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I notice that in a lot of bacteria testing procedures the sample has to be cultured. You place the sample in agar or another growth medium and wait for the bacteria to proliferate before examining them.
Why is this necessary? If the bacteria are present in the sample, can't you just examine the sample directly with a microscope to find them? Is it a needle in a haystack problem?
For example, a listeria bacteria is about 1 micrometer in size and has a positive gram stain. So, at 1000x magnification the organism would be 1mm in size in the objective which is highly visible. Even at 100x magnification you should still be able to see a tiny dot and be able to zoom in on the dot.
With only a microscope, you might be able to find the bacteria, but how do you know what kind of bacterium it is?
Taking Listeria, under a microscope, it looks like a small rod. Well, so do lots of other bacteria. If you only see that one bacterium, you can't take it and do any further tests with it, you can only see it.
So we do cultures, where bacteria multiply and form colonies. Whether they even grow under these conditions already tells you a lot - a lot of bacterial species won't grow colonies on The type of colony is another factor in determining what bacterium we are looking at - does it have fuzzy or clear edges, what color is it, is it "shiny"?
And then, with that culture, further tests can be done, like what chemicals the bacterium in this colony can break down, what they produce, whether they can be stained with certain dyes, etc. Many times this is needed because bacteria look very much alike (round or rods).
For Listeria specifically, there are special plates used as a growth medium that has certain chemicals added that makes their colonies appear red-violet. Other bacterial species won't show this color even if they do grow on the plates.
Cell lines and microorganisms cannot be held in culture indefinitely due to the gradual rise in toxic metabolites, use of nutrients and increase in cell number due to growth. Once nutrients are depleted and levels of toxic byproducts increase, the bacteria in the overnight culture enter the stationary phase, where proliferation is greatly reduced or ceased (the cell density value plateaus). When microorganisms from this overnight culture are transferred into the fresh media, nutrients trigger the growth of the microorganism and it goes through the lag phase, a period of slow growth and adaptation to the new environment, and then the log phase, a period where the cells grow exponentially. 
Subculture is therefore used to produce a new culture with a lower density of cells than the originating culture, fresh nutrients and no toxic metabolites allowing continued growth of the cells without risk of cell death. Subculture is important for both proliferating (e.g. a microorganism like E. coli) and non-proliferating (e.g. terminally differentiated white blood cells) cells. Subculturing can also be used for growth curve calculations (ex. generation time)  and obtaining log-phase microorganisms for experiments (ex. Bacterial transformation). 
Typically, subculture is from a culture of a certain volume into fresh growth medium of equal volume, this allows long-term maintenance of the cell line. Subculture into a larger volume of growth medium is used when wanting to increase the number of cells for, for example, use in an industrial process or scientific experiment.
It is often important to record the approximate number of divisions cells have had in culture by recording the number of passages or subcultures. In the case of plant tissue cells somaclonal variation may arise over long periods in culture. Similarly in mammalian cell lines chromosomal aberrations have a tendency to increase over time. For microorganisms there is a tendency to adapt to culture conditions, which is rarely precisely like the microorganism's natural environment, which can alter their biology.
The protocol for subculturing cells depends heavily on the properties of the cells involved.
Non-adherent cells Edit
Many cell types, in particular, many microorganisms, grow in solution and not attached to a surface. These cell types can be subcultured by simply taking a small volume of the parent culture and diluting it in fresh growth medium. Cell density in these cultures is normally measured in cells per milliliter for large eukaryotic cells, or as optical density for 600nm light for smaller cells like bacteria. The cells will often have a preferred range of densities for optimal growth and subculture will normally try to keep the cells in this range.
Adherent cells Edit
Adherent cells, for example many mammalian cell lines, grow attached to a surface such as the bottom of the culture flask. These cell types have to be detached from the surface before they can be subcultured. For adherent cells cell density is normally measured in terms of confluency, the percentage of the growth surface covered by cells. The cells will often have a preferred range of confluencies for optimal growth, for example a mammalian cell line like HeLa or Raw 264.7 generally prefer confluencies over 10% but under 100%, and subculture will normally try to keep the cells in this range. For subculture cells may be detached by one of several methods including trypsin treatment to break down the proteins responsible for surface adherence, chelating calcium ions with EDTA which disrupts some protein adherence mechanisms, or mechanical methods like repeated washing or use of a cell scraper. The detached cells are then resuspended in fresh growth medium and allowed to settle back onto their growth surface.
