tag:blogger.com,1999:blog-37730529990857664432024-03-13T08:38:42.419+05:30BiotechfrontEasy Notes for Clear ConceptHarshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comBlogger169125tag:blogger.com,1999:blog-3773052999085766443.post-80876545568270286762024-03-04T07:48:00.001+05:302024-03-04T07:48:07.710+05:30Dot Blot Technique Principle, Steps & Applications<p> In the realm of molecular biology, where precision and efficiency are paramount, the dot blot technique stands out as a simple yet powerful method for detecting and analyzing biomolecules. Originally developed in the 1970s by George Stark and colleagues, dot blotting has since become a cornerstone in various research fields, including genetics, immunology, and diagnostics.</p><p><br /></p><h4 style="text-align: left;">Principle of Dot Blotting :</h4><p> At its core, dot blotting involves the immobilization of target molecules, such as DNA, RNA, or proteins, onto a solid support membrane. This membrane, typically made of nitrocellulose or nylon, acts as a platform for subsequent detection and analysis steps.</p><p>The procedure begins with the application of a small volume (usually a few microliters) of the sample directly onto the membrane in the form of discrete dots. The samples can be pure substances, crude extracts, or complex mixtures, depending on the experimental objectives.</p><h4 style="text-align: left;"><b>Steps Involved in Dot Blotting:</b></h4><p><i><b>1.Sample Application: </b></i>The sample is carefully spotted onto the membrane, usually using a pipette or a specialized dot blot apparatus. The arrangement of the dots can be controlled to facilitate comparison and quantification.</p><p><i><b>2.Blocking:</b></i> To minimize nonspecific interactions and reduce background noise, the membrane is incubated with a blocking agent, such as bovine serum albumin (BSA) or non-fat dry milk. This step saturates any unbound binding sites on the membrane surface.</p><p><i><b>3.Primary Antibody Incubation:</b></i> Next, the membrane is exposed to a specific primary antibody that recognizes the target molecule of interest. This antibody binds selectively to its target, forming an antigen-antibody complex.</p><p><b><i>4.Washing:</i></b> Excess primary antibody is removed through several washes with a suitable buffer. This step helps to remove any unbound antibodies and further reduces background signals.</p><p><i><b>5.Detection:</b></i> The presence of the target molecule is visualized by adding a detection reagent that interacts with the primary antibody. This can involve secondary antibodies conjugated to enzymes, fluorophores, or other reporter molecules. The detection reagent generates a signal that can be detected and quantified.</p><p><i><b>6.Analysis:</b></i> The dot blot results are analyzed either qualitatively or quantitatively, depending on the research goals. Quantification can be achieved through densitometry or by comparing signal intensities relative to standards of known concentration.</p><h4 style="text-align: left;">Advantages of Dot Blotting:</h4><p><i><b>- Speed and Simplicity</b></i>: Dot blotting offers a rapid and straightforward alternative to traditional methods such as Western blotting and Southern blotting. The entire procedure can be completed in a matter of hours, making it ideal for high-throughput applications.</p><p><b><i>- Versatility:</i></b> Dot blotting can be adapted to detect a wide range of biomolecules, including DNA, RNA, proteins, antibodies, and antigens. This versatility makes it a valuable tool in various research areas, from gene expression analysis to disease diagnostics.</p><p><b><i>- Sensitivity:</i></b> With proper optimization, dot blotting can achieve high levels of sensitivity, allowing for the detection of low-abundance molecules in complex samples.</p><p><b><i>- Cost-Effectiveness:</i></b> Dot blotting requires minimal specialized equipment and reagents, making it a cost-effective option for many laboratories.</p><h4 style="text-align: left;">Applications of Dot Blotting:</h4><p><b>- Gene Expression Analysis:</b> Dot blotting can be used to study gene expression patterns by detecting mRNA transcripts or specific DNA sequences.</p><p><b>- Protein Detection: </b>Dot blotting is commonly employed to screen for the presence of specific proteins in biological samples, such as cell lysates or tissue extracts.</p><p><b>- Antibody Screening: </b>Dot blotting can rapidly screen for the presence of antibodies in patient sera, facilitating the diagnosis of infectious diseases or autoimmune disorders.</p><p><b>- Quality Control: </b>Dot blotting is often used in quality control processes to assess the purity and identity of biomolecules, such as recombinant proteins or synthetic oligonucleotides.</p><p><br /></p><p><br /></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-40738910985642185312024-02-10T14:10:00.008+05:302024-02-10T14:10:58.126+05:30Application of Biotechnology in various fields <p> </p><p> Biotechnology is a multidisciplinary field that combines principles of biology, chemistry, physics, engineering, and computer science to develop technologies and products that utilize biological systems, living organisms, or derivatives thereof, for various applications. These applications can range from healthcare and agriculture to industrial processes and environmental remediation. In essence, biotechnology harnesses the inherent capabilities of living organisms or their components to solve problems, create new products, or improve existing processes. Examples include genetic engineering, biopharmaceuticals, agricultural biotechnology, biofuels, and environmental bioremediation.</p><h3 style="text-align: left;"><b>Application of Biotechnology in various fields </b></h3><p>Biotechnology, a dynamic field at the intersection of biology and technology, has revolutionized industries ranging from healthcare to agriculture. With its vast array of applications, biotechnology continues to push the boundaries of what is possible in science and innovation.</p><h4 style="text-align: left;">Healthcare Advancements</h4><p> One of the most impactful applications of biotechnology is in healthcare. From personalized medicine to advanced diagnostics, biotechnology plays a pivotal role in improving human health. Genetic engineering techniques, such as CRISPR-Cas9, have opened doors to precise gene editing, offering hope for treating genetic disorders and diseases. Additionally, biopharmaceuticals derived from biotechnology, such as insulin and monoclonal antibodies, have transformed the treatment of various illnesses, including cancer and autoimmune diseases.</p><h4 style="text-align: left;">Agricultural Innovations</h4><p> Biotechnology has also revolutionized agriculture, offering solutions to global challenges such as food security and environmental sustainability. Genetically modified organisms (GMOs) have been developed to enhance crop yield, improve resistance to pests and diseases, and reduce the need for chemical pesticides and fertilizers. Furthermore, biotechnology enables the development of drought-resistant and nutrient-enriched crops, addressing the challenges posed by climate change and malnutrition.</p><h4 style="text-align: left;">Environmental Remediation</h4><p> Biotechnology presents promising solutions for environmental remediation and conservation. Bioremediation techniques utilize microorganisms to degrade pollutants in soil, water, and air, offering eco-friendly alternatives to traditional cleanup methods. Moreover, biotechnology facilitates the development of biofuels, such as biodiesel and bioethanol, derived from renewable sources like algae and agricultural waste, reducing reliance on fossil fuels and mitigating greenhouse gas emissions.</p><h4 style="text-align: left;">Industrial Applications</h4><p> In the industrial sector, biotechnology drives innovation in various fields, including bio-based materials, bioinformatics, and bioprocessing. Bio-based materials, such as bioplastics and biofibers, offer sustainable alternatives to conventional petroleum-based products, reducing environmental impact and promoting circular economy principles. Additionally, bioprocessing techniques enable the production of bio-based chemicals, enzymes, and pharmaceuticals through microbial fermentation and enzymatic catalysis, fostering a greener and more efficient manufacturing industry.</p><h4 style="text-align: left;">Challenges and Ethical Considerations</h4><p> Despite its tremendous potential, biotechnology also raises ethical and societal concerns, particularly regarding genetic engineering, biosecurity, and bioprospecting. The manipulation of genetic material, while offering opportunities for therapeutic interventions and agricultural improvements, raises questions about safety, equity, and unintended consequences. Furthermore, the commodification of biological resources and the potential for biopiracy highlight the importance of ethical frameworks and regulatory oversight to ensure responsible innovation and equitable access to biotechnological advancements.</p><p><br /></p><p><b><i>Conclusion</i></b></p><p>Biotechnology continues to shape the world we live in, offering transformative solutions to global challenges while posing complex ethical dilemmas. As we navigate the evolving landscape of biotechnological innovation, it is crucial to approach its applications with a balanced perspective, weighing the potential benefits against ethical considerations and societal implications. By harnessing the power of biotechnology responsibly, we can unlock its boundless potential to improve lives, safeguard the environment, and drive sustainable development for future generations.</p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-5670140949399000392024-02-10T14:03:00.003+05:302024-02-10T14:06:37.998+05:30Koch's postulates application in Microbiology <p> In the world of microbiology, understanding the causes of infectious diseases is crucial for effective prevention and treatment strategies. One of the fundamental principles in this field is Koch's postulates, named after the German physician and microbiologist Robert Koch, who developed them in the late 19th century. Koch's postulates provide a systematic approach to demonstrate the causative relationship between a microorganism and a disease, laying the groundwork for the field of medical microbiology.</p><p><b>The Four Koch's Postulates:</b></p><p>1. The microorganism must be present in every case of the disease but absent from healthy organisms.</p><p> This postulate emphasizes the importance of identifying a specific microorganism consistently associated with a particular disease. Koch recognized the need to isolate and characterize the microorganism responsible for causing the illness.</p><p>2. The microorganism must be isolated from the diseased organism and grown in pure culture.</p><p> Once the microorganism is identified in a diseased individual, it must be isolated and cultivated in laboratory conditions. This step ensures that the microorganism can be studied and manipulated independently of its host.</p><p>3. The cultured microorganism should cause disease when introduced into a healthy organism.</p><p> To establish a causal relationship, Koch demonstrated that inoculating a healthy organism with the isolated microorganism results in the development of the same disease observed in the original host. This step confirms that the microorganism is indeed responsible for the illness.</p><p>4. The microorganism must be re-isolated from the experimentally infected organism.</p><p> Finally, to complete the chain of evidence, Koch reisolated the same microorganism from the experimentally infected organism. This step confirms that the microorganism retrieved from the experimental host is identical to the one initially isolated from the original diseased individual.</p><p><b>Applications and Limitations:</b></p><p>Koch's postulates have been instrumental in identifying the causative agents of numerous infectious diseases, including tuberculosis, cholera, and anthrax. By rigorously applying these criteria, researchers have been able to establish causal relationships between specific pathogens and their associated diseases, paving the way for the development of vaccines, antibiotics, and other treatments.</p><p><br /></p><p>However, it's important to note that there are limitations to Koch's postulates. For example, some microorganisms cannot be grown in pure culture or do not cause disease when introduced into a healthy host due to complex interactions with the host's immune system or other factors. Additionally, advances in molecular biology and genomics have revealed cases where multiple microorganisms or non-infectious agents contribute to disease pathogenesis, complicating the application of Koch's postulates in certain contexts.</p><p><b>Conclusion:</b></p><p>Despite these limitations, Koch's postulates remain a cornerstone of microbiology and have greatly contributed to our understanding of infectious diseases. They provide a systematic framework for establishing causation and have guided generations of researchers in their quest to unravel the mysteries of microbial pathogenesis. As technology continues to advance, researchers will undoubtedly refine and expand upon Koch's postulates, ensuring their continued relevance in the field of medical microbiology.</p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-80727386294881051012023-12-31T09:45:00.000+05:302023-12-31T09:45:04.621+05:30General Nature and Basic Structure of Enzyme <p> A living cell is capable of performing a multitude of biochemical reactions in order to survive grow and multiply. This involves</p><p dir="ltr"></p><ul style="text-align: left;"><li>
Degradation of complex nutrients into simpler forms so that they can be absorbed by the cell.</li><li>
Uptake of these simple nutrients.</li><li>
Chemical transformation of these simple nutrient molecules into various precursor metabolites, so that they are available for biosynthesis.</li><li>
Generation of ATP and other bio reactive molecules, which co-operate in cellular biochemical reactions. </li><li>
Biosynthesis of cellular molecules and structural components of the cell etc. </li></ul><p></p>
<p dir="ltr"> All these diverse types of chemical reactions can occur with a high degree of specificity and rate, under normal growth conditions for the organism. These chemical changes are brought about by the living cell through the role and participation of a special class of molecules known as enzymes . Enzymes function in the cell by acting as a biocatalyst. </p>
<p dir="ltr"></p><h2 style="text-align: left;"><b>Discovery of Enzymes</b></h2>
The term enzyme was coined by <b>Kuhne</b> in 1878, suggesting <u>in yeast</u> (en = in, zyme = yeast). It was examined that cell free extract from yeasts is capable of causing conversion of sugar into alcohol. This lead to the understanding that the molecules present inside the cells of yeasts are responsible for these chemical transformations. Kuhne called them as <b>enzymes</b>. <p></p>
<p dir="ltr"> Later on, it was established that all biochemical activities of a living cell are attributed to these magic molecules called enzymes. The first enzyme to be discovered was '<b>amylase</b>'. Its presence in malt extract was detected in 1833 by two French chemists <b>Pain</b> and <b>Persuses</b>.</p>
<p dir="ltr"></p><h2 style="text-align: left;"><b>General Nature of Enzymes</b></h2>
Enzymes are regarded as organic biocatalysts which are capable of functioning both extra cellular and intra cellular. Following are the major characteristics of enzymes. <p></p><p dir="ltr">1] With exception of a few catalytic RNA molecules or ribozymes, all enzymes are protein in nature. Over 90 % enzymes are globular proteins. Many of them are conjugated proteins, having non protein component. </p><p dir="ltr">Enzymes may be monomeric or multimeric proteins. As they are proteins, they share all major characteristic of proteins . <br /></p><ul style="text-align: left;"><li>
They are macromolecules with high MW.</li><li>
They are non dialyzable and are unable to pass through semi permeable membrane.</li><li>
They are amphoteric in nature. i.e. they possess both types of lonizable groups : -NH₂ and -COOH. which on ionization . yield positively charged ammonium (NH4+) ion and negatively charged carboxyl (COO-) ion. </li><li>
As they are amphoteric, they possess specific isoelectric pH. At this pH, both -NH₂ and -COOH groups get equally ionized such that their net electrical charge becomes minimum.</li><li>
They show electrophoretic mobility. If the enzyme solution is at pH, above isoelectric value, they acquire negative charge and hence move towards anode and vice versa. </li><li>
They are colloidal in nature.</li><li>
They can be salted out by salts like ammonium sulfate </li><li>
They can be precipitated out by protein denaturing solvents like acetone and alcohol.</li><li>
They can absorb maximum UV light at 280 um wavelength.</li></ul><p></p>
<p dir="ltr">2]. Enzymes are mostly thermo labile and get denatured at high temperature, usually above 60°C. However, some enzymes are found thermo stable and can withstand high temperature up to 70°C - 80°C or even more.</p>
<p dir="ltr">3]. Enzymes are highly specific in action. They can act on a specific substrate molecule to bring about specific biochemical reaction .</p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Catalytic RNA - Ribozyme</b></h3>
Apart from proteins, a few RNA have also been recognized to have catalytic activity. They are called <b><i>ribozymes</i></b> or <i><b>catalytic RNA</b></i>. They were first recognized by <b>Thomas Cech</b> in 1982. The ribozymes have two types of common roles : <br /><ol style="text-align: left;"><li>RNA processing, where the RNA is involved in RNA splicing, RNA ligation and RNA replication. </li><li>Peptide bond formation during protein synthesis. In ribosomes, they function as a part of rRNA and participate in peptide bond formation. </li></ol><p></p>
<p dir="ltr"></p><h2 style="text-align: left;"><b>Basic structure of Enzymes </b></h2>
In general, enzyme molecule consists of two components.<br /><ol style="text-align: left;"><li>Apo enzyme and </li><li>Prosthetic group or cofactor</li></ol><p></p>
<p dir="ltr"><i><b>Apo enzyme</b></i> is protein part of enzyme.<br />
<i><b>Prosthetic group</b></i> or <b><i>cofactors</i></b> are non protein part of enzymes. They consist of vitamin or their derivatives or metal lons. (If these non protein components are bound loosely to protein, they are called <b><i>cofactors</i></b>) and if they are bound firmly (covalently), are called <i><b>prosthetic groups</b></i>. Apo enzyme and prosthetic group form complex to form active form of enzyme, known as <b>Holoenzyme</b>.</p>
<p dir="ltr"><a href="https://www.biotechfront.com/2021/06/enzyme-classification.html" target="_blank">Classification of Enzymes</a></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-86834442792785036622023-05-21T20:39:00.002+05:302024-02-07T10:18:29.965+05:30What is GMP ?<p> Good Manufacturing Practices <b>(GMP)</b> is the minimum standard that a pharmaceutical manufacturer must meet in their production processes. Process must:</p><p dir="ltr"></p><ul style="text-align: left;"><li>
Be of consistent high quality.</li><li>
Be appropriate to their intended use.</li><li>
Meet the requirements of the marketing authorization (MA) and product specification.</li></ul><p></p>
<p dir="ltr"> Pharmaceutical organizations which comply with GMP must have manufacturer license. Regulatory agencies carries out in section on these pharmaceutical organizations to check if manufacturing sites comply with GMP. Sites are inspected when applied for a manufacturer license and then periodically based on risk assessments.</p>
<h3 style="text-align: left;"><b>CGMP Legal Principles</b></h3>
<p dir="ltr"> <b><i>Quality built into product</i></b><br /></p><ul style="text-align: left;"><li>
By "taking care" in making medicine.</li><li>
Quality system is based on the principle of Quality by Design instead of quality by Inspection.</li></ul>
<b><i>Without/Inadequate cGMP</i></b><br /><ul style="text-align: left;"><li>
Product(s) adulterated(defects need not be shown).</li><li>
Firm and its management are responsible.</li></ul>
<b><i>Current = Dynamic</i></b><br /><ul style="text-align: left;"><li>
Standards evolve over time.</li></ul>
<b><i>Good Practices</i></b><br /><ul style="text-align: left;"><li>
Minimal standards.</li><li>
Not "best practices". (Unless "best" is, in fact, current minimal.)</li></ul><p></p>
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<p dir="ltr"></p><h3 style="text-align: left;"><b>Why is GMP so IMPORTANT?</b></h3><ul style="text-align: left;"><li>
Is a legal requirement enforced and mandated through law, regulations and directives by each country government.</li><li>
To protect public health of each and every individual.</li><li>
Prevent contamination and mix-ups.</li><li>
Prevents mislabeling and adulteration.</li><li>
Consistent maintenance of Quality product supply throughout the product life cycle.</li><li>
Ensure high standard quality product supplied into the market is safe and effective.</li><li>
Satisfy stakeholders, customers and consumers.</li><li>
To meet the requirement of the marketing authorization (MA) and product specification.</li><li>
Enhance the organization image and reputation.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Responsibility of GMP</b></h3><ul style="text-align: left;"><li>
Quality and GMP compliance are independent of job title and have no boundaries.</li><li>
Everyone involves in the process regulatory compliance, manufacturing, packing, Quality Control, distribution and supply of pharmaceuticals product has the responsibility to make sure it reaches to the patient with registered quality standards.</li><li>
Building quality into the entire process of an operation makes sustainable compliance more achievable. However, it requires commitment by Senior management and the allocation of adequate resources (personnel, facilities, training, etc.).</li></ul><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-29378379235154497582023-05-16T12:34:00.000+05:302023-05-16T12:34:17.963+05:30Applications of Biotechnology in Various Fields <p> Biotechnology is a branch of biology involving the use of living organisms and bioprocesses in engineering, technology, medicine, and other fields using bioproducts. The term <b>biotechnology</b> indicates the use of living organisms or their products for modifying the human health and environment.</p>
<p dir="ltr"></p><h2 style="text-align: left;"><b>Applications of Biotechnology in various fields </b></h2>
<h3 style="text-align: left;"><b> 1] Medicine: </b></h3>
Modern biotechnology finds promising applications in medicine as in:<p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>i) Drug Production :</b></i></h4><ul style="text-align: left;"><li>
Most of the traditional pharmaceutical drugs used for treating the symptoms of a disease are simpler molecules found through trials and errors. Small molecules are manufactured chemically, but the larger ones are created by human cells, bacterial cells, yeast cells, and animal or plant cells. </li><li>
Modern biotechnology involves the use of genetically altered microorganisms (e.g., <i><a href="https://www.biotechfront.com/2021/03/escherichia-coli-overview.html" rel="nofollow">E.coli</a></i> or yeast) for producing insulin or antibiotics via synthetic means. Modern biotechnology can also be used for producing plant-made pharmaceuticals. Biotechnology is also used in the development of molecular diagnostic devices used to define the target patient population for a given biopharmaceutical. For example, herceptin was the first drug to be used with a matching diagnostic test for treating breast cancer in women whose cancer cells expressed HER2 protein.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>ii) Pharmacogenomics :</b></i></h4><ul style="text-align: left;"><li>
It is the study of how the genetic inheritance of an individual affects his/her body's response to drugs. The term pharmacogenomics was derived from the words <b>pharmacology</b> and genomics, thus it involves studying the relationship between pharmaceuticals and genetics. </li><li>Pharmacogenomics aims to design and produce drugs adapted to each individual's genetic makeup.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>iii) Gene Therapy : </b></i></h4><ul style="text-align: left;"><li>
It is used for the treatment of genetic and acquired diseases like <a href="https://www.biotechfront.com/2020/08/what-are-causes-of-cancer.html" target="_blank">cancer</a> and <a href="https://www.biotechfront.com/2020/08/aids-disease-and-future-treatment.html" target="_blank">AIDS</a>. Gene therapy utilises normal genes for supplementing or replacing the defective genes or for strengthening immunity. This therapy targets either the somatic cells (i.e., body) or the gametes (ie., egg and sperm). </li><li>In somatic gene therapy, the recipient's genome is altered; however, this alteration is not passed on to the next generation. On the other hand, in <b>germline gene therapy</b>, the egg and sperm cells of the parents are altered to be passed on to their offspring.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>iv)</b></i> <b><i>Genetic</i></b><i> </i><b><i>Testing :</i></b> </h4><div style="text-align: left;">This involves direct examination of the DNA, and is used for: </div><ul style="text-align: left;"><li>
Carrier screening, or identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest,</li><li>
Confirming the diagnosis of symptomatic individuals, </li><li>
Determining sex,</li><li>
Forensic/identity testing.</li><li>
New-born screening.</li><li>
Prenatal diagnostic screening. </li><li>
Pre-symptomatic testing for determining the risk of developing adult-onset cancers, and</li><li>
Pre-symptomatic testing for predicting adult-onset disorders.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b> 2] Cloning :</b></h3>
In this method, the nucleus from one cell is removed and is transferred to an unfertilised egg cell whose nucleus has either been deactivated or removed. Cloning can be done in the following two ways:<p></p><p dir="ltr">
</p><h4 style="text-align: left;"><i><b> i) Reproductive Cloning :</b></i></h4><div style="text-align: left;"><ul style="text-align: left;"><li> In this method, the egg cell after a few divisions is transferred to a uterus for its development into a foetus that is genetically identical to the donor of the original nucleus.</li></ul></div><p></p><p dir="ltr">
</p><h4 style="text-align: left;"><i><b>ii) Therapeutic Cloning :</b></i></h4><p></p><p dir="ltr"></p><ul style="text-align: left;"><li> In this method, the egg is placed in a petridish for its development into embryonic stem cells that are potential for treating several ailments.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b> 3] Agriculture : </b></h3>
Biotechnology in the agricultural field is used for the following purposes:<br />
<h4 style="text-align: left;"><i><b>i) Crop Yield : </b></i></h4><ul style="text-align: left;"><li>For increasing the crop yield, one or two genes are transferred to a highly developed crop variety for imparting a new character. The current techniques of genetic engineering are best for the effects controlled by a single gene. Some genetic characteristics related to yield (e.g., enhanced growth) can be controlled by various genes, each posing a nominal effect on the yield.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>ii) Reduced Vulnerability of Crops to Environmental Stresses : </b></i></h4><ul style="text-align: left;"><li>Such crops which can be made resistant to biotic and abiotic stresses can be developed with the help of genes; for example, drought and salty soil are the two limiting factors in crop productivity.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>iii) Increased Nutritional Qualities :</b></i></h4><ul style="text-align: left;"><li>The nutritional value of proteins contained in foods can be enhanced; for example, proteins in legumes and cereals can be transformed such that they also provide amino acids required in the balanced diet of humans.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>iv) Reduced Dependence on Fertilisers :</b></i></h4><ul style="text-align: left;"><li>Modern biotechnology can also be used to reduce the dependence of farmers on agrochemicals; for example, <i><b>Bacillus thuringiensis</b></i> <i><b>(Bt)</b></i> is a soil bacterium that produces a protein having insecticidal properties. Conventionally, these bacteria were used to produce an insecticidal spray by a fermentation process. </li><li>In this form, the Bt toxin occurs as an inactive protoxin, which becomes effective when digested by an insect. There are several Bt toxins and each has a specificity for some target insects.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><i><b>v) Production of Novel Substances in Crop Plants :</b></i></h4><ul style="text-align: left;"><li>Biotechnology is also applied for novel uses apart from food; for example, oilseed is genetically modified to produce fatty acids for detergents, substitute fuels, and petrochemicals. Potatoes, tomatoes, rice tobacco, lettuce, safflowers, and other plants are genetically engineered to produce insulin and certain vaccines.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>4] Biological Engineering :</b></h3> It is a branch of engineering that involves biotechnologies and biological science. It includes different disciplines such as biochemical engineering, biomedical engineering, bio-process engineering, biosystem engineering, etc.<p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-67206930953312395422023-04-01T12:13:00.001+05:302023-04-01T12:13:10.597+05:30Classification of Bacteria Based on Temperature Requirement <p> </p><p dir="ltr"> The temperature at which the growth of organisms is maximum and most rapid is called the <b><i>optimum growth temperature. </i></b>The range of temperature between which the organisms can grow is called the <i><b>temperature range</b></i> for growth. Shift in temperature on either side of optimum value retards growth. Usually the maximum temperature up to which the organisms can grow, is close to the optimum growth temperature. Whereas the minimum temperature required for growth can be much lower than the optimum value. Minimum, optimum and maximum growth temperatures are called <i><b>cardinal temperatures</b></i>.</p><p dir="ltr">
Cardinal temperatures vary greatly between microorganisms. Optima normally range from 0°C to 75°C; whereas growth can take place between -20°C to 100°C. Organisms having a narrow range of growth temperature are called <i><b>stenothermal</b></i> while the organisms having wide range of growth temperature are called <i><b>eurythermal</b></i>.</p>
<p dir="ltr"> Based on the temperature requirements for growth, bacteria can be divided into three groups:<br /></p><ol style="text-align: left;"><li>Psychrophiles</li><li>Mesophiles</li><li>Thermophiles</li></ol><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><br /></h3><h3 style="text-align: left;"><b>Psychrophiles</b></h3>
The organisms able to grow at low temperature i.e. below 10°C, are called <i><b>psychrophiles</b></i>. They can grow even at 0°C or even lower, if the medium is not frozen due to high salt concentration. The optimum temperature for growth is 15°C or lower. The maximum temperature of growth is 20°C. There are two types of psychrophiles.<p></p>
<p dir="ltr"></p><ol style="text-align: left;"><li><b><i>Obligate or true psychrophiles </i>:<i> </i></b> These are the organisms which grow at 0°C or lower with optimum growth temperature 15°C or below. e.g. <i>Vibrio marinus, Vibrio psychroerythreous.</i></li><li><b><i>Facultative psychrophiles or psychrotroph </i>:</b> These are the organisms which can grow at 0°C. But optimum temperature for growth is between 20°C to 30°C. e.g. <i>Pseudoinonas flourescens.</i></li></ol><p></p>
<p dir="ltr"> The physiological factors for the psychrophilic nature of organisms are not much clear. But it is observed that their ribosomes and other cellular enzymes are unstable at high temperature. Hence, they cannot grow at higher temperature. Even their membrane permeability is altered at temperature above optimum value, leading to leakage of cell material, impairment of membrane permeability and hence results in loss of viability.</p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Mesophiles</b></h3>
These are the organisms that can grow at moderate temperatures of incubation, i.e. within range of 25°C to 40°C. Their optimum growth temperature falls within this range. All pathogenic bacteria for humans and warm blooded animals grow best at body temperature, i.e. at 37°C.<p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Thermophiles</b></h3>
The organisms capable of growing best at temperature above 45°C are called thermophiles. They can be grouped into two categories.<p></p><p dir="ltr">
</p><h4 style="text-align: left;"><i><b>1) Facultative thermophiles </b></i></h4>
These organisms can grow even in the mesophilic range of temperature.<p></p><p dir="ltr">
</p><h4 style="text-align: left;"><i><b>2) Stereothermophiles or hyperthermophiles</b></i></h4>
These bacteria cannot grow in the mesophilic range of temperature. They grow well between temperatures 45°C to 70°C. There are some organisms which can grow even at 110°C. These bacteria are also referred to as strict or obligate thermophiles. These bacteria are normally found from hot water springs, salt lakes etc.<br />
e.g. <i>Bacillus coaggulans, B. stereothermophilus. Thermus aquaticus</i> can grow even at 100°C.<p></p>
<p dir="ltr"> The basis of thermal resistance of these bacteria is the thermal stability of most of their cellular proteins. <br /></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-39444233095959771962023-02-15T18:03:00.003+05:302023-02-15T18:11:32.028+05:30Fermentation : Defination, Principle and Batch Fermentation Method <p> The process of fermentation involves biochemical activity of organisms during their growth, development, reproduction, senescence, and death. Fermentation technology employs organisms to produce food, pharmaceuticals, and alcoholic beverages in industries on a large scale.</p><h3 style="text-align: left;"><b>Principle of Fermentation</b></h3><p dir="ltr">
The principle involved in industrial fermentation technology is that organisms are grown under optimum conditions and are provided with raw materials and other necessary requirements like carbon, nitrogen, salts, trace elements, and vitamins. The end products formed due to their metabolism during their life span are released into the media. These end products are extracted by human beings as they are commercially valuable.</p><p dir="ltr"> Some major end products of fermentation produced on a large scale industrial basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins, enzymes, and other beneficial products.</p><p dir="ltr"><br /></p><h3 style="text-align: left;"><b>Batch Fermentation</b></h3><p dir="ltr">
In batch fermentation process, the microorganisms are inoculated in a fixed volume of batch culture medium. The organisms during their growth consume the nutrients, and the growth products (i.e., biomass and metabolites) start accumulating. Since the nutrient environment within the <a href="https://www.biotechfront.com/2021/12/basic-fermenter-design-external.html" target="_blank">Fermenter</a> is continuously changed, the rate of cell metabolism also changes, and ultimately, cell multiplication stops due to limitation of nutrients and accumulation of toxic excreted waste products.</p><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5MREYxd0C1T7qL34EgGc0z39j7-akIrft_C4-lNm0X_SUtWTC9hE9rTEbeca9uNGNpdeUzsYm6lN3hVYL1ZMpZj7_MhGKR-M_hxgu5Wsmf2p6-eN2zVVWprBvF4lzsr58QOSL2hqjdHMBYaGta5AXWsJquom4rj-FtGuvJoIKdpgIRXeHowyjIvc6/s973/IMG_20230215_174233.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" data-original-height="690" data-original-width="973" height="227" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5MREYxd0C1T7qL34EgGc0z39j7-akIrft_C4-lNm0X_SUtWTC9hE9rTEbeca9uNGNpdeUzsYm6lN3hVYL1ZMpZj7_MhGKR-M_hxgu5Wsmf2p6-eN2zVVWprBvF4lzsr58QOSL2hqjdHMBYaGta5AXWsJquom4rj-FtGuvJoIKdpgIRXeHowyjIvc6/s320/IMG_20230215_174233.jpg" width="320" /></a></td></tr><tr><td class="tr-caption" style="text-align: center;">Growth Characteristics in a Batch Culture of a Microorganism. 1) Lag Phase, 2) Transient Acceleration, 3) Exponential Phase, 4) Deceleration Phase, 5) Stationary Phase, 6) Death Phase</td></tr></tbody></table><p dir="ltr"> There is the complex nature of batch growth of microorganisms. In the initial <b><a href="https://www.biotechfront.com/2020/08/normal-growth-curve-of-bacteria.html">lag phase</a></b>, no apparent growth is observed; however, biochemical analyses show metabolic turnover signifying that the cells are acclimatising to the environmental conditions and will start growing. Then comes the transient acceleration phase when the inoculum begins to grow. This phase is quickly followed by the exponential phase where the organisms are growing at fastest rate as the nutrients are in excess, environmental conditions are optimum and growth inhibitors are absent.</p><p dir="ltr"> In the batch fermentation process, the exponential growth occurs for a limited period. With the change in nutrient conditions, the growth rate decreases and begins deceleration phase followed by the stationary phase at which the growth sto completely because of nutrient exhaustion. The death phase when the grow rate has come to an end is the final phase of the cycle. Mostly t biotechnological batch processes are stopped before this stage because decreasing metabolism and cell lysis.</p><h4 style="text-align: left;"><b>Advantages of Batch Fermentation</b></h4><p dir="ltr"></p><ol style="text-align: left;"><li>It Requires less space.</li><li>It Can be easily handled, and </li><li>There is Less chances of contamination.</li></ol><p></p><h4 style="text-align: left;"><b>Disadvantages of Batch Fermentation </b></h4><p dir="ltr">
</p><p dir="ltr"></p><ol style="text-align: left;"><li>It is time consuming method.</li><li>It requires more time for cleaning, sterilisation, and cooling.</li><li>Product yield is low.</li></ol><p></p><p dir="ltr"><br /></p><p dir="ltr"></p><h3 style="text-align: left;"><br /></h3><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-88430081051493247272023-02-11T10:04:00.006+05:302023-02-11T10:04:48.822+05:30Applications of rDNA Technology and Genetic Engineering in Medicine<p></p><p dir="ltr"> Genetic engineering has an important role to play in the production of medicines. Microorganisms and plant-based substances are used for producing various drugs, vaccines, enzymes, and hormones at low costs. Genetic engineering involves the study of inheritance pattern of diseases in man and collection of human genes that provide a complete map for inheritance of healthy individuals.</p>
<p dir="ltr"> </p><h4 style="text-align: left;"><b>Vaccines</b></h4>
Recombinant DNA technology is used for producing vaccines against diseases by isolating antigen or protein present on the surface of viral particles. Vaccines contain a form of an infectious organism that does not cause disease but allow the body immune system to form antibodies against the infective organism.<p></p>
<p dir="ltr"> When an individual receives vaccination against any viral disease, the antigens produce antibodies to act against and inactivate the viral profeins. The scientists with the help of recombinant DNA technology have transferred the genes for some viral sheath proteins to vecinia virus which was used against small pox. Vaccines produced by gene cloning are non-contaminated and safe as they contain only coat proteins against which antibodies are produced. Vaccines against viral hepatitis influenza, herpes simplex virus, virus-induced foot and mouth disease in animals are being produced by gene cloning.</p>
<p dir="ltr"></p><h4 style="text-align: left;"><b>Hormones</b></h4>
Insulin was commercially produced in 1982 through biogenetic or recombinant DNA technology. Its medicinal use was approved by Food and Drug Administration (FDA) of the USA in the same year. The human insulin gene has been cloned in large quantities in E. coli bacterium that can be used for synthesising insulin. Humilin is the commercially available genetically engineered insulin.<p></p>
<p dir="ltr"> </p><h4 style="text-align: left;"><b>Lymphokines</b></h4>
Lymphokines are proteins regulating the immune system in the human body. u. Interferon is an example of lymphokines that are used to fight viral diseases (such as hepatitis, herpes, and common cold) and cancer. Such drugs can be manufactured in large quantities in bacterial cells. Lymphokines are also helpful in AIDS. Interleukin-II is a commercially available genetically engineered substance that stimulates the multiplication of lymphocytes.<p></p>
<p dir="ltr"> </p><h4 style="text-align: left;"><b>Somatostatin</b></h4>
Somatostatin is used in some of the growth related abnormalities. This drug appears to be species specific and the polypeptide obtained from other mammals has no effect on humans, hence it is extracted from the hypothalamus of cadavers. Genetic engineering has helped in the chemical synthesis of gene which is joined to the PBR 322 plasmid DNA and cloned into a bacterium. This transformed bacterium is converted into a somatostatin synthesising factory.<p></p>
<p dir="ltr"> </p><h4 style="text-align: left;"><b>Production of Blood Clotting Factors</b></h4>
Blockage of coronary arteries by cholesterol or blood clots causes heart attack. Plasminogen is a substance found in blood clots. Genetically engineered tissue Plasminogen Activator (tPA) enzyme is used to dissolve these clots in individuals who have suffered heart attacks.<p></p>
<p dir="ltr"> </p><h4 style="text-align: left;"><b>Cancer</b></h4>
