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Beginner question about growing E. coli

Beginner question about growing E. coli



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I have bought a hobbyist kit which involves growing E. coli. The steps said to grow the E. coli on an LB agar Petri dish overnight. No incubation devices were included in the kit.

I let the E. coli grow overnight at room temperature (65-75°F), but there's pretty minimal growth, if any. I found this question which seems to imply that it's hard to grow E. coli without having temperatures near 37°C, which is much warmer than I can reasonably make my house.

Is it possible for me to do this experiment without purchasing an incubator of some sort? If so, does the lack of growth indicate that I've made a mistake somewhere?


The higher the temperature the faster E. coli will grow, with a maximum growth rate around 37 Celsius.

If you grow E coli at lower temperature, the ecoli will grow slower.

At 37°C you will see colonies overnight (16hr).

At 30°C you will see colonies in about 1 and a half days.

At 25°C you will see colonies in about 3-4 days.

You can do experiments at room temperature, but you will be waiting for days for what you will normally see after a 37°C overnight incubation.

As for satellite colonies… yes ampicilin does break down with time and water. You can solve that problem by increasing the amount of Amp you are using from 25ug/ml to 100ug/ml. Or use 75ug/ml Carbenicilin. Or change your selection markers and use a more stable antibiotic like Chloroamphenicol.


Question : 9. In a molecular biology laboratory, a student obtained competent E. coli cells and used a common transformation procedure to induce the uptake of plasmid DNA with a gene for resistance to the antibiotic kanamycin. The results below were obtained. On which petri dish do only transformed cells grow? a. Plate I b. Plate II c. Plate III d. Plate IV 10. In a

9. In a molecular biology laboratory, a student obtained competent E. coli cells and used a common transformation procedure to induce the uptake of plasmid DNA with a gene for resistance to the antibiotic kanamycin. The results below were obtained.

On which petri dish do only transformed cells grow?

10. In a molecular biology laboratory, a student obtained competent E. coli cells and used a common transformation procedure to induce the uptake of plasmid DNA with a gene for resistance to the antibiotic kanamycin. The results below were obtained.

Which of the plates is used as a control to show that nontransformed E. coli will not grow in the presence of kanamycin?


9.1 How Microbes Grow

Jeni, a 24-year-old pregnant woman in her second trimester, visits a clinic with complaints of high fever, 38.9 °C (102 °F), fatigue, and muscle aches—typical flu-like signs and symptoms. Jeni exercises regularly and follows a nutritious diet with emphasis on organic foods, including raw milk that she purchases from a local farmer’s market. All of her immunizations are up to date. However, the health-care provider who sees Jeni is concerned and orders a blood sample to be sent for testing by the microbiology laboratory.

Jump to the next Clinical Focus box

The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as will be explored in a different chapter. Most bacteria have a single circular chromosome however, some exceptions exist. For example, Borrelia burgdorferi , the causative agent of Lyme disease, has a linear chromosome.

Binary Fission

The most common mechanism of cell replication in bacteria is a process called binary fission , which is depicted in Figure 9.2. Before dividing, the cell grows and increases its number of cellular components. Next, the replication of DNA starts at a location on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane. Replication continues in opposite directions along the chromosome until the terminus is reached.

The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis). This process of cytokinesis and cell division is directed by a protein called FtsZ . FtsZ assembles into a Z ring on the cytoplasmic membrane (Figure 9.3). The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells. Additional proteins required for cell division are added to the Z ring to form a structure called the divisome . The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. The daughter cells are separated by the division septum, where all of the cells’ outer layers (the cell wall and outer membranes, if present) must be remodeled to complete division. For example, we know that specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.

Check Your Understanding

  • What is the name of the protein that assembles into a Z ring to initiate cytokinesis and cell division?

Generation Time

In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments. Most pathogens grow rapidly, like E. coli, but there are exceptions. For example, Mycobacterium tuberculosis , the causative agent of tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s disease (leprosy), grows much more slowly, with a doubling time of 14 days.

Micro Connections

Calculating Number of Cells

It is possible to predict the number of cells in a population when they divide by binary fission at a constant rate. As an example, consider what happens if a single cell divides every 30 minutes for 24 hours. The diagram in Figure 9.4 shows the increase in cell numbers for the first three generations.