The importance of bacteria to humans
Milk from a healthy cow initially contains very few bacteria, which primarily come from the skin of the cow and the procedures for handling the milk. Milk is an excellent growth medium for numerous bacteria, and the bacteria can increase rapidly in numbers unless the milk is properly processed. Bacterial growth can spoil the milk or even pose a serious health hazard if pathogenic bacteria are present. Diseases that can be transmitted from an infected cow include tuberculosis (Mycobacterium tuberculosis), undulant fever (Brucella abortus), and Q fever (Coxiella burnetii). In addition, typhoid fever (Salmonella typhi) can be transmitted through milk from an infected milk handler. Pasteurization procedures increase the temperature of the milk to 63 °C (145 °F) for 30 minutes or to 71 °C (160 °F) for 15 seconds, which kills any of the pathogenic bacteria that might be present, although these procedures do not kill all microorganisms.
Certain bacteria convert milk into useful dairy products, such as buttermilk, yogurt, and cheese. Commercially cultured buttermilk is prepared from milk inoculated with a starter culture of Lactococcus (usually L. lactis or L. lactis cremoris). Yogurt and other fermented milk products are produced in a similar manner using different cultures of bacteria. Many cheeses are likewise made through the action of bacteria. Growth in milk of an acid-producing bacterium such as L. lactis causes the casein to precipitate as curd. Following the removal of moisture and the addition of salt, the curd is allowed to ripen through the action of other microorganisms. Different bacteria impart different flavours and characteristics to foods for example, the mixture of Lactobacillus casei, Streptococcus thermophilus, and Propionibacterium shermanii is responsible for the ripening of Swiss cheese and the production of its characteristic taste and large gas bubbles. In addition, Brevibacterium linens is responsible for the flavour of Limburger cheese, and molds (Penicillium species) are used in the manufacture of Roquefort and Camembert cheeses. Other types of bacteria have long been used in the preparation and preservation of various foods produced through bacterial fermentation, including pickled products, sauerkraut, and olives.
The toxins of many pathogenic bacteria that are transmitted in foods can cause food poisoning when ingested. These include a toxin produced by Staphylococcus aureus, which causes a rapid, severe, but limited gastrointestinal distress, or the toxin of Clostridium botulinum, which is often lethal. Production of botulism toxin can occur in canned nonacidic foods that have been incompletely cooked before sealing. C. botulinum forms heat-resistant spores that can germinate into vegetative bacterial cells that thrive in the anaerobic environment, which is conducive to the production of their extremely potent toxin. Other food-borne infections are actually transmitted from an infected food handler, including typhoid fever, salmonellosis (Salmonella species), and shigellosis (Shigella dysenteriae).
Guardians of the Microbial Galaxy
In 1986, Yiu-Kwok Chan from Agriculture Canada identified a new bacterial species. Following standard protocol, he deposited it in the American Type Culture Collection (ATCC), a repository where scientists store novel microbial strains. It sat there for decades until 2020 when it was noticed by Roland Wilhelm, a postdoctoral researcher at Cornell University, for bearing a striking resemblance to a different group of bacteria. Wilhelm obtained a vial of Chan&rsquos strain from the ATCC and used newer DNA sequencing technology to confirm that the 1986 strain was actually a species of the Paraburkholderia bacteria he was currently studying. This revelation was only possible because of the bacterial archive, which served as a pivotal connection between these two researchers across different eras of science.
Keeping track of global microbial evolution is a challenging task. Microbes form new species faster than humans and many other sexually reproducing animals do, and the number of microbial species scientists have discovered has been steadily growing over the years. However, some estimates suggest that bacterial extinction rates are so close to the new species formation rate that most bacterial lineages that ever existed are now extinct. Microbes are known to be essential for nutrient cycling, agricultural productivity and soil health, producing antibiotics and anticancer compounds and protecting our gut health and immune systems. However, we are still exploring and learning about the microbial world, which makes it all the more important to think about microbial conservation.
Culture collections preserve microbial diversity, just as a seed bank preserves plant genetic diversity. The World Data Center for Microorganisms reports a microbial culture collection in almost every part of the world and together, they contain over two million bacterial, fungal and viral cultures. This number is but a small fraction of the Earth&rsquos prolific microbial diversity.
Microbial culture collections can receive samples from anywhere in the world, but some locations yield more microbes than others. The Jena Microbial Resource Collection receives cultures from all over the world but particularly from Asian countries, according to Michael Ramm, staff member at the JMRC. Some countries or institutions are current hotspots of microbial discovery and are home to large-scale isolation efforts. We often hear about biodiversity hotspots and cautionary extinction stories like the dodo bird&rsquos, but microbial conservation is seldom part of the public conversation.
One reason we don&rsquot think about microbial conservation is that most microbes are invisible to the naked eye and hard to grow outside their natural habitats fewer than 2 percent of environmental bacteria can be grown in the lab. This makes storing and culturing microbes a tricky process that requires finding an elusive combination of nutrients, salts and atmospheric conditions. It can take months or even years for scientists to tease a microbe out from its habitat.
Researchers need repositories like global culture collections to ensure the long-term preservation of the precious cultures that can be cultivated. Kirk Broders, curator of the NRRL Culture Collection in Peoria, Ill., is excited about the potential of such collections. &ldquoConnecting with, and providing resources for, researchers from around the world who are conducting cool research . is the most exciting part of my job. There is also the simple joy of cultivating, growing and admiring the colorful menagerie of beautiful fungi and bacteria.&rdquo
On the surface, it might seem like these collections are cataloging cultures much like a microbial museum. However, the true value of these repositories lies in their potential for science the next novel antibiotic, a compound that cures cancer, or a microbe that reduces greenhouse gas emissions could be hiding in those vials. &ldquoIn science, it can be hard to predict what biological strains may become clinically significant,&rdquo says Sarah Alexander, curator of the National Collection of Type Cultures (NCTC). &ldquoWhen a scientist deposits strains, this material is available to the next generation of scientists and will always be retrievable.&rdquo
Collections allow scientists to make sure that the strain they are working with today is the same one that was used in a study 30 years ago, as in Wilhelm&rsquos story. This is why many culture collections are starting to tighten the restrictions for a submitted strain to be recognized as an official member of the collection. In the past, microscopic examination of a culture might have proved sufficient but repositories like the NRRL are now starting to require an additional security measure that prevents contamination: the gene sequence of the submitted strain must match what the scientist found in the lab. Many microbes can also evolve very quickly, and even a few months of living in the lab can make a strain look different from when it was first identified. Once a microbiologist verifies that the gene sequences match, the strains are stored by cryopreservation, the process of long-term storage using ultracold temperatures or flash freezing with liquid nitrogen.
Culture collections are clearly critical entities that help make science more open, collaborative and reproducible. They preserve Earth&rsquos current microbial diversity and may hold the microscopic keys to solving many pressing global challenges. They are also the libraries of the microbial world and every strain has a unique story the first bacterial isolate in the NCTC was isolated from a World War I soldier and is being used to fight dysentery. Alexander is cognizant of the history and promise of the strains. &ldquoMaintaining, preserving, and growing the collection that contains over 6,000 strains from over 900 different bacterial species is a privilege. A culture collection is a biological repository . whereby we can preserve these living exhibits to ensure they are available to research.&rdquo
It is an essential environmental factor that can influence the growth of the organisms. Most of the pathogens grow at 37 0 C (body temperature). Bacteria are categorized under three groups on the basis of the optimum temperature range
- Mesophile: The optimum temperature range for mesophiles is 25 0 C to 40 0 Most of the pathogenic bacteria come under this group.
- Psychrophile: The optimum temperature for psychrophiles is below 20 0
- Thermophile: The optimum temperature range for thermophiles is 55 0 C to 80 0 Eg: Bacillus stearothermophilus.
pH is an essential factor for the growth of the microbes. May bacteria are able to produce several organic acids which reduce the pH of the medium and also restrict the growth of other bacteria. Apart from that some media constituents can be affected by low pH. Therefore, maintain the optimum pH is highly important to obtain adequate growth of the organisms. Pathogens are mostly requiring neutral pH (7.2). However, industrially important bacteria such as Lactobacillus lactis requires a lower pH for optimum growth.
What is Culture?
Microbial culture is a method of culturing and maintaining microorganisms under laboratory conditions for different purposes. Cultures are grown in solid, semi-solid and liquid media based on the type and the purpose of the microorganism culturing. Cultures are provided with necessary nutrients and growth conditions required by the microorganisms. There are different components of a culture medium such as energy source, carbon source, nitrogen source, minerals, micronutrients, water, solidifying agent, etc. Optimum temperature, oxygen and pH should be adjusted according to the type of the microorganism grown.
There are different types of microbial cultures for example, batch culture, continuous culture, stab culture, agar plate culture, broth culture, etc. According to the composition of the growing medium, there are different types of culture media known as synthetic media, semi-synthetic media and natural media. Microbial cultures are prepared under sterile conditions inside a special chamber called laminar air flow. Growing medium and the glassware are sterilized prior to inoculation of the desired microorganism. Under proper sterile conditions, target microorganism is transferred into the sterilized nutrient medium and incubated at optimum temperature. Inside the medium, microorganism will grow and multiply using the provided nutrients.
Figure 3: A bacterial culture on plate
Creation Scientist Focus 3.1
Carl Fliermans and His Research on Legionella
Dr. Carl B. Fliermans is a microbial ecologist with DuPont and is on the technical advisory board at the Institute for Creation Research. He holds a Ph.D. in microbiology from Indiana University, and a post-doctoral fellowship at the National Institutes of Health. Dr. Fliermans is the scientist who first isolated the “Legionnaires’ Disease” bacterium. He has published over 60 works, and is a member of the American Society for the Advancement of Science, the American Institute for the Biological Sciences, and the American Society for Microbiology, among others. Dr. Fliermans is a Christian who believes the Creator guided him in his discovery of Legionella.
August 1976 Newspaper Headline: “Mystery Illness Strikes Legionnaires”
In one of the most dramatic entrances of any disease into the public-health arena, Legionnaires’ disease appeared at the U.S. Bicentennial Convention of the American Legion, July 21–23, 1976, in Philadelphia. Nearly 5,000 Legionnaires attended the three-day meeting, with over 600 staying at the elegant but aging Bellevue Stratford Hotel. Even before checking out of the hotel, several Legionnaires began to feel ill with flu-like symptoms. On Tuesday, July 27, only four days after leaving Philadelphia, an Air Force veteran who had stayed at the Bellevue Stratford during the convention died at a hospital in Sayre, PA. He was the first of more than 30 Legionnaires to eventually succumb to a lethal pneumonia that the news media quickly named “Legionnaires’ disease.” What was the cause of this new disease?
- Was it biological or chemical?
- Where did the pathogen, if there was one, come from?
- How was the disease spread?
- How could the disease be prevented?
These key questions and more became the focus of an intense investigation that resulted in the discovery in January 1977 that a Gram-negative, rod-shaped bacterium caused the disease. The bacterium was named Legionella pneumophila. Good microbiologists like Dr. Joseph McDade and later Dr. Fliermans considered pathogenic sources in nature, routes of transmission to susceptible persons, and means of preventing the spread of pathogens. Their process illustrates how classical techniques are used to prove the cause of a specific disease.
Investigation of the etiology of Legionnaires’ disease and summarizes Koch’s postulates.
The illustration above traces the investigation of the etiology of Legionnaires’ disease and summarizes Koch’s postulates. Legionnaires’ disease was first recognized in August 1976. It was more than six months before Koch’s postulates were fulfilled and Legionella pneumophila was pronounced the etiologic agent of the disease. First, if the etiological agent were biological, then the agent had to be found to be regularly associated with the disease. Tissues from lung biopsies and sputum samples were examined for a recurring microorganism. A Gram-negative rod with a tendency to form long, looping filaments was consistently detected in specimens. Second, the newly discovered bacterium was isolated in pure culture in the laboratory. This necessitated learning L. pneumophila’s nutritional requirements and designing special growth media that would meet these requirements, supporting the bacterium’s growth. Third, a susceptible animal was needed to demonstrate that L. pneumophila could produce disease, particularly a respiratory disease similar to Legionnaire’s disease in humans. The guinea pig proved to be the animal model of choice. Finally, L. pneumophila was recovered from infected guinea pigs to verify that it had established an infection.
Initial Isolation of Causative Agent
Through a series of experiments, Dr. McDade and his team first discovered from clinical specimens in January 1977 the evidence for the existence and pathogenesis of the bacterium that causes Legionnaires’ disease. The first step was to remove lung samples from a deceased Legionnaire. These cells were ground up, injected into chicken eggs, and incubated. After the incubation period, the eggs were cracked, and the yolk sacs extracted and injected into the footpads of guinea pigs. These animals then developed the typical symptoms of Legionnaires’ disease. McDade then drew blood samples from disease survivors, assuming they contained antibodies against the causative agent. He then mixed the samples with the yolk-sac isolates, and they reacted, confirming that the agent in the yolk sacs was the same agent causing disease in the 33 people.
McDade explained that his team had been stumped for months by several unusual characteristics of the bacteria: The bacteria did not grow under typical conditions. The scientists tried to culture the bacterium from the Legionnaires’ blood and tissue samples in a solution filled with standard media fluid used to grow other bacterial varieties. However, nothing grew. This lack of growth led the team to think that the agent was a virus or “Andromeda strain” never seen before. It was not until samples not treated with antibiotics were injected into the eggs that evidence of biological activity was determined.
Another delay was caused by the use of mice in experiments. It was not until the team switched to guinea pigs that their efforts were productive. Though mice are often used as animal models, it turned out that Legionella replicate primarily inside macrophages. Mice macrophages are very inefficient at ingesting this bacterium, so they never got infected from samples from the Legionnaires. In contrast, guinea pigs were susceptible to Legionella and got infected.
The next step for McDade was to determine why this bacterium was so difficult to culture. They soon discovered that the Legionella bacterium has physiological needs. Standard culture media does not promote growth because it requires high levels of the amino acid cysteine, inorganic iron supplements, low sodium concentrations, activated charcoal, and elevated temperatures. Dr. Fliermans was the first to recognize that the lipids of Legionella were very similar to the thermophilic bacteria he discovered in the thermal areas at Yellowstone National Park. Also, the bacterium tends to live in a nutrient-rich, dark environment as a biofilm (scum) associated with selected algae species. These conditions contributed to the difficulty in viewing the organism in its environment using standard microscopy and other techniques.
McDade asked Fliermans to help complete Koch’s postulates for Legionnaires’ disease. Since 1969, Fliermans has been conducting research on microorganisms associated with natural thermal habitats like those at Yellowstone and man-made habitats coming from thermal streams near electrical and nuclear facilities. The microorganisms associated with these habitats were often mesophilic and thermophilic in their physiological response in that their optimal growth temperature was between 30° and 90°C (86º and 194ºF). The second characteristic unusual for these thermophiles was the large number of branched-chained fatty acids they contained, just like the clinical isolates of Legionella. Armed with this information, Fliermans began looking in aquatic habitats, both natural and man-made, both ambient and thermal, for the presence of Legionella. Fliermans’ seminal work demonstrated that Legionella could be isolated from natural habitats not associated with an outbreak of the disease. These findings opened a new area for thinking, experimentation, and understanding. For Fliermans, the question now became: How and where does Legionella fit in the ecological setting?
Although many at the Centers for Disease Control were puzzled as to the origin of Legionella, the epidemiological data lead Fliermans to focus on aquatic niches as the bacterium’s natural habitats. This hypothesis came from examining the fact that initial clinical presentations demonstrated a seasonality of infection. Such a cyclic pattern was very similar to that observed for the growth of aquatic bacteria. Theories as to the cause of the illnesses ranged from nickel carbonyl intoxication and viral pneumonia to a pharmaceutical conspiracy against American veterans. At the CDC, many hypothesized that Legionella may have been genetically engineered as an “Andromeda strain” by the Soviets or as some other Communist plot. After all, it had primarily affected veterans. Reading his Bible one day, Dr. Fliermans observed Ecclesiastes 1:9 : “The thing that hath been, it is that which shall be and that which is done, is that which shall be done: and there is no new thing under the sun.” Dr. Fliermans believed this was true, and soon altered the way his laboratory looked for the organism, since the bacterium was probably not new under the sun. After praying, planning, and exploring, Dr. Fliermans found the bacterium in thermal waters (initially isolated at 45°C [113ºF] at Savannah River Laboratory) discharged from a nuclear reactor and subsequently from natural hot springs in both the Eastern and Western United States. Once isolated from the environment, the next task was to culture it. At first, it grew only in guinea pigs. Koch’s postulates were initially fulfilled in Dr. Fliermans’ daughter’s guinea pig in the summer of 1977 with samples drawn from cooling towers. He was able to develop and deploy a fluorescent antibody test for detecting Legionella.
This electron micrograph depicts an amoeba, Hartmannella vermiformis (lower left) as it entraps a Legionella pneumophila bacterium (upper right) with an extended pseudopod
Legionella was now easily identified in situ, in vivo, and in vitro. Knowing the molecular and ecological basis of a pathogenesis helps one develop new ways to prevent and cure illnesses. For one thing, one can predict conditions under which pathogens are likely to thrive, spread, and cause illness. With improved techniques and molecular tools, fluorescent antibodies aid in diagnosis. A similar medical detective story is also true for the discovery and diagnosis of the agent causing Lyme disease. (See Body by Design, 2002, p. 145, for details.) Being a medical Sherlock Holmes helps one synthesize the diversity of facts into a unity.
Definition of Synchronous Culture
Synchronous culture refers to the growth process of the microbial population, where individual cells show synchrony with the other cells in the same culture medium by growing at the same growth phase for the given generation time.
The main characteristic of synchronous growth is that all the microbial cells are physiologically identical by growing at the same division cycle and same generation time. Therefore, we can say that the entire microbial population remains uniform concerning cell growth and division.
- By using synchronous culture, we can get the idea of the entire cell crop in the particular stage of their life cycle and their interrelations.
- The measurement of microbial growth in synchronos culture is more accessible than the other growth culture techniques, as the results made on such mass culture is analogous to the measurements made on a single bacterial cell.
- In the synchronous culture, we can elucidate the growth behaviour of the bacterial cells in the same stage of growth.
- The entire microbial population tends to show synchrony by altering the physical conditions and chemical constituents of the culture media.
- It is a kind of open cultivation system.
- In synchronous culture, the microbial cells are physiologically similar.
- All the microbial cells in synchronous culture grow at the same generation time and the same divisional cycle.
The Efficiency of Synchronous Culture
The bacterial cells show asynchronous growth in the random culture medium. However, the microbial cells show synchrony through the selection and induction methods. We could determine the efficiency of synchronous growth by comparing the following two parameters:
Let us see the growth pattern of cells by plotting a graph between doubling time vs a logarithmic number of cells and time vs corresponding mitotic indices for a synchronous and random growth, respectively.
Selection of Microorganisms
For the selection of microorganisms and to keep them in synchrony, there have been two approaches employed based upon the mechanical and induction selection methods.
Selection by Mechanical Method
It refers to a physical separation method, in which the selection of synchronous population is carried out according to the age and size.
To perform this method, you need to filter the microbial cells to separate the small and young cells that are metabolically active. A filter retains large-cells that are ready to split.
In this way, the large-cells are collected from the filter to obtain a synchronous growth by a standard technique known as “Helmstetter Cumming technique”.
In this method, You need to pass the entire cell population through a filter whose particle size is small, sufficient to trap bacteria. This method makes the use of cellulose nitrate membrane filter.
Then, invert the filter paper and pass the fresh nutrient medium over it. By doing this, the loosely associated bacteria will wash away through the filter. The large-sized bacteria will remain on the filter paper and they tend to divide.
Then, collect the sample of this stream, which contains all the newly formed and synchronously dividing cells. The method has one limitation that the population size is petite.
Nowadays, density gradient centrifugation is also used instead of filtration, to select microbial cells of the same size and density and which can divide at the same stage of their life cycle.
Selection by Induction Method
The another method, i.e. shock treatment can also maintain cell synchrony and it includes temperature variation, starvation, light exposure, lethal doses of radiation etc.
All these factors can maintain synchrony in the cell population for several generations. Let us discuss some of the factors that can induce synchrony in the cell culture.
Temperature variation: It is the most common factor that induces the cell maturation to the same point of fission.
We could observe the change in culture medium by growing the microbial culture under 37 degrees Celsius and later subjecting the culture medium to 20 degrees Celsius for about 30 minutes.
During this interval, the cells go through maturation to undergo cell division. At 20 degrees Celsius temperature, no bacteria will undergo fission.
But, if you transfer the culture at a temperature of 37 degrees Celsius, all the cells start dividing synchronously. Therefore, the repeated temperature variation can maintain synchrony for a few generations.
Alternations in the media composition: Other than shock treatment, the synchrony can also be induced by changing the media composition of the culture medium.
The microorganisms grown in the culture medium deficient or containing the essential growth factor may either promote or inhibit the cell division, which eventually withheld the fission.
Let us suppose, the bacterial cells are grown in a culture medium that lacks thymine (an essential element). As a result, the process of fission halts for some time. However, the bacterial cells will divide once you transfer the cells to a complete nutrient medium.
Similarly, colchicine is a growth factor inhibiting the cell division in culture medium for a certain period. But the effect can be reversed once you transfer the cells to the culture medium free of colchicine.
Therefore, we can conclude that the synchronous culture are of two kinds, namely induction and selection synchrony. The induction synchrony induces the bacterial cell population to undergo fission synchronously via physical or chemical treatment.
Oppositely, the selection synchrony selects cells at a particular stage of the cycle, and later the fractions of bacterial cells grow through their natural cycle.
Why are bacterial cultures necessary? - Biology
In many distinct areas of microbiology, the ability to identify microorganisms has important application. For example, in food microbiology it is important to be able to accurately identify food spoilage contaminants. In microbial ecology, the identification of microorganisms helps us characterize biodiversity. In the field of medical microbiology, a branch of microbiology that investigates pathogenic microorganisms, the primary focus is to isolate, identify, and study microorganisms responsible for infectious disease.
Many microorganisms are permanent residents, or normal flora, of the human body. Bacteria that are normal flora are important symbionts of the human body, most of which cause no ill effects and some, which are actually beneficial to human health. Only a small percentage, less than 10%, of all known bacteria are pathogenic, or able to cause disease in a susceptible host. In order to identify an unknown in the clinical laboratory, a sample must be collected from the patient. This could be a sample of urine, feces, saliva, or a swab of the throat or skin. Because the clinical samples will most likely contain many microorganisms, both normal flora and pathogens, it is important to isolate the pathogen in a pure culture using various types of selective and differential media. Following isolation, one of the first steps in identifying a bacterial isolate is the Gram stain, which allows for the determination of the Gram reaction, morphology, and arrangement of the organism. Although this information provides a few good clues, it does not allow us to determine the species or even genus of the organism with certainty. Thus, microbiologists use characteristic biochemicalactivities to more specifically identify bacterial species. A Few Biochemical/Physiological Properties Used for identification of bacteria include: nutrient utilization (carbohydrate utilization, amino acid degradation, lipid degradation), resistance to inhibitory substances (high salt, antibiotics, etc.), enzyme production (catalase, coagulase, hemolysins, etc.) and motility.
This series of lab exercises will introduce many of the physiological characteristics/biochemical activities of bacteria commonly encountered in a clinical microbiology laboratory. Knowledge of these key characteristics will enable the identification of unknown bacterial isolates. It is important to thoroughly understand the basis for each biochemical test and know the key physiological characteristics of the bacterial genera and species presented in these labs.
Note: Labs 15-17 will utilize a number of different media and tests that are described in the ATLAS and on the course web page (Summary of Biochemical Tests). Please use these as references for all of these labs and for the investigation of unknowns in Labs 18-21.
S1 Fig. Impaired metabolism leads to an increase in antibiotics survival.
(A) E. coli MG1655 WT, ΔaceA, and Δicd strains were grown 2 hours to 2.5 hours in LB at 37°C to exponential phase and challenged with ciprofloxacin (1 μg/mL). Survival has been assessed by culture plating and CFU counting. Data are the average results from 3 independent experiments performed with 3 biological replicates (n = 8 or 9). Error bars represent standard deviations. (B) Distribution of the iATPSnFr 1.0 488ex/405ex ratio per single cell measured by microscopy in exponential cultures of MG1655, ΔaceA, and Δicd strains grown in LB at 37°C. Thick lanes represent the median, and secondary lanes the quartiles. Significance was determined using Kruskal–Wallis and Dunn’s multiple comparison tests. Observations were performed with a confocal microscope. (C) Bulk ATP levels were measured in MG1655, ΔaceA, and Δicd strains grown in LB 37°C with firefly luciferase assay and normalized with OD600. Data are the average results from 3 independent experiments performed with 3 biological replicates (n = 9). Error bars represent standard deviations. Significance was determined using one-way ANOVA and Tukey’s multiple comparison tests. The underlying data for this figure can be found in S1 Data.
S2 Fig. Growth of the fluorescent reporter strains.
MG1655 Krebs cycle KO strains and W3110D Krebs cycle mVenus fusion strains were grown in LB (A), in MOPS minimum medium with 0.4% sodium pyruvate (B), or 0.4% sodium acetate (C) as sole carbon source at 37°C. (D) MG1655-SB1 strains expressing or not iATPSnFr 1.0 were grown in LB at 37°C. In (A), (B), (C), and (D), each lane represents a biological replicate coming from experiments performed 3 times (n > 6). The underlying data for this figure can be found in S1 Data.
S3 Fig. Fluorescence analysis of the mVenus fusions.
Representative histogram of fluorescence of the mVenus fusions before (A) and after (B) ciprofloxacin treatment of W3110D: black lane, aceA-mVenus: red-lane, icd-mVenus: blue lane, gltA-mVenus: green lane, sucA-mVenus: orange lane. For each of those fusions, (C), (D), (E), (F), respectively, show the histogram of fluorescence before (black lane) and after (red lane) ciprofloxacin treatment for each fusion (left panel), and the post-sorting purity analysis of the Dim (light grey), Middle (grey), and Bright (dark grey) sorted fractions (right panel). The number of events represented in the y axis is normalized for each graph. The underlying data for this figure can be found in S1 Data.
S4 Fig. Reporting ATP in exponential and stationary phase cultures at the single-cell level.
(A) Representative images of exponential (upper panel) and stationary (lower panel) phase cultures of MG1655_iATPSnFr 1.0 cultured in LB at 37°C. The fluorescent signals 405ex and 488ex are false colored in magenta and green, respectively. In the ratiometric 488ex/405ex panel, orange/yellow cells correspond to cells with higher ATP, and blue cells with lower ATP. Scale bar, 5 μm. White arrow shows the presence of a low 488ex/405ex ratio cell within exponential culture. Observations were performed with a confocal microscope. (B) Distribution of the iATPSnFr 1.0 488ex/405ex ratio in exponential and stationary phase cultures. Thick lanes represent the median, and secondary lanes the quartiles. Significance was determined using two-tailed unpaired Mann–Whitney U test. Data are representative of experiments made twice giving similar results. (C) Bulk ATP levels were measured in MG1655 strain grown in the same conditions as in (A) with firefly luciferase assay and normalized with OD600. Data are the average results from 2 independent experiments performed with 3 biological replicates (n = 6). Error bars represent standard deviations. Significance was determined by using two-tailed unpaired Student t test. The underlying data for this figure can be found in S1 Data.
S5 Fig. Kymographs of the different phenotypes observed.
The fluorescent panels represent the addition of iATPSnFr 1.0 405ex and 488ex intensities. Observations were performed with a time-lapse epifluorescence microscope.
S6 Fig. Kymographs of the persisters analyzed.
(A) Left panel: ATP value at the beginning of the experiment in persisters. Data represent the single-cell 488ex/405ex ratio values analyzed for each of the 16 persisters at the first time frame in Fig 3D histogram. The black horizontal dashed line represents the mean 488ex/405ex ratio of the normal cells (0.6303). Right panel: Kymographs of the 16 persisters analyzed in Fig 3C. The iATPSnFr 1.0 ratiometric 488ex/405ex panels represent ATP level. The color code has been scaled individually for each of the 16 kymographs. Persister number 2 harbors a slow growth pattern different than the others which don’t grow in presence of ampicillin. The white rectangles indicate the time frame where the first septal invagination of each persister is observed (time before first division). Observations were performed with a time-lapse epifluorescence microscope. (B) For each persister, time before first division is plotted over the iATPSnFr 1.0 488ex/405ex ratio at the first time frame of the time lapse (t = 1). Correlation was examined by Pearson’s correlation. The underlying data for this figure can be found in S1 Data.
S7 Fig. iATPSnFr 1.0 488ex/405ex ratio in the mother machine.
(A) Each data point represents the iATPSnFr 1.0 488ex/405ex ratio per single cell over time frames (frames interval is 30 minutes) for normal cells (in black) and persister cells (in red). (B) Distribution of the iATPSnFr 1.0 488ex/405ex ratio per single cell for normal cells (in black) and persister cells (in red) according to their position in the mother machine, at the first time frame of the time lapse (t = 1), and (C) immediately before the antibiotic is added (t = 5). Observations were performed with a time-lapse epifluorescence microscope. The underlying data for this figure can be found in S1 Data.
S8 Fig. Persisters, cell size, and ATP.
(A) Distribution of cell size area. (B) Distribution of the iATPSnFr 1.0 488ex/405ex ratio. A mother machine experiment using the same setup as in Fig 3 was performed. The light grey bars represent all non-persister cells (23,766 cells) at the first time frame, immediately after loading in the mother machine from a stationary phase culture. Dark grey corresponds to non-persister cells with sizes smaller than the average persister cells (5,596 cells). The 5 persisters found in this experiment are plotted in red. Observations were performed with a time-lapse epifluorescence microscope. The underlying data for this figure can be found in S1 Data.
S9 Fig. General schematic of the construction of in-frame chromosomal mVenus fusions.
Schematic of the construction of the translational mVenus fusions. Two-round PCRs were performed. The template used was the pKDN31 plasmid (a gift from Barry Wanner), and the polymerase used was the KOD. Final PCRs were precipitated with ethanol, dried up, then dissolved in H2O, before to be used to electrotransform W3110D/pKD46. Electrocompetent cells of W3110D/pKD46 were prepared according to the standard protocol for λ Red recombination with a higher concentration of L-arabinose (10 mM) [59,60]. The double selection was done using resistance to chloramphenicol (25 μg/mL) and sensitivity to ampicillin (100 μg/mL at 30°C), and clones were PCR checked using Left and Down primers (S3 Table). Finally, bar code sequencing was performed after amplification with Left and NYP244 primers (S3 Table).
S10 Fig. Evaluation of photobleaching and photoactivation effects for the iATPSnFr 1.0 sensor.
Stationary phase cells were concentrated and loaded in the device, and fresh EZRDM was flowed until cells are in balanced growth (from time frame 7 to the end of the experiment). Frames were taken 30 minutes apart. Changes in the 405ex and 488ex signals of the iATPSnFr 1.0 sensor (on the right y axis) and in the 488ex/405ex ratio (on the left y axis) were monitored over time. Data represented are the mean of the iATPSnFr 1.0 488ex, 405ex, and 488ex/405ex ratio per single cell over time frames (n = 2,670). Error bars represent standard errors. Observations were performed with a time-lapse epifluorescence microscope. The underlying data for this figure can be found in S1 Data.