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Storage of bacteria

Storage of bacteria


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Why are strains of bacteria stored when they are inactive (frozen)? What is the problem with storing growing cultures for long periods?


Aside from the comment that frozen cells require less manual maintenance and expense (which is a big plus), active cultures can rearrange/recombine plasmids causing mutations and deletions (especially in the case of toxic inserts). For example, see this article:

Peijnenburg AA, Bron S, Venema G. 1987. Structural plasmid instability in recombination- and repair-deficient strains of Bacillus subtilis. Plasmid 17(2):167-170.


Although a bacterial culture on a petri dish, slope or stab culture is in a closed environment and may start healthy, over time, the number of viable cells will decrease to zero as nutrients are being used up. The goal of preserving the cultures in a frozen state is to slow their metabolism and, in relation to that, their death rate so that when the culture is revisited, some of the cells are still viable and available for culturing. The reasons the cells die can be numerous, but in every instance are based on the inherent chemistry of the cells and their environment. If the deleterious chemical reactions can be slowed or halted, then the overall culture will remain viable for a longer period of time.

As a general rule, the viable storage period of bacteria increases as the storage temperature decreases. Once the temperature is below the freezing point, however, cryoprotectants are essential to reduce cell damage caused by the freezing process.

In addition to the other comments and answers, cryopreservation tubes tend to be much smaller and take up far less space than an agar plate or stab cultures.

Here a table that shows approximate storage spans at different conditions:

Sources:
OPS DIagnostics
Thermo Fisher - Storing Bacterial Samples for Optimal Viability


Practical Work for Learning

Notes on maintaining stock cultures, and preparing actively growing cultures for use by students.

Stock cultures

Instead of re-purchasing whenever needed, it may be convenient to maintain a stock of a pure culture. Most of those recommended are relatively easy to maintain on the appropriate growth medium, but maintenance of stock cultures needs to be well-organised with attention to detail.

Date stamp new cultures on arrival from the supplier.

Cultures on streak plates are not suitable as stock cultures. Cultures of bacteria and fungi are normally kept in Universal/ McCartney bottles or (for a short time) in test tubes in which the agar has been allowed to set at a slope. Dispense the agar in the normal way (5 ml is sufficient in a Universal bottle) and, after autoclaving, prop the bottle (or tube) at an angle, for example, around the edges of a pile of bench mats, until the agar has set. Make sure that the sloping agar does not quite reach the neck of the bottle (or tube). Caps should remain loose until the agar slope has solidified. If a lot of slopes have to be prepared, it might be worthwhile making a d-i-y construction to hold the containers at the correct angle.

Slope cultures in bottles with screw-caps (rather than cotton wool plugs or plastic lids) are preferred because the cap reduces evaporation and dehydration and the caps cannot be accidentally knocked off. Slope cultures are also preferred to broth (liquid medium) cultures because the first sign of contamination is more readily noticed on an agar surface.

Prepare two stock cultures a ‘permanent’ stock which is opened once only (to prepare the next two stock cultures) and a ‘working’ stock for taking sub-cultures for class. If a particular culture is used a lot in a short period, it is much more convenient to prepare several working sub-cultures at once.

Incubate at an appropriate temperature until there is good growth. For growing strict aerobes it may be necessary to slightly loosen the cap for incubation (but close securely before storage) if there is insufficient air in the headspace. If you are not sure whether the strain is a strict aerobe or not, leave the cap loose until the culture is established.

As soon as there is adequate growth, and the caps are screwed tight, the cultures may be stored in a refrigerator (one in which human foodstuffs are never kept), but they will remain viable in either a cupboard or a drawer at room temperature. A dark place at a temperature of 10-15 °C is ideal.

Be prepared to transfer most cultures four times a year to maintain viability. Label each culture clearly including the date of transfer. Do not destroy old cultures until the new subcultures have become established. Some bacteria need more frequent sub-culturing to maintain viability (for example Lactobacillus sp).

Checking purity of cultures

It is sensible to check purity on suspicion of contamination of the working stock, and of the permanent stock when preparing new stock cultures. Evidence of purity is given by the uniformity of colony form on a dilution streak plate, cell form on stained microscope slides, and consistency with the appearance of the original culture.

If a culture becomes contaminated, go back to the working stock or permanent stock cultures, or buy in fresh supplies.

Preparing cultures to use in investigations

Microbial cultures cannot be taken from a shelf and instantly be ready for use. It is necessary to begin to prepare cultures well in advance otherwise the outcome might not be as expected. The key is to transfer cultures several times in advance to ensure that they are growing well and are presented as young, fully-active cultures on the day of the practical class.

To transfer cultures, scrape a small amount of bacteria or yeast cells off the agar slope of a culture using a sterile loop. Wipe the loop over a fresh agar slope to seed a new culture directly or, preferably, shake the loop in a small volume of sterile nutrient broth. After allowing the transferred cells time to multiply until the broth becomes cloudy (1-2 days, or longer with slower-growing organisms or large volumes of broth), the microbes are ready to be used for investigations or to prepare new agar-slope cultures.

Progress of growth can be followed by observation with the naked eye, looking for growth on an agar surface or turbidity in a broth culture. It is usual to grow moulds on the surface of an agar medium, allowing an incubation period of several days to a week.

Use a sterile loop, syringe or Pasteur pipette to transfer bacteria or yeast cells from the broth.

The main points to observe are:

  • use of an adequate amount of inoculum
  • an appropriate culture medium
  • an appropriate incubation temperature
  • adequate aeration for a strictly-aerobic organism in a single large volume (more than 20 cm 3 ) of liquid culture.

Suggestions and tips

1 It will save time preparing large numbers of cultures of bacteria and yeast for a class if the inoculum is taken by Pasteur pipette from a well-growing, turbid broth culture. A line of growth on a slope culture inoculated by a wire loop is easy for students to observe – but you can achieve almost the same effect with a pipette. With a broth (rather than a slope) the risk of spills is greater, so slopes are preferred with younger or inexperienced students.

2 Regular practice of aseptic technique is necessary to ensure that the manipulations involved in culture transfer and handling sterile solutions become second nature.

3 The choice of loop or pipette for transfers between test tubes and screw cap bottles depends on whether they contain agar slopes, liquid media or sterile solutions.

4 To inoculate a tube or bottle from a separate colony on a plate, a wire loop is usually satisfactory. A straight wire is sometimes needed for small colonies (for example in pure cultures of Streptococcus and Lactobacillus) or on plates used to isolate cultures from natural samples.

Health & Safety and Technical notes

Carry out a full risk assessment before planning any work in microbiology (see note 1 for more details).

1 Before embarking on any practical microbiological investigation carry out a full risk assessment. For detailed safety information on the use of microorganisms in schools and colleges, refer to Basic Practical Microbiology – A Manual (BPM) which is available, free, from the Society for General Microbiology (email This email address is being protected from spambots. You need JavaScript enabled to view it. ) or go to the safety area of the SGM website (www.microbiologyonline.org.uk/safety.html) or refer to the CLEAPSS Laboratory Handbook, section 15.2 and 15.12.

Web links

www.microbiologyonline.org.uk/sgmprac.htm
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms.

www.microbiologyonline.org.uk
MiSAC (Microbiology in Schools Advisory Committee) is supported by the Society for General Microbiology (see above) and their websites include more safety information and a link to ask for advice by email.

(Websites accessed October 2011)

© 2019, Royal Society of Biology, 1 Naoroji Street, London WC1X 0GB Registered Charity No. 277981, Incorporated by Royal Charter


Multidrug-Resistant Bacteria

Christopher Grace MD, FACP , in Critical Care Secrets (Fifth Edition) , 2013

7 How do bacteria become multiresistant?

Bacteria become resistant to antibiotics by DNA mutation at select points or by insertions or deletions that alter microbial enzymes or the antibiotic targets. Genetic material can be transferred between bacteria by plasmids (extrachromosomal double-stranded circular DNA) via direct cell-to-cell contact. Bacteria may also acquire new resistance genes by infection with bacteriophage viruses that carry resistance genes with them when they infect bacteria. Once bacteria develop or acquire new resistance genes they have a selective advantage when antibiotics are used. As more mutations or transferred genetic material accumulates, the more classes of antibiotics the bacteria become resistant to, inducing MDR.


A Guide to Bacteria Preservation: Refrigeration, Freezing and Freeze Drying

For information of OPS Diagnostics preservation products visit our pages on Microbial Freeze Drying Buffer, Excipients, Serum Vials, Cryogenic Supplies, and Bacterial Freezing Kit.

Between stock cultures, mutant strains, and genetically engineered variants, the number of individual bacterial cultures which any one lab can accumulate can be numerous. Indeed, the number of variations created in the process of engineering one plasmid can be astounding. And most labs will hold on to all those and other variations as you'll never know what you might need tomorrow. Consequently, preserving all those bacterial cultures and genetic variants is something to be approached with thought.

A bacterial culture in a capped tube is in a closed environment. Though the culture may start healthy, given time the number of viable cells will decrease to zero. The goal of preserving the cultures is to slow that death rate so that when the culture is revisited, some of the cells are still viable and available for culturing. The reasons the cells die can be numerous, but in every instance are based on the inherent chemistry of the cells and their environment. If the deleterious chemical reactions can be slowed or halted, then the overall culture will remain viable for a longer period of time.

