Cryopreservation

From Wikipedia, the free encyclopedia

Tubes of biological samples being placed in liquid nitrogen.

 

Cryogenically preserved samples being removed from a liquid nitrogen dewar.

 

Cryo-preservation or cryo-conservation is a process where organelles, cells, tissues, extracellular matrix, organs or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures (typically −80 °C using solid carbon dioxide or −196 °C using liquid nitrogen). At low enough temperatures, any enzymatic or chemical activity which might cause damage to the biological material in question is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing additional damage caused by the formation of ice crystals during freezing. Traditional cryopreservation has relied on coating the material to be frozen with a class of molecules termed cryoprotectants. New methods are constantly being investigated due to the inherent toxicity of many cryoprotectants. By default it should be considered that cryopreservation alters or compromises the structure and function of cells unless it is proven otherwise for a particular cell population. Cryoconservation of animal genetic resources is the process in which animal genetic material is collected and stored with the intention of conservation of the breed.

Natural cryopreservation

Water-bears (Tardigrada), microscopic multicellular organisms, can survive freezing by replacing most of their internal water with the sugar trehalose, preventing it from crystallization that otherwise damages cell membranes. Mixtures of solutes can achieve similar effects. Some solutes, including salts, have the disadvantage that they may be toxic at intense concentrations. In addition to the water-bear, wood frogs can tolerate the freezing of their blood and other tissues. Urea is accumulated in tissues in preparation for overwintering, and liver glycogen is converted in large quantities to glucose in response to internal ice formation. Both urea and glucose act as "cryoprotectants" to limit the amount of ice that forms and to reduce osmotic shrinkage of cells. Frogs can survive many freeze/thaw events during winter if no more than about 65% of the total body water freezes. Research exploring the phenomenon of "freezing frogs" has been performed primarily by the Canadian researcher, Dr. Kenneth B. Storey.

Freeze tolerance, in which organisms survive the winter by freezing solid and ceasing life functions, is known in a few vertebrates: five species of frogs (Rana sylvatica, Pseudacris triseriata, Hyla crucifer, Hyla versicolor, Hyla chrysoscelis), one of salamanders (Hynobius keyserlingi), one of snakes (Thamnophis sirtalis) and three of turtles (Chrysemys picta, Terrapene carolina, Terrapene ornata).^[3] Snapping turtles Chelydra serpentina and wall lizards Podarcis muralis also survive nominal freezing but it has not been established to be adaptive for overwintering. In the case of Rana sylvatica one cryopreservant is ordinary glucose, which increases in concentration by approximately 19 mmol/l when the frogs are cooled slowly.

History

One of the most important early theoreticians of cryopreservation was James Lovelock. In 1953, he suggested that damage to red blood cells during freezing was due to osmotic stress, and that increasing the salt concentration in a dehydrating cell might damage it. In the mid-1950s, he experimented with the cryopreservation of rodents, determining that hamsters could be frozen with 60% of the water in the brain crystallized into ice with no adverse effects; other organs were shown to be susceptible to damage. This work led other scientists to attempt the short-term freezing of rats by 1955, which were fully active 4 to 7 days after being revived.

Cryopreservation was applied to humans beginning in 1954 with three pregnancies resulting from the insemination of previously frozen sperm. Fowl sperm was cryopreserved in 1957 by a team of scientists in the UK directed by Christopher Polge. However, the rapid immersion of the samples in liquid nitrogen did not, for certain samples—such as some types of embryos, bone marrow and stem cells—produce the necessary viability to make them usable after thawing. Increased understanding of the mechanism of freezing injury to cells emphasized the importance of controlled or slow cooling to obtain maximum survival on thawing of the living cells. A controlled-rate cooling process, allowing biological samples to equilibrate to optimal physical parameters osmotically in a cryoprotectant (a form of anti-freeze) before cooling in a predetermined, controlled way proved necessary. The ability of cryoprotectants, in the early cases glycerol, to protect cells from freezing injury was discovered accidentally. Freezing injury has two aspects: direct damage from the ice crystals and secondary damage caused by the increase in concentration of solutes as progressively more ice is formed. During 1963, Peter Mazur, at Oak Ridge National Laboratory in the U.S., demonstrated that lethal intracellular freezing could be avoided if cooling was slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. That rate differs between cells of differing size and water permeability: a typical cooling rate around 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulphoxide, but the rate is not a universal optimum.

Temperature

Storage at very low temperatures is presumed to provide an indefinite longevity to cells, although the actual effective life is rather difficult to prove. Researchers experimenting with dried seeds found that there was noticeable variability of deterioration when samples were kept at different temperatures – even ultra-cold temperatures. Temperatures less than the glass transition point (Tg) of polyol's water solutions, around −136 °C (137 K; −213 °F), seem to be accepted as the range where biological activity very substantially slows, and −196 °C (77 K; −321 °F), the boiling point of liquid nitrogen, is the preferred temperature for storing important specimens. While refrigerators, freezers and extra-cold freezers are used for many items, generally the ultra-cold of liquid nitrogen is required for successful preservation of the more complex biological structures to virtually stop all biological activity.

Risks

Phenomena which can cause damage to cells during cryopreservation mainly occur during the freezing stage, and include: solution effects, extracellular ice formation, dehydration and intracellular ice formation. Many of these effects can be reduced by cryoprotectants. Once the preserved material has become frozen, it is relatively safe from further damage. However, estimates based on the accumulation of radiation-induced DNA damage during cryonic storage have suggested a maximum storage period of 1000 years.

Solution effects

As ice crystals grow in freezing water, solutes are excluded, causing them to become concentrated in the remaining liquid water. High concentrations of some solutes can be very damaging.

Extracellular ice formation

When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing.

Dehydration

Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The associated stresses on the cell can cause damage directly.

Intracellular ice formation

While some organisms and tissues can tolerate some extracellular ice, any appreciable intracellular ice is almost always fatal to cells.

Main methods to prevent risks

The main techniques to prevent cryopreservation damages are a well established combination of controlled rate and slow freezing and a newer flash-freezing process known as vitrification.

