INS 2007-08 Pre-lab IV Questions 1. You are given a test tube containing 10 mL of a solution with 8.4 x 107 cells/mL. You are to produce a solution that contains less than 100 cells/mL. What dilutions must you perform in order to arrive at the desired result? 2. You have a tube containing 1 mL of a solution with 4.3 x 104 cells/mL and you are to produce a solution that contains 43 cells/mL. What dilutions must you perform? 3. You are given a container with 5 mL of a solution containing 5.1 x 103 cells/mL. You are to produce a solution that contains approximately 100 cells/mL. 4. You are given a container of yeast cells in a concentration of 2.6 x106 cells/mL. You are to prepare a suspension which, when you spread 0.1 mL of the suspension on appropriate media, will result in about 100 cells. How will you do this?2 INS 2007-08 Lab IV The Evolution of Antibiotic Resistance The main goal of this lab is to take a look at evolution and how microbes get some genetic variability from mutations and acquired DNA. Mutations are relatively slow and mutations that are favorable are rare. In a rapidly changing environment even a high replication rate is not enough to ensure survival. So, under these circumstances bacteria have another mechanism to obtain and share genetic variability. Lateral or horizontal gene transfer (HGT) is a process whereby genetic material contained in small packets of DNA can be transferred between individual bacteria. There are three possible mechanisms of HGT. These are transduction, transformation or conjugation. Transduction occurs when bacteria-specific viruses or bacteriophages transfer DNA between two closely related bacteria. Transformation is a process where parts of DNA are taken up by the bacteria from the external environment. This DNA is normally present in the external environment due to the death of another bacterium. Conjugation occurs when there is direct cell-cell contact between two bacteria (which need not be closely related) and transfer of small pieces of DNA called plasmids takes place. This is thought to be the main mechanism of antibiotic resistant gene transfer. Plasmids are small circular DNA, contain information for 3-5 genes that code for proteins, and can replicate in the cell independent of cell division. Plasmids are not chromosomes. They are not required for cell survival under optimum growth conditions and will be lost if not needed. Multiple copies of a given plasmid can be found in a bacterium. Some of these copies can be shared with other bacteria that do not possess them. When plasmids were first studied it was believed that they could only be shared between bacteria of the same species. It has since been observed that bacteria can share plasmids with other bacteria that are not closely related. This is important because it means that non-pathogenic bacteria can give plasmids to pathogenic bacteria. Another goal of this lab is to get you comfortable working with molecular and microbiology equipment. Before jumping into our work exploring antibiotic resistance in bacteria, you will first spend some time developing your laboratory skills. You will learn how to accurately pipette, perform sterile techniques, and calculate serial dilutions. I. Micropipettes 101 We will give a short tutorial on how to use our micropipettes and then allow you to practice with colored water on parafilm. You should spend some time using the different micropipettes, comparing droplet sizes from different volumetric settings. Be sure to dispense the liquid properly so that you do not get bubbles. II. Sterile technique A. Flaming and cooling loops SAFETY LESSON! Loops are sterilized by holding them under a flame until they lightly glow, be careful not to burn yourself. Also, be sure to let it cool entirely before touching it to bacteria. Practice this before going on to part B. You can test the temperature of your loop on the very edge of one of your agar plates. If it sizzles, then it is definitely TOO hot. It is likely to be too hot even if it does not sizzle. B. Quadrant Streaks One way in which we transfer a population of bacteria from one source to another is to streak them out onto a growth plate. Although there are different methods for transferring bacteria, one of the most commonly used techniques is a quadrant streak. This technique allows sequential dilution of the original microbial material (broth culture or colonies on a plate) over the entire surface of a fresh plate. The original sample is diluted by streaking it over successive quadrants, decreasing the number of bacteria. Usually by the third or fourth quadrant only a few bacteria are transferred on the inoculating loop and these produce a few isolated colonies. 1. Ask us for a bacterial source for this part of the lab. 2. Select a colony that you would like to test and streak it onto a plate using the quadrant streak method below.3 a) flame the inoculating loop until it is red hot and then allow it to cool. Avoid killing your bacteria by testing the loop on the inside wall of the agar plate! b) remove a small colony with the sterile loop c) immediately streak the loop very gently over a quarter of the plate using a back and forth motion d) flame the loop again and allow it to cool. Go back over the edge of the first streaked area ONCE and extend the streaks into a new quadrant. e) flame the loop again and allow it to cool. Go back over the edge of the second streaked area and extend the streaks into a new quadrant. f) flame the loop again and allow it to cool. Go back over the edge of the third streaked area and extend the streaks into the last quadrant. g) sterilize your loop by flaming it again, close the lid, and let the plate sit a few minutes. It should be incubated upside down to avoid condensation. III. Serial Dilutions A serial dilution is simply a series of simple dilutions which amplifies the dilution factor quickly beginning with a small initial quantity of material (i.e., bacterial culture, a chemical, dye, etc.). The source of dilution material for each step comes from the diluted material of the previous. In a serial dilution the total dilution factor at any point is the product of the individual dilution factors in each step up to it. Final dilution factor (DF) = DF1 * DF2 * DF3 (etc. . .) Example: In the following, a bottle contains an unknown concentration of bacteria ("cfu/ml" means "colony forming units per