FDE 206 FOOD MICROBIOLOGY

PREPARATION OF AGAR MEDIA AND CULTIVATION OF BACTERIA INSTRUCTOR NAME : F.Yeim EKNC

ZUHAL AKGN 280709014

PARTNERS NAME Gnay Yurdakul Melis Yourtu Seda Gamsz Simge Say

SUBMITTED TO Eray Esendir Ezgi zcan Melis Baer Raziye Piranl

Experiment Date:29 February 2012 Submission Date:7 March 2012 Yeditepe University stanbul
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TABLE OF CONTENTS 1.Purpose 2.Theoretical Background 2.1 Nutrient Agar and Medium 2.2 Sterilization and Autoclave 2.3 Growth of Microorganism 2.3.1 Effect of Oxgen 2.3.2 Effect of Temperature 3.Materials 4.Method 5.Result 6.Discussion 7.References 3 3 3 4 6 6 6 7 7 7 8 9

1.PURPOSE The purpose of this experiment is try to prepare the agar media and cultivate bacterias in the same culture media but different incubators. 2.THEORETICAL BACKGROUND Bacteria and fungi are grown on or in microbiological media of various types. The medium that is used to culture the microorganism depends on the microorganism that one is trying tob isolate or identify. Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH indicators are often added for differentiation of microbes based on their biochemical reactions: the indicators may turn one color when slightly acidic,another color when slightly basic. Other added ingredients may be growth factors, NaCl, and pH buffers which keep the medium from straying too far from neutral as the microbes metabolize. 2.1 NUTRIENT AGAR AND MEDIUMS Agar is used to gel bacteriological broths (liquid media) to form slants, and to avoid sloushing so oxygen can't easily get to the bottom of the medim. Agar is obtained from seaweeds called kelps. There are many different grades of agar and most were developed for use in human food. There are several kinds of agar which are long chain polymers made by certain marine alga. Commercial agar is often a mixture of molecules having some what differing chemical structures. Agar from different algae have different structures, and, therefore, have differing properties. While some marine bacteria can use agar as food and form pits when spread on a plate of agar, most bacteria can't damage agar. General Purpose Media are designed to grow most organisms and do not contain growth inhibitors. Standard Methods Agar and Blood Agar Bases are examples of general purpose media. Differential Media contain a component that allows an observable change when a specific chemical reaction takes place. Simmons Citrate Agar is an example of a differential medium. In Simmons Citrate Agar there is a pH indicator that turns from green to blue when citrate is utilized as the sole carbon source.Selective media encourage the growth of some organisms and suppress the growth of others. Dyes, antimicrobials, and salts are all examples of selective agents used for this purpose. Bile Salts are used to inhibit the growth of Gram-positive organisms on MacConkey Agar. Selective / Differential Media can be both selective and differential depending on the formula. Hektoen Enteric Agar is an example of a selective and differential medium. Hektoen Enteric Agar contains Bile Salts, the selective agent used to suppress Gram-positive organisms, and pH indicators, Acid Fuchsin, and Bromthymol Blue Enrichment Media contain nutrients that encourage the growth of organisms. Media containing enrichmentscan be selective or general purpose. Universal Pre-enrichment Broth is a general purpose enrichment broth for Salmonella and Listeria, and is particularly successful at resuscitating low numbers of injured cells. Nutrient media are basic culture media used for maintaining microorganisms, cultivating fastidious organisms by enriching with serum or blood and are also used for purity checking prior to biochemical or serological testing. Nutrient Agar is ideal for demonstration and
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teaching purposes where a more prolonged survival of cultures at ambient temperature is often required without risk of overgrowth that can occur with more nutritious substrate. This relatively simple formula has been retained and is still widely used in the microbiological examination of variety of materials and is also recommended by standard methods. It is one of the several non-selective media useful in routine cultivation of microorganisms.It can be used for the cultivation and enumeration of bacteria which are notm particularly fastidious. Addition of different biological fluids such as horse or sheep blood, serum, egg yolk etc. makes it suitable for the cultivation of related fastidious organisms. Peptic digest of animal tissue, beef extract and yeast extract provide the necessary nitrogen compounds, carbon, vitamins and also some trace ingredients necessary for the growth of bacteria. Sodium chloride maintains the osmotic equilibrium of the medium.
Table 1 : Composition of Nutrient Agar