Slow programmable freezing

A tank of liquid nitrogen, used to supply a cryogenic freezer (for storing laboratory samples at a temperature of about −150 °C)

 

Controlled-rate and slow freezing, also known as slow programmable freezing (SPF), is a set of well established techniques developed during the early 1970s which enabled the first human embryo frozen birth Zoe Leyland during 1984. Since then, machines that freeze biological samples using programmable sequences, or controlled rates, have been used all over the world for human, animal and cell biology – "freezing down" a sample to better preserve it for eventual thawing, before it is frozen, or cryopreserved, in liquid nitrogen. Such machines are used for freezing oocytes, skin, blood products, embryo, sperm, stem cells and general tissue preservation in hospitals, veterinary practices and research laboratories around the world. As an example, the number of live births from frozen embryos 'slow frozen' is estimated at some 300,000 to 400,000 or 20% of the estimated 3 million in vitro fertilisation (IVF) births.

Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. To minimize the growth of extracellular ice crystal growth and recrystallization, biomaterials such as alginates, polyvinyl alcohol or chitosan can be used to impede ice crystal growth along with traditional small molecule cryoprotectants. That rate differs between cells of differing size and water permeability: a typical cooling rate of about 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulfoxide, but the rate is not a universal optimum. The 1 °C / minute rate can be achieved by using devices such as a rate-controlled freezer or a benchtop portable freezing container.

Several independent studies have provided evidence that frozen embryos stored using slow-freezing techniques may in some ways be 'better' than fresh in IVF. The studies indicate that using frozen embryos and eggs rather than fresh embryos and eggs reduced the risk of stillbirth and premature delivery though the exact reasons are still being explored.

Vitrification

Researchers Greg Fahy and William F. Rall helped to introduce vitrification to reproductive cryopreservation in the mid-1980s. As of 2000, researchers claim vitrification provides the benefits of cryopreservation without damage due to ice crystal formation. The situation became more complex with the development of tissue engineering as both cells and biomaterials need to remain ice-free to preserve high cell viability and functions, integrity of constructs and structure of biomaterials. Vitrification of tissue engineered constructs was first reported by Lilia Kuleshova, who also was the first scientist to achieve vitrification of oocytes, which resulted in live birth in 1999. For clinical cryopreservation, vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they decrease the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution becomes an amorphous ice—it vitrifies. Rather than a phase change from liquid to solid by crystallization, the amorphous state is like a "solid liquid", and the transformation is over a small temperature range described as the "glass transition" temperature. 

Vitrification of water is promoted by rapid cooling, and can be achieved without cryoprotectants by an extremely rapid decrease of temperature (megakelvins per second). The rate that is required to attain glassy state in pure water was considered to be impossible until 2005.

Two conditions usually required to allow vitrification are an increase of the viscosity and a decrease of the freezing temperature. Many solutes do both, but larger molecules generally have a larger effect, particularly on viscosity. Rapid cooling also promotes vitrification. 

For established methods of cryopreservation, the solute must penetrate the cell membrane in order to achieve increased viscosity and decrease freezing temperature inside the cell. Sugars do not readily permeate through the membrane. Those solutes that do, such as dimethyl sulfoxide, a common cryoprotectant, are often toxic in intense concentration. One of the difficult compromises of vitrifying cryopreservation concerns limiting the damage produced by the cryoprotectant itself due to cryoprotectant toxicity. Mixtures of cryoprotectants and the use of ice blockers have enabled the Twenty-First Century Medicine company to vitrify a rabbit kidney to −135 °C with their proprietary vitrification mixture. Upon rewarming, the kidney was transplanted successfully into a rabbit, with complete functionality and viability, able to sustain the rabbit indefinitely as the sole functioning kidney.

Freezable tissues

Generally, cryopreservation is easier for thin samples and small clumps of individual cells, because these can be cooled more quickly and so require lesser doses of toxic cryoprotectants. Therefore, cryopreservation of human livers and hearts for storage and transplant is still impractical. 

Nevertheless, suitable combinations of cryoprotectants and regimes of cooling and rinsing during warming often allow the successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include:

• Semen in semen cryopreservation
• Blood
  • Special cells for transfusion
  • Stem cells. It is optimal in high concentration of synthetic serum, stepwise equilibration and slow cooling.
  • Umbilical cord blood
• Tissue samples like tumors and histological cross sections
• Eggs (oocytes) in oocyte cryopreservation
• Embryos at cleavage stage (that are 2, 4 or 8 cells) or at blastocyst stage, in embryo cryopreservation
• Ovarian tissue in ovarian tissue cryopreservation
• Plant seeds or shoots may be cryopreserved for conservation purposes.

Additionally, efforts are underway to preserve humans cryogenically, known as cryonics. For such efforts either the brain within the head or the entire body may experience the above process. Cryonics is in a different category from the aforementioned examples, however: while countless cryopreserved cells, vaccines, tissue and other biological samples have been thawed and used successfully, this has not yet been the case at all for cryopreserved brains or bodies. At issue are the criteria for defining "success". 

Proponents of cryonics claim that cryopreservation using present technology, particularly vitrification of the brain, may be sufficient to preserve people in an "information theoretic" sense so that they could be revived and made whole by hypothetical vastly advanced future technology. Not only is there no guarantee of its success, many people argue that human cryopreservation is unethical. According to certain views of the mind body problem, some philosophers believe that the mind, which contains thoughts, memories, and personality, is separate from the brain. When someone dies, their mind leaves the body. If a cryopreserved patient gets successfully resuscitated, no one knows if they would be the same person that they once were or if they would be an empty shell of the memory of who they once were.

Right now scientists are trying to see if transplanting cryopreserved human organs for transplantation is viable, if so this would be a major step forward for the possibility of reviving a cryopreserved human.

Embryos

Cryopreservation for embryos is used for embryo storage, e.g., when in vitro fertilization (IVF) has resulted in more embryos than is currently needed.

Pregnancies have been reported from embryos stored for 16 years. Many studies have evaluated the children born from frozen embryos, or “frosties”. The result has been uniformly positive with no increase in birth defects or development abnormalities. A study of more than 11,000 cryopreserved human embryos showed no significant effect of storage time on post-thaw survival for IVF or oocyte donation cycles, or for embryos frozen at the pronuclear or cleavage stages. Additionally, the duration of storage did not have any significant effect on clinical pregnancy, miscarriage, implantation, or live birth rate, whether from IVF or oocyte donation cycles. Rather, oocyte age, survival proportion, and number of transferred embryos are predictors of pregnancy outcome.

