Benjamin Holland MD

• Medical Resident, Department of Internal Medicine, University of
• Virginia, Charlottesville, VA, USA

First menstrual yeast infection 0.625 mg premarin with visa, nitrate reagent A (sulfanilic acid) and nitrate reagent B (naphthylamine) are added to test for the reduction of nitrate to nitrite menopause 45 generic premarin 0.625 mg without a prescription. If nitrite is present in the tube women's health group boca raton generic premarin 0.625mg with visa, a red color will develop pregnancy predictor purchase premarin cheap, indicating the reduction of nitrate to nitrite and the presence of nitrate reductase women's health center lattimore road buy premarin. If a red color is not apparent after the addition of nitrate reagents A and B women's health issues-night sweats buy premarin with mastercard, two possibilities exist: either nitrate was not reduced at all, and is still present in the medium, or nitrate was reduced to an end product other than nitrite. To differentiate between these two possibilities, nitrate reagent C (powdered zinc) is added to the medium, which is mixed slightly and allowed to incubate for 5 min. Zinc catalyzes the reduction of nitrate to nitrite and will convert any nitrate in the tube to nitrite, which will in turn produce a red color because of the presence of nitrate reagents A and B. If the medium remains clear after the addition of zinc, then nitrate was reduced to ammonia, hydroxlamine, or a similar nonnitrite compound. After the addition of nitrate reagents A and B to a nitrate reduction test, a red color indicates the presence of a. After incubation, a gas bubble trapped in the Durham tube indicates that denitrification took place, i. If after nitrate reagents A and B are added to the medium a red color appears, then the organism reduced nitrate to nitrite. If the medium remains clear after the addition of nitrate reagents A and B, then zinc is added to catalyze the reduction of nitrate in the tube. A red color at this point indicates that nitrate was not reduced by the organism; if the medium remains clear, then nitrate was reduced to a nonnitrite end product. No red color at this point means that the bacterium has reduced the nitrate to a non-nitrate product, often nitrogen gas. If a nitrate reduction test turns red only after the addition of zinc, the bacterium being studied a. If the tube remains clear, add a small quantity of zinc, mixing gently to disperse the zinc throughout the media. Record the appearance of each of your tubes using the tube outlines provided, being sure to note the point in the analysis of the nitrate broth. What, specifically, does the reagent combination sulfanilic acid (nitrate A) and naphthylamine (nitrate B) detect Explain the reactions you would see in your control tube at each step of the evaluation process. How would you interpret a red color in your control tube after the addition of nitrate reagents A and B Understand the use of the coagulase test to detect the presence of the enzyme coagulase as an aid in the identification of bacterial species. To test for the presence of coagulase, a tube of is inoculated with the bacterium to be tested. A coagulase tube in which the medium has thickened but has not completely solidified represents a. The result of a coagulase test is commonly used to differentiate Staphylococcus aureus from other species of Staphylococcus. Define the following terms using the laboratory exercise or glossary: Fibrin 71 the coagulase test detects whether a particular bacterium produces the enzyme coagulase. The presence of coagulase is commonly used to differentiate Staphylococcus aureus from other species of Staphylococcus. In the body, this thickening of the plasma around bacterial cells provides a great deal of protection, as these cells are then shielded from phagocytosis and other actions of the immune system. The tube is checked hourly for the next several hours and then again 24 h after the initial inoculation. