# What can be used for DNA preservation?

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I am neither a student nor really advanced in biology, I am just writing a tabletop role-playing game scenario that I want as realistic as possible. In the story, the players find an old laboratory where genetic mutations were studied, but as the electricity is off since 40 years, most of the embryos are "rotten". I still need to have some embryos still "fresh" so someone could get DNA from it and continue the experiment. Problem is, I thought about formol (the experiment is supposed to have taken place in the 1980's, so the health problem is not one right now), but I've read that formol alters and destroys the DNA. I've seen older ways with alcohol, but the process doesn't seem as efficient.

What liquid (or technic) could have been used in the 80's to preserve embryos and their DNA so we could still exploit it 40 years later, and with not electricity?

There are a few plausible ways:

As someone mentioned in the comments; Alcohol works well for this. We have samples from a hundred years ago (e.g. Thylacine), which have been stored at room temperature in a museum for that period of time and have had DNA easily extracted from them.

Unfortunately for many species, alcohol preservation fell out of favour due to it shrinking (by dehydration) specimens, so that they no longer look "realistic" in terms of morphology (something that is very important for classical identification methods). In addition, ethanol and other alcohols tend to evaporate fairly fast if not sealed properly. For these reasons aldehyde based solutions came into favour about 100 years ago. The downside to this is that formaldehyde and other aldehydes cross-link the genetic material, making it more or less impossible to extract and use.

Another possible method is dehydration - mummification. Small samples like embryos or tissues can be dehydrated quite fast. In the absence of water, these samples should last more or less forever - we can extract DNA from mummies in many places around the world, but these ones might interest you in particular.

There are also specialist chemical solutions such as RNAlater that are routinely used in labs for preservation of precious genetic material.

Given that we knew these sorts of things in the 1980's and that the lab was staffed with competent scientists, and that they knew that the lab was going to shut down but would re-start at some time in the future, then it is entirely plausible that they would have used alcohol or something like RNAlater.

In the absence of this knowledge, but still competent scientists, it is much more likely that they would have used some sort of cold (frozen) storage method; whether this was cryopreservation (liquid nitrogen), such as is used for storage of cells or a specialist -80 $$^circ$$C freezer or a -20 $$^circ$$C freezer like you might find at your house. Some samples may have been still stored in chemical preservation in the interests of space-saving in freezers though.

## Should Genetic Engineering Be Used as a Tool for Conservation?

Researchers are considering ways to use synthetic biology for such conservation goals as eradicating invasive species or strengthening endangered coral. But environmentalists are worried about the ethical questions and unwanted consequences of this new gene-altering technology.

The worldwide effort to return islands to their original wildlife, by eradicating rats, pigs, and other invasive species, has been one of the great environmental success stories of our time. Rewilding has succeeded on hundreds of islands, with beleaguered species surging back from imminent extinction, and dwindling bird colonies suddenly blossoming across old nesting grounds.

But these restoration campaigns are often massively expensive and emotionally fraught, with conservationists fearful of accidentally poisoning native wildlife, and animal rights activists having at times fiercely opposed the whole idea. So what if it were possible to rid islands of invasive species without killing a single animal? And at a fraction of the cost of current methods?

That’s the tantalizing – but also worrisome – promise of synthetic biology, a Brave New World sort of technology that applies engineering principles to species and to biological systems. It’s genetic engineering, but made easier and more precise by the new gene editing technology called CRISPR, which ecologists could use to splice in a DNA sequence designed to handicap an invasive species, or to help a native species adapt to a changing climate. “Gene drive,” another new tool, could then spread an introduced trait through a population far more rapidly than conventional Mendelian genetics would predict.

Synthetic biology, also called synbio, is already a multi-billion dollar market, for manufacturing processes in pharmaceuticals, chemicals, biofuels, and agriculture. But many conservationists consider the prospect of using synbio methods as a tool for protecting the natural world deeply alarming. Jane Goodall, David Suzuki, and others have signed a letter warning that use of gene drives gives “technicians the ability to intervene in evolution, to engineer the fate of an entire species, to dramatically modify ecosystems, and to unleash large-scale environmental changes, in ways never thought possible before.” The signers of the letter argue that such a “powerful and potentially dangerous technology … should not be promoted as a conservation tool.”

On the other hand, a team of conservation biologists writing early this year in the journal Trends in Ecology and Evolution ran off a list of promising applications for synbio in the natural world, in addition to island rewilding:

• Transplanting genes for resistance to white nose syndrome into bats, and for chytrid fungus into frogs and other amphibians.
• Giving corals that are vulnerable to bleaching carefully selected genes from nearby corals that are more tolerant of heat and acidity.
• Using artificial microbiomes to restore soils damaged by mining or pollution.
• Eliminating populations of feral cats and dogs without euthanasia or surgical neutering, by producing generations that are genetically programmed to be sterile, or skewed to be overwhelmingly male.
• And eradicating mosquitoes without pesticides, particularly in Hawaii, where they are highly destructive newcomers.

Kent Redford, a conservation consultant and co-author of that article, argues that conservationists and synbio engineers alike need to overcome what now amounts to mutual ignorance. Conservationists tend to have limited and often outdated knowledge of genetics and molecular biology, he says. In a 2014 article in Oryx, he quoted one conservationist flatly declaring, “Those were the courses we flunked.” Stanford University’s Drew Endy, one of the founders of synbio, volunteers in turn that 18 months ago he had never heard of the IUCN—the International Union for Conservation of Nature—or its “Red List” of endangered species. “In engineering school, the ignorance gap is terrific,” he adds. “But it’s symmetric ignorance.”

At a major synbio conference he organized last month in Singapore, Endy invited Redford and eight other conservationists to lead a session on biodiversity, with the aim, he says, of getting engineers building the bioeconomy “to think about the natural world ahead of time … My hope is that people are no longer merely naïve in terms of their industrial disposition.”

Likewise, Redford and the co-authors of the article in Trends in Ecology and Evolution, assert that “it would be a disservice to the goal of protecting biodiversity if conservationists do not participate in applying the best science and thinkers to these issues.” They argue that “it is necessary to adapt the culture of conservation biologists to a rapidly-changing reality”—including the effects of climate change and emerging diseases. “Twenty-first century conservation philosophy,” the co-authors conclude, should “embrace concepts of synthetic biology, and both seek and guide appropriate synthetic solutions to aid biodiversity.”

The debate over “synthetic biodiversity conservation,” as the Trends in Ecology and Evolution authors term it, had its origins in a 2003 paper by Austin Burt, an evolutionary geneticist at Imperial College London. He proposed a dramatically new tool for genetic engineering, based on certain naturally occurring “selfish genetic elements,” which manage to propagate themselves in as much as 99 percent of the next generation, rather than the usual 50 percent. Burt thought that it might be possible to use these “super-Mendelian” genes as a Trojan horse, to rapidly distribute altered DNA, and thus “to genetically engineer natural populations.” It was impractical at the time. But development of CRISPR technology soon brought the idea close to reality, and researchers have since demonstrated the effectiveness of “gene drive,” as the technique became known, in laboratory experiments on malaria mosquitoes, fruit flies, yeast, and human embryos.

Burt proposed one particularly ominous-sounding application for this new technology: It might be possible under certain conditions, he thought, that “a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations.” And this is, in fact, likely to be the first practical application of synthetic biodiversity conservation in the field. Eradicating invasive populations is of course the inevitable first step in island rewilding projects.

The proposed eradication technique is to use the gene drive to deliver DNA that determines the gender of offspring. Because the gene drive propagates itself so thoroughly through subsequent generations, it can quickly cause a population to become almost all male and soon collapse. The result, at least in theory, is the elimination of mice, rats, or other invasive species from an island without anyone having killed anything.

Research to test the practicality of the method—including moral, ethical, and legal considerations—is already under way through a research consortium of nonprofit groups, universities, and government agencies in Australia, New Zealand, and the United States. At North Carolina State University, for instance, researchers have begun working with a laboratory population of invasive mice taken from a coastal island. They need to determine how well a wild population will accept mice that have been altered in the laboratory.

