BIOL3422 Experiment 6: Phage Specificity PDF

Experiment 6: Phage Specificity After this lab, students should be able to: Perform a phage plaque assay to quantify phage titer from a provided sample. Demonstrate an understanding of bacteriophage diversity and specificity. Purpose: To determine concentration/titer of a phage sample...

Experiment 6: Phage Specificity After this lab, students should be able to: Perform a phage plaque assay to quantify phage titer from a provided sample. Demonstrate an understanding of bacteriophage diversity and specificity. Purpose: To determine concentration/titer of a phage sample, and to examine specificity of this phage’s ability to infect diBerent bacterial strains. Background Bacteriophages (phage for short) are viruses that infect bacteria. Phage species are as diverse as the bacterial hosts they infect, but they share a conserved structure consisting of a protein capsid surrounding the viral genome, which can be made of either DNA or RNA. Like all viruses, bacteriophage cannot replicate without infection of a bacterial host and must rely on the host’s replication machinery to synthesize viral proteins and nucleic acids for propagation. In order to gain entry into a bacterium, bacteriophage capsids bind to specific bacterial receptors, allowing the phage to inject its nucleic acids through the cell membrane and into the bacterium. The receptors that phages use for attachment are highly specific and vary from one bacterial species to the next. For this reason, bacteriophages have specific host ranges, which will be examined in this lab. Bacteriophage Life Cycles Bacteriophage life cycles have two distinct pathways: the lytic pathway and the lysogenic pathway. In the lytic pathway, the phage uses the host’s replication machinery to produce bacterial capsids and replicate its genome. The nucleic acids are packaged inside the capsids and, when the phage reaches critical mass inside of the bacterium, the bacterial cell is lysed and the phages escape to infect new hosts. In the lysogenic pathway, the phage’s genetic material is integrated into the host’s own genome. The phage’s genetic material, termed a prophage in this state, lies dormant and is replicated with the bacterial genome during production of daughter cells. No phage are produced until an external factor, such as ultraviolet light, induces excision of the prophage from the genome and restarts viral replication via the lytic pathway. Transduction Bacteriophage are also able to transfer genetic material between hosts in a process known as transduction. During phage assembly, it is possible that a phage capsid will take up chromosomal DNA from the host instead of or in addition to phage DNA. The newly assembled phage may then inject this bacterial DNA into a new host, where it may be integrated into the host genome. Many applications in bacterial genetics take advantage of transduction to alter bacterial genomes or introduce genes of interest to a bacterial species. Quantifying Phage Titer In this lab, we will be performing a phage plaque assay. While conceptually similar to the CFU counting we did previously, the process is a visual inversion from the prior experiment: we will be covering plates with a mixture of bacteria and phage and counting the number of plaques—locations where bacteria does not grow as a result of phage activity. By counting the number of plaques made by diBerent dilutions of phage, we can back-calculate the concentration of the original phage sample. To cover the plates evenly, we will use top agar, melted agar that is mixed with a sample before pouring over a regular plate with the aim of evenly distributing the sample across the surface before solidifying. Top agar is kept in a hot water bath to remain liquid and must not be removed from the heat until you are ready to mix and plate your samples. Materials (for each pair of students): One tube of phage One tube of Bacillus subtilis, a known host One tube of a potential host species o We will have several diBerent potential host species for you to choose from – not all of them are susceptible to the phage. Be sure to record which potential host you choose! LB broth for dilutions 8 plates of LB agar 8 tubes of LB top agar (kept in 50°C water bath until use) o Do not remove top agar tubes from the water bath until you are ready to plate your cultures! Procedure: 1. In two fresh 1.5mL tubes, prepare serial dilutions of your phage sample. a) Add 900µL of LB broth to both tubes. b) To the first tube, add 100µL of stock phage to make a 10-fold dilution. Pipet up and down several times to mix. c) To the second tube, add 100µL of the 10-fold dilution to make a 100-fold dilution. Pipet up and down several times to mix. 2. Label eight fresh 1.5mL tubes A-H, and prepare the following mixtures of host and phage: A. 200µL Bacillus subtilis 100µL LB broth B. 200µL Bacillus subtilis 100µL undiluted phage C. 200µL Bacillus subtilis 100µL 10-fold diluted phage D. 200µL Bacillus subtilis 100µL 100-fold diluted phage E. 200µL potential host 100µL undiluted phage F. 200µL potential host 100µL 10-fold diluted phage G. 200µL potential host 100µL 100-fold diluted phage H. 200µL LB broth 100µL undiluted phage  3. Cap tubes and briefly vortex to mix. Incubate at 37°C for 15 minutes. During this incubation step, label your eight agar plates A-H. Be sure to include your initials and the date. 4. After incubation, mix each sample into a tube of melted top agar and pour onto a plate of LB agar. Gently rotate the plate so the mixture evenly covers the surface of the agar. a) It is extremely important that you do not remove the top agar from the water bath before you are ready to plate your sample. The top agar must remain liquid to evenly mix and distribute the sample. b) It is also important to mix the sample thoroughly – you can do this by pipetting up and down a few times, or by swirling the tube. Either way, try to avoid creating bubbles in the mixture. 5. Let your plates sit for 10 minutes until the agar is solidified. Tape them together and set them where directed by your TA. The plates will be incubated at 37°C overnight, and we will examine them for growth and clearance next lab. Data Analysis Examine your control plates. Plate A, which had bacteria but no phage, should have even bacterial growth covering the surface of the agar. Plate H, which had phage but no bacteria, should have no growth. Plates B, C, and D should be covered with varying amounts of bacterial growth, punctuated by circular clearances, or plaques, where the bacteria have been lysed by phage. Count and record the number of plaques on each plate. If there are too many plaques for you to count individual plaques, you can note “TMTC” or “too many to count.” For each plate, calculate the concentration of phage in plaque forming units per milliliter (recall that you added 0.1mL), and back-calculate by the dilution factor to determine the PFU/mL in the original stock. Sample Plaque Count PFU/mL at dilution Dilution Factor Stock PFU/mL Plate B 1 Plate C 10 Plate D 100 Next, examine plates E, F, and G for the presence or absence of plaques. Because of the specificity between phage and host, you may not see any – this simply means that the bacterial strain you chose to investigate is diBerent enough from Bacillus subtilis for the phage to infect. If you do have plaques, count and record them as you did above. Sample Plaque Count PFU/mL at dilution Dilution Factor Stock PFU/mL Plate E 1 Plate F 10 Plate G 100 Questions for Homework: 1. Did your control plates (plates A and H) have the expected results? If not, provide possible explanations as to why they may not have. 2. Report the plaque counts on your Bacillus subtilis plates (B, C, and D), and provide an estimate of the PFU/mL in the provided stock culture based on the averages. Are there any issues with your plates or other reasons to believe your estimate is inaccurate? 3. What bacterial species did you choose to investigate as a potential host for the phage? If you had plaques on the plates with that species (E, F, and G), compare the incidence of plaque formation to that of the B. subtilis plates. 4. Whether or not you had plaques on your “potential host” plates, investigate how closely related Bacillus subtilis is to that potential host. Does the closeness of this relation align with the results of your plaque assay? Environmental Isolate Much can be learned about an unknown bacterium by investigating its metabolic, biochemical, and other phenotypic traits. Across the microbial world, there is a vast array of information that can be used to identify an unknown, including what carbon sources it can use, what environmental conditions it can tolerate, and what hazardous compounds it can survive. This week, we will be performing our first round of phenotypic analysis of our environmental isolates with a commercial test branded BIOLOG. Each of the 96 wells in a BIOLOG plate contains a unique carbon source, antibiotic compound, or environmental growth condition (see below). Each pair of students will inoculate all 96 wells of this plate with their environmental unknown and incubate it overnight. If your bacteria can grow, the cellular respiration will reduce a tetrazolium dye in the well, giving it a purple color. Next week, we will examine the plates to see which wells have color and compare these results to BIOLOG’s library of species results. Figure 5.1: Diagram of BIOLOG Gen III Microplate assays. Sourced from Biolog, Inc. Materials (for each pair of students): 1 BIOLOG 96 well plate. Environmental unknown o You may have more than one environmental unknown – between you and your lab partner, choose one that you want to investigate further in this experiment. The other will be kept as a backup. BIOLOG inoculating fluid. o Most groups will use IF-A. However, if you have a gram-positive rod, you should use IF-B. Sterile cotton-tipped swabs. Plenty of yellow pipet tips. Protocol: 1. If you have more than one, decide which of your environmental unknowns you want to investigate. Discuss with the other students at your lab table what kinds of bacteria you are inoculating – we recommend the plates at each table look at di=erent types of bacteria (e.g. gram-negative vs positive, cocci vs rod). a. Confirm which tube of inoculating fluid you will be using. Most will use IF-A, but those investigating gram-positive rods should use IF-B. 2. Use a cotton-tipped swab to pick up a ~3mm colony from your plate and swirl it into your tube of inoculating fluid. 3. Bring your tube of IF with bacteria to the front of the room, where your TA has set up a turbidimeter. Set the turbidimeter to 100% with a clean, sterile tube of IF, and measure the transmittance of your sample. It should ideally be around 95%, but anywhere in the range of 90- 98% is acceptable. a. If your transmittance is too high, add more bacteria. b. If it is too low, add a small volume of IF from a fresh tube provided by your TA. 4. Inoculate your BIOLOG plate. Each of the 96 wells needs to be filled with 100µL of your sample. Make sure that the sample is placed at the bottom of the well (not sticking to the sides) and that you are not spilling between wells. Change your tip after each sample. 5. Ensure the BIOLOG plate is labeled with your group info and leave it where directed by your TA. They will be incubated at 37°C overnight and examined for color next week.