Microbiology, part 29: Growth - Biofilms & Quorum Sensing

Hi. I'm Cathy with Level Up RN. In this video, I will be discussing measurement of bacterial growth, including both direct and indirect methods of measuring growth. At the end of the video, I'm going to give you guys a little quiz to test your understanding of some of the key facts I'll be talking about, so definitely stay tuned for that. And if you have our Level Up RN microbiology flashcards, go ahead and pull out your flashcards so you can follow along with me. And of note, our flashcards are also available via Flashables, which offers personalized and guided learning as well as progress tracking.

Let's first talk about direct methods for measuring bacterial growth, which includes a direct cell count. With a direct cell count, we count the number of cells in a liquid culture or the number of colonies on a plate. And this, in turn, provides an estimate of how many organisms are present in the sample. When counting cells using a microscope, a sample of the culture is added to a calibrated slide, which is etched with squares of varying sizes. You would count the number of cells in a square and then divide that by the volume of the square. And that would give you the number of cells per milliliter. And then you need to perform the same process using multiple squares and take the average.

In addition to manually counting cells, there are also electronic cell-counting devices, such as a culture counter, which can provide a more rapid and accurate count. One important thing to remember is that a key downside to either of these methods is that you cannot differentiate between live and dead cells. Another direct method of measuring bacterial growth that does estimate the number of live cells in a sample is a plate count. With this method, colonies, which are formed when cells replicate, are counted on a medium. In theory, each viable cell can form a colony on an agar plate. But we know that some cells are grouped or arranged together as a unit, such as gram-positive cocci in pairs, chains, and clusters. So it is not always a single cell that forms a colony, but rather a unit of cells. So as a result, we express plate counts as colony-forming units per milliliter as opposed to cells per milliliter.

To use the plate count method, we would first use the pour plate method or spread plate method to prepare the plate with our sample. However, in many cases, if we inoculate the plate with an undiluted sample, then we're going to end up with a plate with too many colonies to count. Therefore, we need to dilute our sample to obtain a plate with a countable number, ideally between 30 and 300 colony-forming units before we inoculate the plate. So let's take a look at how serial dilution works using an illustration from our microbiology flashcard deck. As I mentioned before, our goal is to have a plate with 30 to 300 colony-forming units. With serial dilution, we are going to perform a number of dilutions in multiples of 10, which simplifies calculations.

So in this example, we first place 1 milliliter of the original sample in a test tube with 9 milliliters of sterile broth, and we mix that together. So in this tube, we have a dilution factor of 10, or 1 to 10, as compared to the original sample. Next, we take 1 milliliter from this tube and place it into the next tube with 9 milliliters of sterile broth. So in this tube, we have a dilution factor of 100, or 1 to 100. And then, as you can see in this example, we do this three more times. And a 1-milliliter sample is taken from each tube and is plated on solid media using the pour plate or spread plate method. So here at the bottom, you can see that the tube with the 1 to 10 dilution was not dilute enough. It produced so many colonies that we cannot count them. The same thing was the case for the tube with the 1 to 100 dilution as well as the tube with the 1 to 1,000 dilution. The fourth tube, however, produced 47 colony-forming units, which is between 30 and 300, which is perfect. So we are going to use this plate to estimate how many colony-forming units are in the original sample. To do the math, we are going to take the number of colony-forming units on this plate, which is 47, multiply that by the dilution factor of the tube, which is 10,000, and then multiply that by the volume of the sample that was inoculated on the plate, which is 1 milliliter. So if you multiply that out, we get 470,000 colony-forming units per milliliter.

Next, we're going to talk about membrane filtration, which is a method used to estimate bacterial growth in very dilute fluid samples, such as drinking water. With this method, a known volume of liquid is vacuum filtered through a membrane. This membrane is then transferred to a petri plate containing a medium. The plate is then incubated, and then the colonies on the plate are counted. So the cell density is calculated as the number of colony-forming units that were counted on the plate divided by the volume of the filtered liquid. The last direct method of measuring bacterial growth that we're going to talk about is the most probable number method, or MPN method. This is a statistical method used to estimate the concentration of bacteria in a sample. It is often used to determine the extent of fecal contamination in a water sample.

So with this method, different amounts of the sample are placed in nutrient broth tubes. These tubes are then incubated, and after incubation, they are examined for microbial growth, which can cause a change in turbidity, a change in color, and/or the presence of gas. Let's take a look at an example of an MPN test from our microbiology flashcard deck that shows how this test is performed. With this MPN test, we put 10 milliliters of the sample into each of the five tubes in this first set of lactose broth tubes. We then put 1 milliliter of the sample into each of the five tubes in the second set of lactose broth tubes. And then finally, we put 0.1 milliliters of the sample into each of the tubes in the last set of lactose broth tubes. All of the tubes are then incubated, and after incubation, they are examined for lactose fermentation, which would cause a change in color and the production of gas, as seen here in the Durham tube. Next, we would count up the number of tubes that exhibited these changes in each of the three sets of tubes.

So in this example, all five tubes in the first set change color and have gas present. In the second set of tubes, two tubes have a color change and the presence of gas. And in the third set of tubes, none of the tubes show a change in color or have gas present. Next, we take those numbers 5, 2, and 0, and we look them up in a special MPN table, which provides an estimate of the number of bacteria present in 100 milliliters of water. So if we look up 5, 2, and 0, we see that this equals approximately 49 bacteria present in 100 milliliters of water.

Now that we've talked about direct methods for measuring bacterial growth, let's now talk about indirect methods. One indirect method is to measure the turbidity or cloudiness of a liquid sample using a device called a spectrophotometer. With this device, light passes through a bacterial suspension into a detector. As the number of bacteria increases in the suspension, this causes an increase in the turbidity of the solution, which in turn causes less light to reach the detector. Another way of indirectly measuring bacterial growth is to measure the dry weight of a sample. With this method, the sample is concentrated, washed, and dried prior to weighing. This method is useful with microorganisms such as filamentous bacteria, which are long and thread-like. These bacteria can grow longer without an increase in the number of cells. So measuring the dry weight is the best way to determine their growth.

And lastly, we can also indirectly measure bacterial growth by monitoring the metabolic activity of the microorganisms in the sample. We do this by monitoring substances produced during bacterial growth or the disappearance of substances used by bacteria during growth. An example of this would be the methylene blue dye reduction test, which is used to determine the amount of bacterial contamination in milk. So this dye is blue in the presence of oxygen, but becomes decolorized when there is less oxygen. So during this test, the blue dye is mixed with milk in a test tube, sealed, and incubated. If there is bacterial growth, which requires oxygen, then oxygen is used up, which will make the dye become decolorized.

All right. It's quiz time, and I have four questions for you. Question number one, a key disadvantage to a direct microscopic count or an electronic cell count is the inability to differentiate between living and dead cells. True or false? The answer is true. Question number two, after serial dilution, 120 colonies are counted on a plate that was inoculated with 1 milliliter from a test tube with a dilution factor of 100. Approximately how many colony-forming units per milliliter are present in the original sample? All right. To calculate this, you take the number of colonies times the dilution factor times the volume of the sample that was inoculated onto the plate. So we have 120 times 100 times 1. So the answer is 12,000 colony-forming units per milliliter. Question number three, which of the following are indirect methods of measuring bacterial growth? Select all that apply. A, measurement of metabolic activity, B, plate count, C, membrane filtration technique, and D, dry weight measurement. The answer is A and D. Question number four, with the MPN method, what characteristics indicate the presence of bacterial growth in the nutrient broth tubes? The answer is a change in color, a change in turbidity, or the presence of gas in the tube.

All right. That's it for this video. I hope it was helpful. Thank you so much for watching, and take care.

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Now that we have talked about direct methods of measuring bacterial growth, let's now learn how to talk in general. The plate is incubated into a detector to reach.