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How do I test if microbes have survived after dehydration?

How do I test if microbes have survived after dehydration?


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I have a solution containing various bacteria and fungi. My aim is to place solution on filter paper, and wait until it dries. I then wish to test if the organisms have survived, either on the dried paper, or by rehydrating it (maybe placing the paper into water). Can someone provide advice on the type of test(s) I should perform to achieve this result? Preferably the test would be easily sourced (purchase online?) and relatively cheap and easy as I am quite inexperienced in this area. Thank you in advance for any advice that you can provide.


Tardigrades turn into glass to survive complete dehydration

They are probably the toughest creatures on Earth, and now we know how they manage to survive years of complete dehydration.

Water bears, or tardigrades, have been recorded surviving the vacuum of space, high doses of radiation and pressure. These water dwelling creatures can also survive dry environments in a shrivelled-up, dormant state for as long as a decade, reviving within an hour when exposed to water.

To pull off this remarkable trick, the animals rely on proteins unique to them, called tardigrade-specific intrinsically disordered proteins (TDPs).

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When there is water around, these anti-dehydration proteins are jelly-like and don’t form into well-defined three-dimensional structures like most known proteins.

But when water bears start to dry out, these proteins turn into a kind of glassy sanctuary that cocoons all dehydration-sensitive materials in the animal from harm.

“When the animal completely desiccates, the TDPs vitrify, turning the cytoplasmic fluid of cells into glass,” says lead author Thomas Boothby of the University of North Carolina at Chapel Hill.

“We think this glassy mixture is trapping [other] desiccation-sensitive proteins and other biological molecules and locking them in place, physically preventing them from unfolding, breaking apart or aggregating together,” says Boothby.


Materials and methods

Insects

Cryptobiotic larvae of Polypedilum vanderplanki Hint. were collected from rock pools in Nigeria in 2000. They were transferred to the laboratory and put into a plastic container (200 mm×300 mm×100 mm)containing water (depth, 20-30 mm) on autoclaved soil (depth, 20-30 mm). The rearing water was aerated continuously. The container was covered with a nylon-mesh cage (200 mm×300 mm×250-300 mm). The larvae were reared for successive generations under controlled light (13 h: 11 h light:dark) and temperature (27°C).

Desiccating procedure

Groups of 3-5 larvae were placed on pieces of filter paper with 0.44 ml of distilled water in a glass Petri dish (diameter 65 mm, height, 20 mm). Two or three of these dishes were immediately transferred to a desiccator (<5%relative humidity) at room temperature (24-26°C) and gradually dried over a period of 48 h (0.22-0.23 ml day -1 ).

Desiccation and recovery of intact and treated larvae

After ligation (applied behind the head or thorax), the head or head and thorax segments were severed from final instar larvae of a similar body mass(approximately 1 mg) in iced water. The remaining body parts were incubated in distilled water for one day, and then completely dried over 3 days in the desiccator. Intact larvae were transferred directly to the desiccator. Subsequently, after being rehydrated by immersion in distilled water, the larvae were observed closely every 0.5-1 h to check their recovery. Larvae were judged to survive if they could repeatedly contract their abdomen. Because insects have an open circulatory system, radical treatments such as ligation and decapitation are routinely used, particularly in the field of insect endocrinology (Wigglesworth,1972).

Sugar and polyol measurements

Each group of 3-5 intact or operated larvae was placed in a desiccating Petri dish or in distilled water for 12, 18, 24, 30, 36, 42, 48 or 72 h, and then homogenized individually with 0.1 mg of sorbitol as an internal standard in 0.2 ml of 90% ethanol. After membrane filtration (pore size 0.45 μm),the supernatant was dried under a stream of nitrogen gas at 60°C and the dried residue dissolved in 500 μl of MilliQ water (Millipore). The samples were analysed on a Shimadzu HPLC system (LC-10A system, Shimadzu, Japan)equipped with a guard column (Shim-pack SCR-C, 4.0 mm×50 mm Shimadzu,Japan) connected to an analytical column (Shim-pack SCR-101C, 7.9 mm×300 mm Shimadzu, Japan) and a reflective index detector (RID-6A Shimadzu,Japan). The columns were heated to 80°C, and MilliQ water as the mobile phase was allowed to flow at the rate of 0.8 ml min -1 . The injection volume was set at 10 or 20 μl. Standard trehalose and sorbitol solutions were prepared in MilliQ water in the range of 1-5,000 μg ml -1 . From the HPLC profile, trehalose and sorbitol could be quantified, at least in the higher range.


1. Bad Breath Is a Possible Warning Sign of Dehydration

Saliva has antibacterial properties, but dehydration can prevent your body from making enough saliva.

“If you’re not producing enough saliva, you can get bacterial overgrowth in the mouth, and one of the side effects of that is bad breath,” says John Higgins, MD, a professor of medicine at the University of Texas in Houston and the chief of cardiology at Lyndon B. Johnson General Hospital in Houston.

It’s the same reason you may wake up with “morning breath”: Saliva production slows down during sleep, notes the Mayo Clinic, leading to an unpleasant taste in the mouth as bacteria grow. So the next time your mouth seems dry and your breath smells less-than-fresh, it may be time to rehydrate.


Genetic mechanism prevents kidney injury after severe dehydration

Millions of people die every year from dehydration as a result of exposure and illness. In humans, even the most minor dehydration can compromise the kidneys causing lifelong, irreparable issues or even death. However, some animals living in desert environments are able to survive both acute and chronic dehydration. While these animals, like cactus mice, have evolved over time to deal with environmental stressors like dehydration, researchers at the University of New Hampshire have found it's not the physical makeup that is helping them survive, but rather their genetic makeup.

"Initially, we thought that maybe their kidneys are structurally different from people, but they're not," said Matt MacManes, assistant professor of genome enabled biology at UNH and lead author of the study. "However, when exposed to acute dehydration, no kidney injury was apparent, which would definitely be the case for humans exposed to similar levels of dehydration, suggesting their genes may be what's preventing widespread kidney damage."

"The kidney is the canary in the coal mine when it comes to dehydration," continues MacManes. "The exciting outcome of this research is that the molecular toolkit of the cactus mouse has orthologues, or related genes, in humans. These provide the potential for development of drugs or other therapies that could help protect the human body from the damages of dehydration." Such a response could be extremely valuable in a wide variety of situations -- for people with renal failure, where water is severally limited due to geography or possibly global climate change, for troops deployed in the desert, and perhaps even in space travel.

To understand how desert-adapted cactus mice (Peromyscus eremicus) survive, the study recently published in the American Journal of Renal Physiology outlines how the researchers modeled a desert-like condition. The mice that went without water for 72 hours lost on average 23 percent of their body weight, which would be fatal for humans. Even though dehydrated, the mice continued to be active, eat, and interact normally. Researchers analyzed several other factors including serum electrolytes (sodium, calcium, bicarbonate ion) as well as blood urea nitrogen (BUN) and creatinine. While both were slightly elevated, gene-based biomarkers for kidney injury, were not, which suggests kidney injury is not occurring.

Further analysis found genes that are important in modulating electrolytes were very active, as were genes responsible for maintaining kidney blood pressure.


The Plasma Membrane

The plasma membrane is a thin lipid bilayer (6 to 8 nanometers) that completely surrounds the cell and separates the inside from the outside. Its selectively-permeable nature keeps ions, proteins, and other molecules within the cell, preventing them from diffusing into the extracellular environment, while other molecules may move through the membrane. The general structure of a cell membrane is a phospholipid bilayer composed of two layers of lipid molecules. In archaeal cell membranes, isoprene (phytanyl) chains linked to glycerol replace the fatty acids linked to glycerol in bacterial membranes. Some archaeal membranes are lipid monolayers instead of bilayers.

Figure (PageIndex<1>): Plasma membrane structure: Archaeal phospholipids differ from those found in Bacteria and Eukarya in two ways. First, they have branched phytanyl sidechains instead of linear ones. Second, an ether bond instead of an ester bond connects the lipid to the glycerol.


Evolution in a test tube: These bacteria survive on deadly copper surfaces

The descendants of regular wild-type bacteria can evolve to survive for a long time on metallic copper surfaces that would usually kill them within a few minutes. An international research team led by Martin Luther University Halle-Wittenberg (MLU) and the Bundeswehr Institute of Microbiology was able to produce these tiny survivalists in the lab and has been able to study them more closely. The team reports on its findings in Applied and Environmental Microbiology.

Bacterial infections are usually treated with antibiotics. However, in recent decades many pathogenic bacteria have developed an increasing tolerance to common drugs. So-called multidrug-resistant bacteria are of particular concern as they can no longer be combated with most antibiotics. Copper surfaces -- for example on door handles -- are a good weapon to fight these germs. "Copper surfaces are a sure-fire way to kill bacteria. Most bacteria die within minutes after landing on a copper surface," explains Professor Dietrich H. Nies, a microbiologist at MLU. Copper is a vital trace element for bacteria -- but only in very small quantities. On the copper surfaces, however, the bacteria are literally flooded to death with copper ions because that they can no longer stave them off using their normal defence strategies.

Nies' research team wanted to find out if and how quickly two typical species of bacteria, Escherichia coli and Staphylococcus aureus, are theoretically able to adapt to survive on copper surfaces. The team therefore placed the bacteria on the surfaces for only a few minutes before returning them to a normal culture medium where they were allowed to recover. This process was repeated several times, with the survivors gradually being exposed to the deadly surface for longer and longer periods of time. Within three weeks, the researchers had produced bacteria that could survive for more than one hour on a copper surface. "Outside the laboratory, conditions are obviously not as ideal. But if copper surfaces are not cleaned regularly, insulating layers of grease can begin to form on them, which could produce a similar development over time," says Nies.

Using comprehensive genetic analyses, the team sought to understand why the bacteria no longer died on the surfaces. "We were unable to find a gene that made them resistant to the deadly effect of metallic copper surfaces," says Nies. Instead, the team observed a phenomenon among the surviving bacteria that was already known for quite some time, although in a slightly different manner: the bacteria's metabolism slowed down to a bare minimum and they fell into a kind of hibernation. Because most antibiotics aim to disrupt the metabolism of growing bacteria, they are almost completely ineffective against these special bacteria, which are also known as "persisters." "No matter how well an antibiotic works, there are always a handful of persisters in every generation," explains Nies. However, these are not considered antibiotic-resistant bacteria, because their offspring are once again susceptible to the drugs.

Normally only a tiny proportion of bacteria become persisters. However, in the case of the isolated bacteria, it was the entire population. Although they were able to grow just as fast as their predecessors, they were also able to rescue themselves by switching rapidly into an early state of persistence under adverse conditions. The scientists were concerned one additional thing they observed: "The bacteria also inherited this capability over 250 generations, even though the offspring had not come into contact with a copper surface," says Nies. The team therefore recommends that copper surfaces be cleaned regularly and thoroughly with special agents so that no persister bacteria can develop in the first place. At the same time, Nies points out that the use of copper surfaces is only one of many ways -- including antibiotics -- to effectively combat harmful bacteria.


The Prokaryotic Cell

All cells share four common components: (1) a plasma membrane, an outer covering that separates the cell’s interior from its surrounding environment (2) cytoplasm, consisting of a jelly-like region within the cell in which other cellular components are found (3) DNA, the genetic material of the cell and (4) ribosomes, particles that synthesize proteins. Prokaryotic cells differ from eukaryotic cells in several key ways.

Figure 2. The features of a typical prokaryotic cell are shown.

A prokaryotic cell is a simple, single-celled (unicellular) organism that lacks a nucleus, or any other membrane-bound organelle. Prokaryotic DNA is found in the central part of the cell: a darkened region called the nucleoid (Figure 2).

Some prokaryotes have flagella, pili, or fimbriae. Flagella are used for locomotion, while most pili are used to exchange genetic material during a type of reproduction called conjugation. Many prokaryotes also have a cell wall and capsule. The cell wall acts as an extra layer of protection, helps the cell maintain its shape, and prevents dehydration. The capsule enables the cell to attach to surfaces in its environment.

Reproduction

Reproduction in prokaryotes is asexual and usually takes place by binary fission. Recall that the DNA of a prokaryote exists as a single, circular chromosome. Prokaryotes do not undergo mitosis. Rather the chromosome is replicated and the two resulting copies separate from one another due to the growth of the cell. The prokaryote, now enlarged, is pinched inward at its equator and the two resulting cells, which are clones, separate. Binary fission does not provide an opportunity for genetic recombination or genetic diversity, but prokaryotes can share genes by three other mechanisms.

In transformation, the prokaryote takes in DNA found in its environment that is shed by other prokaryotes. If a nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and incorporates the new DNA into its own chromosome, it too may become pathogenic. In transduction, bacteriophages, the viruses that infect bacteria, sometimes also move short pieces of chromosomal DNA from one bacterium to another. Transduction results in a recombinant organism. Archaea are not affected by bacteriophages but instead have their own viruses that translocate genetic material from one individual to another. In conjugation, DNA is transferred from one prokaryote to another by means of a pilus, which brings the organisms into contact with one another. The DNA transferred can be in the form of a plasmid, a small circular piece of extrachromosomal DNA, or as a hybrid, containing both plasmid and chromosomal DNA. These three processes of DNA exchange are shown in Figure 3.