Ovarian tissue

Cryopreservation of ovarian tissue is of interest to women who want to preserve their reproductive function beyond the natural limit, or whose reproductive potential is threatened by cancer therapy, for example in hematologic malignancies or breast cancer. The procedure is to take a part of the ovary and perform slow freezing before storing it in liquid nitrogen whilst therapy is undertaken. Tissue can then be thawed and implanted near the fallopian, either orthotopic (on the natural location) or heterotopic (on the abdominal wall), where it starts to produce new eggs, allowing normal conception to occur. The ovarian tissue may also be transplanted into mice that are immunocompromised (SCID mice) to avoid graft rejection, and tissue can be harvested later when mature follicles have developed.

Oocytes

Human oocyte cryopreservation is a new technology in which a woman’s eggs (oocytes) are extracted, frozen and stored. Later, when she is ready to become pregnant, the eggs can be thawed, fertilized, and transferred to the uterus as embryos. Since 1999, when the birth of the first baby from an embryo derived from vitrified-warmed woman’s eggs was reported by Kuleshova and co-workers in the journal of Human Reproduction, this concept has been recognized and widespread. This break-through in achieving vitrification of woman’s oocytes made an important advance in our knowledge and practice of the IVF process, as clinical pregnancy rate is four times higher after oocyte vitrification than after slow freezing. Oocyte vitrification is vital for preservation fertility in young oncology patients and for individuals undergoing IVF who object, either for religious or ethical reasons, to the practice of freezing embryos.

Semen

Semen can be used successfully almost indefinitely after cryopreservation. The longest reported successful storage is 22 years. It can be used for sperm donation where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as chemotherapy, radiation therapy or surgery.

Testicular tissue

Cryopreservation of immature testicular tissue is a developing method to avail reproduction to young boys who need to have gonadotoxic therapy. Animal data are promising, since healthy offspring have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and in vitro maturation (IVM) has proved efficient and safe in humans as yet.

Moss

Four different ecotypes of Physcomitrella patens stored at the IMSC.

 

Cryopreservation of whole moss plants, especially Physcomitrella patens, has been developed by Ralf Reski and coworkers and is performed at the International Moss Stock Center. This biobank collects, preserves, and distributes moss mutants and moss ecotypes.

Mesenchymal stromal cells (MSCs)

MSCs, when transfused immediately within a few hours post-thawing, may show reduced function or show decreased efficacy in treating diseases as compared to those MSCs which are in log phase of cell growth (fresh). As a result, cryopreserved MSCs should be brought back into log phase of cell growth in in vitro culture before these are administered for clinical trials or experimental therapies. Re-culturing of MSCs will help in recovering from the shock the cells get during freezing and thawing. Various clinical trials on MSCs have failed which used cryopreserved products immediately post-thaw as compared to those clinical trials which used fresh MSCs.

Preservation of microbiology cultures

Bacteria and fungi can be kept short-term (months to about a year, depending) refrigerated, however, cell division and metabolism is not completely arrested and thus is not an optimal option for long-term storage (years) or to preserve cultures genetically or phenotypically, as cell divisions can lead to mutations or sub-culturing can cause phenotypic changes. A preferred option, species-dependent, is cryopreservation. Nematode worms are the only multicellular eukaryotes that have been shown to survive cryopreservation.

Fungi

Fungi, notably zygomycetes, ascomycetes and higher basidiomycetes, regardless of sporulation, are able to be stored in liquid nitrogen or deep-frozen. Crypreservation is a hallmark method for fungi that do not sporulate (otherwise other preservation methods for spores can be used at lower costs and ease), sporulate but have delicate spores (large or freeze-dry sensitive), are pathogenic (dangerous to keep metabolically active fungus) or are to be used for genetic stocks (ideally to have identical composition as the original deposit). As with many other organisms, cryoprotectants like DMSO or glycerol (e.g. filamentous fungi 10% glycerol or yeast 20% glycerol) are used. Differences between choosing cryoprotectants are species (or class) dependent, but generally for fungi penetrating cryoprotectants like DMSO, glycerol or polyethylene glycol are most effective (other non-penetrating ones include sugars mannitol, sorbitol, dextran, etc.). Freeze-thaw repetition is not recommended as it can decrease viability. Back-up deep-freezers or liquid nitrogen storage sites are recommended. Multiple protocols for freezing are summarized below (each uses screw-cap polypropylene cryotubes):

Bacteria

Many common culturable laboratory strains are deep-frozen to preserve genetically and phenotypically stable, long-term stocks. Sub-culturing and prolonged refrigerated samples may lead to loss of plasmid(s) or mutations. Common final glycerol percentages are 15, 20 and 25. From a fresh culture plate, one single colony of interest is chosen and liquid culture is made. From the liquid culture, the medium is directly mixed with equal amount of glycerol; the colony should be checked for any defects like mutations. All antibiotics should be washed from the culture before long-term storage. Methods vary, but mixing can be done gently by inversion or rapidly by vortex and cooling can vary by either placing the cryotube directly at −50 to −95 °C, shock-freezing in liquid nitrogen or gradually cooling and then storing at −80 °C or cooler (liquid nitrogen or liquid nitrogen vapor). Recovery of bacteria can also vary, namely if beads are stored within the tube then the few beads can be used to plate or the frozen stock can be scraped with a loop and then plated, however, since only little stock is needed the entire tube should never be completely thawed and repeated freeze-thaw should be avoided. 100% recovery is not feasible regardless of methodology.

Worms

The microscopic soil-dwelling nematoderoundworms Panagrolaimus detritophagus and Plectus parvus are the only eukaryotic organisms that have been proven to be viable after long-term cryopreservation to date. In this case, the preservation was natural rather than artificial, due to permafrost.

Personal identity

From Wikipedia, the free encyclopedia

What does it take for a person to persist from moment to moment—for the same person to exist at different moments?

 

In philosophy, the matter of personal identity deals with such questions as, "What makes it true that a person at one time is the same thing as a person at another time?" or "What kinds of things are we persons?" Generally, personal identity is the unique numerical identity of a person in the course of time. That is, the necessary and sufficient conditions under which a person at one time and a person at another time can be said to be the same person, persisting through time.

In contemporary metaphysics, the matter of personal identity is referred to as the diachronic problem of personal identity. The synchronic problem concerns the question of what features and traits characterize a person at a given time. In continental philosophy and in analytic philosophy, enquiry to the nature of Identity is common. Continental philosophy deals with conceptually maintaining identity when confronted by different philosophic propositions, postulates, and presuppositions about the world and its nature.

Theories

Continuity of substance

Bodily substance

One concept of personal persistence over time is simply to have continuous bodily existence. However, as the Ship of Theseus problem illustrates, even for inanimate objects there are difficulties in determining whether one physical body at one time is the same thing as a physical body at another time. With humans, over time our bodies age and grow, losing and gaining matter, and over sufficient years will not consist of most of the matter they once consisted of. It is thus problematic to ground persistence of personal identity over time in the continuous existence of our bodies. Nevertheless, this approach has its supporters which define humans as a biological organism and asserts the proposition that a psychological relation is not necessary for personal continuity. This personal identity ontology assumes the relational theory of life-sustaining processes instead of bodily continuity. 

Derek Parfit's teletransportation problem is designed to bring out intuitions about corporeal continuity. This thought experiment discusses cases in which a person is teleported from Earth to Mars. Ultimately, the inability to specify where on a spectrum does the transmitted person stop being identical to the initial person on Earth appears to show that having a numerically identical physical body is not the criterion for personal identity

Mental substance

In another concept of mind, the set of cognitive faculties are considered to consist of an immaterial substance, separate from and independent of the body. If a person is then identified with their mind, rather than their body—if a person is considered to be their mind—and their mind is such a non-physical substance, then personal identity over time may be grounded in the persistence of this non-physical substance, despite the continuous change in the substance of the body it is associated with. The mind-body problem concerns the explanation of the relationship, if any, that exists between minds, or mental processes, and bodily states or processes. One of the aims of philosophers who work in this area is to explain how a non-material mind can influence a material body and vice versa. 

However, this is not uncontroversial or unproblematic, and adopting it as a solution raises questions. Perceptual experiences depend on stimuli which arrive at various sensory organs from the external world and these stimuli cause changes in mental states; ultimately causing sensation. A desire for food, for example, will tend to cause a person to move their body in a manner and in a direction to obtain food. The question, then, is how it can be possible for conscious experiences to arise out of an organ (the human brain) possessing electrochemical properties. A related problem is to explain how propositional attitudes (e.g. beliefs and desires) can cause neurons of the brain to fire and muscles to contract in the correct manner. These comprise some of the puzzles that have confronted epistemologists and philosophers of mind from at least the time of René Descartes.

Continuity of consciousness

Locke's conception

An Essay Concerning Human Understanding in four books (1690) by John Locke (1632–1704)

 

John Locke considered personal identity (or the self) to be founded on consciousness (viz. memory), and not on the substance of either the soul or the body. Book II Chapter XXVII entitled "On Identity and Diversity" in An Essay Concerning Human Understanding (1689) has been said to be one of the first modern conceptualizations of consciousness as the repeated self-identification of oneself. Through this identification, moral responsibility could be attributed to the subject and punishment and guilt could be justified, as critics such as Nietzsche would point out. 

According to Locke, personal identity (the self) "depends on consciousness, not on substance" nor on the soul. We are the same person to the extent that we are conscious of the past and future thoughts and actions in the same way as we are conscious of present thoughts and actions. If consciousness is this "thought" which "goes along with the substance [...] which makes the same person", then personal identity is only founded on the repeated act of consciousness: "This may show us wherein personal identity consists: not in the identity of substance, but [...] in the identity of consciousness". For example, one may claim to be a reincarnation of Plato, therefore having the same soul substance. However, one would be the same person as Plato only if one had the same consciousness of Plato's thoughts and actions that he himself did. Therefore, self-identity is not based on the soul. One soul may have various personalities. 

Neither is self-identity founded on the body substance, argues Locke, as the body may change while the person remains the same. Even the identity of animals is not founded on their body: "animal identity is preserved in identity of life, and not of substance", as the body of the animal grows and changes during its life. On the other hand, identity of humans is based on their consciousness.

But this interesting border-case leads to this problematic thought that since personal identity is based on consciousness, and that only oneself can be aware of his consciousness, exterior human judges may never know if they really are judging—and punishing—the same person, or simply the same body. In other words, Locke argues that may be judged only for the acts of the body, as this is what is apparent to all but God; however, are in truth only responsible for the acts for which are conscious. This forms the basis of the insanity defense: one cannot be held accountable for acts from which one was unconscious—and therefore leads to interesting philosophical questions:

...personal identity consists [not in the identity of substance] but in the identity of consciousness, wherein if Socrates and the present mayor of Queenborough agree, they are the same person: if the same Socrates waking and sleeping do not partake of the same consciousness, Socrates waking and sleeping is not the same person. And to punish Socrates waking for what sleeping Socrates thought, and waking Socrates was never conscious of, would be no more right, than to punish one twin for what his brother-twin did, whereof he knew nothing, because their outsides were so like, that they could not be distinguished; for such twins have been seen.

Or again:

PERSON, as I take it, is the name for this self. Wherever a man finds what he calls himself, there, I think, another may say is the same person. It is a forensic term, appropriating actions and their merit; and so belong only to intelligent agents, capable of a law, and happiness, and misery. This personality extends itself beyond present existence to what is past, only by consciousness,—whereby it becomes concerned and accountable; owns and imputes to itself past actions, just upon the same ground and for the same reason as it does the present. All which is founded in a concern for happiness, the unavoidable concomitant of consciousness; that which is conscious of pleasure and pain, desiring that that self that is conscious should be happy. And therefore whatever past actions it cannot reconcile or APPROPRIATE to that present self by consciousness, it can be no more concerned in it than if they had never been done: and to receive pleasure or pain, i.e. reward or punishment, on the account of any such action, is all one as to be made happy or miserable in its first being, without any demerit at all. For, supposing a MAN punished now for what he had done in another life, whereof he could be made to have no consciousness at all, what difference is there between that punishment and being CREATED miserable? And therefore, conformable to this, the apostle tells us, that, at the great day, when every one shall 'receive according to his doings, the secrets of all hearts shall be laid open.' The sentence shall be justified by the consciousness all person shall have, that THEY THEMSELVES, in what bodies soever they appear, or what substances soever that consciousness adheres to, are the SAME that committed those actions, and deserve that punishment for them.

Henceforth, Locke's conception of personal identity founds it not on the substance or the body, but in the "same continued consciousness", which is also distinct from the soul since the soul may have no consciousness of itself (as in reincarnation). He creates a third term between the soul and the body—and Locke's thought may certainly be meditated by those who, following a scientist ideology, would identify too quickly the brain to consciousness. For the brain, as the body and as any substance, may change, while consciousness remains the same. Therefore, personal identity is not in the brain, but in consciousness. 

However, Locke's theory of self reveals debt to theology and to apocalyptic "great day", which by advance excuse any failings of human justice and therefore humanity's miserable state. The problem of personal identity is at the center of discussions about life after death and, to a lesser extent, immortality. In order to exist after death, there has to be a person after death who is the same person as the person who died.

Philosophical intuition

Bernard Williams presents a thought experiment appealing to the intuitions about what it is to be the same person in the future. The thought experiment consists of two approaches to the same experiment. 

For the first approach Williams suggests that suppose that there is some process by which subjecting two persons to it can result in the two persons have "exchanged" bodies. The process has put into the body of person B the memories, behavioral dispositions, and psychological characteristics of the person who prior to undergoing the process belonged to person A; and conversely with person B. To show this one is to suppose that before undergoing the process person A and B are asked to which resulting person, A-Body-Person or B-Body-Person, they wish to receive a punishment and which a reward. Upon undergoing the process and receiving either the punishment or reward, it appears to that A-Body-Person expresses the memories of choosing who gets which treatment as if that person was person B; conversely with B-Body-Person. 

This sort of approach to the thought experiment appears to show that since the person who expresses the psychological characteristics of person A to be person A, then intuition is that psychological continuity is the criterion for personal identity.

The second approach is to suppose that someone is told that one will have memories erased and then one will be tortured. Does one need to be afraid of being tortured? The intuition is that people will be afraid of being tortured, since it will still be one despite not having one's memories. Next, Williams asked one to consider several similar scenarios. Intuition is that in all the scenarios one is to be afraid of being tortured, that it is still one's self despite having one's memories erased and receiving new memories. However, the last scenario is an identical scenario to the one in the first scenario.

In the first approach, intuition is to show that one's psychological continuity is the criterion for personal identity, but in second approach, intuition is that it is one's bodily continuity that is the criterion for personal identity. To resolve this conflict Williams feels one's intuition in the second approach is stronger and if he was given the choice of distributing a punishment and a reward he would want his body-person to receive the reward and the other body-person to receive the punishment, even if that other body-person has his memories.

Psychological continuity

In psychology, personal continuity, also called personal persistence or self-continuity, is the uninterrupted connection concerning a particular person of his or her private life and personality. Personal continuity is the union affecting the facets arising from personality in order to avoid discontinuities from one moment of time to another time. Personal continuity is an important part of identity; this is the process of ensuring that the qualities of the mind, such as self-awareness, sentience, sapience, and the ability to perceive the relationship between oneself and one's environment, are consistent from one moment to the next. Personal continuity is the property of a continuous and connected period of time and is intimately related to do with a person's body or physical being in a single four-dimensional continuum. Associationism, a theory of how ideas combine in the mind, allows events or views to be associated with each other in the mind, thus leading to a form of learning. Associations can result from contiguity, similarity, or contrast. Through contiguity, one associates ideas or events that usually happen to occur at the same time. Some of these events form an autobiographical memory in which each is a personal representation of the general or specific events and personal facts. 

Ego integrity is the psychological concept of the ego's accumulated assurance of its capacity for order and meaning. Ego identity is the accrued confidence that the inner sameness and continuity prepared in the past are matched by the sameness and continuity of one's meaning for others, as evidenced in the promise of a career. Body and ego control organ expressions. and of the other attributes of the dynamics of a physical system to face the emotions of ego death in circumstances which can summon, sometimes anti-theonymistic, self-abandonment.

Identity continuum

It has been argued that from the nature of sensations and ideas there is no such thing as a permanent identity. Daniel Shapiro asserts that one of four major views on identity does not recognize a "permanent identity" and instead thinks of "thoughts without a thinker" − "a consciousness shell with drifting emotions and thoughts but no essence". According to him this view is based on the Buddhist concept of Anatta − "a continuously evolving flow of awareness". Malcolm David Eckel states that "the self changes at every moment and has no permanent identity" − it is a "constant process of changing or becoming", a "fluid ever-changing self".

Bundle theory of the self

A Treatise Of Human Nature: Being An Attempt To Introduce The Experimental Method Of Reasoning Into Moral Subjects. For John Noon, 1739

 

David Hume undertook looking at the mind–body problem. Hume also investigated a person's character, the relationship between human and animal nature, and the nature of agency. Hume pointed out that we tend to think that we are the same person we were five years ago. Though we've changed in many respects, the same person appears present as was present then. We might start thinking about which features can be changed without changing the underlying self. Hume, however, denies that there is a distinction between the various features of a person and the mysterious self that supposedly bears those features. When we start introspecting, "we are never intimately conscious of anything but a particular perception; man is a bundle or collection of different perceptions which succeed one another with an inconceivable rapidity and are in perpetual flux and movement".

It is plain that in the course of our thinking, and in the constant revolution of our ideas, our imagination runs easily from one idea to any other that resembles it, and that this quality alone is to the fancy a sufficient bond and association. It is likewise evident that as the senses, in changing their objects, are necessitated to change them regularly, and take them as they lie contiguous to each other, the imagination must by long custom acquire the same method of thinking, and run along the parts of space and time in conceiving its objects.

Note in particular that, in Hume's view, these perceptions do not belong to anything. Hume, similar to the Buddha, compares the soul to a commonwealth, which retains its identity not by virtue of some enduring core substance, but by being composed of many different, related, and yet constantly changing elements. The question of personal identity then becomes a matter of characterizing the loose cohesion of one's personal experience.

In short, what matters for Hume is not that 'identity' exists but that the relations of causation, contiguity, and resemblances obtain among the perceptions. Critics of Hume state in order for the various states and processes of the mind to seem unified, there must be something which perceives their unity, the existence of which would be no less mysterious than a personal identity. Hume solves this by considering substance as engendered by the togetherness of its properties.

No-self theory

The "no-self theory" holds that the self cannot be reduced to a bundle because the concept of a self is incompatible with the idea of a bundle. Propositionally, the idea of a bundle implies the notion of bodily or psychological relations that do not in fact exist. James Giles, a principal exponent of this view, argues that the no-self or eliminativist theory and the bundle or reductionist theory agree about the non-existence of a substantive self. The reductionist theory, according to Giles, mistakenly resurrects the idea of the self in terms of various accounts about psychological relations. The no-self theory, on the other hand, "lets the self lie where it has fallen". This is because the no-self theory rejects all theories of the self, even the bundle theory. On Giles' reading, Hume is actually a no-self theorist and it is a mistake to attribute to him a reductionist view like the bundle theory. Hume's assertion that personal identity is a fiction supports this reading, according to Giles.

The Buddhist view of personal identity is also a no-self theory rather than a reductionist theory, because the Buddha rejects attempts to reconstructions in terms of consciousness, feelings, or the body in notions of an eternal/permanent, unchanging self since our thoughts, personalities and bodies are never the same from moment to moment.

According to this line of criticism, the sense of self is an evolutionary artifact, which saves time in the circumstances it evolved for. But sense of self breaks down when considering some events such as memory loss, split personality disorder, brain damage, brainwashing, and various thought experiments. When presented with imperfections in the intuitive sense of self and the consequences to this concept which rely on the strict concept of self, a tendency to mend the concept occurs, possibly because of cognitive dissonance.

Mirror test

From Wikipedia, the free encyclopedia

A baboon looking in a mirror

The mirror test – sometimes called the mark test, mirror self-recognition test (MSR), red spot technique, or rouge test – is a behavioral technique developed in 1970 by psychologist Gordon Gallup Jr. as an attempt to determine whether an animal possesses the ability of visual self-recognition. The MSR test is the traditional method for attempting to measure self-awareness. However, there has been agreement that animals can be self-aware in ways not measured by the mirror test, such as distinguishing between their own and others' songs and scents. On the other hand, animals that can pass the MSR do not necessarily have self-awareness.

In the classic MSR test, an animal is anaesthetised and then marked (e.g. painted, or a sticker attached) on an area of the body the animal cannot normally see. When the animal recovers from the anesthetic, it is given access to a mirror. If the animal then touches or investigates the mark, it is taken as an indication that the animal perceives the reflected image as itself, rather than of another animal. 

Very few species have passed the MSR test. As of 2015, only great apes (including humans), a single Asiatic elephant, dolphins, orcas and the Eurasian magpie have passed the MSR test. A wide range of species have been reported to fail the test, including several species of monkey, giant pandas, sea lions, and dogs.

Method and history

The inspiration for the mirror test comes from an anecdote about Charles Darwin and a captive orangutan. While visiting the London Zoo in 1838, Darwin observed an orangutan, named Jenny, throwing a tantrum after being teased with an apple by her keeper. This started him thinking about the subjective experience of an orangutan. He also watched Jenny gaze into a mirror and noted the possibility that she recognized herself in the reflection.

In 1970, Gordon Gallup, Jr., experimentally investigated the possibility of self-recognition with two male and two female wild pre-adolescent chimpanzees (Pan troglodytes), none of which had presumably seen a mirror previously. Each chimpanzee was put into a room by itself for two days. Next, a full-length mirror was placed in the room for a total of 80 hours at periodically decreasing distances. A multitude of behaviors was recorded upon introducing the mirrors to the chimpanzees. Initially, the chimpanzees made threatening gestures at their own images, ostensibly seeing their own reflections as threatening. Eventually, the chimps used their own reflections for self-directed responding behaviors, such as grooming parts of their body previously not observed without a mirror, picking their noses, making faces, and blowing bubbles at their own reflections. 

Gallup expanded the study by manipulating the chimpanzees' appearance and observing their reaction to their reflection in the mirror. Gallup anaesthetized the chimpanzees and then painted a red alcohol-soluble dye on the eyebrow ridge and on the top half of the opposite ear. When the dye dried, it had virtually no olfactory or tactile cues. Gallup then returned the chimpanzees to the cage (with the mirror removed) and allowed them to regain full consciousness. He then recorded the frequency with which the chimpanzees spontaneously touched the marked areas of skin. After 30 minutes, the mirror was re-introduced into the room and the frequency of touching the marked areas again determined. The frequency of touching increased to 4–10 with the mirror present, compared to only 1 when the mirror had been removed. The chimpanzees sometimes inspected their fingers visually or olfactorily after touching the marks. Other mark-directed behaviour includes turning and adjusting of the body to better view the mark in the mirror, or tactile examination of the mark with an appendage while viewing the mirror.

An important aspect of the classical mark-test is that the mark/dye is non-tactile, preventing attention being drawn to the marking through additional perceptual cues (somesthesis). For this reason, animals in the majority of classical tests are anesthetised. Some tests use a tactile marker.

Animals that are considered to be able to recognize themselves in a mirror typically progress through four stages of behavior when facing a mirror:

1. social responses
2. physical inspection (e.g. looking behind the mirror)
3. repetitive mirror-testing behavior
4. realization of seeing themselves

Gallup conducted a follow-up study in which two chimpanzees with no prior experience of a mirror were put under anesthesia, marked and observed. After recovery, they made no mark-directed behaviours either before or after being provided with a mirror.

The rouge test was also done by Michael Lewis and Jeanne Brooks-Gunn in 1979 for the purpose of self-recognition with human mothers and their children.

Animals that have passed

European magpies have demonstrated mirror self recognition

 

A large number of studies using a wide range of species have investigated the occurrence of spontaneous, mark-directed behavior when given a mirror, as originally proposed by Gallup. Most marked animals given a mirror initially respond with social behavior, such as aggressive displays, and continue to do so during repeated testing. Only a small number of species have touched or directed behavior toward the mark, thereby passing the classic MSR test. 

Findings in MSR studies are not always conclusive. Even in chimpanzees, the species most studied and with the most convincing findings, clear-cut evidence of self-recognition is not obtained in all individuals tested. Prevalence is about 75% in young adults and considerably less in young and aging individuals.

Until the 2008 study on magpies, self-recognition was thought to reside in the neocortex area of the brain. However, this brain region is absent in non-mammals. Self-recognition may be a case of convergent evolution, where similar evolutionary pressures result in similar behaviors or traits, although species arrive at them via different routes, and the underlying mechanism may be different.

Mammals

Cetaceans

• Bottlenose dolphin (Tursiops truncatus): Researchers in a study on two male bottlenose dolphins observed their reactions to mirrors after having a mark placed on them. Reactions such as decreased delay in approaching the mirror, repetitious head circling and close viewing of the eye or genital region which had been marked, were reported as evidence of MSR in these species.
• Killer whale (Orcinus orca): Killer whales and false killer whales (Pseudorca crassidens) may be able to recognise themselves in mirrors.

Primates

• Bonobo (Pan paniscus)
• Bornean orangutan (Pongo pygmaeus): However, mirror tests with a juvenile (2-year-old), male orangutan failed to reveal self-recognition.
• Chimpanzee (Pan troglodytes): However, mirror tests with a juvenile (11 months old) male chimpanzee failed to reveal self-recognition. Two young chimpanzees showed retention of MSR after one year without access to mirrors.
• Human (Homo sapiens): Humans begin to show self-recognition in the mirror test when they are about 18 months old, or in what psychoanalysts call the "mirror stage".

Proboscidea

Asian elephant (Elephas maximus): In a study performed in 2006 three female Asian elephants were exposed to a large mirror to investigate their responses. Visible marks and invisible sham-marks were applied to the elephants' heads to test whether they would pass the MSR test. One of the elephants showed mark-directed behavior, though the other two did not. An earlier study failed to find MSR in two Asian elephants; it was claimed this was because the mirror was too small. The study was conducted with the Wildlife Conservation Society (WCS) using elephants at the Bronx Zoo in New York. All three Asian elephants in the study were standing in front of a 2.5 m-by-2.5 m mirror—they inspected the rear and brought food close to the mirror for consumption. Evidence of elephant self-awareness was shown when one (and only one) elephant, Happy, repeatedly touched a painted X on her head with her trunk, a mark which could only be seen in the mirror. Happy ignored another mark made with colorless paint that was also on her forehead to ensure she was not merely reacting to a smell or feeling. Frans De Waal, who ran the study, stated, "These parallels between humans and elephants suggest a convergent cognitive evolution possibly related to complex society and cooperation."

Birds

• Eurasian magpie (Pica pica): The Eurasian magpie is the first non-mammal to have passed the mirror test. Researchers applied a small red, yellow or black sticker to the throat of five Eurasian magpies, where they could be seen by the bird only by using a mirror. The birds were then given a mirror. The feel of the sticker on their throats did not seem to alarm the magpies. However, when the birds with colored stickers caught a glimpse of themselves in the mirror, they scratched at their throats—a clear indication that they recognized the image in the mirror as their own. Those that received a black sticker, invisible against the black neck feathers, did not react.

• Pigeons can pass the mirror test after training in the prerequisite behaviors.

Insects

In a Belgian study from 2015, 23 out of 24 adult ants scratched at small blue dots painted on their clypeus (part of their "face") when they were able to see the dot in a mirror. According to the purported results, the ants were individually tested and were from three species, Myrmica sabuleti, Myrmica rubra and Myrmica ruginodis. None of the ants scratched the clypeus when they had no mirror to see the dot. None tried to scratch the blue dot on the mirror. When they had a mirror and a brown dot similar to their own color, only one of thirty ants scratched the brown dot; researchers said she was darker than average so the dot was visible. They also reacted to the mirror itself. Even without dots, 30 out of 30 ants touched the mirror with legs, antennae and mouths, while 0 of 30 ants touched a clear glass divider, with ants on the other side. Ants a few days old did not react to the dots. These three species have limited eyesight, with 109–169 facets per eye, and the authors suggest doing tests on ants with more facets (some have 3,000) and on bees.

Fish 

Cleaner Wrasse have become the first fish ever to pass the mirror test.

Animals that have failed

A range of species have been exposed to mirrors. Although these might have failed the classic MSR test, they have sometimes shown mirror-related behavior:

Mammals

• Sea lions (Zalophus californianus)
• Giant panda (Ailuropoda melanoleuca): In one study, 34 captive giant pandas of a wide range of ages were tested. None of the pandas responded to the mark and many reacted aggressively towards the mirror, causing the researchers to consider the pandas viewed their reflection as a conspecific.

Primates

• Gibbon (g. Hylobates, Symphalangus and Nomascus)
• Stump-tailed macaque (Macaca arctoides)
• Crab-eating macaque (Macaca fascicularis)
• Rhesus monkey (Macaca mulatta): It has been reported that rhesus monkeys exhibit other behaviors in response to a mirror which indicate self-recognition.
• Black-and-white colobus monkey (Colobus guereza)
• Capuchin monkey (Cebus apella)
• Hamadryas baboon (Papio hamadryas)
• Cotton-top tamarin (Saguinus oedipus)

Birds

• Grey parrot
• New Caledonian crow
• Jackdaw
• Great tit (Parus major) 

Fish 

Daffodil cichlid (Neolamprologus pulcher)

Octopuses 

Octopuses oriented towards their image in a mirror, but there is no difference in their behavior in this condition, compared with a view of other octopuses.

Animals that may pass

Gorillas

Findings for gorillas are mixed. At least four studies have reported that gorillas failed the MSR test. It has been suggested that the gorilla may be the only great ape "which lacks the conceptual ability necessary for self-recognition". Other studies have found more positive results, but have tested gorillas with extensive human contact, and required modification of the test by habituating the gorillas to the mirror and not using anaesthetic. Koko reportedly passed the MSR test, although this was without anaesthetic. In gorillas, protracted eye contact is an aggressive gesture and they may therefore fail the mirror test because they deliberately avoid making eye contact with their reflections. This could also explain why only gorillas with extensive human interaction and a certain degree of separation from other gorillas and usual gorilla behaviour are more predisposed to passing the test.

Fish

Two captive giant manta rays showed frequent, unusual and repetitive movements in front of a mirror suggested contingency checking. They also showed unusual self-directed behaviors when exposed to the mirror.

Other uses for mirrors

Primates, other than the great apes, have so far universally failed the mirror test. However, mirror tests with three species of gibbon (Hylobates syndactylus, H.gabriellae, H. leucogenys) have shown convincing evidence of self-recognition despite the fact that the animals failed the standard version of the mirror test.

Rhesus macaques have failed the MSR test, but use mirrors to study otherwise-hidden parts of their bodies, such as their genitals and the implants in their heads. It has been suggested this demonstrates at least a partial self-awareness, although this is disputed.

Pigs can use visual information seen in a mirror to find food, and show evidence of self-recognition when presented with their reflection. In an experiment, 7 of the 8 pigs tested were able to find a bowl of food hidden behind a wall and revealed using a mirror. The eighth pig looked behind the mirror for the food. BBC Earth also showed the food bowl test, and the "matching shapes to holes" test, in the Extraordinary Animals series.

B. F. Skinner found that Pigeons are capable of passing a highly modified mirror test after extensive training. In the experiment, a pigeon was trained to look in a mirror to find a response key behind it, which the pigeon then turned to peck to obtain food. Thus, the pigeon learned to use a mirror to find critical elements of its environment. Next, the pigeon was trained to peck at dots placed on its feathers; food was, again, the consequence of touching the dot. The latter training was accomplished in the absence of the mirror. The final test was placing a small bib on the pigeon—enough to cover a dot placed on its lower belly. A control period without the mirror present yielded no pecking at the dot. When the mirror was revealed, the pigeon became active, looked in the mirror and then tried to peck on the dot under the bib. However, untrained pigeons have never passed the mirror test.

Manta rays repeatedly swim in front of the mirror, turning over to show their undersides and moving their fins. When in front of the mirror, they blow bubbles, an unusual behavior. They do not try to socially interact with the mirror image, suggesting that they recognize that the mirror image is not another ray. However, a classic mirror test using marks on the rays’ bodies has yet to be done.

Robots

In 2012, early steps were taken to make a robot pass the mirror test.

Criticism

The MSR test has been criticized for several reasons, in particular, because it may result in findings that are false negatives.

The MSR test may be of limited value when applied to species that primarily use senses other than vision. For example, dogs mainly use olfaction and audition; vision is used only third. It is suggested this is why dogs fail the MSR test. With this in mind, the biologist Marc Bekoff developed a scent-based paradigm using dog urine to test self-recognition in canines. He tested his own dog, but his results were inconclusive. Dog cognition researcher Alexandra Horowitz formalized Bekoff's idea in a controlled experiment, reported on in 2016 and published in 2017. She compared the dogs' behavior when examining their own and others' odors, and also when examining their own odor with an added smell "mark" analogous to the visual mark in MSR tests. These subjects not only discriminated their own odor from that of other dogs, as Bekoff had found, but also spent more time investigating their own odor "image" when it was modified, as subjects who pass the MSR test do. A 2016 study suggested an ethological approach, the "Sniff test of self-recognition (STSR)" which may shed light on different ways of checking for self-recognition.

Another concern with the MSR test is that some species quickly respond aggressively to their mirror reflection as if it were a threatening conspecific thereby preventing the animal to calmly consider what the reflection actually represents. It has been suggested this is the reason why gorillas and monkeys fail the MSR test.

In a MSR test, animals may not recognzse the mark as abnormal, or, may not be sufficiently motivated to react to it. However, this does not mean they are unable to recognize themselves. For example, in a MSR test conducted on three elephants, only one elephant passed the test but the two elephants that failed still demonstrated behaviors that can be interpreted as self-recognition. The researchers commented that the elephants might not have touched the mark because it was not important enough to them. Similarly, lesser apes infrequently engage in self-grooming, which may explain their failure to touch a mark on their head in the mirror test.

Finally, it should be noted that there has been controversy over whether self-recognition implies self-awareness. The ant researchers state that many ants, from three species, pass the mirror test, but the researchers do not know that they have self-awareness. Dogs recognize their own scent as different from others' scents, but fail the mirror test.

Rouge test

A human child exploring his reflection

 

The rouge test is a version of the mirror test used with human children. Using rouge makeup, an experimenter surreptitiously places a dot on the face of the child. The child is then placed in front of a mirror and their reactions are monitored; depending on the child's development, distinct categories of responses are demonstrated. This test is widely cited as the primary measure for mirror self-recognition in human children.

Developmental reactions

From the age of 6 to 12 months, the child typically sees a "sociable playmate" in the mirror's reflection. Self-admiring and embarrassment usually begin at 12 months, and at 14 to 20 months most children demonstrate avoidance behaviors. Finally, at 18 months half of children recognize the reflection in the mirror as their own and by 20 to 24 months self-recognition climbs to 65%. Children do so by evincing mark-directed behavior; they touch their own nose or try to wipe the mark off.

It appears that self-recognition in mirrors is independent of familiarity with reflecting surfaces. In some cases the rouge test has been shown to have differing results, depending on sociocultural orientation. For example, a Cameroonian Nso sample of infants 18 to 20 months of age had an extremely low amount of self-recognition outcomes at 3.2%. The study also found two strong predictors of self-recognition: object stimulation (maternal effort of attracting the attention of the infant to an object either person touched) and mutual eye contact. A strong correlation between self-concept and object permanence have also been demonstrated using the rouge test.

Implications

The rouge test is a measure of self-concept; the child who touches the rouge on his own nose upon looking into a mirror demonstrates the basic ability to understand self-awareness. Animals, young children, and people who have their sight restored after being blind from birth, sometimes react to their reflection in the mirror as though it were another individual.

Theorists have remarked on the significance of this period in a child's life. For example, psychoanalyst Jacques Lacan used a similar test in marking the mirror stage when growing up. Current views of the self in psychology position the self as playing an integral part in human motivation, cognition, affect, and social identity.

Methodological flaws

There is some debate as to the interpretation of the results of the mirror test, and researchers in one study have identified some potential problems with the test as a means of gauging self-awareness in young children and animals.

Proposing that a self-recognizing child or animal may not demonstrate mark-directed behavior because they are not motivated to clean up their faces, thus providing incorrect results, the study compared results of the standard rouge test methodology against a modified version of the test.

In the classic test, the experimenter first played with the children, making sure that they looked in the mirror at least three times. Then, the rouge test was performed using a dot of rouge below the child's right eye. For their modified testing, the experimenter introduced a doll with a rouge spot under its eye and asked the child to help clean the doll. The experimenter would ask up to three times before cleaning the doll themselves. The doll was then put away, and the mirror test performed using a rouge dot on the child's face. These modifications were shown to increase the number of self-recognizers.

The results uncovered by this study at least suggest some issues with the classic mirror test; primarily, that it assumes that children will recognize the dot of rouge as abnormal and attempt to examine or remove it. The classic test may have produced false negatives, because the child's recognition of the dot did not lead to them cleaning it. In their modified test, in which the doll was cleaned first, they found a stronger relationship between cleaning the doll's face and the child cleaning its own face. The demonstration with the doll, postulated to demonstrate to the children what to do, may lead to more reliable confirmation of self-recognition.

On a more general level, it remains debatable whether recognition of one's mirror image implies self-awareness. Likewise, the converse may also be false—one may hold self-awareness, but not present a positive result in a mirror test.