Biology

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**BACKGROUND INFORMATION:** There are several types of bacteria that are found in raw meat. One example is **Escherichia coli,** it may be harmful when the beef isn't cooked properly, it usually colonize in the intestines of alive animals and contaminate muscle meat when it is slaughtered. E. coli can lead to diseases such as **bloody diarrhea.** However, it is usually destroyed during proper cooking. Microorganisms that are attached to beef in grocery stores are **psychrotrophic bacteria**, they can survive and keep growing in the cold temperature. Basically, these bacteria are not poisonous but they accelerate the process of oxidation, therefore reducing the life cycle of fresh beef. In most cases, the bacteria are not only present on the surface of the meat but also a few millimeters from the surface.

**MATERIALS:**
 * 225 grams of sirloin meat
 * 9 sterilized petri dishes
 * coffee paper
 * nutrient agar
 * nutrient agar powder (12 grams)
 * distilled water (600 ml)
 * 50 ml beaker
 * 1000 ml beaker
 * magnetic stirrer
 * electronic heater
 * aluminium foil
 * electronic griller
 * incubator
 * clear plastic wrap
 * spirit burner
 * 2 tweezers
 * cutting board
 * knife
 * sterilized cotton
 * electronic mass balance
 * marker
 * labels

__Independent Variable:__ → **meat at different rawness lev****el** : The amount of bacterial growth on the nutrient agar would be investigated using different timed cooked meat. The meat would be grilled with an electronic grill and cooked for times of 0, 2 and 4 minutes.
 * VARIABLES: **

__Dependent Variable:__ → **Amount of bacterial growth on the nutrient agar in the petri dishes** : The amount of bacterial growth would be affected by the different timed cooked meat. The bacteria would be counted from the bacterial colonies, which can be identified where there are clumps of bacteria.

__Controlled:__ →**Mass**: The mass of each piece of meat would all be 25 grams. The bigger the meat, the more bacteria there would be, therefore if we keep the mass constant, the amount of bacteria in each one would roughly be the same and so the results would be more accurate. → **Level of heat in the grill**: The level of the heat of the grill would be set at 4 so that the cooking rate of the meat would be the same. This ensures that at 2 and 4 minutes, the level of rawness of the meat would be relatively the same. → **Time for bacterial growth**: The time we allow the bacteria to grow would be the same which is 22 hours, so the results would be more accurate as to which meat bacterial growth would be the most. Bacteria has a growth curve as time goes on and the phase of growth is called logarithmic or exponential because the rate of increase in cell number is a multiplicative function of cell number. → **Time for meat on nutrient agar:** The time in which we let the meat touch the nutrient would be the same, which is 10 seconds. This is the time for the bacteria from to meat to go to the nutrient agar where it would grow. By leaving it for the same time, it give the same time for the bacteria to be transferred in each trial, making the results more accurate.
 * →Temperature**: The petri dishes would be kept in an incubator with a set temperature of 37°C which is room temperature. This is the temperature in which most bacteria thrive.

**PROCEDURE:**
 * 1) Collect all the needed materials to do the experiment.
 * 2) Wrap the petri dish in coffee paper and sterilize it at a temperature of 120 °C in the sterilizing machine.
 * 3) Start making the nutrient agar
 * 4) Turn on the electronic heater to let it heat up
 * 5) Using a beam electronic beam balance and a 50ml beaker, measure the amount of nutrient agar powder needed (2 grams for every 100 ml)
 * 6) Mix the distilled water and the nutrient agar powder in a beaker big enough to contain the water.
 * 7) Insert the magnetic stirrer
 * 8) Place the beaker on the electronic heater
 * 9) Wait until it boils
 * 10) Once it boils, remove it from the heater and pour the nutrient agar mixture in a bottle. Leave the bottle aside for it to cool.
 * 11) Sterilize the bottle containing the nutrient agar in the sterilizing machine at a temperature of 120 °C
 * 12) When the sterilizing is done, light up the spirit burner, spray and rub alcohol on your hands and open up the petri dishes from the coffee paper and burn the sides by turning it around. Once all the sides has come in contact with fire, open up the petri dish and pour 65 ml of nutrient agar. Leave the agar to set with the cover of the petri dish slightly opened. This takes around 1-2 hours.
 * 13) Then, cut the meat into 25gr pieces (The meat should be in room temperature). Measure it using the electronic mass balance.
 * 14) Cover up these pieces of meat using the plastic wrap
 * 15) Insert a tweezers in a 50 ml beaker and pour 10 ml of alcohol in it.
 * 16) Once the nutrient agar has set, spray and rub alcohol on to your hand again and let the sides of the petri dish come in contact with the fire, turn it around until all the sides has come in contact with the fire.
 * 17) If there is evaporated water inside the petri dish, open that petri dish, get the sterilized cotton and wipe it until it is dry. Close the petri dish and burn the sides again.
 * 18) Open the petri dish and using the tweezers from the beaker, take the meat and place it on the nutrient agar. Wait for 10 seconds then flip the meat to the other side and count for another 10 seconds.
 * 19) Remove the meat and close the petri dish. Burn the sides of the petri dish by turning it for 3-4 times.
 * 20) Cover the sides of the petri dish with the clear plastic wrap until it is tightly sealed.
 * 21) Label the petri dish according to the time the meat was cooked and the actual time.
 * 22) Insert the petri dish upside-down in the incubator, to prevent water condensation is accumulating and disturbing the cultured microbes.
 * 23) Repeat steps 8-14 for the 2 and 4 minutes grilled meat. Create 3 trials for the different-timed cooked meat.
 * 24) Grill the meat on both sides. For the 2-min grilled meat, spend 1 min for each side and for the 4-min grilled meat, spend 2 min for each side.
 * 25) Check the petri dish after 24 hours and by using total bacteria count; count the amount of clumped bacteria colonies there are.
 * 26) Record the amount of bacteria.


 * OBSERVATIONS: **



**DATA COLLECTED:** raw data ** ±0,01 minutes ** |||||| ** Colony ± colony/gr ** || processed data (calculations) Convert the bacteria into colony forming unit form (bacteria/ml)  Colony Forming Unit:
 * ** Time (minute) **
 * ^  || Trial 1 || Trial 2 || Trial 3 ||
 * 0 minutes (raw) || >300 || 119 || 101 ||
 * 2 minutes (grilled) || 35 || 39 || 45 ||
 * 4 minutes (grilled) || 20 || 35 || 31 ||

colony obsreved  beef (gr)

Sample calculations:

300colonies -- = 12colonies/gr 25gr

±1 bacteria/gr  ||
 * Time (Minute) ±0,01min ||||||||||   Colony Forming Unit (bacteria/ml)
 * ^  || Trial 1 || Trial 2 || Trial 3 || Average || SD ||
 * 0 (Raw) ||  12   ||   5   ||   4   ||   7   ||   4,358899   ||
 * 2 (Grilled) ||  1   ||   1   ||   2   ||   1,333333   ||   0,57735   ||
 * 4 (Grilled) ||  1   ||   1   ||   1   ||   1   ||   0   ||


 * GRAPH**

**Sources of Errors:**

 * ====Some bacteria were unable to grow under your conditions - causing your count to be too small ====
 * ====Contamination from dust and bacterias in the room - causing your count to be too large ====
 * ====Some bacteria may be clumped together and grow a single colony - causing your count to be too small ====