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Juvinch R.

Vicente Bio 120 Section 1 TF 7:00-10:00

Date Performed: June 17, 2011 Date Submitted: June 24, 2011

CULTURE MEDIA AND STERILIZATION AND DISINFECTION TECHNIQUES Experiment No. 4 and 5

I. INTRODUCTION In order to keep running, all living things need an unceasing supply of energy and materials. Microorganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have adapted to the habitats most suitable for their needs, in the laboratory, however, these requirements must be met by a culture medium. This is basically an aqueous solution to which all the necessary nutrients have been added. Culture media for microbiological studies have to basic categories, the chemically defined media, and the complex media. A chemically defined medium is one whose exact chemical composition is known. These types of culture media are usually reserved for laboratory experimental work or for the growth of autotrophic bacteria. On the other hand, complex media are usually used for experimental work involving heterotrophic microorganisms such as fungi and other sorts of bacteria. In these types of culture media the combination of extracts and sugar creates a medium which is rich in minerals and organic nutrients, have unknown composition making it complex. In this experiment, we will be preparing one of the most common complex culture medium used for microbiological studies which is the Nutrient Agar. It is a microbiological growth medium commonly used for the routine cultivation of non-fastidious bacteria. Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections. Furthermore, it is useful because it remains solid even at relatively high temperatures. Lastly, Bacteria grown in nutrient agar grows on the surface, and is clearly visible as small colonies making it easy to observe. Technically, Sterilization is the removal or destruction of all forms of microbial life including bacteria, bacterial spores, fungi, and viruses. On the other hand, Disinfection is the removal of vegetative pathogens, and could destroy many microorganisms (bacteria, viruses, fungi) but do not destroy bacterial spores. Both processes could be physically or chemically done.

Heat is the most common medium of sterilization. Heat appears to kill microorganisms by incorporating oxidative effects, coagulation, and denaturation to their proteins. The heat used in killing microorganisms is categorized into two general types; it could be dry heat or moist heat. Dry heat includes several specific techniques such as incineration, flaming, using hot air oven, or by using red flame. On the other hand, moist heat includes pasteurization, boiling, water baths, and by autoclaving. Basically, moist heat varies with dry heat because it uses water (as liquid or steam) during the heating process. Normally, moist heat is much more effective than dry heat. In this experiment, we will be using a specific type of moist heat technique which is by the use of autoclave in sterilizing our glasswares for the next experiment. Most disinfection techniques are done chemically such as the use of alcohols, aldehydes, phenols, halogen, and other use heavy metals.

II. OBJECTIVES At the end of this experiment we are expected to familiarize ourselves in culture media preparation. Also, this experiment is expected to teach us the principles behind Sterilization and Disinfection techniques.

III. MATERIALS AND METHODS y y y y y y Agar Peptone Sodium Chloride Beef extracts E. coli culture Cotton y y y y y y Tissue Petri Dishes Erlenmeyer flask Microwave oven Spatula Autoclave

Culture Media 1. Three sets of culture media were prepared for the whole class, two 200 mL, and one 70 mL culture media. 2. Using a top-loading balance, Beef extract, Agar, Sodium chloride, and peptone were accordingly weighed such that there will be 10g Beef extract, 5g NaCl, 15g Agar, and 10g peptone in every liter of culture media. 3. The weighed Beef extract, NaCl, Agar, and peptone were then mixed inside the prepared Erlenmeyer flasks. 4. After mixing, the mixture was then diluted with sterile distilled water into each appropriate volume. 5. The mixtures were then heated to facilitate the dissolution of the solid particles. The media were heated at a rate of 1min/100mL using Microwave oven. This was done repeatedly after each boiling until all solid particles dissolve. 6. After complete dissolution, the Erlenmeyer flasks containing 200 mL culture media were then covered with the prepared cotton plugs and kept for sterilization. 7. The 70 mL culture media was distributed evenly to the prepared test tubes and then plugged with cotton.

Sterilization and disinfection: 1. All necessary glass wares were prepared for the sterilization process. 2. The Petri dishes were wrapped with scratch paper, while the Erlenmeyer flasks and test tubes than contain the culture media were covered with aluminium foil to avoid introduction of water inside. 3. The glass wares were then placed properly inside the Autoclave. 4. The Autoclave was then covered correctly and heated. 5. After reaching the desired temperature (which is around 120C), the heating process was then held for 15 minutes.

IV. RESULTS AND DISCUSSIONS In the complex media we have prepared, the energy, carbon, nitrogen, and sulphur requirements of the growing microorganism will be provided primarily by protein. Protein is a large, relatively insoluble molecule that a minority of microorganisms can utilize it directly. However, a partial digestion by acids or enzymes reduces protein to shorter chains of amino acids called peptones. These small, soluble fragments can be digested by most bacteria. The beef extract added in the medium provides the vitamins and other organic growth factors. The soluble vitamins and minerals from the beef extract are dissolved in the extracting water. The mixture was then held at high temperature to evaporate water so that the vitamins and minerals will be further concentrated. Another thing, these extracts also supplement the organic nitrogen and carbon compounds requirements of the growing microorganism. Some mineral requirements of the microorganisms to be grown will also be aided by the salt added on the media. The mixture of necessary nutrients can be used as a liquid medium usually in aqueous mixture. If water is needed, it is very much necessary to use sterile water to make sure that the culture will not be contaminated by any microorganisms that is present in water. When it is desirable to grow bacteria on a solid medium, a solidifying agent must be added to the mixture. In the case of our experiment, we have used the most common solidifying agent used for culture media which is Agar. It is a complex polysaccharide derived from marine algae and has long been used as a thickener in foods. Agar has some very important properties that make it valuable to microbiology, and no satisfactory substitute has ever been found. First, only few microbes can degrade agar, so it remains solid. Also, agar liquefies at about 100C, the boiling point of water, and at sea level remains liquid until the temperature drops to about 40C. These properties particularly make Agar very useful in culture media preparation. Autoclave is the technical term for pressure-cooker. From the name itself, this technique utilizes the build up of pressure inside a tightly closed container. While keeping the volume of the boiling water (generating steam) constant, the increase in the pressure inside the container will consequently result to the increase in the systems temperature. This phenomenon follows Gay Lussacs gas law which states that at constant volume, the absolute temperature of a gas is directly proportional to the pressure it exerts to its container. At standard pressure (1 atm) water normally boils at 100C and starts to generate steam with the same temperature. However, at elevated pressure, water boils at higher temperature. In the case of this experiment, the pressure was allowed to build up to around 15 lbs/sq.inch, which consequently resulted to the increase in the boiling point of water at 120C.

Sterilization can be more effectively achieved at this temperature. Also, steam has more penetrating power than dry air (which is used in hot air oven). It moistens the spores of the bacteria which is essential for the coagulation of the proteins. Also, condensation of steam on cooler surfaces releases latent heat, condensation of steam draws in fresh steams which further optimizes its killing power. Though moist heat is more advantageous than dry heat, several drawbacks are still associated with this technique. First, the use of moist heat often causes drenching and wetting of articles being sterilized. Also, trapped air may reduce the efficacy of the process which could result to longer time of sterilization. Lastly, the use of autoclave (especially those old models) is dangerous to the proponent of the sterilization. V. CONCLUSIONS We were able to successfully prepare a culture media for microbial growth. This culture media can support microbial life artificially by providing nutrient requirements of the microorganisms through the different components composing the media. Also, agar was able to act as solidifying agent for the nutrient broth we have made. Sterilization by autoclaving is an efficient way of killing all microorganisms on the articles we have sterilized by exploiting several factors. First, it is a pressurized system making it able to generate steam at temperature higher than the boiling point of water. Also, water is a better conductor of heat than air making moist heat much better than dry heat. Moreover, the use of water as medium for heating efficiently facilitates coagulation of microbial proteins which in turn kills them. Culture media and Sterilization and Disinfection techniques are indeed vital in microbiological studies. While culture media keep microbial life running, sterilization and disinfection techniques provides the protection and isolation of the microorganism being studied by killing possible contaminants. Without these, microbiological studies would not lead to any success. VI. REFERENCES y www.stason.org. June 2011 y www.biosynth.com. June 2011 y Todar K. Online textbook of Bacteriology. June 2011 y Tortora G. Funke B. Case C.: 2008. Microbiology: An introduction. 8thEdition. Published by Pearson Education, Inc.