“The success of this idea depends heavily,” according to gene drive researcher Megan Serr, “on the genetically modified male mice being ‘studs’ with the island lady mice … Will she want a hybrid male that is part wild, part lab?” Beyond that, the research program needs to figure out how many modified mice to introduce to eradicate an invasive population in a habitat of a particular size. Other significant practical challenges will also undoubtedly arise. For instance, a study early this year in the journal Genetics concluded that resistance to CRISPR-modified gene drives “should evolve almost inevitably in most natural populations.”

Political and environmental resistance is also likely to develop. In an email, MIT evolutionary biologist Kevin Esvelt asserted that CRISPR-based gene drives are “not suited for conservation due to the very high risk of spreading” beyond the target species or environment. Even a gene drive system introduced to quickly eradicate an introduced population from an island, he added, “still is likely to have over a year to escape or be deliberately transported off-island. If it is capable of spreading elsewhere, that is a major problem.”

Even “a highly contained field trial on a remote island is probably a decade or so away,” said Heath Packard, of Island Conservation, a nonprofit that has been involved in numerous island rewilding projects and is now part of the research consortium. “We are committed to a precautionary step-wise approach, with plenty of off-ramps, if it turns out to be too risky or not ethical.” But his group notes that 80 percent of known extinctions over the past 500 or so years have occurred on islands, which are also home to 40 percent of species now considered at risk of extinction. That makes it important at least to begin to study the potential of synthetic biodiversity conservation.

Even if conservationists ultimately balk at these new technologies, business interests are already bringing synbio into the field for commercial purposes. For instance, a Pennsylvania State University researcher recently figured out how to use CRISPR gene editing to turn off genes that cause supermarket mushrooms to turn brown. The U.S. Department of Agriculture last year ruled that these mushrooms would not be subject to regulation as a genetically modified organism because they contain no genes introduced from other species.

With those kinds of changes taking place all around them, conservationists “absolutely must engage with the synthetic biology community,” says Redford, “and if we don’t do so it will be at our peril.” Synbio, he says, presents conservationists with “a huge range of questions that no one is paying attention to yet.”

Richard Conniff is a National Magazine Award-winning writer whose articles have appeared in The New York Times, Smithsonian, The Atlantic, National Geographic, and other publications. His latest book is House of Lost Worlds: Dinosaurs, Dynasties, and the Story of Life on Earth. He is a frequent contributor to Yale Environment 360. More about Richard Conniff →

### Step 1. Breaking cells open to release the DNA

The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing , and put into a solution containing salt. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA.

A detergent is then added. The detergent breaks down the lipids in the cell membrane and nuclei . DNA is released as these membranes are disrupted.

### Step 2. Separating DNA from proteins and other cellular debris

To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible. This can be done by a variety of methods. Often a protease ( protein enzyme) is added to degrade DNA-associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample.

### Step 3. Precipitating the DNA with an alcohol

Finally, ice-cold alcohol (either ethanol or isopropanol ) is carefully added to the DNA sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and can be spooled out. If there is lots of DNA, you may see a stringy, white precipitate.

### Step 4. Cleaning the DNA

The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use.

### Step 5. Confirming the presence and quality of the DNA

For further lab work, it is important to know the concentration and quality of the DNA.

Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in your sample and give an indication of its quality.

## What does DNA do?

DNA contains the instructions needed for an organism to develop, survive and reproduce. To carry out these functions, DNA sequences must be converted into messages that can be used to produce proteins, which are the complex molecules that do most of the work in our bodies.

Each DNA sequence that contains instructions to make a protein is known as a gene. The size of a gene may vary greatly, ranging from about 1,000 bases to 1 million bases in humans. Genes only make up about 1 percent of the DNA sequence. DNA sequences outside this 1 percent are involved in regulating when, how and how much of a protein is made.

DNA contains the instructions needed for an organism to develop, survive and reproduce. To carry out these functions, DNA sequences must be converted into messages that can be used to produce proteins, which are the complex molecules that do most of the work in our bodies.

Each DNA sequence that contains instructions to make a protein is known as a gene. The size of a gene may vary greatly, ranging from about 1,000 bases to 1 million bases in humans. Genes only make up about 1 percent of the DNA sequence. DNA sequences outside this 1 percent are involved in regulating when, how and how much of a protein is made.

Diagnosis of certain medical conditions can often be made from DNA extracted from a patient. Conditions that can be diagnosed by genetic testing include cystic fibrosis, sickle-cell anemia, fragile x syndrome, Huntington's disease, hemophilia A, Down's syndrome and Tay-Sachs disease. In addition to diagnosing existing diseases, geneticists also commonly test whether a person is a carrier of a particular genetic condition but does not have any symptoms of the disease.

A well-known use for genetic extraction is genetic fingerprinting, a process that matches genetic material from an individual with other genetic material available. One example is paternity testing, to determine someone’s biological father. Another common use for DNA extraction in identity verification is for forensic purposes. Genetic material from an individual can be compared to genetic material at a crime scene, such as blood, for example. Genetic verification has worked both to place a person at the scene of a crime and to exonerate people falsely convicted of a crime.

## Cryopreservation

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Cryopreservation, the preservation of cells and tissue by freezing.

Cryopreservation is based on the ability of certain small molecules to enter cells and prevent dehydration and formation of intracellular ice crystals, which can cause cell death and destruction of cell organelles during the freezing process. Two common cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol. Glycerol is used primarily for cryoprotection of red blood cells, and DMSO is used for protection of most other cells and tissues. A sugar called trehalose, which occurs in organisms capable of surviving extreme dehydration, is used for freeze-drying methods of cryopreservation. Trehalose stabilizes cell membranes, and it is particularly useful for the preservation of sperm, stem cells, and blood cells.

Most systems of cellular cryopreservation use a controlled-rate freezer. This freezing system delivers liquid nitrogen into a closed chamber into which the cell suspension is placed. Careful monitoring of the rate of freezing helps to prevent rapid cellular dehydration and ice-crystal formation. In general, the cells are taken from room temperature to approximately −90 °C (−130 °F) in a controlled-rate freezer. The frozen cell suspension is then transferred into a liquid-nitrogen freezer maintained at extremely cold temperatures with nitrogen in either the vapour or the liquid phase. Cryopreservation based on freeze-drying does not require use of liquid-nitrogen freezers.

An important application of cryopreservation is in the freezing and storage of hematopoietic stem cells, which are found in the bone marrow and peripheral blood. In autologous bone-marrow rescue, hematopoietic stem cells are collected from a patient’s bone marrow prior to treatment with high-dose chemotherapy. Following treatment, the patient’s cryopreserved cells are thawed and infused back into the body. This procedure is necessary, since high-dose chemotherapy is extremely toxic to the bone marrow. The ability to cryopreserve hematopoietic stem cells has greatly enhanced the outcome for the treatment of certain lymphomas and solid tumour malignancies. In the case of patients with leukemia, their blood cells are cancerous and cannot be used for autologous bone-marrow rescue. As a result, these patients rely on cryopreserved blood collected from the umbilical cords of newborn infants or on cryopreserved hematopoietic stem cells obtained from donors. Since the late 1990s it has been recognized that hematopoietic stem cells and mesenchymal stem cells (derived from embryonic connective tissue) are capable of differentiating into skeletal and cardiac muscle tissues, nerve tissue, and bone. Today there is intense interest in the growth of these cells in tissue culture systems, as well as in the cryopreservation of these cells for future therapy for a wide variety of disorders, including disorders of the nervous and muscle systems and diseases of the liver and heart.

Cryopreservation is also used to freeze and store human embryos and sperm. It is especially valuable for the freezing of extra embryos that are generated by in vitro fertilization (IVF). A couple can choose to use cyropreserved embryos for later pregnancies or in the event that IVF fails with fresh embryos. In the process of frozen embryo transfer, the embryos are thawed and implanted into the woman’s uterus. Frozen embryo transfer is associated with a small but significant increase in the risk of childhood cancer among children born from such embryos.

Profound hypothermia, a form of mild cryopreservation used in human patients, has significant applications. A common use of induction of profound hypothermia is for complex cardiovascular surgical procedures. After the patient has been placed on complete cardiopulmonary bypass, using a heart-lung machine, the blood passes through a cooling chamber. Controlled cooling of the patient may reach extremely low temperatures of around 10–14 °C (50–57 °F). This amount of cooling effectively stops all cerebral activity and provides protection for all the vital organs. When this extreme cooling has been achieved, the heart-lung machine can be stopped, and the surgeon can correct very complex aortic and cardiac defects during circulatory arrest. During this time, no blood is circulating within the patient. After the surgery has been completed, the blood is gradually warmed in the same heat exchanger used for cooling. Gradual warming back to normal body temperatures results in resumption of normal brain and organ functions. This profound hypothermia, however, is far removed from freezing and long-term cryopreservation.

Cells can live more than a decade if properly frozen. In addition, certain tissues, such as parathyroid glands, veins, cardiac valves, and aortic tissue, can be successfully cryopreserved. Freezing is also used to store and maintain long-term viability of early human embryos, ova (eggs), and sperm. The freezing procedures used for these tissues are well established, and, in the presence of cryoprotective agents, the tissues can be stored over long periods of time at temperatures of −14 °C (6.8 °F).

Research has shown that whole animals frozen in the absence of cryoprotective agents can yield viable cells containing intact DNA upon thawing. For example, nuclei of brain cells from whole mice stored at −20 °C (−4 °F) for more than 15 years have been used to generate lines of embryonic stem cells. These cells were subsequently used to produce mouse clones.

Touch DNA, the invisible cells humans transfer to everything we contact, is currently being evaluated for its potential to contaminate crime scene evidence. Though valued for its ability to derive evidence where visible DNA (such as blood, semen, hair, or saliva) can't be found, some researchers are challenging the validity of that evidence because the microscopic genetic markers are transferred so easily.

In a 2015 study entitled "Could Secondary DNA Transfer Falsely Place Someone at the Scene of a Crime?" University of Indianapolis researchers performed experiments to test the sensitivity of Touch DNA transfers. Person A held a handshake with Person B for two minutes, then handled a knife. T he DNA profile of the Person B, who never touched the weapon, was identified on the swab of the weapon handle in 85 percent of the samples. In one-fifth of those experiments, the person who had never directly touched the knife was identified as the main or only contributor of the DNA on the handle, according to the study.

## Sample Preservation

Alcohol is a preservative, not a fixative. Alcohol does not penetrate invertebrate tissue as well as fixatives such as formalin. Therefore, unless alcohol is used in sufficient strength and quantity the sample will decay. We do not use formalin because it is a carcinogen and a mutagen. Please do not use dyes such as Rose Bengal. They do not facilitate sorting of freshwater samples and some dyes are suspected carcinogens.

We recommend using 90-95% denatured ethanol to preserve field samples. Select a technical rather than reagent grade of ethanol to save on cost. Unless you have a special Alcohol, Tobacco and Firearms permit, you can only purchase denatured ethanol. “Denatured” means other alcohols and smelly ketones are blended with the ethanol to keep it from being consumed.

Isopropyl alcohol (rubbing alcohol) can also be used in a pinch. It is available from grocery, drug and discount stores. If you decide to use isopropyl alcohol, purchase 90% rather than 70% concentration.

We recommend Tarr, LLC as a supplier of alcohol in the western United States. A 5-gallon tin of Tarsol E2-190 denatured ethanol costs approximately \$50. See the Tarr website for more information, including their delivery service area. Fisher or VWR Scientific can ship small quantities of denatured ethanol, but for a higher cost.

### How to Preserve your Samples

Never leave unpreserved samples exposed to direct sunlight because the invertebrates will die and decay rapidly. Preserve samples within an hour of collection. Preserved samples do not have to be refrigerated, but do store them in a cool place away from direct sunlight. Be as gentle as possible handling samples so that invertebrates are preserved intact, rather than ground into pieces.

Sample bottles should not be filled more than half full with a sample of sediment, bugs and debris (see photo). Make sure the sample is well drained of water before transferring it to the bottle. Then fill the bottle with 90-95% denatured ethanol or isopropyl alcohol. Invert the bottle several times to ensure the alcohol is well mixed with the sample.

For samples that contain much fine detritus or filamentous algae, which retain a lot of water, we recommend that half the alcohol be decanted off at the end of the day and replaced with fresh alcohol.

## Homogenization Options of Leaf Tissue for Nucleic Acid Isolation

Many methods have been described for preparing leaf tissue for nucleic acid isolation and like most laboratory protocols, there are as many variations as researchers. Generally, leaf tissue is harvested and processed fresh, frozen and processed cryogenically, or frozen, freeze dried, and then homogenized. Each variation can impact the quality of the DNA, such as the size of the fragments isolated. The protocol used for isolating the DNA will also greatly affect DNA quality, especially relating to contaminating polysaccharides and polyphenols. Depending upon the need, harvesting and homogenization are matched for optimal yield.

There are three common routes by which leaf tissue is harvested prior to disruption. The first involves harvesting leaf tissue followed by freezing. Placing the tissue in a -80°C freezer provides a suitably cold environment that preserves DNA and many proteins, but is unsuitable for preserving RNA. Even at -80°C there is sufficient water activity and nuclease action to degrade RNA, albeit slowly. To harvest leaves and preserve RNA, samples must be frozen rapidly, usually by submersing in liquid nitrogen. To preserve the RNA the samples must be held below -120°C, the glass transition temperature of water. At this temperature all biological activity ceases. The second option of preparing leaf tissue prior to homogenization is to harvest, freeze and then freeze dry the samples. Freeze drying allows for long-term storage of DNA and protein (though not all proteins will remain active), but once again RNA typically doesn't survive the freeze drying process. Freeze drying of leaves removes water which if present can alter the concentration of analytes in extractions buffers. The third options for preparing leaf tissue for disruption is to simply harvest the leaves and homogenize them while they are fresh. With the advent of buffers which preserve DNA and RNA, such as Trizol, disrupting leaf tissue when harvested is often practical.

Once leaves are harvested, one of several grinding/homogenization methods is used to break open the cells. Below is a brief summary of each methods.

CryoCooler&trade, a tool for collecting and transporting temperature sensitive samples ( video ).

Grinding in Liquid Nitrogen with Mortar & Pestle

One of the most traditional and common methods for harvesting nucleic acids from plants involves grinding leaves in liquid nitrogen with a mortar and pestle. Either the mortar and pestle can be pre-chilled and the grinding performed dry on frozen leaves, or the leaves can be submersed in liquid nitrogen for the grinding. Cryogenic grinding is a very effective technique for taking hard substances, like plant and animal tissues, and turning them into dust. The tough carbohydrates of plant tissues become very fragile at -196°C and easily shatter. The two concerns with cryogenic grinding is that the sample may warm up, and secondly, throughput is very low. Preventing sample warming can be done be adding additional liquid nitrogen to the mortar while pulverizing the sample. Low throughput is a more difficult issue as a mortar can only be used once before it must be cleaned. As mortar and pestles are typically ceramic, this means that the set must be warmed gradually before it is cleaned. Materials ground into the surface of the mortar may be difficult to remove and hence may act as a contaminant.

Disruption via Homogenizer: Rotor-Stators and Blenders

For samples which are fresh or freeze dried, homogenization can be attained by shearing leaves with a blade. The simplest bladed homogenizer is a blender, which at times can be completely adequate for disrupting samples. Though efficient for milkshakes, blenders are best used for course shearing while rotor-stators are the preferred tool for efficiently disrupting tissues. Rotor-stators have a spinning circular blade called a rotor inside of a tube with slits, known as the stator. As the blade passes the slits it acts like a fine scissor and shears whatever straddles the slit. As the rotors can turn at 20,000 rpm, this makes the rotor-stator very efficient at tearing open cells. The problem with both blenders and rotor-stators is throughput. These homogenizers must be cleaned between use, and in both cases, this may require taking apart the blade assembly.

Where mortar and pestle and homogenizers fall short in throughput, bead beating makes it up. Bead beating is accomplished using a mixer mill, which is basically a machine that rapidly shakes samples which have been mixed with balls. The balls crash around and effectively shear and crack cells and tissues. For microorganisms, small beads of several hundred microns are mixed with the microbe in a microfuge tubes which can be vortexed. Some vortexers will hold multiple tubes making the processing of many samples relatively easy. However, with leaf tissues, it is most practical to use vials or deep well plates and large stainless steel balls. With Vial Sets that use 4 ml polycarbonate vials, the balls are large being 3/8" in diameter. Several hundred milligrams of tissue can be disrupted using a vial. Larger vials can also accommodate up to five grams of leaf tissue. But the most widely used method for homogenizing leaf tissue is to punch leaf holes with a paper punch and drop one disk into a deep well of a microplate along with a 5/32" stainless steel ball. Using a high throughput homogenizer that holds deep well plates, multiple samples can be processed in minutes.

Freezing is often used to store harvested leaves

Harvesting fresh tissue for immediate homogenization is dependent upon throughput and practicality.

## Searching for Evidence

Marilyn T. Miller , Peter Massey , in The Crime Scene , 2016

### Chemical Evidence Visualization and Enhancement

#### Latent Fingerprints and Other Impressions

There are various methods for the visualization and enhancement of latent fingerprints and impression evidence (see Table 6.2 ). Special methods of visualization sometimes will combine the ALS with physical and chemical techniques. These combination methods are especially useful for latent fingerprints. The enhancement reagents are based on their chemical composition and the chemical reactions that occur when reacting with the various types of patterned impression evidence. The enhancement reagents for latent fingerprint enhancement react with chemicals found in the residues deposited on various surfaces.

Table 6.2 . Fingerprint and Impression Visualization and Enhancement

Reagent Use
Blood enhancementAmido black
Coomassie blue
Crowle’s double stain
Diaminobenzidine (DAB)
Fluorescein
Leucocrystal violet
Luminol
Latent printsPowder dusting
Ninhydrin
Superglue (hotshots, cyanowand, pouches)
Sticky surface powder
1,8-Diazafluoren-9-one (DFO)
Small particle reagent
Ardrox
Rhodamine 6G

Latent fingerprints on nonporous surfaces: the secretions from the pores of the friction ridges on skin will contain water, salt, proteins, and oily substances. These secretions will be visible using oblique lighting methods followed by dusting with contrasting colored powders (black powder on light backgrounds or light powder on dark backgrounds). Magnetic and fluorescent powders are also used on this surface. Care must be taken not to “overdust” the surface.

Latent fingerprints on porous surfaces: for this visualization a chemical reaction between the secretions and applied reagent occurs. It is a protein-based color reaction as discussed above. Ninhydrin remains the most successful reagent for this visualization method.

Latent fingerprints on sticky surfaces: this visualization is easily seen with oblique lighting but can be enhanced using a solution of dusting powder in water with a couple drops of a surfactant (dish cleaning soap). Once brushed on the sticky surface a quick rinse with water will visualize the fingerprints or other impression.

Latent fingerprints on a wet surface (porous and nonporous): this visualization and enhancement method is a chemical reaction of the wet surface reagent, small particle reagent, and the secretion chemicals. A black product is formed that visualizes the fingerprint pattern. The reagent is applied by spraying or immersion directly on to the wet surface. Overspraying is not a problem. See Figure 6.12 .

Figure 6.12 . Spraying small particle reagent on wet surfaces—porous and nonporous.

Superglue fuming: for this visualization and enhancement technique cyanoacrylate or superglue is used. It self-polymerizes upon exposure to the air. This polymer will coat the nonporous surface with the latent fingerprint so that it can be powder dusted. Portable fuming tanks can be transported to crime scenes for processing there or larger fixed tanks in laboratories are useful, too. It is possible to overfume the surface that will make dusting of the coated secretions impossible. Additionally, later collection for touch-DNA cannot be done after superglue application. See Figure 6.13 .

Figure 6.13 . Too much superglue.

