GROWTH & DEVELOPMENT

1. GROWTH & DEVELOPMENT

2. To grow organisms must draw all the substances from the environment they require for the synthesis of their cell materials and for energy generation. These substances are known as nutrients. Numerous media have been developed for bacterial cultivation. Because the nutritional requirements of bacteria vary widely, there are great differences in the chemical compositions of the media used in the laboratory. Bacteria also exhibit wide differences with respect to the physical conditions favouring their growth, such as temperature, pH and gaseous environment.

3. All forms of life, from microbes to human beings share certain nutritional requirements for growth and normal functioning. The general requirements are- 1.All organisms require a source of energy. Some rely on chemical compounds for their energy and they are called chemotrophs. Others can utilise radiant or light energy and are known as phototrophs. Both chemotrophs and phototrophs exist among bacteria. Chromatiumokenii is an example of phototroph, Escherichia coli is an example of chemotroph.

4. 2.All organisms require a source of electrons for their metabolism. Some rely on reduced inorganic compounds as electron donors and are known as lithotrophs (some may be chemolithotrophs and others may be photolithotrophs). Other organisms use organic compounds as electron donors and are known as organotrophs (some are chemoorganotrophs and others are photoorganotrophs). Nitrosomonaseuropaea is lithotrophic whereas Escherichia coli is organotrophic.

5. 3.All organisms require carbon in some form for use in synthesising cell components. All of them require at least small amounts of carbon dioxide. However some use carbon dioxide as their major or even sole source of carbon, such organisms are autotrophs. Others require organic compounds as their carbon source and are known as heterotrophs. Pseudomonas pseudoflava is an autotroph and Escherichia coli is a heterotroph

6. 4.All organisms require nitrogen in some form for cell components. Bacteria are extremely versatile in this respect. Unlike eucaryotes, some bacteria can utilise atmospheric nitrogen. Others thrive on inorganic nitrogen compounds such as nitrates, nitrites and ammonium salts. Still others derive nitrogen from organic compounds such as amino acids.

7. 5.All organisms require oxygen, sulphur and phosphorus for cell components. Oxygen is provided in various forms such as water; component atoms of various nutrients or molecular oxygen. Sulphur is needed for the synthesis of certain amino acids (cysteine, cystine and methionine).Some bacteria require organic sulphur compounds, some are capable of utilising inorganic sulphur compounds and some can also use elemental sulphur. Phosphorus, usually supplied in the form of phosphates , is an essential component of nucleotides, nucleic acids , phospholipids , teichoic acids and other components.

8. 6.All living organisms require metal ions , such as K+, Ca2+, Mg2+ and Fe2+ for normal growth. Other metal ions are also needed but usually at very low concentrations, such as Zn2+, Cu2+, Mn2+, Mo6+,etc and they are known as trace elements. • Most bacteria donot require Na+ but certain marine bacteria, cyanobacteria and photosynthetic bacteria do require it.

9. 7.All living organisms contain vitamin and vitamin like compounds. These function either as coenzymes for several enzymes or as the building blocks for coenzymes. Some bacteria are capable of synthesising their entire requirement of vitamins from other compounds in the culture medium, but others cannot do so .They will not grow unless the required vitamins are supplied previously in the medium. For example, Bacillus anthracis requires Thiamin or vitamin B1, Lactobacillus spp require Pyridoxine or vitamin B6,etc.

10. Nutritional needs of microbial cells are supplied through a variety of media. The following list illustrates the nutritional diversity that exists among microbes. • 1.Carbon-Most essential and central atom common to all cellular structures and functions. Among the microbial cells, two carbon dependent types are: • a) Autotrophs-These organisms can be cultivated in a medium consisting solely of inorganic compounds, specifically they use inorganic carbon in the form of CO2. • b) Heterotrophs-They don’t grow in a medium containing only inorganic compounds, media must be supplied with organic nutrients, particularly glucose.

11. 2.This is also an essential atom in many cellular macromolecules, particularly proteins and nucleic acids. Proteins serve as structural molecules forming the fabric of the cell and as functional molecules , enzymes that are responsible for the metabolic activities of the cell. Nucleic acids include DNA, the genetic basis of cell life and RNA which plays an active role in protein synthesis within the cell. Some microbes use atmospheric nitrogen, others rely on inorganic compounds such as ammonium or nitrate salts, still others require nitrogen containing organic compounds such as amino acids.

