TASK 1 MICROBIOLOGY PHARMACY

Growth Media
WRITTEN BY : IKA YULIA RIZKI 1111012053

Lecturer : Prof. DR. Marlina, M.Farm, Apt

PHARMACY FACULTY ANDALAS UNIVERSITY PADANG 2012

MEDIA CLASSIFICATION AND BACTERIAL NUTRITIONAL REQUIREMENTS

Defenition
Media is a material consisting of a mixture of nutrient (nutrients) that are useful for culturing microbes. By using a variety of media to do the isolation, propagation, testing and calculation of a number of physiological properties of microbes. So that microbes can grow well in a medium, the medium must meet the requirements, among others: should contain all the nutrients are easily used by microbes, must have an osmotic pressure, surface tension and pH in accordance with the needs of the microbes that will grow, not contain substances that can inhibit microbial growth, must be in sterile condition before being used, so that the microbe can be grown successfully grown
media serves to cultivate microbes, isolation, increase the number, examine the properties and the calculation of the amount of microbial physiology, which should be sterilized in the manufacturing process and apply the aseptic methods to avoid contamination of the media. Nutrient medium that is common to test the water and dairy products. NA is also used for the majority of the growth of microorganisms that are not selective, in terms of heterotrophic microorganisms. Media is a simple media made from beef extract, peptone, and agar. NA is one of the media commonly used in bacteriological procedures such as regular testing of water, sewage, food products, to bring the stock culture, to the growth of bacteria on the test samples, and to isolate the organism in pure culture with caradisterilisasi by autoclaving at 121 C for 15 minutes (Fathir, 2009).

Using Selective, Differential and Selective-Differential Media in the isolation and identification of individual bacteria
Bacteria must be isolated from their natural environments before they can be characterized. As bacteria exist in mixed populations in the soil, water, food and within or on the human body, specialized media are often used to isolate individual bacteria from these populations and to characterize the isolated bacteria. Selective media are used to select for the growth of some bacteria while inhibiting the growth of others. Such media take advantage of the differences in the nutritional needs of individual bacteria or exploit the ability of some organisms to grow in the presence of a noxious compound. For example, Pseudomonas aeruginosa is able to grow in the presence of a variety of antibiotics (compounds that inhibit bacterial growth or kill bacteria) while organisms like Staphylococcus aureus or Escherichia coli are sensitive to these antibiotics. 1

Differential media allow a variety of organism to grow but contain substances that allow the student to distinguish between the different types of bacteria growing on the media. Starch Agar: This is a differential medium used to determine whether a given bacterium is able to use starch as a carbon source and an energy source. Starch is a polymer (polysaccharide) of repeating glucose monomers and is too large to be transported into the cell. Organisms that use starch produce an exoenzyme called alpha-amylase that breaks the bonds between the glucose monomers such that the glucose monomers can be taken up into the cell and catabolized. Alpha-amylase is secreted outside of the cell but is still active at this location. To determine if the organism produces this exoenzyme, one adds Grams Iodine to the plate that turns the intact starch in the medium purple. If the organism utilizes starch, the area around the colony will be clear because, the starch has been broken down into glucose monomers (starch positive). If the organism does not utilize starch then the media surrounding the organism will be dark purple (starch negative.

MacConkey Lactose Agar: This is a selective-differential media that selects for the growth of enteric bacteria. Enteric bacteria are gram-negative rods that are facultatively anaerobic. Enterics are most commonly found in the gastro-intestinal tract of humans and include E. coli, Serratia spp. and Salmonella spp. Enterics grow on MacConkey media because the bile salts that are present in the media inhibit the growth of non-enteric organisms while the crystal violet inhibits the growth of Gram positive organisms that might otherwise grow on this media. MacConkey media also differentiates between the noncoliforms and the coliforms that also grow on this medium. Enteric bacteria can be coliforms or noncoliforms. Enteric bacteria that ferment lactose to form acid and gas are coliforms, enteric bacteria that do not ferment lactose are noncoliforms. Coliforms will produce bright pink colonies on MacConkey lactose agar because a pH indicator (methyl red) is present in the media. The non-coliforms will grow on the media but the colonies will appear either light yellow or colorless.