Antibodies cloned from a single source and targeted for a specific antigen (monoclonal antibodies) have proved useful in the treatment of cancer. Monoclonal antibodies have been targeted with radioactive elements of cytotoxins (e.g., Ricin from castor seed) to make them more deadly. Such antibodies seek cancer cells and kill them with their radioactivity or toxin.<p></p><br /><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-19231538632726475272022-10-22T18:41:00.004+05:302022-10-22T18:41:24.482+05:30Bergey's Manual of Systemic Bacteriology<p> <span style="font-size: medium;">Bergey's manual is an accepted reference on the identification of bacteria. It has undergone gradual transformations and expansion since the time of its first publication. </span></p><p><span style="font-size: medium;"> The American Society of Microbiology published first edition of Bergey's Manual of Determinative Bacteriology in 1923. Professor <b>David H. Bergey</b> (Chair Person) and other four colleagues acted as the members of the editorial board. Afterwards, there was a sequel of eight editions, an abridged version and several supplements. At present, ninth edition (published in 1994) is available. It is used to classify bacteria based on their structural and functional attributes by arranging them into specific familial orders. However, this process has become more empirical in recent years.</span></p>
<p dir="ltr"></p><h2 style="text-align: left;"><span style="font-size: medium;"><b>Bergey's Manual of Systematic Bacteriology Manual of Systematic Bacteriology</b></span></h2><span style="font-size: medium;">
It is the main resource for determining the identity of prokaryotic organisms, emphasizing bacterial species, using every characterizing aspect. <b>First edition</b> of this manual consists of four volumes. It's first volume was published in 1984, the second in 1986 and final two volumes in 1989. This manual has much broader scope. It includes all information of earlier manuals. In addition it includes the information on taxonomy, ecology, cultivation, maintenance and the preservation of organisms. It includes many kinds of information such as... <br /><ol style="text-align: left;"><li><span style="font-size: medium;">Descriptions and photographs of species, </span></li><li><span style="font-size: medium;">
Test of distinguish to distinguish among genera and species, </span></li><li><span style="font-size: medium;">
DNA relatedness among organisms and </span></li><li><span style="font-size: medium;">
Various taxonomic studies. </span></li></ol>
There are four divisions of kingdom Procaryotae according to <b>Bergey's Manual of Systematic Bacteriology</b>.<br /></span><p></p><p style="text-align: left;"><span style="font-size: medium;"><b>
Division I </b>: <i>Gracilicutes </i></span></p><blockquote style="border: none; margin: 0 0 0 40px; padding: 0px;"><p style="text-align: left;"><span style="font-size: medium;">Includes prokaryotes with thin cell walls e.g. <a href="https://www.biotechfront.com/2020/11/gram-positive-vs-gram-negative-kye.html" target="_blank">Gram negative bacteria</a>.</span></p></blockquote><p dir="ltr"><span style="font-size: medium;"><b>
Division II</b> : <i>Firmicutes </i><br />
</span></p><blockquote style="border: none; margin: 0 0 0 40px; padding: 0px;"><p style="text-align: left;"><span style="font-size: medium;">Includes prokaryotes with thick cell wall e.g. <a href="https://www.biotechfront.com/2020/11/gram-positive-vs-gram-negative-kye.html" rel="nofollow" target="_blank">Gram positive bacteria</a>. </span></p></blockquote><p dir="ltr"><span style="font-size: medium;"><b>
Division III</b> : <i>Tenericutes </i><br />
</span></p><blockquote style="border: none; margin: 0 0 0 40px; padding: 0px;"><p style="text-align: left;"><span style="font-size: medium;">Includes the prokaryotes that lack cell wall.</span></p></blockquote><p dir="ltr"><span style="font-size: medium;"><b>
Division IV</b> : <i>Mendosicutes</i><br />
</span></p><blockquote style="border: none; margin: 0 0 0 40px; padding: 0px;"><p style="text-align: left;"><span style="font-size: medium;">Includes the prokaryotes lacking peptidoglycan in their <a href="https://www.biotechfront.com/2020/07/the-cell-wall-of-bacteria.html" target="_blank">cell wall</a>. </span></p></blockquote>
<p dir="ltr"> <span style="font-size: medium;"><b>Second edition</b> of Bergey's Manual of systematic Bacteriology consists of five volumes. Volume I was published in 2001, Volume II in 2005 and the other three volumes in 2007. In comparison to the first edition, second edition has certain changes. e.g ., <br /></span></p><ol style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;"><b>Volume - I</b> includes The <b>Archaea</b> and The Deeply Branching And <a href="https://www.biotechfront.com/2020/07/nutritional-types-of-bacteria.html" target="_blank">Phototrophic bacteria</a>.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;"><b>Volume II</b> includes the Gram-negative <b><i>Proteobacteria</i></b>. It includes medically important genera are <i><a href="https://www.biotechfront.com/2021/03/escherichia-coli-overview.html" target="_blank">Escherichia</a>, Neisseria, Pseudomonas, Rhizobium, Rickettsiae, Salmonella</i> and <i>Vibrio</i>.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;"><b>Volume - III</b> includes the Gram- positive bacteria with low G + C content in their <a href="https://www.biotechfront.com/2020/10/nucleic-acids-DNA-RNA.html" target="_blank">DNA</a>. They are the members of phylum <b>Firmicutes</b> . It includes rods and cocci and also pleomorphic <i>Mycoplasma</i>. They may form endospores. Its classes are <i>Clostridia, Mollicutes</i> and <i>Bacilli</i>.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;"><b>Volume - IV</b> includes the Gram - positive bacteria with high G + C content in their DNA. They have more than 50- 50 % G + C content. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;"><b>Volume - V</b> includes ten phyla. They are located here for convenience. Includes morphologically diverse gram - negative organisms. They may not be related. Organisms included are <i>Plantomycetes, Chlamydia, Spirochaetes, Bacteroilds, Fusobacteria, Chlamydiae, Acidobacteria, Verrucomicrobia </i>and<i> Pictyoglomus</i>.</span></span></li></ol><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-55352890311390964412022-06-27T14:05:00.005+05:302023-05-22T08:54:40.216+05:30Effect of pH on Microbial growth <p> The self-ionization of water is a continuous process when it is in pure form. In this process, when there is collision of two H₂O molecules they dissociate into H<sup>+</sup> and OH<sup>-</sup> ions. There is no free hydrogen ions in the water. Because the free hydrogen ions have tendency to attach to the H₂O molecule to form hydronium ion. This means, you will always find H₃O⁺ or hydronium ions in water, instead of free H+ ions.</p>
<p dir="ltr"></p><p style="text-align: left;"><b>What is pH ?</b></p>
pH stands for <b>potential of hydrogen</b>, or power of hydrogen. pH can be defined as measurement of hydrogen ion concentration in a solution, or mathematically it can be defined as negative logarithm of hydrogen ion concentration. <br />
<br />
Formula of pH : <b>-log [H+]</b><p></p>
<p dir="ltr"> pH is applied only to aqueous solutions. That means where there is water, there is pH. pH was first described by <b>Sorensen</b> in 1909. pH reveals the acidity or basicity of water. <br />
The pH scale ranges from 0 to 14 where the value 7 indicates the neutral pH. pH less than 7 indicate acidic conditions, and greater than seven indicate basic or alkaline. In other word pH is about calculating the free hydronium ions and free hydroxyl ions in a given solution. </p>
<p dir="ltr">A solution with more H+ ions is acidic and gives a pH value less than 7 Similarly, a solution with more OH- ions, is basic and gives a value greater than seven.</p>
<h3 style="text-align: left;"><b>Importance of pH</b></h3>
<p dir="ltr"> Certain reactions in our body require certain value of pH. Anything higher or lower value may cause damage. For example, if too much of hydrochloric acid is produced in the stomach, the patient will be advised to take antacids like magnesium hydroxide in order to neutralize the excess pH. <br />
In the same way plants cannot grow if there is continuous rising and falling of pH of soil. Animals in the river cannot survive when acid rains fall on the river.<br />
Similarly, pH plays an important role on <b>microbial growth</b>. </p>
<p dir="ltr"> There were cases where microbes did not show growth in the absence of right pH conditions, even they were supplied with all the required nutrients.<br />
The pH value where a microbe can grow its best, is called the <b>optimum pH</b>. <br />
Most bacteria grow best at a pH value near to 7, which means, most bacteria are <b>neutrophils</b>. <br />
Some bacteria can grow at a pH range between 3 and 4. These are called <b>acedophiles</b>. <br />
<b>Alkaliphiles</b> are the bacteria that can tolerate pH between 8 and 11. </p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Impact of pH on Microbial growth </b></h3>
The bacterial cell consists of several protein molecules, lipids, and nucleic acids. And the cell hosts several biochemical reactions. All these activities are regulated when there is optimum pH. <br />
In lower pH conditions, the increased hydrogen ions break the weak hydrogen bonds of protein side chains and finally change the shape of the protein.<br />
When a protein is not in its original shape, it cannot perform its routine function, and ultimately the bacteria cannot survive. <p></p>
<p dir="ltr">There are two methods to measure pH in a microbiology lab. The first one is using pH paper. The pH paper changes its color when it is dipped into a solution. This color change is based on acidity and basicity of the sample solution. <br />
Later, the color of the paper will be compared with the color chart provided along with the paper. These papers are coated with a pigment called <b>Flavin</b>, which is extracted from red cabbage. Flavin has ability to change color when it comes in contact with an acid or base. This method will not provide the exact pH value. However, this will provide a value which is closer to the actual pH value. <br />
The other method to measure pH that gives an accurate value is using a pH meter. <br />
</p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-3129584799100110632022-06-19T17:35:00.000+05:302022-06-19T17:35:01.139+05:30INTRODUCTION TO BIOGECHEMICAL TRANSFORMATIONS IN SOIL : MINERALIZATION AND IMMOBILIZATION OF ELEMENTS<p> Erth is a closed system, where the over all quantity of matter remains constant. Microorganisms need electron, energy and nutrients to grow. They are responsible for cyclic transformation of compounds, and therefore they are called <b>biogeochemical agents</b>. They carryout transformation of carbon, nitrogen, sulphur, phosphorus, iron etc. This cycling of elements is called <b>biogeochemical cycling</b>. Both biological and chemical process are involved in biogeochemical cycling.</p>
<p dir="ltr"> The oxidation reduction reactions are mainly responsible for biogeochemical cycling of compounds. This changes the chemical and physical characteristics of different compounds. These cyclic turnover of elements are brought about by different types of microorganisms resulting into continuous change in chemical states of matter.</p>
<p dir="ltr"> The life of earth depends on cyclic conversion of chemicals from inorganic state to organic (complex state) to the elemental state. The break in the cycle at any point would dramatically affect all life forms. Various processes carrying out these transformation include :</p>
<p dir="ltr"></p><h2 style="text-align: left;"><b>Mineralization :</b> </h2>
It is a process of conversion of complex organic compounds to simple inorganic forms. Many <a href="https://www.biotechfront.com/2020/07/nutritional-types-of-bacteria.html" target="_blank">heteroprophic microbes</a> play role in mineralization. <br />
The resultant simple compounds are made available to plants and microbes. Energy is released in the process. The process of mineralization is very important as it increases soil fertility.<p></p>
<p dir="ltr"></p><p style="text-align: left;"><i><b><u>Carbon mineralization :</u></b></i></p><ul style="text-align: left;"><li>
Organic carbon is mineralized to inorganic state.</li><li>
Under aerobic condition the main products of carbon mineralization are CO₂ and water. </li><li>
In absence of O₂ organic carbon is incompletey metabolized to produce organic acids, alcohols and gases. </li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><b><u><i>Assimilation :</i></u></b></p><ul style="text-align: left;"><li>
It is the process of conversion of substrate elements to protoplasmic elements. </li><li>
Microbes take up the simple materials from the environment (soil) and convert them into cellular materials. It is known as <b>assimiliation </b>or<b> biosynthesis</b>. </li><li>
In this process synthesis of energy and cellular material takes place. Through assimilation microbes store the excess of simple inorganic chemicals, and prevent their loss due to erosion.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Immobilization :</b></h3>
It is a process in which the quantity of plant available nutrients are reduced in soil by microorganisms. The nutrient assimilation is an important method of immobilization.<br />
The uptake of various elements like carbon, nitrogen, phosphorus, sulphur, etc. cause immobilization. <p></p>
<p dir="ltr"> Intermediary substances accumulate abundant quantities of CH4 and smaller amount of H₂ is evolved. The factors affecting mineralization are level of organic matter, temperature, moisture, pH, depth and aeration. <br />
All these factors affect the growth and metabolism of microbes and the process of mineralization. </p>
<p dir="ltr"></p><ul style="text-align: left;"><li>In nitrogen mineralization ammonium, nitrite and nitrates are accumulated from organic nitrogenous compounds like proteins, nucleic acids, etc.</li><li>
In phosphorus mineralization organic phosphorus present in nucleic acid, phytin, lecithin, etc. are converted to inorganic phosphorus. </li><li>
Sulfur mineralization involves aerobic breakdown of sulfur containing amino acids : cystine, cysteine, methionine, and vitamins, thiamine, biotin, thioctic acid releasing sulfates. Whereas in absence of oxygen , H₂S and odoriferous mercaptans accumulate in soil.</li></ul><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-23843864630675752842022-04-07T12:36:00.003+05:302022-04-10T07:43:49.937+05:30Lytic cycle: Multiplication of T4 Bacteriophage<p dir="ltr">T4 coliphage is a phage that infect coliform bacteria especially <i>E.coli</i>. T4 is a double stranded DNA phage, it has a contractile sheath and an unique base 5-hydroxyl methyl cytosine (5-HMC). It is the best known member of large virulent phages.</p>
<p dir="ltr">The lytic cycle of T4 phage involve following steps: ADSORPTION <br />
PENETRATION <br />
BIOSYNTHESIS <br />
ASSEMBLY <br />
RELEASE</p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>1]. Adsorption :</b></h3><ul style="text-align: left;"><li>
The lytic cycle begins when a bacteriophage comes in contact with a susceptible host cell by random collision. </li><li>
Phage possess an adsorption organs or anti-receptors and host cells possess receptors. </li><li>
Host cell surface components -Flagella, Pilli , Teichoic acids, Proteins, Carbohydrates , LPs and Lipopolysaccharides serves as receptors.</li><li>
Phage components such as tail fibers, tail proteins and spikes serves as adsorption organs or anti-receptors. </li><li>
Each phage has its specific receptor to which it adsorbs.</li><li>
Adsorption takes place only when the anti-receptor is chemically complementary to the receptor.</li><li>
T4 phage possess tail fibers that serves as an adsorption organ or anti-receptor.</li><li>
Normally, tail fibers are present in folded form around the tail. </li><li>
Whiskers hold the tail fibers in folded form. </li><li>
When phage come in contact with host, tail fibers unfold. </li><li>
Unfolding of fibers requires tryptophan & co-factors - Mg++ & Ca++.</li><li>
Thus, the phage and host binding is favoured by ionic environment.</li><li>
T4 host <i>E.coli</i> possess outer membrane protein C (OmpC) – lipopolysaccharide complex as receptor. </li><li>
Initial attachment occurs when tail fibers attach to the OmpC- lipopolysaccharide complex. </li><li>
Initial adsorption is weak and reversible. </li><li>
It becomes irreversible when tail pins attach to lipopolysaccharide.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>2]. Penetration : </b></h3><ul style="text-align: left;"><li>
Once attached, the bacteriophage injects DNA into the bacterium.</li><li>
Bacteria possess rigid cell wall and therefore the phages directly cannot penetrate into the bacterial cells. </li><li>
They inject only their nucleic acids inside the host cell. </li><li>
In the T-even phage, irreversible binding of the phage to host results in the contraction of the sheath and the hollow tail tube is inserted through host cell wall.</li><li>
Some phages have enzymes like lysozyme that digest the cell wall components of the bacterial cell.</li><li>
The penetration of T4 phage DNA occurs when - </li></ul><ol style="text-align: left;"><li>There is irreversible attachment of phage to host cell,</li><li>Contraction of sheath, pushing tail tube through cell envelope</li><li>Injection of DNA into cell like injection of vaccine/drug by a syringe</li></ol><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>3]. Biosynthesis :</b></h3>
Biosynthesis divided into three steps: <br /><ol style="text-align: left;"><li>Formation of immediate early and delayed early protein </li><li>Replication of phage genome</li><li>Formation of late proteins</li></ol><p></p>
<p dir="ltr"></p><p style="text-align: left;"><b>i) Formation of immediate early and delayed early protein : </b></p><ul style="text-align: left;"><li>
Part of phage DNA is immediately transcribed by host RNA polymerase to form immediate early m-RNAS. </li><li>
These early m-RNA translate to following enzyme proteins -</li><li>
a) <b>Nucleases</b> - Breaks down host DNA & make nucleotides available for its own synthesis. </li><li>
b) <b>α-subunit modifying enzyme</b> - modifies <b>α</b>-subunit of host RNA polymerase. </li><li>
Modified host RNA polymerase transcribes part of viral genome to delayed early m-RNAS. </li></ul>Delayed early mRNAs are translated to following enzymes-<p></p>
<p style="text-align: left;"></p><ul style="text-align: left;"><li> a) Phage enzymes that produce 5-hydroxyl methyl cytosine (5-HMC), a unique base in phage DNA</li><li>
b) Polymerases and ligases - that play role in phage DNA replication and recombination. </li><li>
c) Glucosylation enzyme-adds glucose to HMC & protects phage DNA from host restriction endonuclease </li><li>
d) σ-subunit modifying enzyme - modifies σ-factor of RNA polymerase so that is transcribes late mRNAs.</li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><b>ii) Replication of Phage Genome :</b></p>
Two modes have been proposed for the replication of T4 phage DNA. <br /><b><i>
By bi-directional mode - at early stage <br />
By rolling circle mode - at later stage</i></b><p></p>
<p dir="ltr"></p><ul style="text-align: left;"><li>Initial replication is <b>bi-directional</b> and semi-discontinuous.</li><li>
Leading strand is synthesized continuously and lagging strand is synthesized discontinuously leading to the formation of eye structure.</li><li>
Bi-directional replication is initiated at several origins along the DNA and is catalyzed by phage coded enzymes.</li></ul><ul style="text-align: left;"><li>In the <b><a href="https://www.biotechfront.com/2021/06/rolling-circle-model-of-replication.html">rolling circle</a> </b>mode of replication, a cut is made in one of the DNA strands by a specific endonuclease and 3'end is made free. </li><li>
DNA polymerase extends the free 3'OH end by adding complementary bases.</li><li>
Intact strand serves as template for addition of complementary bases. </li><li>
Due to extension of 3'OH end, the 5'end is displaced.</li><li>
Displaced strand is synthesized discontinuously by adding Okazaki fragments. </li><li>
This mechanism produces multi-genome length molecules.</li><li>
Such molecules are referred to as concatemers.</li><li>
The concatemers are later cleaved to head sized molecules by headful cutting mechanism.</li></ul><b><u>III] Formation of late proteins </u></b><br /><ul style="text-align: left;"><li>Soon after the replication of phage DNA, transcription of late m-RNAs occur. </li></ul><p></p>
<p dir="ltr"></p><ul style="text-align: left;"><li>
These late m-RNAs translate to different proteins.</li><li>
These proteins include the structural proteins.</li><li>
They are proteins involved in phage assembly and an enzyme lysozyme that degrades the peptidoglycan layer of bacterial cell wall. </li><li>