#### Gunshot Residue

When a firearm is discharged, it creates gases, soot, and burned or partially burned gunpowder particles that are deposited on any and all surfaces at the crime scene, but especially on the shooter’s hand and the intended target. GSR comes from detonation of the primer, gunpowder, lubricants, or components of the projectile. These materials are propelled forward with the projectile toward the target and many will fall on the surfaces of any nearby objects. Searching for GSR is done for two reasons: (1) to determine if an individual fired or handled a recently discharged firearm and (2) to analyze the pattern of GSR for the purpose of determining the muzzle-to-target distance. Any test designed to detect GSR must be used in a manner that minimizes the potential for damaging the GSR pattern. GSR is not evidence that will remain for a long period of time especially if it lands on a target that may move. For this reason the visualization and enhancement of GSR must be done within 2–4 h of firearm discharge. Documentation, preservation, and collection by correct methods should follow immediately. See Figure 6.14 .

Figure 6.14 . Gunshot residue on clothing and enhancement.

These visualization and enhancement reagents react with nitrate and nitrite compounds in GSR, yielding a colored reaction or pattern. The modified Greiss reagent, diphenylamine, and sodium rhodizonate reagents will produce colored products when reacting with GSR components. Reagents can be used as swabbed color tests or by a spraying application. Attention to false positives from cigarette smoke, urine, and fertilizer is important to consider before interpretation is attempted.

#### Explosive Residues

Explosives are chemical substances, which are unstable in their natural form. When heated, shocked, or ignited, they are capable of rapid chemical reaction, producing an explosion by the liberation of large quantities of heat and gas often followed by fire. Explosive residues may be encountered in numerous forms at scene. Explosive residues may be located on the hands or clothes of a suspect, at storage or production scenes, and in vehicles or containers used to transport the explosive material. At postblast crime scenes there may be both unexploded explosive material and products of the explosion.

Searching for explosive residues can be done by portable hydrocarbon or ion “sniffers.” Canine programs have reported significant success detecting minute amounts of explosive materials. The visualization and enhancement of explosive residues is not usually done but preliminary testing for screening purposes can be done. The color reagents that give characteristic colors are the same as the GSR reagents. These reagents react with nitrate and nitrite compounds found in all three categories of explosives to produce color reactions. As with the GSR these reagents can be used as swabbed color tests or by a spraying application. Positive reactions are only indicative and require collection for laboratory confirmation.

#### Controlled Substances and Drugs

Crime scenes are often part of drug investigations like clandestine labs or other crimes relating to drug cases. As such, crime scene investigators may be asked to search the scenes for controlled substances on a variety of surfaces. For these reasons, there is often a need for a quick screening test or field test to analyze a material suspected of being a drug or controlled substance. In many situations, this field test helps provide the necessary probable cause to substantiate an arrest for sale and/or possession of a controlled substance.

Certain drugs will react with selected chemical reagents to give characteristic color changes or precipitates. Easy-to-use kits with widespread use are commercially available which contain these reagents in convenient single-test vials. Some commonly used drug-screening reagents and their characteristic reactions are shown in Table 6.3 .

Table 6.3 . Screening Tests for Drug Substances

ReagentApplicationResult
MarquisLarge variety general reagentOpiates = purple
PCP = colorless to light pink
Phenethylamines = orange to brown
LSD = orange/brown/purple
Mescaline and psilocybin = orange
MandelinVariety phenethylaminesOpiates = blue-gray
Ampethamines = green
LSD = orange/green/gray
Psilocybin = green
Conc. nitric acidMorphine vs heroinMorphine = orange to red
Heroin = yellow to green
DuquenoisMarihuana THCMarihuana, THC, and Hashish = blue-violet
Cobalt thiocyanateCocaine and derivatives“Cocaines” = blue precipitate
Dille–KoppanyiBarbituratesRed-violet

#### Portable Instrumentation for Visualization and Enhancement

There are many different instruments that have moved out of the forensic laboratory and into the field and crime scenes. For many years the only instrument at the crime scene was the metal detector, but now it is common to find portable ground penetrating radar, portable X-ray detectors, and a variety of classic lab instruments like Fourier transform infrared spectroscopy and Raman spectroscopy, even gas chromatography–mass spectroscopy. The negative for these portable instruments remains the availability of the scientist to get to the scene to operate the instrument. See Figure 6.15 .