Hektoen Enteric Agar: This is also a selective-differential media that contains bile salts to select for enteric organisms but distinguishes between these enterics by virtue of their abilities to ferment lactose, salicin or sucrose and to reduce sulfur to hydrogen sulfide gas. In addition to these fermentable sugars, H-E Agar contains ferric ammonium citrate that reacts with the hydrogen sulfide to form a black precipitate and the dyes acid fuchsin and bromothymol blue that are color indicators. This media is often used to isolate Salmonella and Shigella spp from other enterics. The enterics that ferment the sugars produce acids and form yellow to pink colonies on this medium. Shigella and Salmonella spp.do not ferment these sugars and produce blue colonies. Salmonella spp. (but not Shigella spp.) reduce sulfur to hydrogen sulfide forming colonies containing a black precipitate.

Selective-Differential Media have characteristics of both selective media and differential media. These media only allow a subset of bacteria to grow and allow the student to distinguish between the different types of bacteria that are able to grow on these media. For example, one can distinguish between bacteria that ferment lactose and those that do not in MacConkey Agar by adding a carbon 2

source (lactose) and a pH indicator (methyl red)bacteria that ferment lactose produce acids that imparts a color change to the media surrounding the individual bacterial colony.

Type of Media
Media is divided into: 1. Media General, the media can be used for the growth and proliferation of one or more groups of microbes in general. 2. Media add-on, which is used to mean "an opportunity" to a species or group of microbes to grow rapidly. 3. Selective media, ie media that can only be covered by one or more types of specific microbes but will inhibit or shut off for other types. 4. Differentiation media, the media used for testing a compound or a particular object with the help of microbes. 5. Media testers, the media used for testing a compound or a particular object with the help of microbes. 6. Enumeration media, ie media that is used to calculate the number of microbes in a material. (Suriawiria, 2005). Based on the consistency or density, the medium is divided into three types, namely: a. Liquid medium / broth / liquid medium Example: water peptone, nutrient broth, lactosa b. Semi-solid medium (semi-solid medium) Example: sim agar, and brain in order to cary c. Solid medium (solid medium) Example: endo agar, PDA, Nutrient order

The outline of the manufacture of medium that is composed of several ingredients are as follows: a. Mixing materials - materials, dissolved in distilled water and heated in a water heater so that a homogeneous solution. b. Filter with filter paper, cloth, or cotton. To order or gelatin media, filtering should be done in hot conditions. c. Determine and adjust the pH. d. Insert media into a certain place. Before sterilized, media inserted into the erlenmeyer or other clean container, then close the cover of cotton or paper (parchment paper) that is not wet when sterilized. e. Sterilization is generally performed with hot air in the autoclave at a temperature of 1210 C for 15-30 minutes (Sutedjo, 1991). PH is a factor that greatly affects the success in the manufacture of medium so that the pH is too alkaline or too acidic medium is not suitable to serve as microbes because microbes can not live in these 3

conditions. Medium silenced or kept for 2 x 24 hours to assure that the medium is sterile, because in addition to pH as a determinant of the growth of microbes, tools and a sterile medium also determines (Dwidjoseputro, 1994).

Preparation of Media
To investigate the bacteria in the laboratory, we must be able to grow them in pure culture. To do this, one must understand the types of physical environments that provide the optimum conditions for growth. In the laboratory, the physical environment in question is the culture media. Sterile culture media is the medium used to grow microorganisms. Culture media consisting of an organic salt, a source of energy (carbon), vitamins and growth regulators (PGR). Moreover, it can also add other components such as organic compounds and other complex compounds (Soeryowinoto, 1985). Wide diversity in terms of nutrients between bacteria type, offset by the availability of various media for many kinds kultuivasinya. Kinds of media can be divided based on the shape and composition. based on form, media divided over medical liquid, semi liquid and solid. Being according to its composition, the media can be divided into complex medium and synthetic media. As in this experiment, the type of media used is a type of media PDA (Potato dextrose order) that are classified as solid media and complex media. Culture media capable of supporting the optimization of growth milroorganisme should be able to meet the requirements of nutrients for microorganisms. element in the form of organic salts, energy sources (carbon), vitamins and growth regulators (PGR). Moreover, it can also add other components such as organic compounds and other complex compounds (Soeryowinoto, 1985). Types that include inorganic salts, especially potassium nitrate form of nitrogen (KNO3), sulfur (inorganic sulfur), phosphorus and inorganic metal elements such as sodium, potassium, kalsiuum, magnesium, manganese, iron, zinc, copper, and cobalt . The element carbon that is used by microorganisms can be either emitted or light dinamankan fototrof, and the other is kind of kemototrof using the oxidation of chemical compounds in the media to obtain energy (Hadioetomo, 1986). Function of vitamins in the culture media to form a substance that activates the enzyme. Microorganisms show different symptoms making patterns dala nutrients. Although all microorganisms require vitamins in metabolic processes, but some types of microorganisms Nampu synthesize their own vitamin requirements of other compounds in the medium. These vitamins are often added in the culture medium are: niacin, glycine, pyridoxine, hydrochloride, Thiamine, folic acid, etc. (Soeryowinoto, 1985) On plant growth regulators are organic compounds rather than nutrients, which in small amounts but can support, inhibit, and alter the plant physiology. Growth regulator is required as a component for the growth and differentiation medium, without the addition of growth regulators in the medium, the 4

