The vitamins are certain organic compounds that are needed by the body for healthy life but that cannot be manufactured by the body. They mainly serve as catalysts for certain reactions in the body. The amounts of vitamins required are very small, perhaps hundredths of grams. Do you know that vitamins are mainly obtained from our foods? As a health professional, I know vitamins are needed for a healthy life. In this article, you are going to learn Classification of Vitamins Vitamin Sources RDA (Required Daily Allowance) Diseases that can occur due to Vitamin Deficiency Vitamin Toxicity Let us start with the vitamins classification. I know most of us do not really remember basic knowledge of vitamins. You may also be interesting in reading 05 Vitamins for good hair growth. Classification of Vitamins Based on solubility Vitamins are classified as either fat-soluble (lipid-soluble) or water-soluble. Vitamins A, D, E, and K are fat-soluble Vitamin C and B is water-soluble. All types of vitamins are needed for a healthy life. Water Soluble Vitamins 1. B Complex Vitamins Eight of the water-soluble vitamins are known as the vitamin B-complex group: thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6 (pyridoxine), folate (folic acid), vitamin B12, biotin, and pantothenic acid. The B vitamins are widely distributed in foods and their influence is felt in many parts of the body. They (vitamins) function as coenzymes that help the body obtain energy from food which is needed for a healthy life. The B vitamins are also important for normal appetite, good vision, and healthy skin, the nervous system, and red blood cell formation. A. Vitamin B1 (Thiamin) Thiamin, or vitamin B1, helps to release energy from foods, promotes normal appetite, and is important in maintaining proper nervous system function for a healthy life. Food Sources Sources include peas, liver, and legumes. Most commonly, thiamin is found in whole grains and fortified grain products such as cereal enriched products like bread, pasta, rice, The process of enrichment adds back nutrients that are lost when grains are processed. Among the nutrients added during the enrichment process are thiamin (B1), niacin (B3), riboflavin (B2), folate, and iron. RDA (Required Daily allowance) Males: 1.2 mg/day; Females: 1.1 mg/day Thiamin Deficiency Under-consumption of thiamin is rare due to the wide availability of enriched grain products. However, low-calorie diets, as well as diets high in refined and processed carbohydrates, may place one at risk for thiamin deficiency. Alcoholics are especially prone to thiamin deficiency because excess alcohol consumption often replaces food or meals. Symptoms of thiamin deficiency include mental confusion, muscle weakness, wasting, water retention (edema), impaired growth, and the disease known as beriberi. Thiamin deficiency is currently not a problem in the United States. Thiamin toxicity No problem with overconsumption is known for thiamin. B. Vitamin B12 (Riboflavin) Riboflavin, or vitamin B2, helps to release energy from foods, promotes good vision, and healthy skin. It also helps to convert the amino acid tryptophan (which makes up protein) into niacin. Food Sources Sources include liver, eggs, dark green vegetables, legumes, whole and enriched grain products, and milk. Ultraviolet light is known to destroy riboflavin, which is why most milk is packaged in opaque containers instead of clear. RDA (Required Daily Allowance) Males: 1.3 mg/day; Females: 1.1 mg/day Riboflavin Deficiency Under consumption of riboflavin is rare. However, it has been known to occur with alcoholism, malignancy, hyperthyroidism, and in the elderly. Symptoms of deficiency include cracks at the corners of the mouth, dermatitis on the nose and lips, light sensitivity, cataracts, and a sore, red tongue. Riboflavin toxicity No problems with overconsumption are known for riboflavin. C. Vitamin B3 (Niacin, Nicotinamide, Nicotinic Acid) Niacin, or vitamin B3, is involved in energy production, normal enzyme function, digestion, promoting normal appetite, healthy skin, and nerves. Food Sources Sources include liver, fish, poultry, meat, peanuts, whole, and enriched grain products. RDA (Required Daily Allowance) Males: 16 mg/day; Females: 14 mg/day Niacin Deficiency Niacin deficiency is known to occur with alcoholism, protein malnourishment, low-calorie diets, and diets high in refined carbohydrates. Pellagra is a disease state that occurs as a result of severe niacin deficiency. Symptoms include cramps, nausea, mental confusion, and skin problems. Niacin toxicity Consuming large doses of niacin supplements may cause flushed skin, rashes, or liver damage. Overconsumption of niacin is not a problem if it is obtained through food. D. Vitamin B6 (Pyridoxine, Pyridoxal, Pyridoxamine) Vitamin B6, otherwise known as pyridoxine, pyridoxal, or pyridoxamine, aids in protein metabolism and red blood cell formation. It is also involved in the body’s production of chemicals such as insulin and hemoglobin. Food Sources Sources include meats, whole grains and cereals, legumes, and green, leafy vegetables. RDA (Required Daily Allowance) The RDA for vitamin B6 is 1.3 mg/day for adult males and females through age fifty. Vitamin B6 Deficiency Deficiency symptoms include skin disorders, dermatitis, cracks at the corners of the mouth, anemia, kidney stones, and nausea. A vitamin B6 deficiency in infants can cause mental confusion. Vitamin B6 Toxicity Overconsumption is rare, but excess doses of vitamin B6 over time have been known to result in nerve damage. E. Folate: (Folic Acid, Folacin) Folate, also known as folic acid or folacin, aids in protein metabolism, promoting red blood cell formation, and lowering the risk for neural tube birth defects. It may also play a role in controlling homocysteine levels, thus reducing the risk for coronary heart disease. Food Sources Sources of folate include liver, kidney, dark green leafy vegetables, meats, fish, whole grains, fortified grains and cereals, legumes, and citrus fruits. Not all whole grain products are fortified with folate.. RDA (Required Daily Allowance) The RDA for folate is 400 mcg/day for adult males and females. Pregnancy will increase the RDA for folate to 600 mcg/day. Folate Deficiency Folate deficiency affects cell growth and protein production, which can lead to overall impaired growth. Deficiency symptoms also include anemia and diarrhea. A folate deficiency in women who are pregnant or of childbearing age
