Mark A. Graber, MD

• Professor
• Departments of Family Medicine and Emergency Medicine
• Roy J. and Lucille A. Carver College of Medicine
• University of Iowa
• Iowa City, Iowa

Iron deficiency was very common in prehistoric populations and is associated with characteristic pitting of the roof of the orbit (cribra orbitalia) treatment diabetic neuropathy diamox 250mg line. Copper is needed in trace amounts to assist in normal cell function and is also a cofactor in enzymes responsible for bone collagen synthesis and crosslinking medicine vs medication buy diamox 250mg overnight delivery. However symptoms gallbladder problems buy online diamox, fluoride incorporated into hydroxyapatite also makes the crystal-and bone-more brittle treatment zoster purchase diamox with amex, causing it to break more easily medicine park oklahoma diamox 250mg, even though it is denser and stronger (recall in Chapter 7 that strength is defined by maximum load but is not necessarily related to fracture resistance). Because of this, fluoride was discarded as a treatment for osteoporosis and replaced with more effective therapies. Nevertheless, fluoridation of drinking water in appropriate amounts has been a significant public health accomplishment as it enhances mineralization of tooth enamel and greatly reduces incidence of dental caries. While these claims are unsubstantiated, a true concern is the risk of fluorosis (fluoride toxicity), which results in discoloration and pitting of tooth enamel and occurs with overfluoridation of drinking water or overingestion of fluoride-containing products such as toothpaste. In addition, overfluoridated drinking water has been associated with bone fractures. Therefore, it is important that drinking water be fluoridated to levels set by the Environmental Protection Agency to reap the benefits of reducing both dental caries and fracture risk. Strontium, boron, and lead are examples of minerals that are not essential nutrients but that can play roles in bone. Strontium can replace calcium ions in hydroxyapatite and also acts to suppress bone resorption. Additionally, strontium in high quantities can lead to osteomalacia, especially in growing bone. Lead can also replace calcium ions in hydroxyapatite, but lead accumulation is toxic and environmental exposure is of concern, especially in children. Lead is sequestered into bone and may lead to impaired bone growth and increased susceptibility to fracture. Lead levels in food additives have been greatly reduced as methods of detection have become more sensitive and Food Chemical Codex guidelines of quality and purity of food ingredients set by the Institute of Medicine have become more restrictive. Lead in the water supply remains of concern in some regions and in older buildings and houses in which lead pipes are used for plumbing. Animal studies that have used highly purified diets to deplete boron levels have been shown to be detrimental to bone, particularly reduced trabecular bone properties and impaired alveolar bone repair. However, the levels of boron in the normal human diet are not likely to compromise bone because boron is quite ubiquitous in the food supply and is only needed in trace amounts. In addition to these minerals, several essential vitamins have been identified for their role in bone health, particularly vitamins C, K, A, and D. Vitamin C functions as a cofactor in enzymes responsible for hydroxylation of lysine and proline residues in bone collagen and is therefore essential for collagen formation and cross-linking in bone. Vitamin C also stimulates alkaline phosphatase production, which is important for bone formation. Vitamin K is a cofactor of vitamin K-dependent gamma-carboxylase, an enzyme required for the -carboxylation (activation) of osteocalcin produced by osteoblasts and involved in bone formation and mineralization. The nuclear receptors for vitamin A have been identified in both osteoblasts and osteoclasts, thus implicating vitamin A action in bone remodeling. Epidemiological evidence points toward a U-shaped risk relationship between vitamin A intake and bone, with the most beneficial intake being around the current recommended intake level (Table 14. Vitamin D and mineral homeostasis is discussed in greater detail in Chapter 13, and the effects of 1,25-dihydroxyvitamin D on bone cells are discussed in Chapter 15. Protein is an essential macronutrient, which is important for bone health as a part of the organic bone collagen matrix. Low dietary protein intakes have been associated with fractures in older adults, whereas high-protein + high-calcium diets are associated with reduced hip fractures. But, high protein intake is also associated with higher urinary calcium excretion, purportedly due to increased metabolic acid load from sulfur-containing amino acids, and has therefore been tagged as a risk factor for osteoporosis. However, urine calcium can increase not only as a result of higher bone resorption or lower renal calcium reabsorption but also from greater renal-filtered load from increased intestinal calcium absorption. Indeed, calcium isotope tracer studies in postmenopausal women have shown that high-protein + adequate-calcium diets increase intestinal calcium absorption, which accounts for the increase in urine calcium observed, rather than calcium loss from bone (see section below, "Perturbing Calcium Metabolism"). However, excessive protein intake in the context of inadequate calcium intake can increase urine calcium losses without a substantial offset by increased calcium absorption, resulting in bone loss. Recent evidence shows there is an interaction between calcium and protein intake such that high protein intake is associated with increased hip fracture when calcium intake is low but is protective when calcium intake is adequate. Additionally, adequate protein intake helps in muscle growth and maintenance, thus indirectly influencing bone through muscle­bone interactions. Periods of rapid bone turnover occur during life stages associated with significant hormonal changes such as the pubertal growth spurt, pregnancy (and lactation), and menopause. During growth, bone formation exceeds resorption, resulting in bone accumulation, whereas in later life, bone resorption often exceeds formation, resulting in bone loss. Adequate nutrition may have the greatest impact during these periods of rapid bone turnover, and a variety of nutrient inadequacies early in life can compromise the achievement of peak height and bone mass, and exacerbate bone loss with aging, menopause, or lactation. For example, protein-calorie malnutrition and mineral deficiencies such as calcium or zinc can lead to growth stunting; vitamin D or calcium deficiency can lead to rickets in children; and calcium deficiency can worsen age-related bone loss. However, supplementing with only the apparently deficient nutrients may be an overly simplistic approach, as a nutrient deficiency may actually be reflective of broader inadequate nutrition/malnutrition. On the other hand, the influence of hormonal changes occurring in a particular life stage may overwhelm any effects of nutrition on bone. For example, calcium supplementation does not prevent the rapid bone loss associated with menopause, but it can help slow the more gradual bone loss that occurs 5­10 years postmenopause. Another example is during lactation when adequate nutrition is generally helpful for retaining bone mass, but even supplementation with calcium and vitamin D does not prevent the bone loss that occurs during this time under powerful hormonal control. Instead, the postweaning hormonal changes increase net bone formation until prepregnancy bone mass (or greater) in the mother is achieved. Thus, despite loss of bone during lactation, the compensation that occurs post weaning results in overall beneficial effects of lactation on bone. So, while supplementation with individual nutrients can have positive effects on bone mass or reduced rates of fractures, the whole diet should be considered (see below). Absorbed calcium can reenter the gut (endogenous secretion), be excreted via the kidney into the urine, or transferred to bone. As bone is always remodeling (except in cases of adynamic bone), some calcium is always leaving and entering bone. Measurement of tracers in blood, urine, and stools over time can be used in a computer software program to determine all of the rates of transfer indicated. Improving calcium and vitamin D status are associated with increasing calcium absorption, but only if intakes are suboptimal before supplementation. If already adequate, these nutrients are not limiting and do not further increase calcium absorption efficiency. It is also important to note that there is a limit to the extent that vitamin D supplementation can compensate for low calcium intakes. When calcium intake is low, improving vitamin D status can increase calcium absorption efficiency by increasing the calcium transport proteins involved in transcellular transport (See Chapter 13). However, while calcium absorption efficiency (% absorbed) may be increased by improving vitamin D status, the absolute amount of calcium absorbed will continue to be low if the total amount of calcium consumed is low. Conversely, high calcium intakes can prevent the adverse effects on bone metabolism and mass that occur from vitamin D deficiency, as has been demonstrated in vitamin D receptor knockout mice fed high-calcium diets. The absolute calcium absorbed increases with increasing calcium intake, predominantly by the paracellular pathway, and excess is then excreted into the urine, thus controlling increases in net calcium retention. Some compounds bind calcium and form insoluble salts, thereby reducing intestinal calcium absorption and increasing calcium excretion in the feces. Oxalates, found in many plants including rhubarb, spinach, and legumes, are the strongest inhibitors of calcium absorption in the diet. Phosphates, such as those used in many processed foods, also inhibit calcium absorption. Phytate, the storage form of phosphorus in seeds, also forms insoluble salts with cations such as calcium and magnesium, but they bind more strongly to zinc and iron. These cations can be freed from the negatively charged phosphate groups on phytates by fermentation, such as occurs during leavening, from hydrolysis by phytases present in yeast. The adverse effects of caffeine consumption on bone are associated with a small increase in urinary calcium loss, easily offset by 5­10 mLs of milk. Adverse effects during growth are usually related to displacement of milk by caffeinated beverages. The Food and Drug Administration recently commissioned a systematic review of the adverse effects of caffeine consumption across the lifespan including bone outcomes. The conclusion was that the comparator of 400 mg caffeine/ day was not associated with adverse events. Green arrows indicate a stimulatory effect; red stop-lines indicate an inhibitory effect. Unabsorbed calcium is excreted in the feces and absorbed calcium from the gut enters the exchangeable pool. From there it can reenter the gut with endogenous secretions, be excreted in the urine via the kidney, or enter bone mineral during bone formation. Dietary intakes of some nutrients can alter the transport efficiency of some of these pathways, as indicated and described in the text. Dietary potassium can decrease calcium lost in the urine and improves calcium balance when sufficiently increased to levels approaching the recommended intakes of 4700 mg/day. In contrast, sodium increases calcium excretion via the kidney and reduces calcium retention. As the kidneys excrete excess sodium, calcium is also lost into the urine by solvent drag. Dietary protein also has a calciuric effect, but this can be offset by a concurrent increase in calcium absorption from increased protein intake. Modeling calcium movement (using stable or radioactive calcium tracers) into bone assesses bone formation rate, and movement of calcium out of bone reflects bone resorption rate. Thus, net calcium balance reflects net bone balance and is the difference between bone formation rate and bone resorption rate. Net calcium retention can be determined by metabolic balance studies, in which diets are controlled and intake and excretion of calcium are measured. Transfer of calcium movement in the body can be assessed using calcium isotopic tracers. Oral and intravenous calcium tracers are measured in blood, excreta, and sometimes saliva to estimate calcium absorption and excretion. Use of kinetic modeling programs allows a determination of transfer rates of calcium to and from different body "pools". On the same dietary calcium intakes, black adolescents retain higher amounts of calcium than do whites. This is attributable to both higher intestinal calcium absorption and lower urinary calcium excretion in blacks. Accordingly, fracture rates are lower in blacks than in whites, and a difference in calcium handling at the time of peak bone mass accrual may be responsible, at least in part. Asians also have higher calcium retention than do whites on the same calcium intakes, due to higher intestinal calcium absorption efficiency and lower urinary calcium excretion. However, calcium intakes among many Asians are so low that calcium retention, and consequently peak bone mass, is far below the genetic potential. Despite lower peak bone mass, Asians have a lower risk of hip fracture, partly related to a shorter femoral neck, which provides a more favorable hip geometry for fracture resistance by reducing bending stresses, especially during a fall. Differences in calcium retention between adolescent boys and girls have also been observed, with boys having higher calcium retention resulting from higher intestinal calcium absorption and lower urinary calcium excretion. For example, human milk is the perfect single food for nourishing term infants during the first few months of life. Nutrient requirements for infancy are based on the composition of human milk, and infant formulas are made generally to emulate the composition of human milk. Newer research is probing the oligosaccharides in milk for their unique biological roles such as serving as a substrate that establishes the dominant gut microbiota. Human milk must be augmented after 6 months to meet the energy, iron, and fiber needs of the growing child. It has become the standard of care for physicians to recommend calcium supplements to patients with osteopenia and for subjects in randomized control trials to receive calcium supplementation in both the placebo and treatment arms. However, calcium supplements are not necessary to meet calcium requirements if the Dietary Guidelines for Americans of three cups of low-fat milk/day are consumed. This recommendation is based not only on meeting calcium requirements but also for helping to meet requirements for vitamin D (when fortified), magnesium, potassium, protein, and riboflavin. A calcium supplement may have as much bioavailable calcium as a serving of milk, but it does not have all of the nutrients known to be essential to bone health. Vitamin D is sometimes added to calcium supplements to improve calcium absorption, which can improve status for absorbing future calcium but not for coingested calcium. Many calciumfortified foods are available in the marketplace, and this has helped to close the gap between intakes and requirements. Some calcium-fortified foods, including orange juice, soy beverages, and tofu, which contain as much calcium as a glass of milk, have been tested for comparable calcium bioavailability against milk and have a similar profile of the major nutrients resembling milk. Not all nutrients are in a bioavailable form, either from the diet or from nutritional supplements. The choice of form of nutrient used for fortifying foods or in supplements is usually determined by cost, compatibility with the matrix, proportion of nutrient in the compound, and bioavailability. For example, calcium carbonate mined from the ground is the most common calcium salt used for this purpose because of its low cost, high availability, large proportion of calcium salt (which means the least amount of compound required), and comparable absorbability to calcium in milk. However, calcium carbonate has a relatively low solubility in water at neutral pH. Solubility is important in foods and beverages to ensure that the mineral dissolves uniformly.

