When microbes cannot be completely eliminated from a material, such as food products that cannot be heated to high temperatures, measures can be taken to mitigate the growth of microbes. Recognizing how temperature impacts growth, supports the importance of refrigeration. As mentioned, cold temperatures slow the growth of microbes, so refrigeration can delay the growth of microbes in these food products.
As described above, microbes can replicate as quickly as every 20 minutes leading to visible growth within only a few hours. At a lower temperature, the cells may divide only once every few hours and it will take multiple days to see visible growth. Alternatively, when we want to take advantage of microbes, we try to optimize the conditions for their growth. This is why yeasted dough is left at a warm temperature to allow the yeast to grow rapidly.
If the dough is refrigerated, it takes much longer to rise. Similarly, to use E. Continuing to better understand microbial growth will help us live safely with the microbes in our community and make use of their unique capabilities.
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Notify me of follow-up comments by email. Notify me of new posts by email. Currently you have JavaScript disabled. In order to post comments, please make sure JavaScript and Cookies are enabled, and reload the page. Click here for instructions on how to enable JavaScript in your browser. Skip to content by Molly Sargen figures by Molly Sargen and Nicholas Lue Microbes also known as microorganisms are everywhere: on surfaces we touch, in the air we breathe, and even inside us.
Figure 1: Microscopy reveals the intricate features of microbes. It takes at X magnification to see these organisms clearly with a microscope. Image sources: S. Figure 2: Features of a Microbial Cell. This diagram of a bacterial cell shows the essential features of a microbial cell including DNA, a cell membrane, and the essential components within the cell.
This cell has a cell wall and also flagella an appendage some bacteria use for movement. Mechanisms of microbial growth Microbial growth refers to an increase in number of cells rather than an increase in cell size. Figure 3: The population increases exponentially as cells divide. Microbes with different shapes divide similarly. Figure 4: Some cells use budding to produce daughter cells. A parent cell produces small protrusions called buds.
Factors affecting microbial growth All types of microbial growth are heavily impacted by environmental conditions. Figure 5: Microbes grow well within a specific range of conditions for multiple environmental variables. Some microbes can tolerate a wide range of conditions, while others require a specific condition to grow well. Sometimes the conditions that permit growth overlap.
Homepage Blog listing How do your microbes grow? Previous post Next post. How do your microbes grow? The fraction of cells with a red dot tells you how many generations have passed.
Share this page:. However, newly developed fluorescence staining techniques make it possible to distinguish viable and dead bacteria. These viability stains or live stains bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane. The primary stain, which fluoresces green, can penetrate intact cytoplasmic membranes, staining both live and dead cells.
The secondary stain, which fluoresces red, can stain a cell only if the cytoplasmic membrane is considerably damaged. Thus, live cells fluoresce green because they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids Figure 8.
Another technique uses an electronic cell counting device Coulter counter to detect and count the changes in electrical resistance in a saline solution. A glass tube with a small opening is immersed in an electrolyte solution. A first electrode is suspended in the glass tube. A second electrode is located outside of the tube.
As cells are drawn through the small aperture in the glass tube, they briefly change the resistance measured between the two electrodes and the change is recorded by an electronic sensor Figure 9 ; each resistance change represents a cell. The method is rapid and accurate within a range of concentrations; however, if the culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results.
This method also does not differentiate between live and dead cells. Direct counts provide an estimate of the total number of cells in a sample. However, in many situations, it is important to know the number of live, or viable, cells. Counts of live cells are needed when assessing the extent of an infection, the effectiveness of antimicrobial compounds and medication, or contamination of food and water. Figure 9. A Coulter counter is an electronic device that counts cells.
It measures the change in resistance in an electrolyte solution that takes place when a cell passes through a small opening in the inside container wall.
A detector automatically counts the number of cells passing through the opening. The viable plate count, or simply plate count, is a count of viable or live cells. It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen. Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse and a single colony may represent several cells. Some cells are described as viable but nonculturable and will not form colonies on solid media.
For all these reasons, the viable plate count is considered a low estimate of the actual number of live cells. These limitations do not detract from the usefulness of the method, which provides estimates of live bacterial numbers. Microbiologists typically count plates with 30— colonies. Also, counts in this range minimize occurrences of more than one bacterial cell forming a single colony.
Thus, the calculated CFU is closer to the true number of live bacteria in the population. There are two common approaches to inoculating plates for viable counts: the pour plate and the spread plate methods. Although the final inoculation procedure differs between these two methods, they both start with a serial dilution of the culture. The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method.
The goal of the serial dilution process is to obtain plates with CFUs in the range of 30—, and the process usually involves several dilutions in multiples of 10 to simplify calculation. The number of serial dilutions is chosen according to a preliminary estimate of the culture density.
Figure 10 illustrates the serial dilution method. Figure Serial dilution involves diluting a fixed volume of cells mixed with dilution solution using the previous dilution as an inoculum. The result is dilution of the original culture by an exponentially growing factor. A fixed volume of the original culture, 1. This step represents a dilution factor of 10, or , compared with the original culture.
From this first dilution, the same volume, 1. The dilution factor is now compared with the original culture. This process continues until a series of dilutions is produced that will bracket the desired cell concentration for accurate counting. From each tube, a sample is plated on solid medium using either the pour plate method Figure 11 or the spread plate method Figure The plates are incubated until colonies appear.
Two to three plates are usually prepared from each dilution and the numbers of colonies counted on each plate are averaged. In all cases, thorough mixing of samples with the dilution medium to ensure the cell distribution in the tube is random is paramount to obtaining reliable results.
This process is repeated for each serial dilution prepared. The resulting colonies are counted and provide an estimate of the number of cells in the original volume sampled. In the spread plate method of cell counting, the sample is poured onto solid agar and then spread using a sterile spreader. The resulting colonies are counted and provide an estimate of the number of cells in the original volume samples. The dilution factor is used to calculate the number of cells in the original cell culture.
In our example, an average of 50 colonies was counted on the plates obtained from the , dilution. Because only 0. The colony count obtained from the dilution was , well below the expected for a fold difference in dilutions.
This highlights the issue of inaccuracy when colony counts are greater than and more than one bacterial cell grows into a single colony. A very dilute sample—drinking water, for example—may not contain enough organisms to use either of the plate count methods described. In such cases, the original sample must be concentrated rather than diluted before plating.
This can be accomplished using a modification of the plate count technique called the membrane filtration technique. Known volumes are vacuum-filtered aseptically through a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a Petri plate containing an appropriate growth medium.
Colonies are counted after incubation. Calculation of the cell density is made by dividing the cell count by the volume of filtered liquid. The number of microorganisms in dilute samples is usually too low to be detected by the plate count methods described thus far.
For these specimens, microbiologists routinely use the most probable number MPN method, a statistical procedure for estimating of the number of viable microorganisms in a sample. Often used for water and food samples, the MPN method evaluates detectable growth by observing changes in turbidity or color due to metabolic activity.
A typical application of MPN method is the estimation of the number of coliforms in a sample of pond water. Coliforms are gram-negative rod bacteria that ferment lactose. The presence of coliforms in water is considered a sign of contamination by fecal matter. For the method illustrated in Figure 13, a series of three dilutions of the water sample is tested by inoculating five lactose broth tubes with 10 mL of sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.
The lactose broth tubes contain a pH indicator that changes color from red to yellow when the lactose is fermented. After inoculation and incubation, the tubes are examined for an indication of coliform growth by a color change in media from red to yellow. The first set of tubes mL sample showed growth in all the tubes; the second set of tubes 1 mL showed growth in two tubes out of five; in the third set of tubes, no growth is observed in any of the tubes 0.
The numbers 5, 2, and 0 are compared with Figure 1 in Mathematical Basics , which has been constructed using a probability model of the sampling procedure. From our reading of the table, we conclude that 49 is the most probable number of bacteria per mL of pond water. In the most probable number method, sets of five lactose broth tubes are inoculated with three different volumes of pond water: 10 mL, 1 mL, and 0.
Bacterial growth is assessed through a change in the color of the broth from red to yellow as lactose is fermented. Besides direct methods of counting cells, other methods, based on an indirect detection of cell density, are commonly used to estimate and compare cell densities in a culture.
The foremost approach is to measure the turbidity cloudiness of a sample of bacteria in a liquid suspension. The laboratory instrument used to measure turbidity is called a spectrophotometer Figure In a spectrophotometer, a light beam is transmitted through a bacterial suspension, the light passing through the suspension is measured by a detector, and the amount of light passing through the sample and reaching the detector is converted to either percent transmission or a logarithmic value called absorbance optical density.
As the numbers of bacteria in a suspension increase, the turbidity also increases and causes less light to reach the detector. The decrease in light passing through the sample and reaching the detector is associated with a decrease in percent transmission and increase in absorbance measured by the spectrophotometer.
Measuring turbidity is a fast method to estimate cell density as long as there are enough cells in a sample to produce turbidity. It is possible to correlate turbidity readings to the actual number of cells by performing a viable plate count of samples taken from cultures having a range of absorbance values. Using these values, a calibration curve is generated by plotting turbidity as a function of cell density. Once the calibration curve has been produced, it can be used to estimate cell counts for all samples obtained or cultured under similar conditions and with densities within the range of values used to construct the curve.
The spectrophotometer allows choice of the wavelength of light to use for the measurement. The optical density turbidity of the sample will depend on the wavelength, so once one wavelength is chosen, it must be used consistently.
The filtered light passes through the sample or a control with only medium and the light intensity is measured by a detector.
The light passing into a suspension of bacteria is scattered by the cells in such a way that some fraction of it never reaches the detector. This scattering happens to a far lesser degree in the control tube with only the medium. Measuring dry weight of a culture sample is another indirect method of evaluating culture density without directly measuring cell counts.
The cell suspension used for weighing must be concentrated by filtration or centrifugation, washed, and then dried before the measurements are taken. The degree of drying must be standardized to account for residual water content. This method is especially useful for filamentous microorganisms, which are difficult to enumerate by direct or viable plate count.
As we have seen, methods to estimate viable cell numbers can be labor intensive and take time because cells must be grown. I miss a more thorough walk-through of ho An excellent introduction to mircrobiology w to classify microbes, but I guess that would require a more advanced course. All in all, a very good course.
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Build your knowledge with top universities and organisations. Learn more about how FutureLearn is transforming access to education. Learn more about this course. How Do Microbes Grow and Replicate? Microbes have the ability to replicate rapidly. Find out how in this article. Microbiology small and mighty 14 Sep, Really loved this course and found it really fascinating. Visit the course. Yes the level was a 16 May, Yes the level was a right and I really love it.
So interesting 31 Oct,
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