There are two basic approaches to slowing the rate of deleterious reactions in a culture of bacteria. The first is to lower the temperature which decreases the rate of all chemical reactions. This can be done using refrigerators, mechanical freezers, and liquid nitrogen freezers. The second option is to remove water from the culture, a process which can be tricky and involves sublimation of water using a lyophilizer.

    • Storage of Bacterial Cultures at 4°C- Protocol on how to refrigerate bacterial cultures.
    • Protocol of Freezing Bacteria in Glycerol- Preserving bacteria by freezing in glycerol.
    • Bacteria Freeze Drying Protocol- Outlining key considerations on how to freeze dry bacterial cultures.

    Following is a brief discussion of the major options for preserving bacteria. The strengths and weakness of each option is reported.

    Bacteria can survive for a short period of time at 4°C. For strains that are used daily or weekly, cultures grown on agar slants or plates can be stored in a refrigerator assuming that precaution has been taken to avoid contamination. Cultures should be prepared using standard techniques and then sealed before storing. For slants, we recommend using screw capped tubes. For cultures on Petri dishes, the plates need to be sealed with Parafilm. Sealing the plates not only helps to prevent molds from sneaking into the plates, but it slows the agar from drying. For anything over a week or two, cultures can be stored as stabs in small, flat-bottomed screw capped vials. In this technique, vials are filled with a small amount of agar medium (e.g., 1 ml) and sterilized. Bacteria are then introduced into the solidified agar with a sterile needle. The culture is incubated overnight with loose caps and then stored at 4°C with tight caps. Cultures stored in stabs are more resistant to drying and contamination, but they will lose viability more quickly than frozen stocks. The length of time a stab can remain viable is dependent upon the strain. Some manuals claim that stabs are good for a year however it is unwise to make that assumption unless it is tested.

    Freezing is a good way to store bacteria. Generally, the colder the storage temperature, the longer the culture will retain viable cells. Freezers can be split into three categories: laboratory, ultralow, and cryogenic. The problem faced by bacteria (and other cells) stored in freezers is ice crystals. Ice can damage cells by dehydration caused by localized increases in salt concentration. As water is converted to ice, solutes accumulate in the residual free water and this high concentration of solutes can denature biomolecules. Ice can also rupture membranes, though this problem is more often associated with cells lacking walls, such as cultured animal cells. To lessen the negative effects of freezing, glycerol is often used as a cryoprotectant. Glycerol is produced by many fish and insects to defend against cold temperatures by depressing the freezing point of the cells, enhancing supercooling, and by protection from ice. With bacteria, adding glycerol to final concentration of 15% will help to keep cells viable under all freezing conditions (see this link for a protocol or this link for a ready to use freezing tube ). The following are some specifics for each freezer category.

    Laboratory freezers are those that can pull temperatures down to -20 to -40°C. These are single stage systems (one compressor) and often called general purpose freezers. Bacteria can be stored for moderate periods of time, e.g., 1 year, in general purpose freezers. It is best to use freezers without frost-free temperature cycling as this can wreak havoc on cells and other temperature sensitive biomolecules. General purpose freezers are inexpensive and found in most labs, thus they are readily available for storing cultures. The downside is that they are not sufficiently cold for long-term storage.

    Ultralow freezers are two stage systems (two compressors each having a different refrigerant) which pull down to around -86°C. Ultralow freezers are very prevalent, but space in them can sometimes be limited and competitive. Ultralow freezers also are much more expensive to purchase, run and maintain. The upside is that cells stored at -80°C tend to remain viable for several years. The lower temperature generated by ultralow freezers substantially reduces chemical reactions within the culture. However, molecular motion still occurs in frozen cells and thus the viability of the culture will decline. It is important to regularly monitor cultures to assess their level of viability.

    Cryogenic freezers are very cold and rely on liquid nitrogen or specialized mechanical systems to operate. For biological samples, cryogenic storage should be below -130°C. At this temperature, the molecular motion of water is halted and cells are trapped in a glass-like matrix. Bacteria stored in cryogenic freezers retain their viability for many years. In our laboratory bacterial and yeast cultures have been maintained at -140°C for 15 years without significant loss of viability. Storing cells in cryogenic freezers is the most effective and, as compared to freeze drying, the easiest method for long-term storage. The downside is cost and potential vulnerability of stocks to power outages, mechanical failures, and failed deliveries of liquid nitrogen. Additionally, tubes should never be stored in tanks submersed in liquid nitrogen. Screw cap tubes leak and will pull the nitrogen into the tube along with contaminants ( see link for more information ). Liquid nitrogen vapor phase freezers will effectively avoid this problem, but these freezers are very expensive (upwards of $10K) and require large volumes of liquid nitrogen. An alternative is mechanical cryogenic freezers that can go as low as -150°C, but these are also very expensive to purchase (about $20K). Both cryogenic freezers will cost several hundred dollars a month to operate.

    In an aqueous system, such as a living cell, water not only serves as the medium for enzymatic reactions, but also spontaneous negative reactions such as free radical formation. Removing water halts both enzymatic and non-enzymatic reactions. Freeze drying is one method of removing this water. Many bacteria can be preserved very effectively by freeze drying. By freezing the cells in a medium that contains a lyoprotectant (usually sucrose) and then pulling the water out using a vacuum (sublimation), cells can be effectively preserved. This method is laborious and requires specialized equipment, but it has the advantage of generating stock cultures that are unaffected by power outages and empty liquid nitrogen tanks. Furthermore, if cultures are routinely shipped to other labs, freeze dried cultures do not require special handling. The downside on freeze drying is that not all cultures react the same way thus some experimentation is required to optimize the process for each strain. For any lab which is serious about producing and maintaining a culture collection, then freeze drying should be included as a major method for preservation.

    Details on freeze drying bacteria can be found on the webpage Bacteria Freeze Drying Protocol.


    INTERNAL ORGANIZATION OF BACTERIA

    Cell membrane or plasma membrane is present beneath the cell wall. It is very thin and flexible. It completely surrounds the cytoplasm. Chemically, it is composed of phospholipids and proteins. Plasma membrane is very delicate in nature. Any damage to it results in death of the organisms. The bacterial cell membrane is different from the eukaryotic membrane. It lacks sterols like cholesterol. Cell membrane regulates the transport of proteins, nutrients, sugar, electrolytes and other metabolites. The plasma membrane also contains enzymes for respiratory metabolism.

    The cell membranes fold to form two types of structures:

    (a) Mesosomes: The cell membrane invaginates to form

    mesosomes. Mesosomes are in the tbrin of vesicles. tubules or lamellae. Mesosomes are involved in DNA replication and cell division. Some mesosomes are also involved in the export of exocellular enzymes. Respiratory enzymes are also present on the mesosomes.

    (b) Photosynthetic membranes: The membrane forms tubular or sheet like infoldings in photosynthetic bacteria. These infoldings contains enzymes for photosynthesis.

    (2) Cytoplasmic Matrix

    The cytoplasmic matrix is a substance present between the plasma membrane and the nucleoid. The membranous bound organelles and cytoskeleton (microtubules) are absent in the prokaryotic cytoplasm. It has gel like structure. Small molecules can move through it rapidly. The plasma membrane and everything present in it is called protoplast. Thus the cytoplasmic matrix is a major part of the protoplasm. Other structures like chromatin/nuclear body, ribosomes, mesosomes, granule and nucleoid are present in this matrix.

    (3) Microcompartments

    Micro-compartments like carboxysome provide a further level of organization. These are compartments within bacteria that are surrounded by polyhedral protein shells, rather than by lipid

    membranes. These polyhedral organelles localize and compartmentalize bacterial metabolism. This function is performed by the membrane-bound organdies in eukaryote

    (4) Nucleoid and plasmids

    The nuclear material or DNA of bacteria aggregates to form irregular shaped dense body called nucleoid. Nuclear membrane is absent in bacterial cell. The order Planctomycetes is an exception. They have a membrane around their nucleoid. The nuclear material or DNA is present near the centre of the cell. This nuclear material is composed of single, circular and double stranded DNA molecule. Very little protein is associated with DNA. Bacterial chromosome is called gonophore. Nucleoid is also called nuclear body, chromatin body or nuclear region. It has very long molecule of DNA. This DNA is tightly folded and fit inside the cell components. Bacteria has single chromosome. So bacteria are haploid. Nucleoid is visible in the light microscope after staining with FeuIgen stain.

    Plasmids are circular, double stranded extra chromosomal DNA molecules in bacteria. Many bacteria contain plasm ids in addition to chromosomes. They are self – replicating bodies. Plasmids are not essential for the bacterial growth and metabolism. Plasmids contain drug and heavy . metals resistant genes. Disease and insect resistant genes are also present on them. Plasmids play an important role in conjugation.

    Ribosomes are composed of RNA and protein. Some ribosomes are also loosely attached with the plasma membrane. Ribosomes are protein factories. There are thousands of ribosomes in each healthy growing cell. The ribosome of bacteria (705) is smaller than the ribosomes of eukaryotes (80S).

    Bacteria live in an environment where nutrients are in short supply. The bacteria try to store extra nutrients when possible. This storage material may be glycogen, sulphur, fat and phosphate. The cell also contains waste material. This waste material is excreted later on. Common waste materials are alcohol, lactic acid and acetic acid.

    The metabolically dormant (inactive) bodies with thick wall are called spores. Certain species of bacteria produces spores. Spores have a central core of cytoplasm containing DNA and ribosomes. It is surrounded by a cork”, layer and protected by an impermeable and rigid con There are two types of spores:

    (a) Etospores: These are produced out side the vegetative cells.

