behavior

JoVE行動学は、人間や動物の行動を理解するための観察および実験手法を公開しています。このセクションには、内外の刺激に対する遺伝的原因と生理学的反応を分析する方法と、関係する脳領域を調べる方法が含まれています。

行動学

biochemistry

JoVE生化学は、生体分子（タンパク質、核酸、炭水化物、脂質、代謝物など）の構造と機能、および生物学的プロセス中に起こるそれらの相互作用と変換についての理解を深める方法を出版しています。

生化学

bioengineering

JoVE生物工学は、物理学と生命科学を融合して基本的な生物学的プロセスと機能を理解する方法と技術を出版しています。このセクションでは、病気の測定、診断、治療を改善するための物理科学的ツールとアプローチについても特集します。

生物工学

biology

JoVE生物学は、細胞生物学、分子生物学、生命体生物学の分野で使用される方法を出版しています。これは、標準的な技法の新しいアプリケーションから、生物の基本的な生物学的機能を理解することを目的とした新規のアプローチにまで及びます。

生物学

cancer research

JoVEがん研究は、がん生物学の研究で使用される技術を出版しています。このセクションでは、発がん、薬剤耐性のメカニズム、診断、転移、革新的な治療法に取り組む研究方法論を取り上げます。

がん研究

chemistry

JoVE化学は、分析化学、有機化学、高分子化学、物理化学、無機化学など、幅広い分野の方法を出版しています。このセクションには、高度なまたはユニークなアプリケーション向けの材料および低分子の設計・調整も含まれています。

化学

developmental biology

JoVE発生生物学では、分子、細胞、組織、器官、および生物全体のレベルにおける、試験管内および/または生体内での生物発生を研究するのに使用される方法と技術を出版しています。

発生生物学

engineering

JoVE工学では、工学と応用物理学で利用される方法と技術を出版しています。方法には、機械、電気、化学のエンジニア、物理学者、材料科学者からの寄稿が含まれます。

工学

environment

JoVE環境学は、地球のプロセスと生態系を研究し、環境問題に対処するための研究方法と技術を出版しています。このセクションでは、農業および食品科学だけでなく、幅広い学際的な環境研究分野からの実験室およびフィールドベースの方法を取り上げます。

環境学

genetics

JoVE遺伝学は、ゲノミクス、遺伝子発現、遺伝子工学、後生遺伝学など、遺伝学のすべての分野（すべての生物）に取り組む方法を出版しています。このセクションでは、塩基配列決定技術とその応用の進歩についても特に取り上げます。

遺伝学

immunology and infection

JoVE免疫学と感染では、病原体に対する生物学的反応、感染に対する免疫反応、基本的な微生物学、および感染性微生物に対する新しい治療法を評価するための方法と技術を出版しています。

免疫・感染学

medicine

JoVE医学では、生物医学研究方法と臨床応用を出版しています。このセクションでは、疾患の動物モデル、医療処置、手術、患者のケーススタディ、および臨床試験の方法論について説明します。

医学

neuroscience

JoVE神経科学では、脳と神経系の構造、機能、生理学、および病態生理学を研究するための方法を出版しています。このセクションには、神経疾患および神経障害の潜在的な治療基盤と外科技術も含まれています。

神経科学

jove journal

JoVE Journal

科学ビデオジャーナル。査読された論文。学際的。

https://cloudfront.jove.com/files/banners/journal/journal_lander.jpg

Research

Psychosurgery

Psychosurgery, the surgical alteration or permanent removal of brain tissue to alleviate severe psychological conditions, stands as one of the most radical and controversial treatments in the history of mental health care. Its development and application have evolved significantly, marked by dramatic shifts in scientific understanding and ethical perspectives.
Historical Development of Psychosurgery
In the 1930s, Portuguese neurologist Antonio Egas Moniz introduced a surgical procedure designed to sever neural pathways between the frontal lobe and thalamus. This intervention aimed to mitigate severe mental health disorders, including schizophrenia and severe anxiety. Despite its crude methodology, the surgeries gained recognition, and Moniz was awarded the Nobel Prize in Physiology or Medicine in 1949.
Walter Freeman, an American neurologist, popularized and coined the term prefrontal lobotomy in the United States, introducing a simplified and faster technique that involved accessing the brain through the eye socket using a surgical instrument similar to an ice pick. Freeman performed over 3,000 lobotomies, often in public demonstrations, and fervently advocated for the procedure as a solution to various psychological disorders. However, his approach often overlooked long-term outcomes and ethical considerations.
Decline of Traditional Lobotomies
The widespread adoption of lobotomies from the 1930s to the 1960s resulted in significant and irreversible harm to many patients. Adverse effects included severe cognitive impairments, memory loss, emotional flatness, and personality changes. Coupled with the advent of psychiatric medications in the mid-1950s, which offered non-invasive alternatives, lobotomies fell out of favor. Reports of poor outcomes and heightened ethical scrutiny further discredited the procedure.
Modern Psychosurgical Techniques
Contemporary psychosurgery employs far more precise and controlled methods, targeting specific brain regions such as the amygdala or anterior cingulate cortex. Techniques like cingulotomy and capsulotomy are used to treat intractable psychiatric conditions such as severe obsessive-compulsive disorder (OCD) and major depressive disorder. These procedures are guided by advanced imaging technologies to minimize damage to surrounding tissues.
Stringent regulations, such as institutional review boards, now govern psychosurgery, ensuring thorough patient evaluation, informed consent, and surgeon&#39;s competency. These measures aim to balance the potential benefits of psychosurgery with its inherent risks, emphasizing its use as a last resort when all other treatments have failed.
Ethical and Scientific Considerations
Despite technological advances, psychosurgery remains a contentious field. Critics point to the procedure&#39;s historical misuse and potential for unintended consequences, while proponents argue for its value in treating otherwise untreatable conditions. Ongoing research seeks to refine surgical techniques and deepen understanding of the brain, underscoring the need for a cautious and ethical approach to this powerful intervention.

Psychosurgery involves removing or permanently destroying brain tissue to treat severe psychological conditions, making it a controversial biomedical ...

Interpersonal Psychotherapy

Interpersonal psychotherapy (IPT) is a structured, time-limited therapeutic approach initially developed to treat depression. It integrates key concepts from psychodynamic, humanistic, and cognitive-behavioral therapies, making it a uniquely eclectic framework. The therapy is rooted in the interpersonal theories of Adolph Meyer and Harry Stack Sullivan, as well as John Bowlby&#39;s attachment theory, and focuses on the interplay between interpersonal relationships and emotional well-being.
Foundations of Interpersonal Psychotherapy
The foundational premise of IPT is that interpersonal relationships significantly influence mood and psychological health. Sullivan emphasized the collaborative role of the therapist as a &#34;participant observer,&#34; working to identify and address maladaptive interpersonal patterns in the client&#39;s daily life. This focus on relationship dynamics distinguishes IPT from other therapeutic models. While it draws on psychodynamic concepts, IPT diverges from traditional psychodynamic therapy by explicitly emphasizing practical strategies to improve interpersonal functioning.
Structure and Scope of IPT
IPT is typically delivered in a short-term format, encompassing 12&#8211;16 sessions. The approach aims to enhance clients&#39; social skills, resolve interpersonal conflicts, and assist with life transitions, such as childbirth, retirement, or bereavement. The therapy is structured around specific problem areas identified during the initial assessment, such as role disputes, role transitions, unresolved grief, or interpersonal deficits. By addressing these areas, IPT helps clients develop healthier relationships and coping mechanisms.
Effectiveness Across Conditions
Originally designed to treat depression, IPT has demonstrated efficacy in combination with pharmacological treatments and as a standalone intervention. Clinical studies have shown its effectiveness in treating various conditions, including substance use disorders and eating disorders, with outcomes comparable to those achieved through cognitive-behavioral therapy. IPT&#39;s success lies in its ability to adapt its core principles to address the unique challenges associated with different mental health disorders.
The integration of theoretical insights with practical strategies makes IPT a versatile and evidence-supported approach, offering a collaborative pathway to improved interpersonal functioning and psychological health.

Interpersonal psychotherapy, or IPT, is an insight-based therapy focusing on the relationship between an individual&#39;s mood and their interpersonal ...

Couples Therapy

Couples therapy is a therapeutic approach designed to help individuals in intimate relationships address conflicts, improve communication, and foster healthier dynamics. It is appropriate for couples at various stages, including those who are dating, married, or in long-term partnerships, and aims to support partners in navigating their unique relational challenges.
Core Principles and Techniques
Couples therapy often incorporates cognitive-behavioral principles to identify and modify negative patterns within relationships. Therapists guide partners in understanding how their individual backgrounds, beliefs, past experiences, and behaviors influence their interactions. This insight is crucial in addressing issues such as poor communication, infidelity, jealousy, sexual difficulties, substance abuse, and the complexities of blended families or two-career households.
Therapists use various techniques such as validation, reframing, structural change, and detriangulation to promote relational growth. Validation involves acknowledging each partner&#39;s emotions and beliefs, as well as fostering mutual respect and understanding. Reframing helps couples view their challenges as shared problems rather than assigning blame, encouraging collaboration. The structural change focuses on reorganizing interaction patterns to create a supportive and functional environment, while detriangulation addresses external conflicts that may be diverting attention from the relationship itself.
Navigating High-Conflict Relationships
High-conflict relationships present unique challenges due to heightened emotions and volatility. Therapists act as mediators, helping partners navigate intense disagreements and work toward constructive resolutions. These situations require a nuanced approach to de-escalate tensions and facilitate productive dialogue.
Goals and Outcomes
The primary goal of couples therapy is to equip partners with tools to enhance understanding and foster healthier interactions. For some, this process strengthens the relationship, while for others, therapy may reveal irreconcilable differences, leading to a mutual decision to separate. Regardless of the outcome, therapy emphasizes clarity and informed decision-making, enabling couples to navigate their circumstances with greater insight and confidence.
By addressing relational dynamics and promoting constructive change, couples therapy supports partners in building more harmonious connections or reaching resolutions that align with their well-being.

Couples therapy helps individuals in intimate relationships resolve conflicts and improve dynamics. Couples may be dating, married, or in long-term ...

Self-Help Support Groups

Self-help support groups are voluntary, community-based organizations that provide a platform for individuals with shared concerns to exchange support, insights, and practical strategies for coping with life challenges. Typically led by group members or paraprofessionals, these groups form a cornerstone of mental health care, especially in reaching populations that are underserved by traditional healthcare systems.
Accessibility and Cost-Effectiveness
One of the primary strengths of self-help groups is their accessibility. By leveraging community resources and operating at a low cost, they serve individuals who might otherwise face barriers to mental health services, such as those with limited financial means or lower levels of education. These groups offer an inclusive space where participants can openly share experiences, seek advice, and explore solutions in a non-judgmental environment.
The Power of Shared Experience
The effectiveness of self-help groups is rooted in their emphasis on shared experience. Participants gain unique insights from peers who have faced similar challenges, fostering a sense of credibility and understanding that professionals may not always provide. For instance, a rape survivor might find meaningful support and guidance from another survivor who has successfully navigated a similar recovery process. These interactions not only validate individual experiences but also enhance emotional resilience and promote self-efficacy.
Applications in Mental Health and Beyond
Self-help groups address a wide array of issues, including substance abuse, chronic illnesses such as heart disease or cancer, and psychological trauma. Alcoholics Anonymous (AA) exemplifies the impact of these groups, with research highlighting its role in reducing alcohol consumption through mechanisms such as enhanced social networks, improved self-efficacy, increased spirituality, and alleviation of negative emotions.
The Role of Online Platforms
Advances in technology have expanded the reach of self-help groups through online platforms, offering greater accessibility for individuals unable to attend in-person meetings. However, virtual settings pose unique challenges, including the risk of misinformation. In the absence of guidance from a trained professional, members may lack the expertise and knowledge to provide optimal advice. This increases the potential for harmful group dynamics, as evidenced by certain pro-anorexia communities. Effective moderation by group leaders can mitigate these risks, ensuring a safe and constructive environment for all members.
Self-help groups exemplify the value of peer-led initiatives in mental health care, bridging gaps in accessibility and fostering collective resilience through shared understanding and mutual support.

Self-help support groups are voluntary organizations where individuals with common interests meet regularly to discuss shared concerns and provide mutual ...

Family Therapy

Family therapy conceptualizes psychological challenges as arising from dysfunctional interactions within the family unit, rather than as isolated issues within individuals. This approach seeks to address and transform the patterns of communication, roles, and relationships within families to promote healthier dynamics and emotional well-being for all members.
Strategic Family Therapy
Strategic family therapy emphasizes resolving communication barriers and improving problem-solving abilities among family members. Therapists employ carefully planned tasks, known as directives, to alter dysfunctional interaction patterns. One notable technique is the use of paradoxical requests, where problematic behaviors are reframed in unexpected ways to elicit change. For example, a couple prone to frequent arguments might be instructed to argue more intentionally, thereby reframing their conflict as an expression of connection. This paradoxical approach often leads to reduced conflict and an improved relationship dynamic by altering the way family members perceive and engage in their interactions.
Structural Family Therapy
Structural family therapy focuses on reorganizing the family&#39;s hierarchical structure and boundaries to enhance functionality. Therapists actively participate in family activities to observe and modify interactions, fostering healthier dynamics. For instance, helping a teenager who initially sought their family&#39;s attention by refusing to eat to express their needs verbally, ultimately leading them to stop using food refusal as a way to seek affection. This direct engagement strengthens communication, reinforces healthy boundaries, and empowers the family to support each member&#39;s needs effectively.
Effectiveness and Applications
Research demonstrates that family therapy is as effective as individual therapy, offering distinct advantages for families dealing with relational or systemic issues. By addressing problems within their broader context, family therapy promotes growth and healing not just for individuals but for the family as a cohesive unit. Approaches such as strategic and structural therapy exemplify the flexibility and efficacy of this modality, making it a valuable tool in fostering healthier familial relationships and emotional resilience.

Family therapy views psychological problems as rooted in dysfunctional family systems rather than in an individual.
The primary focus of treatment is the ...

Community Based Intervention

Community-based interventions in mental health represent a paradigm shift from institution-centered care to treatments embedded within the fabric of local communities. By prioritizing inclusion and leveraging existing societal structures, this approach fosters a supportive environment conducive to addressing mental health challenges while promoting individual dignity and agency.
Foundations of Community Mental Health Programs
Central to the success of community-based interventions is the training of local stakeholders such as teachers, nurses, and community members. These individuals are equipped with fundamental counseling skills and are often involved in workshops aimed at stress management, substance abuse reduction, and interpersonal skill development. Such initiatives help extend mental health care beyond specialized professionals, making it more accessible and reducing stigma.
The deinstitutionalization movement of the 1960s marked the beginning of this transition. It was driven by concerns over the ethical failings of institutional care, which often subjected individuals to neglect and abuse, and by economic considerations regarding the unsustainable costs of long-term institutionalization. As individuals were reintegrated into communities, the focus shifted to creating support systems that address mental health in a holistic, culturally sensitive manner.
Empowerment and Sustainability
Community-based mental health programs emphasize empowerment, enabling individuals to actively engage in their recovery and integrate fully into familial and societal contexts. This approach, championed by community psychologists, seeks to cultivate long-term well-being by embedding care within the individual&#39;s social and cultural milieu.
However, these programs require robust support in terms of local resources and government funding. Sustainable initiatives are essential to ensure continuity in care and to prevent adverse outcomes, such as homelessness, for individuals with chronic mental health conditions. Investment in community-based interventions not only alleviates the strain on formal healthcare systems but also promotes a more inclusive and equitable approach to mental healthcare.

Community-based intervention provides care within society and family, fostering inclusion and treating mental health in familiar settings like community ...

Electroconvulsive Therapy

Electroconvulsive therapy (ECT), or shock therapy, remains a critical biomedical intervention for severe, treatment-resistant depression. While its origins can be traced back to Hippocrates&#39; observations that malaria-induced convulsions alleviated mental illness, modern ECT has evolved significantly from its earlier, more primitive applications. First introduced in 1938 by Ugo Cerletti and his colleagues, ECT involves inducing controlled seizures using electrical currents. In its early years, patients were subjected to the procedure without anesthesia, leading to severe bodily convulsions and a barbaric reputation. Today, however, ECT is far more refined and safer due to anesthesia, muscle relaxants, and targeted stimulation.
The current ECT procedure involves administering a brief electric pulse, typically lasting less than a second, through electrodes placed on the patient&#39;s scalp. This electrical current triggers a seizure lasting approximately 60 seconds. Patients are rendered unconscious with anesthesia and muscle relaxants to prevent physical convulsions, ensuring minimal discomfort. Increasingly, the stimulation is applied to the brain&#39;s right hemisphere, reducing the risk of memory loss and cognitive impairments, which are among ECT&#39;s most debated side effects. Patients awaken shortly after the procedure with no memory of the treatment, and recovery occurs within 30 minutes.
ECT is primarily used for individuals with severe depression who do not respond to medication or psychotherapy. It is particularly effective for cases involving acute suicidality, offering rapid mood improvement. Studies report that after two to four weeks of treatments &#8212; typically three sessions per week &#8212; approximately 70% of patients experience significant relief. This success has been attributed to ECT&#39;s ability to reduce overactivity in brain regions associated with depression and potentially stimulate neurogenesis, promoting the growth of new neurons and synaptic connections.
Despite its effectiveness, ECT remains controversial due to its side effects, including memory loss and cognitive impairment. Though modern advancements, such as unilateral stimulation and refined protocols, have reduced these risks, they still warrant careful consideration. Long-term efficacy also remains uncertain, as relapses can occur, necessitating maintenance therapies like antidepressants or exercise. Nonetheless, ECT continues to save lives, especially in severe cases of depression where other treatments fail. For many, its benefits far outweigh the risks.

Electroconvulsive therapy, or ECT, commonly known as shock therapy, originated from Hippocrates&#39; observations in ancient Greece, who noted that ...

Cognitive Therapy

Cognitive therapy, pioneered by Aaron T. Beck in the 1960s, is a structured approach to addressing psychological distress by focusing on the influence of thoughts on emotions and behaviors. All cognitive therapies involve the basic assumption that human beings have control over their feelings, and that how individuals feel about something depends on how they think about it. Unlike psychoanalytic methods that delve into unconscious processes or humanistic approaches emphasizing self-actualization, cognitive therapy targets distorted or dysfunctional thought patterns, aiming to restructure these to alleviate maladaptive behaviors and emotional responses.
Principles of Cognitive Therapy
Central to cognitive therapy is the concept of cognitive restructuring, which involves identifying and modifying distorted thinking patterns (cognitive distortions). For example, a client may interpret a single academic failure as indicative of global incompetence, leading to feelings of worthlessness. Through guided questioning, therapists challenge these irrational beliefs and help clients develop more accurate, rational perspectives. This active and structured process distinguishes cognitive therapy as an action-oriented approach rather than one focused on uncovering historical psychological roots.
Application to Depression and Other Disorders
Initially developed to treat depression, cognitive therapy has demonstrated effectiveness across a range of psychological disorders. Beck&#39;s work emphasized the role of self-defeating attitudes and negative automatic thoughts in perpetuating depressive states, even amidst positive life circumstances. By addressing these maladaptive cognitions, clients can reframe their interpretations of situations, reducing distress and promoting healthier emotional responses.

Cognitive therapy is a form of psychotherapy that emphasizes the role of thoughts in causing emotional distress and maladaptive behaviors.
It aims to ...

Rational Emotive Behavior Therapy

Cognitive-behavioral therapies (CBTs) are grounded in the belief that our thoughts profoundly influence our emotions and actions. Advocates of CBT emphasize three core assumptions: first, that cognitions are identifiable and measurable; second, that they are central to psychological functioning; and third, that irrational or maladaptive beliefs can be replaced with rational and adaptive ones. This transformative approach to therapy has paved the way for specific models such as Albert Ellis&#39;s Rational Emotive Behavior Therapy (REBT), which integrates both cognitive and behavioral elements to foster mental well-being.
The ABCs of Rational Emotive Behavior Therapy
Ellis introduced REBT in the mid-1950s as a pioneering cognitive-behavioral framework. At its core lies the ABC model, which outlines the interaction between activating events (A), belief systems (B), and emotional and behavioral consequences (C). For instance, two individuals receiving the same test score may exhibit vastly different responses due to disparities in their belief systems. Rational beliefs, characterized by flexibility and self-acceptance, contrast sharply with irrational beliefs, which often involve unrealistic demands and catastrophic thinking.
To address irrational beliefs, Ellis extended the ABC model to include disputation (D), effective new beliefs (E), and fostering desired feelings and behaviors (F). Therapists employing REBT actively challenge clients&#39; maladaptive beliefs and encourage rational rethinking. Assignments or &#8220;homework&#8221; often reinforce this process, such as tasks designed to confront and disprove fears tied to irrational thoughts.
Cognitive-Behavioral Therapy: Expanding on Rationality
CBT builds upon the principles of cognitive psychology and behaviorism, focusing on present cognitive and behavioral interactions rather than past influences. It posits that psychological disorders stem from illogical thought patterns, which, when restructured into logical and rational frameworks, lead to symptom alleviation. The three overarching goals of CBT include symptom relief, equipping clients with coping strategies, and fostering a shift from self-defeating to self-enhancing thoughts.
By blending cognitive interventions with behavioral techniques, CBT offers a robust, action-oriented approach to mental health, emphasizing the transformative power of rational thinking and adaptive behavior.

In the mid-1950s, Albert Ellis developed rational emotive behavior therapy or REBT, a key example of a cognitive-behavioral approach.
He introduced the ...

Group Therapy

Group therapy is a sociocultural approach to psychological treatment, where individuals with shared psychological challenges come together under the guidance of a mental health professional. This therapeutic modality offers unique opportunities for individuals to connect, share, and grow within the context of a supportive group. By fostering mutual understanding and collaboration, group therapy can address a range of psychological concerns effectively, often complementing or surpassing the benefits of individual therapy.
Benefits of Group Therapy
Group therapy promotes universality, helping participants realize that others share similar struggles, which can reduce feelings of isolation and shame. For instance, a child dealing with poor social skills may benefit from observing and practicing interpersonal techniques within a structured group setting. Similarly, a woman with postpartum depression might feel less guilt and more connection in a group of others facing the same challenges.
Altruism is another key feature: participants provide support, advice, and empathy to one another, which can enhance feelings of competence and efficacy. Additionally, the group environment fosters interpersonal learning, where individuals receive feedback from peers, enabling them to refine social behaviors and develop healthier interaction patterns. For those with specific needs, such as sexual abusers, group therapy offers a safe space for practicing social skills, modeling openness, and confronting harmful patterns.
Challenges and Considerations
While group therapy has significant advantages, it is not without challenges. Participants may initially feel reluctant to share personal issues with strangers, and confidentiality concerns can arise if members disclose private information outside the group. Personality conflicts and disagreements within the group can also present obstacles. Despite these challenges, effective facilitation ensures a balanced and inclusive environment where everyone benefits from the group dynamic.
Applications and Formats
Group therapy can adopt various forms, including psychodynamic, humanistic, behavioral, or cognitive approaches. Psycho-educational groups address specific topics, such as coping with a family member&#39;s illness or overcoming bullying, often within school settings. Structured groups have defined goals and activities, while unstructured groups allow members to determine the focus collaboratively. These diverse formats cater to different therapeutic needs, making group therapy an adaptable and powerful tool for psychological growth and healing.

Group therapy is a sociocultural approach to treating psychological disorders, where individuals with similar issues participate in guided sessions led by ...

Beck's Cognitive Therapy

Cognitive therapy is a psychological approach designed to address distortions in thinking, which can lead to negative emotions and unrealistic beliefs. These cognitive distortions often influence how individuals interpret and respond to situations, exacerbating emotional distress. Below are some prevalent cognitive distortions, their characteristics, and examples of how they manifest in thought processes.
Arbitrary Inference
Arbitrary inference involves making conclusions without sufficient evidence, often based solely on personal assumptions. This type of distortion disregards facts and relies on conjecture. For example, an individual may interpret a canceled lunch appointment as evidence that their friend is seeing someone else, despite having no supporting evidence. This tendency to jump to conclusions can lead to unwarranted anxiety or mistrust.
Selective Thinking
Selective thinking occurs when a person fixates on one aspect of a situation, ignoring other relevant details that might provide balance or perspective. For instance, a student might receive praise for their work but focus entirely on a minor critique, concluding that their performance was poor. This cognitive bias limits the individual&#39;s ability to see the broader context of their achievements or the constructive nature of feedback.
Overgeneralization
Overgeneralization involves drawing broad, often exaggerated conclusions based on a single incident. For example, someone reprimanded at work might irrationally assume that their work relationships are doomed and envision a cascade of negative outcomes. This distortion perpetuates feelings of hopelessness and unwarranted despair.
Magnification and Minimization
In this distortion, individuals amplify the significance of negative events while downplaying positive ones. For example, a student who consistently excels academically might catastrophize a single mediocre grade as a sign of inevitable failure, disregarding their overall success. This imbalance skews self-assessment and fosters unwarranted negative self-perceptions.
Personalization
Personalization involves taking unwarranted responsibility for external events. For example, a person might attribute their partner&#39;s bad mood to their actions, ignoring external factors such as work-related stress. This distortion often results in misplaced guilt or self-blame.
Cognitive therapists aim to address these distortions by encouraging clients to evaluate their beliefs critically. Through structured questioning, such as examining the origin or evidence of a belief, clients are guided to identify and challenge illogical patterns. This process fosters more balanced and realistic thinking, improving emotional well-being and resilience.

Cognitive therapy, developed by Aaron T. Beck, addresses distorted thinking and unrealistic beliefs, particularly in depression.
Some of the common ...

Operant Conditioning Intervention

Operant conditioning serves as a foundational principle in therapeutic interventions aimed at modifying maladaptive behaviors. Central to this approach is the notion that behaviors, both adaptive and maladaptive, are learned through reinforcement. By analyzing the environmental factors that reinforce problematic behaviors, clinicians can design interventions to weaken these reinforcements and replace maladaptive behaviors with healthier alternatives.
In operant conditioning, behaviors that are reinforced tend to recur, whereas those that are not reinforced eventually diminish or become extinct. For example, obsessive-compulsive disorder (OCD) behaviors, such as compulsive checking rituals, are maintained through negative reinforcement. A compulsive action, like repeatedly checking if a door is locked, alleviates anxiety and prevents anticipated negative outcomes, reinforcing the behavior. Therapy based on operant conditioning addresses this by extinguishing avoidance learning. The individual is gradually exposed to situations without performing the compulsion, demonstrating that the feared outcomes do not occur. Simultaneously, relaxation training and alternative coping mechanisms are introduced to reduce anxiety, breaking the cycle of compulsive reinforcement.
Behavioral therapies based on operant principles are particularly effective for populations with limited cognitive abilities, such as children or adults with developmental disabilities. In treating autism spectrum disorder (ASD), behavioral therapies have proven highly effective. It is used to address self-injurious behaviors, promote communication, and enhance cognitive and language development. Research indicates that intensive early interventions can significantly improve outcomes for children with ASD.
One widely implemented operant conditioning technique is the token economy. This method is commonly used in controlled environments, such as psychiatric hospitals, to encourage positive behaviors. Tokens, which serve as secondary reinforcers, are awarded for desirable actions and can be exchanged for privileges or rewards. For instance, patients may earn tokens for completing daily tasks or engaging in social interactions, which they can later trade for amenities like extra leisure time or preferred activities. The structured reinforcement system of token economies fosters sustained behavioral improvements across diverse clinical settings.

Operant conditioning suggests that behaviors are shaped by their consequences.
Behaviors followed by positive outcomes are reinforced, such as a child ...

Humanistic Therapy

Humanistic therapies emphasize personal growth, self-understanding, and the fulfillment of human potential. Rooted in the belief that individuals inherently strive toward self-actualization, these approaches encourage clients to explore their feelings and experiences in a supportive, nonjudgmental environment. Humanistic therapies differ from psychodynamic approaches by focusing on conscious experiences, present circumstances, and the potential for self-improvement rather than past conflicts and unconscious drives.
Carl Rogers&#39; client-centered therapy is a cornerstone of humanistic psychotherapy. It aims to foster self-acceptance and unity between the client&#39;s actual self and ideal self. Therapists create a therapeutic environment characterized by three essential elements:
<ol>
	<li>Unconditional Positive Regard: Therapists affirm the inherent worth of clients, offering nonjudgmental acceptance to promote self-acceptance.</li>
	<li>Empathy: By deeply understanding and reflecting on the client&#39;s emotions, therapists help clients feel heard and validated.</li>
	<li>Genuineness: Therapists are authentic and transparent, fostering a genuine connection with the client.</li>
</ol>
Active listening and reflective speech are central techniques that allow clients to identify and articulate their feelings. For example, a therapist might mirror a client&#39;s grief or anger, encouraging deeper emotional exploration.
Gestalt therapy, developed by Fritz Perls, focuses on integrating conflicting aspects of the self to achieve wholeness. Techniques like the two-chair dialogue allow clients to confront and synthesize opposing parts of their personality, fostering authenticity and emotional balance. Gestalt therapy emphasizes the &#34;here and now,&#34; urging clients to take responsibility for their feelings and actions. Techniques such as the two-chair method in Gestalt therapy, where clients alternate speaking from different perspectives to address and resolve internal conflicts, help promote self-awareness and integration.
Existential therapy, exemplified by Viktor Frankl&#39;s logotherapy, explores meaning-making as a response to life&#39;s challenges. Frankl believed that even in dire circumstances, individuals could find purpose and maintain dignity. Existential therapists help clients confront themes like freedom, isolation, and mortality, encouraging personal responsibility and resilience.
Humanistic principles are integral to various contemporary approaches, including motivational interviewing, which leverages empathy and reflective listening to encourage behavior change. Whether addressing mental health conditions or lifestyle modifications, humanistic therapies remain influential in fostering emotional awareness, self-empowerment, and authentic living.

Humanistic therapy focuses on self-healing, conscious thoughts, the present, and self-fulfillment, encouraging individuals to realize their potential and ...

Behavior Therapy

Behavior therapy incorporates diverse techniques rooted in classical conditioning principles to address maladaptive behaviors and anxiety disorders. These methods aim to reduce avoidance behaviors, foster adaptive coping mechanisms, and alter associations between stimuli and responses, making them effective in a wide range of therapeutic contexts.
Exposure therapy is a cornerstone of behavioral treatment for anxiety disorders. It involves systematic exposure to feared stimuli, either in real life, through visualization, or via virtual reality, to desensitize the individual. Gradual exposure, beginning with the least anxiety-provoking scenarios and progressing to more challenging ones, is designed to help clients build tolerance.
Systematic desensitization builds upon this by pairing exposure with relaxation techniques. For instance, someone with a fear of public speaking may practice relaxation while initially speaking to a single person and then gradually progressing to larger audiences, reducing anxiety through reciprocal inhibition.
Flooding involves directly confronting the most anxiety-inducing stimuli in a controlled setting to extinguish avoidance behaviors. Unlike graded exposure, flooding does not follow a hierarchical approach. For example, an individual with a phobia of dogs might be guided to interact with a calm and friendly dog immediately, enabling them to experience the absence of feared consequences, which helps diminish their fear response.
Aversive conditioning is employed to deter undesirable behaviors by pairing them with unpleasant experiences. For example, to address overeating, therapists may use aversive stimuli, such as a bitter taste, to create a negative association with the behavior. This method conditions the individual to avoid the maladaptive behavior due to its unpleasant consequences.
Relaxation techniques, including deep breathing and progressive muscle relaxation, are integral to these therapies. They work through reciprocal inhibition, whereby a physiological state incompatible with anxiety, such as relaxation, counteracts the anxious response. By combining relaxation with gradual or direct exposure, individuals can recondition their responses to previously feared stimuli, fostering long-term behavioral change.

In behavior therapy, a therapist employs principles of learning, such as classical and operant conditioning, to help clients change undesirable 
...

Drug Therapy

The advent of drug therapy has profoundly shaped modern mental health care, providing targeted treatments for a range of psychological disorders. Psychotherapeutic drugs, classified into antianxiety, antidepressant, and antipsychotic medications, address symptoms across anxiety disorders, mood disorders, and schizophrenia. While these medications have transformed patient outcomes, they require careful management due to their potential side effects and limitations.
Antianxiety Medications
Antianxiety drugs, commonly known as tranquilizers, include benzodiazepines such as Xanax, Valium, and Librium. These medications alleviate anxiety by binding to neurotransmitter receptors that are hyperactive during periods of distress. Although benzodiazepines act rapidly and are effective for short-term relief, they pose risks of addiction, drowsiness, impaired coordination, and potentially harmful interactions with alcohol and other drugs. Alternatives like buspirone (BuSpar) offer less addictive solutions but may take longer to provide symptomatic relief. Caution is essential in prescribing these medications, particularly for long-term use, due to their addictive potential.
Antidepressant Drugs
Antidepressants regulate mood by influencing brain neurotransmitters, particularly serotonin and norepinephrine. Types of antidepressants such as tricyclics and tetracyclics, named for their molecular structures, have demonstrated efficacy but come with significant side effects such as dizziness, memory issues, and faintness. Monoamine oxidase (MAO) inhibitors, though effective, are less commonly used due to dietary restrictions and risks of high blood pressure. More commonly prescribed are selective serotonin reuptake inhibitors (SSRIs), which target serotonin reabsorption, offering fewer side effects and broad applications for mood, anxiety, and even chronic pain disorders. However, withdrawal from SSRIs requires careful management to avoid severe symptoms.
Mood Stabilizers Medications
Lithium remains a cornerstone treatment for bipolar disorder, effectively managing manic episodes and stabilizing mood. Its therapeutic benefits are tempered by a narrow safety margin, requiring careful blood monitoring to avoid toxicity. Side effects such as weight gain, thyroid issues, and kidney complications are common, highlighting the need for patient adherence and regular medical supervision.
Antipsychotic Medications
Antipsychotics have revolutionized schizophrenia treatment by reducing symptoms such as hallucinations and agitation. Neuroleptics, the most widely used class, modulate dopamine pathways but are associated with severe side effects, including tardive dyskinesia, a condition characterized by involuntary facial and limb movements. Newer atypical antipsychotics, such as Clozaril and Risperdal, also target serotonin and have fewer side effects, but challenges remain in patient adherence and long-term symptom management. Combining medication with psychotherapy and skill-building therapies improves outcomes, emphasizing the importance of holistic care.
Drug therapies, while pivotal in psychiatric treatment, require personalized and cautious administration. Side effects, risk of dependency, and adherence issues necessitate a balanced approach, often integrating pharmacological interventions with psychotherapy for optimal results.

The development of psychotherapeutic drugs revolutionized mental healthcare, offering effective treatments for anxiety, mood disorders, and 
...

Psychodynamic Therapy

Psychodynamic therapies emphasize the exploration of unconscious processes and early childhood experiences as fundamental contributors to psychological difficulties. These therapies, deeply rooted in Freud&#39;s psychoanalytic theory, aim to uncover and resolve unconscious conflicts, granting individuals insights that promote emotional and behavioral healing. Contemporary psychodynamic approaches have evolved, integrating a broader range of influences and methodologies while still valuing the unconscious mind and the therapeutic relationship.
Freud&#39;s psychoanalysis is the cornerstone of psychodynamic therapies. He proposed that unconscious conflicts, often originating in childhood, manifest as present-day psychological problems. Through techniques such as free association, dream analysis, and interpretation, psychoanalysts endeavor to illuminate these hidden struggles.
Free Association encourages clients to articulate thoughts without censorship, facilitating the emergence of repressed emotions or memories. Freud viewed moments of resistance &#8212; when clients hesitated or avoided certain topics &#8212; as indicators of unresolved conflicts.
Dream Analysis involves interpreting dreams&#39; manifest content (the recalled narrative) to reveal their latent content or hidden unconscious meanings. Freud believed that dreams serve as a disguised outlet for repressed wishes, often rooted in sexual or aggressive instincts.
Interpretation entails uncovering symbolic meanings in clients&#39; words and actions and offering hypotheses about unconscious motivations that drive behaviors.
Freud highlighted the importance of transference, where clients project feelings toward significant figures in their lives onto the therapist. This phenomenon provides a window into relational patterns and unresolved emotions, which therapists can help clients process. Conversely, resistance reflects unconscious defense mechanisms that hinder therapy progress. Psychoanalysts work to address and understand these resistances, aiding clients in confronting difficult truths.
Modern psychodynamic therapies have shifted focus, often prioritizing the conscious mind and current relationships over Freud&#39;s emphasis on sexual conflicts. They explore recurring life patterns, significant past events, and distressing emotions to provide insight. Therapists, influenced by figures like Heinz Kohut, emphasize empathy and healthy therapeutic relationships as a means to foster self-understanding and emotional resilience. Contemporary practices also adapt to practical constraints, favoring weekly sessions over the intensive regimens of Freud&#39;s era.
This evolution reflects an enduring commitment to exploring the depths of the human psyche while embracing contemporary perspectives on mental health.

Psychodynamic therapy explores the unconscious mind, such as repressed memories, conflicts, and early childhood experiences, to understand current ...

Modeling in Therapy

Modeling, a key technique in therapy, uses observational learning to help clients acquire and practice new skills by watching therapists demonstrate desired behaviors. This approach, rooted in Albert Bandura&#39;s concept of vicarious learning, plays a significant role in therapeutic interventions for various psychological conditions, including social anxiety, ADHD, and depression.
Participant Modeling
Participant modeling involves therapists demonstrating calm and effective behaviors in situations that clients find challenging. The therapist first models a constructive approach to the feared object or scenario and then guides the client through the same process step-by-step until the client gains the confidence to manage independently. This technique helps clients confront their fears in a controlled and supportive environment.
Assertion Training
Assertion training incorporates modeling to help individuals express their thoughts and feelings assertively while avoiding extremes of submissiveness and aggressiveness. This training teaches clients to advocate for themselves respectfully and effectively, ensuring that their needs and rights are recognized. The focus is on fostering assertiveness as the balance between passivity and hostility, a skill particularly valuable for those with social anxiety or interpersonal challenges.
Behavioral Rehearsal
Behavioral rehearsal, often integrated with assertion training, employs role-playing exercises to enhance the client&#39;s skills. The therapist assumes the role of a significant figure in the client&#39;s life, such as a supervisor or family member, allowing the client to practice responses in a simulated interaction. As a result, role reversal enables the therapist to model ideal verbal and nonverbal communication, illustrating not just what to say but also how to say it effectively. Feedback and coaching refine the client&#39;s performance, helping them build confidence and competence.
Applications Beyond Therapy
These techniques extend beyond therapy sessions by encouraging clients to apply learned skills in real-life situations. By practicing outside the therapeutic setting, clients consolidate their learning and improve their ability to handle social interactions. Modeling and social skills training are instrumental in managing conditions such as schizophrenia, autism spectrum disorders, and ADHD, where improved interpersonal and coping skills can significantly enhance quality of life.
Through structured observation, practice, and application, modeling serves as a powerful tool in helping clients overcome barriers and adopt constructive behaviors.

Modeling allows clients to learn by watching therapists demonstrate positive behaviors, such as maintaining eye contact during conversations.
Similarly, ...

Treatment Strategies for Psychological Disorders

Treatment approaches for psychological disorders fall into three main categories: psychological, biological, and sociocultural. Each approach targets different aspects of mental health, requiring varying levels of education and training.
Psychological therapies focus on modifying emotions, thoughts, and behaviors through talking, interpreting, listening, rewarding, challenging, and modeling. Clinical psychologists, counselors, and social workers commonly practice psychotherapy. Clinical psychologists typically hold a Ph.D. or Psy.D., which involves years of graduate education, supervised clinical practice, and a clinical internship. Licensing and certification are two processes through which society regulates psychotherapy practitioners. Specific laws govern the requirements for obtaining a license or certification, ensuring that professionals meet specific educational and training standards. These regulations outline the necessary qualifications and mandate formal examinations to assess the practitioner&#39;s skills and competency before allowing them to practice.
Biological therapies, or biomedical therapies, aim to treat psychological disorders by altering physical body functions, often through medications. This approach views mental health disorders similarly to physical diseases, requiring medical intervention. Practitioners of biological therapies are generally medical doctors (MDs), with psychiatrists specializing in this field. Psychiatrists undergo medical school, residency, and specialized training in psychiatry. While both psychologists and psychiatrists can administer psychotherapy, only psychiatrists can prescribe medication. If medication is necessary, a psychotherapist may refer clients to a psychiatrist for prescriptions.
Sociocultural therapies emphasize the impact of social and cultural factors on mental health. This approach considers the individual&#39;s relationships, societal roles, and cultural influences in developing and treating psychological issues. Practitioners include psychotherapists, social workers, and psychiatric social workers, who are trained to intervene in the context of these social systems. Social workers typically hold a master&#39;s degree in social work (MSW), and psychiatric social workers receive additional training in mental health. Paraprofessionals, individuals without formal mental health qualifications but trained by professionals, can also assist, often bringing personal experience with specific disorders.

Treatment strategies for psychological disorders fall under three broad approaches - psychological, biological, and sociocultural.
The psychological ...

Elements Crucial for Effective Psychotherapy

Research has highlighted several critical factors that influence the effectiveness of psychotherapy, such as the therapeutic alliance, the therapist, and the client.
The Therapeutic Alliance
The therapeutic alliance refers to the relationship between the therapist and the client. The alliance strengthens when the therapist and the client engage in a nurturing, supportive, trusting, empathetic, and respectful relationship, improving therapeutic outcomes. Therapists must monitor this relationship continuously, as studies suggest that clients who rate the alliance poorly are more likely to discontinue therapy or have negative experiences. Conversely, those who share a strong bond with their therapist are more likely to see successful outcomes.
The Therapist
The effectiveness of psychotherapy can depend significantly on the therapist&#39;s skill set and expertise. Experienced therapists deeply understand psychological issues and are committed to adapting their approach based on ongoing progress. Research also suggests that the compatibility between the therapist&#39;s style and the client&#39;s personality plays a crucial role in therapy success. For example, a more flexible and laid-back therapist may succeed with a client who resists change, while a directive approach may work better for others.
The Client
Another major determinant of therapeutic outcomes is the client&#39;s role in the process. Meta-analyses suggest that the client&#39;s participation is one of the most critical factors in determining success. While clients often seek therapy for life challenges, their strengths, skills, and motivation largely contribute to their progress. Research has consistently shown that therapy&#39;s success depends significantly on the client&#39;s engagement, and clients who fail to participate actively are more likely to experience unsatisfactory results.

Effective psychotherapy depends on three crucial elements&#8212; the therapeutic alliance, the therapist, and the client.
The therapeutic alliance refers ...

Psychotherapy

Psychotherapy is a versatile, nonmedical approach aimed at helping individuals address emotional, behavioral, and interpersonal issues to enhance their overall well-being. It can involve one-on-one sessions, couples counseling, or small group discussions with a therapist. The therapeutic process includes various techniques such as open discussion, interpretation of thoughts and behaviors, active listening, positive reinforcement, and role modeling. Psychotherapy aims to support individuals in understanding themselves better, whether or not they have diagnosed mental health conditions.
Insight Therapies
Insight therapies focus on uncovering the psychological motives behind behavior. By exploring unconscious processes, emotions, and past experiences, these therapies aim to help individuals better understand their actions and thought patterns. Psychodynamic therapy, based on Freud&#39;s psychoanalytic principles, delves into unresolved conflicts stemming from childhood. Humanistic therapy, developed by Carl Rogers, emphasizes personal growth and self-actualization, encouraging clients to explore their potential. Existential therapy addresses questions of meaning, freedom, and responsibility, while group therapy fosters interpersonal learning through shared experiences within a group setting.
Action Therapies
Action therapies, on the other hand, focus on modifying harmful thoughts and behaviors without delving deeply into their origins. Cognitive and cognitive-behavioral therapies (CBT) are the most widely used action therapies. Cognitive therapy helps individuals identify and challenge distorted or irrational thought patterns, while CBT combines this cognitive restructuring with behavioral techniques. The aim is to replace negative thought patterns with healthier, more adaptive ones and to encourage positive behavioral changes. These therapies are highly effective in treating conditions such as depression, anxiety disorders, and phobias.

Psychotherapy is a nonmedical approach designed to help individuals recognize and address emotional, behavioral, and interpersonal problems to improve ...

Conduct Disorder

Conduct disorder is a complex mental health diagnosis characterized by a repetitive and persistent pattern of behavior that violates societal norms, the rights of others, or age-appropriate rules. The diagnostic criteria for conduct disorder require the presence of at least three problematic behaviors within the past 12 months, with at least one occurring in the past six months. These behaviors are grouped into four categories: aggression toward people and animals; destruction of property; deceitfulness or theft; and serious violations of rules. The severity of symptoms, age of onset, and associated features all play crucial roles in understanding the disorder&#39;s impact and prognosis.
Diagnostic Features and Severity
The behaviors defining conduct disorder range from bullying, physical cruelty, and theft, to truancy and property destruction. Severity is categorized as mild, moderate, or severe, based on the number of symptoms and their impact on others. Childhood-onset conduct disorder, marked by symptoms before age 10, is associated with a poorer prognosis compared to adolescent-onset conduct disorder. Those with the specifier &#34;limited prosocial emotions&#34; &#8212; such as lack of remorse, callousness, or shallow affect &#8212; exhibit a more severe and persistent course.
Associated Factors and Risks
A combination of genetic, environmental, and temperamental factors influences conduct disorder. Risk factors include parental neglect, inconsistent discipline, exposure to violence, and association with delinquent peers. Neurobiological findings, such as reduced autonomic fear responses, and structural differences in brain regions associated with emotional regulation, have been observed but are not diagnostic. Co-occurring conditions, including ADHD, substance use disorders, and mood disorders, are common and can exacerbate the symptoms of conduct disorder.
Developmental Trajectory
Conduct disorder typically emerges during middle childhood or adolescence, often following oppositional defiant disorder. Symptoms evolve with age, beginning with less severe behaviors such as lying and escalating to more severe actions like theft and aggression. While the majority of cases remit by adulthood, childhood-onset conduct disorder is strongly predictive of antisocial personality disorder and other psychosocial difficulties later in life.
Sociocultural and Gender Considerations
The expression of conduct disorder behaviors differs by gender, with boys more likely to exhibit physical aggression and girls more often engaging in relational aggression or truancy. Cultural and environmental contexts, such as systemic oppression or high-crime areas, may influence the prevalence and manifestation of conduct disorder, necessitating careful diagnostic evaluation to avoid bias.
Conduct disorder presents significant challenges for affected individuals, families, and communities. Its association with academic failure, legal issues, and health risks underscores the importance of early intervention and comprehensive treatment approaches tailored to the individual&#39;s developmental and social context.

Conduct disorder involves a repetitive pattern of behavior violating others&#39; rights or societal norms, typically emerging in childhood or 
...

Intellectual Disability

Intellectual disability (ID) is a neurodevelopmental condition characterized by deficits in intellectual and adaptive functioning that manifest during the developmental period. This condition encompasses challenges in reasoning, memory, problem-solving, and learning, accompanied by impairments in everyday life skills, such as communication, self-care, and social interactions. Intellectual disability affects approximately 1% of the population in the United States, impacting an estimated 5 million individuals.
Assessment and Diagnosis
Historically, the diagnosis of intellectual disability was heavily reliant on IQ scores, with an IQ below 70 considered a diagnostic threshold. However, recent standards, such as those outlined in the DSM-5, prioritize adaptive functioning over rigid IQ thresholds. Current assessments evaluate impairments across three domains: conceptual, social, and practical skills. Conceptual abilities include literacy, numeracy, and time management, while social skills cover empathy, interpersonal communication, and compliance with social norms. Practical skills pertain to personal care, job responsibilities, and daily routines.
Adaptive functioning is evaluated in the context of the individual&#39;s life circumstances. For example, two individuals with similar IQ scores might differ significantly in functionality &#8212; one might live independently with minimal support, while the other might require institutional care. This shift in diagnostic criteria emphasizes functional capabilities rather than test scores, allowing for a more nuanced understanding of intellectual disability.
Causes and Classifications
The etiology of intellectual disability can be organic or cultural-familial. Organic causes include genetic disorders, such as Down syndrome and Fragile X syndrome, and prenatal exposure to alcohol or toxins,&#160; which result in fetal alcohol syndrome. Fetal alcohol syndrome is a condition that results from exposing a developing embryo to alcohol, and intelligence levels can range from below average to levels associated with intellectual disability. Environmental factors, such as malnutrition, lack of stimulation, and inadequate healthcare, often underlie cultural-familial intellectual disability. This type of intellectual disability, with no identifiable physical damage, typically results in milder impairments.
Classifications range from mild to profound, based on the severity of deficits. Individuals with mild ID may achieve partial independence with support, while those with profound ID often remain fully dependent on caregivers due to severe limitations in all adaptive domains.
Holistic Perspectives
While intellectual disability affects cognitive and adaptive skills, it does not diminish the individual&#39;s capacity for forming meaningful relationships and contributing to their community. Understanding and addressing their diverse needs through tailored support and interventions can significantly enhance their quality of life and social integration.

Intellectual disability, previously termed as mental retardation, has been defined by significant cognitive deficits&#8212;which have been established ...

Learning Disabilities

Learning disabilities are cognitive disorders caused by neurological impairments that affect cognitive functions like language and reading, without indicating overall intellectual or developmental challenges. These disabilities differ from global intellectual or developmental disabilities as they are limited to distinct cognitive functions. Common learning disabilities include dysgraphia, dyslexia, and dyscalculia, each of which impacts unique aspects of learning.
<h4>Dyslexia</h4>
Dyslexia is a learning disability characterized by difficulties with reading fluency and comprehension despite normal intelligence. Individuals with dyslexia often struggle to decode and interpret written text. They may require additional time for reading and have difficulty associating letters with their corresponding sounds. Research has shown that dyslexia is independent of general cognitive ability, occurring across a broad spectrum of IQ levels.
<h4>Dysgraphia</h4>
Dysgraphia is marked by difficulty in writing legibly and coherently, making the physical act of writing a significant challenge. This impairment often contrasts with the individual&#39;s overall intellectual potential. Children with dysgraphia may struggle to translate their thoughts into written words and face spatial organization challenges, affecting handwriting consistency. These difficulties often require academic accommodations, such as assistive technologies or alternative assessment methods, to ensure success in educational settings.
<h4>Dyscalculia</h4>
Dyscalculia affects a child&#39;s ability to learn and understand arithmetic. Early signs include difficulty recognizing the quantity of small groups of objects without counting. Other challenges involve memorizing basic math facts, organizing numerical information, and distinguishing between numerical symbols and their verbal equivalents.

Learning disabilities are cognitive disorders caused by neurological impairments that affect cognitive functions like language and reading without ...

Personality Disorders: Dependent and Obsessive-Compulsive

Dependent personality disorder and obsessive-compulsive personality disorder are two separate psychological conditions that influence behavior, relationships, and overall life functioning. Though both involve maladaptive behaviors, their core characteristics and motivations differ significantly.
&#160;Dependent Personality Disorder
Dependent personality disorder is characterized by an excessive reliance on others to manage various aspects of life. Individuals with this disorder often struggle with decision-making, requiring constant reassurance and advice before making even minor choices. Their persistent fear of separation and abandonment leads them to exhibit submissive and clingy behaviors, as they prioritize maintaining relationships over asserting independence.
A key feature of dependent personality disorder is a profound lack of self-confidence, which contributes to feelings of helplessness when alone. Individuals with this disorder tend to perceive others as more competent and powerful while viewing themselves as childlike, weak, and incapable of functioning independently. This reliance on external support can lead them to tolerate mistreatment or remain in unhealthy relationships to avoid being alone.
Furthermore, their reluctance to take the initiative often results in an inability to complete tasks without assistance. They may avoid responsibilities that require self-sufficiency, preferring to defer to others for guidance. As a result, they may struggle in professional and personal settings where autonomy is needed.
Obsessive-Compulsive Personality Disorder
Obsessive-compulsive personality disorder is characterized by a pervasive preoccupation with perfectionism, order, and control, often at the expense of task completion, relationships, and overall well-being. Individuals with this disorder are excessively focused on details, rules, and schedules, hindering productivity as they struggle to overcome minor imperfections. Their rigid adherence to structure and standards often leads to inefficiency, as they prioritize precision over practicality.
A defining feature of obsessive-compulsive personality disorder is an intense devotion to work and productivity, often at the expense of leisure activities and personal relationships. These individuals may neglect social interactions and relaxation, believing that time not spent on work or structured tasks is wasted. Their inflexible mindset makes it difficult for them to delegate tasks, as they insist that things be done precisely their way. This insistence on control can strain professional and personal relationships, as they impose strict expectations on themselves and others.
Furthermore, individuals with this disorder tend to exhibit a rigid and stubborn disposition, resisting changes or alternative approaches. Their perfectionistic tendencies extend to financial habits, often manifesting as excessive frugality. They may be reluctant to spend money, even on necessities, due to an ingrained need for control and order in all aspects of life.

Dependent personality disorder is characterized by an excessive reliance on others to meet emotional and physical needs.
Individuals with this disorder ...

Creating a Winogradsky Column: A Method to Enrich the Microbial Species in a Sediment Sample

Source: Elizabeth Suter1, Christopher Corbo1, Jonathan Blaize1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
The Winogradsky column is a miniature, enclosed ecosystem used for enriching sediment microbial communities, especially those involved in sulfur cycling. The column was first used by Sergei Winogradsky in the 1880s and has since been applied in the study of many diverse microorganisms involved in biogeochemistry, such as photosynthesizers, sulfur oxidizers, sulfate reducers, methanogens, iron oxidizers, nitrogen cyclers, and more (1,2).
The majority of microorganisms on Earth are considered unculturable, meaning that they cannot be isolated in a test tube or on a petri dish (3). This is due to many factors, including that microorganisms depend on others for certain metabolic products. The conditions in a Winogradsky column closely mimic a microorganism&#39;s natural habitat, including their interactions with other organisms, and allows for them to be grown in a lab. Therefor, this technique permits scientists to study these organisms and understand how they are important to Earth&#39;s biogeochemical cycles without having to grow them in isolation.
Earth&#39;s environments are full of microorganisms which thrive in all types of habitats, such as soils, ocean water, clouds, and deep-sea sediments. In all habitats, microorganisms depend on each other. As a microorganism grows, it consumes particular substrates, including carbon-rich fuels like sugars as well as nutrients, vitamins, and respiratory gases like oxygen. When these important resources run out, different microorganisms with different metabolic needs can then bloom and thrive. For example, in the Winogradsky column, microbes first consume the added organic material while depleting the oxygen in the bottom layers of the column. Once the oxygen is used up, anaerobic organisms can then take over and consume different organic materials. This consecutive development of different microbial communities over time is called succession (4). Microbial succession is important in a Winogradsky column, where microbial activity changes the chemistry of the sediment, which then affects activity of other microbes and so on. Many microorganisms in soils and sediments also live along gradients, which are transitional zones between two different types of habitats based on the concentrations of substrates (5). At the correct spot in the gradient, a microbe can receive optimal amounts of different substrates. As a Winogradsky column develops, it begins to mimic these natural gradients, particularly in oxygen and sulfide (Fig. 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10506/10506fig1.jpg" /> 
Figure 1: A representation of the oxygen (O2) and sulfide (H2S) gradients that develop in a Winogradsky column.
In a Winogradsky column, mud and water from a pond or wetland are mixed in a transparent column and allowed to incubate, typically in the light. Additional substrates are added to the column to give the community sources of carbon, usually in the form of cellulose, and sulfur. Photosynthesizers typically start to grow in the top layers of the sediment. These photosynthetic microorganisms are largely composed of cyanobacteria, which produce oxygen and appear as a green or red-brown layer (Fig. 2, Table 1). While photosynthesis produces oxygen, oxygen is not very soluble in water and it decreases below this layer (Fig. 1). This creates a gradient of oxygen, ranging from high concentrations of oxygen in the top layers to zero oxygen in the bottom layers. The oxygenated layer is called the aerobic layer and the layer without oxygen is called the anaerobic layer.
In the anaerobic layer, many different microbial communities can proliferate depending on the type and amount of substrates that are available, the source of the initial microbes, and the porosity of the sediment. At the bottom of the column, organisms that anaerobically break down organic matter can thrive. Microbial fermentation produces organic acids from the breakdown of cellulose. These organic acids can then be used by sulfate reducers, which oxidize those organics using sulfate, and produce sulfide as a byproduct. The activity of sulfate reducers is indicated if the sediment turns black, because iron and sulfide react to form black iron-sulfide minerals (Fig. 2, Table 1). The sulfide also diffuses upward, creating another gradient in which sulfide concentrations are high in the bottom of the column and low in the top of the column (Fig. 1).
Near the middle of the column, sulfur oxidizers take advantage of the supply of oxygen from above and sulfide from below. With the right amount of light, photosynthetic sulfur oxidizers can develop in these layers. These organisms are known as green and purple sulfur bacteria, and often appear as green, purple, or purple-red filaments and blotches (Fig. 2, Table 1). Green sulfur bacteria have a higher tolerance for sulfide and usually develop in the layer directly below purple sulfur bacteria. Above the purple sulfur bacteria, purple nonsulfur bacteria may also develop. These organisms photosynthesize using organic acids as electron donors instead of sulfide and often appear as a red, purple, orange, or brown layer. Nonphotosynthetic sulfur oxidizers can develop above the purple nonsulfur bacteria, and these usually appear as white filaments (Fig. 2, Table 1). Additionally, bubbles may also form in the Winogradsky column. Bubbles in the aerobic layers indicates the production of oxygen by the cyanobacteria. Bubbles in the anaerobic layers are likely due to the activity of methanogens, organisms which anaerobically break down organic matter and form methane as a byproduct.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Position in Column</td>
			<td>Functional group</td>
			<td>Organism Examples</td>
			<td>Visual Indicator</td>
		</tr>
		<tr>
			<td>Top</td>
			<td>Photosynthesizers</td>
			<td>Cyanobacteria</td>
			<td>Green or reddish-brown layer. Sometimes bubbles of oxygen.</td>
		</tr>
		<tr>
			<td rowspan="5"></td>
			<td>Nonphotosynthetic sulfur oxidizers</td>
			<td>Beggiatoa, Thiobacilus</td>
			<td>White layer.</td>
		</tr>
		<tr>
			<td>Purple nonsulfur bacteria</td>
			<td>Rhodomicrobium, Rhodospirilum, Rhodopseuodmonas</td>
			<td>Red, purple, orange, or brown layer.</td>
		</tr>
		<tr>
			<td>Purple sulfur bacteria</td>
			<td>Chromatium</td>
			<td>Purple, or purple-red layer.</td>
		</tr>
		<tr>
			<td>Green sulfur bacteria</td>
			<td>Chlorobium</td>
			<td>Green layer.</td>
		</tr>
		<tr>
			<td>Sulfate Reducing Bacteria</td>
			<td>Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfuromonas</td>
			<td>Black layer.</td>
		</tr>
		<tr>
			<td>Bottom</td>
			<td>Methanogens</td>
			<td>Methanococcus, Methanosarcina</td>
			<td>Sometimes bubbles of methane.</td>
		</tr>
	</tbody>
</table>
Table 1: The main groups of bacteria that may appear in a classical Winogradsky column, from top to bottom. Examples of organisms from each group are given, and the visual indicators of each layer of organisms are listed. Based on Perry et al. (2002) and Rogan et al. (2005).

Winogradsky Column to Enrich Microbial Species in a Sample

ウィノグラツキーカラムの作成:堆積物サンプル中の微生物種を濃縮する方法

Source: Elizabeth Suter1, Christopher Corbo1, Jonathan Blaize1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 
...

Serial Dilutions and Plating: Microbial Enumeration

Source: Jonathan F. Blaize1, Elizabeth Suter1, and Christopher P. Corbo1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Quantitative assessment of prokaryotes can be onerous given their abundance, propensity for exponential proliferation, species diversity within a population, and specific physiological needs. Compounding this challenge, is the four-phase nature in which bacteria replicate (lag, log, stationary and death). The ability to accurately estimate microorganism concentration is necessary for successful identification, isolation, cultivation, and characterization (6). As such, microbiologists have employed serial dilution and various plating techniques for over a century to reliably quantify bacterial and viral load in clinical, industrial, pharmaceutical, and academic laboratory environments (2,4,6). Descriptions of this methodology first appeared in 1883 when the German scientist and physician Robert Koch published his work on infectious disease-causing agents (2). Often referred to as the father of modern bacteriology, Koch&#39;s forenamed techniques have become the gold standard for enumeration of microorganisms, culturable or otherwise, throughout the world.
Serial dilution is a systematic reduction of a known or unknown entity (a solute, organism, etc.) through successive re-suspension of an initial solution (solution0) into fixed volumes of a liquid diluent (blanks). These blanks usually consist of 0.45% saline, though the composition can be varied (7). While an experimenter can choose any volume for each diluent, it is most often a multiple of 10, facilitating logarithmic reduction of the sample. For example, solution0 contains a total of 100 E. coli cells suspended in 10 mL of nutrient broth. If 1 mL of solution0 is removed and added to 9 mL of saline (diluent1), the new solution (solution1) would contain 1/10th of the initial concentration of E. coli. In this example, the new solution (solution1) would contain 10 E. coli cells. Repeating this process by removing 1 mL of solution1 and adding it to another 9 mL of saline (diluent2) would yield solution2, containing only a single E. coli cell. Since each new solution (9 mL of diluent + 1 mL of solution) contains a total of 10mL, we can conclude that the dilution factor for this reduction is 10 or that this was a 10-fold serial dilution (Figure 1). Since we only began with 100 cells in this example and we are diluting by a factor of 10, only two steps are required to reach the absolute minimum concentration of 1 cell.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10507/10507fig1.jpg" /> 
Figure 1: Serial dilution of a stock solution. A 1 mL aliquot of the stock solution (solution0) is added to tube 1 which contains 9 mL of 0.45% saline (dilent1); the product of this mixture is solution1. Repeat by aliquoting 1 mL of the newly created solution1 and adding it to tube 2. Aliquoting and resuspension continues in this fashion until the final tube is reached, diluting the stock concentration by a factor of 10 each with each step. <a href="https://www.jove.com/files/ftp_upload/10507/10507fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Serial dilution is the simplest technique for obtaining manageable concentrations of a desired organism and it is complemented by petri dish streaking and spreading, just two of many plating techniques used by microbiologists. This benefit of this approach is that the experimenter can harvest pure strains of a single species or separate strains from a mixed population (7). Streaking is accomplished by introducing an organism to a solid medium (generally consisting of agarose) that it will grow upon if the appropriate nutrients are available. Gently sweeping a sterile inoculating loop across the medium (so that a subtle streak remains) in a rigid sinusoidal pattern will distribute the organism proportionally to the frequency of the experimenter&#39;s waveform. Dividing the Petri dish into thirds or fourths (quadrant streak) and decreasing the frequency of each streak as a new region of the dish is entered will gradually reduce the number of microorganisms that can occupy that region, producing single colonies instead of an unquantifiable bacterial lawn. Spread plating does not additionally dilute samples; a sterile glass spreader is used to distribute an aliquot of suspension media across an entire petri dish (Figure 2). The colonies that grow on the spread plate arise from a single cell and each colony on the dish can be counted to estimate the number of colony forming units per milliliter (CFU) in a given suspension, represented as CFU/mL (6) (Figure 3) Soft agar and replica plating are variations of the aforementioned techniques and allow for isolation of bacteriophage and mutant screening, respectively (1,7).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10507/10507fig2.jpg" /> 
Figure 2: Plate streaking for bacterial enumeration and strain isolation. Label the bottom of a petri dish with identification information (bacteria name, date, media) and divide into thirds. After selecting an appropriate dilution of the stock sample, take a sterile (disposable or flamed) inoculating loop and submerge it the test tube (here, T3). Slightly raise the petri dish cover on one side so that only the inoculating loop can access the agar. Glide the inoculating loop across the top of the media in a zig-zag fashion being careful not to compromise the agar. Rotate the plate by roughly 1/3rd (~118&#176;) and reduce the frequency of zig-zag motion. Rotate a final time and reduce zig-zag frequency once more. <a href="https://www.jove.com/files/ftp_upload/10507/10507fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10507/10507fig3.jpg" /> 
Figure 3: Spread plating. 1 g of the aerobic zone was resuspended in T1 and then serially diluted. A sterile glass or plastic disposable spreading rod is used to distribute inoculum throughout each dish. This was repeated with 1 g of the anaerobic zone. <a href="https://www.jove.com/files/ftp_upload/10507/10507fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As with serial dilutions, a logarithmic scale is employed to express organismal concentration. The number of colonies grown in standard petri dishes measuring 100mm x15mm can be enumerated manually (or automated with the aid of computational processing) by identifying isolated clusters of growth. Counts that total fewer than 30 or greater than 300 should be defined as too few to count (TFTC) or too numerous to count (TNTC), respectively. In the case of the latter, a serial dilution should be performed to reduce concentration before restreaking a new petri dish. Averaging the number of self-contained colonies identified from three separate petri dishes and multiplying the mean by the dilution factor will yield CFU/mL; plotting the log10 of CFU/mL against time will reveal the mean generation time of the organism (7).

シリアル希釈とめっき:微生物列挙

Source: Jonathan F. Blaize1, Elizabeth Suter1, and Christopher P. Corbo1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island ...

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

エンリッチメント培養:選択的および差動媒体における好気性微生物と嫌気性微生物の培養

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...

Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Seemingly impossible to determine, microbial biodiversity is truly astounding with an estimated one trillion coexisting species (1,2). Although particularly harsh climates, like the acidic environment of the human stomach (3) or the subglacial lakes of Antarctica (4), may be dominated by a specific species, bacteria are typically found in mixed cultures. As each strain may influence the growth of another (5), the ability to separate and cultivate &#34;pure&#34; (consisting only of one type) colonies has become essential in clinical and academic settings alike. Pure cultures enable further genetic (6) and proteomic examinations (7), analysis of sample purity and, perhaps more noteworthy, the identification and characterization of infectious agents from clinical samples.
Bacteria have a wide range of growth requirements and there are numerous types of nutrient media designed to sustain both the un-demanding and the fastidious species (8). Growth media can be prepared either in liquid form (as a broth) or in a typically agar-based (a gelling agent derived from red algae) solid form. Whereas direct inoculation into broth carries the risk of generating a genetically diverse or even mixed bacterial population, plating and re-streaking creates a purer culture where each cell has a highly similar genetic makeup. The streak plate technique is based on progressive dilution of a sample (Figure 1), with the aim of separating individual cells from one another. Any viable cell (hereafter referred to as a colony forming unit, CFU) sustained by the media and designated environment can subsequently found an isolated colony of daughter-cells through binary fission. In spite of the rapid mutation rates within bacterial communities, this cell-group is generally regarded as clonal. Harvesting and re-streaking this population consequently ensures that subsequent work involves only a single bacterial type.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10509/10509fig1.jpg" /> 
Figure 1: A streak plate is based on progressive dilution of the original sample. I) The inoculum is initially dispersed using a zig-zag motion, creating an area with a relatively dense bacterial population. II-IV) Streaks are drawn from the preceding area, using a sterile inoculation loop each time, until the fourth quadrant is reached. V) A final zig-zag motion directed towards the middle of the plate forms a region where the inoculum has been markedly diluted, allowing colonies to appear separate from one another.
The streak plate technique can also be combined with the use of selective and/or differential media. A selective medium will inhibit the growth of certain organisms (e.g. through addition of antibiotics) whilst a differential medium will solely help distinguish one from another (e.g. through hemolysis on blood agar plates).
Underlying all work in microbiology is the use of aseptic (sterile) techniques. Every bacterial culture should be considered potentially pathogenic as there is a risk of unintended growth of treacherous strains, aerosol formation and contamination of equipment/personnel. To minimize these risks, all media, plastic-, metal- and glass-ware are typically sterilized through autoclaving before and after use, subjecting them to high-pressure saturated steam at around 121&#176;C that effectively wipes out any lingering cells. The work space is generally disinfected using ethanol both prior to, and after, use. Lab coat and gloves are always worn during work with infectious agents.

Pure Cultures and Streak Plating: Isolation of Bacterial Colonies

純粋な培養物と縞めっき:混合サンプルからの単一細菌コロニーの単一の分離

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Planet Earth is a habitat for millions of bacterial species, each of which has specific characteristics. Identification of bacterial species is widely used in microbial ecology to determine biodiversity of environmental samples and medical microbiology to diagnose infected patients. Bacteria can be classified using conventional microbiology methods, such as microscopy, growth on specific media, biochemical and serological tests, and antibiotic sensitivity assays. In recent decades, molecular microbiology methods have revolutionized bacterial identification. A popular method is 16S ribosomal RNA (rRNA) gene sequencing. This method is not only faster and more accurate than conventional methods, but also allows identification of strains that are difficult to grow in laboratory conditions. Furthermore, differentiation of strains at the molecular level enables discrimination between phenotypically identical bacteria (1-4).
16S rRNA joins with a complex of 19 proteins to form a 30S subunit of the bacterial ribosome (5). It is encoded by the 16S rRNA gene, which is present and highly conserved in all bacteria due to its essential function in ribosome assembly; however, it also contains variable regions which may serve as fingerprints for particular species. These features have made the 16S rRNA gene an ideal genetic fragment to be used in identification, comparison, and phylogenetic classification of bacteria (6).
16S rRNA gene sequencing is based on the polymerase chain reaction (PCR) (7-8) followed by DNA sequencing (9). PCR is a molecular biology method used to amplify specific fragments of DNA through a series of cycles that include:
i) Denaturation of a double stranded DNA template 
ii) Annealing of primers (short oligonucleotides) that are complementary to the template 
iii) Extension of primers by the DNA polymerase enzyme, which synthesizes a new DNA strand
A schematic overview of the method is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10510/10510fig1.jpg" /> 
Figure 1: Schematic overview of the PCR reaction. <a href="https://www.jove.com/files/ftp_upload/10510/10510fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several factors that are important for a successful PCR reaction, one of which is quality of the DNA template. Isolation of chromosomal DNA from bacteria can be performed using standard protocols or commercial kits. Special care should be taken to obtain DNA that is free of contaminants that can inhibit the PCR reaction.
Conserved regions of the 16S rRNA gene permit the design of universal primer pairs (one forward and one reverse) that can bind to and amplify the target region in any bacterial species. The target region can vary in size. While some primer pairs can amplify most of the 16S rRNA gene, others amplify only parts of it. Examples of commonly used primers are shown in Table 1 and their binding sites are depicted in Figure 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer name</td>
			<td>Sequence (5&#39;&#8594;3&#39;)</td>
			<td>Forward/ reverse</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>8F b)</td>
			<td>AGAGTTTGATCCTGGCTCAG</td>
			<td>forward</td>
			<td>-1</td>
		</tr>
		<tr>
			<td>27F</td>
			<td>AGAGTTTGATCMTGGCTCAG</td>
			<td>forward</td>
			<td>-10</td>
		</tr>
		<tr>
			<td>515F</td>
			<td>GTGCCAGCMGCCGCGGTAA</td>
			<td>forward</td>
			<td>-11</td>
		</tr>
		<tr>
			<td>911R</td>
			<td>GCCCCCGTCAATTCMTTTGA</td>
			<td>reverse</td>
			<td>-12</td>
		</tr>
		<tr>
			<td>1391R</td>
			<td>GACGGGCGGTGTGTRCA</td>
			<td>reverse</td>
			<td>-11</td>
		</tr>
		<tr>
			<td>1492R</td>
			<td>GGTTACCTTGTTACGACTT</td>
			<td>reverse</td>
			<td>-11</td>
		</tr>
	</tbody>
</table>
Table 1: Examples of standard oligonucleotides used in amplification of 16S rRNA genes a). 
a) The expected lengths of the PCR product generated using the different primer combinations can be estimated by calculating the distance between the binding sites for the forward and the reverse primer (see Figure 2), e.g. the size of the PCR product using primer pair 8F-1492R is ~1500 bp, and for primer pair 27F-911R ~900 bp. 
b) also known as fD1
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10510/10510fig2.jpg" /> 
Figure 2: Representative figure of the 16S rRNA sequence and the primer-binding sites. Conserved regions are colored in grey and variable regions are filled with diagonal lines. To allow for the highest resolution, primer 8F and 1492R (name based on location on rRNA sequence) are used to amplify the whole sequence, allowing for the sequencing of several variable regions of the gene. <a href="https://www.jove.com/files/ftp_upload/10510/10510fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cycling conditions for PCR (i.e. the temperature and time required for the DNA to be denatured, annealed with primers, and synthesized) are dependent on the type of polymerase that is used and the properties of the primers. It is recommended to follow the manufacturer&#39;s guidelines for a particular polymerase.
After the PCR program is completed, the products are analyzed by agarose gel electrophoresis. A successful PCR yields a single band of expected size. The product must be purified prior to sequencing to remove residual primers, deoxyribonucleotides, polymerase, and buffer which were present in the PCR reaction. The purified DNA fragments are usually sent for sequencing to commercial sequencing services; however, some institutions perform DNA sequencing at their own core facilities.
The DNA sequence is automatically generated from a DNA chromatogram by a computer and must be carefully checked for quality, as manual editing is sometimes needed. Following this step, the gene sequence is compared with sequences deposited in the 16S rRNA database. The regions of similarity are identified, and the most similar sequences are delivered.

16S rRNA Sequencing: Identifying Bacterial Species by PCR

16S rRNAシーケンシング:細菌種を同定するPCRベースの技術

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, ...

Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements

Source: Andrew J. Van Alst1, Rhiannon M. LeVeque1, Natalia Martin1, and Victor J. DiRita1 
1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
Growth curves provide valuable information on bacterial growth kinetics and cell physiology. They allow us to determine how bacteria respond in variable growth conditions as well as to define optimal growth parameters for a given bacterium. An archetypal growth curve progresses through four stages of growth: lag, exponential, stationary, and death (1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10511/10511fig1.jpg" /> 
Figure 1: Bacterial growth curve. Bacteria grown in batch culture progress through four phases of growth: lag, exponential, stationary, and death. Lag phase is the period of time it takes for the bacteria to reach a physiological state capable of rapid cell growth and division. Exponential phase is the stage of fastest cell growth and division during which DNA replication, RNA transcription, and protein production all occur at a constant, rapid rate. Stationary phase is characterized by a slowing down and plateauing of bacterial growth due to nutrient limitation and/or toxic intermediate accumulation. Death phase is the stage during which cell lysis occurs as a result of severe nutrient limitation.
Lag phase is the period of time it takes for the bacteria to reach a physiological state capable of rapid cell growth and division. This lag occurs because it takes time for bacteria to adjust to their new environment. Once the necessary cellular components are generated in lag phase, bacteria enter the exponential phase of growth where DNA replication, RNA transcription, and protein production all occur&#160;at a constant, rapid rate (2). The rate of rapid cell growth and division during the exponential phase is calculated as the generation time, or doubling time, and is the fastest rate at which the bacteria can replicate under the given conditions (1). The doubling time can be used to compare different growth conditions to determine which is more favorable for bacterial growth. The exponential growth phase is the most reproducible growth condition as bacterial cell physiology is consistent throughout the entire population (3). Stationary phase follows the exponential phase where cell growth plateaus. Stationary phase is brought on due to nutrient depletion and/or accumulation of toxic intermediates. Bacterial cells continue to survive in this stage, although the rate of replication and cell division is drastically reduced. The final phase is death, where severe nutrient depletion leads to the lysing of cells. Features of the growth curve that provide the most information include the duration of lag phase, the doubling time, and the maximum cell density reached.
Quantification of bacteria in batch culture can be determined using both colony forming units and optical density measurements. Enumeration by colony forming units (CFU) provides a direct measurement of bacterial cell counts. The standard unit of measure for CFU is the number of culturable bacteria present per 1 mL of culture (CFU/mL) determined by serial dilution and spread plating techniques. For each timepoint, a 1:10 dilution series of the batch culture is performed and 100 &#181;l of each dilution is spread plated using a cell spreader.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10511/10511fig2.jpg" /> 
Figure 2. Serial dilution plating schematic. General flowthrough for dilution plating from batch culture. The batch culture is serially diluted 1:10 by transferring 1 mL &#160;of the previous dilution into the subsequent tube containing 9ml PBS. From each dilution tube, 100 &#181;l is spread plated using a plate spreader which is an additional dilution of 1:10 as it is 1/10th the volume of 1 mL volume when calculating CFU/mL. Plates are incubated and enumerated once clonal colonies grow on the plates.
The plates are then incubated overnight and clonal colonies enumerated. The dilution plate which grew 30-300 colonies is used to calculate the CFU/mL for the given timepoint (4, 5). Stochastic variation in colony counts under 30 are subject to greater error in the calculation of CFU/mL and counting colonies greater than 300 can be underestimated due to colony crowding and overlapping. Using the dilution factor for the given plate, the CFU of the batch culture can be calculated for each timepoint.
Optical density gives an instant approximation of bacterial cell count measured using a spectrophotometer. The optical density is a measure of absorbance of light particles that pass through 1cm of culture and detected by a photodiode sensor (6). The optical density of a culture is measured in relation to a media blank and increases as bacterial density increases. For bacterial cells, a wavelength of 600 nm (OD600) is typically used when measuring optical density (4). By generating a standard curve relating colony forming units and optical density, the optical density measurement can be used to readily approximate the bacterial cell count of a batch culture. However, this relationship begins to deteriorate as early as 0.3 OD600 as cells begins to change shape and accumulate extracellular products in the media, influencing the optical density reading as it relates to CFU (7). This error becomes more pronounced during stationary and death phases.
Here, Escherichia coli is grown in Luria-Bertani (LB) broth at 37&#176;C over the course of 30 hours (7). Both CFU/mL and optical density growth curves have been generated&#160;as well as the standard curve relating optical density to CFU.&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10511/10511fig3.jpg" /> 
Figure 3. Escherichia coli optical density at 600 nm wavelength (OD600) growth curve. Optical density values were taken directly from the spectrophotometer after blanking with sterile LB media. OD600 values greater than 1.0 were diluted 1:10 by combining 100 &#181;l culture with 900 &#181;l fresh LB, again measured, and then multiplied by 10 to obtain the OD600 value. This step is taken as the accuracy in measurement of the spectrophotometer is reduced at high cell density. From the curve, lag phase extends to around 1h of growth, transitions to exponential phase from 2h to 7h, then begins to plateau, entering stationary phase. Death phase is not a stark transition, however, as optical density gradually begins to decline after 15h.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10511/10511fig4.jpg" /> 
Figure 4. Escherichia coli colony forming unit per milliliter (CFU/mL) growth curve. CFU/mL values for each timepoint were calculated from the dilution plate that contained 30-300 colonies. From the curve, lag phase extends out to around 2h of growth, transitions to exponential phase from 2h to 7h, then begins to plateau, entering stationary phase. Death phase is not a stark transition, however, as CFU/mL gradually begins to decline after 15h from a peak of 2 x 109 to approximately 5 x 108 at 30 hours.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/10511/10511fig5.jpg" /> 
Figure 5. Standardization curve for CFU/mL versus OD600. A linear regression can be used to relate these units so that optical density may be used to approximate bacterial cell density. Optical density can be used to provide and instant approximation of the CFU/mL of the batch culture. Here, only the first six timepoints are plotted as the relationship between OD600 and CFU/mL is less accurate beyond 1.0 OD600 as cell shape and extracellular products begin to accumulate as the bacteria enter stationary phase, which occurs shortly after reaching 1.0 OD600. Changes in cell shape and extracellular products in the media influence the optical density reading and therefore the relationship between optical density and the number of bacteria in the culture is also impacted.
The doubling time has also been determined to be 15 minutes and 19 seconds. From this data, the capacity for growth in LB for E. coli can be visualized and be used for comparison between different media or bacteria.

Growth Curves, CFU and Optical Density Measurements

成長曲線:コロニー形成単位と光学密度測定を用いて成長曲線を生成する

Source: Andrew J. Van Alst1, Rhiannon M. LeVeque1, Natalia Martin1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy

Source: Anna Bl&#228;ckberg1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund Sweden
Knowledge of the interactions between antibiotics and bacteria is important in understanding how microbes evolve antibiotic resistance. In 1928, Alexander Fleming discovered penicillin, an antibiotic that exerts its antibacterial function by interfering with cell wall regeneration (1). Other antibiotics with diverse mechanisms of action have subsequently been discovered, including drugs that inhibit DNA replication and protein translation in bacteria; however, no new antibiotics have been developed in recent years. Resistance to current antibiotics has been increasing, resulting in severe infectious diseases that cannot be effectively treated (2). Here, we describe several methods to assess antibiotic resistance in bacterial populations. Each of these methods works, regardless of the mechanism of action of the antibiotics used, because bacterial death is the measured outcome. Antibiotic resistance is not only rapidly disseminated specifically through hospital settings, but also throughout society. In order to investigate such means of resistance, different methods have been developed including the Epsilometer test (E-test) and the broth dilution test (3).
The E-test is a well-established method and is a cost-effective tool that quantifies Minimal Inhibitory Concentration (MIC) data, the lowest concentration of an antimicrobial that inhibits visible growth of a microorganism. Depending on the bacterial strain and antibiotics used, the MIC value can vary between sub &#956;g/mL to &#62;1000 &#956;g/mL (4). The E-test is performed using a plastic strip containing a predefined antibiotic gradient, which is imprinted with the MIC reading scale in &#956;g/mL. This strip is directly transferred on the agar matrix when applied to the inoculated agar plate. After incubation, a symmetrical elliptic inhibition zone is visible along the strip as bacterial growth is prevented. MIC is defined by the area of inhibition, which is the endpoint where the ellipse intersects the strip. Another common method to determine MIC is the microbroth dilution method. Microbroth dilution incorporates different concentrations of the antimicrobial agent added to a broth medium containing inoculated bacteria. After incubation, MIC is defined as the lowest concentration of antibiotic that prevents visible growth (5). It is also a quantitative method and can be applied to several bacteria. Disadvantages of this method include the possibility of errors when preparing the concentrations of the reagents and the large number of reagents required for the experiment. Measuring antibiotic resistance is imperative from both a clinical and research perspective, and these in vitro methods of investigating resistance are discussed and showcased below.
The profile of resistance for a specific bacterium can be applied in order to optimize antibiotic treatment to determine whether a patient would benefit from combination treatment versus single therapy. For the usage of more than one antibiotic at a single time, it is imperative to know their interactions with each other and if they have an additive, synergistic, or antagonistic effect. An additive effect can be seen when the joint effect of the antibiotics equals the potency of the individual antibiotics given at an equal dose. Synergy between antibiotics, on the other hand, is present when the joint effect of the antibiotics is more potent than if the drug would be given alone (6). Applying combinations of antimicrobial treatment is used to avoid the occurrence of antimicrobial resistance thus to enhance the effect of the individual antibiotic treatment (7). Knowledge of antagonism is also as important to prevent unnecessary use of antimicrobial combinations. E-test methodology offers simple and several ways to determine possible synergy and antagonism between different antimicrobial agents. In order to face the proliferation of antibiotic resistant pathogens, knowledge of possible synergistic and antagonistic mechanisms of certain antibiotics is important resulting in clinical efficacy and battling multidrug-resistance.
Determination of synergy using E-tests can be divided into two broad approaches: cross and non-cross testing. While both synergy tests rely on the previous knowledge of individual MIC values, the two approaches are slightly different in methodology and conceptual approach. In a non-cross synergy test, the first antibiotic in the pair to be tested is placed on an agar plate inoculated with bacteria. After allowing the antibiotics from the first strip infuse the plate (e.g. after 1 hour), the strip is removed and a new strip containing the second antibiotic is placed on the exact same spot as the first, making sure to place the two individual MIC values on top of each other. The resulting inhibition zone can then be analyzed as described above, and synergy calculated based on the Equation 1.
Equation 1 - Fractional Inhibitory Concentrations (FIC)
<img src="/files/ftp_upload/10512/10512eq1.jpg" />
Values &#62;0.5 demonstrates synergy.
While rewarding the examiner with easy-to-analyze plates, the method is somewhat laborious and time-consuming due to the change of strips, as well as the need of using two plates per experiment. Instead, a cross test is often employed. Instead of adding the two different E-test strips subsequently on top of each other (after removal of the first), both are placed simultaneously but in the form of a cross (<img align="center" src="/files/ftp_upload/10512/10512eq3.jpg" /> 90&#176; angle), with the two previously determined MIC values forming the 90&#176; angle. By this approach only one plate is needed per synergy test, as well as less work, making it a preferred choice despite being slightly more difficult to analyze. The new MIC values in the combined antibiotics approach can be visualized as the modified inhibition zones, after which synergy can be determined by Equation 1.
Instead of using an agar plate approach, a microbroth approach can often be preferential due to its higher flexibility (e.g. ability to choose specific concentrations of antibiotics outside the limits of an E-test strip). Further, microbroth tests are suggested to be more sensitive due to their even distribution of antibiotics in a liquid solution, not depending on the dissociation within a solid phase (agar plate). Wells in a 96-well microplate will be inoculated with a set number of bacteria (106 cfu/mL: bacterial concentration can be estimated by OD600 nm measurements, turbidity standards, or by spread plating samples from 10x bacterial serial dilutions), and antibiotics in different dilutions will be added to the wells. Similarly, to the E-test strips MIC is determined as the intersection (well/spot) with the lowest concentration of antibiotics inhibiting visible growth of bacteria.
Experimental Aim
<ul>
	<li>The project below describes strategies to determine MIC-values of penicillin G and gentamicin of Streptococcus group G by two different methods, E-test and microbroth dilution. For E-test, Mueller-Hinton agar plates inoculated with Streptococcus group G were used in combination with gradient strips of penicillin G and/or gentamicin; while MH-broth with 50% lysed horse blood and 20 mg/mL &#946;-NAD were used with soluble antibiotics together with Streptococcus group G in a microbroth approach.</li>
</ul>
Materials
<ul>
	<li>Bacterial colonies on a blood agar plate, stored &#60;7 days in 4&#176;C</li>
	<li>Blood agar plates</li>
	<li>0.5 McFarland standard
	<ul>
		<li>1% BaCl2</li>
		<li>1% H2SO4</li>
	</ul>
	</li>
	<li>Saline tube (2 mL)</li>
	<li>Cotton tipped applicator</li>
	<li>Mueller-Hinton agar plates (MHA plates)</li>
	<li>MH broth with 50% lysed horse blood and 20 mg/mL &#946;-NAD (MH-F)</li>
	<li>E-test penicillin/gentamicin (or antibiotics of interest) (BioMerieux, Marcy l&#39;Etoile, France, Sweden)</li>
	<li>Antibiotics penicillin/gentamicin (or antibiotics of interest (powder/solution))</li>
</ul>
Note: The specific media used for bacterial growth may vary for different species.

Antibiotic Susceptibility Testing, MIC and Antibiotic Synergy

抗生物質感受性試験:2つの抗生物質のMIC値を決定し、抗生物質の相乗効果を評価するエプシロメーター試験

Source: Anna Bl&#228;ckberg1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 ...

Microscopy and Staining: Gram, Capsule, and Endospore Staining

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1 
1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
Bacteria are diverse microorganisms found nearly everywhere on Earth. Many properties help distinguish them from each other, including but not limited to Gram-staining type, shape and arrangement, production of capsule, and formation of spores. To observe these properties, one can use light microscopy; however, some bacterial characteristics (for example size, lack of coloration, and refractive properties) make it hard to distinguish bacteria solely with a light microscope (1, 2). Staining bacteria is necessary when distinguishing bacterial types with light microscopy. The two main types of light microscopes are simple and compound. The main difference between them is the number of lenses used to magnify the object. Simple microscopes (for example a magnifying glass) have only one lens to magnify an object, while compound microscopes have several lenses to enhance magnification (Figure 1). Compound microscopes have an objective lens close to the object which collects light to create an image of the object. This is then magnified by the eyepiece (ocular lens) which enlarges the image. Combining the objective lens and eyepiece allows for higher magnification than using a single lens alone. Typically, compound microscopes have multiple objective lenses of varying powers to allow for different magnification (1, 2). Here, we will discuss visualizing bacteria with Gram stains, Capsule stains, and Endospore stains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10513/10513fig1.jpg" /> 
Figure 1: A typical compound microscope. The most important parts of the microscope are labeled.
The Gram stain, developed in 1884 by the Danish bacteriologist Hans Christian Gram (1), differentiates bacteria based on the composition of the cell wall (1, 2, 3, 4). Briefly, a bacterial smear is placed on a microscope slide and then heat-fixed to adhere the cells to the slide and make them more readily accepting of stains (1). The heat-fixed sample is stained with Crystal Violet, turning the cells purple. The slide is flushed with an Iodine solution, which fixes the Crystal Violet to the cell wall, followed by a decolorizer (an alcohol) to wash away any non-fixed Crystal Violet. In the final step, a counterstain, Safranin, is added to color cells red (Figure 2). Gram-positive bacteria stain purple due to the thick peptidoglycan layer which is not easily penetrated by the decolorizer; Gram-negative bacteria, with their thinner peptidoglycan layer and higher lipid content, destain with the decolorizer and are counterstained red when Safranin is added (Figure 3). Gram staining is used to differentiate cells into two types (Gram-positive and Gram-negative) and is also useful to distinguish cell shape (spheres or cocci, rods, curved rods, and spirals) and arrangement (single cells, pairs, chains, groups, and clusters) (1, 3).

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10513/10513fig2.jpg" /> 
Figure 2: Schematic of the Gram Staining Protocol. The left column shows how Gram-negative bacteria react at each step of the protocol. The right column shows how Gram-positive bacteria react. Also, shown are two typical bacterial cell shapes: the bacilli (or rods) and the cocci (or spheres).

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10513/10513fig3.jpg" /> 
Figure 3: Gram Staining Results. A Gram stain of a mixture of Staphylococcus aureus (Gram-positive purple cocci) and Escherichia coli (Gram-negative red rods).
Some bacteria produce an extracellular viscous outer layer called a capsule (3, 5). Capsules are protective structures with various functions, including but not limited to adherence to surfaces and other bacteria, protection from desiccation, and protection from phagocytosis. Capsules are typically composed of polysaccharides containing more than 95% water, but some may contain polyalcohols and polyamines (5). Due to their mostly non-ionic composition and tendency to repel stains, simple staining methods do not work with capsule; instead, capsule staining uses a negative staining technique which stains the cells and the background, leaving the capsule as a clear halo around the cells (1, 3) (Figure 4). Capsule staining involves smearing a bacterial sample into an acidic stain on a microscope slide. Unlike Gram staining, the bacterial smear is not heat-fixed during a Capsule Stain. Heat-fixing can disrupt or dehydrate the capsule, leading to false negatives (5). Furthermore, heat-fixing can shrink cells resulting in a clearing around the cell which can be mistaken as a capsule, leading to false positives (3). The acidic stain colors the slide background; while follow up with a basic stain, Crystal Violet, colors the bacterial cells themselves, leaving the capsule unstained and appearing as a clear halo between the cells and the slide background (Figure 5). Traditionally, India ink has been used as the acidic stain because these particles cannot penetrate the capsule. Therefore, neither the capsule nor the cell is stained by India ink; instead, the background is stained. Congo Red, Nigrosin, or Eosin can be used in place of India ink. Capsule staining can help doctors diagnose bacterial infections when looking at cultures from patient samples and guide appropriate patient treatment. Common diseases caused by encapsulated bacteria include pneumonia, meningitis, and salmonellosis.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10513/10513fig4.jpg" /> 
Figure 4: Schematic of the Capsule Staining Protocol. The top panel shows the slide smear prior to any stain application. The middle panel shows how the slide and bacteria look after the primary stain, Congo Red. The final panel shows how the slide and bacteria look after the counterstain, Crystal Violet.

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/10513/10513fig5.jpg" /> 
Figure 5: Capsule Staining Results. Capsule staining of encapsulated Acinetobacter baumannii (denoted with black arrows) and non-encapsulated Escherichia coli (denoted with white arrows). Notice the background is dark and A. baumannii cells are stained purple. The capsule around A. baumannii cells is evident as a halo, while E. coli has no halo.
In adverse conditions (for example nutrient limitation, extreme temperatures, or dehydration), some bacteria produce endospores, metabolically inactive structures that are resistant to physical and chemical damage (1, 2, 8, 9). Endospores allow the bacterium to survive harsh conditions by protecting the genetic material of the cells; once conditions are favorable for growth, spores germinate, and bacterial growth continues. Endospores are difficult to stain with standard staining techniques because they are impermeable to dyes typically used for staining (1, 9). The technique routinely used to stain endospores is the Schaeffer-Fulton Method (Figure 6), which uses the primary stain Malachite Green, a water soluble stain that binds relatively weakly to cellular material, and heat, to allow the stain to break through the cortex of the spore (Figure 7). These steps color the growing cells (termed vegetative cells in the context of endospore biology), as well as endospores and any free spores (those no longer within the former cell envelope). Vegetative cells are washed with water to remove Malachite Green; endospores retain the stain due to heating the Malachite Green within the spore. Finally, the vegetative cells are counterstained with Safranin to visualize (Figure 8). Staining for endospores helps differentiate bacteria into spore formers and non-spore formers, as well as determines whether spores are present in a sample which, if present, could lead to bacterial contamination upon germination.

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/10513/10513fig6.jpg" /> 
Figure 6: Schematic of the Endospore Staining Protocol. The left column shows how spore forming bacteria react at each step of the protocol. The right column shows how non-spore forming bacteria react.

<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/10513/10513fig7.jpg" /> 
Figure 7: Diagram of Endospore Structure. Bacterial cell containing an endospore with the various spore structures labeled.

<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/10513/10513fig8.jpg" /> 
Figure 8: Endospore Staining Results. A typical staining of endospores of Bacillus subtilis. The vegetative cells (denoted with the white arrows) are stained red, while the endospores (denoted with the black arrows) are stained green.

Microscopy and Staining Techniques in Bacteria

顕微鏡検査と染色:グラム、カプセル、内胞染色

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d&#39;H&#233;relle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8).
Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cannot be cultivated on nutritional agar plates like bacteria, as they need host cells to prey on.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10514/10514fig1.jpg" /> 
Figure 1: The morphology of a bacteriophage, here exemplified by an E. coli phage, can be studied using scanning electron microscopy. Most bacteriophages belong to Caudovirales (tailed bacteriophages). This particular phage has a very short tail structure and an icosahedral head, placing it in the family of Podoviruses.
The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell. With each lysed cell, multiple adjacent ones become immediately infected. Several cycles in, a clear zone (a plaque) can be observed in the otherwise turbid plate (Figure 2B/Figure 3A), indicating the presence of what was initially a single bacteriophage particle. The number of plaque forming units per volume (i.e. PFU/mL) of a sample, can thus be determined from the number of plaques generated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10514/10514fig2.jpg" /> 
Figure 2: Testing for plaque forming units (PFU) is a common method for determining the number of bacteriophages in a sample. (A) The base of a sterile Petri dish is covered with an appropriate solid nutrient medium, followed by a mixture of soft media, susceptible host cells and a dilution of the original bacteriophage sample. Note that the phage-suspension could, in some cases, also be evenly spread-out across the surface of already solidified soft agar. (B) Growth of the host bacteria forms a lawn of cells in the top agar layer. Bacteriophage replication generates clear zones, or plaques, caused by host cell lysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10514/10514fig3.jpg" /> 
Figure 3: Results from PFU-testing show multiple plaques generated by bacteriophages. Due to lysis of susceptible host cells, plaques can be seen as clearing zones in the bacterial lawn, either with (A) full clearance, or (B) partial re-growth caused by generation of resistant bacteria (or possibly by temperate phages in the lysogenic cycle).
Certain temperate phages can adopt what is referred to as a lysogenic lifecycle, in addition to the formerly described lytic growth. In lysogeny, the virus assumes a latent state through incorporation of its genetic material into the genome of the host cell (9), often conferring resistance to further phage infections. This is sometimes revealed through a slight clouding of the plaque (Figure 3B). It is worth noting however, that plaques can also appear blurred due to re-growth of bacteria that have evolved resistance to the phage independent of previous phage infections.
Viruses can attach, or adsorb, to only a limited range of host bacteria (10). Host ranges are further limited by intracellular anti-viral strategies such as the CRISPR-Cas system (11). The resistance/sensitivity towards specific phages displayed by bacterial subgroups has historically been used to categorize bacterial strains into different phage types (Figure 4). Although the effectiveness of this method has now been surpassed by novel sequencing techniques, phage typing can still provide valuable information about bacteria-phage interactions, for instance, facilitating the design of a phage cocktail for clinical usage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10514/10514fig4.jpg" /> 
Figure 4: Phage sensitivity of different bacterial strains. Soft agar plates with Cutibacterium acnes strain (A) AD27 and (B) AD35, were spotted with 21 different C. acnes bacteriophages. Only phage 11 were able to infect and kill AD27 while strain AD35 showed sensitivity towards all phages. This technique, termed phage typing, can be used to divide bacterial species and strains into different subgroups based on phage susceptibility.

Determining Viral Titer as Plaque Forming Units PFU

プラークアッセイ:プラーク形成単位(PFU)としてウイルス定数を決定する方法

Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1 
1 Department of Microbiology and Molecular Genetics, Michigan State University
Bacteria have the ability to exchange genetic material (DeoxyriboNucleic Acid, DNA) in a process known as horizontal gene transfer. Incorporating exogenous DNA provides a mechanism by which bacteria can acquire new genetic traits that allow them to adapt to changing environmental conditions, such as the presence of antibiotics or antibodies (1) or molecules found in natural habitats (2). There are three mechanisms of horizontal gene transfer: transformation, transduction, and conjugation (3). Here we will focus on transformation, the ability of bacteria to take up free DNA from the environment. In the laboratory, the transformation process has four general steps: 1) Preparation of competent cells, 2) Incubation of competent cells with DNA, 3) Recovery of cells, and 4) Plating of the cells for growth of the transformants (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10515/10515fig1.jpg" /> 
Figure 1: General steps of the transformation process. The transformation process has four general steps: 1) Preparation of competent cells, 2) Incubation with DNA, 3) Recovery of the cells and 4) Plating cells for growth of the transformants.
For transformation to occur, the recipient bacteria must be in a state known as competence. Some bacteria have the ability to become naturally competent in response to certain environmental conditions. However, many other bacteria do not become competent naturally, or the conditions for this process are yet unknown. The ability to introduce DNA into bacteria has a range of research applications: to generate multiple copies of a DNA molecule of interest, to express large amount of proteins, as a component in cloning procedures, and others. Because of the value of transformation to molecular biology, there are several protocols aimed to make cells artificially competent when conditions for natural competence are unknown. Two main methods are used to prepare artificially competent cells: 1) through chemical treatment of cells and 2) exposing the cells to electric pulses (electroporation). The former uses different chemicals depending on the procedure to create attraction between the DNA and the cell surface, while the latter uses electric fields to generate pores in the bacterial cell membrane through which DNA molecules can enter. The most efficient approach for chemical competence is incubation with divalent cations, most notably calcium (Ca2+) (4,5) Calcium-induced competence is the procedure that will be described here (6). This method is mainly used for transformation of Gram-negative bacteria, and that will be the focus of this protocol.
The procedure of chemical transformation involves a series of steps in which cells are exposed to cations to induce chemical competence. These steps are subsequently followed by a temperature change - heat shock - that favors the uptake of foreign DNA by the competent cell (7). The bacterial cell envelopes are negatively charged. In Gram-negative bacteria like Escherichia coli, the outer membrane is negatively-charged due to the presence of lipopolysaccharide (LPS) (8). This results in repulsion of the similarly negatively-charged DNA molecules. In chemical competence induction, positively-charged calcium ions neutralize this charge repulsion enabling DNA absorbance onto the cell surface (9). Calcium treatment and incubation with DNA are done on ice. Subsequently, an incubation at higher temperatures (42&#176;C), the heat shock, is performed. This temperature imbalance further favors the DNA uptake. Bacterial cells need to be at the mid-exponential growth phase to withstand the heat shock treatment; in other growth stages the bacterial cells are too sensitive to the heat resulting in loss of viability which significantly decreases transformation efficiency.
Different DNA sources can be used for transformation. Typically, plasmids, small circular, double-stranded DNA molecules, are used for transformation in most laboratory procedures in E. coli. For plasmids to be maintained in the bacterial cell after transformation, they need to contain an origin of replication. This allows them to be replicated in the bacterial cell independently from the bacterial chromosome. Not all the bacterial cells get transformed during the transformation procedure. Thus, transformation yields a mixture of transformed cells and non-transformed cells. To distinguish between these two populations, a selection method to identify the cells that have acquired the plasmid is used. Plasmids usually contain selectable markers, which are genes encoding a trait that confers an advantage for growth (i.e. resistance to an antibiotic or chemical or rescue from a growth auxotrophy). After transformation, bacterial cells are plated on selective media, which only allows for growth of the transformed cells. In the case of cells transformed with a plasmid conferring resistance to a given antibiotic, the selective media will be growth media containing that antibiotic. Several different methods can be used to confirm that the colonies grown in the selective media are transformants (i.e. have incorporated the plasmid). For example, plasmids can be recovered from these cells using plasmid preparation methods (10) and digested to confirm plasmid size. Alternatively, colony PCR can be used to confirm the presence of the plasmid of interest (11).
The aim of this experiment is to prepare E. coli DH5&#945; chemically competent cells, using an adaptation of the calcium chloride procedure (12), and to transform them with the plasmid pUC19 to determine transformation efficiency. The E. coli strain DH5&#945; is a commonly used strain in molecular biology applications. Because of its genotype, specifically the recA1 and endA1, this strain allows for increased insert stability and improve the quality of plasmid DNA in subsequent preparations. Since the transformation efficiency decreases with increasing size of the DNA, the plasmid pUC19 was used in this protocol because of its small size (2686 bp) (see https://www.mobitec.com/cms/products/bio/04_vector_sys/standard_cloning_vectors.html for a vector map). pUC19 confers resistance to ampicillin and thus, this was the antibiotic used for selection.

Transformation of E. coli: Adapted Calcium Chloride Procedure

適応塩化カルシウム手順を用いて大腸菌細胞の形質転換

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
First discovered by Lederberg and Tatum in 1946, conjugation is a form of horizontal gene transfer between bacteria that relies on direct physical contact between two bacterial cells (1). Unlike other forms of gene transfer, such as transformation or transduction, conjugation is a naturally occurring process in which DNA is secreted from a donor cell to a recipient cell in a unidirectional manner. This directionality and the ability for this process to increase the genetic diversity of bacteria has given conjugation the reputation as a form of bacterial &#34;mating,&#34; which is believed to have contributed greatly to the recent rise in antibiotic resistant bacteria (2, 3). By using selective pressures, for example the use of antibiotics, conjugation has been manipulated for use in the laboratory setting, making it a powerful tool for horizontal gene transfer between bacteria, and in some cases from bacteria to yeast, plant, and animal cells (4). Apart from applications in the laboratory, bacterium-eukaryote gene transfer by conjugation is an exciting avenue of DNA transfer with a multitude of possible biotechnical applications and naturally occurring implications (5).
Conjugation is thought to work by a &#34;two-step mechanism&#34; (6). First, before any DNA can be transferred, the donor cell must make direct cell-to-cell contact with the recipient. This process has been characterized best in gram-negative bacteria, the most studied of which is Escherichia coli. Cell-to-cell contact is established by the presence of a complex network of extracellular filaments on the donor known as the sex pilus, a conjugative element encoded for by the transferable gene known as the F (fertility) factor (7, 8). In addition to establishing contact between donor and recipient, several proteins are transported via the sex pilus to the recipient cytoplasm, forming a type IV secretion system (T4SS) conduit between the two cells, a necessary structure for the second step of conjugation, DNA transfer (6). By combining this function of the sex pilus with rolling circle replication of DNA, the donor cell is able to transfer DNA in the form of a transposable element, such as a plasmid or transposon, to the recipient by a &#34;shoot and pump&#34; model (6). In this case, the &#34;shooting&#34; is the transport of the pilot protein, with linked DNA, by the T4SS into the recipient cell, and the &#34;pumping&#34; is the active transport of DNA to the recipient, a process reliant on the T4SS and catalyzed by coupling proteins (6). The machinery used in this this process is composed of an origin of transfer sequence (oriT), which must be provided by the DNA in cis and trans genes, which encode a relaxase, mate pair formation complex, and type IV coupling protein, and can be present in cis or trans (9). This relaxase cleaves the nic site within the oriT sequence and covalently attaches to the 5&#39; end of the transferred strand to produce the relaxosome, a single-stranded DNA-relaxase complex with other auxiliary proteins (9). Once formed, the relaxosome connects to the mating pair formation complex, via the type IV coupling protein, which allows for the transfer of the ssDNA-relaxase complex into recipient cells by the T4SS (10). Once in the cytoplasm of the recipient, the DNA can integrate into the recipient genome or exist separately in the form of a plasmid, either of which allow for the expression of its genes.
In this experiment, the widely used conjugation donor strain E. coli WM3064 was used to transfer the gene encoding for ampicillin resistance to the recipient strain E. coli J53. While both strains of the gram-negative bacteria were resistant to tetracycline, only the donor strain WM3064 had the gene for ampicillin resistance, encoded for in the pWD2-oriT shuttle vector, and was auxotrophic to diaminopimelic acid (DAP) (11-13). This experiment consisted of two main steps, the preparation of donor and recipient strains, followed by the transfer of the ampicillin resistance gene from donor to recipient by conjugation (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10516/10516fig1.jpg" /> 
Figure 1: Conjugation schematic. This schematic shows the successful transfer of a plasmid, only one example of a transposable DNA element, from a donor cell to a recipient cell using conjugation. Upon contact with the recipient cell by the donor cell via the sex pilus, the plasmid replicates by rolling circle replication, moves through the multiprotein complex joining the two cells, and forms a new full-length plasmid in the recipient cell.
By incubating a mixture of donor and recipient cells, then successively plating these cells in the presence of tetracycline and DAP, this allowed for the successful transfer of the ampicillin resistance gene. Successively plating cells grown from this mixture in the presence of tetracycline and ampicillin, removed all donor cells due to the lack of DAP and any recipient cells that may not have gained the ampicillin resistance gene, yielding strictly recipient J53 strain bacteria that acquired ampicillin resistance (Figure 2). Once carried out, the successful transfer of the ampicillin resistance gene was confirmed by PCR. Since conjugation was successful, the J53 strain of E. coli contained pWD2-oriT and was resistant to ampicillin, and the gene encoding for this resistance is detectable by PCR. However, if unsuccessful there would have been no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10516/10516fig2.jpg" /> 
Figure 2: Protocol schematic. This schematic shows an overview of the presented protocol.
<img alt="Figure 3A" class="xfigimg" src="/files/ftp_upload/10516/10516fig3a.jpg" /> 
Figure 3A: The confirmation of successful conjugation by PCR. A) Freezer stocks of the conjugated and negative control samples were streaked out on agar plates and a colony was selected (red) for DNA isolation.

Conjugation: Transferring Ampicillin Resistance in E. coli

結合:アンピシリン耐性をドナーからレシピエント大腸菌に移す方法

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
Transduction is a form of genetic exchange between bacteria that utilizes bacteriophages, or phages, a class of virus that infects exclusively prokaryotic organisms. This form of DNA transfer, from one bacterium to another by way of a phage, was discovered in 1951 by Norton Zinder and Joshua Ledererg, who termed the process &#34;transduction&#34; (1). Bacteriophages were first discovered in 1915 by British bacteriologist Frederick Twort, then independently discovered again in 1917 by French-Canadian microbiologist Felix d&#39;Herelle (2). Since then, the structure and function of these phages have been widely characterized (3), dividing these phages into two classes. The first of these classes are the lytic phages which upon infection multiply within the host bacterium, disrupting the bacterial metabolism, lysing the cell, and releasing progeny phage (4). As a result of this anti-bacterial activity and the increasing prevalence of antibiotic-resistant bacteria, these lytic phages have recently proved useful as a substitute treatment for antibiotics. The second of these classes are the lysogenic phages which can either multiply within the host via the lytic cycle or enter a quiescent state in which their genome is integrated into that of the host (Figure 1), a process known as lysogeny, with the ability for phage production to be induced in multiple later generations (4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10517/10517fig1.jpg" /> 
Figure 1: Infection of host cell by bacteriophage. Adsorption by the phage to the bacterial cell wall via interactions between the tail fibers and receptor (purple). Once on the cell surface, the phage is irreversibly attached to the bacterial cell using the base plate (black) which is moved to the cell wall by the contractile sheath (yellow). Phage genome (red) then enters the cell and integrates into the host cell genome.
While bacterial transduction is a naturally occurring process, using modern technology it has been manipulated for the transfer of genes into bacteria in the laboratory setting. By inserting genes of interest into the genome of a lysogenic phage, such as phage, one is able to transfer these genes into the genomes of bacteria and consequently express them within these cells. While other methods of gene transfer, such as transformation, use a plasmid for gene transfer and expression, the insertion of the phage genome into that of the recipient bacterium not only has the potential to confer new traits to this bacterium, but also allows for naturally occurring mutations and other factors of the cellular environment to alter the function of the transferred gene.
Compared to other methods of horizontal gene transfer, such as conjugation, transduction is fairly flexible in the criteria required for donor and recipient cells. Any genetic element that can fit inside the genome of the phage being used can be transferred from any strain of donor bacteria to any strain of recipient bacteria as long as both are permissive to the phage, requiring the expression of necessary phage receptors on the cell surfaces. Once this gene is moved out of the donor genome and packaged into the phage, it can be transferred to the recipient. Following transduction, it is necessary to select for recipient bacteria that contain the gene of interest have to be selected for. This could be done by use of a genetic marker, such as a FLAG-tag or polyhistidine-tag, to mark the gene of interest, or the intrinsic function of the gene, in the case of genes that encode for antibiotic resistance. Additionally, PCR could be used to further confirm successful transduction. By using primers for a region within the gene of interest and comparing signal to a positive control, bacteria that has the gene of interest, and a negative control, bacteria that underwent the same steps as the transduction reaction with no phage. While bacterial transduction is a useful tool in molecular biology, it has and continues to play an important role in the evolution of bacteria, particularly with regard to the recent rise of antibiotic resistance.
In this experiment, bacterial transduction was used to transfer the gene encoding for resistance to the antibiotic ampicillin from the W3110 strain of E.coli to the J53 strain via the P1 bacteriophage (5). This experiment consisted of two main steps. First, the preparation of P1 phage containing the ampicillin resistance gene from the donor strain. Second, the transfer of this gene to the recipient strain by transduction with the P1 phage (Figure 1). Once carried out, the successful transfer of the ampicillin resistance gene could be determined by qPCR (Figure 2). If transduction was successful, the J53 strain of E. coli would be resistant to ampicillin, and the gene conferring this resistance detectable by qPCR. If unsuccessful, there would be no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10517/10517fig2.jpg" /> 
Figure 2: The confirmation of successful transduction by qPCR. By comparing the Cq values detected for the gene of interest from the transduction reaction and the negative control reaction, and normalizing these values against a housekeeping gene, one was able to confirm that bacterial transduction was successful.

Education Microbiology

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Education Microbiology

@media screen and (min-width: 320px){.css-u03n47{font-size:var(--chakra-fontSizes-xs);}}Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)

Science Education (高等生物学)