12. 3.Non-metallic elements required for cellular nutrition are – • a) Sulphur-this is integral to some amino acids and is a component of proteins. Sources include organic compounds such as sulphur containing amino acids, inorganic compounds as sulphates and elemental sulphur. • b)Phosphorus-this is required for the formation of nucleic acids DNA and RNA and also for the synthesis of high- energyorganic compound ATP. Phosphorus is supplied in the form of phosphorus salts for use by all microbial cells.

13. 4.Metallic elements:Ca2+ ,Zn2+ ,Na+ , K+,Cu2+, Mn2+,Mg2+ and Fe2+ are needed forcontinuous efficient performance of variety of cellular activities like osmoregulation, regulation of enzyme activities, electron transport during biooxidation. • 5.Vitamins-These organic substances contribute to cellular growth. They are needed in minute concentrations for cellular activities and are also sources of coenzymes. • 6.Water- All cells require water in the medium so that low molecular weight nutrients can cross the cell membrane.

14. 7.Energy- two bioenergetic types of microbes are there, • a) Phototrophs- they use radiant energy as their sole energy source. • b) Chemotrophs- they depend on oxidation of chemical compounds as their energy source. Some microbes use organic molecules like glucose, others utilise inorganic compounds such as H2S or NaNO2.

15. Nutritional types of bacteria • Bacteria can be divided into many groups on the basis of their nutritional requirements. The major separation is into two groups, phototrophs and chemotrophs. • Among the phototrophic bacteria are species that use inorganic compounds as their source of electrons (i.e.photolithotrophs).For example Chromatiumokenii uses H2S as its electron donor, oxidising it to elemental sulphur . • Some phototrophic bacteria use organic compounds such as fatty acids and alcohols as electron donors and so they are photoorganotrophs. For example Rhodospirillumrubrum can use succinate as an electron donor.

16. Chemotrophs rely on chemical compounds rather than light for their energy , so under certain circumstances a phototrophic bacterium can grow as a chemotroph.For example, in the absence of oxygen under anaerobic conditions R.rubrum is dependent on light as its source of energy and lives as a photoorganotroph. However in the presence of oxygen it can grow in the dark as a chemoorganotroph. • Among the chemotrophic bacteria are species that use inorganic compounds as their source of electrons (i.e.chemolithotrophs).For example bacteria of the genus Nitrosomonas use ammonia as their electron source , obtaining energy by oxidising ammonia to nitrite.

17. Many other chemotrophic bacteria use organic compounds such as sugars and amino acids as electron donors and are called chemoorganotrophs. • Certain bacteria can grow as either chemolithotrophs and chemoorganotrophs. For example, Pseudomonas pseudoflava can use either the organic compound glucose or the inorganic compound H2 as its source of electrons.

18. Autotrophs- The chemolithotrophic bacteria of the genus Nitrosomonas are able to oxidise ammonia to nitrite , thereby obtaining sufficient energy to assimilate the carbon of CO2 into cell components ( CO2 fixation) .Organisms that can use CO2 as their sole source of carbon for assimilation are called autotrophs.

19. Until recently it was thought that all chemolithotrophic bacteria were autotrophs. Although this is true for most chemolithotrophs , a few of them are now recognised to be chemolithotrophicheterotrophs(mixotrophs) i.e. they obtain their energy by utilising inorganic electron donors , but obtain most of their carbon from organic compounds. One such organism is Desulfovibriodesulfuricans , which uses electrons from H2 for the reduction of sulphate , yet derives most of its carbon from organic compounds in the culture media.

20. Some autotrophs are facultative autotrophs i.e. they can live either as autotrophs, deriving their carbon from CO2, or they can live as heterotrophs deriving their carbon from organic compounds. For example P.pseudoflava can live as a heterotroph, using glucose as a source of carbon for assimilation . However if H2 is provided as the electron source , then it can use CO2 as sole carbon source and can grow as an autotroph.

21. Why do we need microbial control? • To control the growth of microorganisms of different types , for preservation of food and environment, particularly for the disinfection of water, prevention of spoilage of articles of economic importance and also control of diseases caused by microbes.