2.2 STERILIZATION AND THE AUTOCLAVE When microbiological media has been made, it still has to be sterilized because of microbial contamination from air, glassware, hands, etc. Within a few hours there will be thousands of bacteria reproducing in the media so it has to be sterilized quickly before the microbes start using the nutrients up. The sterilization process is a 100% kill, and guarantees that the medium will stay sterile unless exposed to contaminants by less than adequate aseptic technique to exposure to air. Media sterilization is carried out with the autoclave, basically a huge steam cooker. Steam enters into a jacket surrounding the chamber. When the pressure from the steam is at a certain point in the jacket, a valve allows the steam to enter the chamber. The pressure will go up over 15 pounds per square inch (psi): at this point the timer begins to count down--- usually for 15 minutes, depending on the type of media. The high pressure in a closed container allows the temperature to go above the highest temperature one could get by just boiling, around 121 degrees C. Therefore, the parameters for sterilization with an autoclave are 121 C at >15 psi for 15 minutes. Fifteen minutes is the thermal death time for most organisms. The prepared media is distributed in different ways, depending on the form one is making.Broths and agar deeps are dispensed into tubes and then sterilized. Agar slant tubes are sterilized and then the rack is tilted to allow the agar to solidify in a slanted fashion. Agar medium which will be poured into plates is sterilized in a flask, and then poured afterward. Not all media or solutions can be sterilized via an autoclave. Certain high-protein solutions such as urea, vaccines, and serum will denature in the extreme heat, and so they may have to be filter-sterilized without heat.

Figure 1: The working principle of autoclave

Figure 2 : 4 phases of autoclave machine

2.3 GROWTH OF MICROORGANISMS Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. The chemicals and elements of this environment that are utilized for bacterial growth are referred to asnutrients or nutritional requirements. In the laboratory, bacteria are grown in culture media which are designed to provide all the essential nutrients in solution for bacterial growth. The procaryotes exist in nature under an enormous range of physical conditions such as O2 concentration, Hydrogen ion concentration (pH) and temperature. The exclusion limits of life on the planet, with regard to environmental parameters, are always set by some microorganism, most often a procaryote, and frequently an Archaeon. Applied to all microorganisms is a vocabulary of terms used to describe their growth (ability to grow) within a range of physical conditions. A thermophile grows at high temperatures, an acidophile grows at low pH, an osmophile grows at high solute concentration, and so on. This nomenclature will be employed in this section to describe the response of the procaryotes to a variety of physical conditions. 2.3.1 The effect of Oxygen:Oxygen is a universal component of cells and is always provided in large amounts by H2O. However, procaryotes display a wide range of responses to molecular oxygen O2.Obligate aerobes require O2 for growth; they use O2 as a final electron acceptor in aerobic respiration.Obligate anaerobes do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which either kills or inhibits their growth. Facultative anaerobes are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions they grow by fermentation or anaerobic respiration, but in the presence of O2 they switch to aerobic respiration.Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O2. 2.3.2 The effect of Temperature:Temperature is probably the most important environmental factor affecting growth. If temperature is too hot or too cold microorganisms will not grow.The minimum and maximum temperatures for microbial growth vary widely among microorganisms and is usually a reflection of the temperature range and average temperature of their habitat.

