microbial_growth_requirements

Microbial growth requirementsDefinition of growthOrderly increase in the sum of all the components of an organism, cell multiplication is a sequence of growth; in unicellular organism, growth leads to an increase in the number of an individuals making up a population or culture.Nutrition al requirements for growthBacteria differ widely in there nutritional requirement, some bacteria can synthesized the entire requirement from the simplest elements.Other including the most pathogenic bacteria are unable to do this, they need a ready made slowly of some organic compound required for there growth.
ElementsBacterial structural components and the macromolecules for cell metabolism are synthesized from elements *tableFor the most part, organic matter is macromolecules formed byanhydride bondsbetween building blocks synthesis of these compounds needs energy provided by ATP (adenosinetripophosphate). And additional energy required to maintain the cytoplasm composition during growth which derived fromproton motiveforce (is an electrochemical gradient with two components a difference in pH (hydrogen ion concentration) and difference in ionicchargeSources of metabolic energyThe three major mechanisms for generating metabolic energy areFermentationRespirationPhotosynthesis
Table 2. Major nutritional types of procaryotes
FermentationFermentation is characterized by a substrate (phosphorylationoxidativeoxidativephosphorylationthe formation of high-energy phosphate bonds byphosphorylationof ADP to ATP coupled to the transfer of electrons from reduced coenzymes to molecular oxygen via the electron transport chain; it occurs in themitochondria.thephosphrelaytedintermediated are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, orarginin.Respiration is analogous to the coupling of an energy-dependant process to the discharge of a batteryPhotosynthesis is similar to respiration in that the reduction of an oxidant via a specific series of electron carries establishes a proton motive force. The differences in the two process is that in the photosynthesis the reluctant and oxidant are created photochemical by light energy absorbed pigment in the membrane it can be continues as long as the source of sunlight.
NutritionAll organism require source of energy, some rely on chemical compounds for there energy and called asChemotrophes.Other utilize radiant energy (light) are calledphototrophsAll require source of electrons for there metabolism, by reducing inorganic compounds as electron donorschemolithotrophicOr using organic compounds as electron donors and calledchemo-organotrophsCarbonsourceAll organism require carbon in synthesizing cell component. All organism at least small amount of CO2. , those organism using CO2as a major source know asautorophs. Other require organic compounds as there carbon source and called asheterotrophsNitrogen source
Nitrogen is a major component of proteins and nucleic acid about 10 % of dry weight, nitrogen may be supplied in a number different forms and microorganism vary in their ability to assimilate nitrogen The end products of all pathways for nitrogen assimilation is the most reduced form of the elements ammonium ion (NH4+).Many microorganism possess the ability to assimilate nitrate (NO3-) and nitrite (NO2-) reductively by conversion these ions to ammonia (NH3)The ability to assimilate nitrogen gas reductively by ammonia which his called nitrogen fixation is properties unique to prokaryotes. Most microorganisms can use NH4+ as a source for nitrogen source and many organism posses ability t o produce ammonium ions from amines or amino acids.Sulfur sourcelike nitrogen is component of many inorganic cells substances it forms part of several coenzymes most of microorganism can uses sulfate and sulfur source reducing sulfate to level of hydrogen sulfide (h2s)
Phosphorus sourcePhosphate is required as a component of ATP nucleic acids and such as coenzyme NAD NADP andflavines, metabolites lipid, cell wall, capsular polysaccharides. It are always assimilated as a free inorganic phosphateMineral sourcesnumerous minerals are required for enzyme function as, metal ions K+, Ca+, Mg+ ,Fe+and others trace elements.Water: all living microorganism require waterGrowth factorGrowth factor is an organic compound (amino acids,puriensandpyramidines, vitamins) which a cell must contain in order to grow but which is unable to synthesize.many micro-organism when provided with the listed above are able to synthesize all of the building blocks for macromolecules ( amino acid,purine,pyrimidine, and pentose) all are metabolic precursors for the nucleic acids then incorporated into DNA, additionalcho. precursors for polysaccharides and fatty acids
When organism undergo a gene mutation so the chain is broken and no longer there is a products, so the organism must obtain that compound from the environment the compound has become as a growth factor for the organismNutritional types of bacteriaPhototrophs: uses inorganic compounds astheresource of electrons e.g.chromatiumokeniiusesH2S S+2e +2H+Asan electron donor oxidizing it to elemental sulfurOtheruses organic compoundssuchas fatty acid andalcholesas electrondonorsChemotrophsthat uses inorganic compounds astheresource of electrons e.g. ammonia astheirelectronsourceobtainingthere energyby oxidizing ammonia to nitriteAutotrophic andheterotrophicOrganism can utilize and uses for examplecharbohydratesand CO2astheirsource of carbonObligatparasite those bacteria whichcannotbe cultivated artificially onartificialmedia
Hydrogen ion concentration (pH)Most organism have optimal narrowpHmostof organism areneutrophilgrow best at pH 6.0-8.0Otheacidophoilslow pH 3Alkaliphileshigh pH 10.5TemperatureDifferent microbial species varywidelyin their optimal temperature ranges for growth:Psychrophilicforms grows best at lowtemperatures(15-20oC)Mesophilicgrow best at (30-37oC)*Thermophilicgorwat (50-60oC)
Hydrogen ion concentration (pH)Most organism have optimal narrowpHmostof organism areneutrophilgrow best at pH 6.0-8.0Other acidophiluslow pH 3Alkaliphileshigh pH 10.5TemperatureDifferent microbial species varywidelyin their optimal temperature ranges for growth:Psychrophilicforms grows best at lowtemperatures(15-20oC)Mesophilicgrow best at (30-37oC)*Thermophilicgorwat (50-60oC)

Hydrogen ion concentration (pH)Most organism have optimal narrowpHmostof organism areneutrophilgrow best at pH 6.0-8.0Otheacidophoilslow pH 3Alkaliphileshigh pH 10.5TemperatureDifferent microbial species varywidelyin their optimal temperature ranges for growth:Psychrophilicforms grows best at lowtemperatures(15-20oC)Mesophilicgrow best at (30-37oC)*Thermophilicgrow at(50-60oC)

Heat shock response: when organisms exposed to a sudden rise in temperature above growth optimal, theses proteins appears to be unusually heat resistant to stabilize the heat-sesitveproteinsCold shock; a number of compound protects cells from either freezing or cold shock (glycerol addimethylsulphoxideare mist common used.Cold shock; a number of compound protects cells from either freezing or cold shock (glycerol addimethylsulphoxideare mist common used.AerationMany of organism are obligate aerobic others are facultative aerobic and anaerobic.The natural products of aerobic metabolism are the reactive compound hydrogen peroxide (H2O2), Andsuperperoxide(O2)These products can damage any biological macromolecules2O2 + 2H O2 + H2O2Many aerobes and anaerobic are protected from these products by the presences ofsuperperoxieddismutase enzyme that catalysis the reaction (Catalaseenz).

2H2O2 2H2O2+O2Some fermentation organism doesn’t contains either ofenz.Oxygen is not reduced therefore there will be no productsFor anaerobic organism have a considerable tolerance to oxygen as a result of their ability to produce high level of anenzy(NADHoxidase) that reduces oxygen to waterNADH + H +1/2 O2 NAD + H2OHydrogen peroxide owes much of its toxicity to the damage it causes to DNA


Hydrogen peroxide owes much of its toxicity to the damage it causes to DNAObligates anaerobic present a problem in oxygen exclusion using reducing agent such asthioglycolatecan be added to liquid medium or the medium sealed with a layer of petrolatum and paraffinIonic strength and osmotic pressureOrganism require high salt concentration know ashallophilicThose requiring high osmotic pressure are calledosmophilicMost of bacteria are able to tolerate external osmotic pressure and ionic strength because of their ability to regulate internalosmolalityand ion concentration

Culturing of microorganismCultureteachniqueused to isolate pathogens in pure culture so that they can be identified , andifindicated,testedforthieresensitivity (Susceptibility to antimicrobials)Most of bacteria can be cultured artificially providing:The culture medium contains the required nutrients in the correct amounts and the osmotic pressure and pH of the medium also correctThe microorganism are incubated in atmosphere and temperature most suited to there metabolismMicrobial growth requirementApproximately 80% of the living weight of bacterial cell is water and the rest is of dry weight 2-5% is phosphorus , minerals oxygen and hydrogen inorganic compounds

Culturing of microorganismCultureteachniqueused to isolate pathogens in pure culture so that they can be identified , andifindicated,testedforthieresensitivity (Susceptibility to antimicrobials)Most of bacteria can be cultured artificially providing:The culture medium contains the required nutrients in the correct amounts and the osmotic pressure and pH of the medium also correctThe microorganism are incubated in atmosphere and temperature most suited to there metabolismMicrobial growth requirementApproximately 80% of the living weight of bacterial cell is water and the rest is of dry weight 2-5% is phosphorus , minerals oxygen and hydrogen inorganiccompounds

