Lab 7: Public Health Microbiology and Bacterial GrowthExercise 17: The bacterial growth curveIn Exercise 17, we’ll use optical density (OD) measurements to record bacterial growth, plot a growth curve, and calculate the growth rate and generation time.To set up the growth curve experiment, we need to inoculate a batch culture. (Remember that a batch culture is a closed system culture in which no new nutrients are added and no wastes removed after the culture has been inoculated.) This batch culture will be very simple: 10 ml of T-soy broth in a screw-capped test tube, which will be inoculated with 200 ?l of a young E. coli culture.Because the E. coli starter culture is young (in early to mid log phase), I expect that the lag phase is going to be very short, or even non-existent. Why? Because young vigorous cells should already be growing (and therefore don’t need to do anything to prepare themselves for growth.) Chances are, we’ll skip the lag phase altogether, and proceed directly into the log phase. That’s great, because it’s the log phase in which exponential growth occurs. And so this is the phase we need to measure the growth rate.Watch the following video to see how I inoculate the batch culture to start the growth curve, and then how I use the spectrophotometer to take OD readings.So that’s how we do it. In the lab, we take measurements every 30 minutes, so the growth curve takes 2 hours to complete. (As you can imagine there are some people who aren’t quite thrilled about waiting around for E. coli to grow.) Lucky for you, you don’t have to wait. I’ve done the work for you, and collected all of the data. Now you just need to plot a growth curve and calculate the growth rate and generation time.Use the data in the following table to fill in the results on page 104 of your lab manual.Time (min)Absorbance measurement at 540 nm00.007300.026600.077900.1751200.622Next, plot a growth curveYou can plot your growth curve using Excel. For this curve, assume that there is no lag phase (the cells were already young and growing when we started the batch culture.)Plot the data on a semi-log scale, and use a line-of-best-fit to find the trend shown by the data (also known as a ‘trend line’ in Excel.) Instructions for plotting a growth curve using Excel are available in the lab manual, and there is a walk-through video in the Lab 7 folder on UM Learn. Note that at the time of this writing, it is not possible to make this graph using the online version of Excel. (Because the online version doesn’t have the trendline function.) You will need to use an installed version of Excel, which all U of M students can download from the IST website. opens in new windowNext, calculate the growth rateUse the formula k = Log A2 ? Log A1(0.301)(?t){“version”:”1.1″,”math”:”http://www.w3.org/1998/Math/MathML”> mathvariant=”normal”>k = Log A2 – Log A1(0.301)(?t)”} to calculate growth rate from the data above. Note that this is the same formula used in the lecture. The variables have just been changed to A1 and A2, because in this lab we measured absorbance with the spectrophotometer.To calculate the growth rate successfully, you must use the slope of the best fit line from the graph you plotted above. You cannot just insert two of the data points from the table into the formula because they won’t give the same slope that a best fit line will give.Generation timeOnce you’ve calculated growth rate (k), you’re ready to calculate the generation time (g). Use the formula g =1k{“version”:”1.1″,”math”:”http://www.w3.org/1998/Math/MathML”> mathvariant=”normal”>g =1k”} to calculate the generation time for the culture. Give your final answer in min/gen.Exercise 18: The Disk Diffusion Test In this exercise, we use the Kirby-Bauer disk diffusion assay to determine the antimicrobial activity of a number of household products, including antibacterial hand soaps, hand sanitizers, cleaners, and even antibacterial toothpaste. Antimicrobial productsAntimicrobial products meant for use on humans must be registered with Health Canada, and will have a unique Drug Identification Number (DIN) that can be used to look up the product on the Health Canada website opens in new window. This includes hand soaps and lotions, sanitizers, toothpastes and mouthwashes that contain antimicrobial chemicals. (Note that cleaners don’t have a DIN because they’re not meant for use on humans. Non-antimicrobial products, like regular non-antibacterial hand soaps, are also not regulated by Health Canada, and so they also do not have a DIN.)The following picture shows some of the antimicrobial products that we have available for testing in the lab. Some of these products have active ingredients different from those listed in the lab manual. And some of the products don’t contain any antimicrobial chemical at all (the so called-green products usually don’t have any active antimicrobial ingredients.) You should be familiar with the four types of antimicrobial chemicals listed in the lab manual (Alcohols, QUATs, Chlorine