EXERCISE # 1 GROWING MICROBES: THE USE OF CULTURE MEDIA I.

OBJECTIVES Practice aseptic techniques in handling cultures and sterile materials. To perform a media preparation and moist heat sterilization e.g. autoclave. Learn transfer techniques from a different culture media with bacteria to the prepared culture media. Observe bacteria in colonies, their formation and color. II. MATERIALS & REAGENTS Reagents:

Apparatus/Materials:

0.72 grams beef extract 7.2 grams peptone 3.75 agar 250 mL and 90 mL distilled water Bacterium M. Luteus

Test tubes Test tube rack Inoculating loop/needle Alcohol lamp Beaker Erlenmeyer flask Wax paper

Top loading balance Matches Spoontula Graduated cylinder Pipette Cotton plugs Autoclave

III. METHODS/PROCEDURE The first part of the experiment we did in day 1 was media preparation. We first calculated the proportion of each component for a given volume of water for each of the mediums. Calculations will be presented in the data sheet attached. The third group prepared the liquid medium they only added 2.16 grams of broth into 270 mL hot water. After computing, the components were weighed in a wax paper and then placed in the beaker. Components were mixed until all granules were dissolved. Agar was cooked in the microwave. While waiting for it to be cooked then cooled we labelled all the tubes according to what medium will be placed in it, date, our section and group number. When agar was cooled it was then transferred into an Erlenmeyer flask and five test tubes (6 mL for each tube). The groups shared on the liquid medium prepared. We cannot put exactly 6 mL on each of our fifteen test tubes because the other two groups left us less than 90 mL so we just estimated the liquid medium to fill every test tube. After filling each test tube it was covered with cotton plug immediately. All tubes were in the rack and were placed into an autoclave-safe plastic and were sealed with a rubber band for sterilizing. After a week, it was day 2 of the exercise. The group practiced transfer techniques but before that the teacher taught aseptic techniques where we always flame the mouths of the tubes and always heat the inoculating loop/needle every time we use it. This was done to ensure that we have prevented microbes from air from entering the sterile medium. To start with, only inoculating loop was used. It was heated until the wire turns red, then the mouth of the tube containing the specimen to be transferred was introduced to flame for a while then the inoculating loop was dipped to the tube which was immediately closed. All tubes were labelled,

this is important in every experiment because without a label we tend to mismatch the apparatus/specimen that we are using. The blank liquid mediums mouth was heated then the loop was dipped into it. This process was for the broth to broth transferring where all media used was liquid. For the slant to broth inoculation the process was the same except for the dipping of the loop into the tube with specimen rather it was just touched at the slant with growth. In slant to slant method, again the inoculating loop and tube was heated and the loop was touched into the part of the medium with growth then the blank mediums mouth was heated and the loop touched the agar and was slid in a zigzag manner to spread the bacterium M. luteus. In transferring from a colony in a petri dish into the broth, you first choose a colony to get. The loop was again heated but this time it was not the mouth of the plate heated but the sides it was turned, opened then heated until all sides were introduced to the flame. Pick the colony you wish gently not to distort the agar on the plate then transfer the bacteria into the liquid medium with the mouth already heated. IV. RESULTS Data sheet is attached below after the pictures. Table 1. Observations of the majority of the tubes per transfer technique. Description Turbidity Sediment Surface Growth BB + + + SB Not quite + + SS N/A N/A + AB + Not quite +

Figure 1. Broth to broth replicates.

Figure 3. Slant to broth replicates.

Figure 2. Colony to broth replicates.

Figure 4. Slant to slant replicates.

Figure 5. From top left to bottom right; BB SS AB SB after 42 hours.

V. DISCUSSION Media must be prepared in such a way that it is sterile prior to being inoculated with a bacterial sample, so that when a particular type of bacteria is cultured (cultivated) on that medium, it is the only type of bacteria present (Bauman, 2007). Some media formulations are very specific recipes in which certain ingredients must be present in specific amounts. These defined media (also known as synthetic media) are used to grow bacteria that have very particular needs (Perry and Stanley, 1997). Cultivation of microorganisms depends on a number of important factors: Proper nutrients must be available. Oxygen or other gases must be available, as required. Moisture is necessary. The medium must have an appropriate pH. Proper temperature relations must prevail. The medium must be free of interfering bioburden. Contamination must be prevented. A satisfactory microbiological culture medium must contain available sources of: Carbon Nitrogen Inorganic phosphate and sulfur Trace metals Water Vitamins