    (b)Endospores: These are present within the vegetative cells. Its example is Bacillus.

    They are produced at later stages of growth. Spores are resistant to adverse environmental conditions like light, high temperature, desiccation, pH and chemical agents. They grow under favorableconditions and form new vegetative cells.

    Cysts: Cysts are dormant, thick- walled and desiccating resistant structure. These are present in certain bacteria like Azotobacter. They develop during reproduction of vegetative cells. Such cells germinate only under suitable conditions. They are not heat resistant structures.

    Shapes of Bacteria

    Bacteria are classified in to three categories on the basis of their shapes. These shapes are cocci, bacilli and spiral. Most of the bacteria have constant shapes. Some bacterial cells are pleomorphic and exist in different shapes.

    Cocci are spherical or oval bacteria. They have different arrangements. These arrangements are based on their plane of division.

    1. Diplococcus: In this case, division occur in one plane and cocci occur in pairs.
    2. Strptococcus: In this case, division also occurs in one plane but the cocci forms long chain of cells.
    3. Tetrad: When division of cells occurs in two planes, it will produce a tetrad arrangement. A tetrad is a square of 4 cocci.
    4. Sarcina: When division occurs in three planes, it will produce a sarcina arrangement. Sarcina is a cube of 8 cocci.
    5. Staphylococcus: When division occurs in random planes, it will produce a staphylococcus arrangement. In this case, the cocci are arranged irregularly like clusters of grapes.

    Examples: Diplococcus Pneumoniae. Staphylococcus aureus.

    Bacilli are rod shaped bacteria. Bacillus is a single cell of bacteria. There are following arrangements of bacilli.

    a) Streptobacillus: Streptobacillus is a chain of bacilli.

    b) Diplobacilli: When bacilli occur in pairs, then the arrangement is called diplobacilli.

    Examples of bacilli: Escherichia coil, Bacillus subtilis, Pseudomonas.

    c) Spiral

    The spiral shaped bacteria are spirally coiled. There are following forms of spirals:

    a) Vibrio: It is a curved or comma-shaped spiral.

    b) SpinBum: It is a thick rigid spiral.

    c) Spirochete: It is thin, flexible spiral.

    Examples of spiral bacteria: Vibrio, Hyphomicrobium.

    Arrangement of Bacteria

    Different types of bacteria arranged in different manners to form different structures. Some of these are:

    sheath that contains many individual cells. Certain types like genus Nocardia, even form complex, branched filaments. These filaments similar in appearance to fungal mycelia.


    Storage stability of freeze–dried Lactobacillus acidophilus (La-5) in relation to water activity and presence of oxygen and ascorbate ☆

    Storage stability of freeze–dried Lactobacillus acidophilus was found to depend on water activity (0.11–0.43), oxygen level (atmospheric oxygen level and <4% oxygen compared) and presence of sodium ascorbate (0% and 10% (w/w)). Increasing water activities decreased bacterial survival, and a reduced oxygen level (<4% oxygen) improved the storage stability, which strongly indicates a connection between oxidative reactions and bacterial instability. The detrimental effect of atmospheric oxygen was reduced by including ascorbate in the freeze drying medium. However, when ascorbate was present a pink/red colour was observed on the surface of the dried samples increasing with the water activity and oxygen level. Increased water activity lead to increased browning also for samples without ascorbate. Free radicals were detected in the dried bacteria by ESR spectroscopy (broad single-peak ESR spectra), where the shape and the g-value was found to depend on the presence of ascorbate and the extent of browning. For increasing water activities the content of radicals increased to a certain level, after which it levelled off and/or decreased. The highest concentrations of radicals were detected in the dried bacteria with highest survival for a given water activity, i.e. low oxygen level and presence of ascorbate, pointing towards a role of semi-stable ascorbyl radicals as a “dead end” for otherwise detrimental free radical reactions.


    Tech Turns to Biology as Data Storage Needs Explode

    Researchers have decoded the genomes of mammoths and a 700,000-year-old horse using DNA fragments extracted from fossils in the past few years. DNA clearly persists far longer than the bodies for which it carries the genetic codes.

    Computer scientists and engineers have long dreamed of harnessing DNA&rsquos tininess and resilience for storing digital data. The idea is to encode all those 0s and 1s into the molecules A, C, G, and T that form the twisted, ladder-shaped DNA polymer&mdashand this decade&rsquos advances in DNA synthesis and sequencing have bought the technology forward by leaps and bounds. Recent experiments indicate that we might one day be able to encode all the world&rsquos digital information into a few liters of DNA&mdashand read it back after thousands of years.

    Now interest from Microsoft and other tech companies is energizing the field. Microsoft Research announced last month that it would pay synthetic biology start-up Twist Bioscience an undisclosed amount to make 10 million DNA strands designed by Microsoft&rsquos computer scientists to store data. Top memory manufacturer Micron Technology is also funding DNA digital storage research to determine whether a nucleic acid&ndashbased system can expand the limits of electronic memory. This influx of money and interest could lead to research and progress that eventually drive down today&rsquos prohibitively high costs and make DNA data storage possible within the decade, researchers say.

    Humans will generate more than 16 trillion gigabytes of digital data by 2017, and much of it will need to be archived: Think: legal, financial and medical records as well as multimedia files. Data is stored today on hard drives, optical disks or tapes in energy-hogging, warehouse-size data centers. These media last anywhere from a few years to three decades at most. Plus, says Microsoft Research computer architect Karin Strauss, &ldquowe&rsquore producing a lot more data than the storage industry is producing devices for, and projections show that this gap is expected to widen.&rdquo

    Enter DNA. It lasts for centuries if kept cold and dry. And it could in theory pack billions of gigabytes of data into the volume of a sugar crystal. Magnetic tapes, today&rsquos densest storage medium, hold 10 gigabytes in the same amount of space. &ldquoDNA is an unbelievably dense, durable, nonvolatile storage medium,&rdquo says Olgica Milenkovic, an electrical and computer engineering professor at the University of Illinois at Urbana&ndashChampaign.

    That is because each of its four building-block molecules&mdashadenine (A), cytosine (C), guanine (G) and thymine (T)&mdashis only a cubic nanometer in volume. Using a coding system&mdashat its simplest, say A represents bits &lsquo00,&rsquo C represents &lsquo01&rsquo and so on&mdashscientists can take the strings of 0s and 1s that form digital data files and design a DNA strand that maps an image or video. (Of course, the actual coding techniques scientists use are much more complex.) Synthesizing the designer DNA strand is the data-writing part. Scientists can then read the data by sequencing the strands.

    Harvard University geneticist George Church jump-started the field in 2012 by encoding 70 billion copies of a book&mdashone million gigabits&mdashin a cubic millimeter of DNA. A year later researchers at the European Bioinformatics Institute showed that they could read, without any errors, 739 kilobytes of data stored in DNA.

    A few teams have demonstrated fully functioning systems in the past year. In August researchers at E.T.H. Zurich encapsulated synthetic DNA in glass, exposed it to conditions simulating 2,000 years and recovered its coded data accurately. In parallel, Milenkovic and her colleagues reported storing the Wikipedia pages of six U.S. universities in DNA and&mdashby giving the sequences special &ldquoaddresses&rdquo&mdashselectively reading and editing parts of the written text. Such random access to data is critical to avoid having to &ldquosequence a whole book to read just one paragraph,&rdquo she says.

    In April Microsoft&rsquos Strauss and computer scientists Georg Seelig and Luis Ceze at the University of Washington reported being able to write three image files, each a few tens of kilobytes, in 40,000 strands of DNA using their own encoding scheme&mdashand then reading them individually with no errors. They presented this work in April at an Association for Computing Machinery conference. With the 10 million strands Microsoft is buying from Twist Bioscience, the team plans to prove that DNA data storage can work on a much larger scale. &ldquoOur goal is to demonstrate an end-to-end system where we encode files to DNA, have the molecules synthesized, store them for a long time and then recover them by taking DNA out and sequencing it,&rdquo Strauss says. &ldquoStart with bits and go back to bits.&rdquo

    Memory maker Micron is exploring DNA as a post-silicon technology. The company is funding work by Harvard&rsquos Church and researchers at Boise State University to explore an error-free DNA storage system. &ldquoThe rising cost of data storage will drive alternate solutions, and DNA storage is one of the more promising solutions,&rdquo says Gurtej Sandhu, director of Advanced Technology Development at Micron.

    These researchers are still looking into cutting the error rates in encoding and decoding data. But the major pieces of the technology are in place. So what is keeping us from shoe box&ndashsize data vaults containing DNA-loaded glass capsules? Cost. &ldquoThe writing process is about a million times too expensive,&rdquo Seelig says.

    Here&rsquos why: Making DNA involves stringing together its nanometers-size molecules one by one with high precision&mdashnot an easy task. And although the cost of sequencing has plummeted due to the booming demand for medical applications such as disease screening and diagnostics, DNA synthesis has not had a similar market driver. Milenkovic paid about $150 to get a string of 1,000 nucleotides synthesized. Sequencing a million nucleotides costs about a cent.

    Interest in data storage from Microsoft and Micron might be just the kind of impulse needed to start lowering costs, Seelig says. Clever engineering and new technologies such as microfluidics and nanopore DNA sequencing, which help miniaturize and speed things up, will also be key. Right now it takes several hours to sequence a few hundred nucleotide pairs&mdashdays to synthesize them&mdashusing multiple instruments and manual preparation of DNA. &ldquoYou&rsquod want all of this in a pretty small box, otherwise you&rsquod lose the advantage of DNA&rsquos storage density,&rdquo Seelig explains.

    If it all works out, Microsoft&rsquos Strauss imagines companies offering archival DNA storage services within the next decade. &ldquoYou could open your browser and upload files to their site or get your bytes back, like cloud storage,&rdquo she says. Or, with as yet unrealized breakthroughs in DNA synthesis and sequencing, &ldquoyou could buy a DNA drive instead of a disk drive.&rdquo


    Bacteria storage - (Oct/12/2005 )

    Is there any way to store bacteria that doesn't involve making an overnight broth culture first? To make a glycerol stock do you need the overnight culture? Or can you just take colonies from an agar plate?

    you want good log phase cells in suspension

    if you want to do it in one day, inoculate a small amount of broth early in the morning (assuming e coli or something that grows fast) and make glycerol stocks in the afternoon

    if you use colonies, many of those cells are in stationary phase and it is harder to get good recovery from the freezer.

    Aimikins is right, the best is to prepare from a fresh culture, but if you are really in a hurry and find no colleague to do it for you, you can always resuspend a colony in 15% glycerol (adding salt like 0.8% NaCl or 4g/l Na Metaphosphate help too) and freeze that.

    Another solution is to prepare conservation medium. You just pick your colony deep into the tube and you can keep your tubes in the dark at room temperaturen for years. But I admit that even if I had succes ressucisitating some culture from the 70's stored this way, I never prepared any, and wouldn't know (or even want) to do that.

    I think mel4n6 is referring to what we used to call stab cultures. There are also slants. Any fairly good intro to micro book will show you how to make them (it's essentailly solid media in a cryo tube), and they do work well.

    it is always better to preserve the fresh grown culture. But it is not always that we need a liquid (broth) culture. you can take colonies from a plate better if these are not very old. then suspend these colonies in a glycerol stock (10% ) and can be preserved for long time.


    Long-term Storage of Bacterial Strains

    Materials and Equipment

    • Permanent marker
    • Fresh liquid bacterial culture for most bacteria, a culture grown overnight, or for approximately 8&ndash12 hours, works best for freezing
    • Micropipette (P1000) (1) and sterile tips (2)
    • Sterile microcentrifuge or screw-cap tube (1)
    • Sterile glycerol (autoclave to sterilize)
    • Liquid nitrogen (optional)
    • Resealable plastic bag
    • Freezer

    Procedure

    1. Using a permanent marker, label a sterile microcentrifuge or screw-cap tube with the date and the name of the bacteria.
    2. Using a micropipette, add 150 µl of sterile glycerol to the tube.
    3. With a new tip, use the micropipette to transfer 850 µl of the bacterial culture to the same tube.
    4. Cap the tube and invert it several times to thoroughly mix the glycerol and bacteria.
    5. If you are going to store the bacteria in a special -80°C freezer, you should first snap-freeze the bacterial stock by dropping it in a container of liquid nitrogen. If you are storing the bacteria in a regular -20°C freezer, the bacterial stock can be placed there with no further treatment.

    Microbiology Laboratory: 15 Infrastructure Components of a Microbiology Laboratory

    Some of the infrastructure components of a microbiology laboratory are: 1. Main Laboratory 2. Instrument Room 3. Stock Culture Room 4. Electron Microscopy Chamber 5. Inoculation Chamber 6. Store Room 7. Laboratory Animal House 8. Store for Animal Feeds, Medicines etc 9. Chamber of the Laboratory Head 10. Staff Room 11. Office Room 12. Library-cum-Reading Room 13. Seminar Hall-cum-Display Room 14. Toilets 15. Garage.

    Aim: To study the infrastructure components of a microbiology laboratory.

    An ideal sophisticated laboratory should have the following infrastructures, which should preferably be designed as shown in Figure 3.1.

    1. Main Laboratory:

    Most of the laboratory activities are performed in the main laboratory. It should have sufficient space inside. A concrete shelf with glazed tile or marble top should project from the wall for keeping the equipments and for performing routine works.

    Few sun mica-top laboratory tables with shelves, sinks and gas connections should be kept in the lab. Prepared chemicals and reagents should be kept on the shelves of the tables. Disinfectant solution should be kept on each table in a bottle along with a sponge pad. Before and after each experiment, the table-top must be cleaned with the disinfectant solution using the sponge pad.

    A dispose jar containing germicide solution such as lysol with a thick layer of cotton at its bottom should be kept on or near each table for the pipettes to be disposed into it after use. At one corner of the lab, an autoclave, preferably a double-jacketed horizontal autoclave should be kept.

    Space should be left out on the concrete shelf for different purposes labeled accordingly such as, ‘waste to be sterilized’, ‘sterilized materials’, ‘materials to be sterilized’, ‘light microscopy’, ‘glassware cleaning’ etc.

    Equipments, such as oven, microbiological incubator, BOD incubator, single- pan balance, double-pane electrical balance, ultrapure water purification system, distilled water plant, shaking water bath, TLC apparatus, UV lamp, magnetic stirrer homogenizer etc. should be kept on the shelf as shown in Figure 3.1.

    2. Instrument Room:

    This room should be kept neat and clean, as most of the sophisticated instruments are kept in it. It should be air-conditioned to make it dust-free and to reduce humidity as well as to avoid high ambient temperatures. Otherwise, accumulation of dust particles on and inside the instruments as well as exposure to high room temperatures decreases the longevity of the instruments.

    At the same time, high humid conditions lead to the rusting of their metallic components and fungal growth on optical parts, such as microscope lenses and phototubes of spectrophotometers. The instruments to be kept on the floor of the room include fridge, refrigerated centrifuge and ultracentrifuge.

    Other equipments, such as single-pan precision balance, Quebec colony counter, electronic colony counter, particle counter, electrophoresis apparatus, UV-cum-visible double beam spectrophotometer, computer, gas chromatography (GC), high performance liquid chromatography (HPLC), pH meter, trinocular research microscope with photomicrography attachment, projection microscope, fluorescence microscope, dark-field microscope, phase-contrast microscope and PCR thermo cycler should be arranged on a continuous concrete shelf projecting from the wall as shown in Figure 3.1.

    3. Stock Culture Room:

    In routine microbiological analysis, very often it is required to isolate different types of bacteria in samples, to maintain the isolated bacteria as pure cultures and to identify them in subsequent days by performing several tests.

    After identification, it is required to compare them with standard pure cultures of the same species obtained from international standard microbiology laboratories such as, American Type Culture Collection (ATCC) of the USA National Collection of Type Cultures (NCTC) of England and Pasteur Culture Collection (PCC) of France.

    These laboratories maintain and supply the standard pure cultures of known bacteria. If a pure culture of unknown bacteria is found to differ from similar standard pure cultures of known bacteria in morphology, staining reactions, biochemical reactions and serological tests, then it is sent to the international standard laboratories.

    After thorough testing in these laboratories, if it is found to differ from the known bacteria, then it is declared ‘new’ and is published in the International Journal of Systematic Bacteriology of the American Society of Microbiologists. It is given a new name (a new genera, or new species or new strain) based on its closeness to the known bacteria.

    The standard pure cultures of known bacteria obtained from the international laboratories are maintained in the stock culture room. Isolated pure cultures of unknown bacteria (stock cultures) are also maintained in this room for further identification. Great care is taken in this room, so that the pure cultures do not get contaminated with other bacteria, which, otherwise, would lead to erroneous results.

    4. Electron Microscopy Chamber:

    The electron microscope is installed in this room and the room is air-conditioned.

    5. Inoculation Chamber:

    This room is meant for inoculation of bacteria i.e. Transfer of bacteria from one container to another. Sometimes, unwanted microbes, usually floating in air on dust particles, may enter into the containers and contaminate the pure stock culture as well as the inoculated ones. To overcome this, the room is kept extremely hygienic. The walls should be plastic-painted and the room should be air-conditioned.

    There should be a laminar flow chamber with gas connection, for inoculation of bacteria. A bottle of disinfectant solution, a sponge pad and a dispose jar should be kept beside the laminar flow chamber as in case of tables in the main lab.

    6. Store Room:

    The store room should be closed from all sides with entrance into the main lab. There should be no window otherwise the chemicals may get spoiled. There should be a number of concrete shelves for storage of chemicals, reagents, glassware’s and other such items. The room should not be opened unless needed. The chemicals should be arranged alphabetically on the shelves for easy location.

    7. Laboratory Animal House:

    Laboratory animals, such as guinea pig and rabbit are required for in-vivo (in natural condition) studies as well as to get blood. These animals are reared in the animal house located slightly away from the main building. The house is designed in such a way that it is well-ventilated and can be easily cleaned daily. This keeps the room hygienic and the animals remain disease-free.

    8. Store for Animal Feeds, Medicines etc:

    It is a small room adjacent to the animal house where animal feeds, medicines and other such items required for rearing the animals are stored.

    9. Chamber of the Laboratory Head:

    This room is meant for the head of the unit. It should have excellent official interior decoration, which should be maintained properly.

    10. Staff Room:

    This room is meant for the scientists and other research personnel.

    11. Office Room:

    This room is meant for the administrative staff of the laboratory.

    12. Library-cum-Reading Room:

    In this room the books should be kept on shelves or inside glass-fitted cupboards (almirahs). There should be a catalogue in wooden box for easy reference. Computer-assisted cataloguing should also be done. Recent journals should be displayed on slanting display boards. Tables and chairs should be arranged for reading inside the library.

    13. Seminar Hall-cum-Display Room:

    The seminar hall should be furnished with required furniture. Besides, there should be a black board, a projection screen, an LCD projector, an overhead projector, a slide projector, an epidiascope, video equipments and audio systems.

    Important charts, diagrams, photos, particularly those highlighting the achievements of the laboratory and ongoing research works should be displayed on the walls. The windows should have deep color curtain to make the room dark, when required for visual presentations.

    14. Toilets:

    The chamber of the head of the laboratory, office room, staff room and seminar hall should have separate toilets in addition to a common toilet.

    15. Garage:

    There should be a garage behind the building for parking of the vehicles, so as to avoid erratic parking. The space surrounding the building should have lawns with tall plants like deodar or pine near the boundary walls.


    Contents

    The word bacteria is the plural of the New Latin bacterium, which is the latinisation of the Greek βακτήριον (bakterion), [17] the diminutive of βακτηρία (bakteria), meaning "staff, cane", [18] because the first ones to be discovered were rod-shaped. [19] [20]

    The ancestors of modern bacteria were unicellular microorganisms that were the first forms of life to appear on Earth, about 4 billion years ago. For about 3 billion years, most organisms were microscopic, and bacteria and archaea were the dominant forms of life. [21] [22] Although bacterial fossils exist, such as stromatolites, their lack of distinctive morphology prevents them from being used to examine the history of bacterial evolution, or to date the time of origin of a particular bacterial species. However, gene sequences can be used to reconstruct the bacterial phylogeny, and these studies indicate that bacteria diverged first from the archaeal/eukaryotic lineage. [23] The most recent common ancestor of bacteria and archaea was probably a hyperthermophile that lived about 2.5 billion–3.2 billion years ago. [24] [25] The earliest life on land may have been bacteria some 3.22 billion years ago. [26]

    Bacteria were also involved in the second great evolutionary divergence, that of the archaea and eukaryotes. Here, eukaryotes resulted from the entering of ancient bacteria into endosymbiotic associations with the ancestors of eukaryotic cells, which were themselves possibly related to the Archaea. [27] [28] This involved the engulfment by proto-eukaryotic cells of alphaproteobacterial symbionts to form either mitochondria or hydrogenosomes, which are still found in all known Eukarya (sometimes in highly reduced form, e.g. in ancient "amitochondrial" protozoa). Later, some eukaryotes that already contained mitochondria also engulfed cyanobacteria-like organisms, leading to the formation of chloroplasts in algae and plants. This is known as primary endosymbiosis. [29] [30]

    Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are about one-tenth the size of eukaryotic cells and are typically 0.5–5.0 micrometres in length. However, a few species are visible to the unaided eye—for example, Thiomargarita namibiensis is up to half a millimetre long [31] and Epulopiscium fishelsoni reaches 0.7 mm. [32] Among the smallest bacteria are members of the genus Mycoplasma, which measure only 0.3 micrometres, as small as the largest viruses. [33] Some bacteria may be even smaller, but these ultramicrobacteria are not well-studied. [34]

    Most bacterial species are either spherical, called cocci (singular coccus, from Greek kókkos, grain, seed), or rod-shaped, called bacilli (sing. bacillus, from Latin baculus, stick). [35] Some bacteria, called vibrio, are shaped like slightly curved rods or comma-shaped others can be spiral-shaped, called spirilla, or tightly coiled, called spirochaetes. A small number of other unusual shapes have been described, such as star-shaped bacteria. [36] This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton, and is important because it can influence the ability of bacteria to acquire nutrients, attach to surfaces, swim through liquids and escape predators. [37] [38]

    Many bacterial species exist simply as single cells, others associate in characteristic patterns: Neisseria form diploids (pairs), Streptococcus form chains, and Staphylococcus group together in "bunch of grapes" clusters. Bacteria can also group to form larger multicellular structures, such as the elongated filaments of Actinobacteria, the aggregates of Myxobacteria, and the complex hyphae of Streptomyces. [39] These multicellular structures are often only seen in certain conditions. For example, when starved of amino acids, Myxobacteria detect surrounding cells in a process known as quorum sensing, migrate towards each other, and aggregate to form fruiting bodies up to 500 micrometres long and containing approximately 100,000 bacterial cells. [40] In these fruiting bodies, the bacteria perform separate tasks for example, about one in ten cells migrate to the top of a fruiting body and differentiate into a specialised dormant state called a myxospore, which is more resistant to drying and other adverse environmental conditions. [41]

    Bacteria often attach to surfaces and form dense aggregations called biofilms, and larger formations known as microbial mats. These biofilms and mats can range from a few micrometres in thickness to up to half a metre in depth, and may contain multiple species of bacteria, protists and archaea. Bacteria living in biofilms display a complex arrangement of cells and extracellular components, forming secondary structures, such as microcolonies, through which there are networks of channels to enable better diffusion of nutrients. [42] [43] In natural environments, such as soil or the surfaces of plants, the majority of bacteria are bound to surfaces in biofilms. [44] Biofilms are also important in medicine, as these structures are often present during chronic bacterial infections or in infections of implanted medical devices, and bacteria protected within biofilms are much harder to kill than individual isolated bacteria. [45]

    Intracellular structures

    The bacterial cell is surrounded by a cell membrane, which is made primarily of phospholipids. This membrane encloses the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential components of the cytoplasm within the cell. [46] Unlike eukaryotic cells, bacteria usually lack large membrane-bound structures in their cytoplasm such as a nucleus, mitochondria, chloroplasts and the other organelles present in eukaryotic cells. [47] However, some bacteria have protein-bound organelles in the cytoplasm which compartmentalize aspects of bacterial metabolism, [48] [49] such as the carboxysome. [50] Additionally, bacteria have a multi-component cytoskeleton to control the localisation of proteins and nucleic acids within the cell, and to manage the process of cell division. [51] [52] [53]

    Many important biochemical reactions, such as energy generation, occur due to concentration gradients across membranes, creating a potential difference analogous to a battery. The general lack of internal membranes in bacteria means these reactions, such as electron transport, occur across the cell membrane between the cytoplasm and the outside of the cell or periplasm. [54] However, in many photosynthetic bacteria the plasma membrane is highly folded and fills most of the cell with layers of light-gathering membrane. [55] These light-gathering complexes may even form lipid-enclosed structures called chlorosomes in green sulfur bacteria. [56]

    Bacteria do not have a membrane-bound nucleus, and their genetic material is typically a single circular bacterial chromosome of DNA located in the cytoplasm in an irregularly shaped body called the nucleoid. [57] The nucleoid contains the chromosome with its associated proteins and RNA. Like all other organisms, bacteria contain ribosomes for the production of proteins, but the structure of the bacterial ribosome is different from that of eukaryotes and Archaea. [58]

    Some bacteria produce intracellular nutrient storage granules, such as glycogen, [59] polyphosphate, [60] sulfur [61] or polyhydroxyalkanoates. [62] Bacteria such as the photosynthetic cyanobacteria, produce internal gas vacuoles, which they use to regulate their buoyancy, allowing them to move up or down into water layers with different light intensities and nutrient levels. [63]

    Extracellular structures

    Around the outside of the cell membrane is the cell wall. Bacterial cell walls are made of peptidoglycan (also called murein), which is made from polysaccharide chains cross-linked by peptides containing D-amino acids. [64] Bacterial cell walls are different from the cell walls of plants and fungi, which are made of cellulose and chitin, respectively. [65] The cell wall of bacteria is also distinct from that of Archaea, which do not contain peptidoglycan. The cell wall is essential to the survival of many bacteria, and the antibiotic penicillin (produced by a fungus called Penicillium) is able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan. [65]

    There are broadly speaking two different types of cell wall in bacteria, that classify bacteria into Gram-positive bacteria and Gram-negative bacteria. The names originate from the reaction of cells to the Gram stain, a long-standing test for the classification of bacterial species. [66]

    Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. Most bacteria have the Gram-negative cell wall, and only the Firmicutes and Actinobacteria (previously known as the low G+C and high G+C Gram-positive bacteria, respectively) have the alternative Gram-positive arrangement. [67] These differences in structure can produce differences in antibiotic susceptibility for instance, vancomycin can kill only Gram-positive bacteria and is ineffective against Gram-negative pathogens, such as Haemophilus influenzae or Pseudomonas aeruginosa. [68] Some bacteria have cell wall structures that are neither classically Gram-positive or Gram-negative. This includes clinically important bacteria such as Mycobacteria which have a thick peptidoglycan cell wall like a Gram-positive bacterium, but also a second outer layer of lipids. [69]

    In many bacteria, an S-layer of rigidly arrayed protein molecules covers the outside of the cell. [70] This layer provides chemical and physical protection for the cell surface and can act as a macromolecular diffusion barrier. S-layers have diverse but mostly poorly understood functions, but are known to act as virulence factors in Campylobacter and contain surface enzymes in Bacillus stearothermophilus. [71]

    Flagella are rigid protein structures, about 20 nanometres in diameter and up to 20 micrometres in length, that are used for motility. Flagella are driven by the energy released by the transfer of ions down an electrochemical gradient across the cell membrane. [72]

    Fimbriae (sometimes called "attachment pili") are fine filaments of protein, usually 2–10 nanometres in diameter and up to several micrometres in length. They are distributed over the surface of the cell, and resemble fine hairs when seen under the electron microscope. Fimbriae are believed to be involved in attachment to solid surfaces or to other cells, and are essential for the virulence of some bacterial pathogens. [73] Pili (sing. pilus) are cellular appendages, slightly larger than fimbriae, that can transfer genetic material between bacterial cells in a process called conjugation where they are called conjugation pili or sex pili (see bacterial genetics, below). [74] They can also generate movement where they are called type IV pili. [75]

    Glycocalyx is produced by many bacteria to surround their cells, and varies in structural complexity: ranging from a disorganised slime layer of extracellular polymeric substances to a highly structured capsule. These structures can protect cells from engulfment by eukaryotic cells such as macrophages (part of the human immune system). [76] They can also act as antigens and be involved in cell recognition, as well as aiding attachment to surfaces and the formation of biofilms. [77]

    The assembly of these extracellular structures is dependent on bacterial secretion systems. These transfer proteins from the cytoplasm into the periplasm or into the environment around the cell. Many types of secretion systems are known and these structures are often essential for the virulence of pathogens, so are intensively studied. [78]

    Endospores

    Certain genera of Gram-positive bacteria, such as Bacillus, Clostridium, Sporohalobacter, Anaerobacter, and Heliobacterium, can form highly resistant, dormant structures called endospores. [79] Endospores develop within the cytoplasm of the cell generally a single endospore develops in each cell. [80] Each endospore contains a core of DNA and ribosomes surrounded by a cortex layer and protected by a multilayer rigid coat composed of peptidoglycan and a variety of proteins. [80]

    Endospores show no detectable metabolism and can survive extreme physical and chemical stresses, such as high levels of UV light, gamma radiation, detergents, disinfectants, heat, freezing, pressure, and desiccation. [81] In this dormant state, these organisms may remain viable for millions of years, [82] [83] [84] and endospores even allow bacteria to survive exposure to the vacuum and radiation in space, possibly bacteria could be distributed throughout the Universe by space dust, meteoroids, asteroids, comets, planetoids or via directed panspermia. [85] [86] Endospore-forming bacteria can also cause disease: for example, anthrax can be contracted by the inhalation of Bacillus anthracis endospores, and contamination of deep puncture wounds with Clostridium tetani endospores causes tetanus. [87]

    Bacteria exhibit an extremely wide variety of metabolic types. [88] The distribution of metabolic traits within a group of bacteria has traditionally been used to define their taxonomy, but these traits often do not correspond with modern genetic classifications. [89] Bacterial metabolism is classified into nutritional groups on the basis of three major criteria: the source of energy, the electron donors used, and the source of carbon used for growth. [90]

    Bacteria either derive energy from light using photosynthesis (called phototrophy), or by breaking down chemical compounds using oxidation (called chemotrophy). [91] Chemotrophs use chemical compounds as a source of energy by transferring electrons from a given electron donor to a terminal electron acceptor in a redox reaction. This reaction releases energy that can be used to drive metabolism. Chemotrophs are further divided by the types of compounds they use to transfer electrons. Bacteria that use inorganic compounds such as hydrogen, carbon monoxide, or ammonia as sources of electrons are called lithotrophs, while those that use organic compounds are called organotrophs. [91] The compounds used to receive electrons are also used to classify bacteria: aerobic organisms use oxygen as the terminal electron acceptor, while anaerobic organisms use other compounds such as nitrate, sulfate, or carbon dioxide. [91]

    Many bacteria get their carbon from other organic carbon, called heterotrophy. Others such as cyanobacteria and some purple bacteria are autotrophic, meaning that they obtain cellular carbon by fixing carbon dioxide. [92] In unusual circumstances, the gas methane can be used by methanotrophic bacteria as both a source of electrons and a substrate for carbon anabolism. [93]

    Nutritional types in bacterial metabolism
    Nutritional type Source of energy Source of carbon Examples
    Phototrophs Sunlight Organic compounds (photoheterotrophs) or carbon fixation (photoautotrophs) Cyanobacteria, Green sulfur bacteria, Chloroflexi, or Purple bacteria
    Lithotrophs Inorganic compounds Organic compounds (lithoheterotrophs) or carbon fixation (lithoautotrophs) Thermodesulfobacteria, Hydrogenophilaceae, or Nitrospirae
    Organotrophs Organic compounds Organic compounds (chemoheterotrophs) or carbon fixation (chemoautotrophs) Bacillus, Clostridium or Enterobacteriaceae

    In many ways, bacterial metabolism provides traits that are useful for ecological stability and for human society. One example is that some bacteria have the ability to fix nitrogen gas using the enzyme nitrogenase. This environmentally important trait can be found in bacteria of most metabolic types listed above. [94] This leads to the ecologically important processes of denitrification, sulfate reduction, and acetogenesis, respectively. [95] [96] Bacterial metabolic processes are also important in biological responses to pollution for example, sulfate-reducing bacteria are largely responsible for the production of the highly toxic forms of mercury (methyl- and dimethylmercury) in the environment. [97] Non-respiratory anaerobes use fermentation to generate energy and reducing power, secreting metabolic by-products (such as ethanol in brewing) as waste. Facultative anaerobes can switch between fermentation and different terminal electron acceptors depending on the environmental conditions in which they find themselves. [98]

    Unlike in multicellular organisms, increases in cell size (cell growth) and reproduction by cell division are tightly linked in unicellular organisms. Bacteria grow to a fixed size and then reproduce through binary fission, a form of asexual reproduction. [99] Under optimal conditions, bacteria can grow and divide extremely rapidly, and bacterial populations can double as quickly as every 9.8 minutes. [100] In cell division, two identical clone daughter cells are produced. Some bacteria, while still reproducing asexually, form more complex reproductive structures that help disperse the newly formed daughter cells. Examples include fruiting body formation by Myxobacteria and aerial hyphae formation by Streptomyces, or budding. Budding involves a cell forming a protrusion that breaks away and produces a daughter cell. [101]

    In the laboratory, bacteria are usually grown using solid or liquid media. Solid growth media, such as agar plates, are used to isolate pure cultures of a bacterial strain. However, liquid growth media are used when the measurement of growth or large volumes of cells are required. Growth in stirred liquid media occurs as an even cell suspension, making the cultures easy to divide and transfer, although isolating single bacteria from liquid media is difficult. The use of selective media (media with specific nutrients added or deficient, or with antibiotics added) can help identify specific organisms. [103]

    Most laboratory techniques for growing bacteria use high levels of nutrients to produce large amounts of cells cheaply and quickly. However, in natural environments, nutrients are limited, meaning that bacteria cannot continue to reproduce indefinitely. This nutrient limitation has led the evolution of different growth strategies (see r/K selection theory). Some organisms can grow extremely rapidly when nutrients become available, such as the formation of algal (and cyanobacterial) blooms that often occur in lakes during the summer. [104] Other organisms have adaptations to harsh environments, such as the production of multiple antibiotics by Streptomyces that inhibit the growth of competing microorganisms. [105] In nature, many organisms live in communities (e.g., biofilms) that may allow for increased supply of nutrients and protection from environmental stresses. [44] These relationships can be essential for growth of a particular organism or group of organisms (syntrophy). [106]

    Bacterial growth follows four phases. When a population of bacteria first enter a high-nutrient environment that allows growth, the cells need to adapt to their new environment. The first phase of growth is the lag phase, a period of slow growth when the cells are adapting to the high-nutrient environment and preparing for fast growth. The lag phase has high biosynthesis rates, as proteins necessary for rapid growth are produced. [107] [108] The second phase of growth is the logarithmic phase, also known as the exponential phase. The log phase is marked by rapid exponential growth. The rate at which cells grow during this phase is known as the growth rate (k), and the time it takes the cells to double is known as the generation time (g). During log phase, nutrients are metabolised at maximum speed until one of the nutrients is depleted and starts limiting growth. The third phase of growth is the stationary phase and is caused by depleted nutrients. The cells reduce their metabolic activity and consume non-essential cellular proteins. The stationary phase is a transition from rapid growth to a stress response state and there is increased expression of genes involved in DNA repair, antioxidant metabolism and nutrient transport. [109] The final phase is the death phase where the bacteria run out of nutrients and die. [110]

    Most bacteria have a single circular chromosome that can range in size from only 160,000 base pairs in the endosymbiotic bacteria Carsonella ruddii, [111] to 12,200,000 base pairs (12.2 Mbp) in the soil-dwelling bacteria Sorangium cellulosum. [112] There are many exceptions to this, for example some Streptomyces and Borrelia species contain a single linear chromosome, [113] [114] while some Vibrio species contain more than one chromosome. [115] Bacteria can also contain plasmids, small extra-chromosomal molecules of DNA that may contain genes for various useful functions such as antibiotic resistance, metabolic capabilities, or various virulence factors. [116]

    Bacteria genomes usually encode a few hundred to a few thousand genes. The genes in bacterial genomes are usually a single continuous stretch of DNA and although several different types of introns do exist in bacteria, these are much rarer than in eukaryotes. [117]

    Bacteria, as asexual organisms, inherit an identical copy of the parent's genomes and are clonal. However, all bacteria can evolve by selection on changes to their genetic material DNA caused by genetic recombination or mutations. Mutations come from errors made during the replication of DNA or from exposure to mutagens. Mutation rates vary widely among different species of bacteria and even among different clones of a single species of bacteria. [118] Genetic changes in bacterial genomes come from either random mutation during replication or "stress-directed mutation", where genes involved in a particular growth-limiting process have an increased mutation rate. [119]

    Some bacteria also transfer genetic material between cells. This can occur in three main ways. First, bacteria can take up exogenous DNA from their environment, in a process called transformation. [120] Many bacteria can naturally take up DNA from the environment, while others must be chemically altered in order to induce them to take up DNA. [121] The development of competence in nature is usually associated with stressful environmental conditions, and seems to be an adaptation for facilitating repair of DNA damage in recipient cells. [122] The second way bacteria transfer genetic material is by transduction, when the integration of a bacteriophage introduces foreign DNA into the chromosome. Many types of bacteriophage exist, some simply infect and lyse their host bacteria, while others insert into the bacterial chromosome. [123] Bacteria resist phage infection through restriction modification systems that degrade foreign DNA, [124] and a system that uses CRISPR sequences to retain fragments of the genomes of phage that the bacteria have come into contact with in the past, which allows them to block virus replication through a form of RNA interference. [125] [126] The third method of gene transfer is conjugation, whereby DNA is transferred through direct cell contact. In ordinary circumstances, transduction, conjugation, and transformation involve transfer of DNA between individual bacteria of the same species, but occasionally transfer may occur between individuals of different bacterial species and this may have significant consequences, such as the transfer of antibiotic resistance. [127] [128] In such cases, gene acquisition from other bacteria or the environment is called horizontal gene transfer and may be common under natural conditions. [129]

    Movement

    Many bacteria are motile (able to move themselves) and do so using a variety of mechanisms. The best studied of these are flagella, long filaments that are turned by a motor at the base to generate propeller-like movement. [130] The bacterial flagellum is made of about 20 proteins, with approximately another 30 proteins required for its regulation and assembly. [130] The flagellum is a rotating structure driven by a reversible motor at the base that uses the electrochemical gradient across the membrane for power. [131]

    Bacteria can use flagella in different ways to generate different kinds of movement. Many bacteria (such as E. coli) have two distinct modes of movement: forward movement (swimming) and tumbling. The tumbling allows them to reorient and makes their movement a three-dimensional random walk. [132] Bacterial species differ in the number and arrangement of flagella on their surface some have a single flagellum (monotrichous), a flagellum at each end (amphitrichous), clusters of flagella at the poles of the cell (lophotrichous), while others have flagella distributed over the entire surface of the cell (peritrichous). The flagella of a unique group of bacteria, the spirochaetes, are found between two membranes in the periplasmic space. They have a distinctive helical body that twists about as it moves. [130]

    Two other types of bacterial motion are called twitching motility that relies on a structure called the type IV pilus, [133] and gliding motility, that uses other mechanisms. In twitching motility, the rod-like pilus extends out from the cell, binds some substrate, and then retracts, pulling the cell forward. [134]

    Motile bacteria are attracted or repelled by certain stimuli in behaviours called taxes: these include chemotaxis, phototaxis, energy taxis, and magnetotaxis. [135] [136] [137] In one peculiar group, the myxobacteria, individual bacteria move together to form waves of cells that then differentiate to form fruiting bodies containing spores. [41] The myxobacteria move only when on solid surfaces, unlike E. coli, which is motile in liquid or solid media. [138]

    Several Listeria and Shigella species move inside host cells by usurping the cytoskeleton, which is normally used to move organelles inside the cell. By promoting actin polymerisation at one pole of their cells, they can form a kind of tail that pushes them through the host cell's cytoplasm. [139]

    Communication

    A few bacteria have chemical systems that generate light. This bioluminescence often occurs in bacteria that live in association with fish, and the light probably serves to attract fish or other large animals. [140]

    Bacteria often function as multicellular aggregates known as biofilms, exchanging a variety of molecular signals for inter-cell communication, and engaging in coordinated multicellular behaviour. [141] [142]

    The communal benefits of multicellular cooperation include a cellular division of labour, accessing resources that cannot effectively be used by single cells, collectively defending against antagonists, and optimising population survival by differentiating into distinct cell types. [141] For example, bacteria in biofilms can have more than 500 times increased resistance to antibacterial agents than individual "planktonic" bacteria of the same species. [142]

    One type of inter-cellular communication by a molecular signal is called quorum sensing, which serves the purpose of determining whether there is a local population density that is sufficiently high that it is productive to invest in processes that are only successful if large numbers of similar organisms behave similarly, as in excreting digestive enzymes or emitting light. [143] [144]

    Quorum sensing allows bacteria to coordinate gene expression, and enables them to produce, release and detect autoinducers or pheromones which accumulate with the growth in cell population. [145]

    Classification seeks to describe the diversity of bacterial species by naming and grouping organisms based on similarities. Bacteria can be classified on the basis of cell structure, cellular metabolism or on differences in cell components, such as DNA, fatty acids, pigments, antigens and quinones. [103] While these schemes allowed the identification and classification of bacterial strains, it was unclear whether these differences represented variation between distinct species or between strains of the same species. This uncertainty was due to the lack of distinctive structures in most bacteria, as well as lateral gene transfer between unrelated species. [147] Due to lateral gene transfer, some closely related bacteria can have very different morphologies and metabolisms. To overcome this uncertainty, modern bacterial classification emphasises molecular systematics, using genetic techniques such as guanine cytosine ratio determination, genome-genome hybridisation, as well as sequencing genes that have not undergone extensive lateral gene transfer, such as the rRNA gene. [148] Classification of bacteria is determined by publication in the International Journal of Systematic Bacteriology, [149] and Bergey's Manual of Systematic Bacteriology. [150] The International Committee on Systematic Bacteriology (ICSB) maintains international rules for the naming of bacteria and taxonomic categories and for the ranking of them in the International Code of Nomenclature of Bacteria. [151]

    The term "bacteria" was traditionally applied to all microscopic, single-cell prokaryotes. However, molecular systematics showed prokaryotic life to consist of two separate domains, originally called Eubacteria and Archaebacteria, but now called Bacteria and Archaea that evolved independently from an ancient common ancestor. [1] The archaea and eukaryotes are more closely related to each other than either is to the bacteria. These two domains, along with Eukarya, are the basis of the three-domain system, which is currently the most widely used classification system in microbiology. [152] However, due to the relatively recent introduction of molecular systematics and a rapid increase in the number of genome sequences that are available, bacterial classification remains a changing and expanding field. [153] [154] For example, Cavalier-Smith argued that the Archaea and Eukaryotes evolved from Gram-positive bacteria. [155]

    The identification of bacteria in the laboratory is particularly relevant in medicine, where the correct treatment is determined by the bacterial species causing an infection. Consequently, the need to identify human pathogens was a major impetus for the development of techniques to identify bacteria. [156]

    The Gram stain, developed in 1884 by Hans Christian Gram, characterises bacteria based on the structural characteristics of their cell walls. [66] The thick layers of peptidoglycan in the "Gram-positive" cell wall stain purple, while the thin "Gram-negative" cell wall appears pink. By combining morphology and Gram-staining, most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli). Some organisms are best identified by stains other than the Gram stain, particularly mycobacteria or Nocardia, which show acid-fastness on Ziehl–Neelsen or similar stains. [157] Other organisms may need to be identified by their growth in special media, or by other techniques, such as serology. [158]

    Culture techniques are designed to promote the growth and identify particular bacteria, while restricting the growth of the other bacteria in the sample. Often these techniques are designed for specific specimens for example, a sputum sample will be treated to identify organisms that cause pneumonia, while stool specimens are cultured on selective media to identify organisms that cause diarrhea, while preventing growth of non-pathogenic bacteria. Specimens that are normally sterile, such as blood, urine or spinal fluid, are cultured under conditions designed to grow all possible organisms. [103] [159] Once a pathogenic organism has been isolated, it can be further characterised by its morphology, growth patterns (such as aerobic or anaerobic growth), patterns of hemolysis, and staining. [160]

    As with bacterial classification, identification of bacteria is increasingly using molecular methods. Diagnostics using DNA-based tools, such as polymerase chain reaction, are increasingly popular due to their specificity and speed, compared to culture-based methods. [161] These methods also allow the detection and identification of "viable but nonculturable" cells that are metabolically active but non-dividing. [162] However, even using these improved methods, the total number of bacterial species is not known and cannot even be estimated with any certainty. Following present classification, there are a little less than 9,300 known species of prokaryotes, which includes bacteria and archaea [163] but attempts to estimate the true number of bacterial diversity have ranged from 10 7 to 10 9 total species—and even these diverse estimates may be off by many orders of magnitude. [164] [165]

    Phylogenetic tree

    According to the phylogenetic analysis of Zhu (2019), the relationships could be the following: [166]

    Despite their apparent simplicity, bacteria can form complex associations with other organisms. These symbiotic associations can be divided into parasitism, mutualism and commensalism. Due to their small size, commensal bacteria are ubiquitous and grow on animals and plants exactly as they will grow on any other surface. However, their growth can be increased by warmth and sweat, and large populations of these organisms in humans are the cause of body odour. [168]

    Predators

    Some species of bacteria kill and then consume other microorganisms, these species are called predatory bacteria. [169] These include organisms such as Myxococcus xanthus, which forms swarms of cells that kill and digest any bacteria they encounter. [170] Other bacterial predators either attach to their prey in order to digest them and absorb nutrients, such as Vampirovibrio chlorellavorus, [171] or invade another cell and multiply inside the cytosol, such as Daptobacter. [172] These predatory bacteria are thought to have evolved from saprophages that consumed dead microorganisms, through adaptations that allowed them to entrap and kill other organisms. [173]

    Mutualists

    Certain bacteria form close spatial associations that are essential for their survival. One such mutualistic association, called interspecies hydrogen transfer, occurs between clusters of anaerobic bacteria that consume organic acids, such as butyric acid or propionic acid, and produce hydrogen, and methanogenic Archaea that consume hydrogen. [174] The bacteria in this association are unable to consume the organic acids as this reaction produces hydrogen that accumulates in their surroundings. Only the intimate association with the hydrogen-consuming Archaea keeps the hydrogen concentration low enough to allow the bacteria to grow. [175]

    In soil, microorganisms that reside in the rhizosphere (a zone that includes the root surface and the soil that adheres to the root after gentle shaking) carry out nitrogen fixation, converting nitrogen gas to nitrogenous compounds. [176] This serves to provide an easily absorbable form of nitrogen for many plants, which cannot fix nitrogen themselves. Many other bacteria are found as symbionts in humans and other organisms. For example, the presence of over 1,000 bacterial species in the normal human gut flora of the intestines can contribute to gut immunity, synthesise vitamins, such as folic acid, vitamin K and biotin, convert sugars to lactic acid (see Lactobacillus), as well as fermenting complex undigestible carbohydrates. [177] [178] [179] The presence of this gut flora also inhibits the growth of potentially pathogenic bacteria (usually through competitive exclusion) and these beneficial bacteria are consequently sold as probiotic dietary supplements. [180]

    Pathogens

    If bacteria form a parasitic association with other organisms, they are classed as pathogens. Pathogenic bacteria are a major cause of human death and disease and cause infections such as tetanus (caused by Clostridium tetani), typhoid fever, diphtheria, syphilis, cholera, foodborne illness, leprosy (caused by Micobacterium leprae) and tuberculosis (caused by Mycobacterium tuberculosis). A pathogenic cause for a known medical disease may only be discovered many years later, as was the case with Helicobacter pylori and peptic ulcer disease. Bacterial diseases are also important in agriculture, with bacteria causing leaf spot, fire blight and wilts in plants, as well as Johne's disease, mastitis, salmonella and anthrax in farm animals. [181]

    Each species of pathogen has a characteristic spectrum of interactions with its human hosts. Some organisms, such as Staphylococcus or Streptococcus, can cause skin infections, pneumonia, meningitis and sepsis, a systemic inflammatory response producing shock, massive vasodilation and death. [182] Yet these organisms are also part of the normal human flora and usually exist on the skin or in the nose without causing any disease at all. Other organisms invariably cause disease in humans, such as the Rickettsia, which are obligate intracellular parasites able to grow and reproduce only within the cells of other organisms. One species of Rickettsia causes typhus, while another causes Rocky Mountain spotted fever. Chlamydia, another phylum of obligate intracellular parasites, contains species that can cause pneumonia or urinary tract infection and may be involved in coronary heart disease. [183] Finally, some species, such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Mycobacterium avium, are opportunistic pathogens and cause disease mainly in people suffering from immunosuppression or cystic fibrosis. [184] [185]

    Bacterial infections may be treated with antibiotics, which are classified as bacteriocidal if they kill bacteria or bacteriostatic if they just prevent bacterial growth. There are many types of antibiotics, and each class inhibits a process that is different in the pathogen from that found in the host. An example of how antibiotics produce selective toxicity are chloramphenicol and puromycin, which inhibit the bacterial ribosome, but not the structurally different eukaryotic ribosome. [186] Antibiotics are used both in treating human disease and in intensive farming to promote animal growth, where they may be contributing to the rapid development of antibiotic resistance in bacterial populations. [187] Infections can be prevented by antiseptic measures such as sterilising the skin prior to piercing it with the needle of a syringe, and by proper care of indwelling catheters. Surgical and dental instruments are also sterilised to prevent contamination by bacteria. Disinfectants such as bleach are used to kill bacteria or other pathogens on surfaces to prevent contamination and further reduce the risk of infection. [188]

    Bacteria, often lactic acid bacteria, such as Lactobacillus and Lactococcus, in combination with yeasts and moulds, have been used for thousands of years in the preparation of fermented foods, such as cheese, pickles, soy sauce, sauerkraut, vinegar, wine and yogurt. [189] [190]

    The ability of bacteria to degrade a variety of organic compounds is remarkable and has been used in waste processing and bioremediation. Bacteria capable of digesting the hydrocarbons in petroleum are often used to clean up oil spills. [191] Fertiliser was added to some of the beaches in Prince William Sound in an attempt to promote the growth of these naturally occurring bacteria after the 1989 Exxon Valdez oil spill. These efforts were effective on beaches that were not too thickly covered in oil. Bacteria are also used for the bioremediation of industrial toxic wastes. [192] In the chemical industry, bacteria are most important in the production of enantiomerically pure chemicals for use as pharmaceuticals or agrichemicals. [193]

    Bacteria can also be used in the place of pesticides in the biological pest control. This commonly involves Bacillus thuringiensis (also called BT), a Gram-positive, soil dwelling bacterium. Subspecies of this bacteria are used as a Lepidopteran-specific insecticides under trade names such as Dipel and Thuricide. [194] Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators and most other beneficial insects. [195] [196]

    Because of their ability to quickly grow and the relative ease with which they can be manipulated, bacteria are the workhorses for the fields of molecular biology, genetics and biochemistry. By making mutations in bacterial DNA and examining the resulting phenotypes, scientists can determine the function of genes, enzymes and metabolic pathways in bacteria, then apply this knowledge to more complex organisms. [197] This aim of understanding the biochemistry of a cell reaches its most complex expression in the synthesis of huge amounts of enzyme kinetic and gene expression data into mathematical models of entire organisms. This is achievable in some well-studied bacteria, with models of Escherichia coli metabolism now being produced and tested. [198] [199] This understanding of bacterial metabolism and genetics allows the use of biotechnology to bioengineer bacteria for the production of therapeutic proteins, such as insulin, growth factors, or antibodies. [200] [201]

    Because of their importance for research in general, samples of bacterial strains are isolated and preserved in Biological Resource Centers. This ensures the availability of the strain to scientists worldwide. [202]

    Bacteria were first observed by the Dutch microscopist Antonie van Leeuwenhoek in 1676, using a single-lens microscope of his own design. [203] He then published his observations in a series of letters to the Royal Society of London. [204] [205] [206] Bacteria were Leeuwenhoek's most remarkable microscopic discovery. They were just at the limit of what his simple lenses could make out and, in one of the most striking hiatuses in the history of science, no one else would see them again for over a century. [207] His observations had also included protozoans which he called animalcules, and his findings were looked at again in the light of the more recent findings of cell theory. [208]

    Christian Gottfried Ehrenberg introduced the word "bacterium" in 1828. [209] In fact, his Bacterium was a genus that contained non-spore-forming rod-shaped bacteria, [210] as opposed to Bacillus, a genus of spore-forming rod-shaped bacteria defined by Ehrenberg in 1835. [211]

    Louis Pasteur demonstrated in 1859 that the growth of microorganisms causes the fermentation process, and that this growth is not due to spontaneous generation (yeasts and molds, commonly associated with fermentation, are not bacteria, but rather fungi). Along with his contemporary Robert Koch, Pasteur was an early advocate of the germ theory of disease. [212] Before them, Ignaz Semmelweis and Joseph Lister had realised the importance of sanitized hands in medical work. Semmelweis ideas was rejected and his book on the topic condemned by the medical community, but after Lister doctors started sanitizing their hands in the 1870s. While Semmelweis who started with rules about handwashing in his hospital in the 1840s predated the spread of the ideas about germs themselves and attributed diseases to "decomposing animal organic matter", Lister was active later. [213]

    Robert Koch, a pioneer in medical microbiology, worked on cholera, anthrax and tuberculosis. In his research into tuberculosis Koch finally proved the germ theory, for which he received a Nobel Prize in 1905. [214] In Koch's postulates, he set out criteria to test if an organism is the cause of a disease, and these postulates are still used today. [215]

    Ferdinand Cohn is said to be a founder of bacteriology, studying bacteria from 1870. Cohn was the first to classify bacteria based on their morphology. [216] [217]

    Though it was known in the nineteenth century that bacteria are the cause of many diseases, no effective antibacterial treatments were available. [218] In 1910, Paul Ehrlich developed the first antibiotic, by changing dyes that selectively stained Treponema pallidum—the spirochaete that causes syphilis—into compounds that selectively killed the pathogen. [219] Ehrlich had been awarded a 1908 Nobel Prize for his work on immunology, and pioneered the use of stains to detect and identify bacteria, with his work being the basis of the Gram stain and the Ziehl–Neelsen stain. [220]

    A major step forward in the study of bacteria came in 1977 when Carl Woese recognised that archaea have a separate line of evolutionary descent from bacteria. [3] This new phylogenetic taxonomy depended on the sequencing of 16S ribosomal RNA, and divided prokaryotes into two evolutionary domains, as part of the three-domain system. [1]

    Adams, Casey J. Meade, Thomas J. (2021). "Chapter 15. Imaging Bacteria with Contrast-Enhanced Magnetic Resonance". Metal Ions in Bio-Imaging Techniques. Springer. pp. 425–435. doi:10.1515/9783110685701-021.


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