Transferrin is a 1-globulin responsible for transporting Fe3+ throughout the circulation medicine 5000 increase generic diamox 250mg with visa, with the liver and bone marrow as ultimate destination targets symptoms 0f parkinsons disease order 250mg diamox free shipping. As a component of cytochromes and non-heme Fe proteins medications list diamox 250 mg buy line, it is required for oxidative phosphorylation reactions treatment 02 bournemouth buy cheapest diamox. Myeloperoxidase medications keppra diamox 250 mg order on line, a lysosomal enzyme, requires Fe for proper phagocytosis and oxidation of bacteria by neutrophils. The mechanism of Fe toxicity is linked to its ability to exist in different oxidation states and its key role of participation in several redox reactions. Hydrogen peroxide oxidizes reduced form of iron (ferrous) to the oxidized form (ferric), thus generating hydroxy radicals. Oxidative damage is attributed to the ability of iron to participate in the Fenton reaction in its ferrous form and the Haber­Weiss cycle in its ferric form, both of which lead to oxidative stress within the cell. Once transferrin is saturated with bound Fe, the unbound freely circulating Fe in blood is redistributed and deposited in other target organs, resulting in additional oxidative damage. Furthermore, excess Fe deposited in cells induces intracellular changes, forcing the cells to release H+ ions, whose ultimate destination is participation in the formation of life-threatening metabolic acidosis. Organ and cell damage arising from chronic Fe overload affects the liver, the heart, and pancreatic -cells. Hemosiderosis is a rare condition related to excess Fe intake or improper Fe metabolism. The reason for large numbers of premature deaths in individuals with hematochromatosis, the hereditary form of hemosiderosis, is the development of complications from hepatic cirrhosis and hepatoma. In addition, iron accumulation within cellular lysosomal compartments sensitizes the organelles to loss of integrity and rupture. The release of lysosomal enzymes into the cytoplasm of cells induces autophagocytosis, apoptosis, or necrosis. Physiologically, net Fe loss is less than 1 mg/day, but its absorption is usually poor. It is generally present in red meat products and dark green leafy vegetables in the ferric form bound to proteins and organic acids. While only 10% of iron ingested in the diet is absorbed, severe deficiency increases absorption to about 30%. Under the influence of apoferritin in the intestinal wall, Fe2+ is transformed back to its oxidized state, Fe3+ moiety, and eventually enters the plasma. Transferrin transports ferric iron to the liver, where it is bound to ferritin and hemosiderin. From these storage depots, iron is transported out as needed via transferrin to the bone marrow to produce hemoglobin and myoglobin and to other tissues for incorporation into cytochromes and non-heme iron. This stage is relatively asymptomatic, and patients either recover or progress to Stage 3. For patients who progress to the third stage of Fe toxicity, major organ collapse occurs progressively without any observable signs and symptoms. The third stage, or shock stage, is the result of prolonged acute systemic toxicity resulting in extensive vasodilation, poor cardiac output, and metabolic acidosis. Cardiogenic shock usually occurs 26 to 48 hours after ingestion and represents a depressant effect of Fe on myocardial cells. It typically occurs 2­3 days after ingestion of high levels of Fe and is a consequence of extensive oxidative damage to the liver. Patients present with partial or complete bowel obstruction and formation of Fe concretions as the initial injury to the gut lumen heals following scarring and stenosis. Chronic Fe accumulation has been associated with infection with Yersinia enterocolitica, which requires Fe as its essential growth factor. Metals 423 amine is an Fe chelator and is the treatment of choice for acute Fe overload. In patients suspected with signs and symptoms of contamination, cephalosporin or fluoroquinolone classes of antibiotics are the treatment of choice along with adequate fluid and electrolyte replenishment. Serum ferritin correlates directly with Fe stores, but it can also be elevated in liver disease, inflammatory conditions, and malignant neoplasms. Excessive Fe stores are calculated using histological Fe stains of bone marrow and liver biopsies. The technique is primarily performed to determine the amount of available Fe for erythropoiesis. At normal atmospheric pressure, Pb has a melting point of 327°C and a boiling point of 1620°C. It is seldom found alone, and its compounds are widely distributed throughout the world. The main use of Pb is in the fabrication of storage batteries and in sheathing electric cables. It is also useful as protective shielding from common X-rays and from radiation emitted from nuclear reactors. Pb compounds are frequently used as pigments in paint, putty, and ceramic and as insecticides. For decades, Pb was incorporated in gasoline as an "antiknock" agent, until it was banned as an environmental pollutant in the United States in the 1970s. Pb is incorporated into Ca2+-selective molecular structures and mimics its action, interfering with vital proteins. Thus, it is capable of binding to sulfhydryl, amine, phosphate, and carboxyl groups. Consequently, Pb increases intracellular levels of Ca2+ in brain capillaries, neurons, hepatocytes, and arteries, which triggers smooth muscle contraction, thereby inducing hypertension. The effects of Pb on blood formation and heme biosynthesis have been extensively documented. The final heme structure is composed of four N-containing pyrrole rings linked by methylene bridges. After the incorporation of Fe2+, four heme molecules are positioned, one into each of four polypeptide chains, two and two, of the globin molecule in hemoglobin. The effects of Pb on heme synthesis also impact skeletal, renal, and neurological functions. In bone, Pb alters circulating levels of 1,25-dihydroxyvitamin D, affecting Ca homeostasis and osteocyte function. Pb deposits in renal nephrons, eventually revealing itself in glomerulonephritis and glomerular atrophy. In the nervous system, Pb substitutes for Ca2+ as a secondary messenger in neurons, blocking voltagegated Ca2+ channels, inhibiting influx of Ca2+, and obstructing subsequent release of neurotransmitter. Pb inhibits glutamate uptake and glutamate synthetase activity in astroglia, thus inhibiting the regeneration of glutamate, a major excitatory neurotransmitter. Immediately following ingestion, Pb is distributed widely to plasma and soft tissue and redistributes and accumulates in bone. Freely circulating Pb is uncommon; thus, the measurement of blood Pb levels is often poor or inaccurate. In children, Fe deficiency correlates with higher blood Pb levels, suggesting that Fe may affect Pb absorption. Redistribution to Ca2+-containing structures explains the high Pb concentrations found in bone. Interestingly, Pb does not distribute characteristically in bone but will accumulate in the form of lead phosphate in those regions undergoing active calcification at the time of exposure. Inorganic Pb is not metabolized or biotransformed but forms complexes with a variety of protein and nonprotein ligands. Organic Pb is metabolized in the liver by an oxidative dealkylation reaction catalyzed by cytochrome P450. The latter is accompanied by nausea, vomiting, and bloody black stools, with the patient complaining of an oral metallic taste. Headache, confusion, stupor, coma, seizures, and optic neuritis are all manifestations of Pb neurotoxicity. Although initial signs and symptoms are subtle, the child complains of fatigue, muscular weakness, and incoordination. Blue-gray pigmentation of the gingiva ("Burtonian lines" or "lead lines") is also a diagnostic sign. Pb encephalopathy, fatal in 25% of untreated or poorly treated patients, describes the cerebral and neuromuscular effects of chronic lead intoxication and is characterized by irritation, memory loss, and ataxia, followed by delirium, convulsions, and coma. Demyelination of the radial nerve of the forearm typifies lead palsy, demonstrated by wrist drop,* while demyelination of the femoral nerve results in foot drop. Penicillamine has been used as an oral chelating agent for * An inability to freely flex the hand­wrist with a normal hinge reflex movement. In patients with kidney impairment, dimercaprol is recommended, since excretion is primarily in bile rather than urine. Patients diagnosed with elevated Pb concentrations should be relocated from the worksite into well-ventilated surroundings. It is important to note that eating and drinking are prohibited inside Pb-exposed worksites. The treatment protocol for symptomatic patients is similar to that for acute poisoning (see earlier). For two decades, this assay was used to determine the accumulation of protoporphyrin in erythrocytes. The assay, however, proved to be insensitive to Pb levels in the 10­25 g/dl range; it is not recommended for tracking childhood Pb exposure. The evolution of more sensitive testing techniques has made measuring Pb in blood more feasible. As Pb reallocates to bone after ingestion, the use of radiographic techniques also occasionally proves useful for its detection. It is volatile at room temperature and has a low enough vapor pressure to vaporize. Hg compounds are considered major pollutants of the biosphere, with organic mercurials being the most toxic. The chemical properties, characteristics, and pathophysiologic consequences of the different forms of Hg are summarized in Table 26. This natural source of contamination is estimated at 25,000 to 150,000 tons of Hg per year, which binds to organic or inorganic particles and to sediment that has a high sulfur content. Since 1973, approximately 5000­10,000 tons of Hg per year has been discharged from burning coal, natural gas, and the refining of petroleum products, with one-third of the atmospheric Hg due to industrial releases. The element is used in a number of products, including thermometers, barometers, electrical apparatus, paints, and pharmaceuticals. Regardless of the source, both organic and inorganic Hg undergoes environmental transformation. The conversion of inorganic Hg to methyl Hg results in its release Metals 429 from sediment at a relatively fast rate and leads to its wider distribution. With the latter, the absorption of methyl Hg is faster than inorganic Hg, and its clearance rate is slower, resulting in high methyl Hg concentrations. This is of biological as well as public health interest, since fish enter the food chain. Pollution of the environment with Hg compounds has resulted in an increased level of neurotoxicity, referred to as Minamata disease, named after a historic outbreak of Hg poisoning in Japan. Two poisonings, one in Minamata Bay (1956­1975) and one in Niigata (1964), occurred as a consequence of industrial releases of Hg compounds into Minamata Bay and the Agano River. An additional outbreak occurred in Iraq (1971­1972), which resulted from eating bread made from seed grain coated with a methyl Hg fungicide. Most human exposure to Hg is via inhalation, since the highly lipid-soluble compound readily diffuses across the alveolar membrane. Inactivation of various enzymes and structural proteins and alterations of cell membrane permeability contribute to severe toxic effects. The absorption rate for inorganic mercuric salts varies greatly and is dependent largely on the chemical form. Although all forms of Hg have the potential to accumulate in the kidney, inorganic mercuries have a propensity for kidney accumulation. Because of the high lipophilicity of metallic Hg, transfer through the placenta and the blood­brain barrier is thorough. Inorganic Hg compounds have lower lipophilicity, and although there is distribution to most organs, penetration through biomembranes is not as efficient. Metallic Hg is oxidized to the divalent form by the catalase pathway; the divalent ion is reduced to the metallic form. Inorganic Hg is eliminated in urine and feces, while organic Hg is eliminated primarily in the feces. Inorganic Hg is preferentially excreted via the feces and subsequently through the renal route. The five syndromes resulting from Hg intoxication are described accordingly in Table 26. Accidental acute exposure to high concentrations of metallic Hg vapor has resulted in human fatalities. The most commonly reported symptoms of acute inhalation exposure are cough, dyspnea, tightness and burning pain in the chest. Renal effects resulting in proteinuria, hematuria, and oliguria have been demonstrated, and severe neurotoxic reactions are noted as behavioral, motor, and sensory disruptions. A latency period of weeks or months is characterized from the time of exposure until the development of symptoms (onset). Some pathological features include degeneration and necrosis of neurons in focal areas of the occipital cortex and in the granular layer of the cerebellum.

