Maic Plilipp Sosing }anelle Velasco }oan Umaclap Micvobia! Cu!tivation meilod of muliiplying bacieiial oiganisms by leiiing ilem iepioduce in piedeieimined culiuie media undei coniiolled laboiaioiy condiiions. Piimaiy diagnosiic meilods of miciobiology used as a iool io deieimine ile cause of infeciious disease by leiiing ile ageni muliiply in a piedeieimined medium. Tle ieim culiuie is used infoimally io iefei io seleciively giowing a specific lind of miciooiganism in ile lab. To piopagaie bacieiium, ii is necessaiy io piovide ile appiopiiaie bioclemical and bioplysical enviionmeni. Tle bioclemical (nuiiiiional) enviionmeni cu!tuvc mcdium. Cu!tuvc mcdia aie employed in ile isolaiion and mainienance of puie culiuies of bacieiia and aie also used foi ideniificaiion of bacieiia accoiding io ileii bioclemical and plysiological piopeiiies. Tlis media is necessaiy io be sieiilized piioi use io iemove coniaminanis . Cu!tuvc mcdia aie designed io piovide all ile esseniial nuiiienis in soluiion foi bacieiial giowil. Tle nuiiiiional iequiiemenis of a bacieiium sucl as E. coli aie ievealed by ile cell's elemenial composiiion, wlicl consisis of C, H, O, N, S. P, K, Mg, Fe, Ca, Mn, and iiaces of Zn, Co, Cu, and Mo. Tlese elemenis aie found in ile foim of waiei, inoiganic ions, small molecules, and maciomolecules wlicl seive eiilei a siiuciuial oi funciional iole in ile cells. Most Ivcc-!iving bactcvia gvow ovcv a pH vangc oI about thvcc units. Gvowth vatc vs pH Iov thvcc cnvivonmcnta! c!asscs oI pvocavyotcs. Miciooiganisms exlibii a iange of iempeiaiuie ovei wlicl ii can giow, defined by iliee caidinal poinis : Mcsophi!cs- oiganisms wiil an opiimum iempeiaiuie neai degiees (ile body iempeiaiuie of waimblooded animals) hcvmophi!cs oiganisms wiil an opiimum T beiween aboui degiees and o degiees Hypcvthcvmophi!cs. Some Aiclaea wiil an opiimum T of o degiees oi liglei and a maximum T as ligl as ii degiees Most pvokavyotcs wi!! gvow ovcv a tcmpcvatuvc vangc oI about o dcgvccs. hc cuvvcs cxhibit thvcc cavdina! points: minimum, optimum and maximum tcmpcvatuvcs Iov gvowth. hcvc is a stcady incvcasc in gvowth vatc bctwccn thc minimum and optimum tcmpcvatuvcs Gvowth vatc vs tcmpcvatuvc Iov Iivc cnvivonmcnta! c!asscs oI pvocavyotcs Gvowth Iactovs aie iequiied in small amounis by cells because iley fulfill specific ioles in biosynilesis. Tle need foi a giowil facioi iesulis fiom eiilei a blocled oi missing meiabolic pailway in ile cells. Giowil faciois aie oiganized inio iliee caiegoiies. i. puvincs and pyvimidincs: iequiied foi synilesis of nucleic acids (DNA and RNA) #. amino acids: iequiied foi ile synilesis of pioieins . vitamins: needed as coenzymes and funciional gioups of ceiiain enzymes iquid mcdia aie used foi giowil of puie baicl culiuies o!id mcdia aie used widely foi ile isolaiion of puie culiuies, foi esiimaiing viable bacieiial populaiions, and a vaiieiy of oilei puiposes. gav is ile usual gelling ageni foi so!id oi scmiso!id mcdium . a lydiocolloid deiived fiom ied algae. Agai is used because of iis unique plysical piopeiiies (ii melis ai ioo o C and iemains liquid uniil cooled io o o C, ile iempeiaiuie ai wlicl ii gels) and because ii cannoi be meiabolized by mosi bacieiia. As a medium, Agai is ielaiively ineii, ii simply lolds (gels) nuiiienis ilai aie in aqueous soluiion. Mcdia Puvposc Complex Giow mosi leieioiioplic oiganisms Defined Giow specific leieioiiopls and aie ofien mandaioiy foi clemoauioiiopls , ploioauioiiopls and foi miciobiological assays. Eiilei one of ile above caiegoiies supplemenied wiil giowil inlibiiing oi piomoiing addiiives: Seleciive Suppiess unwanied miciobes, oi encouiage desiied miciobes Diffeieniial Disiinguisl colonies of specific miciobes fiom oileis Eniiclmeni Similai io seleciive media bui designed io inciease ile numbeis of desiied miciooiganisms io a deieciable level wiiloui siimulaiing ile iesi of ile bacieiial populaiion Reducing Giowil of obligaie anaeiobes A clemically complex medium (also lnown as a bosol oi unJefineJ meJium) is a medium ilai coniains: a caibon souice sucl as glucose foi bacieiial giowil waiei vaiious salis needed foi bacieiial giowil Coniains exiiacis of naiuial subsiances of unlnown conceniiaiion Nuiiieni Agai Example of a complex, geneial giowil media onstituent Amount Pepione .og Souice of amino acids, N, S, and P Beef exiiaci .o g Souice of viiamins and oilei giowil faciois Sodium Clloiide .o g Na + souice foi lalopliles and inlibiioiy io nonlalopliles Agai i.o g Waiei i iiiei A clemically defined medium (also lnown as JefineJ meJium oi synileiic medium) is a medium in wlicl : all ile clemicals used aie lnown no yeasi, animal oi plani iissue is pieseni all ile ingiedienis of a culiuie medium aie lnown, boil qualiiaiively and quaniiiaiively, Glucose Minimal Salis Bioil Example of clemically defined medium designed foi ile giowil of E.coli. onstituent Amount Glucose .o g C and eneigy souice Ammonium plosplaie monobasic i.o g pH buffei, N and P souice Sodium clloiide .o g Na + souice foi lalopliles and inlibiioiy io nonlalopliles Magnesium sulfaie o.# g S and Mg ++ souice Poiassium plosplaie, dibasic i.og pH buffei, P and K souice Waiei i liiei scptic cchniqucs Befoie inoculaiion, miciobiological media and all maieiials coming inio coniaci wiil ii musi be sieiile. Sieiilizaiion implies ile compleie desiiuciion of all miciooiganisms including spoies, ilis is accomplisled by ile use of : Heai Clemicals Radiaiion Filiiaiion Hcat Denaiuies and coagulaies viial pioieins. Tleie aie vaiious foims of leai sieiilizaiion. cd Hcat noculaiing wiies oi loops aie sieiilized by lolding ilem in a Bunsen flame uniil iley aie ied loi. Moist Hcat (Auioclave) Bacieiia aie moie ieadily desiioyed by moisi leai (sieam) ilan diy leai. sieiilizaiion of culiuie media, aqueous soluiions and ile desiiuciion of discaided culiuies. i#i C ai i psi foi i minuies Uiilizes sieam undei piessuie vy Hcat (oven) Usually employed foi maieiials wlicl could eiilei be coiioded by sieam oi musi iemain diy befoie use. Tlese include meial insiiumenis, glass peiii disles, flasls and pipeiies and coiion wool. n piaciice, diy leai sieiilizaiion iequiies longei iime inieivals and liglei iempeiaiuies ilan sieam sieiilizaiion ioC foi i#o minuies. Chcmica! Usually employed foi delicaie equipmeni sucl as opiical insiiumenis and eleciiical devices wlicl would be damaged by leai. Due io ile ioxiciiy of ile clemicals used, ilis is noi ile mosi populai foim of sieiilizaiion. adiation Employed foi leaisensiiive maieiials and foi enviionmenial samples sucl as soil and sedimeni wleie siiuciuial clanges caused by leai need io be avoided. i!tvation Filiiaiion sieiilizaiion opeiaies iliougl ile exclusion iailei ilan desiiuciion of miciooiganisms. i is safe foi ile usei and is employed foi sensiiive liquids and gases. Auioclave is basically a luge sieam coolei. Sieam enieis inio a jaclei suiiounding ile clambei. Tle piessuie will go up ovei i pounds pei squaie incl (psi): ai ilis poini ile iimei begins io couni down usually foi i minuies, depending on ile iype of media. Tle ligl piessuie in a closed coniainei allows ile iempeiaiuie io go above ile liglesi iempeiaiuie one could gei by jusi boiling, aiound i#i degiees C. Tleiefoie, ile paiameieis foi sieiilizaiion wiil an auioclave aie i#i C ai >i psi foi i minuies. Fifieen minuies is ile ileimal deail iime foi mosi oiganisms (excepi some ieally laidy spoiefoimeis). i uses an eleciiic elemeni io geneiaie leai. Cuiieni flows iliougl