The number of cells increases exponentially and can be expressed as 2 n , where n is the number of generations. If cells divide every 30 minutes, after 24 hours, 48 divisions would have taken place. If we apply the formula 2 n , where n is equal to 48, the single cell would give rise to 2 48 or 281,474,976,710,656 cells at 48 generations (24 hours). When dealing with such huge numbers, it is more practical to use scientific notation. Therefore, we express the number of cells as 2.8 × 10 14 cells.

In our example, we used one cell as the initial number of cells. For any number of starting cells, the formula is adapted as follows:

Nn is the number of cells at any generation n, N0 is the initial number of cells, and n is the number of generations.

Check Your Understanding

  • With a doubling time of 30 minutes and a starting population size of 1 × 10 5 cells, how many cells will be present after 2 hours, assuming no cell death?

The Growth Curve

Microorganisms grown in closed culture (also known as a batch culture ), in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve . An example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment. The culture density is defined as the number of cells per unit volume. In a closed environment, the culture density is also a measure of the number of cells in the population. Infections of the body do not always follow the growth curve, but correlations can exist depending upon the site and type of infection. When the number of live cells is plotted against time, distinct phases can be observed in the curve (Figure 9.5).

The Lag Phase

The beginning of the growth curve represents a small number of cells, referred to as an inoculum , that are added to a fresh culture medium , a nutritional broth that supports growth. The initial phase of the growth curve is called the lag phase , during which cells are gearing up for the next phase of growth. The number of cells does not change during the lag phase however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium. If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase. The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum.

The Log Phase

In the logarithmic (log) growth phase , sometimes called exponential growth phase , the cells are actively dividing by binary fission and their number increases exponentially. For any given bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate . During the log phase, the relationship between time and number of cells is not linear but exponential however, the growth curve is often plotted on a semilogarithmic graph, as shown in Figure 9.6, which gives the appearance of a linear relationship.

Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.

Stationary Phase

As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. Waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth. The total number of live cells reaches a plateau referred to as the stationary phase (Figure 9.5). In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying thus, the total population of living cells is relatively stagnant. The culture density in a stationary culture is constant. The culture’s carrying capacity, or maximum culture density, depends on the types of microorganisms in the culture and the specific conditions of the culture however, carrying capacity is constant for a given organism grown under the same conditions.

During the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids thus, stationary cultures are less susceptible to antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. Secondary metabolites, including antibiotics, are synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.

The Death Phase

As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells (Figure 9.5). This is the aptly named death phase , sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. A few cells, the so-called persisters , are characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.

Sustaining Microbial Growth

The growth pattern shown in Figure 9.5 takes place in a closed environment nutrients are not added and waste and dead cells are not removed. In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in industries that harvest microbial products. A chemostat (Figure 9.7) is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.

Check Your Understanding

  • During which phase does growth occur at the fastest rate?
  • Name two factors that limit microbial growth.

Measurement of Bacterial Growth

Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists. The number of bacteria in a clinical sample serves as an indication of the extent of an infection. Quality control of drinking water, food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of disease. Two major approaches are used to measure cell number. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells. Both direct and indirect methods have advantages and disadvantages for specific applications.

Direct Cell Count

Direct cell count refers to counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample. Let’s look first at a simple and fast method that requires only a specialized slide and a compound microscope.

The simplest way to count bacteria is called the direct microscopic cell count , which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope. The calibrated slide is called a Petroff-Hausser chamber (Figure 9.8) and is similar to a hemocytometer used to count red blood cells. The central area of the counting chamber is etched into squares of various sizes. A sample of the culture suspension is added to the chamber under a coverslip that is placed at a specific height from the surface of the grid. It is possible to estimate the concentration of cells in the original sample by counting individual cells in a number of squares and determining the volume of the sample observed. The area of the squares and the height at which the coverslip is positioned are specified for the chamber. The concentration must be corrected for dilution if the sample was diluted before enumeration.

Cells in several small squares must be counted and the average taken to obtain a reliable measurement. The advantages of the chamber are that the method is easy to use, relatively fast, and inexpensive. On the downside, the counting chamber does not work well with dilute cultures because there may not be enough cells to count.

Using a counting chamber does not necessarily yield an accurate count of the number of live cells because it is not always possible to distinguish between live cells, dead cells, and debris of the same size under the microscope. 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 9.9).

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 9.10) 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.