When flaming the loop symptoms zenkers diverticulum 250mg diamox purchase with visa, you will have put the agar plate back on the bench with the lid on medications i can take while pregnant cheap diamox express. If you go over the same streaks symptoms after conception buy diamox 250mg low cost, you will end up with growth in one place and no growth in another symptoms 5-6 weeks pregnant diamox 250mg purchase amex. Try tilting the plate in the light; this can often help you see where the last streak was symptoms rsv proven 250 mg diamox. If you do not move the loop through the end of the previous streaks after sterilising it, you will end up with no growth on those areas of the plate. You can minimise the chances of losing your place by lining up the initial inoculum with the writing on the bottom of the plate when you start the process. You can also try tilting the plate in the light, and sometimes you can see where you have run the loop along the surface of the agar. If you forget to sterilise the loop, you will not be diluting the bacteria and will end up with strong growth on all streaks and will not get single colonies. To help with this, a range of special media were designed that can differentiate, select, or enrich populations of bacteria. These media, like the ones discussed previously, contain all nutrients needed to grow bacteria, but they will also have added components that allow you to isolate or identify specific bacteria or groups of them. Selective media can be useful, especially if you are trying to isolate one group of bacteria from another. This is normally achieved when the media contain a substance that will support the growth of one type of bacteria but inhibits the growth of other types. You might use this if, for example, you were trying to isolate all staphylococcal species from a skin swab but do not want gram-negative bacteria to grow. Differential media, in contrast, allow more than one type of bacteria to grow but help you to distinguish between two or more groups of bacteria based on their biochemical capabilities, usually through a colour change in the media of bacterial colonies. The selective part of this agar is the high level of salt, which will allow the growth of staphylococci but will inhibit most other bacteria from growing. The differential part of this medium is the phenol red, which will change colour depending on the pH of the medium, which is altered in different ways by different bacteria. If you wanted to check whether you had Staphylococcus aureus or Staphylococcus epidermidis within that sample, you could use this medium to answer the question. The plate on the left shows the typical yellow colouration produced after incubation with S. MacConkey agar is another example of a selective and differential medium that is commonly used. Selective parts of the agar are the bile salts and crystal violet, which allow the growth of enteric organisms but inhibit the growth of gram-positive bacteria. The differential part of the agar is lactose coupled with a pH indicator called neutral red. Bacteria that can ferment lactose, but not as strongly, will cause a pH drop in the agar, turning the bacterial colonies pink-red, but will not cause the agar surrounding the bacteria to change colour. Bacteria that cannot metabolise lactose, such as Salmonella spp or Pseudomonas aeruginosa, will still be able to grow on the agar and will use the peptone available in the agar. If you are trying to isolate and differentiate between lactose fermenting and non-lactose fermenting enteric bacteria, this would be an agar worth considering in your work. The plate on the left shows typical growth on the plate after incubation with non-lactose fermenting P. These are organisms that do not ferment lactose and are normally not faecal coliforms. Lots of chromogenic agars are available on the market that will differentiate among bacteria by using chromogenic substrates that are activated when those substrates are cleaved by specific enzymes, so it is worth looking around if you are doing experiments that require isolating and growing specific bacteria. These media will come with instructions that help you 36 Bacteriology Methods for the Study of Infectious Diseases understand what the different colours of colony mean. Media have also been designed to enrich the growth of bacteria by providing a rich source of nutrients, which might be helpful if you are trying to culture a mixed sample of bacteria or a single type that is fastidious, such as Haemophilus influenzae, which will not grow on non-enriched media. Commonly used enriched media include, among others, both blood and chocolate agar (often horse, sheep or rabbit blood). These contain nutrients to support many types of bacteria and blood and heat-treated (lysed) blood, respectively. You can buy pre-poured agar plates that already contain the blood, or you can buy the blood and add it to the base media yourself. With both the premade plates and the blood, it is important to note the expiration date and storage conditions. If you buy it in powdered form and make it up yourself rather than buying premade plates or broth, it is also important to check how to store not only the original product (often at room temperature) but also the plates and broths once they are made up. If you purchase premade plates, check the storage conditions and shelf life for those products (often fridge storage). In general bacteriology, enumeration is frequently used and is particularly important when investigating the number of bacteria found in water or food samples. It is also commonly employed in other strands of research for many purposes from standardising the starting inoculum to determining how many bacteria in a population have survived a novel antimicrobial treatment. When turbidity is dense enough to be detected by eye, this phenomenon is Bacterial growth in solid and liquid media 37 caused by light scattering off cells present in the media. You cannot tell by eye how many cells are in the population, but you can use a spectrophotometer to estimate the cell number or to follow the growth pattern of the culture over time. Spectrophotometry is often used in research, especially before inoculating an experiment, to ensure that every experiment has the same number of cells inoculated into it at the beginning. This standardisation of the starting culture allows you to minimise the effect of the starting inoculum on the results. Before starting an experiment that requires a spectrophotometer, make sure your laboratory has one and that you have cuvettes that fit the spectrophotometer that is in stock. If your laboratory does not already have cuvettes in stock, you can get both macro- and microcuvettes (which can be reusable or disposable). Once you have checked that you have the right equipment, you can use the spectrophotometer to set up lots of experiments, including basic growth curves and antimicrobial susceptibility assays, or to establish which in media your bacteria grows best. For example, if using the spectrophotometer to normalise cell numbers at the beginning of an experiment, you might follow a procedure like the one described next. The following day, from your now turbid culture, take 1 mL of the overnight culture and inoculate a fresh 10-mL nutrient broth and incubate it for a couple of hours at 37°C so that the cells reach log phase growth before you start working with them. Turn on the spectrophotometer at the wall and make sure it is switched on at the machine if there is another button there. Remove your culture from the incubator, pipette a portion of the cells into the cuvette, make sure you know what volume your cuvette holds and ensure when pipetting that you do not over fill the cuvette. Do not forget to keep working aseptically and place the lid back on the cells you are not putting into the cuvette. It is important when handling the cuvette that you do not accidently get material on the part of the cuvette through which light will pass. Because light passes through the cuvette in this orientation, you should try to keep this area as clean as possible so no dirt will alter the results. Most cuvettes have a mark on the top that indicates which way round in the spectrophotometer it will go. You can also wear gloves to prevent fingerprints from transferring onto the cuvette. In addition to a cuvette containing the cells, you will need a second cuvette that contains a control (blank) liquid. In this example, it would be nutrient broth with no cells, but you should use whatever media you are growing your cells in. Once both cuvettes have the correct volume of liquid, you can measure the turbidity of your cells. You will need to select the wavelength of light that you are using on the spectrophotometer. Some spectrophotometers read only at set wavelengths, so check what these are before you use the machine. If you are working with bacteria, 600 nm is commonly used and could be taken as your starting point if this technique has not been employed before in your laboratory. Other wavelengths are used fairly commonly with unpigmented bacteria, such as 480, 540 and 660 nm, so it is worth checking whether your machine is set to one of these or whether the bacteria with which you are working produce pigment that might require a specific wavelength. Normally, you close the lid of the spectrophotometer and then select zero on the machine. You can then place the cuvette containing the bacterial culture into the machine, making sure the cuvette is in the correct orientation, and close the lid. Record that number in your laboratory notebook and do not forget to remove the cuvette from the machine and throw it away once you have the reading. If you are testing lots of samples, it is a good idea to re-blank the machine between each sample to be sure you obtain accurate readings. If you are doing something like a growth curve, you take the reading at each time point and plot it on a graph. This way, even if you need to carry out the experiment again months later, you can make sure that the starting inoculum is similar. Machines that work with these plates are designed to read multiple wells at the same time and can be used to take end point readings of cell growth in much the same way as the cuvette spectrophotometer (so you could read at 0 and 20 h). Some can be used to hold a plate for a period of time (so you could add the plate to the machines and set it to read every 30 min for 20 h). In this case, inoculate the plate with the bacterial culture (checking the initial inoculum using the spectrophotometer, as discussed earlier). Then, insert the microtitre plate into the spectrophotometer, It is a good idea to take a reading of the plate at this point (a 0-h reading) because this can be subtracted from any readings you take later, which allows variations in initial turbidity or the colour of the broth to be removed from the growth curve. These microplate spectrophotometers often have heating and shaking capabilities, so you can tailor the incubation to your needs. Some spectrophotometers will also allow you to select how often and where in the well you take your readings from. They can be useful for generating growth data, especially if you are looking at the effect of a compound on the bacteria, or at how different mutations have affected growth. For example, Row 1 could be your negative control (growth media, no bacteria), Row 2 could have an inhibitor, Row 3 could have a supplement and so on. Sometimes the spectrophotometer can be used to look at end point readings of stained bacteria. For instance, if you were measuring biofilm growth (details are provided in Chapter 6) using crystal violet, the wavelength is set to 570 nm. Rows A­H can hold replicates of the sample whereas Columns 1­12 contain both negative and positive controls as well as a different test condition in each column from 2 to 11. Using the spectrophotometer to read the plate at various time points, you can see when the inhibitor has an impact on cell growth. You can buy McFarland standards at different levels of turbidity that approximate different numbers of bacteria. To test the culture, hold the culture tubes up alongside the McFarland standard against a white background with black lines to check turbidity. The culture should be as turbid as the McFarland standard to have the number of bacteria approximated by the McFarland standard in it. If you want to check how accurate the enumeration of the culture is and do not have access to a spectrophotometer, you can plate out some of the culture as a total viable cell count (see direct enumeration, discussed subsequently) just before or after setting up the experiment. This way, you can incubate the cell count plate overnight and check the cfu per millilitre of the sample the next day to confirm that you started with the right number of cells. You will probably use both the spectrophotometer and the microplate spectrophotometer a lot when conducting experiments, because they provide quick and easy ways to check that you are using a reproducible number of cells in your experiments. However, the two methods mentioned earlier do not provide a direct count of bacterial cell numbers and only estimate the numbers of cells in the culture. You may encounter some issues when using them, depending on the type of bacteria and the experiment you are performing. When looking at turbidity, either by eye or with a spectrophotometer, you cannot tell whether the turbidity you can see is being generated by live or dead cells, which can lead to an overestimation of numbers if there are many dead cells in the culture. The spectrophotometer readings could also be misleading if Bacterial growth in solid and liquid media 43 the cells have produced extracellular material that can also scatter light and may lead to an overestimation of cell numbers. Some bacteria clump; this can lead to odd readings when using the spectrophotometer. For example, if you want to plot a growth curve but you want a count of viable cells in the culture rather than an estimation from the spectrophotometer, you can use a viable cell count method. This technique is based on the Miles and Misra method first described in 1938 and uses a serial dilution technique. The methods allows you to dilute the bacteria, enabling you to obtain a viable count. This is important because bacterial growth is rapid, and if you are counting bacteria from an overnight incubation or even at multiple points for a growth curve, soon there will be too many bacteria to count accurately if they are plated undiluted onto agar. You need to make sure you have ready everything you need and that you work aseptically when carrying out the viable count. It is worth checking that the agar plates are fully dry before you start this work, because if they are wet it can affect the results and delay work. It is also a good idea to warm the plates to room temperature before you plate the cultures onto them. To start, take the bacterial culture (working a blue Bunsen flame) and remove 1 mL of the culture. Mix well (this is important; if you do not mix well, the results will be inaccurate). If using a vortex or mixing by hand, try not to get the culture in the lid of the culture vessel; otherwise, you might get it on your hand or drip it on the bench when you remove the lid. Bacteria are diluted from the neat overnight culture (far left) down to a 10-7 dilution (far right) by pipetting 1 mL of bacteria from each tube into the next. It is a good idea to prepare duplicate plates to ensure you get reproducible cell counts. It also means that if you contaminate a plate or have a wet plate that does not grow the bacteria properly, you might still get a usable count from the other plate.