Treatment Strategies for COVID-19
Coronavirus disease (COVID-19) is a life-threatening disease caused by a deadly coronavirus. In this article, you will learn about the treatment strategies for COVID-19 Most people infected with the COVID-19 virus will experience mild to moderate respiratory illness and recover without requiring special treatment. Aged people and those with some serious diseases like cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more prone to develop life-threatening illnesses. Most people recover at home after becoming ill with COVID-19. Following things you can do to feel better if you have the flu getting enough rest, staying well hydrated, and taking medications to relieve fever and pains — also help with COVID-19. COVID-19 Current Treatment Strategies The current methodology to treat COVID-19 involves using antiviral medications, anti-inflammatory medications, and immune system modulators, including remdesivir, glucocorticoids, and tocilizumab. Dexamethasone Many doctors around the world, including those in the UK & United States, have been treating very serious COVID-19 patients with corticosteroids since the start of the pandemic. The strategy behind this is to minimize a hyper-immune response (a cytokine storm) in COVID-19 patients. As we all know that in these cases, it is the immune system’s overworking that is denaturing the lungs and other organs, and too often leading to death. Some of the potent anti-inflammatory drugs include Dexamethasone and other corticosteroids (prednisone, methylprednisolone). These drugs are frequently available and inexpensive. According to the NIH COVID-19 treatment guidelines, the use of dexamethasone in certain people hospitalized with severe COVID-19 is highly recommended. These recommendations were based on results from the RECOVERY trial. In the recovery trial study, above 6,000 patients hospitalized with COVID-19 randomly received either standard treatment or dexamethasone. The result of the study showed that patients who required supplemental oxygen or ventilators and who received dexamethasone were less likely to die within 28 days than those who received standard care. The study confirms that Dexamethasone did not have a benefit in patients who did not need respiratory support. Remdesivir The FDA approved the antiviral drug Remdesivir in October 2020 to treat COVID-19. The drug can be used to treat adults and children ages 12 and older. Those who weigh at least 88 pounds, who have been hospitalizing for COVID-19. Clinical trials suggest that in these patients, remdesivir may modestly speed up recovery time. Baricitinib in combination with remdesivir In November 2020, the Food and Drug Administration (FDA) issued an emergency use authorization (EUA) for the use of baricitinib in combination with remdesivir. This drug allow administering in hospitalized adults and children 2 years and older who require respiratory support. However, there is not yet enough evidence to support the use of this therapy instead of dexamethasone with or without remdesivir. Anticoagulation drugs (“blood thinners”) Almost all people admitted to the hospital with COVID receive medications to help prevent blood clots. Doctors usually prescribe low-dose heparin or enoxaparin. However, some patients require full doses of anticoagulants. If they already have developed blood clots or have a high risk of doing so. Doctors always need to balance the risk of dangerous bleeding when prescribing full doses. Conclusion Treatment strategies for Covid-19 change as the various clinical studies results are publishing. I have covered the latest treatment strategies going on in the world. Please share your valuable comments below if you know any current strategies.
BLISTER PACKING MACHINE:
The Ultimate Guide For Learners And Buyers Are you planning to buy a blister packing machine? We know that buying a blister packing machine can be a tough job especially when you do not have expertise in it. Interesting thing is that – you are not alone. After helping 1000+ customers buying the right blister packing machine, we have planned to make the most detailed guide on how to buy a blister packing machine without having technical knowledge. The guide is easy to read and follow whether you are in your early 20’s or 50+ age. Anyhow, if you still need our help in selecting the right model of blister packing machine, our technical team is available 24/7 for your help free. Which things do you need to know before buying a blister packing machine? A brief knowledge about blister packing machines and their applications Your careful attention for 15 minutes to read this guide. Yes, only 15 minutes you need to understand, how to buy a blister packing machine and you will pass through the whole steps. In this guide, you will cover. Chapter 1: Blister packing machine overview Chapter 2: Working principle of blister packing machine Chapter 3: Types of blister packing machine based on Operation Method Technology (Packing Material Used) Chapter 4: Main parts of blister packing machine Chapter 5: Materials used in the blister packaging process Chapter 6: Quality parameters checking of blister packing machine Chapter 7: Troubleshooting of common problems, which occurs during operation Chapter 8: Factors you need to consider while importing a blister packing machine Chapter 9: Merits and demerits of blister packing machine CHAPTER 1 Blister Packing Machine Blister packing machine is equipment, which consists of stationary (non-movable), and moving components. These components aid to seal final products in the pockets or cavities. By doing several mechanical steps, it forms pockets or cavities on forming material fills the pockets or cavities and covers them with a fitting material. Hence it assists with setting up the medicine and nutraceutical items for safe conveyance. Reading further this guide, you will learn the features of this machine, which can enhance productivity in Chapter 4. CHAPTER 2 Working principle of Blister packing machine You have taken the blister packing machine overview in Chapter 1. Now after reading you must be thinking that How does this machine work? How various parts of this machine work together? Well, that is what you are going to learn in this chapter. For your ease, only the general working principle is covered in this guide. Now let’s start! The machine works on the rotary sealing