22. Conditions influencing the effectiveness of antimicrobial agents: • 1.Population size- Because an equal fraction of a microbial population is killed during each interval, a larger population requires a longer time to die than a smaller one. • 2.Population composition- The effectiveness of an agent varies greatly with the nature of the organisms being treated because microbes differ markedly in susceptibility. Bacterial endospores are much more resistant to most anti-microbial agents than the vegetable forms. Young cells are more readily destroyed than mature organisms.

23. Conditions influencing the effectiveness of antimicrobial agents: • 3.Concentration and intensity of an antimicrobial agent- Often but not always the more concentrated a chemical agent or intense a physical agent , the more rapidly the microbes are destroyed. However, effectiveness of the agent is usually not directly related to the concentration or intensity. • 4.Duration of exposure-The longer the population is exposed to a microbicidal agent , the more number of organisms are killed.

24. Conditions influencing the effectiveness of antimicrobial agents: • 5.Temperature-An increase in the temperature at which a chemical acts often enhances its activity. Frequently a lower concentration of disinfectant or sterilising agent can be used at a higher temperature. • 6.Local environment- The population to be controlled is not isolated but surrounded by environmental factors that may either offer protection or aid in its destruction. For example, as heat kills more readily at an acidic pH, acidic foods and beverages such as fruits and tomatoes are easier to pasteurise than foods with higher pHs like milk.

25. Physical methods of control • Heat- High temperature combined with high moisture is one of the most effective methods of killing microbes. Moist heat kills microbes by coagulating their proteins .Dry heat destroys them by oxidising their chemical constituents. Spores of Clostridium botulinum are killed in (4-20) mins by moist heat at 120 degree centigrade, whereas a 2 hour exposure to dry heat at the same temperature is required. Vegetative cells are more sensitive than spores. • Moist heat- Steam under pressure-use of autoclave.

26. Fractional sterilisation- • Some microbiological media, chemical solutions and biological materials cannot be heated above 100 degrees without being damaged. However if they can withstand the temperature of free-flowing steam they are sterilised by fractional sterilisation or tyndallisation. The material is heated at 100 degrees on 3 successive days with incubation periods in between. Resistant spores germinate during the incubation periods on subsequent exposure to heat and the vegetative cells are destroyed.

27. Boiling water- • This destroys the vegetative cells of disease producing organisms, it doesn’t carry out sterilisation. • Pasteurisation- • Milk, cream and certain alcoholic beverages like beer and wine are subjected to controlled heat treatment known as pasteurisation. Only some specific microbes are killed , all organisms are not destroyed.

28. Incineration • - Destruction of microbes by burning at a very high temperature is routinely practised in the laboratory when the transfer needle is introduced into the flame of Bunsen burner. By this method infected laboratory animals, etc are destroyed. • Temperature below the optimum for growth depress the rate of metabolism. If the temperature is sufficiently low, growth and metabolism stops. Low temperature is useful for preservation of cultures, since microbes have a unique capacity for surviving extreme cold. • Inactivation of organisms in foodstuff processing. High pressure processing is a non-thermal process capable of inactivating and eliminating pathogenic and food spoilage organisms.

29. Filtration- • Bacteriological filters are used to remove microbes from liquids or gases. The filters are made of different materials – an asbestos pad in the Seitz filter, diatomaceous earth in the Berkefeld filter, porcelain in the Chamberland Pasteur filter, etc. The mean pore diameter in these filters range from 1 to several micrometers. Porosity alone is not the only factor preventing the passage of organisms, other factors like electric charge of the filter , electric charge carried by the organisms, nature of the fluid being filtered can influence the efficiency of filtration. Sterilisation of heat-sensitive biological fluids and air disinfection is possible by this method.

30. Desiccation- • In this method, cessation of metabolic activities of the microbial cells occur, followed by a decline in the total viable population. The time of survival of micro-organisms after desiccation varies, depending on the following factors-the kind of microbes, the material on which the organism is dried, completeness of the drying process, physical conditions like light, temperature, humidity, etc to which the dried organisms are exposed.

31. Osmotic pressure- • The solute concentrations within the microbial cells is approximately .95%. If the cells are exposed to solutions with higher solute concentration, water will be drawn out of the cell. This process is plasmolysis. The passage of water from a low solute concentration into the cell is plasmoptysis. The pressure built up within the cell as a result of this water intake is osmotic pressure. Plasmolysis leads to the dehydration of the cells and metabolic processes are retarded partially or completely. The antimicrobial effect is similar to that caused by desiccation.

32. Radiation- • Electromagnetic radiation- UV light, x-rays, acoustic radiation, sound waves, etc are used in cold sterilisation process because ionising radiations produce relatively little heat in the material being irradiated. • The uv portion of the spectrum includes all radiations from (150-3900) Angstrom. Sunlight under certain conditions has microbicidal capacity but to a limited degree. Germicidal lamps emitting UV radiations are widely used to reduce microbial populations in hospital operating rooms, asceptic filling rooms , in the pharmaceutical industries, food and dairies, etc.

33. Mode of action- • Nucleic acids of the cells absorb UV light, the pyrimidines of the nucleic acid forms pyrimidinedimers in which 2 adjacent pyrimidines become bonded. DNA replication is thus inhibited and mutations result. X-rays, gamma rays, cathode rays also carry out microbial controls in packaged food, medical devices, etc.

34. Chemical methods of control • Phenol and phenoliccompounds- Phenol has the additional distinction of being the standard against which the other disinfectants of a similar chemical structure are compared to determine their antimicrobial activity. The used procedure is called the phenol-coefficient technique. • They cause cellular disruption, cell protein precipitation, enzyme inactivation, leakage of amino acids from the cells. The lethal effect is associated with the physical damage to the membrane structures in the cell surface which initiates further deterioration.

35. Alcohol • -Ethyl alcohol, methyl alcohol they are used for reducing the microbial flora of skin, disinfection of clinical thermometers, etc. • They are protein denaturants, alcohols are lipid solvents, so they damage lipid complexes of cell membrane. They are also dehydrating agents. Severe dehydration leads to bacteriostatic action. Alcohol has a cleansing or detergent action that results in the mechanical removal of microbes.

36. Halogens- • Iodine- Tincture of iodine, pure iodine is a bluish-black crystalline element with a metallic lustre, slightly soluble in water but readily soluble in alcohol and aqueous K or NaI. Iodine is also used in the form of substances known as iodophors. It is a highly effective bactericidal agent, killing bacteria .It also possesses sporicidal activity, its used for the disinfection of skin. • Iodine is an oxidising agent, so can irreversibly oxidise and thus inactivate essential metabolic compounds like proteins with –SH groups.

37. Chlorine and chlorinated compounds- • Hypochlorites , Ca(OCl)2 , NaOCl, chloramines, etc. Products containing Ca(OCl)2 are used for sanitising dairy equipment and eating utensils in the restaurants. Chlorine compounds are used to disinfect open wounds, to treat athlete’s foot, etc. • The antimicrobial action of chlorine and its compounds come through the hypochlorous acid formed when free chlorine is added to water. The nascent oxygen released in this reaction is a strong oxidising agent, it acts on cellular constituents.

38. Heavy metals- • Mercury- Inorganic compounds HgCl2, Hg2Cl2, HgO, ammoniated mercury they are bactericidal in dilutions of 1:1000, limited use because of corrosive action, high toxicity to animals, used in ointments and antiseptics. • Organic compounds are mercurochrome, metaphen, merthiolate, etc. They are less irritating, less toxic than the inorganic mercury compounds used as antiseptics on cutaneous and mucosal surfaces.

39. Silver-Colloidal silver compounds, AgNO3, Ag-lactate, Ag-picrate, they are bacteriostatic or bactericidal in action. AgNO3 is germicidal in action and is used most widely. • Copper- CuSO4 , it is effective against algae and molds more than bacteria. Bordeaux mixture contains copper, it is a fungicide that prevents plant diseases. CuSO4 is used in swimming pools and open water reservoirs • .

40. Mode of action- In the case of all the 3 heavy metals, the mode of action is almost the same. These heavy metals and their compounds act by combining with cellular proteins and inactivating them.For HgCl2 , inhibition is directed at enzymes containing –SH groups. • The inhibitory effects of heavy metals against microbes is oligodynamic action.