The Lubbock method also allows media to be changed for longterm growth experiments medications prednisone cheap diamox 250 mg buy line, and the position of the biofilm enables topical treatments such and antimicrobial dressings to be applied medications during labor order 250mg diamox otc. Thus treatment pancreatitis buy cheap diamox 250 mg line, essentially medicine 360 250mg diamox buy with mastercard, you could use a nutrient agar plate as a starting point and perhaps choose different media or nutrients for later experiments to investigate how this might affect biofilm growth medicine 8 pill diamox 250mg purchase on line. This method is best-suited to study established biofilm rather than early biofilm formation, and so it is advisable to allow the biofilm to grow for a minimum of 48 h in the first instance. Fresh media can be introduced to the system by moving the filter on which the biofilm is growing onto a fresh agar plate. It is possible in this manner to change the media or nutrients on which the biofilm is growing, if this is an aspect of biofilm growth in which you are interested. This is important because most environments in which bacterial biofilms are found are not static, even within the human body. Many are available commercially, but the costs can be prohibitive; others can be constructed in part by the user. Consider whether your studies would benefit from using a flow device, and whether you have any of these items at your disposal during your research project. Biofilm flow devices work in a variety of ways that might make them more or less relevant to your research. Each peg can be removed without disrupting the flow of media or the ongoing experiment, and several biofilms can be cultured under the same conditions simultaneously. The device can be set up and an experiment conducted for long periods of time providing there are sufficient media to flow through the system (more fresh media can be added as the experiment progresses). With regard to specific projects, it is important to consider what factors would make this a useful tool. Biofilms are submerged by the media at all times, and the flow can be varied depending on the environment you are trying to mimic. It is possible to look at a time course for biofilm development, but it is not easy to include treatments that cannot be incorporated into the media that are feeding the biofilms. Thus, two separate experiments would need to be performed to test whether a disinfectant impaired biofilm formation, for example. Flow systems are also available that allow bacteria to be grown at an air­ liquid interface rather than being submerged. These might better represent oral or wound biofilms, compared with those of the gastrointestinal tract, for example. This allows multiple biofilms to be grown on coupons that are located on a central rotating disk. It is possible to remove the coupons from the device as the experiment progresses, to analyse the biofilm composition and structure. Many studies have produced excellent confocal images of complex bacterial biofilms cultured this way. Drip-flow reactors can also be used to culture biofilms at an air­liquid interface. Drip-flow reactors are available commercially and can be purchased or they can be constructed in-house by the user. Waste media flow out of the device by gravity, owing to the angle at which the biofilm is grown. Devices can be used to culture single or multiple biofilms, so that it is possible to prepare replicates. Where separate reactors are present in one device, it is possible to remove biofilms at given time points. Like previous models, these devices are good for studying established biofilms, and once cultured, biofilms can be removed and treated with antimicrobial agents: for example, by immersion into a solution or by applying a topical treatment. This will ascertain how tolerant an established biofilm is to any given antimicrobial intervention. Smaller flow cells are available that are designed for real-time visualisation of biofilm development by microscopy. These types of flow cells are available commercially or they can be constructed by the user; however, this requires some degree of technical expertise (see Tolker-Nielsen and Sternberg, 2011 for how to construct a flow cell). Each flow cell has three channels, which means that three identical biofilms can be simultaneously cultured, each submerged in the flowing media. Again, consider the biofilm model you need relevant to the organism and system you are trying to mimic. If you gather information about biofilm development using a flow cell, the next experiment might be to flow through a compound with disinfectant properties to see how effective it is at removing established biofilm, and how quickly. Like other flow devices, media are pumped through the system using a peristaltic pump via an inlet and outlet port. The agar plugs are permeable to nutrients, and so the biofilm is fed in this manner. Whereas the device can be fed by only one type of media at any one time, the three separate channels allow for the growth of three different bacteria without risking cross-contamination. Filters can be removed from the device as the experiment progresses without disrupting the flow of the experiment, and it is possible to apply a topical antimicrobial such as a wound dressing to the biofilm. The device can be used to study early stages of biofilm growth and established mature biofilms. Details of this and how to set up and run the system can be found in the following publication: Duckworth et al. When it comes to deciding whether a biofilm flow device is useful for your research, you need to think carefully about whether flow is relevant to your study and what sort of questions you are looking to answer by employing a biofilm flow device. Given the availability, cost and technical expertise required to use some of the available devices, you must also consider whether the equipment is available, whether you will be able to use it effectively and whether you can afford it according to your project budget. There might be equipment available in your place of study, or your project supervisor might have contacts who have access to the equipment you need. This approach allows a high degree of sensitivity and can provide insight into the regulation of bacteria under varying conditions. The equipment and techniques to measure gene expression are widely available and can be adapted to answer many experimental questions. Although it is limited in the sense that changes in gene expression do not always translate into a phenotypic change, this technique provides the researcher with a great deal of useful information. This article offers details on the basics of sample preparation and extraction, methodologies to allow the successful measurement of gene expression and information on how best to analyse the data generated by these techniques. Ideally, the extractions should be performed in an organised and clean fashion, because the contamination of samples could lead to erroneous