These were originally supplied in the form of meat infusion. Beef or yeast extracts frequently replace meat infusion in culture media. The addition of peptones, which are digests of proteins, provides readily available sources of nitrogen and carbon. Solidifying agents, including agar, gelatin and albumin, can be added to a liquid medium in order to change the consistency to a solid or semisolid state. Peptone, protein hydrolysates, infusions and extracts are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material; e.g., meat, milk (Zimbro, et. al., 2009). The bacterium to be studied in this exercise is Micrococcus luteus. The microbe can be found in a variety of environments including soil, water, animals, and some dairy products. Micrococcus is generally thought to be a saprotrophic or commensal organism, though it can be an opportunistic pathogen. This is particularly true in hosts with compromised immune systems. Being Gram-positive, these organisms appear blue to violet when stained using a Gram-stain technique. M. luteus is an aerobe, which means that it thrives in an oxygen rich environment, and is normally found living in the human mouth, mucosal linings of the upper pharynx, and respiratory tract. Those who may be immunocompromised such as AIDS patients, or those on chemotherapy, need to be concerned about this bacterium; however, these organisms are usually considered non-pathogenic (Gwartney, 2012).

Answers to study questions: 1# Aside from the fact that broth is a liquid medium while a slant is made out of agar that makes it solid, the growth of microbes in a slant and in a broth medium has many differences. Microbes do not suspend inside up to the bottom of the slant while it can in a liquid medium. On the other hand there are no stroke patterns that are formed in the broth which you can clearly see in the slant. Turbidity is not applicable in the slant. Even if they have differences both of them are capable of surface growth. 2# Sterilization by Filtration. Filtration is a useful method for sterilizing liquids and gases. Filtration excludes microorganisms rather than destroying them. Two major types of filters may be used, depth filters and membrane filters. Chemical Sterilization. Chemical sterilization employs gaseous and liquid sterilants for certain medical and industrial instruments. The gases include ethylene oxide, formaldehyde and beta-propiolactone. The liquid sterilants include glutaraldehyde, hydrogen peroxide, peracetic acid, chlorine dioxide and formaldehyde. Chemical sterilization is not employed in the preparation of culture media due to unfavorable affects upon performance. Radiation Sterilization. Radiation sterilization is an optional treatment for heat-sensitive materials. This includes ultraviolet light and ionizing. Dry Heat Sterilization. Dry heat is employed for materials such as metal instruments that could be corroded by moist heat, powders, ointments and dense materials that are not readily penetrated by steam. Because dry heat is effective only at considerably higher temperatures and longer times than moist heat, dry heat sterilization is restricted to those items, unlike culture media, that will withstand higher temperatures.

3# Nutrient gelatin is useful in bacterial identification. Gelatin is a protein that can be broken down by enzymes secreted by some bacteria. Bacteria that secrete these enzymes are identified when the nutrient gelatin they are grown on undergoes liquefaction. VI. CONCLUSION Aseptic technique should always be practiced during handling microbes in the laboratory same as in chemistry, to be safe it is needed to be cautious since there are toxic chemicals, well in microbiology we should treat each organism handled as pathogenic. In transferring it is necessary to heat every apparatus mouth/ side in order to kill the microbes in the air that potentially might contaminate the desired culture. Practicing the techniques makes one better in handling such stuff. Always use distilled water in media preparations, etc. if necessary to avoid contamination and use face masks and gloves therefore in coming to class one should always be prepared. In preparing media, one should have the skill for calculation and analysis. After preparing, sterilization should be done in order to be sure that the media to be used is conducive for culture. There are differences in using a liquid and solid media but both have the same goal, to culture the microbe/s desired. In inoculation hands should be calm while eyes should inspect how coordinated you are. This technique is important to be mastered because it will be encountered

again and again in the course. The bacterias growth in the tubes is directly proportional of the number of days. 2 days is not enough to see best results. It is important to observe sharply the happenings and describe the microbe studied since it is one of the goals. Therefore in order to have desired results with less error, contamination should be avoided, measure accurately, think twice before doing a move and always look at everything in your reach. Keep eyes open, mouth shut and ears alert. VII. REFERENCES Book Sources Zimbro, M. J., Power, D. A., Miller, S. M., Wilson, G. E., and Johnson, J. A. (2009). Difco & BBL Manual: Manual of Microbiological Culture Media. 2nd ed. United States of America. Becton, Dickinson and Company. Pp. 4-5. Bauman, R. (2007). Microbiology with Diseases by Taxonomy. Pearson Benjamin Cummings. Perry, J. and Stanley, J. (1997) Microbiology Dynamics & Diversity. Saunders College Publishing. Greenblatt, C.L., Baum, J. and Klein, B. (2004). Micrococcus luteus Survival in Amber. Microbial Ecology. 48. Pp. 120-127. Internet Sources http://www.microbeworld.org/component/jlibrary/?view=article&id=8054 http://www.scienceprofonline.org/microbiology/types-culture-media-for-growing-bacteria.html http://www.alpenacc.edu/faculty/milostanm/html_ppt02/solidifying_agents.htm http://www.microrao.com/micronotes/culture_media.pdf