and Triclosan) and you should find at least one kind of product that contains each of them.Here are some of the products that I will test when I set up the disk diffusion assay:COLGATE TOTAL TOOTHPASTEPURELL HAND-SANITIZERDIAL ANTIBACTERIAL HAND SOAPCLOROX BLEACH DISINFECTING SPRAYCREST PRO-HEALTH MOUTHWASHWhat is an example of a product that contains Alcohol as the active ingredient? What is the concentration of alcohol in this product?What is an example of a product that contains Chlorine bleach? What is the actual chemical ingredient in this product?What is an example of a product that contains triclosan?What is a potential danger of using products containing triclosan?What are two examples of products that contain QUATs? (Choose two products that have different uses.)Inoculate disk diffusion plate and apply productsWhen you’re familiar with the antimicrobial chemicals, it’s time to do a disk diffusion test. I’ll begin my disk diffusion test by inoculating a T-soy plate with a thin even film of bacteria. (Notice that the way I inoculate the plate is very different from the streak plate technique used to achieve isolated colonies.) In this case I want to grow a ‘lawn’ of bacteria. That means that after the plate has been incubated, I want to see thick growth of bacteria that covers the plate evenly, all the way to the edges. (I don’t want colonies!)Watch this video to see how I cover the plate with a thin even film of bacteria (that can grow into a lawn), and then how I apply my antimicrobial products. Once the plate has been inoculated, and the antimicrobial products have been placed, the plate is placed in the incubator for about 24 hours. The thin even film of bacteria will grow to form a lawn that covers the surface, except where the growth is inhibited by the products diffusing out of the disks.Exercise 19: Food Microbiology and the Pour Plate TechniqueIn this exercise, we’ll attempt to perform a viable count of the bacteria in a food sample. (ie. a standard plate count.) It will be very similar to the plate count we did in lab 2, except that this time our sample is a solid (not a liquid culture of bacteria) so we need to begin by homogenizing the sample in water.Food microbiologists have invented all kinds of different machines for freeing bacteria from food so that they can be counted. But I’m going to keep things simple and just homogenize my food in a blender to free the bacteria and resuspend them in liquid.Pour Plate Method for Enumerating Bacteria in a Food Sample Once the food sample has been homogenized, it’s time to perform our serial dilution. As you watch the following video, you should notice a few things:The dilution series is a bit more complicated than the series I used in lab period 2. In this dilution series I’ll be using different sized flasks, bottles and test tubes.First, the blended food sample was made by blending 20g of carrots into 180ml of water. This is itself a dilution, and represents the first dilution of the series. If you blend 20g of food into 180ml of water, how much have you diluted the food sample?Then I’ll be transferring 1 ml of the blended carrots into a 99ml water blank. If we transfer 1 ml of an already diluted food sample into 99ml of water, what will the new dilution be? (Hint: you need to multiply the dilutions together. So if the the food sample was a ____ dilution, and the 99ml water blank dilutes it a further 1/100, then _____ x 10-2 = ____)After that, I’ll start using the same 9ml water blanks you saw in Lab 2, so each new step will dilute the sample a further 1/10.The other thing you’ll see is different, is that I decided to use the P1000 mechanical pipette to perform my serial dilution. Pay attention carefully to see what I do differently when I’m doing a serial dilution with the mechanical pipette, as compared to a full sized plastic or glass pipette. (Hint: in Lab 2, you’ll remember that at each step of the serial dilution, I drew liquid into my pipette and flushed it back out several times to mix the contents of the tube, and to rinse out my pipette before continuing on with the serial dilution.) In this video, you’ll see that I am not flushing my pipette tip the same way. Watch carefully to see what I do instead.Instead of drawing liquid up and down in the pipette after each transfer, what do I do instead? Pouring platesOnce my dilution series is finished, I’m ready to pour plates to complete this pour plate technique. Watch this video to see how I make sure that the agar is the correct temperature for pouring plates, and how I perform the pour plate technique.Once my plates have solidified, they’re ready to be turned upside down and placed in the incubator. Why do we always incubate our plates upside down? (Check back to Lab 1 for a review, if necessary.)Before you see the pictures of how the plates turned out, you should ask yourself, what do we expect to see on these plates? Will we get colonies of bacteria? And how many? Why was the spinach sample diluted further than the other foods?