principle. This film is moving ahead from a reel and enters in a blister forming unit where the film is softened by heat and forms blisters, then the filling process starts, and after filling the package in blisters, sealing part of the machine sealed the blisters. At the end printing of batch number, start in the printing section and the blistering process complete. The above summary of the working principle consists of 06 steps. Let us briefly go through all these steps. Step 1: Turning on the machine As you turn on the machine, you will notice that feeding rollers and other parts of the machine start moving. At this stage, the forming film moves to the first stage. Step 2: Heating Station Processing Thermoforming blister packing machine includes this heat-processing unit. At this stage, the machine starts to heat the forming film to the desired temperature until it is soft enough to form cavities. The set temperature is always base on the type of forming material you are using. Step 3: Film Cavities Forming In the third step, two mechanisms are used to form desire cavities i.e. compressed air or die plates. These cavities form the blister packing machine will cool down these cavities to give them a rigid shape. Step 4: Product filling and Empty cavities checking In this stage, the blister packing machine will fill the cavities with the final product. The filling step will be either manual or automatic, which totally depends on the type of machine you have selected. You know that aluminum is opaque (you cannot see through) in nature so it is mandatory to look for empty cavities. Therefore, this empty pocket detector is placed at the end of the filling station. Step 5: Blister Sealing At this stage, the filled cavities are ready for sealing. To seal the blister lidding material is used. It is very important for a machine that lidding material is in place timely as forming film reaches the sealing station. Moving further the lidding material cover the fill cavities. The heat-sealing station comes next which seals the product in the cavities. To make sure that sealing is up to the mark both high temperature and pressure are applied. Step 6: Printing & trimming of product This is the last step of blister packing. The seal pack moves to the next stage i.e. printing station. At this point machine, emboss the set message in it. After that machine, push forward the blister pack to the cutting or trimming station. This trimmer trims it into desire units. The leftover lidding and forming material is rolled into a scrap collector. CHAPTER 3: Types of blister packing machine Dear readers, it is very important for you to know about various types of blister packing machines. As you have already study about its overview and its working principle in chapter 1 and 2 now we will discuss its various types. It is also very important for you to know every machine’s technical specifications which you will read in Chapter 12. Without wasting any time now let us dig into it. There are several ways to classify these machines. However, in this guide, you will learn about the types based on their operation and the packing material used. There are the following three types of packing machines based on their operation Thermoforming Blister Packing Machine Thermoforming
Learn HPLC: The Ultimate Guide for Learners & Buyers
Are you planning to learn or buy an HPLC (High Performance Liquid Chromatography)? Buying an HPLC can be a tough job especially when you do not have expertise in it. The interesting thing for you is that we are with you. After helping 1000+ buyers buying the right HPLC, we have planned to make the most detailed guide on how to buy an HPLC without having technical knowledge. The guide is easy to read and follow whether you are in your early 20’s or 50+ age. Anyhow, if you still need our help in selecting the right model of HPLC, our technical team is available 24/7 for your help free. Which things you need to need to know before buying a HPLC? A brief knowledge about HPLC and its applications Your careful attention for 15 minutes to read this guide. Yes, only 15 minutes you need to understand, how to buy HPLC and you will pass through the whole steps. In this guide, you will cover. HPLC Overview Working Principle of HPLC Types of HPLC based on Operational Method Main Parts of HPLC Quality Parameters Checking of HPLC Trouble Shooting of Common Problems, which occurs during Operation. HPLC Applications Merits and demerits of HPLC Famous brands of HPLC High Performance Liquid Chromatography (HPLC) In this chapter you are going to learn about chromatography and its types, its major concern is about High performance liquid chromatography (HPLC). Chromatography is a separating technique, in which separation is done on the basis of the structure of the molecule in two-phase (i.e. Mobile phase and Stationary phase) The stationary phase is a solid or a liquid material packed in a column and a mobile phase is a liquid or a gas that flows through the stationary phase. The mobile phase flows through the stationary phase and carries the components of the mixture with it. The component of a mixture, which shows stronger interactions with the stationary phase, will retain in the stationary phase while the component of the mixture, which dissolves in the mobile phase, will move with it and elute first. HPLC stands for High performance Liquid chromatography or high-pressure liquid chromatography as it uses a high-pressure liquid mobile phase than gases used in Gas chromatography. HPLC is an analytical technique used for the separation of components of the organic mixture of compounds when such compounds are nonvolatile, thermally unstable, and have relatively high molecular weights. Working principle of HPLC In the previous chapter you learn about the introduction of HPLC, now in this chapter, you will learn about the working principle of HPLC. The working principle of HPLC is based on the distribution of the sample between a mobile phase (eluent) and a stationary phase (packing material of the column). Molecule retarded in stationary phase and separate on the basis of difference in retention time. The component of a mixture which retain in the stationary phase elute later, as compared to that which dissolves in the mobile phase. Types of HPLC This section will help you to learn different types of HPLC. Types of HPLC, Based on mode of separation 1. Normal Phase HPLC In this type of HPLC, components are separated on the basis of polarity. In the Normal phase HPLC, the polar stationary phase is used with the non-polar mobile phase. The stationary phase is usually silica and the mobile phase is Hexane, Methylene Chloride, Chloroform, Diethyl ether, and mixtures of these. As the Stationary phase is polar in normal phase HPLC therefore, the Retention time of Polar samples on the polar surface of the column is longer and they elute later than less polar materials. 2. Reverse Phase HPLC In this type of HPLC, the stationary phase is non-polar in nature, while the mobile phase is a polar liquid i.e. mixtures of water and Methanol or Acetonitrile. It works on the principle of hydrophobic interactions hence retention time of non-polar material is longer than polar material. Note: Reverse phase chromatography is more commonly used as drugs are usually Hydrophilic. 3. Size-exclusion HPLC In this type of HPLC, the Column used is filled with a material having control pore size and separate the components on the basis of size. Large size molecules pass through the column while small size molecules penetrate inside the pores of the packing material and due to having large retention time elute later. 4. Ion-Exchange HPLC In this type of HPLC, The stationary phase has a charged surface of opposite charge to the sample ions. The sample with a strong charge will be attracted to the ionic surface, due to this attraction its retention time will increase and elute later. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time. (Learn) Types of HPLC Based on Elution technique: Isocratic Elution: A separation in which the composition of the mobile phase remains the same throughout the procedure. In isocratic elution, peak width increases due to high retention time which directly relates to the number of theoretical plates. This leads to the disadvantage that late eluting peaks become broad and flat. Gradient Elution: A separation in which the composition of the mobile phase is changed during the procedure. Gradient elution decreases the retention of later-eluting components and gives narrow peaks and also improves the peak shape and height. Main parts of HPLC Now it is the time to learn about some main parts of HPLC. The components of HPLC are following, 1. Solvent Reservoir The mobile phase contained in a glass reservoir. In HPLC, the mobile phase is normally a mixture of polar and non-polar liquid components whose compositions are changed by changing the composition of the sample. 2. Pump A pump sucks the mobile phase from the solvent reservoir and forces it to pass through the system’s column and detector. The pressure of the pump depends on a number of factors including column dimensions, the particle size of the stationary phase, the
Fungi Staining Technique: Learning Guide
Staining of Fungi Direct microscopic examination without stain lacks sensitivity, especially when hyphae are sparse in the specimen. A variety of differential stains are commonly used like Gram, Giemsa, India Ink Stain, Lactophenol cotton blue stain, etc to stain fungi. To Study Bacterial Staining Technique Click Here Methods of Staining Fungi India Ink stain Introduction This stain was previously known as the “Nigrosin stain”. India ink staining is a negative staining technique used to determine an organism’s cellular morphology. The background is stained whereas the organism remains unstained and the morphology is not distorted in any way. Capsules displace the dye and appear as halos surrounding the organism. This fungi stain provides a high degree of contrast not available in most other staining procedures. This technique is particularly recommended for the demonstration of the capsule of the yeast, Cryptococcus neoformans and it can also be used to demonstrate the presence of bacterial capsules. Method place a drop of India ink on to a clean glass slide Add 1 drop of the specimen or liquid culture or rub a speck of material on the slide surface just beside the ink before mixing it into the ink. Sputum or pus can be cleared with KOH and heat and then mixed with India ink Note: If preparation is too dark, it may be diluted with a small drop of water Place a cover slip over the smear avoiding air bubbles, press it down gently through a sheet of blotting paper so that the film becomes very thin and pale in colour Examine with a high-power lens (phase-contrast microscope) for the presence of encapsulated cells. Bright field microscopy may also be used Interpretation Positive result Organisms possessing a capsule appear highly refractile, surrounded by a clear zone or halo against a dark background. Leucocytes may also appear haloed due to leakage of the cytoplasm but the halo has a fuzzy, irregular appearance at the periphery and the cell within the halo has a paler cell wall. Note: Some Cryptococcus neoformans strains have been reported to be India ink negative. Negative result No clear zone around the organism is observed. Technical information Sensitivity The cryptococcal latex antigen test has been proven to be significantly more sensitive than the India ink preparation and is therefore recommended for the initial diagnosis of cryptococcal disease. Errors with India ink stain Common errors with this stain are: The use of diluted ink. The correct concentration of India ink is critical for showing the capsular zone The smear on the slide is too thick. Some practice is required by laboratory staff in making satisfactory smears Lactophenol cotton blue stain Introduction The lactophenol cotton blue (LPCB) stain is the most widely used staining solution in the examination of yeasts and molds and serves as both a mounting fluid in wet mounts and a stain. It is simple to prepare. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls. Upon the addition of lactophenol cotton blue, fungi stain blue allowing for easier visualization and examination. Other alternative stains that can be used are the Lactofuchsin or aniline blue stains and these have the same principles as the LPCB stain. The Lactofuchsin stain, if performed correctly, can preserve the structure and arrangement of the hyphae, if present for several weeks. Safety considerations Lactophenol cotton blue is acidic while Lactofuchsin is corrosive. They can be toxic if inhaled, in contact with skin and if swallowed. Adhesive tape preparations from fungal cultures must be prepared under a biological safety cabinet to ensure safety of laboratory personnel. Attention must be paid to patient travel history and any suspected Hazard Group 3 organisms must be processed in a Class 1 exhaust protective cabinet in a Containment Level 3 room. Phenol kills any fungus present, allowing the microscope preparations to be examined out with the biological safety cabinet. Skin scraping, Fluid exudate or Tissue Mix the specimen whether a skin scraping, fluid exudate or tissue with two drops of 10% KOH on a clean slide. add one, or at most two drops of the lactophenol cotton blue mountant/stain to the slide Press a cover slip gently to make a thin mount avoiding air bubbles. Gentle warming can also aid in clearing the mount Examine the prepared slide under low power (x10) with reduced lighting. Switch to high power (x40) to check for the presence of suspected fungal structures. Culture If examining a fungal culture, direct microscopic mounts can be made. Alternatively, the adhesive tape method may be used. Direct microscopic mount place one drop of lactophenol cotton blue mountant to a microscope slide using a mounted needle, gently remove a small portion of the colony and place in the LPCB drop cover with a coverslip, pressing gently to make a thin mount avoiding air bubbles blot off any excess LPCB stain examine the prepared slide under low power (x100) with reduced lighting. Switch to high power (x400) to examine the fungal structures in more detail Adhesive tape This technique may be a quick and easy alternative to direct preparation; it often allows fungal structures to remain intact in the slide preparation. There are many variations of this technique; the standard procedure is given below: Place one or two drops of lactophenol cotton blue mountant to a clean glass microscope slide Take a 40mm length of clear adhesive tape and place the sticky side on to the surface of the culture, gently applying pressure allowing fungal elements to become attached to the tape. Forceps may be used. Carefully lift the tape and place on to the LPCB mountant on the slide gently pressing down Note: The preparation may also be examined directly without the use of a coverslip; however, another drop of LPCB stain may be added on top of the tape and a cover glass placed on top examine the prepared slide under low power (x100) with reduced lighting. Switch to
Bacterial Staining Technique: Learning Guide
Staining is a valuable technique used in microscopy to enhance contrast in the microscopic image. Stains are used to highlight structures in clinical specimens, often when viewed with the aid of different microscopes. Stains have different affinities for different organisms and are used to differentiate types of organisms or to view specific parts of organisms. Staining involves the sample preparation onto slides, fixation (which aims to preserve the shape of the cell), the staining with dyes, and the observation under the microscope. Common Errors During Staining Procedure The duration of each step may vary depending on the concentration and formulation of staining solutions and other reagents. Follow the manufacturer’s instructions where possible. Rinsing stepThe use of tap water is not recommended when making the smears or whenperforming rinse steps in some staining protocols, for example, in the Ziehl-Neelsen protocol, Mycobacterium gordonae has been found in tap water and may interfere with the accurate assessment of the specimen to be stained. Deionized or distilled water is recommended.Excess rinsing between steps could also cause error in a staining procedure. Decolourising stepMany laboratories do not adhere to a fixed decolorizing time for staining protocols and so results may vary. In some laboratories, laboratory staff is taught to add the decolorizing reagent drop by drop until it runs clear. Difficulties in interpreting stain resultsThe staining technique is one factor that affects results. This may be due to differences in applying the steps in the protocols which might warrant analysis if problems in interpretation persist. Standardization of the protocols will minimize variation in results. Other issues that may affect results are:• when cultures have not been sufficiently mixed to break up clumps of cells, the resulting smear can be difficult to read because individual cells are not discernible • partially acid-fast bacteria may also contribute to confusion during a smear evaluation• the type and quality of specimen/smear. Smears that are too thick will not be readable and those that are too thin may result in false negatives or result in the need to repeat the procedure• expired reagents• preparation of reagents – this includes confirming the expiration dates ofreagents and confirming protocols to ensure proper reagent concentrations.Difficulty in reading stains can occur when reagents are not prepared to their right concentrations• improper operation of the microscope Bacteria Staining Procedures 1 Auramine-phenol stain – 1 (acid fast bacilli) This staining technique is used to demonstrate the presence of acid-fast bacilli (Mycobacterium species). These organisms have waxy envelopes that make them difficult to stain and decolorize. A fluorescent stain is used in this method. Auramine stain show higher sensitivity and specificity than Ziehl-Neelsen’s method. It is a better method for screening samples from suspected cases of tuberculosis especially pulmonary and extrapulmonary cases where bacilli count is usually low. Method • prepare smear and heat to fix• flood the slide with Auramine-phenol (1:10v/v) and leave for 10min• gently rinse with water (ensure water is either deionized or distilled)• decolorize with 1% acid alcohol for 3-5min• gently rinse with water as above• repeat acid alcohol step until no further stain seeps from the film • counterstain with 0.1% potassium permanganate or thiazine red for 15sec (this ensures a dark background for the fluorescing alcohol and acid-fast bacilli (AAFB) which are easier to see). KMNO4 stains all epithelial cells making it more difficult to see AAFB• gently rinse with water as above and air dry. Do not blot dry• examine slides using ultraviolet epi-fluorescence microscopy at 25 x or 40 x magnification (the use of a 40 x magnification non-cover-glass (NCG) objective lens will avoid the need to apply a cover glass) InterpretationPositive resultAcid fast bacilli vary from 0.5-10µm in length and stain bright yellow-green against a dark background.Negative resultNo fluorescence observed. Non-acid-fast cells appear dark. Quality control organismsPositive controlMycobacterium species.Negative controlA proven negative smear may be used as a negative control. 2 Gram stain The Gram stain is complex and differential staining technique that remains a useful test performed in microbiology laboratories. The staining procedure differentiates organisms of the domain bacteria according to the cell wall structure. Organisms are classified according to their Gram staining reaction – Gram-positive and Gram-negative. The name “Gram” comes from its inventor, Hans Christian Gram. Gram-positive bacteria have thicker and denser peptidoglycan layers in their cell walls. Iodine penetrates the cell wall in these bacteria and alters the blue dye to inhibit its diffusion through the cell wall during decolorization. Gram-positive bacteria must have an intact cell wall to produce a positive reaction. Gram-negative cells which do not retain the methyl/crystal violet are stained by a counterstain. Neutral red, safranin, or carbol-fuchsin may be used as the counterstain. This technique has also been used for staining of certain fungi such as Candida and Cryptococcus which are observed as Gram-positive yeasts. MethodHucker’s modification of Gram staining technique for the examination of smears• prepare a smear and heat gently to fix• flood the slide with 0.5% crystal violet and leave for 30sec• tilt the slide, and rinse slide gently with water• flood on sufficient (1%) Lugol’s iodine (also known as Gram’s iodine) to rinse off excess water, cover with fresh iodine and allow to remain for 30sec• tilt the slide and wash off the iodine with water• decolorize with 95 – 100% ethanol or acetone until color ceases to run out of the smear• rinse with water• flood the slide 0.1% counterstain safranin and leave to act for about 30sec to 1min Note: It can be counterstained for longer if using other dyes, for example,neutral red for about 2min• wash briefly with water and blot dry• examine the slide using an oil immersion objective to observe cell morphology and Gram reaction InterpretationPositive resultGram-positive organisms stain deep blue/purple.Negative resultGram-negative organisms stain pink/red.Note: Other counterstains (such as carbol fuchsin) used may give more intense colours.Quality control organismsA culture containing Gram-positive and Gram-negative organisms may be used for quality control. Common errors in the Gram staining procedureThese are the errors that arise depending on the method and techniques
Minimum Inhibitory Concentration (MIC) Learning Guide
The minimum inhibitory concentration (MIC) is the smallest concentration of an antimicrobial agent that inhibits the growth of bacteria. The value is obtained in a highly mechanized fashion, but this procedure interval-censored reading. It is often of interest to use data collected from complex experiments to see how the mean MIC is affected by different factors. Principles: The Minimum Inhibitory Concentration Assay is a technique used to determine the lowest concentration of a particular antibiotic needed to kill bacteria. This assay is typically performed on planktonic (free-floating) bacterial cells. Methods for determination of MIC: Serial/Broth Dilution method Agar Dilution Method Material used: Laminar air flow Petri dishes Tips Micropipettes Muller Hinton Broth and Agar 1.5% Vortex Mixer Multi-inoculator Procedure: Serial/Broth Dilution Broth dilution testing allows the option of providing both quantitative (MIC) and qualitative (category interpretation) results. MIC can be helpful in establishing the level of resistance of a particular bacterial strain and can substantially affect the decision to use certain antimicrobial agents. Broth dilution can again be performed by 2 ways Macro dilution: Uses broth volume of 1 ml in standard test tubes . Microdilution: Uses about 0.05 to 0.1 ml total broth volume and can be performed in a microtiter plate or tray . The procedure for both macro and microdilution are same except the volume of the broth. For example, let’s say you wish to determine the MIC of an antibiotic on a bacterium. You decide to test 3 concentrations (10 µg/ml, 1 µg/ml and 0.1 µg/ml). Each of these tubes have growth media inoculated with a standard concentration of bacteria and the respective antibiotic concentration (Figure 1). The tubes are allowed to incubate overnight. Broth tubes that appear turbid are indicative of bacterial growth while tubes that remain clear indicate no growth. The MIC of the antibiotic is the lowest concentration that does NOT show growth. After incubating the tubes overnight, you observe the tubes in Figure 2: From this example, Tube C, (with 0.1 ug/ml of antibiotic) did not inhibit bacterial growth. Tubes A and B, on the other hand, did inhibit growth. Tube B has the minimum inhibitory concentration because B is the lowest concentration of the antibiotic that inhibited cell growth. Therefore, the MIC for this bacterium is 1 µg/ml. Agar dilution MIC of an antibiotic using broth dilution method is determined by using the following procedure Preparation of antibiotic stock solution Preparation of antibiotic dilution range Preparation of agar dilution plates Preparation of inoculum Inoculation Incubation Reading and interpreting results Antibiotic stock solution can be prepared by commercially available antimicrobial powders (with given potency). The amount needed and the diluents in which it can be dissolved can be calculated by using either of the following formulas to determine the amount of antimicrobial powder (1) or diluent (2) needed for a standard solution: Prepare antimicrobial agent stock solutions at concentrations of at least 1000 μg/mL (example: 1280 μg/mL) or 10 times the highest concentration to be tested, whichever is greater. Because microbial contamination is extremely rare, solutions that have been prepared aseptically but not filter-sterilized are generally acceptable. If desired, however, solutions may be sterilized by membrane filtration. Dispense small volumes of the sterile stock solutions into the sterile glass, polypropylene, polystyrene, or polyethylene vials; carefully seal; and store (preferably at −60 °C or below, but never at a temperature warmer than −20 °C and never in a self-defrosting freezer). Vials may be thawed as needed and used the same day. Preparation of antibiotic dilution range Use sterile 13- x 100-mm test tubes to conduct the test. If the tubes are to be saved for later use, be sure they can be frozen. Close the tubes with loose screw-caps, plastic or metal closure caps, or cotton plugs. Prepare the final two-fold (or other) dilutions of antimicrobial agent volumetrically in the broth. A minimum final volume of 1 mL of each dilution is needed for the test. Note: For, microdilution, only 0.1 ml is dispensed into each of the 96 wells of a standard tray. Preparation of inoculum Prepare the inoculum by making a direct broth suspension of isolated colonies selected from an 18- to 24-hour agar plate (use a non-selective medium, such as blood agar). Adjust the suspension to achieve turbidity equivalent to a 0.5 McFarland turbidity standard. This results in a suspension containing approximately 1 to 2 x 10^8 colony forming units (CFU)/mL for Escherichia coli ATCC® 25922. Compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines. Optimally within 15 minutes of preparation, dilute the adjusted inoculum suspension in broth so, after inoculation, each tube contains approximately 5 x 10^5 CFU/mL. Note: This can be accomplished by diluting the 0.5 McFarland suspension 1:150, resulting in a tube containing approximately 1 x 10^6 CFU/mL. The subsequent 1:2 dilution in step 3 brings the final inoculum to 5 x 10^5 CFU/mL. Inoculation Within 15 minutes after the inoculum has been standardized as described above, add 1 ml of the adjusted inoculum to each tube containing 1 ml of the antimicrobial agent in the dilution series (and a positive control tube containing only broth), and mix. This results in a 1:2 dilution of each antimicrobial concentration and a 1:2 dilution of the inoculums. Incubation: Incubate the inoculated tubes at 35 ± 2 ºC for 16 to 20 hours in an ambient air incubator. To maintain the same incubation temperature for all cultures, do not stack microdilution trays more than four high. Interpretation Compare the amount of growth in the wells or tubes containing the antimicrobial agent with the amount of growth in the growth-control wells or tubes (no antimicrobial agent) used in each set of tests when determining the growth endpoints. For a test to be considered valid, acceptable growth (≥ 2 mm button or definite turbidity) must occur in the growth-control well. Quality Control Use reference bacterial strains that are genetically stable and have well-defined MICs that are in the middle range of the expected MICs of each antimicrobial agent to be tested. A dilution series should include at least two concentration
Antiviral Drugs A Quick Review
Antiviral drugs are prescription medicines (pills, liquid, an inhaled powder, or an intravenous solution) that fight against flu viruses in your body. Antiviral drugs are not sold over-the-counter. You can only get them if you have a prescription from a health care provider. Antiviral drugs are different from antibiotics, which fight against bacterial infections. If you get sick with flu, antiviral drugs are a treatment option. Check with your doctor promptly if you are at high risk of serious flu complications and you develop flu symptoms. Flu signs and symptoms can include feeling feverish or having a fever, cough, sore throat, runny or stuffy nose, body aches, headache, chills, and fatigue. Your doctor may prescribe antiviral drugs to treat your flu illness. Benefits of Antiviral Drugs Antiviral treatment works best when started soon after flu illness begins. When treatment is started within two days of becoming sick with flu symptoms, antiviral drugs can lessen fever and flu symptoms, and shorten the time you are sick by about one day. They also may reduce the risk of complications such as ear infections in children, respiratory complications requiring antibiotics, and hospitalization in adults. For people at high risk of serious flu complications, early treatment with an antiviral drug can mean having milder illness instead of more severe illness that might require a hospital stay. For adults hospitalized with flu illness, some studies have reported that early antiviral treatment can reduce their risk of death. Classification of Antiviral Drugs Drug Mechanism of Action Therapeutic Uses Key Notes Neuraminidase Inhibitors Oseltamivir Zanamivir -Prevent the release of new virions and their spread from cell to cell -Given prior to exposure they prevent infection -Given24-48 hours after onset of infection, they effect intensity and duration of symptoms -Type A and B influenza viruses -Orthomyxoviruses need neuraminidase for life cycle of the virus -Sialic acid analogs -Do not interfere with response immune response to influenza A vaccine -Zanamivir should be avoided in pts with severe reactive asthma or COPD Amantadine Rimantadine -Block the viral membrane matrix protein (M2) which is required for the fusion of viral membrane with cell membrane Preventing formation of endosome Interferes with viral uncoating -70 to 90% effective in preventing infection if tx begun at time of or prior to exposure to infection -Given w/in first 48 hours, they reduce duration and severity of systemic symptoms -Influenza A viruses -Amantadine: Parkinson’s disease -Do not impair the response of influenza A vaccine -Amantadine may accumulate to toxic levels in pts with renal failure -Cautious use in pregnant and nursing mothers (embryotoxic and teratogenic) -Cross-resistance occurs between the two drugs Ribavirin -Converted to 5’-phosphate derivative forming ribavirin-triphosphate Inhibits guanosine triphosphate formation Prevents viral mRNA capping, blocking RNA-dependent RNA polymerase -Infants and young children: severe RSV infections -Chronic hepatitis C (w/ IFN) -Reduces mortality and viremia of Lassa fever -Guanosine analogs -Contraindicated in pregnancy due Interferon -Interfere with ability of viruses to infect cells -Induce host cell enzymes that inhibit viral RNA translation Degradation of viral mRNA and tRNA -IFN-α-2b: hepatitis B, C -Condylomata acuminate -Hairy cell leukemia -Kaposi’s sarcoma-IFN-β: MS -Interferes with metabolism of THEOPHYLLINE -Potentiates the bone marrow suppression caused by other agents Lamivudine -Inhibitor of both HBV DNA polymerase and HIV reverse transcriptase -Must be phosphorylated by host cell enzymes to active form (triphosphate) -HBV -HIV -Cytosine analog -Chronic treatment reduces hepatic inflammation -Dose reductions required in moderate renal insufficiency Adefovir dipivoxil -Phosphorylated to adefovir