Figure 3 : Temperature and Bacterias

3.MATERIALS Powder Nutrient Agar Weighing Dish Top Balance Magnetic Stirerr Bottle Spoon Petri Dishes Distilled Water Graduated Cylinder Gloves

4.METHOD For the culture media preparation we started with the powder nutrient agar. The amount of powder that must be used written in the label. According to the label, for preparing 1L of nutrient agar media 23 gr of powder must be weighed. For our experiment, 200ml nutrient agar media murt be prepared. According to this information, 4,6 grams of powder weighed in the top balance with the weighing dish.Then, 200 ml distilled water put in the graduated cylinder. After this measurements ,the mixture of powder and distilled water gently shaked.Usingg the magnetic stirrer, the mixture will be mix until the color of the suspension take a clear colour. After all these preparation steps, the bottle will autoclaved at 121C 15 minutes. The aim is to autoclave the equipments, to sterilize them. The autoclave machine has 4 steps : Stand by-Heating-Exhausting- Cooling.When the machine shows 70C, the equipments must be canceled from the machine and put on the water bath which in 50C.Then, the mixture pour down the petri dishes. The poured mixture freezed in the petri dishes. The last step is labelling which is very important. The second part of the experiment is to observe the microbial growth in different incubators. The nutrient agar again use for this aim. The samples put by the students.( Salivia,Finger Touching, Hair etc..). After 1 day, the growth is observed in different incubators. 5.RESULTS The results taken 1 day After from the experiment. The Table below shows the microbial growth in the petri dishes in different incubators.
Table 2 : Microbial Growth in different incubators

1 2 3 4 5

Group Member Gnay Melis Seda Simge Zuhal

Sample touch of finger touch of finger touch of finger touch of finger touch of finger with gloves

Incubator 37C ,5 % CO2 Room temperature 25C Anaerobic jar 37C 37C

Microbial Growth growth observed. growth did not observed growth did not observed growth did not observed growth observed. 7

6.DISCUSSION In this experiment we try to observe the growth of microorganisms in different incubators. There are growth in the 37C 5% carbondioxide concentration and 37C incubators. There wasnt observe growth in room temperature,anaerobic jar and 25C. The important thing is the group member who touch to the nutrient agar with the glove in her hand. That means the media that we are doing the experiment is not sterile enough. The bacterias may be classified as a mesophilic bacteria which is defined as a microorganism with a growth optimum around 20 to 45oC a minimum of 15 to 20oC and a maximum of about 45oC or lower. The Examples include Listeria monocytogenes, Psudomonas maltophilia, Thiobacillus novellus, Staphylococcus aureus, Streptococcus pyrogenes, Streptococcus pneumoniae, Escherichia coli, and Clostridium kluyveri.These are the bacterias can be growth in the incubator which is 37C .The other organisms that can be growth in the 37C 5% carbondioxide concentration defined as aerotolerant anaerobes. Like Obligate anaerobes, cannot use oxygen to transform the energy but can grow in presence. They obtain energy only by fermentation. The examples can be genus of gram negative bacillus bacteria or clostridium species or peptostreptococcus.. The growth observe in this incubator. The glove is play an important role in this growth because it shows that the safety is important in the laboratory. The benches that is not sterilized well can be contamination source. Gloves prevent the contamination of our body or experiment materials. And this can be gives error for our experiment. The safety conditions must be carefully applied by the lab students. This helps to prevent the contamination.

7.REFERENCES
http://textbookofbacteriology.net/themicrobialworld/nutgro.html http://archive.food.gov.uk/hea/teachers/plainenglish/part2.html http://secure.sciencecompany.com/How-To-Grow-Bacteria-in-Agar-Petri-Dishes-W54.aspx http://bioweb.wku.edu/courses/Biol208/Lab_Manual/208-4%20week%205.pdf http://www.fsis.usda.gov/ophs/Microlab/Mlgchp12.pdf

Lengeler, J.W., Drews, G, and Schlegel. H.C., Biology of the Prokaryotes, Blackwell Science, New York, 1999, pp. 59.110,163. Gottschalk, 0, Bacterial Metabolism, 2nd ed., Academic Press, New York, 1986, pp. 13, 96, 141, 210. Rose, A.H., Chemical Microbiology, Butterworths, London, 1965, pp. 79, 85, 94. Jr. and Nottingham, P.M., Temperature. in Microbial Ecology of Foods, Vol. 1, Silliker, J.H., Bd., Academic Press, New York, 1980, p. 1. Tortora, 0.1., Funke, B.R., and Case, CL.Microbiology: All Introduction, 4th ed., Benjamin Cummings, Park, CA, 155, 1992.