plants are buried or clumped in the soil, they all disappedred, transformed into the ssubstances that make up the soil, and all these changes are made by microorganism present in prexisiing soil, i.e. the conucrion of organic matter in to simple inorganic substances that provide nutrient material for the palent word. Thus microorganisms play a key role in maintaining life on the earth as we know it. immunity they immanity. 6. Immunology Immunology is that branch of microbiology in which we study that how the immune system of the body protect the body from pathogens and the response of infectious agents or it is the study of specific resistance offered by the body to further infection y a particular microorganisms when microorganism enter into the body, they body produces antibodies against it and kill them, they WBCs also kill them for the prevention of the body. Through the vaccination we can also kill the microbr and prevent the body. In old age antibodies production decrease so infection increases. 7. Industrial Microbiology In industrial microbiology the microorganisms are used to make products much as antibodies, vaccines, steroids, alcohols, and other solvents, vitamins amino acids and emyones. Essential nutrient are also synthesized by the microorganisms The microorganisms produce. i. ii. primary metabalites metabolic products secondary metabalites. resistance offered by the host to this effect of pathogenic microbiology infrcton is called that result in food poisoning. Still some microorganisms are used in the preparation and prsevation of food products such as

Primary metabolites i.e. compounds not required for cell growth. We can prodced industrially. Isulin & growth harmonic by fixing gene in bacterial or other microorganisms.48

8. Genetic engineering Microorganisms In the genetic engineering micriology we study that genes can be trnaser from on organism to other organisms by producing the desired result. This process is called recombinant DNA technology and in this process the mcoroorganism act as gene transferfactors, e.g. specifi type of gene recombine in bacteria from thes plant of normal environment when this bacteria ifect the clesert plant wo n the characteristics of nomal environment plent can be seen n desert plant. By this technique it amy be possible to give concern and wheat nitrogen fixation genes so they will not require nitrogen fertilizers 9. Agricultural Microbiology Agricultural microbiology is concerned with the impact of microorganisms on agriculture. Here we study palent disease caused by microogansisms. Methods to increase the fertility of the soil of carp yields. 10. Microbial Ecology The branch of microbial ecology concerned with the realtinship of microbs with ther livng and non living environment or habitats. It includes the contribution of microorganinsms to carbon nintrogen and sulfer cycles in soil and freshwater. Scientific Methods Although biologist employ a variety of approaches in conducting oriente biologist often ues the gernela approach know as the scientific method. Whenever the biologist come across a problem the following sequence of biological methods to solve it. 1. hypothesis 2. deduction 3. experimentation 4. theory 1. Hypothesis The biologist first gather observation of the problem to be studied and then deuclop a testative hypothesis on educated guess to explain the observation so hypothesis is a statement given by abiologist on the basis of the observations collected about the problem This step is creative or nductiv because there is no deteviled. Automatic technique for genevating hypotheses. The hypothesis may be possible answer of the proble, but at this

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stage ist si in raw form and willl still undergo otherer scientific methods, at this stage the hypothesis may be true or false, For example hypothesis about malaria is that marleria is caused by plasmodium 2. Dedication A dedication is a logyicl consequence of a hypothesis the biologists after making the hypothesis the hypthesis the biologists collect the information rquied to tst the hypothesis and to deduce the resalts from htypothesis, through observation or carefully designed experiments. On the basis of the information clected the biologist decide whether the hypethesis has bene supported or rfalshified. If it has failed to pass the test, the hypothesis is rejected, and an new explaination or hypothesis is contracted. If the hypothesis passes the test I t is subjected to more secro testing. This general approach is often called as hypothetic deductive method. 3. Experimentation After deduction experiments are conducted by the biologists to test the hypothesis and deduction. Whin carrying out and experiment, ist is essentilla to use a contol group as well as an experimental group. If the hypothesis and result deduction are proved to be true by the experiments thy hypothesis is regarded as true. If the experiments do not support the hypothesis it is rejected. 4. Theory If a hypothesis continues to sunive testing, if may be asscepted as a valid theory. A theory is a set of concepts and proposition that provides a reliable systematic account of an aspect of nature The main difference between the hypothesis and the theory is that. A hypothesis is a statement given by a biologist after on the basis of simple observation about the problem while a theory is a set of concept which are made form the hypothesis after they had undergone many tests and experiment for acceptance. It is importance to note that hypothesis and theories are never absolutcly proven scientists simply gain more and more confidence in thie accuracy as they continues to service testing, fit with new observation and experiments and satisfactory explain the observation phenomena.