Check Your Understanding

  • Why would you count the number of cells in more than one square in the Petroff-Hausser chamber to estimate cell numbers?
  • In the viability staining method, why do dead cells appear red?

Plate Count

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. The results are usually expressed as colony-forming unit s per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the same spot to give rise to a single colony. 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 30–300 colonies. Samples with too few colonies (<30) do not give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual 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.

Serial Dilution

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 30–300, 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 9.11 illustrates the serial dilution method.

A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the first dilution tube solution, which contains 9.0 mL of sterile broth. This step represents a dilution factor of 10, or 1:10, compared with the original culture. From this first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution. The dilution factor is now 1:100 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 9.12) or the spread plate method (Figure 9.13). 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.

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 1:10,000 dilution. Because only 0.1 mL of suspension was pipetted on the plate, the multiplier required to reconstitute the original concentration is 10 × 10,000. The number of CFU per mL is equal to 50 × 10 × 10,000 = 5,000,000. The number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the 1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights the issue of inaccuracy when colony counts are greater than 300 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.

Link to Learning

Watch this video for demonstrations of serial dilutions and spread plate techniques.

The Most Probable Number

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 9.14, 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 0.1 mL of sample. 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 (10-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 (0.1-mL dilution). The numbers 5, 2, and 0 are compared with Figure B1 in Appendix B, 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 100 mL of pond water.no lo

Check Your Understanding

  • What is a colony-forming unit?
  • What two methods are frequently used to estimate bacterial numbers in water samples?

Indirect Cell Counts

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 9.15). 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.

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. Recently, indirect ways of measuring live cells have been developed that are both fast and easy to implement. These methods measure cell activity by following the production of metabolic products or disappearance of reactants. Adenosine triphosphate (ATP) formation, biosynthesis of proteins and nucleic acids, and consumption of oxygen can all be monitored to estimate the number of cells.

Check Your Understanding

  • What is the purpose of a calibration curve when estimating cell count from turbidity measurements?
  • What are the newer indirect methods of counting live cells?

Alternative Patterns of Cell Division

Binary fission is the most common pattern of cell division in prokaryotes, but it is not the only one. Other mechanisms usually involve asymmetrical division (as in budding) or production of spores in aerial filaments.

In some cyanobacteria , many nucleoids may accumulate in an enlarged round cell or along a filament, leading to the generation of many new cells at once. The new cells often split from the parent filament and float away in a process called fragmentation (Figure 9.16). Fragmentation is commonly observed in the Actinomycetes , a group of gram-positive, anaerobic bacteria commonly found in soil. Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the giant bacterium Epulopiscium . Several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells to the environment. Other species may form a long narrow extension at one pole in a process called budding . The tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast (Figure 9.16), but it is also observed in prosthecate bacteria and some cyanobacteria.

The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in fungi, resulting in long cells with multiple nucleoids. Environmental signals, probably related to low nutrient availability, lead to the formation of aerial filaments. Within these aerial filaments , elongated cells divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give rise to new colonies.

Check Your Understanding

Biofilms

In nature, microorganisms grow mainly in biofilms , complex and dynamic ecosystems that form on a variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds. Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a biofilm. Microbial mats that float on water, for example, are biofilms that contain large populations of photosynthetic microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species. Regardless of the environment where they occur, biofilms are not random collections of microorganisms rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.

Biofilm Structure

Observations using confocal microscopy have shown that environmental conditions influence the overall structure of biofilms. Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-moving fluids. The streamers are anchored to the substrate by a “head” and the “tail” floats downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like shape. The structure of biofilms may also change with other environmental conditions such as nutrient availability.

Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal clusters of microorganisms embedded in a matrix interspersed with open water channels. The extracellular matrix consists of extracellular polymeric substances (EPS) secreted by the organisms in the biofilm. The extracellular matrix represents a large fraction of the biofilm, accounting for 50%–90% of the total dry mass. The properties of the EPS vary according to the resident organisms and environmental conditions.

EPS is a hydrated gel composed primarily of polysaccharides and containing other macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of the biofilm. Channels in the EPS allow movement of nutrients, waste, and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).

Biofilm Formation

Free-floating microbial cells that live in an aquatic environment are called planktonic cells. The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate, where they become sessile (attached to a surface). This occurs in stages, as depicted in Figure 9.17. The first stage involves the attachment of planktonic cells to a surface coated with a conditioning film of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including an extensive matrix and water channels. Appendages such as fimbriae , pili , and flagella interact with the EPS, and microscopy and genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites. This stage is referred to as dispersal .

Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that promote the growth of anaerobes. This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens.

The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum sensing . Quorum sensing—which can occur between cells of different species within a biofilm—enables microorganisms to detect their cell density through the release and binding of small, diffusible molecules called autoinducers . When the cell population reaches a critical threshold (a quorum), these autoinducers initiate a cascade of reactions that activate genes associated with cellular functions that are beneficial only when the population reaches a critical density. For example, in some pathogens, synthesis of virulence factors only begins when enough cells are present to overwhelm the immune defenses of the host. Although mostly studied in bacterial populations, quorum sensing takes place between bacteria and eukaryotes and between eukaryotic cells such as the fungus Candida albicans , a common member of the human microbiota that can cause infections in immunocompromised individuals.

The signaling molecules in quorum sensing belong to two major classes. Gram-negative bacteria communicate mainly using N-acylated homoserine lactones, whereas gram-positive bacteria mostly use small peptides (Figure 9.18). In all cases, the first step in quorum sensing consists of the binding of the autoinducer to its specific receptor only when a threshold concentration of signaling molecules is reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in gene expression. The result is the activation of biological responses linked to quorum sensing, notably an increase in the production of signaling molecules themselves, hence the term autoinducer.

Biofilms and Human Health

The human body harbors many types of biofilms, some beneficial and some harmful. For example, the layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off infections by pathogens. However, other biofilms in the body can have a detrimental effect on health. For example, the plaque that forms on teeth is a biofilm that can contribute to dental and periodontal disease. Biofilms can also form in wounds, sometimes causing serious infections that can spread. The bacterium Pseudomonas aeruginosa often colonizes biofilms in the airways of patients with cystic fibrosis , causing chronic and sometimes fatal infections of the lungs. Biofilms can also form on medical devices used in or on the body, causing infections in patients with in-dwelling catheters , artificial joints, or contact lenses .

Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating counterparts. Several hypotheses have been proposed to explain why. Cells in the deep layers of a biofilm are metabolically inactive and may be less susceptible to the action of antibiotics that disrupt metabolic activities. The EPS may also slow the diffusion of antibiotics and antiseptics, preventing them from reaching cells in the deeper layers of the biofilm. Phenotypic changes may also contribute to the increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of efflux pumps , membrane-embedded proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an important mechanism of antibiotic resistance among biofilm-associated bacteria. Finally, biofilms provide an ideal environment for the exchange of extrachromosomal DNA , which often includes genes that confer antibiotic resistance.


Questions & Answers: Sickness caused by E. coli

E. coli is a common kind of bacteria that lives in the intestines of animals and people. There are many strains of E. coli. Most are harmless. However, one dangerous strain is called E. coli O157:H7. It produces a powerful poison. You can become very sick if it gets into your food or water.

In 1999 it was estimated that about 73,000 people in the U.S. got sick each year from E. coli. About 60 died. It&rsquos believed that the number of illnesses and deaths has been dropping since then.

How is E. coli O157:H7 spread?

Outbreaks often are caused by food that has gotten the bacteria, E coli, in it. Bacteria can get accidentally mixed into ground beef before packaging. Eating undercooked meat can spread the bacteria, even though the meat looks and smells normal. E. coli can also live on cows&rsquo udders. It may get into milk that is not pasteurized.

Raw vegetables, sprouts, and fruits that have been grown or washed in dirty water can carry E. coli O157:H7. It can get into drinking water, lakes, or swimming pools that have sewage in them. It is also spread by people who have not washed their hands after going to the toilet.

E. coli can be spread to playmates by toddlers who are not toilet trained or by adults who do not wash their hands carefully after changing diapers. Children can pass the bacteria in their stool to another person for 2 weeks after they have gotten well from an E. coli O157:H7 illness. Older children and adults rarely carry the bacteria without symptoms.

What are the signs of E. coli O157:H7 sickness?

Bloody diarrhea and stomach pain are the most common signs of E. coli O157:H7 sickness. People usually do not have a fever, or may have only a slight fever.

Some people, especially children under 5 and the elderly, can become very sick from E. coli O157:H7. The infection damages their red blood cells and their kidneys. This only happens to about 1 out of 50 people, but it is very serious. Without hospital care, they can die. See a doctor right away if you think you may have gotten sick from E. coli O157:H7.