So the media should contains water, source of nitrogen, carbon, minerals , and essential vitamins. Other substances may be included according to the species requirements.Common ingredient of culture mediaPeptone:This is a general term for the water soluble products obtained from the breakdown (hydrolysis) of animal or plant proteins.The proteins are commonly those frommeat, milk, and soyabean meal. Theyare hydrolyzed by acids or by enzymessuch as pepsin,trypsin, andpapain. The products are free amino acids, peptides (polymers of amino acids) andproteoses(large size peptides).All forms of peptone are not coagulated by heat.

Peptone providesnitrogenfor growingmicroor-ganisms. Plant proteins such assoyapeptonealso providecarbohydrates,and most peptones containnucleic acid fractions, minerals and vitamins.*Peptone powder should be light in color, dry, and have a neutralpH.The concentration and form of peptone used depend on the uses of individual culture media, for example peptones with a high tryptophan content are used inindoletesting media,proteosepeptone is used in media for bacterial toxin production,tryptosein enriched media, andtryptonewhich is particularly rich in amino acids is added to several media including blood culture media.

2H2O2 2H2O2+O2Some fermentation organism doesn’t contains either ofenz.Oxygen is not reduced therefore there will be no productsFor anaerobic organism have a considerable tolerance to oxygen as a result of their ability to produce high level of anenzy(NADHoxidase) that reduces oxygen to waterNADH + H +1/2 O2 NAD + H2OHydrogen peroxide owes much of its toxicity to the damage it causes to DNAObligates anaerobic present a problem in oxygen exclusion using reducing agent such asthioglycolatecan be added to liquid medium or the medium sealed with a layer of petrolatum and paraffinIonic strength and osmotic pressureOrganism require high salt concentration know ashallophilicThose requiring high osmotic pressure are calledosmophilicMost of bacteria are able to tolerate external osmotic pressure and ionic strength because of their ability to regulate internalosmolalityand ion concentration

Culturing of microorganismCultureteachniqueused to isolate pathogens in pure culture so that they can be identified , andifindicated,testedforthieresensitivity (Susceptibility to antimicrobials)Most of bacteria can be cultured artificially providing:The culture medium contains the required nutrients in the correct amounts and the osmotic pressure and pH of the medium also correctThe microorganism are incubated in atmosphere and temperature most suited to there metabolismMicrobial growth requirementApproximately 80% of the living weight of bacterial cell is water and the rest is of dry weight 2-5% is phosphorus , minerals oxygen and hydrogen inorganic compounds

So the media should contains water, source of nitrogen, carbon, minerals , and essential vitamins. Other substances may be included according to the species requirements.Common ingredient of culture mediaPeptone:This is a general term for the water soluble products obtained from the breakdown (hydrolysis) of animal or plant proteins.The proteins are commonly those frommeat, milk, and soyabean meal. Theyare hydrolyzed by acids or by enzymessuch as pepsin,trypsin, andpapain. The products are free amino acids, peptides (polymers of amino acids) andproteoses(large size peptides).All forms of peptone are not coagulated by heat.Peptone providesnitrogenfor growingmicroor-ganisms. Plant proteins such assoyapeptonealso providecarbohydrates,and most peptones containnucleic acid fractions, minerals and vitamins.

Peptone powder should be light in color, dry, and have a neutralpH.The concentration and form of peptone used depend on the uses of individual culture media, for example peptones with a high tryptophan content are used inindoletesting media,proteosepeptone is used in media for bacterial toxin production,tryptosein enriched media, andtryptonewhich is particularly rich in amino acids is added to several media including blood culture media.Meat extractsBeef extract such asLabLemcoprovides organisms with a further supply of amino acids, and also with essential growth vitamins and mineral salts including phosphates andsulphates. It is an ingredient of many culture media including nutrient agar and nutrient broth.Trypsindigested meat extracts are also usedYeast extractThis is contained in many culture media as a bacterial growth stimulant, for example inxyloselysinedeoxycholate(XLD) medium, modified New York City (MNYC) medium, andthiosulphatecitrate bile salt sucrose (TCBS) medium.

Mineral saltsFor cell growth,sulphatesare required as sources ofsulphurand phosphates as sources of phosphorous. Culture media should also contain traces of magnesium, potassium, iron, calcium and other elements which are required for bacterial enzyme activity. Sodium chloride is also an essential ingredient of most culture media.CarbohydratesSimple or complex sugars are added to many culture media to provide bacteria with sources of carbon and energy.Carbohydrates are also added to media to assist in the differentiation of bacteria, for example lactose is added toMacConkeyagar anddeoxycholatecitrate agar to differentiateenterobacteria, and sucrose to TCBS agar to differentiateVibriospecies. Fermentation of the sugar with acid production is detected by a change incolourof the indicator. Fermentation is often accompanied by the production of gas (carbon dioxide and hydrogen).



Besides being used to solidify culture media, agar also provides microorganisms with calcium and other organic ions.WaterThis is essential for the growth of all microorganisms. It must be free from any chemicals which inhibit bacterial growth.Deionizedor distilledwater must be used in the preparation of culture media if the local water supply has a high mineral content.TYPES AND SELECTION OF CULTURE MEDIAThe main types of culture media are:BasicEnriched and enrichmentSelectiveDifferentialTransportBasic media

These are simple media such as nutrient agar and nutrient broth that will support the growth of microorganisms that do not have special nutritional requirements. ,They are often used in the preparation of enriched media, to maintain stock cultures of control strains of bacteria, and forsubculturingpathogens from differential or selective media prior to performing biochemical and serological identification tests.Enriched mediaThese are media that are enriched with whole blood,lyzedblood, serum, extra peptones, special extracts, or vitamins to support the growth of pathogens that require additional nutrients or growth stimulants.Enriched media are required for the culture ofHaemophilusinfluenzae, pathogenicNeisseria, and severalStreptococcus species. Blood agar andtryptonesoya media are used to produce a better and more rapid growth of a wide range of pathogens.

Note: The term enrichment is used to describe a fluid medium that increases the numbers of a pathogen by containing enrichments, and, or substances that discourage the multiplication of unwanted bacteria. For example,seleniteF broth is used as an enrichment medium for salmonellae infaecesor urine prior tosubculturingonxyloselysinedeoxycholate(XLD) agar or other enteric selective medium.Selective mediaThese are media which contain substances that prevent or slow down the growth of microorganisms other than the pathogens for which the media are intended. For example, XLD agar selects forsalmonellaeandshigellaeby containing bile salts that inhibit the growth of manyfaecalcommensals.In recent years, antimicrobials have become increasingly used as selective agents in culture media. Examples of antimicrobial selective media include modified New York City (MNYQ medium for isolatingNeisseriagonorrhoeaefromurogenitalspecimens, andButzlermedium for isolatingCampylobacterspecies fromfaeces.

Selective media are available for isolating most of the important pathogens.Differential (indicator) mediaThese are media to which indicators, dyes, or other substances are added to differentiate microorganisms, for example TCBS agar contains the indicatorbromothymol. blue which differentiates sucrose fermenting from non-sucrose fermentingVibriospecies.

Most, but not all differential media distinguish between bacteria by an indicator which changescolourwhen acid is produced following carbohydrate fermentation. Blood agar, however, can also be described as a differential medium when it differentiateshaemolyticfrom non-haemolyticbacteria.As shown in the Chart on p. 43-44, many culture media are both differential and selective such as TCBS agar,MacConkeyagar, XLD agar and DCA. Enriched media may also be madeselective and, or, differential. For example, crystal violet blood agar is an enriched, selective, and differential medium for Streptococcuspyogenes(Group A Streptococcus).Transport mediaThese are mostly semisolid media that contain ingredients to prevent the overgrowth ofcommensalsand ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured soon after collection. Their use is particularly important when transporting microbiological specimens from healthcentresto the district microbiology laboratory.

Examples of transport media include Cary-Blair medium for preserving enteric pathogens (see p. 405) andAmiestransport medium (see p. 402) for ensuring the viability of gonococci and other pathogens in specimens collected on swabs. Other transport media are listed in the Chart on p. 43-44.Choice of culture mediaThe selection of culture media to use in district microbiology laboratories will depend on:- The major pathogens to be isolated, their growth requirements, and the features by which they are recognized.- Whether the specimens being cultured are from sterile sites or from sites having a normal microbial flora.

