Compiled by : Hastya Afinia Rusyda P278340110018 Regular Class

!R!"A# A#A$%" &'"'HA(A# "!RA)A*A P+$%('&#%& &'"'HA(A# ,'PAR('-'# &'"'HA(A# "!RA)A*A 2011 . 2012

Culture Media Preparation

-ost of t/e laboratory e0periments in t/is manual 1ill utili2e ba3teriologi3al media4 (/e 3ir3umstan3es may arise 1/en you 1ill need some spe3ial medium t/at is not already prepared5 and it 1ill be up to you to put it toget/er4 (/e first portion is dis3ussing about t/e different types of media and /o1 t/ey relate to t/e needs of mi3roorganisms4 (/e last part is dis3ussing about t/e a3tual me3/ani3s of ma6ing up a bat3/ of medium4 Media Consistency A mi3robiologi3al medium also known as media5 in plural5 is t/e food for 3ulturing ba3teria5 molds5 and ot/er mi3roorganisms4 (/ere are t/ree 3onsisten3ies : Liquid media, are used for t/e propagation of large numbers of organisms5 fermentation studies5 and 7arious ot/er tests4 #utrient brot/5 3itrate brot/5 glu3ose brot/5 litmus mil65 et3 are in3luded to t/e li8uid media4 Solid media, are made by adding a solidifying agent to a li8uid medium4 #utrient agar5 blood agar5 and "abouraud9s agar are e0ample of solid media t/at are used for de7eloping surfa3e 3olony gro1t/ of ba3teria and molds4 Semisolid media, are parti3ularly useful in determining 1/eter 3ertain ba3teria are motile4 (/ese media fall in bet1een li8uid and solid media4 Nutritional Needs of Bacteria -edium t/at is to be suitable for a spesifi3 group of organisms must ta6e into a33ount t/e follo1ing se7en fa3tors4 (/at fa3tors are : Water (/e 8ualify of 1ater used in preparing media is important4 Hard tap 1ater5 /ig/ in 3al3ium and magnesium ions5 s/ould not be used4 (/e best poli3y is to always use distilled water. Carbon 1it/ respe3t to t/eir sour3es of 3arbon5 organisms are di7ided into t1o groups4 Autotrophs and Heterotrhops. Autotrophs 3an utili2e t/e 3arbon in t/e 3arbon dio0ie for synt/esis of all 3ell materials4 Heterotrophs must /a7e one or more organi3 3ompounds for t/eir 3arbon sour3e4 Energy +rganisms t/at /a7e pigments t/at enable t/em to utili2e solar energy are 3alled photoautotrophs4 Autotrop/s t/at 3annot utili2e solar enegry but are able to o0idi2e simple inorgani3 substan3es for anergy are 3alled chemoautotrophs. Category t/at re8uire an organi3 sour3e of energy5 su3/ as glu3ose or amino a3ids are 3alled chemoheterotrophs. +rganisms t/at /a7e p/otosynt/eti3 pigments are enable t/em to utili2e sunlig/t for energy are 3alled photoheterotrophs5 t/eir 3arbon sour3e must be an organi3 3ompound su3/ as al3o/ol4 Nitrogen autotrop/i3s organisms 3an utili2e inorgani3 sor3es of nitrogen5 t/e /eterotrop/s get t/eir nitrogen from amino a3ids and intermediate protein 3ompounds su3/ as peptides5 proteoses5 and peptones4 Mineral all organisms re8uire se7eral metalli3 elements su3/ as sodium5 potassium5 3al3ium5 magnesium5 manganese5 iron5 2in35 3opper5 p/osp/orus5 and 3obalt4 Growth Factors 3ontain of any essential 3omponent of 3ell material4 Hydrogen on Concentration (/e en2ymes of mi3roorganisms are greatly affe3ted by t/is fa3tor4 Exact Composition Media -edia 3an be prepared to e0a3t spe3ifi3ations so t/at t/e e0a3t 3omposition is 6no1n4 "u3/ media are readily reprodu3ible 6no1n as synthetic media! -edia t/at 3ontain ingredients of impre3ise 3omposition are 3alled nonsynthetic media! )ot/ t/e beef e0tra3t and peptone in nutrient brot/ are ine0tra3t in 3omposition4