Reproduction can be very rapid: a few minutes for some species. This short generation time coupled with mechanisms of genetic recombination and high rates of mutation result in the rapid evolution of prokaryotes, allowing them to respond to environmental changes (such as the introduction of an antibiotic) very quickly.

Figure 3. Besides binary fission, there are three other mechanisms by which prokaryotes can exchange DNA. In (a) transformation, the cell takes up prokaryotic DNA directly from the environment. The DNA may remain separate as plasmid DNA or be incorporated into the host genome. In (b) transduction, a bacteriophage injects DNA into the cell that contains a small fragment of DNA from a different prokaryote. In (c) conjugation, DNA is transferred from one cell to another via a mating bridge that connects the two cells after the sex pilus draws the two bacteria close enough to form the bridge.

The Evolution of Prokaryotes

How do scientists answer questions about the evolution of prokaryotes? Unlike with animals, artifacts in the fossil record of prokaryotes offer very little information. Fossils of ancient prokaryotes look like tiny bubbles in rock. Some scientists turn to genetics and to the principle of the molecular clock, which holds that the more recently two species have diverged, the more similar their genes (and thus proteins) will be. Conversely, species that diverged long ago will have more genes that are dissimilar.

Scientists at the NASA Astrobiology Institute and at the European Molecular Biology Laboratory collaborated to analyze the molecular evolution of 32 specific proteins common to 72 species of prokaryotes. [1] The model they derived from their data indicates that three important groups of bacteria—Actinobacteria, Deinococcus, and Cyanobacteria (which the authors call Terrabacteria)—were the first to colonize land. (Recall that Deinococcus is a genus of prokaryote—a bacterium—that is highly resistant to ionizing radiation.) Cyanobacteria are photosynthesizers, while Actinobacteria are a group of very common bacteria that include species important in decomposition of organic wastes.

The timelines of divergence suggest that bacteria (members of the domain Bacteria) diverged from common ancestral species between 2.5 and 3.2 billion years ago, whereas archaea diverged earlier: between 3.1 and 4.1 billion years ago. Eukarya later diverged off the Archaean line. The work further suggests that stromatolites that formed prior to the advent of cyanobacteria (about 2.6 billion years ago) photosynthesized in an anoxic environment and that because of the modifications of the Terrabacteria for land (resistance to drying and the possession of compounds that protect the organism from excess light), photosynthesis using oxygen may be closely linked to adaptations to survive on land.


How Salt Kills Bacteria

It's this process of osmosis that makes high concentrations of salt kill bacteria. When there are high salt concentrations outside of a bacterial cell, water from inside the bacteria diffuses out of the cell in order to reach equilibrium and equalize the salt concentration. When bacterial cells lose all of their water like this, it:

  • Dehydrates the cell
  • Causes the loss of the cell's structure
  • Leads to enzyme and protein malfunction
  • Eventually leads to cell death

Simply put: Salt sucks all of the water out of the bacteria, which leads to cell death. However, some bacteria are tolerant of salty conditions. These types of bacteria are called halotolerant.


Endospores and Epulopiscium

Some Epulopiscium-like surgeonfish symbionts form mature endospores at night. These spores possess all of the characteristic protective layers seen in B. subtilis endospores and also contain large amounts of dipicolinic acid. These are the largest endospores described thus far, with the largest being over 4000 times larger than a Bacillus subtilis endospore.

The formation of endospores may help maintain the symbiotic association between these Epulopiscium-like symbionts and their surgeonfish hosts. Since endospore formation coincides with periods in which the host surgeonfish is not actively feeding, the cells do not need to compete for the limited nutrients present in the gut at night. The protective properties of the endospores also allow them to survive passage to new surgeonfish hosts. The fish may also benefit from this relationship because it is able to maintain stable microbial populations that assist in digestion and may receive a nutritional gain from microbial products released during mother cell death and spore germination.

Daily life cycle of endospore-forming Epulopiscium-like symbionts.

Endospore formation in some Epulopiscium-like symbionts follows a daily cycle:
A) Polar septa are formed at the poles of the cell.
B) Forespores become engulfed.
C) Forespores gradually increase in size within the mother cell through the day.
D) In late afternoon, final preparations for endospore dormancy.
E) Endospores mature and remain dormant throughout most of the night.
F) Just before sunrise, the endospores germinate and are released from mother cell to repeat the cycle.



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