Lab 7: Results of Disk diffusions and food sample plate countsExercise 18: The Disk Diffusion TestIn Exercise 18, we set up a disk diffusion plate to test the effectiveness of four different anti-microbial products against the common skin bacterium Staphylococcus epidermidis.Examine the following plates:The first plate is one that was prepared by Dr. Chris. It shows four completely different antimicrobial products, each with a different active ingredient. You can use these results to fill in Table 18.1 on page 112 of your lab manual.You can use these results to fill in the “product”, “zone of inhibition”, and “effectiveness” columns in the table. More information about the products, including their active ingredients, can be found on the first day of this lab.Here are some additional pictures that were taken of plates made in the last lab of Winter 2020 These were made in the last in-class lab period before we shut down our labs and went home due to the COVID-19 pandemic. They show some of the antimicrobial products shown in the picture in the first part of this lab, as well as some of the non-antimicrobial products for comparison sake.Note that I did not try the ‘Crest Pro-health’ mouthwash shown on the first page. But I believe that it’s the one shown on the bottom left of these pictures. (The are real student plates, and students don’t always label their plates very well. So it’s tough to tell!) Now, answer the practice questions on page 113 of your lab manual.In addition to the questions in the lab manual, you might want to think about the following questions as you look at the plates above.Why did the Purell hand-sanitizer fare so poorly in this experiment? Is it because hand-sanitizer doesn’t work? Or could there be another reason? (Look at the description of alcohols on page 109 for a hint.)The disk dipped in “Softsoap” seems to have a yellow ring around it, inside the zone of clearing produced by the antimicrobial agent. What could this ring be? (Look at the description of QUATs on page 109 for a hint. Ewww! Gross! I think I’ll be throwing the Softsoap out as soon as I return to the lab!)The Colgate Total contains Triclosan, which seems to be the run-away winner of antibacterials (at least against S. epidermidis, the bacterium that we inoculated our disk diffusion plates with.) Why doesn’t Triclosan get used in hand soaps anymore? How do you feel about it’s continued use in toothpaste?Exercise 19: Food Microbiology and the Pour Plate TechniqueIn Exercise 19 we looked at our second standard plate count. This time from a solid food sample, rather than from a liquid culture of bacteria. The serial dilution was a little bit more complicated, which should give you some extra practice figuring out dilution questions. (These are always some of the worst answered questions on the lab quizzes, by the way.)The food sample dilutions were then plated using a pour plate technique. If the serial dilution and pour plate worked out, we’ll now have well isolated colonies that can be used to figure out how many bacteria were in the original food sample.Counting platesYou can use the results for the fresh carrots in the pictures and table below to fill in the results table on page 118 of the lab manual. Then use these numbers to calculate the average cfu/g in the sample of carrots.FRESH UNPEELED CARROTSThe above figure shows the four pour plates prepared from the blended carrots (shown in the video in the first part of this lab.) Each plate was made by transferring 1 ml of the corresponding dilution into an empty Petri dish, which was then covered with molten agar media (ie. the dilutions were plated using the pour plate technique). The plates were then incubated at 28°C for 2 days for colonies to form.OBTAIN A SECOND DATA SETBecause plate counts are inherently unreliable, it’s always better to have more than one replicate. In the lab, you would find someone else who sampled the exact same food sample that you did (and performed an identical serial dilution and plate count) to share data with.You can use the data in the following table to fill in the second part of the table: Replicate 2 (counts obtained from someone else at your work bench.)Food sample10-310-410-510-6CarrotsTNTC51126621
CALCULATE THE CONCENTRATION OF BACTERIA IN THE FOOD SAMPLENow use all of the data recorded in the table on page 118 to calculate the average cfu/g of bacteria in the sample of fresh unpeeled carrots.Make sure that you are using only significant plate counts as part of your calculation. (You can find the statistically significant range for plate counts on page 34 of the lab manual, if you’ve forgotten it.)To avoid introducing averaging error: Calculate cfu/g for each of the significant plate counts separately, and then average them together in one step at the end. (Never take an average of an average!)To avoid introducing rounding error, wait until you have a final answer before rounding. (ie. after you’ve taken the average of the cfu/g values)Don’t forget that we always round cfu counts to one decimal place (review page 35, if necessary.)Are you surprised by the final answer? Did you think that there would be so many bacteria in a raw food, like unpeeled carrots?
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