Although a selective medium is usually more expensive than a non-selective one, the use of a selective medium often avoidssubculturing, isolates a pathogen more quickly, and makes it easier to differentiate and interpret bacterial growth especially by laboratory staff with limited experience.- The cost, availability, and stability of differentmedia in tropical and developing countries.- The training and experience of laboratory staffin preparing, using, and controlling culture media.Note: Information regarding the preparation and control of culture media can be found in 48: 1.

- The cost, availability, and stability of differentmedia in tropical and developing countries.- The training and experience of laboratory staffin preparing, using, and controlling culture media.Note: Information regarding the preparation and control of culture media can be found in 48: 1.SOLID, SEMISOLID AND FLUID CULTURE MEDIACulture media can be used in three forms:SolidSemisolid

FluidSolid culture mediaThis form of media is used mainly inpetridishes as plate cultures. It can also be used in bottles or tubes as stab (deep) or slope cultures. The inoculation of plates, slopes, and deepsWhen grown on solid media, microorganisms multiply to form visible colonies. Colonial appearances and any changes in the surrounding medium help to identify bacteria and differentiatecommensalsfrom pathogens. Some cultures also have a distinctive smell, for example those of Proteus and Pseudomonasaeruginosa.Colonial appearancesBacterial colonies should be examined in a good light. A low power magnifying lens is required to see morphological details.

When viewed from above, colonies may appear round, irregular,crenated, or branching. They may be transparent or opaque and their surface may be smooth or rough, dull or shiny. The colonies of capsulated species appearmucoid. Mature colonies ofpneumococcihave a ringed appearance.When viewed from the side, colonies may appear flat or raised in varying degrees sometimes withbevellededges or with a central elevation or depression.

When touched with a wire loop, some colonies are soft and easily emulsified such as Staphylococcusaureus, whereas others are difficult to break up such as Streptococcuspyogenes.Thecolourof colonies also helps to identify bacteria, especially when using differential media containing indicators.Medium changesThese includehaemolyticreactions, pigment production,colourchanges surrounding carbohydrate fermenting colonies, and blackening due to hydrogensulphideproduction.Am example of a pigment-forming organism is Pseudomonasaeruginosawhich produces a yellow-greencolourin media such as blood agar andMacConkeyagar.

When touched with a wire loop, some colonies are soft and easily emulsified such as Staphylococcusaureus, whereas others are difficult to break up such as Streptococcuspyogenes.Thecolourof colonies also helps to identify bacteria, especially when using differential media containing indicators.Medium changesThese includehaemolyticreactions, pigment production,colourchanges surrounding carbohydrate fermenting colonies, and blackening due to hydrogensulphideproduction.Am example of a pigment-forming organism is Pseudomonasaeruginosawhich produces a yellow-greencolourin media such as blood agar andMacConkeyagar.

examples of carbohydrate fermenting bacteria that produce color changes in media include sucrose fermentingVibriocholeraethat gives a yellowcolourin TCBS agar, lactose fermentingClostridiumperfringensthat produces a pink-red color in lactose egg yolk milk agar, andmanitolfermenting Staphylococcusaureusthat gives ayellow color inmannitolsalt agar.Blackening in the medium due to hydrogensulphideproduction is seen with many salmonellae cultured inKligleriron agar.

Haemolyticreactions in blood agar are seen ,%ithbeta-haemolyticstreptococci andalphahaemolyticpneumococci.Fluid culture mediaThe growth and multiplication of bacteria in a fluid medium is usually described in four stages, or phases, as follows:- Lag phase, during which the organisms adjustto their new surroundings.- Logarithmic phase, during which the bacteria reproduce rapidly. In multiplying, the organisms use up the food substances in the medium and introduce into it toxic products of metabolism.- Stationary phase, during which there is no further increase in the concentration of living bacteria in the medium. There is a balance reached between the number of bacteria dying and those being produced.

Decline phase, during which the concentrationof living organisms is reduced as the number of dyingbacto,'fiaout-number the living bacteria in the medium.The inoculation of fluid culture media is described in 35:4. Growth is shown by a turbidity in the medium. A surface growth is shown by some organisms, for examplevibriosin alkaline peptone water.Fluid media are used mainly as enrichment media, biochemical testing media, and blood culture media (see Chart at the end of this subunit)Semisolid culture mediaThis form of medium is prepared by adding a small amount of agar (0.4-0.5% w/v) to a fluid medium.Semisolid media are used mainly as transport media and for motility testing. Examples are given in the following Chart: