Name:______Seat # ______Do not write in pencil (use pen). Do not use Liquid Paper (draw one line through error). Date: ____/____/____ Periods______Please staple all work.

Solutions and Making Media Lab 2 Lab Number

Purpose: The purpose of this lab is to learn how to make reagents that are needed for labs and media in order to grow and identify various microorganisms.

Materials: Balance Cotton Disinfecting Weighing Paper Safety gloves Biohazard containers wipes Spatula Autoclave Instrument 500 mL Erlenmeyer / Florence Flask Tubes for broth + slants NaCl Stirring Rods Test tube rack Parafilm Distilled Water Slant rack Plastic pipette 500ml graduated cylinder Funnel Beaker of H2O (1000 mL) Dry Media Working Mat Safety glasses Petri dish(es) Laboratory Coat Procedure Part 1: 1. Calculate how to make the following mixtures :500 mL of .850% NaCl in the raw data section 2. Clean your safety glasses and put them on. 3. Zero scale. 4. Tare paper or container (If you can). 5. Take solute (NaCl) out of container with spatula (do not use hands) until desired mass is obtained. 6. Carefully place solute into the mouth of volumetric flask by bending weighing container or weighing paper over the mouth of volumetric flask. 7. Place the funnel over the mouth of the volumetric flask. 8. Pour in a small amount of water and gently swirl flask to mix. Add more water and mix. Repeat this process until the desired amount of water is obtained. Be careful not to go past the fill line. Use a plastic pipette to add water when close to fill line. If you go past the fill line, start over. 9. Place parafilm or lid over the mouth of the volumetric flask and mix gently by inverting the flask upside down several times. Be careful that the parafilm doesn’t have holes. If using a lid, hold the lid when mixing. Procedures Part 2: Students will work together to get steps done. 1. Tare weighing paper. 2. Weigh out the appropriate grams of powder with spatula (see media container) 3. Measure the proper amount of distilled water using a graduated cylinder, and put it in one of the other empty flasks. 4. With a funnel pour a small amount of distilled water into another flask, just enough to cover the bottom of the flask. 5. Pour powder into the Erlenmeyer flasks that contain the small amount of water by bending your weighing container, and putting it over the mouth of the flask. Tap gently to put powder in flask. 6. Make a paste with the powder and water. (Slowly mix well as water is added, until all the water is added) 7. After mixture has completely dissolved (you will have to heat most media to dissolve), pour (1/2) of the media into the other flask (only if you are making a full batch.) 8. Put flask on hot plate and heat according to instructions on container. 9. Top the mouth of the flask with cotton before putting flask in the autoclave. 10. Autoclave according to instructions on container. 11. Label the fat side of the petri dishes with media’s abbreviated name and date. 12. Spread petri dishes all over a table one inch apart with small side on top. 13. Fill (1/2 to 2/3) of petri dish with media. *** BE CAREFUL MIXTURE WILL BE HOT*** 14. Place petri dish cover over plate leaving the cover (1/8) inch open so condensation will not build up on media. (close cover after media cools and solidifies.) 15 Invert plates and stack plates in the center of your table. Plates will be boxed and refrigerated for 72 hrs. 16. Replace and dispose of all supplies and equipment according to instructor (also see lab disposal book). 17. Clean work area. Raw Data/Calculations: (20 points) Show your work for the following calculations here. 1) 500 mL of .850% NaCl.

2) Calculate how much water is needed if a full batch of media requires 1Liter, and the lab requires making: a. 1/2 batch = ______ml b. 1/4 batch = ______ml c. 1/8 batch = ______ml Results (20 points): ______Complete name of media Abbreviated name of media

Normal Range: N/A

Conclusion (20 points): Write a sentence about how your media turned out according to the instructor.

Clinical Significance (20 points): Write a sentence stating the importance of making media.

Questions: Clinical Significance (20 points): Please complete prior to completing the lab in class.

1. What is Agar?______2. How is a slant made? ______3. Why is media autoclaved?______4. Why is media stored inverted after it is made?______5. What are the three forms of media? ______6. What is the pressure, temperature, and time for autoclaving media?______7. If _____grams are needed to make a full batch of SIM media, how many grams are needed for: a. 1/2 batch = _____g b. 1/4 batch = _____g c. 1/8 batch= _____g 8. If _____grams are needed to make a full batch of MR-VP media, how many grams are needed for: a. 1/2 batch = _____g b. 1/4 batch = _____g c. 1/8 batch= _____g 9. If _____grams are needed to make a full batch of Simmons Citrate media, how many grams are needed for: a. 1/2 batch = _____g b. 1/4 batch = _____g c. 1/8 batch= _____g 10. If _____grams are needed to make a full batch of Muller Hinton media, how many grams are needed for: a. 1/2 batch = _____g b. 1/4 batch = _____g c. 1/8 batch= _____g