Special Media Contain of sele3ti7e and differential media4 Selecti"e Media are media t/at allo1 only 3ertain types of organisms to gro1 in or on t/em be3ause 1: t/e absen3e of 3ertain 3riti3al nutrients5 or 2: t/e presen3e of in/ibitory substan3es4 #i$$erential Media are media t/at 3ontain substan3es t/at 3ause some ba3teria to ta6e on a different appearan3e from ot/er spe3ies4 Dehydrated Media ,e/ydrated media /a7e re7olutioni2ed media preparation te3/ni8ues in mu3/ t/e same 1ay ta/t 3ommer3ial 3a6e mi0es /a7e ta6en o7er in t/e 6it3/en4 Media Preparation Assignment (est tubes t/at t/e most generally used are eit/er 1;mm or 20mm diameter and 1<3m long4 Large tubes =20mm dia: : for all pours: i4e45 nutrient agar5 "abouraud9s agar5 '-) agar5 et34 Small tubes =1;mm dia: : for all broth, deeps, and slants4 %f t/e tubes need 3leaning5 s3rub out t/e insides 1it/ 1arm 1ater and detergent5 using a test>tubes brus/4 Rinse t1i3e5 first 1it/ tap 1ater5 and finally 1it/ distilled 1ater to rid t/em of all tra3es of detergent4 Pla3e t/em in a 1ire bas6et or ra365 in7erted5 so t/at t/ey 3an drain4 ,o not dry 1it/ a to1el4 Meausurement and mi%ing (/e amount of media s/ould be determined to a7oid s/ort>age or e03ess4 Materials graduate5 bea6er5 glass stirring rod bottles of de/ydrated media5 bunsen burner and tripod5 or /ot plate 14 -easure t/e 3orre3t amount of 1ater needed4 24 Consult t/e label on t/e bottle to determine /o1 mu3/ po1der is needed4 34 $ the medium contains agar, /eat t/e mi0ture o7er a )unsen burner or on an ele3tri3 /ot plate until it 3omes to a boil4 Caution & 1e must be sure to 6eep stirring from t/e bottom 1it/ a glass stirring rod5 so t/e medium does not 3/ar on t/e bottom of t/e bea6er 44 C/e36 t/e le7el of t/e medium 1it/ t/e mar6 on t/e bea6er4 'd(usting the )H -a6e pH ad?usment as follo1s : Materials bea6er of medium5 a3id and base 6its5 glass stirring rod5 pH papers5 pH meter 14 ,ip a pie3e of pH test paper into t/e medium4 24 $ the )H is too high, add a drop of 1# HCl4 !se a glass stirring rod to mi0 t/e solution4 34 $ the )H is too low, add #a+H4 !se a glass stirring rod to mi0 t/e solution4 Filling The Test Tu es !se t/e follo1ing pro3edure 1/en filling tubes 1it/ a funnel assembly4 Materials ring stand assembly5 funnel assembly5 graduate in small si2e 14 @ill one test tube 1it/ measured amount of medium4 24 @ill t/e funnel and pro3eed to fill t/e test tube to t/e proper le7el4 34 &eep t/e bea6er of medium o7er /eat if 3ontains agar4 44 %f fermentation tubes are to be used5 add one to ea3/ tube at t/is time4 Ca))ing the *ubes

(/e last step before sterili2ation is to pro7ide a 3losure for ea3/ tube4 Plasti3 =polypropylene: 3aps are suitable in most 3ases4 "terili2ation "terili2ation must be done in an auto3la7e4 (/e follo1ing 3onsiderations are important in using an auto3la7e : Complete sterili2ation o33urs at 2<0A @ =1214;A C:4 Dont overload the chamber. Pro7ide ample sap3e bet1een bas6ets of media to allo1 for 3ir3ulation steam4 Adjust the time of sterilization to the size of load. An auto3la7e full of media may re8uire 30 minutes for 3omplete sterili2ation4 After "terili2ation Slants it is ne3essary to lay t/e tubes do1n in a near>/ori2ontal manner as soon as t/ey are remo7ed from t/e auto3la7e +ther Media s/ould be allo1ed to 3ool to roon temperature after remo7al from t/e auto3la7e4 After t/ey 3olled do1n5 pla3e t/em in a refrigerator or 3old>storage room4 Storage 1/en stored for long periods of time at room temperature media tend to lose moisture4 At refrigerated temperatures media 1ill 6eep for mont/s4 ,e$lection % t/in6 in t/is e0er3ise about 3ulture preparation media5 t/ere are some 1ords t/at easy to understand and also t/ere are some 1ords t/at diffi3ult to understand4 Benerally5 some 1ords t/at diffi3ult to understand are t/e scientific 1ord4 )ut all of t/is e0er3ise are useful for me4 )e3ause % 3an 6no1 e7ery steps t/at used in preparing media4