Name: _______________________________________ Section:_________ Microbiology Selective and Differential Media Materials required: Misc. equipment and supplies as needed MacConkey Agar 1 Mannitol salt agar (MSA) 1 Colistin-Nalidixic acid agar (CNA) 1 TSA (3) Organisms: o Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris o Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis Directions: 1. Work in pairs. Label the plates with the organisms listed below and your names. Inoculate your plates only after proper instruction. It is imperative that you use good technique to ensure the success of this procedure. Be sure that your loop is sterilized, but don't "hot loop" the specimen. There is no need to shovel the inoculum. Refer to tables below for organisms to use. 2. Incubate under standard conditions. 3. For reliable references concerning media purposes and reagents, search the following websites CNA agar: http://www.bd.com/ds/technicalCenter/inserts/L007370(08)(0806).pdf MSA agar: http://www.bd.com/ds/technicalCenter/inserts/L007389(07)(0907).pdf MacConkey Agar: http://www.bd.com/ds/technicalCenter/inserts/L007388(08)(1006).pdf Trypticase Soy Agar (TSA): http://www.vgdusa.com/Spreadsheets/tryptic-trypticase-soy-agar-broth-difco-bacto-bd.pdf 1 Rev 2/2012 Name: _______________________________________ Section:_________ Record your observations in the tables below. Columbia CNA Agar: Organism Growth Interpretation of growth on CNA CNA TSA Staphylococcus aureus Enterococcus faecalis Escherichia coli Mannitol Salt Agar (MSA): Organism Growth MSA Growth Interpretation of growth on MSA MSA TSA Color (Y/R) Staphylococcus aureus Staphylococcus epidermidis Escherichia coli 2 Rev 2/2012 Name: _______________________________________ Section:_________ MacConkey Agar (MAC): Organism Growth Mac Growth Interpretation of growth on MAC MacConkey TSA Color (P/C) Escherichia coli Klebsiella pneumoniae Proteus vulgaris Enterococcus faecalis Based upon your observations and research, answer the following questions: 1. What is the purpose of including selective and differential media in the primary setup of clinical specimens? (2pt) 2. CNA contains __________ and ___________ that inhibit gram negative organisms. Describe how each of these agents work. (2 pt) 3. Earlier formulations of CNA were made using slightly higher concentrations of the inhibitory agents, but this was changed to improve recovery of gram positive organisms. List some reasons why the higher concentration was less effective at allowing growth. 1 pt) 4. What purpose does the TSA plates serve for these exercises? In what way does it increase the validity of the test result? (1 pt) 3 Rev 2/2012 Name: _______________________________________ Section:_________ 5. What would be the likely consequence of omitting the NaCL in Mannitol Salt Agar? Crystal violet in MacConkey Agar? (1 pt) 6. Would omitting the reagents in #5 alter the medium’s specificity or sensitivity? Explain. Your answer is either specificity or sensitivity (not both) and why.(1 pt.) 7. The organisms that grow on MSA utilize which ingredients as their carbon source or nitrogen source. Refer to hyperlinks listed on page one to determine which reagents supplies the carbon and which supply the nitrogen. a) Carbon (1 pt.) b) Nitrogen (1 pt.) 8. With diversity of microorganism in the world, how can a single test, such as MSA, be used to confidently identify Staphylococcus aureus? (2 pt.) 9. With respect to MacConkey Agar, what would be the consequence of: a) replacing lactose with glucose? (Describe the color of the organisms on the plate). (1 pt) E. coli K. pneumoniae P. vulgaris E. faecalis b) replacing neutral red with phenol red? Describe the color of the organisms on the revised media. (1 pt) E. coli K. pneumoniae P. vulgaris E. faecalis 4 Rev 2/2012 Name: _______________________________________ Section:_________ 10.MacConkey is a selective media. Is it also a defined or an undefined media? Why is that formulation desirable? (1 pt) 5 Rev 2/2012 .