41. Dyes • 2 classes of dye compounds are anti-microbially active. • Triphenylmethane dyes-malachite green, brilliant green, crystal violet dyes are used extensively in public health microbiology, where detection of E.coli is important. They exert their inhibitory effect by interfering with cellular oxidation processes. • Acridine dyes- Acriflavine and tryptoflavine have selective inhibition against bacteria, specially staphylococci, gonococci. They are used for the treatment of burns , wounds, ophthalmic applications, etc

42. Synthetic detergents- • They are surface tension depressants and are used for cleaning surfaces. Soap is an example. • They are of 3 types- • Anionic detergents-sodium lauryl sulphate, • Cationic detergents-cetylpyridinium chloride. • Non-ionic ones-they donot ionise, are amphipathic molecules, disrupts membranes by intercalating into phospholipidbilayers and solubilising lipids and proteins. Ionic detergents sodium deoxycholate and sodium dodecyl sulphate(SDS) contain a charged group, non-ionic detergents Tritin-X-100 and Octylglucoside lack a charged group.

43. Quarternary ammonium salts- • Thebactericidal power of the quarternaries is high against gm+ve bacteria and also some gm-ve ones. The combined properties of the germicidal activity and detergent action, other features such as low toxicity, high solubility in water, stability in solution, non-corrosiveness- these properties are used as sanitising agents. They are used to control microbes on the floors, walls, hospitals, nursing homes, food utensils, etc. • Mode of action involves protein denaturation, interference with glycolysis, membrane damage. They alter the vital permeability features of the cell structure

44. Aldehydes • - Two antimicrobial aldehydes are formaldehyde, glutaraldehyde. HCHO is used for the sterilisation of instruments. In the gaseous form it is used for disinfection and sterilisation of enclosed areas. Vegetative cells are more quickly killed than spores. In order to sterilise an enclosure , the temperature must be room temperature, about 22degrees and relative humidity (60-80) %.

45. Disadvantage of this process is the limited ability of formaldehyde vapours to penetrate covered surfaces. HCHO and glutaraldehyde are extremely reactive chemicals, they combine readily with vital organic nitrogenous compounds like proteins, nucleic acids, etc and thereby inactivating them probably by cross linking and alkylating molecules.

46. Gaseous agents- • Gaseous sterilisation method is used to sterilise plastic syringes, blood transfusion apparatus, etc. The main agents are ethylene oxide, beta-propiolactone, formaldehyde, etc. • The mode of action of ethylene oxide is believed to be alkylation reactions with organic compounds such as enzymes and other proteins. Many heat sensitive items such as disposable plastic petri dishes and syringes, heart-lung machine components, sutures and catheters are sterilised with ethylene oxide gas. Ethylene oxide is both microbicidal and sporicidal, it kills by combining cell proteins and particularly effective as a sterilising agent because it rapidly penetrates packing materials and even plastic wrappers.

47. Organic chemicals- • 2 important chemicals are alcohol and phenol. The physicians clean the skin by rectified spirit before injection. The effective concentration of alcohol needed is 70% by weight or 77% by volume for sterilisation. This concentration of alcohol sterilises the dry surface and that for weight surface 90% by weight has been recommended. The aliphatic alcohols are also germicidal, egs methyl alcohol, ethyl alcohol, etc

48. . The germicidal action of aliphatic alcohols increase with the number of carbon atoms in the chain upto C5, from C5 to C8 it remains quite stationary and after that there is a sharp fall of germicidal action of alcohol. Higher the solubility of the alcohol lower is its antimicrobial property. Some water must be present for alcohols to disinfect because they act by coagulating proteins. Water is needed for coagulation reactions.70% alcohol-water mixture penetrates more deeply than pure alcohol into most of the materials that has to be disinfected.

49. The mode of action involves denaturation of bacterial proteins. Protein denaturation is activated only in the presence of moisture and so absolute alcohol is less effective than rectified spirit that contains some amount of water.

50. Among glycols, only triethylene glycol and propylene glycols are effective. The glycols are effective in the form of fumes. Fumes sterilise a medium by killing microbes. • Action of dyes towards killing of microbes- The gm+ve organisms are most susceptible to the action of crystal violet than the gm-ve organisms because the gm+ve ones retain the crystal violet dye whereas the gm -ve ones donot.