How will my doctor know if E. coli O157:H7 made me sick?

Your doctor will test to see if your sickness was caused by E. coli by sending a stool sample to a lab. The lab will test for the bacteria.

Anyone who suddenly has diarrhea with blood in it should call or see a doctor.

How is it treated?

Your doctor will tell you what is best. Taking medicine on your own may not help you get better, and it could make things worse. Do not take antibiotics or diarrhea medicine like Imodium® unless your doctor tells you to.

Will E. coli O157:H7 infection cause problems for me later?

People who have only diarrhea and stomach ache usually get completely well in 5-10 days. They do not have problems later.

For those people who get very sick and have kidney failure, about 1 out of 3 may have kidney problems later. In rare cases, people have other problems like high blood pressure, blindness, or are paralyzed. Talk to your doctor if you have questions about this.

What is the U.S. government doing to keep food safe from E. coli O157:H7?

New laws have helped keep food from being contaminated with E. coli O157:H7. They keep meat safer during slaughter and grinding, and vegetables safer when they are grown, picked, and washed. But there is still a chance that E. coli O157:H7 could reach your food, so you should take the precautions listed below.

What can I do to stay safe from E. coli O157:H7?

  • During an outbreak: Carefully follow instructions provided by public health officials on what foods to avoid in order to protect yourself and your family from infection.
  • Cook all ground beef thoroughly. During an outbreak of E. coli O157:H7, vegetables should be boiled for at least 1 minute before serving.
  • Cook ground beef to 160° F Test the meat by putting a food thermometer in the thickest part of the meat. Do not eat ground beef that is still pink in the middle.
  • If a restaurant serves you an under-cooked hamburger, send it back for more cooking. Ask for a new bun and a clean plate, too.
  • Don&rsquot spread bacteria in your kitchen. Keep raw meat away from other foods. Wash your hands, cutting board, counter, dishes, and knives and forks with hot soapy water after they touch raw meat, spinach, greens, or sprouts.
  • Never put cooked hamburgers or meat on the plate they were on before cooking. Wash the meat thermometer after use.
  • Drink only pasteurized milk, juice, or cider. Frozen juice or juice sold in boxes and glass jars at room temperature has been pasteurized, although it may not say so on the label.
  • Drink water from safe sources like municipal water that has been treated with chlorine, wells that have been tested or bottled water.
  • Do not swallow lake or pool water while you are swimming.

Page last modified December 10, 2006
Content source: CDC Clear and Cultural Communications


2.4: Review Questions

  • Contributed by Joan Petersen & Susan McLaughlin
  • Associate Professors (Biological Sciences and Geology) at Queensborough Community College

1. Based on your results and the information in the lab manual, fill in the table below.

Predicted Gram Reaction Ability to ferment lactose/sucrose Ability to ferment mannitol Coliform or Non-coliform
S. aureus
E. coli
P. aeruginosa
M. luteus

2. What general type of growth medium would you use to:

(a) grow one type of bacteria but inhibit the growth of another type?

(b) discriminate between different types of bacteria?

3. Why is it necessary to sterilize the loop between streaks when streaking for single colonies?

4. Define and/or explain the use of the following:

5. A bacterial species is inoculated on EMB agar.

(a) The bacteria do not grow. Why?

(b) If the bacteria ferment lactose, what would you expect to see?

(c) The bacteria produce clear colonies. Why?

6. What medium would you use (TSA, EMB, MS) if you wanted to determine if a Staphylococcus isolate could ferment mannitol? Describe what you would see on this medium.

7. If you were testing water for the presence of fecal coliforms, what sort of medium would you use: TSA, EMB agar or MS agar? If fecal coliforms were present, what would their growth characteristics be on this medium?


[ If E. coli was allowed to grow for 80 minutes then what would be the proportions of light and hybrid densities DNA molecule?) * Very similar exneriments invobing use of rodinnotive thumiding to

Which of the following statements is incorrect regarding the structure of a typical bacterial cell? (a) Cells possess naked circular DNA which is folded to form nucleoid. (b) Cells are surrounded by a peptidoglycan cell wall and a mucilaginous sheath (c) Cells possess well developed membrane bound cell organelles (d) Ribosomes in these cells are 705 in nature.

