Bacteriology The Study of Bacteria

Transcription

1 Name Bacteriology The Study of Bacteria Observing bacterial structure differential Gram stain To observe the structure of bacteria, we often use a process called the Gram stain developed in Europe by Hans Christian Gram. Place one drop of water on a clean slide. Using a sterile loop, remove a healthy loopful of bacteria from the stock culture. Stir the loop in the drop of water to make a nice thin suspension of cells on the slide. Flame the loop to clean it. Allow the slide to air dry you can apply gentle heat from the alcohol lamp to speed this process. After the film is completely dry, pass the slide about three times through the flame. You are just trying to attach the cells to the glass NOT trying to cook them! Flood the dry smear with a drop or two of Crystal violet (aka gentian violet) stain and allow it to stand for 10 seconds. The dye will pass into the cells attached to the glass. Flood the slide with Gram iodine for another 10 seconds. This will also pass into the cells and form complexes with the violet stain inside the cells. Flood the slide with 95% alcohol to destain the smear. The thinnest areas on the slide should lose virtually all of their color! Continue to destain until you reach that point. Gram+ bacteria will likely retain more color Gram- bacteria will destain quite easily and completely. Flood the slide with Safranin to counterstain the smear for 10 seconds. Blot dry with a towel. Observe your smear in the compound light microscope. Adding a drop of immersion oil if needed. If your staining technique is good, Gram+ bacteria will appear to be purple and Gram- bacteria will be pink. If you are stuck waiting for some other part of the worksheet, go ahead and analyze some of our other stock cultures and add to your analysis. Bacillus cereus Bacillus megaterium Citrobacter freundii Enterococcus faecalis Escherichia coli Micrococcus luteus Pseudomonas fluorescens Rhodospirillum rubrum Serratia liquifaciens Serratia marcescens Cell Shape coccus bacillus spirillum vibrio Cell Grouping uni diplo strepto staphylo Gram Result + - Page 1

2 Fermentation Bacteria have metabolism All organisms must obtain nutrients for their growth. Heterotrophic bacteria rely upon other organisms or sources for carbon and energy. Some of the more primitive metabolic pathways to process these external molecules are called fermentation. These processes convert sugars into acids and sometimes also carbon dioxide. To detect acids we can use Phenol red in the medium; if acids are produced, the pink color will change toward yellow. As we prepare the medium we can also add a very small upside-down test tube to trap any gas being made by the bacteria growing in the medium. This arrangement is called a Durham Fermentation Tube. Your group will receive 12 tubes of medium: 4 of phenol red lactose broth, 4 of phenol red glucose broth, and 4 of phenol red sucrose broth. We will test whether each organism can ferment lactose, glucose, or sucrose. Our four organisms are: Citrobacter freundii, Enterococcus faecalis, Micrococcus luteus, Serratia liquefaciens. From the stock culture of each organism, inoculate a tube of lactose, a tube of dextrose, and a tube of sucrose broth. Flame and cool the loop before each inoculation. Leave these tubes in the rack as you inoculate them. Next week we will observe the color of the broth and the presence of gas in the tube. Record the medium color and +/- gas in the tables below. Citrobacter freundii Color Gas Enterococcus faecalis Color Gas Micrococcus luteus Color Gas Serratia liquefaciens Color Gas Cross-feeding pathways dealing with excess nitrogenous waste Serratia marcescens D1 is a well-known pigmented bacterium synthesizing the red pigment, prodigiosin. This pigment is the result of a reasonably long biochemical pathway in which portions of several amino acids are assembled into the final prodigiosin chemical structure. Some of the intermediates are volatile and others are soluble in the medium. The details of the pathway are still not completely understood, but today you will learn a few things about it. In a pathway we generally think of a cascade of steps: a---->b---->c (red) Each arrow in this pathway represents a chemical reaction probably catalyzed by an enzyme that is coded in the genes of the organism. Now if a gene is mutant and thus produces an ineffective protein, then the pathway cannot be completed and the final product is not made. So in one organism the mutation might be in the gene for the first enzyme in the pathway: a--//-->b---->c (not red) In such an organism, chemical a would accumulate and b and c would not be produced at all. In a second organism the mutation might be in the gene for the second enzyme in the pathway: Page 2

3 a---->b--//-->c (not red) This second organism would accumulate b but would not make c. Now if c is a red pigment, then both mutant organisms would lack the pigment. But what if b not only accumulates in the second organism, but also leaches out into the air or medium around the second organism? We could grow the two mutants close together and we should see that the second mutant would feed the first with b needed to complete the pathway: a---->b--//-->c (not red) \ a--//-->b---->c (red!) So today you will try to see how two mutant strains of Serratia marcescens interact in a crossfeeding experiment. Our two strains are named 933 and WCF. Streak these in the pattern diagrammed on the whiteboard...being careful not to allow any of the strokes to actually touch. The medium is Luria Broth Agar (LB). In one dish, pair D1 with 933. In a second dish, pair D1 with WCF. In a third dish, pair 933 with WCF. Make a sketch of the results below after a few days of incubation. Be sure your dishes are incubated with the cover down (medium up) so that the dish can trap volatiles as needed. Decide what you think your results mean about the pathway to prodigiosin and where the 933 and WCF mutations block the path. Present this in a short paragraph explaining your logic. Make a sketch to elucidate this path. Page 3

4 UV light exposure interaction of light and genes Now, that we know just a tiny bit about the pigmentation of Serratia marcescens D1, we will now see if we can mutate the bacteria with exposure to ultraviolet light to create our own colorless mutant bacteria. You will make your plate by spreading bacteria from a broth culture over the solid LB medium. This project should be carried out in the laminar flow hood. Use a sterile yellow pipette tip and a pipetter set for 100 µl. Place the 100 µl drop in the center of each of two nutrient agar plates. A spreader is standing in a beaker of alcohol. Remove this from the beaker and flame it off in the alcohol lamp. CAUTION: do not put the spreader back in the alcohol because there may be flames still on the spreader could start a very LARGE fire! Gently lower the spreader onto the agar and DO NOT push down! Instead let the weight of the spreader rest on the agar. Rotate the plate so that the spreader coats the surface of the agar with the 100µL of broth culture. Tape the cover to the bottom of the dish. Tape a sunglasses lens to the lid of one plate. Place this plate in the designated place for exposure to the UV lamp for 15 minutes. After exposure, trace around the sunglasses lens and remove it. The control plate will be grown on without light exposure. After a few days of incubation, make a sketch of the results here. Be sure to show where the sunglass lens was located by drawing its outline. Endospore project response to temperature How do bacteria respond to temperature extremes? Some have evolved to tolerate extreme temperature very well. In fact Lactobacillus thermophilus, is famous for having evolved to grow in very warm milk. Other species are quite labile to high temperature. There is very good reason for our bodies to exhibit a fever as it battles bacterial infections! We shall try to test the capacity of some species to tolerate high temperature. LB broth (with 0.5% dextrose) 4-day cultures are available for Bacillus cereus, Micrococcus luteus, and Pseudomonas fluorescens. This project should be set up in the laminar flow hood. Using a sterile pipette tip, dispense and spread 100µL of the culture over one plate of LB medium as your untreated control. Place the three stock culture tubes into the 85 C temperature block for 15 minutes. Then, again using a sterile pipette tip and spreader, spread 100µL of a culture onto a second LB plate. Label this dish heat treated. Page 4

5 Both plates for each stock culture will be incubated until the next week. Growth of heat-treated bacteria would indicate tolerance to heat. This is sometimes accomplished by production of endospores. Sketch the results and label below: Page 5