Culture Media Preparation

1. A microbiological medium is the food that we use for culturing bacteria, molds, adan other microorganism. It can exist in three consistencies : liquid, solid, semisolid. Liquid media include nutrient broth, citrate broth, glucose broth, litmus milk, etc. These media are used for the propagation of large number of organisms, fermentation studies, and arious other tests. !olid media are made b" adding solidif"ing agent, such as agar, gelatin or silica gel, to a liquid medium. A good solidif"ing agent is one that is not utili#ed b" microorganisms, does not inhibit bacterisl growth, and does not liquef" at room temperature. $utrient agar, blood agar, and !abouraud%s agar are examples of solid media that used for de eloping surface colon" growth of bacteria and molds !emisolid media fall in between liquid and solid media. Although the" are similar to solid media in that the" contain solidif"ing agent such as agar and gelatin, the" are more &ell"like due to lower percentages of these solidifiers. These media are particularl" useful in determining whether certain bacteria are motile.

'. An" medium that is to be suitable for a specific group of organisms must take into account the following se en factors : water, carbon, energ", nitrogen, minerals, growth factor, and p(. The role of each one of these factors follows. )ater *rotoplasm consists of +,- to ./- water. The water in a single celles organism is continuous with the water of its en ironment. The qualit" of water used in preparing media is important. The best polic" is to alwa"s use distilled water. 0arbon 1rganisms are di ided into two groups with respect to their sources of carbon. Those that can utili#ed the carbon in carbon dioxide for s"nthesis of all cell materials are called autotrophs. If the" must ha e one or more organic compounds for their carbon source, the" are called heterotrophs. In addition to organic sources of carbon, the heterotrophs are also dependent on carbon dioxide. If this gas is completel" excluded from their en irontment, their growth is greatl" retarded, particularl" in the earl" stages of starting a culture. 2nerg" 1rganisms that ha e pigments that enable them to utili#e solar energ" are called photoautotrophs. Autotrophs that cannot utili#e soolar energ" but are able to oxidi#e simple inorganic substances for energ" are called chemoautotrophs . The essential energ"3"ielding sustance for these organisms ma" be elemental as nitrite, nitrate, or sulfide. A small number of bacteria are classified as photoautotrophs. These organisms ha e photos"nthetic pigments that enable them to utili#e sunlight for energ". $itrogen Although autrotophs organisms can utili#e inorganic sources of nitrogen, the heterotrophs get their nitrogen from amino acid and intermediate protein compounds such as peptides, proteoses, and peptones. 4inerals all organic require se eral metallic element such as sodium, potassium, calcium, magnesium, manganese,iron, #inc, copper, phosphorus, and cobalt for normal growth.

5rowth factors an" essential component of cell material that in organisms is unable to s"nthesi#e from its basic carbon and nitrogen sources is classifiedas being a growth factros, example amino acids or itamins. (idrogen Ion 0oncentration The en#"mes of microorganisms are greatl" affectedb" this factor. !ince most bacteria grow best around p( +or slightl" lower.

6. These media are generall" made from chemical compounds that are highl" purified and precisel" defined. !uch media are readil" reproducible, the" are known as s"nthetic media. 4edia such as nutrient broth that contain ingredients of imprecise composition are called nons"nthetic media. 7. Two kinds of special media that will be widel" used in this manual are selecti e and differential media. !electi e media are media that allow onl" certain t"pes of organisms to grow in. 8ifferential media are media that contain substances that cause some bacteria to take on different appearance from other species, allowing one of differentiate one species from another. /. 8eh"drated media ha e re olutioni#ed media preparation techniques in much the same wa" that commercial cake mixes ha e taken o er in the kitchen. 9or most routine bacteriological work , media preparation has been simplified to the extent that all that is necessar" is to dissol e a measured amount of deh"drated medium in water, ad&ust the p(, dispense into tubes, and sterili#e. :. Test tubes that the most generall" used are either 1:mm or ',mm diameter and 1/cm long. Large tubes ;',mm dia< : for all pours: i.e., nutrient agar, !abouraud%s agar, 24= agar, etc. Small tubes ;1:mm dia< : for all broth, deeps, and slants. If the tubes need cleaning, scrub out the insides with warm water and detergent, using a test3tubes brush. >inse twice, first with tap water, and finall" with distilled water to rid them of all traces of detergent. *lace them in a wire basket or rack, in erted, so that the" can drain. 8o not dr" with a towel. Meausurement and mixing The amount of media should be determined to a oid short3age or excess. Materials graduate, beaker, glass stirring rod bottles of deh"drated media, bunsen burner and tripod, or hot plate 1. 4easure the correct amount of water needed. '. 0onsult the label on the bottle to determine how much powder is needed. 6. If the medium contains agar, heat the mixture o er a =unsen burner or on an electric hot plate until it comes to a boil. Caution : we must be sure to keep stirring from the bottom with a glass stirring rod, so the medium does not char on the bottom of the beaker 7. 0heck the le el of the medium with the mark on the beaker. Adjusting the pH

4ake p( ad&usment as follows : Materials beaker of medium, acid and base kits, glass stirring rod, p( papers, p( meter 1. 8ip a piece of p( test paper into the medium. '. If the pH is too high, add a drop of 1$ (0l. ?se a glass stirring rod to mix the solution. 6. If the pH is too low, add $a1(. ?se a glass stirring rod to mix the solution. illing !he !est !ubes ?se the following procedure when filling tubes with a funnel assembl". Materials ring stand assembl", funnel assembl", graduate in small si#e 1. 9ill one test tube with measured amount of medium. '. 9ill the funnel and proceed to fill the test tube to the proper le el. 6. @eep the beaker of medium o er heat if contains agar. 7. If fermentation tubes are to be used, add one to each tube at this time. Capping the !ubes The last step before sterili#ation is to pro ide a closure for each tube. *lastic ;pol"prop"lene< caps are suitable in most cases. !terili#ation !terili#ation must be done in an autocla e. The following considerations are important in using an autocla e : 0omplete sterili#ation occurs at '/,A 9 ;1'1.:A 0<. Dont overload the chamber. *ro ide ample sapce between baskets of media to allow for circulation steam. Adjust the time of sterilization to the size of load. An autocla e full of media ma" require 6, minutes for complete sterili#ation. After Sterili"ation Slants it is necessar" to la" the tubes down in a near3hori#ontal manner as soon as the" are remo ed from the autocla e #ther Media should be allowed to cool to roon temperature after remo al from the autocla e. After the" colled down, place them in a refrigerator or cold3storage room. Storage when stored for long periods of time at room temperature media tend to lose moisture. At refrigerated temperatures media will keep for months.

$eflection I think in this exercise about culture preparation media, there are some words that eas" to understand and also there are some words that difficult to understand. 5enerall", some words that difficult to understand are the scientific word. =ut all of this exercise are useful for me. =ecause I can know e er" steps that used in preparing media.