How many of the given statements are incorrect? A. Nucleolus and nuclear membrane disappear in prophase. B. Synaptonemal complex formation occurs during zygotene. C. The enzyme responsible for crossing over in pachytene is recombinase. D. Chiasmata formation occurs in diakinesis.

8. कॉलमों के बीच मिलान कीजिए और सही विकल्प चुनिए- DNA की कालम-I कालम-II A. थायलेकॉइड (i) गॉल्जी उपकरण में डिस्कनुमा कोष B. क्रिस्टी (ii) संघनित संरचना C. सिस्टी (iii) स्ट्रोमा में चपटे झिल्लीमय को D. क्रोमैटिन (iv) माइटोकॉन्ड्रिया में अंतर्वलन A B C D (a) (iii) (iv) (1) (i (b) (iii) (i) (iv) (iv) (ii c) (ii) (iv) (ii) (ii) (1) (2015) Civi


Protocol for S30-ribosome-lysate

(adapted from Kigawa et al. (2004), Preparation of Escherichia coli cell extract for highly productive cell-free protein expression, J. Struct. Fun. Gen., 5, 63-68)

E. coli strain: BL21(DE3), BL21(DE3) CodonPlus RIL or variants (not pLysS!)

! around 7 ml of S30-ribosome-lysate can be isolated from 1 litre of culture !

Per litre medium:

5.6 g KH2PO4
28.9 g K2HPO4
10 g yeast extract
15 mg Thiamine add after sterilisation (filtered sterile)
40 ml 25% Glucose add after sterilisation (filtered sterile)

All buffers and stock solutions should be prepared with diethylpyrocarbonate (DEPC)-treated H2O (DNAse/RNAse-free).
All stock-solution can be stored @ -20°C for weeks except creatine kinase. Creatine kinase has to be dissolved in 30 mM Glycin, pH = 9.0 + 20 mM DTT. After freezing in liquid nitrogen, it is stable @ -80°C for weeks.
All individual amino acids have to be dissolved as described on package. The mixture contains 5 mM of each amino acid with a pH of 7.5 adjusted with KOH.


Pre-lab Activity: Think Pair Share - Discuss What You Already Know

Prior to the lab period you should read the material, take some notes on your thoughts on the following questions (Think). In lab, you will be asked to discuss with your partner your thoughts and hear theirs (Pair). Take additional notes on new insights during this small discussion. Finally, you may be asked to discuss your ideas with the class (Share). It is not expected that you know all the answers before the class discussion but by the end of the activity, you should have the questions answered completely and correctly.

  1. What is the term for bacterial reproduction? Describe this process.
  2. What roles do proteins play within cells?
  3. What happens to protein function if a protein loses, or never correctly achieves, the prescribed conformation?
  4. How does the order of amino acids relate to protein conformation and thus protein function?
  5. Since we can control when to &ldquoturn on&rdquo or express our gene, when is the best time to do so? To help you decide ask yourself:
    1. Would producing protein take energy?
    2. Would a greater number of cells produce more of your protein of interest than fewer cells?
    3. Can proteins degrade if left too long?

    Protein Purification

    Transformed bacteria can multiply in culture and produce the protein of interest. If this protein is to be used therapeutically, it will need to be purified. This means that other cellular components, including other proteins, must be separated from your protein. Column chromatography is a common method to separate proteins.

    Proteins are made of amino acids. Individual amino acids have different properties such as hydrophobicity (water-hating) or hydrophilicity (liking water), ionic charge, or the ability to form weak or strong bonds with other amino acids. When a protein is first made in a cell, it is a long chain of amino acids in an order determined by the gene. The order of the amino acids in a protein determines how the chain will fold to produce a three dimensional protein conformation (See Figure 2). This specific conformation will have different amino acids interacting with each other in specific ways. Amino acids facing the environment in a folded protein can interact with other molecules. Specific groups of amino acids near each other can form binding sites to interact with other specific molecules. Overall, these relationships determine protein structure and thus protein function. Imagine proteins involved in enzymatic reactions, as channels in membranes, in transporting other molecules, or for binding DNA. These proteins all have very specific binding interactions determined by amino acids in specific locations in a folded protein.

    Figure 2. The folding of a protein

    QUESTION: If individual amino acids are swapped or deleted in an amino acid chain, do you imagine this would affect the function of a protein?