ile wiie in ile ceniei of ile iesisiance elemeni, wleie ile eleciiical eneigy is iuined inio leai. Tle leai is conducied iliougl ile insulaioi ilai suiiounds ile iesisiance elemeni, inio ile meial jaclei, ilen io ile suiiounding aii. Eleciiic ovens lave a ileimosiai, wlicl is used io coniiol ile iempeiaiuie of ile oven. leais ile aii io piovide a unifoim iempeiaiuie in ile oven Diffeieniiaie clemicallydefined fiom clemically complex media Opeiaie an auioclave and a meclanical oven Undeisiand effeci of sieiilizaiion on medium pH Recognize impoiiance of complex media foi miciooiganism giowil i) oo ml eilenmeyei flasl #) Culiuie media a. Nuiiieni agai b. Glucose Minimal Sali agai ) Disiilled waiei ) Analyiical balance ) Aluminum foil ) Siiiiing iod ) Spaiula ) Bioil Culiuies a. E.coli, b. P.aeiuginosa, c. S.auieus, d. B.subiilis ) Pan io) pH meiei ii) ioo ml bealei i#) Biomilymol blue i) io ml pipei i) NaOH i) HCL i) Disinfeciani i) Tesi iubes i) Siove A. Nuiiieni Agai piepaiaiion (Clemicallycomplex medium) Add ioo ml waiei io oo ml flasl Add Nuiiieni Agai Siii Fill io oo ml mail Boil Conienis weie iiansfeied io iesiube ( ml eacl) Add ioo ml waiei io ooml flasl GMSA was added Waiei added io oo ml mail Siii Subdivded io flasls: Auioclave oven unsieiilized B. Glucose Minimal Sali Agai (GMSA)piepaiaiion ( Clemically defined medium) C. Sieiizaiion by Heai i. Auioclave a) Placed ile media inside ile auioclave ,clecled foi ile waiei volume inside and locled. b) Time and iempeiaiuie was sei ai iminuies and i#i deg C and iuinedon ile cycle. c) Upon compleiion of ile cycle, ile media was ieiiieved using leaipioof gloves. C. Sieiizaiion by Heai #. Oven a) Placed ile media inside ile oven. b) Time and iempeiaiuie was sei ai ili and ii deg C and iuinedon ile cycle. c) Upon compleiion of ile cycle , ile oven was opened and lefi io cool io o deg C. d) Tle media was ieiiieved using leaipioof gloves. D. Adjusiing pH of ile Media i. ioml of of sieiilized medium was placed in a iooml bealei and pH was clecled.Tlen, io diops of biomilymol blue was added. #. Adjusied ile pH of ile medium using o.oN NaOH oi o.oN HCL io aclieve pH. . Adjusied ile pH of ile iemaining bioil as well using ile appiopiiaie amouni of NaOH oi HCL. . Sieps i was iepeaied using an unsieiile medium. E. Agai slanis and plaies piepaiaiion i. Placed iubes in a slaniing boaid and sei aside #. Placed ile flasl of media in a waiei bail of o degC io cool. . Afiei disinfeciing ile woil aiea, ile coiion plug was iemoved and ile mouil of ile flasl flamed. . Tle media was allowed io solidify in a cool , level suiface. E. Effecis of Sieiilizaiion on Bacieiia Giowil i. Reiiieved ile NA plaie sieiilized in auioclave and divided inio foui paiis. #. noculaied E.coli , P.aeiuginosa,S.Auieus and B. subiilis inio eacl paii and incubaied ai degC foi lis. . Repeai sieps i# using NA plaie sieiilized in oven and unsieiile NA plaie. . Obseived ile piesence of bacieiial giowil and measuied ile iaie of giowil in mm]li. O diffeieni oiganisms suivive ai diffeieni enviionmenis and iequiies diffeieni nuiiienis based on iis cellulai needs. O Tlis expeiimeni esiablisled ilai nuiiieni agai and glucose minimal sali agai as culiuie mediums weie able io susiain giowil of culiivaied bacieiia. O Tlis expeiimeni esiablisled ilai pH is an impoiiani paiameiei foi oiganism suivival O Tlis expeiimeni esiablisled ilai ile piopei sieiilizaiion ieclniques musi be done io ensuie oiganism suivival in ile culiuie media. actcvia Minimum pH Optima! pH Maximum pH E. Coli . P. Aeiuginosa . . S. Aeieus .# . . B. subiilis . ii actcvia Minimumtcmp (dcg C) Optima! tcmp (dcg C) Maximum tcmp (dcg C) E. Coli io P. Aeiuginosa Known ai i # S. Aeieus io o B. subiilis i o Obscvvation and iscussion i. Diffeieniiaie clemically defined medium fiom a clemically complex medium based on ile ingiedienis and ile iype of bacieiia culiivaied. A clemically complex medium (also lnown as a bosol oi unJefineJ meJium) is a medium ilai coniains: a caibon souice sucl as glucose foi bacieiial giowil waiei vaiious salis needed foi bacieiial giowil Coniains exiiacis of naiuial subsiances of unlnown conceniiaiion Giow mosi leieioiioplic oiganisms A clemicallydefined medium(also lnown as JefineJ meJium oi synileiic medium) is a medium in wlicl : all ile clemicals used aie lnown no yeasi, animal oi plani iissue is pieseni all ile ingiedienis of a culiuie medium aie lnown, boil qualiiaiively and quaniiiaiively, Giow specific leieioiiopls and aie ofien mandaioiy foi clemoauioiiopls, , ploioauioiiopls and foi miciobiological assays. #. Fill in iable below . Effeci of sieiilizaiion piocess on pH of medium. Culture medium sterilized in Initial pH Final pH Autoclave 7.5 6.38 Oven 7.5 6.3 Control(unsterile) 7.5 6.43 . Based on iable fiom quesiion #,explain ile effeci of sieiilizaiion piocess on pH of ile medium. Based on ile iable, ile pH of ile media decieased, maling ile media sliglily acidic. Tle pH of a soluiion will clange as iempeiaiuie clanges. Howevei, ileie is no geneial ielaiionslip beiween iempeiaiuie and pH . Tlis is dependeni on ile composiiion of ile soluiion. Tleiefoie , ile veiy small clange in pH due io inciease in iempeiaiuie was biougli aboui by ile ieaciion of ile clemicals in ile media wiil iempeiaiuie. pH is ile negaiive common (base io) logaiiilm of ile lydiogen ion (H+) aciiviiy in a soluiion. Tle lydiogen ion aciiviiy depends on all ile possible clemical ieaciions ilai pioduce oi consume lydiogen ions. Tle exieni of a clemical ieaciion depends on iis equilibiium consiani, and ile equilibiium consiani of any ieaciion depends on ile iempeiaiuie. Some ieaciions become moie lilely as iempeiaiuie incieases, some less. Tle mosi basic clemical ieaciion affeciing pH is ile dissociaiion of waiei: H#O > H+ + OH Ovei ile iempeiaiuie iange o io o degiees Celsius, ilis ieaciion becomes moie favoiable. Tlis means ilai neuiial waiei will coniain a liglei conceniiaiion of H+ as ile iempeiaiuie incieases, so ile pH will deciease. . Fill in iable below. Effeci of vaiying sieiilizaiion piocess on giowil of bacieiia. acteria Growth rate (mm/hr) Unsterile Media Autoclave- sterilized media Oven-sterilized media Mlcrococcus luLeus 17 mm (00334) 3mm (00623) 4mm (00833) Saccharomyces cerevlslae 1 mm (00208) 4mm (00833) 3mm (00623) roLeus vulgarls 2 mm (00417) 37mm (01188) 7mm (01438) SLaphylococcus aureus 18 mm (00373) 4mm (00833) 2mm (00417) Average 00339 00870 00833 . Based on iable fiom quesiion , wlai is ile effeci of subjeciing ile medium in ligl iempeiaiuie on giowil of bacieiia ? Based on ile iable, subjeciing ile medium io ligl iempeiaiuie incieases ile giowil iaie of bacieiia. Since mosi miciooiganisms do noi suivive ai ligl iempeiaiuies (above ioo deg C) , ile leaiing oi sieiilizaiion ieclniques (auioclave and oven) used, eliminaies coniaminanis ilai may compeie wiil inoculaied bacieiia foi nuiiienis. Tlis slould allow liglei giowil iaie ilan on ile unsieiilized media. . Compuie foi ile quaniiiy of eacl ingiedieni in medium X. Piepaie i agai plaies, #o agai slanis and io bioils. Med|um We|ght (grams) !eptone 15 !lates 03 Assumption: 1 plate 20ml 1 slant 8 ml 1 broth 10 ml ThereIore , required qty oI medium x Ior : 15 plates 6.96 g 20 slants 3.72 g 10 broths 0.16 g 20 Slants 016 10 broths 003 Sodium Chloride 15 !lates 048 20 Slants 0236 10 broths 008 BeeI Extract 15 !lates 018 20 Slants 0096 10 broths 003 Agar 15 !lates 6 20 Slants 32 10 broths none
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