diphosphate Incorporated into viral DNA Termination of further DNA synthesis and prevents viral replication -Causes decreased viral load and improved liver function -Nucleotide analog -Cautious use in pts with existing renal disease Entecavir -Intracellular phosphorylation to triphosphate Competes for viral reverse transcriptase -LAMIVUDINE-resistant HBV -Guanosine analog -Improves liver scarring and inflammation -Drugs that have renal toxicity should be avoided Telbivudine -Phosphorylated intracellularly to triphosphate Competes with endogenous thymidine triphosphate for incorporation into DNA Terminates further elongation of DNA -HBV -Thymidine analog -Dose should be adjusted in renal failure -Can be combined with LAMIVUDINE Acyclovir -Monophosphorylated in cell by thby thymidine kinase -Acyclovir triphosphate: competes with deoxyguanosine triphosphate as a substrate for viral DNA polymerase Incorporates into viral DNA Premature DNA-chain termination -HSV 1 and 2 -Varicella-Zoster virus -EBV -DOC: HSV encephalitis -Prophylactic to sero+ pts before BM or heart transplant Accumulates in pts with renal failure -Valacyclovir: hydrolyzed to Acyclovir -Resistance occurs due to altered or deficient to thymidine kinase and DNA polymerase -Cross-resistance to other cyclovirs occurs Cidofovir -Inhibits viral DNA synthesis -CMV-induced retinitis in pts w/ AIDS -Nucleotide analog -Eliminates the permanent venous access used for GANCICLOVIR therapy -CI in pts with renal impairment or taking nephrotoxic drugs -PROBENECID can be given to reduce risk of nephrotoxicity Fomivirsen -Directed against CMV mRNA -CMV retinitis -Antisense oligonucleotide -Should be used 2-4 weeks after CIDOFOVIR to reduce toxicity Foscarnet -Reversibly inhibits viral DNA and RNA polymerases Interferes with viral DNA and RNA synthesis -CMV retinitis in immunocompromised pts -ACYCLOVIR-resistant HSV and Herpes Zoster infections -Phosphonoformate Ganciclovir -Activated through conversion to nucleotide triphosphate Nucleotide competitively inhibits viral DNA polymerase -CMV retinitis in immunocompromised pts -CMV prophylaxis in transplant pts -Analog of ACYCLOVIR -Accumulates in pts with renal failure-Valganciclovir: rapid hydrolysis in intestine and liver Peniciclovir -Triphosphate inhibits HSV DNA polymerase -Pain and healing are shortened -HSV 1 and 2 -Varicella Zoster -Acyclic guanosine nucleoside Famciclovir -Prodrug metabolized to active PENCICLOVIR -Acute herpes zoster -In animals: increased mammary adenocarcinomas and testicular toxicity Vidarabine (ara-A) -Converted in cells to ara-ATP Inhibit viral DNA synthesis -Tx of immunocompromised pts with herpetic and vaccinial keratitis -HSV kerato- conjunctivitis -Adenosine analog Trifluridine -Converted to triphosphate Competitively inhibits incorporation of thymidine triphosphate into viral DNA Defective DNA -Irreversible inhibitor of viral thymidine HSV 1 and 2 -Vaccina virus -DOC: HSV keratoconjunctivitis and recurrent epithelial keratitis -Fluorinated pyrimidine nucleoside analog NRTIs Zidovudine Stavudine Didanosine Tenofovir Lamivudine Emtricitabine Zalcitabine Abacavir -After entering a cell, they are phosphorylated to corresponding
Common Antidote & Drug-Related Side Effects
Antidote An antidote is a drug, chelating substance, or a chemical that counteracts (neutralizes) the effects of another drug or a poison. There are dozens of different antidotes; however, some may only counteract one particular drug, whereas others (such as charcoal) may help reduce the toxicity of numerous drugs. Most antidotes are not 100% effective, and fatalities may still occur even when an antidote has been given. Some examples of antidotes include: Toxicity Antidote Arsenic Dimercaprol Benzodiazepines Flumazenil Acetaminophen N-acetylcysteine Heparin Protamine Sulfate Warfarin Vitamin K or Fresh-frozen plasma Tissue Plasminogen Activator Aminocaproic acid Methotrexate Leucovarin (Folinic acid) Sodium Nitroprusside Sodium Thiosulfate β-blockers Glucagon Carbon Monoxide 100% Oxygen Opioid Naloxone, Naltrexone Methanol Ethanol Organophosphates Atropine, PAM Iron Desferoxamine Cyclophosphamide Mesna Lead D-penicillamine Common Antidotes of Poisoning Drug Related Side Effects Side effects, also known as adverse events, are unwanted or unexpected events or reactions to a drug. Side effects can vary from minor problems like a runny nose to life-threatening events, such as an increased risk of a heart attack. Several things can affect who does and does not have a side effect when taking a drug – age, gender, allergies, how the body absorbs the drug, other drugs, vitamins and dietary supplements that you may be taking. Common side effects include upset stomach, dry mouth, and drowsiness. A side effect is considered serious if the result is: death; life-threatening; hospitalization; disability or permanent damage; or exposure prior to conception or during pregnancy caused birth defect. Side effects can happen when you start taking a new drug, dietary supplement, or vitamin/mineral stop taking a drug that you’ve been on for a while, or when you increase or decrease the amount of a drug that you take. Side Effect Causative Drugs Pulmonary Fibrosis BleomycinAmiodaroneBusulfan Pseudomembranous Colitis ClindamycinAmpicillinAmoxicillin Tendonitis Fluoroquinolones Interstitial Nephritis NSAIDsMethicillin Cholestatic Jaundice Macrolides Coronary Vasospasm CocaineSumatriptan Gout Thiazides Disulfiram-like reactions ChlorpropamideMetronidazoleCefoxitinCefamandole Atropine-like reactions TCAs Cough ACE Inhibitors Aplastic Anemia CarbamazepineChloramphenicolGold compounds EPS Antipsychotics Fatal Hepatotoxicity HalothaneValproic acidThiazolidinedionesAcetaminophen Gray Baby Syndrome Chloramphenicol Gynecomastia CimetidineSpironolactoneAlcohol Hand Foot Syndrome 5-FU Hemolytic anemia w/ G6PD deficiency PrimaquineDapsoneSulfa drugsNitrofurantoinChloramphenicol Hot Flashes NiacinTamoxifen Postural Hypotension α-Blockers Myelosuppression Alkylating cancer drugs SLE-like reactions ProcainamideHydralazineMethyldopa Red Man Syndrome Vancomycin Optic Neuritis Ethambutol Ototoxicity AminoglycosidesLoop diuretics Pancreatitis Didanosine Adrenal Insufficiency Steroid-withdrawal Thrombocytopenia HeparinRifampin Rebound Hypertension Clonidine-withdrawalα-Methyldopa Severe HTN w/ Tyramine MAO inhibitors Agranulocytosis Clozapine Cinchonism Quinidine Antidotes and Drugs Related To Famous Side Effects