    The rules for protein folding are not perfectly understood and is an area of active scientific investigation. However, a few basic rules have been discovered. Factors that cause proteins to fold in specific ways include:

    1. Weak bonds will form between amino acids with a negative and a positive charge.
    2. Strong (covalent) bonds will form between sulfur-containing amino acids. These are called disulfide bridges.
    3. Hydrophilic amino acids locate to the outer surfaces of proteins because they interact with the cell environment, which is mostly water. Hydrophobic amino acids hide on the inside of proteins or embed within cell membranes to avoid contact with the water in the environment.

    Depending on the content of amino acids in a specific protein, overall it will take on a hydrophobic or hydrophilic character. Column chromatography can separate hydrophilic and hydrophobic proteins from the rest of the cell contents. Small beads coated with a material called a resin are packed into a column. The resin attracts proteins which will bind to the resin as other cell contents flow past. For hydrophobic proteins to stick, they must be treated to expose the typically buried hydrophobic amino acids. Buffer solutions can be used to cause proteins to denature (unfold) and expose the amino acids that will be attracted to the resin.

    Different buffers are passed over the column in an order determined to best separate the proteins of interest from the rest of the cell contents. Figure 14.4 shows three solutions used to separate green fluorescent protein from the rest of the cell. The binding buffer denatures proteins so that the hydrophobic amino acids stick to the resin. The wash buffer removes loosely adherent proteins and material from the column leaving the more strongly attached protein of interest. Finally, the elution buffer, which has a low buffer concentration, causes the protein to begin to refold to hide the hydrophobic amino acids which releases the protein from the resin coated beads in the column. The portion or fraction of fluid exiting the column that contains your protein can be captured in a container and saved.

    Figure 4. Separation of green fluorescent protein by hydrophobicity using column chromatography

    QUESTION: Do you believe that all types of protein would use the same types of resin-coated beads and the same types of buffers to become purified? Explain your answer.


    E. coli and Food Safety

    E. coli are bacteria found in the intestines of people and animals and in the environment they can also be found in food and untreated water.

    Most E. coli are harmless and are part of a healthy intestinal tract. However, some cause illnesses that are sometimes severe, such as diarrhea, urinary tract infections, respiratory illness, and bloodstream infections. The types of E. coli that cause diarrheal illness are spread through contaminated food or water and through contact with animals or people.

    Who is more likely to get an E. coli infection?

    Anyone can get sick from E. coli, but some people have an increased chance of infection. These people are:

    • Adults aged 65 and older
    • Children younger than 5 years of age
    • People with weakened immune systems, including pregnant women
    • People who travel to certain countries

    What are the symptoms of E. coli infections?

    Most people have diarrhea, which can be bloody, and most have stomach cramps that may be severe. Some also have vomiting. A high fever is uncommon. Symptoms usually last 5&ndash7 days.

    About 5&ndash10% of people diagnosed with a type of E. coli called Shiga toxin-producing E. coli O157 develop hemolytic uremic syndrome (HUS)&mdasha type of kidney failure that can be life-threatening.

    Contact your healthcare provider if you have diarrhea or vomiting that lasts for more than 2 days, bloody stools, a fever higher than 102°F, or signs of dehydration (including little or no urination, excessive thirst, a very dry mouth, dizziness or lightheadedness, or very dark urine).

    How can I prevent E. coli infection?

      thoroughly with soap and running water.
  6. Follow the four steps to food safety when preparing food: clean, separate, cook, and chill.
  7. Use a food thermometer to make sure meat has reached a safe minimum cooking temperature external icon :
    • Cook ground beef, pork, and lamb to an internal temperature of at least 160°F (70°C). The best way to check the temperature of patties is to insert the thermometer from the side until it reaches the center.
    • Cook steaks and roasts of beef to an internal temperature of at least 145°F (62.6°C) and allow the meat to rest for 3 minutes after you remove it from the grill or stove. Check the temperature in the thickest part of steaks or roasts.

    How can I prevent E. coli infection from animals?

    Play it safe around animals, including those at petting zoos, farms, fairs, and even in your backyard.


    Examiners report

    Question 2 on the Meselson and Stahl investigation was generally problematic and very poorly answered though a few candidates did score high marks.

    Question 2 on the Meselson and Stahl investigation was generally problematic and very poorly answered though a few candidates did score high marks.

    The majority of candidates described the shading and densities as opposed to explaining the pattern in terms of the N present in the DNA.

    Many candidates scored 2 marks distinguishing between semi-conservative and conservative replication.