For example - head (capsid) proteins, tail tube protein, sheath proteins, collar, whiskers, base plate, tail fiber, tail pins, lysozyme etc.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>4. Assembly of Phages :</b></h3><ul style="text-align: left;"><li>
Assembly of new phage particles begins after accumulation of structural proteins and nucleic acid molecules in the cell.</li><li>
Process of assembling phage particles is known as known as maturation. </li><li>
There are four different pathways that lead to the formation of phage particles. </li><li>
These include base plate, tail tube & tail sheath, tail fibers and head.</li><li>
About 50 genes take part in the morphogenesis of T4 phage.</li><li>
Subunits of base plate assemble to form a base plate. </li><li>
Then tail tube and sheath subunits polymerize on base plate to form mature tail. </li><li>
The subunits of head assemble together to form <i><b>prohead</b></i> and then DNA is inserted in the prohead to form complete head.</li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>5. Release :</b></h3><ul style="text-align: left;"><li>
The release of newly synthesized phages occurs by sudden explosion or bursting (lysis) of bacterial cell. </li><li>
Lysis begins after about 22 minutes. </li><li>
One of the gene products involved in the process include lysozyme. </li><li>
It cleaves glycosidic bonds in the peptidoglycan making the cell wall susceptible to the rupture.</li><li>
There is another protein termed as <b>holin</b> that make holes in the cell membrane and makes the way for lysozyme action.</li></ul>
<p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-70796172807033633942022-03-05T12:36:00.010+05:302024-02-07T10:19:07.164+05:30Eijkman Test Principle and Procedure<p dir="ltr"> The <a href="https://www.biotechfront.com/2022/03/indoe-methylred-vogesprokauer-citrateutilization-tests-principles-procedures-results.html" target="_blank">IMViC test</a> has two drawbacks. The first, It has many controversial procedures and second is test results do not give satisfactory differentiation between fecal and non-fecal coliforms. In 1904 <b>Eijkman</b> proposed another test to differentiate fecal and non-fecal coliform. </p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Principle of Eijkman Test</b></h3><ul style="text-align: left;"><li>
Only fecal coliforms of warm blooded animals grow at 46°C and ferment lactose with Acid & Gas production </li><li>
Most strains of fecal <i>E.coli</i> can ferment lactose in a special buffered broth when incubated at 45°C, where as very few or less frequently the <i>Enterobacter aerogenes</i> do so. </li><li>
The test is called as Eijkman test or elevated temperature test. </li></ul><p></p>
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<p dir="ltr"></p><h3 style="text-align: left;"><b>Procedure of Eijkman Test</b></h3><ul style="text-align: left;"><li>
A buffered tryptose lactose broth in tubes with inverted <b>Durham's tube</b> is inoculated with a culture of coliforms. </li><li>
It is then incubated in water jacketed incubator at 45°C for 48 hours.</li><li>
Gas production after incubation constitutes a positive test for fecal coliforms. </li><li>
Another method, instead of tryptone lactose broth, buffered boric acid lactose broth (BALB) medium is used. </li><li>
The advantage is that, the medium used is <b>selective for fecal </b><b><i>Escherichia</i></b>. </li><li>
It selectively inhibits growth and gas production by Enterobacter and other intermediate members of coliforms.</li><li>
Sterile medium is first warmed to 37°C and then inoculated with culture & incubated at 45°C for 48 hrs.</li><li>
Gas production indicates positive test. </li><li>
Eijkman test gives better result than <a href="https://www.biotechfront.com/2022/03/indoe-methylred-vogesprokauer-citrateutilization-tests-principles-procedures-results.html" target="_blank">IMViC tests. </a></li><li>
Therefore, it is generally preferred in water examination.</li></ul><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-26108720937623106282022-03-05T12:21:00.011+05:302024-02-07T10:20:15.628+05:30IMViC Biochemical Tests: Principles Procedures and Results<p dir="ltr">IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. In this the first test is Indole test, the second one is Methyl Red, the third one is Voges Proskauer and the fourth one is Citrate Utilization test.</p>
<p dir="ltr"></p><ul style="text-align: left;"><li>In the quantitative test for coliforms if completed test is positive, further testing is essential to differentiate fecal and nonfecal coliforms. </li><li><i>Escherichia coli</i> and <i>Enterobacter aerogens</i> are the important contaminants of water respectively.</li><li><i>Escherichia coli</i> is a fecal coliform as it is mainly found in human feces while <i>Enterobacter aerogenes</i> is considered as non fecal as it also occurs in soil & plant material.</li><li>
They closely resemble each other in their morphological and cultural characteristics. </li><li>
Therefore, the biochemical tests are performed to differentiate them. </li><li>
Tests are collectively designated as the <b>IMViC tests</b>.</li><li>
The name was coined by <b>Parr</b> from the first letters of the four tests namely - <b>I</b> for Indole, <b>M</b> for Methyl Red, <b>V</b> for Voges Proskauer and <b>C</b> for Citrate Utilization test. </li><li>
The letter <b>i</b> between V and C is added solely for euphony.</li></ul>
<p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b><u>Indole Test</u></b></h3><ul style="text-align: left;"><li>
Indole Test is used to detect <b><i>indole production</i></b> from amino acid tryptophan.</li><li><i>E. coli</i> has the ability to breakdown the tryptophan by enzyme <b>tryptophanase</b> with release of indole, pyruvic acid and ammonia. </li><li><i>Enterobacter</i> doesn't produce enzyme tryptophanase. Therefore, they are not producing indol from an amino acid tryptophan.</li><li>
Test is performed by inoculating the test organism in 1 % tryptone water or 2 % peptone water, incubation at 37°C for 24 hrs.</li><li>
Indole production can be detected by adding few drops of <b>xylene and Kovac's</b> or <b>Ehrlich's reagent</b> which contains p-dimethyl aminobenzaldehyde.</li><li>
This p-dimethyl aminobenzaldehyde reacts with the indole and produce a <b>cherry red</b>(pink) coloured compound. This is a reduction type of reaction.</li><li>
Xylene extracts the indole in upper layer of the medium.</li></ul>
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<p dir="ltr"></p><h3 style="text-align: left;"><u><b>Methyl Red Test</b></u></h3><ul style="text-align: left;"><li>
Methyl Red Test is carried out to detect acid production ability of test organism from glucose.</li><li>
It is performed by inoculating test organism in <b>glucose phosphate broth tube</b> and incubating at 37°C for 24 hrs </li><li>
Methyl red indicator is then added to detect acid production which gives <u>red colour in positive reaction and yellow in negative</u>. </li><li><i>Escherichia coli</i> rapidly ferments glucose with production of acids and reduce the pH to about 5.0</li><li>
This pH / acidity prevents further growth of <i>E.coli </i>in<i> </i>glucose phosphate broth tube.</li><li><i>Enterobacter aerogens</i> initially produce acids but later on it is converted to non acid products such as ethanol, acetyl-methyl-carbinol (acetoin) and 2, 3- butane-di-ol ( reduction product of acetoin).</li><li>
Due to this, <i>E.aerogens</i> continues to grow without producing its limiting pH. </li><li>
Thus, <i>Escherichia coli</i> gives positive methyl red test while <i>Enterobacter aerogeys</i> gives negative test. </li><li>
Methyl red is a pH indicator which is <b>red</b> at pH 4.4 while yellow at pH 6.2</li></ul>
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<p dir="ltr"></p><h3 style="text-align: left;"><b><u>Voges Proskauer</u></b></h3><ul style="text-align: left;"><li>
The test used for the detection of acetyl methyl carbinol (acetoin) production from glucose, by the test organisms.</li><li>
It is also performed by inoculating the test organisms in glucose phosphate broth medium and incubating at 37°C for 24 hrs.</li><li>
This is followed by addition of 40 % potassium hydroxide and 5 % a-naphthol solution with the shaking of the tube.</li><li>
For the Detection of acetoin requires its further oxidation to diacetyl, In presence of catalyst α-naphthol, alkali(KOH) and air, acetoin is further oxidised to diacetyl. </li><li>
Diacetyl in presence of peptone, gives a red colour. </li><li>
The constituent of peptone responsible for red colour is guanidine nucleus of the amino acid arginine. </li><li>
Thus, a positive test is indicated by development of red colour.</li><li><i>Enterobacter aerogenes</i> produces acetoin from pyruvic acid(Positive test) while <i>Escherichia coli</i> doesn't produce it(Negative test).</li></ul>
<p></p><p dir="ltr"></p><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEisofB0q2ZdoMX746Q-nhuRnedrofD-QaJxdnqYA63gVh060tYJwIQhxTe2UqtudFnXxJ35J_6sF4CtRKvOp2JRJjOOoA4PGx47HRz7nd7ASzJEtqIoHhr7N8zKb8vkBJr6a6EWWQ_in68/s1600/1646463110053978-0.png" style="margin-left: auto; margin-right: auto;" width="400" /></td></tr><tr><td class="tr-caption" style="text-align: center;">IMViC tests Results</td></tr></tbody></table><div class="separator" style="clear: both; text-align: center;">
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<p dir="ltr"></p><h3 style="text-align: left;"><b><u>Citrate Utilization Test</u></b></h3><ul style="text-align: left;"><li>
Citrate Utilization test is carried out to detect the ability of organism to utilize citrate as a sole source of carbon and energy.</li><li>
The utilization of citrate depends upon the enzyme <b>citrate permease</b> that facilitates citrate transport into the cell.</li><li><b>E. aerogenes</b> produce citrate permease and are able to utilize citrate as the sole source of carbon while <i>E.coli</i> do not produce </li><li>
The test is performed by inoculating the test organisms in <b>Koser's citrate medium</b> which sodium citrate as the sole source of carbon; and incubating at 37°C for 24 hrs.</li><li>
Ability to use citrate is indicated by the development of <b>turbidity</b> in medium.</li><li>Citrate Utilization test can also be performed by inoculating the test organisms on the Simmon's citrate agar slant and incubating at 37°C for 24 hrs.</li><li>
The Simmon's citrate agar is the modified processed agar media which contains bromothymol blue as a pH indicator </li><li><i>Enterobacter</i> converts citrate to oxaloacetate and acetic acid by enzyme citrase.</li><li>
These products are further converted to pyruvic acid and CO2.</li><li>
The CO2 reacts with sodium and water to form sodium carbonate </li><li>
Sodium carbonate is an alkaline product and it raises the pH of medium Bromothymol blue (pH indicator) is blue in alkaline and green in acidic condition.</li><li>
The change in colour of slant from green to blue indicates positive test.</li></ul>
<p></p>
<h4 style="text-align: left;"><b> Results :</b></h4><div class="separator" style="clear: both; text-align: center;">
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<p></p><div><b>The IMViC test has two drawbacks as-</b> </div><div><ol style="text-align: left;"><li>It has many controversial procedures </li><li>Test results do not give satisfactory differentiation between fecal and non-fecal coliforms.</li></ol></div>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-28271794481873862552022-02-12T20:20:00.074+05:302022-02-12T22:00:36.334+05:30Microbial Metabolism<p dir="ltr"> Every living organism has the fundamental capability to grow and synthesize new cell material. This requires processing of the nutrient molecules taken up by the cell and involves a series of biochemical transformations. The set of biochemical reactions occurring in cell includes degradation, synthesls as well as modification of the molecules.<br /></p>
<p dir="ltr"> These chemical reactions, which operate by the living cells are collectively referred to as metabolic reactions and the phenomenon is called <b>metabolism</b>. Thus, metabolism is defined as the sum total of all biochemical reactions carried out by a living cell.</p>
<p dir="ltr">The metabolic reactions are further categorized as<br /></p><ol style="text-align: left;"><li>
Catabolism </li><li>
Anabolism </li><li>
Primary metabolism </li><li>
Secondary metabolism</li><li>
Intermediary metabolism</li></ol><p></p>
<h2 style="text-align: left;"><b>Catabolism</b></h2>
<p dir="ltr"> Catabolism includes the set of biochemical reactions which involve degradation of the molecules taken up by the cell and generation of substances essential for biosynthesis of cell constituents. The products of catabolism are <b><i>catabolites</i></b>. </p>
<p dir="ltr"> They, generally, involve formation of various precursor metabolites, energy rich compounds and reducing power. Hence, these metabolic reactions are often called as <i><b>fuelling reactions</b></i>, which provide essential fuels required for cellular synthesis. The precursor metabolites provide basic carbon skeleton for the synthesis of building blocks of the called cellular macromolecules.</p>
<p dir="ltr"> The energy rich compounds are ATP, GTP, CTP, TTP, UTP and acetyl CoA, which provide necessary form of biochemical energy required to drive various energy requiring blochemical reactions. On the hydrolysis of high energy bond of these compounds, necessary free energy is available for the purpose. <br />
<br />
Reducing power, generated during the catabolism. is in form of reduced pyridine compounds. NADH and NADPH. They provide essential reducing conditions required for several blosynthetic as well as assimilatory reactions of the cell.</p>
<p dir="ltr"></p><h2 style="text-align: left;"><b>Anabolism</b></h2>
Anabolism includes the set of blochemical reactions which involve synthesis cellular molecules. <br />
These include blosynthesis of <br /><ol style="text-align: left;"><li>Building blocks of cellular macromolecules e.g. amino acids, nucleotides, fatty acids, sugars etc. </li><li>Vitamins and coenzymes, which are essential for driving various enzyme catalyzed reactions.</li><li>Cellular macromolecules such as proteins, lipids, nucleic acids, polysaccharides as well as synthesis of cell structural compounds.</li></ol>
These anabolic reactions are fuelled by the products of catabolism. <p></p>
<h2 style="text-align: left;"><b>Primary metabolism</b></h2>
<p dir="ltr"> The part of cellular metabolism which is very much essential for cell growth is termed as the <b><i>primary metabolism</i></b>. The products of primary metabolism are called <b><i>primary metabolites</i></b>.<br />
<br />
The primary metabolism includes the metabolisms associated with generation of energy rich compounds, reducing power, precursor metabolites as well as the synthesis of bullding blocks of cellular macromolecules. </p>
<p dir="ltr">The products of primary metabolism include <br /></p><ol style="text-align: left;"><li>Energy rich compounds such as ATP and others. </li><li>Organic acids such as lactic acid, citric acid, acetic acid etc.</li><li>Organic alcohols and solvents such as ethanol, glycerol. acetone, butanol etc. </li><li>Amino acids, vitamins coenzymes, nucleotides etc.</li></ol><p></p>
<p dir="ltr"> The primary metabolism, in general, is found operative lin the cell during <a href="https://www.biotechfront.com/2020/08/normal-growth-curve-of-bacteria.html" target="_blank">log phase</a> of the growth.</p>
<h2 style="text-align: left;"><b>Secondary metabolism </b></h2>
<p dir="ltr"> The part of cellular metabolism, which is not essential for cellular growth is called <b><i>secondary metabolism</i></b>. Products of secondary metabolism are called <i><b>secondary metabolites</b></i>.</p>
<p dir="ltr"> It becomes active during late log phase and <a href="https://www.biotechfront.com/2020/08/normal-growth-curve-of-bacteria.html" target="_blank">stationary phase</a> of the growth. It involves utilization of the excess remains of carbon precursors and energy for synthesis of new molecules which may have secondary role in growth of a cell. <br />
e.g. Antibiotic synthesis, which is not essential for growth. but does help the organisms to survive in the environment in the presence of various antagonistic organisms by destroying them.</p>
<h2 style="text-align: left;"><b>Intermediary metabolism</b></h2>
<p dir="ltr"> The part of cellular metabolism which occurs after the entry of nutrients into the cell, leading to the synthesis of bullding blocks of the cellular macro molecules is generally referred to as the <i><b>intermediary metabolism</b></i>. </p>
<h2 style="text-align: left;"><b>Central metabolic pathways </b></h2>
<p dir="ltr"> The central metabolic pathways are basically catabolic in nature. These pathways Include <a href="https://www.biotechfront.com/2020/11/glycolysis-metabolism-and-its-steps.html" target="_blank">Glycolysis</a>, pentose phosphate pathway and <a href="https://www.biotechfront.com/2020/08/krbes-cycle-products.html" target="_blank">TCA Cycle</a>.They participate in generation of energy, reducing power and precursor metabolites required for cellular synthesis. </p>
<h3 style="text-align: left;"><b>Role of reducing power in metabolism</b></h3>
<p dir="ltr"> All elements, except phosphorus, present in cell are in reduced form. Carbon exists in organic form. Nitrogen is present as amino group. sulfur as úSH group etc. Therefore, all these elements must be reduced at the cellular level of reduction, before they are assimilated in to cell during biosynthetic reactions. </p>
<p dir="ltr"> Most of these elements exist in oxidized state in nature. Therefore, before they are assimilated in the cell, they must be reduced. This requires avallability of sultable reducing power. NADPH serves as the principle reducing power in all such assimilatory and biosynthetic reactions of anabolism. In addition to NADPH, flavin coenzyme, FADH2 can also serve as immediate reducing agent in biochemical reactions.</p>
<p style="text-align: left;"><b>Generation of reducing power :</b></p>
<p dir="ltr"> Reducing power is generated on oxidation of a suitable electron donor during catabolic reactions of metabolism. These electron donors can be either organic or inorganic or both depending on the type of organism. NADPH acts as the reducing power in all anabolic reactions. In addition to NADPH, flavin coenzyme, FADH2 can also serve as reducing power in certain biochemical reactions.</p>
<p dir="ltr"> NADPH is generated during oxidative pentose phosphate pathway, where glucose 6 PO4 is oxidized to pentose phosphate in all most all living organisms. However in arche bacteria, where pentose phosphate pathway does not function, alternate glycolytic pathway function to generate NADPH.</p>
<p dir="ltr">NADPH can also be generated in cell by transhydrogenase reaction, where reduced NAD will participate in reduction of NADP. This reaction also helps in maintaining adequate cellular levels of NADH / NADPH ratio. NADH mainly participates In ATP formation, by transferring electron through electron transport chain. </p>
<p dir="ltr"></p><h3 style="text-align: left;"><b>Role of precursor metabolites</b></h3>
Precursor metabolites are the intermediate molecules in the metabolic pathways. They are produced during operation of catabolic pathways. The precursor metabolites can <br /><ol style="text-align: left;"><li>Provide basic carbon skeleton for the synthesis of all the building blocks required to synthesize macromolecules.</li><li>Undergo oxidation via catabolic pathways to provide ATP and other energy rich compounds that fuel anabolic pathways. </li></ol><p></p>
<p dir="ltr">About 150 different low molecular. weight compounds are required for cellular synthesis. They include <br />1. Building blocks for synthesis of cellular macromolecules. They include<br /></p><ul style="text-align: left;"><li>
Amino acids for synthesis of proteins. </li><li>
Fatty acids for synthesis of lipids.</li><li>
Monosccharides for synthesis of polysaccharides.</li><li>
Purines and pyrimidines for synthesis of Nucleic acids </li></ul>2. Soluble molecules required for cellular metabolic activities. They include vitamins, co-enzymes and polyamines.<p></p>
<p dir="ltr"> There are only 12 compounds, which act as precursor metabolites. They are virtually the same in all living organisms. They include <br /></p><ul style="text-align: left;"><li>Acetyl CoA </li><li>Pyruvate </li><li>Phospho enol pyruvate (PEP) </li><li>3 phospho glyceraldehydes (3PGAL)</li><li>Dihydroxy acetone phosphate (DHAP) </li><li>Glucose 6 Phosphate </li><li>
Fructose 6 Phosphate </li><li>
Erythrose 4 Phosphate </li><li>Ribose 5 Phosphate </li><li>Xylulose 5 Phosphate </li><li>Aplha Keto glutaric Acid (Alpha KG) </li><li>Succinate</li></ul><p></p>
<p dir="ltr">The precursor metabolites are the intermediates of three Indispensible pathways of catabolism <br /></p><ol style="text-align: left;"><li>TCA cycle </li><li>Glycolytic or gluconeogenic pathways </li><li>Pentose phosphate pathway</li></ol><p></p>
<h3 style="text-align: left;"><b>Role of energy rich compounds</b></h3>
<p dir="ltr"> To perform all cellular activities, a suitable form of blochemical energy is required by the cell. This biochemical energy is obtalned as the energy rich compounds, which possess high energy rich chemical bonds. <br />
The necessary energy required to drive a blochemical reaction is released on hydrolysis of this energy rich bond. In living cells, a variety of energy rich compounds are formed, which are utilized for general purpose or to drive a specific biochemical set of reactions. </p>
<p style="text-align: left;"><b>Energy rich compounds of cell</b></p>
<p dir="ltr"> There are mainly two classes of energy rich compounds formed in the cell, which satisfy need of energy requiring reactions. They are <br /></p><ol style="text-align: left;"><li>Compounds having high energy anhydrous phosphoester bond.</li><li>Compounds having high energy thiolester bond.</li></ol><p></p>
<p dir="ltr"> <b><i>Compounds having high energy anhydrous phosphoester bond</i></b><br />
Most energy rich compounds of the cell belong to this category. They are obtained as nucleoside triphosphate derivatives. These include <br /></p><ol style="text-align: left;"><li>ATP Adenosine triphosphate</li><li>GTP Guanosine triphosphate</li><li>
CTP Cytidine triphosphate </li><li>TTP Thymidine triphosphate</li><li>
UTP Uridine triphosphate </li></ol><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b>ATP and its role </b></h4>
ATP is considered as one of the most commonly used energy currency of the cell. It possesses two energy rich anhydrous phosphoester bonds.<p></p>
<p dir="ltr">Hydrolysis of each of this energy rich bond releases 7.3 Keal energy. </p>
<p dir="ltr">ATP + H₂O ➞ ADP + Pi + 7.3 Kcal.<br />
ADP + H₂O ➞ AMP + Pi + 7.3 Kcal.<br />
AMP + H₂O ➞ Adenosine + Pi+ 4 Kcal. <br />
ATP is most commonly required for <br /></p><ol style="text-align: left;"><li>Uptake of nutrients</li><li>Activation of most substrate molecules so that they are able to enter the cell metabolism</li><li>Biosynthesis of most cellular molecules, nucleic acids, chromosome replication and cell division. </li></ol><p></p>
<p dir="ltr"></p><p style="text-align: left;"><b>Other energy rich compounds and their role</b></p>
Apart from ATP, various other kinds of energy rich compounds are formed in the cell. They have specialized role in cellular metabolism. Their specialized utilization in specific metabolic reactions may be considered useful for adequate supply of energy for the concerned biosynthetic reactions, so that they can operate at optimal level in the cell. These energy rich compounds and their role are summarised in below table.<br /><p></p><p dir="ltr"></p><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2qM24gKJ8ZzkFqsJ-WZ5RgXZ95zPQa_9x70YNBJeU1AOsjjfSwiz_nq2l7ttszF1iThuC2W34bTJGuGKLPfN_vD6RIMa79j7c979ID3C2cA7EIFeNEvfU2eovuwVvF5mZJKLhse_lpfc/s1600/1644677420261871-0.png" style="margin-left: auto; margin-right: auto;" width="400" /></td></tr><tr><td class="tr-caption" style="text-align: center;">Energy rich compounds and their role in metabolism.</td></tr></tbody></table><div class="separator" style="clear: both; text-align: center;">
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</div><br /><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-53119886852720097892022-01-19T15:04:00.004+05:302022-01-21T10:49:41.947+05:30Mechanism and Specificity of Enzyme Action<p dir="ltr"> <span style="font-size: large;">Enzymatic reactions include the formation or the destruction of chemical bonds. When two or more substrate molecules are Joined, chemical bonds are formed. When a complex molecule is split into simpler compounds chemical bonds are destroyed.</span></p>
<p dir="ltr"><span style="font-size: large;"> Both the formation and destruction of bonds generally requires the prior stretching or bending of existing bonds, creating a transition (Intermediate) state. The energy required to acquire transition state is called <b>activation energy</b>. Enzymes acts as catalyst and lowers the requirements for this activation energy. Therefore, the reaction can occur even at normal temperature and pressure.</span></p><p dir="ltr"></p><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><span style="font-size: large;"><img border="0" src="https://blogger.googleusercontent.com/img/a/AVvXsEiX6GUCiEJX41ZlBnxjomvn1QT_w1SaCNj2IoYNQom4cy2HQGO2Ye5D2MA9W_mMKU8gifo3lbo19kSK64pqvnyIudKEvMHho_yRj2Nd-mS5xdtZea8EoQLdrVyXKM1SV8pHicDO_tjOr3tc2nI6idh5qI8X_nIRsqnv4PvHE7DAb9CNcJ7kfx1Sv2IV" style="margin-left: auto; margin-right: auto;" width="400" /></span></td></tr><tr><td class="tr-caption" style="text-align: center;"><span style="font-size: large;">Enzyme catalyzed reaction requires a lower activation energy as compared to uncatalyzed reaction.</span></td></tr></tbody></table><div class="separator" style="clear: both; text-align: center;">
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<p dir="ltr"><span style="font-size: large;"> Enzymes act by reacting with substrate through its active site. They are specific in action. Only a specific substrate can bind at the active site of the enzyme to form ES complex, which finally yields product. This can be expressed as under. <br />
E+S ➞ ES ➞ ES* ➞ EP ➞ E + P.</span></p>
<p dir="ltr"></p><ol style="text-align: left;"><li><span style="font-size: large;">First the substrate binds to the active site of enzyme to form ES complex. </span></li><li><span style="font-size: large;">Now the ES complex gets structurally Induced in such a manner that it is able to convert into product and forms EP complex. This involves a chemical change. The reaction energy required for this chemical conversion is lowered dowm by the enzyme.</span></li><li><span style="font-size: large;">Now, the product dissociates from the complex and the enzyme molecule is made free. So that It is able to react with another substrate molecule again.</span></li></ol><p></p>
<p dir="ltr"></p><h2 style="text-align: left;"><b><span style="font-size: large;">Specificity of enzyme action :</span></b></h2><div style="text-align: left;"><span style="font-size: large;"> Enzymes are highly specific in their action. They are specific for the substrate on which they attack and the reaction they catalyze. The basis of this specificity is their active site. The enzyme specificity, therefore, can be divided into two types as under: </span></div><span style="font-size: large;">
1. Substrate specificity <br />
2. Reaction specificity</span><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: large;"><b>Substrates specificity </b></span></h3><span style="font-size: large;">
Enzymes are specifie for the substrate on which they attack. This substrate specificity may further be categorized</span> <span style="font-size: large;">in to different types. <br />
1. Absolute specificity<br />
2. Group specificity <br />
3. Stereo specificity </span><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><span style="font-size: large;"><b>Absolute specificity</b></span></h4><span style="font-size: large;">
When the enzyme possesses specificity for the entire substrate molecule, the specificity is called absolute specificity. <br />
e.g. Urease acts on urea to degrade It into CO2 and NH3.</span><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><span style="font-size: large;"><b>Group specificity</b></span></h4><span style="font-size: large;">
Enzymes may be specifie for a particular group or chemical bond within the substrate and attack on them. This kind of specificity is called group specificity. <br />
e.g. Transaminase acts only on (NH2) group of substrate and cause Its transfer. </span><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><span style="font-size: large;"><b>Stereo specificity</b></span></h4><span style="font-size: large;">
Almost all enzymes are able to recognize orientation of groups within the structure of molecule. Therefore, they are able to distinguish structural as well as optical Isomers and attack them specifically. Such specificity is called stereo specificity. <br />
e.g. Alanine recemase. It causes isomerization of alanine and is able to convert L-alanine to D-alanine.</span><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: large;"><b>Reaction specificity </b></span></h3><span style="font-size: large;">
Enzymes are also specific for the reaction they catalyze. One particular enzyme will catalyze one particular type of blochemical reaction only. Such specificity is called reaction specificity. This kind of specificity is given prime</span> <span style="font-size: large;">importance for <a href="https://www.biotechfront.com/2021/06/enzyme-classification.html" target="_blank">enzyme classification by IUB</a>. Accordingly, they are divided Into six classes: <br /></span><p></p><ol style="text-align: left;"><li><span style="font-size: large;">Oxidoreductases</span></li><li><span style="font-size: large;">Transferases</span></li><li><span style="font-size: large;">
Hydrolases</span></li><li><span style="font-size: large;">
Lyases</span></li><li><span style="font-size: large;">
Isomerases</span></li><li><span style="font-size: large;">
Ligases</span></li></ol><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-39768213093081517952021-12-25T10:46:00.006+05:302022-03-13T19:00:51.581+05:30What is Sterilization in Place (SIP)<p dir="ltr"> <span style="font-size: medium;">Most pharmaceutical GMP systems available today incorporate some form of sterilization using steam. In situ sterilization of equipment using a medium called Pure Saturated Steam. The installation comprises of one or more pieces of processing equipment<br /></span></p><p dir="ltr">
<span style="font-size: medium;"><i>Example</i> • Fermentor or a centrifugal separator to handle harvests, connected by rigid stainless steel or flexible Teflon-lined piping</span></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b><span style="font-size: medium;">Why is there a need for sterilization?</span></b></h4><ul style="text-align: left;"><li><span style="font-size: medium;">To increase the level of sterility assurance associated with those products made by aseptic processing. </span></li><li><span style="font-size: medium;">The size and configuration of large number of process equipment utilized in the production of Biological/parenteral restricts them to be placed inside an autoclave for sterilization.</span></li><li><span style="font-size: medium;">To assure a higher degree of sterility assurance for these items, they should be sterilized in situ rather than sanitized.</span></li><li><span style="font-size: medium;">SIP is required as a result of desire for enhanced sterility assurance for aseptically produced materials.</span></li><li><span style="font-size: medium;">Steam SIP is in daily use in the Biological & parenteral industry.</span></li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b><span style="font-size: medium;">What makes SIP a critical process?</span></b></h4><ul style="text-align: left;"><li><span style="font-size: medium;">Steam-in-place sterilization enables the entire processing system to be sterilized as a single entity thereby reducing the need for aseptic connections. </span></li><li><span style="font-size: medium;">Criticality of system design to achieve sterility with SIP is considered more closely than the steam sterilizer.</span></li><li><span style="font-size: medium;">SIP employs the same moist heat mechanism as steam sterilization in autoclaves.</span></li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b>SIP Fundamentals</b></h4><ul style="text-align: left;"><li>SIP differs only slightly from steam sterilization in autoclaves.</li><li>To achieve effective SIP, the elements necessary for process effectiveness inherent in the autoclave design and operation must be provided in the SIP system.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b>What is a saturated pure steam?</b></h4><ul style="text-align: left;"><li>Saturated steam is a steam-water mixture in which the vapour phase (steam) is in equilibrium with the liquid phase (water or condensate).</li><li>The addition of heat to saturated steam can result in its de-saturation (or superheating).</li><li>The loss of heat from saturated steam will result in its <i><b>condensation</b></i>.</li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b>SIP Design Considerations</b></h4><ul style="text-align: left;"><li>Complete displacement and elimination of entrapped air.</li><li>Constant bleeds of steam at all low points to eliminate condensate build-up Heating large masses of stainless steel from ambient temperature to 121°C results in the creation of large quantities of condensate.</li><li>Strict adherence to the sterilization procedures.</li><li>Proper maintenance of the sterility after the process.</li></ul><p></p>
<p dir="ltr"></p><ul style="text-align: left;"><li>SIP system design concepts is achieved through a review of the equipment elements that make up that system.</li></ul>
• The Components of an SIP system:<br />
Pressure Vessels<br />
Piping system<br />
Filters<p></p>
<p dir="ltr"></p><ul style="text-align: left;"><li>The system must be designed in a way that condensate can be readily removed.</li><li>To achieve this objective, system is sterilized in multiple patterns, in which each pattern sterilizes a portion of the larger system.</li><li>Some portions of the system must be sterilized more than once to assure that all portions of the system are fully covered.</li></ul><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-53399774624001225202021-12-19T08:10:00.064+05:302024-02-07T10:22:10.823+05:30Control System Devices in Fermenter <p dir="ltr"> <span style="font-size: medium;">Fermenter provides defined conditions for the formation of biomass and product in Control temperature, pH, degree of agitation, oxygen concentration, foaming, etc Requires careful monitoring<br /></span></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: medium;">pH control device </span></b></h3><ul style="text-align: left;"><li><span style="font-size: medium;">In batch culture the pH of an actively growing culture will not remain constant for very long.</span></li><li><span style="font-size: medium;">The pH controlling device (probe) checks the pH of the fermentation media at specific intervals of time and adjusts the pH to its optimum level by addition of acids or aikalis for maximum yield of the product.</span></li><li><span style="font-size: medium;">
Maintaining pH to its optimum level is very important for growth of micro-organism to obtain a desired product. </span></li><li><span style="font-size: medium;">
The control of pH values is ensured with the help of peristaltic pumps, releasing out acid/alkali via silicone tubes. </span></li><li><span style="font-size: medium;">
For pH, only sterilisable electrodes are used.</span></li><li><span style="font-size: medium;">
The electrodes used are mainly combined glass reference electrode that can withstand repeated sterilization at 121°C :</span></li><li><span style="font-size: medium;">> Silver/silver chloride electrodes with <b>KCI</b> as electrolyte. </span></li><li><span style="font-size: medium;">> calomel/ mercury electrodes</span></li><li><span style="font-size: medium;">
The electrode is connected via leads to a pH meter/ controller.</span></li></ul><p></p><p dir="ltr"></p><div class="separator" style="clear: both; text-align: center;">
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<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: medium;">Temperature control device</span></b></h3><div style="text-align: left;"><ul style="text-align: left;"><li><span style="font-size: medium;"> Temperature control device generaly contains a thermometer or probe, a heating element and cooling coils or jackets around fermenter.</span></li><li><span style="font-size: medium;">
These are on and off depending on the need for heating or cooling. </span></li><li><span style="font-size: medium;">
During fermentation process, various reactions take place in a <a href="https://www.biotechfront.com/2021/12/basic-fermenter-design-external.html" target="_blank">fermenter</a> which leads to the generation of heat in the fermentation media. The leads to increase in temperature which is detrimental for the growth of microorganisms and may slow down the fermentation process.</span></li><li><span style="font-size: medium;">
Temperature probe is used to measure the temperature of the culture broth in the fermenter vessel like stainless steel <b>Pt100 sensors</b> (platinum resistance electrode). </span></li><li><span style="font-size: medium;">
A temperature probe is a type of temperature sensor. Some temperature probes can measure temperature by being placed onto the surface. Others will need to be inserted or immersed in liquid to be able to measure the temperature. </span></li></ul></div><p></p>
<p dir="ltr"><span style="font-size: medium;">Other temperature measurement devices include:<br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;">
>Glass thermometers with mercury </span></li><li><span style="font-size: medium;">
>Pressure bulb thermometers </span></li><li><span style="font-size: medium;">
>Thermistors (Metal resistant thermometers) </span></li><li><span style="font-size: medium;">
> Bimetallic thermometers >Thermocouples</span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: medium;">Pressure gauge</span></b></h3><ul style="text-align: left;"><li><span style="font-size: medium;"> Industrial fermenters are designed to withstand a specific working pressure.</span></li><li><span style="font-size: medium;">
Pressure measurements are required as a factor of safety. </span></li><li><span style="font-size: medium;">
It is important to fit the equipment with devices that sense, indicate and control pressure. </span></li><li><span style="font-size: medium;">
The correct pressure is maintained by regulatory valves controlled by associated pressure gauges.</span></li><li><span style="font-size: medium;">
Gauges help to ensure there are no leaks or pressure changes that could affect the operating condition of the bioreactor.</span></li></ul><span style="font-size: medium;">
Following are some pressure measuring sensors: <br /></span><ul style="text-align: left;"><li><span style="font-size: medium;">
> Bourdon tube pressure gauge </span></li><li><span style="font-size: medium;">
> Diaphragm gauge </span></li><li><span style="font-size: medium;">
> Piezoelectric transducer</span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: medium;">Dissolved oxygen probe</span></b></h3><ul style="text-align: left;"><li><span style="font-size: medium;"> It monitors the dissolved Oxygen in the system. It is measured by DO probe. </span></li><li><span style="font-size: medium;">
DO electrodes measure partial pressure of dissolved oxygen.</span></li><li><span style="font-size: medium;">
In case of low oxygen tension in broth, more oxygen is purged in fermenter and/or agitator speed is increased. </span></li></ul><span style="font-size: medium;">
The electrodes used to measure DO are: <br /></span><ul style="text-align: left;"><li><span style="font-size: medium;">
> Polarographic electrodes </span></li><li><span style="font-size: medium;">
> Phase fluorometric oxygen sensor</span></li></ul><p></p>
<p dir="ltr"> </p><h3 style="text-align: left;"><b><span style="font-size: medium;">Rotameter</span></b></h3><ul style="text-align: left;"><li><span style="font-size: medium;">It is a device that measures the flow rate of air. </span></li><li><span style="font-size: medium;">It indicates rate of air flow into vessel. </span></li><li><span style="font-size: medium;">It is attached to air sparger</span>. </li><li><span style="font-size: medium;">A pressure valve is attached to rotameter for safer operation.</span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: medium;">Foam control device </span></b></h3><ul style="text-align: left;"><li><span style="font-size: medium;">
Foam is produced during the fermentation process as a result of continuous agitation of the fermentation broth.</span></li><li><span style="font-size: medium;"> This may lead to spill out of the media out of the fermenter and cause media loss as well as contamination.</span></li><li><span style="font-size: medium;">
Therefore, it is necessary to remove/break or neutralize this foam with the help of anti-foaming agents. </span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;">Foams can be eliminated by two methods: <br /></span></p><ol style="text-align: left;"><li><span style="font-size: medium;">By the use of antifoam agents</span></li><li><span style="font-size: medium;">By mechanical breaking of foam</span></li></ol><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b><span style="font-size: medium;">i]. By the use of antifoam agents</span></b></h4><ul style="text-align: left;"><li><span style="font-size: medium;">The set up includes a sensor and a small tank containing anti-foam agent. </span></li><li><span style="font-size: medium;">
A probe (foam sensing and control unit) is inserted through the top plate. </span></li><li><span style="font-size: medium;">
It is set at a defined level abuve the broth surface, when the foam rise and touches the probe surface/tip, a current is passed through the circuit which activates the pump and antifoam is released within seconds. </span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><u><b>Examples:</b></u> <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;">Insaluble oils, polydimethy siloxanes and other silicones, certain alcohols, stearates and glycols.</span></li></ul><p></p>
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<p dir="ltr"></p><h4 style="text-align: left;"><b><span style="font-size: medium;">Characteristics of a perfect antifoam agent :</span></b></h4><ul style="text-align: left;"><li><span style="font-size: medium;">
Long durability (slower consumption). </span></li><li><span style="font-size: medium;">
Low viscosity (ease of pumping & dosing) </span></li><li><span style="font-size: medium;">Stability of the product as supplied (for storage) </span></li><li><span style="font-size: medium;">
Stability of the product as dispersed into the foamant (for effectiveness).</span></li><li><span style="font-size: medium;">
High activity (low dosing) </span></li><li><span style="font-size: medium;">
It should be Effective at low & high temperatures </span></li><li><span style="font-size: medium;">It should be Effective at low & high pH </span></li><li><span style="font-size: medium;">It should be Effective in high salt concentrations</span></li><li><span style="font-size: medium;">
It should be not toxic to culture Microorganisms.</span></li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><b><span style="font-size: medium;">ii]. By mechanical breaking of foam </span></b></h4><ul style="text-align: left;"><li><span style="font-size: medium;">Mechanical antifoam devices like discs, propellers, brushes or hollow cones are attached to the impellors shaft above the broth surface. </span></li><li><span style="font-size: medium;">Foam is braken down when it is thrown against the walls of the fermenter.</span></li></ul>
<p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-65200016479260642042021-12-18T11:39:00.017+05:302024-02-07T10:24:04.043+05:30Basic Fermenter Design : External, Agitation & Aeration, Inlets and Outlets<p dir="ltr"> <span style="font-size: large;">De Becze and Liebmann (1944) used the first large scale (above 20 litre capacity) fermenter for the production of yeast. But it was during the First World War, a British scientist named Chain Weizmann (1914-1918) developed a fermenter for the production of acetone.</span></p><p dir="ltr"><span style="font-size: large;">
Since importance of aseptic conditions was recognised, hence steps were taken to design and construct piping, joints and valves in which sterile conditions could be achieved and manufactured when required. <br />
The first pilot fermenter was erected in India at Hindustan Antibiotic Ltd., Pimpri, Pune in the year 1950.</span></p>
<h4 style="text-align: left;"><b><span style="font-size: large;">Basic criteria of the fermenter </span></b></h4>
<p dir="ltr"></p><ul style="text-align: left;"><li><span style="font-size: large;">The vessel should be capable of being operated aseptically for a number of days and should be reliable in long-term operation and meet the requirements of containment regulations. </span></li><li><span style="font-size: large;">Adequate aeration and agitation should be provided to meet the metabolic requirements of the microorganism. </span></li><li><span style="font-size: large;">However, the mixing should not cause damage to the organism.</span></li><li><span style="font-size: large;">Power consumption should be as low as possible. </span></li><li><span style="font-size: large;">A system of temperature control should be provided. </span></li><li><span style="font-size: large;">A system of pH control should be provided. </span></li><li><span style="font-size: large;">Sampling facilities should be provided. </span></li><li><span style="font-size: large;"><span>Evaporation</span> <span>losses from the fermenter should not be excessive.</span></span></li><li><span style="font-size: large;">The vessel should be designed to require the minimal use of labour in operation, harvesting, cleaning and maintenance. </span></li><li><span style="font-size: large;">Ideally the vessel should be suitable for a range of processes, but this may be restricted because of containment regulations. </span></li><li><span style="font-size: large;">The vessel should be constructed to ensure smooth internal surfaces. </span></li><li><span style="font-size: large;">The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant to facilitate scale-up. </span></li><li><span style="font-size: large;">The cheapest materials which enable satisfactory results to be achieved should be used. </span></li><li><span style="font-size: large;">There should be adequate service provisions for individual plants.</span></li></ul><p></p>
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<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: large;">Parts of fermenter</span></b></h3><span style="font-size: large;">
1. Materials used for body construction.<br />
2. Seals <br />
3. Mixing components <br /></span><ul style="text-align: left;"><li><span style="font-size: large;"> Impellers </span></li><li><span style="font-size: large;"> Spargers </span></li><li><span style="font-size: large;"> Baffles </span></li></ul><span style="font-size: large;">
4. Sampling point <br />
6. Bottom drainage system (outlet)<br />
7. Air filter.</span><p></p><p dir="ltr"></p><div class="separator" style="clear: both; text-align: center;">
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<h3 style="text-align: left;"><b><span style="font-size: large;">1. Material used for fermenter</span></b></h3>
<p dir="ltr"><span style="font-size: large;">Properties of the material used for fermenter designing : <br /></span></p><ul style="text-align: left;"><li><span style="font-size: large;">Non-corrosive </span></li><li><span style="font-size: large;">Non-toxic </span></li><li><span style="font-size: large;">Tolerable to repeated steam sterilization Cycles </span></li><li><span style="font-size: large;">Withstand high pressure </span></li><li><span style="font-size: large;">Resist pH changes</span></li><li><span style="font-size: large;">Selection of the material also depends on the type of fermentation process to be carried.</span></li></ul><span style="font-size: large;"><span>
Mainly two types of materials</span> <span>are used worldwide for the manufacture of fermenters:<br /><ol style="text-align: left;"><li><span>Glass </span></li><li><span>Stainless steel</span></li></ol></span></span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>i]. Glass fermenters </b></span></p><span style="font-size: large;"><ul style="text-align: left;"><li><span>On a small scale (Laboratory scale) (1 to 30 dm³), it is possible to use <b>glass</b>. </span></li><li><span>Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of the vessel. </span></li><li><span>These can be of two types: </span></li></ul><ol style="text-align: left;"><li><span>
> A <i><b>glass vessel</b></i> with a round or flat bottom and a top flange carrying plate. All vessels of this type have to be sterilized by autoclaving. </span></li><li><span>
> A <b><i>giass cylinder</i></b> with <b><i>stainless-steel</i></b> top flange carrying and bottom solid plates. Vessels with two stainless steel plates cost approximately 50% more than those with just a top plate. </span></li></ol><ul style="text-align: left;"><li><span>These top flange carrying plates provides port for the entrance of media, inoculum, buffers and anti-foam agents.</span></li><li><span>At pilot and large scale, when all fermenters are sterilized in situ, any materials used will have to be assessed on their ability to withstand pressure sterilization and corrosion and on their potential toxicity and cost.</span></li></ul></span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>ii]. Stainless steel fermenters</b></span></p><span style="font-size: large;"><ul style="text-align: left;"><li><span>Fermenters are normally constructed of stainiess steel or at least have a stainless-steel cladding (or layering) to limit corrosion. </span></li><li><span>The <b>American Iron and Steel Institute (AISI)</b> states that steels containing less than 4% chromium are cliassified as <i><b>steel alloys</b></i> and those containing more than 4% are classified as stainiess steels. </span></li><li><span>Mild steel coated with <i><b>glass or phenolic epoxy</b></i> materials has occasionally been used. </span></li><li><span>Stainless steel - 304 and 316 grade coated with epoxy or glass lining are into use. </span></li><li><span>The corrosion resistance of stainless steel is thought to depend on the existence of a thin <b><i>hydrous oxide film</i></b> on the surface of the metal. </span></li><li><span>The composition of this film varies with different steel alloys and different manufacturing process treatments such as rolling, pickling (removal of impurities, such as stains, inorganic contaminants, rust) or heat treatment. </span></li><li><span>The film is stabilized by <b>chromium</b> and is considered to be continuous, non-porous, insoluble and self healing. </span></li><li><span>If damaged, the film will repair itself when exposed to air or an oxidizing agent.</span></li><li><span>The minimum amount of chromium needed to resist corrosion will depend on the corroding agent in a particular environment, such as acids,</span> <span>alkalis, gases, soil, salt or fresh water.</span></li><li><span>Increasing the chromium content enhances resistance to corrosion, but only grades of steel containing at least 10 to 13% chromium develop an effective film.</span></li><li><span> The inclusion of <b>nickel</b> in high percent chromium steels enhances their resistance and improves their engineering properties.</span></li><li><span> The presence of <b>molybdenum</b> improves the resistance of stainless steels to solutions of halogen salts and pitting (small holes) by chloride ions in brine or sea water.</span></li><li><span>Corrosion resistance can also be improved by tungsten, silicone and other elements.</span></li><li><span>AISI grade 316 steels which contain 18% chromium, 10% nickel and 2-2.5% molybdenum are now commoniy used in fermenter construction.</span></li><li> Increasing the chromium content enhances resistance to corrosion, but only grades of steel containing at least 10 to 13% chromium develop an effective film.</li><li><span>The inclusion of <b>nickel</b> in high percent chromium steels enhances their resistance and improves their engineering properties. </span></li><li><span>The presence of <b>molybdenum</b> improves the resistance of stainless steels to solutions of halogen salts and pitting (small holes) by chloride ions in brine or sea water. </span></li><li><span>Corrosion resistance can also be improved by tungsten, silicone and other elements. </span></li><li><span>AISI grade 316 steels which contain 18% chromium, 10% nickel and 2-2.5% molybdenum are now commonly used in fermenter construction.</span></li></ul></span><p></p>
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<h3 style="text-align: left;"><b><span style="font-size: large;">2. Types of Seals </span></b></h3>
<p dir="ltr"><span style="font-size: large;"> It is important to consider the ways in which a reliable aseptic seal is made between glass and glass, glass and metal or metal and metal joints such as between a fermenter vessel and a detachable top or base plate. <br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">Between a fermenter vessel and a detachable top or base plate. </span></span></li><li><span><span style="font-size: large;">Sealing assembly for stirrer shaft.</span></span></li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>i]. Between a fermenter vessel and a detachable top or base plate </b></span></p><span style="font-size: large;">
a) Gasket seals are suitable with glass to glass joints. <br /><ul style="text-align: left;"><li><span>Nitryl or butyl rubbers are normally used for these seals as they will withstand fermentation process conditions. </span></li></ul>
b) Lip seals are suitable with glass metal joints. <br /><ul style="text-align: left;"><li><span>These are generally made of silicone and are fluorosilicone elastomers. </span></li></ul>
c) O ring seals are suitable with metal to metal joints. <br /><ul style="text-align: left;"><li><span>These are made of Polytetrafluoroethylene (PTFE) and Neoprene.</span></li></ul></span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>ii]. Sealing assembly for stirrer shaft </b></span></p><span style="font-size: large;">
• Stirrer shaft is a device providing agitation. <br />
• It must be sealed properly ensuring a long term aseptic operation</span><p></p><p dir="ltr"><span style="font-size: large;">There are various types of sealing assembly such as:</span></p><p style="text-align: left;"></p><ul style="text-align: left;"><li><span style="font-size: large;">Mechanical seal</span></li><li><span style="font-size: large;">Packed gland seal</span></li><li><span style="font-size: large;">Magnetic drive.</span></li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>3. Mixing components </b></span></p><span style="font-size: large;">
• Structural components involved in mixing such as :<br /><ol style="text-align: left;"><li><span style="font-size: large;">Impellor for agitation </span></li><li><span style="font-size: large;">Sparger for aeration </span></li><li><span style="font-size: large;">Baffles for breaking vortex </span></li></ol></span><p></p>
<p dir="ltr"><span style="font-size: large;"><b>Importance of Agitation and Aeration </b><br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">Important factor in a fermenter. </span></span></li><li><span><span style="font-size: large;">Provides a provision for adequate mixing of the contents of a fermenter.</span></span></li><li><span style="font-size: large;">Mixing helps to disperse the air bubbles, suspend cells, enhance heat and mass transfer in the medium.</span></li></ul><p></p>
<p dir="ltr"><span style="font-size: large;">Location of mixing components are<br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">Impeller : shaft in the centre of the fermenter </span></span></li><li><span><span style="font-size: large;">
Sparger : below the impeller (at base) </span></span></li><li><span><span style="font-size: large;">Baffles : Along the side walls of the fermenter</span></span></li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>i]. Impellors or agitators </b></span></p><span style="font-size: large;">
Agitation should ensure that a uniform suspension of microbial cells is achieved in a homogenous nutrient medium. <br /><ul style="text-align: left;"><li><span>Agitation is done using <b>impellers</b>. </span></li><li><span>These are mounted on the drive shaft and into the fermenter through its lid (flange). </span></li><li><span>These are made up of impellor biades and the position may be varying according to the need and size of the fermenter. </span></li><li><span>These impellors or blades are attached to a motor on lid.</span></li><li><span>Impellors may not be required in small scale fermenters.</span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: large;"><span>The major function of an impellor is to aid in proper mixing of the following components uniformly</span> <span>throughout the fermentation vessel : <br /></span></span></p><ol style="text-align: left;"><li><span><span style="font-size: large;">
> Suspended microorganisms (microbial cells), </span></span></li><li><span><span style="font-size: large;">
> Media components </span></span></li><li><span><span style="font-size: large;">
> Oxygen (air bubbles) </span></span></li><li><span><span style="font-size: large;">
> Heat transfer. </span></span></li></ol><ul style="text-align: left;"><li><span><span style="font-size: large;">Impellor blades reduce the size of the air bubbles by breaking them and distributes them uniformly into the fermentation media. </span></span></li><li><span><span style="font-size: large;">Impellors also helps in breaking of foam bubbles in the head space of fermenter. </span></span></li><li><span><span style="font-size: large;">This foam formed during fermentation process can cause a major problem of contamination. Therefore, it is important to breakdown these foam so that they don't over flow out of the fermenter and cause contamination.</span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: large;"><b><u>Impellors classification : Basic impellors</u></b></span></p><p dir="ltr"><span style="font-size: large;">
<b>a] Disc turbines or Rushton</b> <b>turbines</b> consists of a disc of a disc with a series of rectangular vanes set in a vertical plate around the circumference.</span></p><p dir="ltr"><span style="font-size: large;">
<b>b] Vaned discs</b> has a series of rectangular vanes attached vertically to the underside. Air from the sparger hits the underside of the disc and is displaced towards the dreamstim vanes where the air bubbles are broken up into smaller bubbles.</span></p><p dir="ltr"><span style="font-size: large;">
<b>c] Open turbines of variable pitch</b></span></p><p dir="ltr"><span style="font-size: large;"><span>
<b>d] Marine propellerss</b></span> <span>- The vanes of a variable pitch open propeller are attached directly to a boss on the agitator shaft. In this case the air bubbles do not initially hit any surface before dispersion by the vanes or blades.</span></span></p>
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<p dir="ltr"><span style="font-size: large;"><span><u><b><i>Modern Agitator Developments</i></b></u></span><span> </span></span></p><p dir="ltr"><span style="font-size: large;"><span>Four other modern agitator developments are derived from <b>open turbines</b>.</span> The new turbine designs make it possible to replace Rushton turbines by :</span></p><p dir="ltr"></p><ul style="text-align: left;"><li><span style="font-size: large;">
larger low power agitators which do not lose as much power when aerated, </span></li><li><span style="font-size: large;">
Which are able to handle higher air volumes without flooding and give better bulk blending and heat transfer characteristics in more viscous media.</span></li><li><span style="font-size: large;">
Good mixing and aeration in high viscosity broths may also be achieved by a dual impeller combination, where the lower impeller acts as the gas disperser and the upper impeller acts primarily as a device for aiding circulation of vessel contents.</span></li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>ii]. Spargers or aerators </b></span></p><span style="font-size: large;"><ul style="text-align: left;"><li><span> A sparger is an aeration system through which sterile air is introduced in the medium of fermentation tank.</span></li><li><span> Spargers are located at the bottom of the fermentation tank.</span></li><li><span>
Glass wool filters are used in a sparger for sterilization of air and other gases. </span></li><li><span>
The sparger pipes contain small holes of about 5-10 mm. </span></li><li><span>
Through these small holes pressurized air is released in the aqueous fermentation media. </span></li><li><span>
The air released is the form of tiny air bubbles. </span></li><li><span>
These air bubbles helps in mixing of media. </span></li><li><span>
Impeller blades disperses air released through sparger into medium.</span></li></ul></span><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><span style="font-size: large;"><b>Types of spargers </b></span></h4><span style="font-size: large;">
<i><b>1. Porous sparger</b></i><br /><ul style="text-align: left;"><li><span>These type of spargers are used mainly in laboratory scale non- agitated fermenter (without impellors). </span></li><li><span>These are mainly made up of ceramics. </span></li><li><span>Pressure drop occurs across the sparger. </span></li><li><span>Fine holes may become blocked due to microbial growth around holes with low pressure. </span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: large;"><b><i>2. Orifice sparger (a perforated pipe)</i></b> <br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">These are used in small or large scale, agitated/non- agitated fermenters. </span></span></li><li><span><span style="font-size: large;">Spargers have holes of at least 6mm diameter. </span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: large;"><i><b>3. Nozzle sparger (an open or partially dosed pipe)</b></i> <br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">It is used in most modern mechanically stirred fermenter design from lab to industrial scale. </span></span></li><li><span><span style="font-size: large;">It has a single open or partially closed pipe to provide the stream of air bubbles.</span></span></li></ul><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>iii]. Baffles </b></span></p><span style="font-size: large;">
Baffles are mounted on the walls of a fermenter. <br /><ul style="text-align: left;"><li><span>4, 6 or 8 baffles may be used in a fermenter. </span></li><li><span>Baffles are metal strips roughly 1/10th of vessel diameter and attached radially to the fermenter wall. </span></li><li><span>The major function of baffles is to <b>break the vortex</b> formed during agitation process by the impellors.</span></li><li><span>If the vortex, formed during agitation, is not broken, the fermentation media may spill out of fermenter and be a major cause of contamination. Therefore, it is important to break the vortex using a barrier in the form of baffles.</span></li></ul></span><p></p><div class="separator" style="clear: both; text-align: center;">
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It is recommended that baffles should be installed so that a gap existed between them and the vessel wall, so that there is a scouring action around and behind the baffles thus minimizing microbial growth on the baffles and the fermenter walls.</span><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: large;"><b>4. Ports</b></span></h3><span style="font-size: large;">
Ports are located on the top flange carrying plate of the fermentation vessel.<br />
Different ports are present for the supply of different components via silicone tubes: <br /><ul style="text-align: left;"><li><span><b>Feeding port:</b> for fermentation media (in case where media is sterilized ex-situ), </span></li><li><span><b>Inoculation port :</b> for seed culture (microorganism).</span></li><li><span><b>Buffer port :</b> for buffers (acid/ alkali) </span></li><li><span><b>Anti-foam port:</b> for anti-foaming agents. </span></li></ul>
Care should be taken that the port provides aseptic transfer.<br />
The reservoirs for the nutrients and inoculum and associated piping are steam sterilized in situ.<br />
Addition is done using peristaltic pump only after aseptic connection has been established.</span><p></p>
<p dir="ltr"><span style="font-size: large;"><b>5. Sampling point </b><br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">
Sampling point is used for time to time withdrawal of samples to monitor fermentation process and quality control.</span></span></li><li><span><span style="font-size: large;">
This sampling point should provide aseptic withdrawal of sample.</span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: large;"><b>6. Bottom drainage system </b><br /></span></p><ul style="text-align: left;"><li><span><span style="font-size: large;">It is aseptic outlet present at the bottom of fermenter for removal of entire fermented media and products formed after the fermentation process is completed. </span></span></li><li><span><span style="font-size: large;">
It is different from the sampling point.</span></span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: large;"><b>7. Air Filter </b></span></h3><span style="font-size: large;"><ul style="text-align: left;"><li><span>Oxygen-enriched air is used for sparging fermenters, to achieve the desired broth aeration and dissolved oxygen levels.</span></li><li><span>Fermenter air should be free of unwanted airborne microorganisms and <a href="https://www.biotechfront.com/2021/02/lysogenic-cycle-of-bacteriophage.html" target="_blank">bacteriophage</a> contamination. </span></li><li><span>It is important to prevent the entry of unwanted microorganisms from the environment, that could interfere with the growth and multiplication of the selected fermentation organisms; contamination would impact fermentation yield and compromise product quality. </span></li><li><span>Depending on the nature of the fermenter microbial</span> <span>cultures in use, it may also be important to remove undesirable microorganisms from fermenter air exhaust before releasing it into the environment. <br />Fermenter air filtration involves filtration of both inlet and exhaust air with pre-filtration and final filtration procedure.</span></li><li><span>Fermenter air filtration involves filtration of both inlet and exhaust air with pre-filtration and final filtration procedure.</span></li></ul></span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: large;"><b>i]. Inlet air filter </b></span></p><span style="font-size: large;">
It is used to filter the air that is coming inside the fermenter to prevent any contamination of the fermentation broth. <br />
It consists of following components: <br /><ul style="text-align: left;"><li><span>
> <b>Compressor :</b> A compressor is a mechanical device that increases the pressure of a gas by reducing its volume. </span></li><li><span>
> <b>Conditioner :</b> a system for controlling the humidity and temperature.</span></li><li><span>
> <b>Pre-filters :</b> Fermenter air pre-filtration of compressed air protects downstream final air filters. It is composed of fibrous materials which removes solid particulars such as dust pollen, mould and bacteria from air. </span></li><li><span>
> <b>Final sterile filters :</b> Final fermenter air filtration provides a sterile barrier between compressed inlet gas supply to fermenters and fermenter broth contents via sparger.</span></li></ul></span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><b><span style="font-size: large;">II). Exhaust air filter </span></b></p><span style="font-size: large;">
<span>It is used to filter the air that is going out of the fermenter to prevent the environment from being contaminated by any biohazards. <br /><ul style="text-align: left;"><li><span>
><b>Pre-filters :</b> Removes undesirable microorganisms to avoid their escaping out of the fermenter. </span></li><li><span>
> <b>Final sterile filters :</b> Final fermenter air filtration provides a sterile barrier between fermenter air exhaust and the surrounding environment.</span></li></ul><div><br /></div><div><u>Also read</u></div><div><u><br /></u></div><div><a href="https://www.biotechfront.com/2021/12/control-system-devices-in-fermenter.html" target="_blank">Control System Devices in Fermenter</a></div></span></span>
<p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-72590978153336358442021-12-07T15:51:00.003+05:302021-12-07T15:58:48.786+05:30Major Differences between Antigens (Ags) & Antibodies (Abs)<p dir="ltr"><span style="font-size: medium;"> An antigen is any foreign molecule that interact with cells of <a href="https://www.biotechfront.com/2020/11/introduction-of-immune-cystem-cells-and.html">immune system</a> and could induce an immune response.<br />
Antibodies are globular proteins or immunoglobulins that react specifically with antigen that stimulated their production. <br />
These are the basic difference between Antigens and Antibodies.</span></p>
<h2 style="text-align: left;"><b><span style="font-size: medium;">Major differences between Antigen and Antibody</span></b></h2><p dir="ltr"><b></b></p><div class="separator" style="clear: both; text-align: center;"><b>
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</b></div><h3 style="text-align: left;"><span style="font-size: medium;"><b>Difference No.1: Definition</b></span></h3><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>Antigen</u></b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
Antigens are any substance or molecules that interact with antibodies or by T cell receptor when complexed with <a href="https://www.biotechfront.com/2021/12/what-is-major-histocompatibility-complex.html">MHC</a> are called antigens.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It include components of <a href="https://www.biotechfront.com/2020/07/the-cell-wall-of-bacteria.html">cell walls</a>, capsules, <a href="https://www.biotechfront.com/2020/11/ultra-structure-of-flagella-.html">flagella</a>, toxins, viral proteins, bacteria and other microorganisms.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
Theoretically any foreign body that is capable of inducing an immune response can be called as an antigen.</span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><u><b>Antibodies</b></u><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">Antibodies are globular proteins or immunoglobulins synthesized by plasma cells of B cells that react specifically with <a href="https://www.biotechfront.com/2021/06/antigen-and-its-epitope.html">antigen</a> that stimulated antibody production.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
There are millions of antibodies inside our body that is specifically designed against different antigens in the surrounding.</span></span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Difference No.2 Chemical Nature</b></span></h3><p style="text-align: left;"><span style="font-size: medium;"><b><u>Antigen</u></b></span></p><span style="font-size: medium;"><ul style="text-align: left;"><li><span style="font-size: medium;">Antigens are generally proteins but can be carbohydrates, lipids or even nucleic acids.</span></li><li><span style="font-size: medium;">
In the case of virus carbohydrate polysaccharide can be antigen.</span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>Antibody</u></b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">Antibodies are glycoproteins made up of amino acids and small amount of carbohydrates.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">Antibodies also called immunoglobulins.</span></span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Difference No.3 : Basic Structure</b></span></h3><p style="text-align: left;"><span style="font-size: medium;"><b><u>Antigen</u></b></span></p><span style="font-size: medium;"><ul style="text-align: left;"><li><span style="font-size: medium;">
Antigenic structure is highly complex in structure and composition. </span></li><li><span style="font-size: medium;">Antigens can be Simple to complex.</span></li><li><span style="font-size: medium;">
Each antigen will interact with a specific antibody, this interactions is highly specific.</span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>Antibody</u></b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
Antibody's '<b></b><a href="https://www.biotechfront.com/2020/07/structure-of-antibody-molecule.html">Y' shaped structure</a> consisting of 4 polypeptide chains, 2 heavy chains (H-chains) and 2 light chains (L-chains) joined by <b>disulphide bonds</b>.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
Each antibody contains two antigen binding site at the Fab region (variable region).</span></span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Difference No.4 : Interactions</b></span></h3><p style="text-align: left;"><span style="font-size: medium;"><b><u>Antigen</u></b></span></p><span style="font-size: medium;"><ul style="text-align: left;"><li><span style="font-size: medium;">The region of the antigen that interacts with the antibodies is called <a href="https://www.biotechfront.com/2021/06/antigen-and-its-epitope.html">epitopes</a>. </span></li><li><span style="font-size: medium;">A pathogen has many epitopes.</span></li><li><span style="font-size: medium;">
This epitope interaction specifically with different antibodies.</span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: medium;"><u><b>Antibody</b></u><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">The variable region of the antibody that specifically binds to an epitope of antigen is called <b>paratope</b>. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">Generally, a Y shaped antibody like <a href="https://www.biotechfront.com/2021/06/igg-structure-property-and-machanism-of.html">IgG</a> has 2 identical paratopes.</span></span></li></ul><p></p>
<h3 style="text-align: left;"><b><span style="font-size: medium;">Difference No.5 : Role in Immune System</span></b></h3>
<p dir="ltr"><span style="font-size: medium;"><b><u>Antigens</u></b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
Antigens causes disease or allergic reaction.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
E.g. Cholera, Malaria, Polio, HIV etc.</span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>Antibody</u></b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
Antibodies are involved in our defence system.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
Protects the system by immobilisation or lysis, agglutination, precipitation of antigenic material.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
Antibodies are the master molecules that is protecting us from diseases.</span></span></li></ul><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-56998497382975768472021-12-04T13:32:00.033+05:302021-12-04T13:45:51.387+05:30Alkaline Phosphates (ALP) : Level, Quantification principle, Isoenzyme & Clinical Symptoms<p dir="ltr"> <span style="font-size: medium;">Alkaline phosphates enzyme plays an important role in the growth and development of bones and teeth. It is a <b>hydrolase</b> enzyme responsible for removing phosphate groups from many types of molecules.<br /></span></p><p dir="ltr"><span style="font-size: medium;">
It is produced by <b>osteoblasts</b> of the bone and is associated with calcification process. It is localized in cell membranes and hence an <b>ecto-enzyme</b>.</span></p>
<p dir="ltr"><span style="font-size: medium;"> Alkaline phosphatase hydrolase enzyme <i><b>catalyzes hydrolytic removal of phosphate group</b></i> from mono-phosphoric esters including glycerophosphates, phenyl-phosphates, nucleotides, and proteins at alkaline pH (9 and 10) as its name indicates.<br />
It is activated by magnesium (Mg++) and manganese(Mn++).</span></p>
<p dir="ltr"><span style="font-size: medium;"> Alkaline phosphatase (ALP) is an enzyme present in the canalicular and sinusoidal membranes of the liver and also active in many other tissues, particularly in bone, kidney, intestine and placenta. It is useful diagnostic test in bone and liver disorders.</span></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Principle of Quantitative Analysis of ALP</b></span></h3><span style="font-size: medium;">
The principle for quantitative analysis of ALP in lab is Alkaline phosphatase in serum acts on the substrate paranitrophenyl phosphate buffered at pH 10 at 37°C to liberate paranitrophenol which gives yellow colour complex and this is measured at 405 nm.</span><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Isoenzymes of Alkaline Phosphates</b></span></h3><span style="font-size: medium;">
It exists in six forms of isoenzymes. The isoenzymes are due to variation in carbohydrate content (sialic acid content), so they can also be called as isoforms. Affinity electrophoresis using polyacrylamide gel is used in separation and identification of fraction of ALP isoenzymes to differentiate liver or bone disorders.</span><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><span style="font-size: medium;"><b>The six isoenzymes of ALP are: </b></span></h4><span style="font-size: medium;">
<u><b>1. Alpha-1ALP</b></u><b>:</b><br /><ul style="text-align: left;"><li><span style="font-size: medium;"> Alpha-1 ALP is 10% of total ALP.</span></li><li><span style="font-size: medium;"> It moves in alpha 1 position in electrophoresis.</span></li><li><span style="font-size: medium;">
It is synthesized by epithelial cells of biliary canaliculi and increased in obstructive jaundice, biliary cirrhosis and to some extent in metastatic carcinoma of liver.</span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>2. Alpha-2, heat labile ALP</u></b><b>:</b> <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">It is 25% of total ALP. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">It is alpha 2 heat labile, stable at 56°C but loses its activity when kept at 65°C for 30 min. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is synthesized by hepatic cells and increased in hepatitis.</span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>3. Alpha-2, heat stable ALP</u></b><b>:</b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
It is only 1% of total ALP. It is also alpha 2 but heat stable isoenzyme. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is not destroyed at 65°C but inhibited by phenylalanine. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is of placental origin and found in blood in normal pregnancy. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It increases in 2nd and 3rd trimester of normal pregnancy and its decrease indicates placental insufficiency and foetal death. </span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>4. Pre-Beta ALP</u></b><b>:</b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
It constitutes 50% of total ALP. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is pre-beta ALP and is most heat labile, loses its activity at 56°C within 10 min. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It originates from the bone and is increased in rickets and Paget's disease.</span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><u><b>5. Gamma ALP</b></u>: <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
It is 10% of total ALP. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is gamma ALP and is inhibited by phenylalanine. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is synthesized by intestinal cells and is increased in ulcerative colitis and after gastrectomy surgery. </span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;"><u><b>6. Leukocyte ALP</b></u>: <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
It constitutes 4% of total ALP. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It originates from leukocytes. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
It is increased in lymphomas and significantly decreased in chronic myeloid leukaemia.</span></span></li></ul><p></p>
<p dir="ltr"></p><h4 style="text-align: left;"><span style="font-size: medium;"><b>Abnormal ALP Isoenzymes</b></span></h4><span style="font-size: medium;">
<b><u>Regan isoenzyme (carcino-placental isoenzyme)</u></b><b>:</b> <br /><ul style="text-align: left;"><li><span style="font-size: medium;">
It is named after the first patient in whom it was detected. </span></li><li><span style="font-size: medium;">
It is an abnormal ALP isoenzyme closely resembling placental ALP isoenzyme. </span></li><li><span style="font-size: medium;">
It is seen in about 15% cases of carcinoma of lung, liver and gut. Chronic heavy smoking also increases regan isoenzyme in blood. </span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: medium;"><b><u>Nagao isoenzyme</u></b><b>: </b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
It is an abnormal ALP isoenzyme present in carcinomas and metastasis.</span></span></li></ul><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Normal Levels of ALP</b></span></h3><span style="font-size: medium;"><ul style="text-align: left;"><li><span style="font-size: medium;">
Normal level of ALP in Adult is 40-125 IU/L.</span></li><li><span style="font-size: medium;">
Normal level of ALP in Children is 42-362 IU/L.</span></li><li><span style="font-size: medium;">
In children, ALP levels are more because of the increased osteoblastic activity in children.</span></li></ul></span><p></p>
<p dir="ltr"><span style="font-size: medium;"><b>Clinical Significance of Alkaline Phosphates</b><br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">
Moderate (2-3 times) increase in ALP level is seen in hepatic diseases such as infective hepatitis, alcoholic hepatitis or hepatocellular carcinoma.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
Very high levels of ALP (10-12 times of upper limit) may be noticed in extrahepatic obstruction (obstructive jaundice) caused by gallstones or by pressure on bile duct by carcinoma of head of pancreas.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
Intrahepatic cholestasis may be due to virus (infective hepatitis) or by drugs (chlorpromazine).</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">
ALP is produced by epithelial cells of biliary canaliculi and obstruction of bile with consequent irritation of epithelial cells leads to secretion of ALP into serum.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">Drastically high levels of ALP (10-25 times of upper limit) are seen in bone diseases where osteoblastic activity is enhanced such as Paget's disease (osteitis deformans), rickets, osteomalacia, osteoblastoma, metastatic carcinoma of bone and hyperparathyroidism.</span></span></li></ul><p></p><p dir="ltr"><br /></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-59163263425929908202021-12-01T15:38:00.002+05:302021-12-01T15:40:46.369+05:30What is Major Histocompatibility Complex ?<p dir="ltr"> <span style="font-size: medium;">Antigens induce an immune response inside the body. Most of these antigens are large protein molecules some are polysaccharides and a few are glycoproteins or nucleoproteins.</span></p>
<p dir="ltr"><span style="font-size: medium;"> Self antigens are the antigens which are found on the membranes of almost all the cells of human body and other vertebrate animals. These self antigens are known as <b>major histocompatibility antigens. </b>Major histocompatibility antigens are glycoproteins in chemical nature. major histocompatibility antigens are also known as histocompatibility antigens.</span></p>
<p dir="ltr"></p><h3 style="text-align: left;"><span style="font-size: medium;"><b>Discovery of Major Histocompatibility Antigens</b></span></h3><span style="font-size: medium;">
These antigens were discovered when scientists were doing transplantation experiments on mice. Scientists found that sometimes the transplant tissue from the donor mouse was accepted by the recipient mouse but at other times it was rejected by the recipient Mouse. </span><p></p>
<p dir="ltr"><span style="font-size: medium;"> Now the question was why the tissues from one individual of a species were destroyed when introduced into member of the same species. <br />
The answer was given by an American Mouse geneticist <b>George Snell.</b> He gave the reason that this rejection was because the tissues of the donor and the recipient were incompatible. </span></p><p dir="ltr"><span style="font-size: medium;"> In other words it was</span> <span style="font-size: medium;">discovered that if the animals were not closely related the recipients rejected the grafts. So if the donor Mouse in the recipient Mouse are of the same genetic background or same inbred strain the transplanted tissue is accepted by the recipient. However if the donor and recipient Mouse are of genetically different backgrounds or different inbred mouse strains the transplanted tissue is rejected by the recipient.</span></p>
<p dir="ltr"><span style="font-size: medium;"> A group of closely related genes was the cause of the rejection. This cluster of gene was named the <b>Major Histocompatibility Complex</b> or <b>MHC.</b> The major histocompatibility complex got its name from the fact that, The genes in this region encode proteins which determine whether a tissue transplanted between two individuals will be accepted or rejected. </span></p>
<h3 style="text-align: left;"><b><span style="font-size: medium;">Major Histocompatibility Complex Genes</span></b></h3><p dir="ltr"><span style="font-size: medium;">
It was discovered that these genes encode proteins which act as self antigens on the surface of the cells. These proteins are known as major histocompatibility antigens.<br />
These antigens gives the answer why rejection of the transplanted tissue happened when a tissue was transplanted from donor mouse to recipient Mouse.<br />
The Hajor Histocompatibility Antigens of donor Mouse were perceived as foreign or non-self by the recipient Mouse. As a result the immune system of recipient Mouse mount an immune response</span> <span style="font-size: medium;">and result in the destruction of the transplanted tissue.</span></p>
<h3 style="text-align: left;"><b><span style="font-size: medium;">Major Histocompatibility Complex</span></b></h3><p dir="ltr"><span style="font-size: medium;">
In the term histocompatibility "histo" means <i><b>tissue</b></i> and "compatibility" means <b><i>getting along</i></b> or <i><b>agreeable.</b></i> The MHC got its name from the fact that the genes in this region encode proteins that determine whether a tissue transplanted between two individuals will be accepted or rejected.</span></p>
<p dir="ltr"><span style="font-size: medium;"> The term complex represents that these genes are localised to a large genetic region containing multiple loci. Since the molecules encoded by these genes were found to have main effects on histocompatibility to distinguish them from other molecules encoded by genome having minor effects on histocompatibility. <br />
They were called major histocompatibility molecules and the genes encoding these molecules were called major histocompatibility complex. </span></p>
<p dir="ltr"><span style="font-size: medium;">Major histocompatibility complex is defined as -<i> A tightly linked cluster of genes whose products play important role in intercellular recognition and in discrimination between self and non-self.</i></span></p>
<p dir="ltr"><span style="font-size: medium;"> Major histocompatibility complex genes are present on <b>chromosome 6</b> in humans and they are known as <b>human leukocyte antigens (HLA)</b> complex. In case of mice they are present on <b>chromosome 17</b> and are known as <b>H-2 complex</b> in mice.</span></p>
<p dir="ltr"><span style="font-size: medium;"> MHC molecules are found on <b>all nucleated cells</b> in the body and they play an important role in the development of both humoral and cellular immunity. B cells react with antigen on their own but T cells recognize antigens only in peptide form and that too when combined with an MHC molecule.</span></p>
<p dir="ltr"><span style="font-size: medium;"> The main function of MHC molecules is to bring <a href="https://www.biotechfront.com/2021/06/antigen-and-its-epitope.html">antigen</a> to the cell surface for recognition by T cells in humans. The genes coding for MHC molecules are found on short arm of chromosome 6. </span></p>
<p dir="ltr"><span style="font-size: medium;">The MHC molecules are divided into three classes namely <br />
MHC Class 1 <br />
MHC Class 2 <br />
MHC Class 3 </span></p><p dir="ltr"></p><div class="separator" style="clear: both; text-align: center;">
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<p dir="ltr"><span style="font-size: medium;"> MHC class 1 molecules are coded at three different locations or low sigh termed a b and c. These glycoproteins are <b>expressed on all nucleated cells.</b></span></p>
<p dir="ltr"><span style="font-size: medium;"> MHC class 2 genes are situated in the D region and there are several different loci known as DR DQ and DP. These glycoproteins appear only on cells that can process non-self materials and present antigen to other cells.</span></p>
<p dir="ltr"><span style="font-size: medium;"> The area between the class MHC 1 and MHC class 2 regions on chromosome 6 is of MHC class 3 genes. These genes code for complement proteins and cytokines which take part in immune response but these molecules are not expressed on cell surface.</span><br />
</p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-69244901355063832352021-11-30T12:05:00.003+05:302021-11-30T13:01:48.749+05:30Hypersensitivity Reaction Type 4<p dir="ltr"> <span style="font-size: medium;">Type 4 hypersensitivity reactions are <b>cell mediated hypersensitivity</b> reactions that result in damage to host cells and tissues. These reactions are initiated by T-cells. The main T-cell types involved are T-helper type 1 cells, Th17 cells and killer or cytotoxic T-cells.</span></p>
<p dir="ltr"><span style="font-size: medium;"><a href="https://www.biotechfront.com/2021/06/antigen-and-its-epitope.html" target="_blank">Antigens</a> are presented to these cells by <b>APCs</b> such as dendritic cells. The damage to hosts is caused by activated macrophages and other leukocytes such as neutrophils and natural killer cells. </span></p>
<p dir="ltr"><span style="font-size: medium;"> Antigens<b> triggering</b> these reactions can be <b>foreign agents</b> that alter self antigens once inside the body. <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">These are basically chemicals that covalently bind to normal glycoproteins present on skin cells. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">Example of such chemical is <b>Urushiol.</b></span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">It is present in the surface oils of the leaves of <b>poison ivy</b> that cause contact hypersensitivity. </span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;">These antigens can also be <b>auto antigens</b> that are recognized by autoreactive T-cells. <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">Autoreactive T-cells can be present in case of failure of self tolerance mechanisms. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">Antigens derived from intracellular pathogens can also trigger type 4 hypersensitivity reactions. </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">Mostly these microbes are those that escape elimination by immune mechanisms and cause prolonged infections. For example Mycobacterium (Tuberculin test)</span></span></li></ul><p></p>
<p dir="ltr"></p><h2 style="text-align: left;"><span style="font-size: medium;"><b>Type 4 Hypersensitivity</b></span></h2><span style="font-size: medium;">
Like all hypersensitivity reactions type 4 hypersensitivity also develops into two stages. <i><b>Sensitization stage</b></i> and <i><b>Effector stage</b></i>.</span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: medium;"><b>Sensitization stage</b></span></p><span style="font-size: medium;"><ul style="text-align: left;"><li><span style="font-size: medium;">Sensitization stage refers to the first or primary contact with the antigen.</span></li><li><span style="font-size: medium;">During sensitization stage T-cells are sensitized and antigen specific memory T-cells are generated.</span></li><li><span style="font-size: medium;">Sensitization in type 4 hypersensitivity occurs in a period of 7 to 10 days. </span></li></ul></span><p></p>
<p dir="ltr"></p><p style="text-align: left;"><span style="font-size: medium;"><b>Effector stage</b></span></p><span style="font-size: medium;"><ul style="text-align: left;"><li><span style="font-size: medium;">Effector stage refers to the secondary or subsequent contact with the antigen.</span></li><li><span style="font-size: medium;">
During the effector stage the host tissue damage takes place.</span></li><li><span style="font-size: medium;">
This damage is apparent only after 1 to 2 days of second exposure.</span></li></ul>
This delay in the manifestation of type 4 hypersensitivity reactions is the hallmark of these reactions. This delay is due to the time taken by T-cells for activation differentiation, cytokine and chemokines secretion, Also the recruitment of macrophages and other leukocytes to the site of antigen exposure takes time. For this reason type 4 hypersensitivity reactions are also known as <b>delayed type hypersensitivity</b> (DTH).</span><p></p>
<h3 style="text-align: left;"><b><span style="font-size: medium;">Mechanism of Type 4 Hypersensitivity Reactions</span></b></h3>
<p dir="ltr"><span style="font-size: medium;"> Type 4 hypersensitivity reactions are initiated by T-cells. There are two main t-cell subtypes <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">CD4 positive T-cells that differentiate into <b>T helper cells</b> and </span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">CD8 positive T-cells that differentiate into <b>cytotoxic</b> or <b>killer T-cells</b>. </span></span></li></ul><p></p>
<p dir="ltr"><span style="font-size: medium;">Which T-cell will initiate the reaction depends on how the antigens are presented to these naive T-cells. <br /></span></p><ul style="text-align: left;"><li><span style="font-size: medium;"><span style="font-size: medium;">If peptide fragments derived from antigens are presented in complex with MHC 2 molecules CD4 positive or helper T-cells are activated.</span></span></li><li><span style="font-size: medium;"><span style="font-size: medium;">On the other hand if antigens are presented in complex with MHC1 molecules CD8 positive or cytotoxic T-cells are activated.</span></span></li></ul><p></p>
<p dir="ltr"> <span style="font-size: medium;"><b>Contact sensitivity</b> caused by poison ivy involves CD8 positive T-cells. These cytotoxic T-cells are sensitized during primary contact with the antigen and on secondary contact activated cytotoxic T-cells use their cytotoxic mechanisms to damage the skin cells and cause local inflammation. </span></p>
<h4 style="text-align: left;"><b><span style="font-size: medium;">Sensitization Stage Reactions</span></b></h4>
<p dir="ltr"><span style="font-size: medium;"> Suppose an intracellular pathogen enters the body for the first time. They infect the local host cells at the site of entry. These antigens are taken up by dendritic cells, which process them and display them as peptide MHC 2 complex on their surface.</span></p>
<p dir="ltr"><span style="font-size: medium;"> These dendritic cells migrate to nearby <b>lymph node</b> and interact with naive CD4 positive T-cells. In the presence of cytokines secreted by dendritic cells and resident macrophages, CD4 positive T-cells get activated and become <b>T-helper type one cells(Th1)</b>.</span></p>
<p dir="ltr"><span style="font-size: medium;"> These cells undergo proliferation and differentiation to form effector T-helper type one(Th1) cells and antigen specific memory T-cells. </span></p>
<p dir="ltr"><span style="font-size: medium;"> Next they migrate to the site of infection and works towards the elimination of the pathogens by cell mediated responses. </span></p>
<p dir="ltr"><span style="font-size: medium;">All these events during the sensitization stage require at least <b>1 to 2 weeks</b>. Now the person is sensitized and antigen specific memory T-cells are present in the body.</span></p>
<h4 style="text-align: left;"><b><span style="font-size: medium;">Effector Stage Reactions</span></b></h4>
<p dir="ltr"><span style="font-size: medium;"> When the individual is exposed to the same antigen for the second time effector stage results. This time antigen specific memory T-cells are already present. <br />
Dendritic cells take up these pathogens process them and present them in complex with MHC 2 molecules. </span></p>
<p dir="ltr"><span style="font-size: medium;"> The resident macrophages also get activated by pathogen and they start releasing cytokines such as interleukin (IL12). </span></p>
<p dir="ltr"><span style="font-size: medium;"> Memory T-cells interact with the antigens presented by dendritic cells and in the presence of cytokines released by activated macrophages they proliferate and differentiate into effector T helper type one cells.</span></p>
<p dir="ltr"><span style="font-size: medium;"> These cells further release cytokines such as interferon gamma<b>(IFN-γ)</b>, tumor necrosis factor beta<b>(TNF-β)</b> and interleukin 2<b> (IL2).</b> These accumulated cytokines at the site of infection. </span></p>
<p dir="ltr"><span style="font-size: medium;"> Now recruit monocytes from circulation to the site. Monocytes differentiate into macrophages when they migrate from blood to tissues. These macrophages also get activated and they further secrete cytokines and chemokines that recruit more monocytes, neutrophils, natural killer cells to the site of infection.</span></p>
<p dir="ltr"><span style="font-size: medium;"> All these activated effector cells release inflammatory mediators that damaged host cells at the site of infection. Together the immune cells and the mediators released by them result in the extensive amplification of the response.</span></p>
<p dir="ltr"><span style="font-size: medium;"> The events of effector stage takes <b>1 to 2 days</b> and only after that the damage to the host is evident. <br />
As the reaction fully develop the majority of participating cells are macrophages and other innate immune cells. Only about 5% cells are antigen specific Th1 cells.</span></p>
<p dir="ltr"><span style="font-size: medium;"> T-helper type 1 (Th-1) cells are the important initiators of type 4 hypersensitivity reactions. Activated macrophages are the principal effector cells of these reactions. The damage is caused to the host because of heightened phagocytic activity and nonspecific destruction of host cells by neutrophils, natural killer cells etc. </span></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.comtag:blogger.com,1999:blog-3773052999085766443.post-87105504204340385412021-11-14T10:38:00.004+05:302024-02-07T10:24:40.248+05:30Bacterial Cell Wall Staining by Chance's Method<p dir="ltr"> <span style="font-size: large;">Bacterial cell is consisting of various structural components. Cell wall is one of the most important component. Cell wall present outside of the bacterial cell membrane. It gives rigidity, protection and shape to the bacterial cell.<br /></span></p><p dir="ltr"><span style="font-size: large;">
Based on the structure of cell wall, all bacteria are divided into two groups as<br /></span></p><ul style="text-align: left;"><li><span style="font-size: large;">
Gram positive and </span></li><li><span style="font-size: large;">
Gram negative. </span></li></ul><span style="font-size: large;">
Cell wall of Gram positive cell is monolayered while cell wall of gram negative bacteria is bilayered.<br /><a href="https://www.biotechfront.com/2020/07/the-cell-wall-of-bacteria.html" target="_blank">Bacterial cell wall</a> can be demonstrated by various special staining methods like Chance's method, Ringer's method & Dyers method. The most common cell wall staining method is Chance's method.</span><p></p>
<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: large;">Requirements :</span></b></h3><ul style="text-align: left;"><li><span style="font-size: large;">Clean grease free slide</span></li><li><span style="font-size: large;">Nichrome wireloop </span></li><li><span style="font-size: large;">24 hrs old culture of bacteria </span></li><li><span style="font-size: large;">0.5% New fuchsin/Basic fuchsin solution (Basic Stain) </span></li><li><span style="font-size: large;">0.5 % Condo red solution (Acidic Stain)</span></li></ul><p></p>
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<p dir="ltr"></p><h3 style="text-align: left;"><b><span style="font-size: large;">Procedure & Steps </span></b></h3><ul style="text-align: left;"><li><span style="font-size: large;">Prepare the smear on the slide under aseptic conditions with the help of wire loop.</span></li><li><span style="font-size: large;">Air dry the smear, but do not heat fix (Because the heat fixation changes the structure of capsule. therefore heat fixation is avoided here).</span></li><li><span style="font-size: large;">Apply 0.5% <b>New Fuchsin</b> for 3 minutes.</span></li><li><span style="font-size: large;">Remove the excess stain (but do not water wash)</span></li><li><span style="font-size: large;">Apply 0.5 % Congo Red for 4 minutes.</span></li><li><span style="font-size: large;">Gentle wash with water </span></li><li><span style="font-size: large;">Air dry and observe under oil immersion objective lens.</span></li></ul><p></p>
<h3 style="text-align: left;"><b><span style="font-size: large;">Machanism and Principle of chance's cell wall staining method</span></b></h3>
<p dir="ltr"><span style="font-size: large;"> New Fuchsin stain is a basic stain. Therefore it is stained cell wall as well as cytoplasm. Cell wall and cytoplasm both are acidic in nature and they are having negative charge on their surface. However here the strong staining of cell wall take place. Because cell wall is more acidic than cytoplasm due to the presence of free carboxylic groups on its surface.</span></p>
<p dir="ltr"><span style="font-size: large;"> Congo red is a acidic stain and this acidic stain bind with basic stain which is already present on the cell and removes that basic stain. That means here Congo red acts as a decolorizer. but the removal of basic stain(New Fuchsin) takes place only from the cytoplasm and not from the cell wall, because it is strongly bound to the cell wall.</span></p><p dir="ltr"><span style="font-size: large;"> Therefore after water wash cytoplasm becomes colourless while cell wall becomes pink coloured. that means here the role of Congo red is to carry out the decolorization, But the decolorization of only cytoplasm takes place and decolorization of cell wall does not take place.</span></p>
<p dir="ltr"><b><span style="font-size: large;">Microscopic Observation</span></b></p><p dir="ltr"><span style="font-size: large;"><b></b></span></p><table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto;"><tbody><tr><td style="text-align: center;"><span style="font-size: large;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEixwsBYTrje5J2T7WA-JnRj8yq4ilIXU0k7rE0MUB9jkBBMBCtNDNBuxMPWAqYQy9YJLWi_2-T4538kfOg7hPeE1sfYIbqldJdMe42YEdFyWAtAxEkDGt5KvHHo-HgB7ArC1YfZb2ClPrY/s1600/1636866485692589-0.png" style="margin-left: auto; margin-right: auto;" width="400" /></span></td></tr><tr><td class="tr-caption" style="text-align: center;"><span style="font-size: large;">Spherical and rod shaped bacterial call wall Pink in colour.</span></td></tr></tbody></table><div class="separator" style="clear: both; text-align: center;"><b><span style="font-size: large;">
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</span></b></div><span style="font-size: large;"> Under the microscope you will observed pink colour cell wall and colourless cytoplasm.</span><b><br /></b><p></p>Harshil Sardharahttp://www.blogger.com/profile/11701395443408821569noreply@blogger.com