growth of microorganisms is hampered even may not be able to grow at all (Gardener, 1993). PDA medium (Potato Dextrose order) are the sorts of substances used in organic nutisi culture medium. This material is commonly used in the composition of the culture lanortorium! 0:1:1 potato, dextrose and order in 1 liter of distilled water .PDA (Potato Dextrose order) is a common medium used in cultivation of bacteria. In addition to its manufacturing process is easy, also because it has a complex composition for the growth of bacteria, namely nerupa (Hendaryono, 1994). Culture media there is a solid, liquid and semi solid. Solid media is a media culture that solidified with agar, there is a reversible (reversible) such as nutrient agar, and there are ireversible (not reversible) such as blood serum terkoagulasi. In medicine, solid media are used most often irreversible. Nutrient being so widely used in other media. Other forms of media in the form of a liquid is a mixture of the components of certain chemicals with distilled water, the media is physically an intermediate between liquid and solid media, such as for software. Media is complex and there are synthetic. Synthetic media is media that all components are chemicals that all components are chemical-specific tangible and less obvious in terms of its content or can not be ascertained, often termed a complex medium. Complex nutrient medium such as that prepared by dissolving each ingredient is needed or even easier by adding water to a commercial product that has been shaped powder medium contains all the nutrients it needs. On practical, all nutrients are commercially in powder form and also in the form of ready-made in a Petri dish, cup, jar or bottle (Hasioetomo, 1986)

Experiment Methodology
Tools
The tools used in these experiments is Erlenmeyer, volume pipettes, autoclave, measuring cups, analytical balance, magnetic stirer, hot plate, and test tubes.

Materials
The materials used in these experiments is the media Nutrient Agar, Malt Extract Agar, Potato Destroxe order, and distilled water.

Work Procedures
a. Preparation of Media Nutrient Agar (NA) A. Created NA media (20 g / L) was mixed with 5 grams peptone, meat extract as much as 3 grams, and order as much as 12 grams into the erlenmeyer. 2. Added 50 mL of distilled water. produced by NA as much as 1 gram. 3. Heated on a hot plate to boil while stirring with a magnetic stirer until homogeneous, then left for some time. 4. Put into 3 test tubes of each of 4 mL 5. Capped reaction tubes using a cotton mouth 5

6. Included in the autoclave to be sterilized along with the tools to be used at temperatures of 121oC and a pressure of 15 psi. b. Making Potato Destroxe Agar (PDA) A. Created media PDA (39 g / L) was mixed with potato as much as 4 grams, as much as 20 grams of glucose, and order as much as 15 grams into the erlenmeyer. 2. Added 50 mL of distilled water. Produced up to as much as 1.95 grams of PDA. 3. Heated on a hot plate to boil while stirring with a magnetic stirer until homogeneous, then left for some time. 4. Put into 3 test tubes of each of 4 mL. 5. Capped reaction tubes using a cotton mouth 6. Included in the autoclave to be sterilized along with the tools to be used at temperatures of 121oC and a pressure of 15 psi. c. Media Making Malt Extract Agar (MEA) A. Created MEA medium (48 g / L) was mixed with as much as 3 grams of peptone, meat extract as much as 30 grams, and the order of 15 grams into the erlenmeyer. 2. Added 50 mL of distilled water. Until the MEA produced as much as 2.4 grams. 3. Heated on a hot plate to boil while stirring with a magnetic stirer until homogeneous, then left for some time. 4. Put into 3 test tubes of each of 4 mL 5. Capped reaction tubes using a cotton mouth 6. Included in the autoclave to be sterilized along with the tools to be used at temperatures of 121oC and a pressure of 15 psi.

Reference
Barker, Kathy. 1998. At The Bench, A Laboratory Navigator. Cold Spring Harbor Laboratory Press, New York. Barrow, G.I. and R.K.A. Feltham. 1993. Manual for the Identification of Medical Bacteria. Cambridge University Press, New York Beishir, Lois. 1991. Microbiology in Practice : A Self Instructional Laboratory Course. Harper Collins Publisher Inc., New York www.microbiology.mtsinai.on.ca