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Microbiology (Microbial Growth) The term growth may be defined as an increase in population and size of cell or Growth may be defined as and increase cellular constituents. But in case of microorganisms, the term growth is usually confined to the increase in cell population. It leads to a rise in cell number when microorganisms reproduce by processes like badding or binary fission. It is usually not convenient to investigate the growth and repodiciton of individual microorganisms because of their small size, there fore when studing growth, microbiologist normally follow changes in the total population number. When microorganisms are grown in a closed system populiton growth remains expenentional for only a few generation and theme enters as stationery phase due to factor such as nutrient limitation and waste accumulation, in an open system with continulal nutrisent addintion and waste removed, the expeonential phase can be maintained for long time.51

A wide variety of techniques can be used to study microbial growth by following changes in the total cell number population of viable microorganisms or the cell mass Growth also results when cell simple become loger of lagrge. If the microorganism is coenocytic i.e. a multinucleate organisms in which nuclear number. The growth curve Growth of population is studied by analyzing the growth curve of a microbial culture. When microorganism is cultivated liquid medium, they usually are grownin ab batch culture or closed system.eventually the microorganisms will undergo binary fission and a period of reped growth will encure in which the cells multiply ot and exponential rate. The growth of microorganisms reproducing by binary fission can be plotted as the logrithem of the number of cell versus the incubation time. As a result the cure si botianed called growth curve. This resulting curve has four distance phases (a) (b) (c) (d) lag phase log phase OR exponential phase. Stationary phase Death Phase or Decline phase division are not accompanied by cell division, growth results in an increase in cell size but not cell

(a) Lag Phase52

When the microorganisms are introduced into fresh culture medium occure and there is no immediatedths period is called as log phases although in lag phase the cell division does not take place and there is no net increase in mass but the cll in synthesizing new components. A log phase prior to the start of ell division can be necessary for a variety of reasons, which are as follows,. 1. 2. The cells are may be old and depleted of ATP, essential co factored and The medium is (or may by) different for that are in which the ribosomes so these must be synthesized before growth can begin. microorganisms were growing previously, hence new enzymes could be synthesized for the metabolism of the nutrienst of the new medium. 3. The microorganisms may have been injured and required time to recovery the log phase varies considerable in length with the condition of the microorganisms and the nature of the medium. This phase may be quite long if the inoculum is form and old culture or non that has been refrigerated. On the other hand, when a young, growing exponential phase culture is transferred to fresh medium of the same composition the lag phase will be short or absent. (b) Exponential phase or log phase During the log phase microorganisms growing and dividing at masimumj possible rate under the provision of genetic potential, the nature of the medium and other condition. The growth rate is constant during the exponential phases i.e. microorganisms are dividing & and doubling in number at regular intervals of time because each individual dived at a slightly darent moment growth nature rises smoothly rather then in descrete jumpe and produce two urgency. These tow agin become four and so on, is resulting in as exponential increase cell number e.g. No. of cells = 1, 2, 4, 8, 16 ..n Example: = 20, 21, 22, 23, 24..2n Exponential growth is balanced gowth. That is all cellular constituents are manufactured at constant rates relative to each other. Then if the nutrient levels or other environmental conditions changes, unbalanced growth may observed in a shift up experiemtsn in which bacteria (microbes) are transferred fro a nutritionally poor medium to a richer one. The cells first construct new ribosomes to enchain their capacity for protein synthesis this is fallowed by increase in protein and DNA synthesis. Finally, the expected rise in reproductive rate take place. The unbalanced growth also53

result when microbes are shifted from a rich medium to poor one. These shift up and shift downexperimetns demonstrate that microbial growth is under precise, coordinated control and responds quickly to changes in environmental conditions. Aft4er a time in log phase the growth rate decreases due to the accumulation of metabolism, products and other factors, so the culture passes into the stationary phase. (c) Stationary Phase In stationary phase the total number of viable (living) microorganisms remain constant and he growth curve becomes horizontal. This may result from a balance between the rate of cell divison and the rate of cell death or the populiaton simply cease to dived thourgh remaining metabolically active. This stationyary phase is usually attained by bacterial at a populaitn level of around to cells per ml. there are many reasons due to which activity growing microbes enter into this phase . 1. The major factor is nutrient limitation if an essential nutrient is severly deplete, the population growth will slow. The aerobic organisms are affected by oxygen unavailability. So the unavailability of oxygen to them will slow down their growth. 2. Population growth may also cease down due to the accumualiton of various toxic waste products. This factor mainly affects the growth of anacrobic cultres e.g. steptocci can produce so much lacteiacid and other organic acids form sugar fermaentaion that their mediumbecomes acids and growth is imhinited. Some of the species of bacteria like e. cali bacillus and dsostridum etc adepts some physiologyica adaptation in the stationay phase for their servial during tough times like channing their cell shape and spore formation etc. the length of this phase is deepening upon the particular microorganisms and condition of the medium. (d) Death phase or decline phase The final phase of the growth cycle is the death phase o rlognartihmic decline phase in which there is decrease in the not no of living organisms (microbes). The death phase is a physialogcal point or stage at which cell death rate exc3eds their birth rate cell death is defined as the irreversible loss of ability to reproduce is called cell death theis may occur due to detrimental environmental changes like nutersn deprivation and the building of toxi wasts. If the incubation of a culture containous for long period of time eventullay cell number begin to decline. It occurs due to DNA or protein dameg or54

perhaps decrease in nutrients and increase in toxic substance. The death of am microbial population is usually laog arithmic just like its growth during exponential phases i.e a constant proportion of cells dies every hour. Often the only way of deciding whether a bacterial calls is viable (living) is by incubating it in fresh medium if it does not grow and reproduce so it is considered as to be deed. It should be noted that the cultures go through death phase because of the limited conditions under which they are forced to grow. If precautions are taken to neutralize or ramous waste products and to provide a constant supply of fresh nutrient medium, it is