results, wasting both time and reagents. It is important to have everything in a place and easy to reach to provide a logical workflow and minimise the risk for contamination. In this section, a rapid, cheap and easy method is outlined that involves extraction using Chelex resin. When handling Chelex (for example, when transferring it in aliquots to sterile containers), it is important to keep agitating it. Chelex is dense and if it is not mixed immediately before pipetting, the Chelex will sink to the bottom. It is easy to see this happening if you keep it in a clear-walled container, so check before pipetting. When heating the bacteria, a heating block or waterbath can be used, and if a shake option is available on the heating block, this can be turned on for 15 min incubation. Each step can then be checked off as the process progresses, which reduces the likelihood of a step being missed or accidently repeated. The list for the reaction mixture given here provides an outline of all necessary components that can then be tailored for specific experiments: 1. A working stock of 100 pmol/mL should be aliquoted out and used for experiments to prevent contamination of the original stock. Buffers may be purchased that already contain these; they are often used and are available commercially. Reaction buffer: Usually available as a solution containing the enzyme at a concentrated level, which is diluted upon addition to the reaction mixture. Ensure you read the instructions when purchasing it so that it is correctly diluted and check the contents. Various types are available that can be used depending on whether you are amplifying long or short sequences; a Taq polymerase is a good starting point for most reactions. As with any of these components, it is best to aliquot it if it is going to be used on multiple occasions, to minimise the chance of contamination. Often, when making up the reaction mixture it will be for several reactions, in which case it is worth making up slightly more mixture than is needed on paper, because some of the mixture will be lost through pipetting. When using small volumes, it can be helpful to pipette the liquid onto the back of the container in which the mixture is being made. This can be achieved by capping the tube or plate and gently centrifuging for a few seconds. Reaction mixtures can be bought premade from various suppliers such as Fisher Scientific (fishersci. In the case of well-researched genes, sometimes it is possible to find primer sequences for the gene of interest in the bacteria being used in a published article. In either case, it is important to make sure that primers you design or use are well-designed. To design primers, there needs to be an area within a gene or genes of interest that can be amplified. If the bacteria being researched are fully sequenced, this can make the job of designing primers easy, because it will be possible to check that the sequence chosen for the primers is within the genetic code of the bacteria. If the bacteria being used are not fully sequenced, it is possible to use online databases to give partial sequences for what should be in the bacteria of interest. There are also likely to be fully sequenced bacteria of the same species as the those being used in freely accessible online databases, but it depends on which bacteria are being researched. These online sequences can be used as a template for the primer design, but in both cases where the sequence for the bacteria being investigated is unavailable, the risk is that there may be absent or changed sequences that render primers designed on the basis of another bacterium of the same species ineffective. When designing the primers, it is important to understand what is going to be amplified. It is a short section of sequence within a particular gene, a whole gene or several genes within an operon. For very long sequences (greater than 10 kb), specific enzymes might be required for the reaction, because it can be more difficult to amplify these sequences. When amplifying within a gene, try to make the product larger than 100 base pairs (bp). Then, looking at the available gene sequence for the gene of interest, pick a starting point for the forward and reverse primers. When selecting the region to be amplified, take care not to select an area with long runs of one base or highly repetitive areas. It is also possible to use the filters on the right side of the page to narrow the search to a specific set of parameters such as organism or species. Once the search is complete, it will generate a list of genome sequences, some of which will not be the one of interest but that may contain the information you need. Underneath this should be a list of the specific genes within the genome provided. Generally, the nucleotide code is pasted into a template box on these primer design webpages. The nucleotide code should be entered unbroken and with no annotation into these boxes or it will not work. Within the primer sequences, try to avoid repeats of bases of one type (try for no more than three of any one base in a row) to prevent secondary structures from occurring. Try to avoid more than three bps of intra-primer homology to prevent double-strand structure formation. Finally, avoid homologous regions between primers in a pair to prevent them from binding to one another (primer dimers). The melting temperature of the primers should be as similar as possible, preferably within 2°C of one another. There are two primers in each reaction, and they will need to work under the same reaction conditions. In this example, the location of the gene name has been highlighted in yellow and the gene link that should be selected next to generate the nucleotide code is highlighted in blue. The nucleotide code for the gene of interest has been entered into the template box. Below this are several other parameters that can be altered depending on the needs of the experiment. Gene expression analysis 149 Keep these points in mind when undertaking primer design. Sometimes it is impossible to fulfil all of these conditions when designing primers. At the end of this process, you should have several sequences for primers that could amplify the gene of interest. There are multiple ways of getting to this point and lots of online sites that can help with finding the template nucleotide code and primer design. The ones suggested here may provide a starting point for this process if you have not carried it out before. As suggested, it is also always worth checking published studies to see whether anyone has already designed the primer pairs needed for your reaction. Whichever way the primer sequences are decided upon, they will then need to be ordered. The most important step in this ordering process is ensuring that the primer sequence is entered into the ordering form correctly. Moreover, when the primers arrive, it is good practice to check primer sequences that were delivered before starting to use the primers.

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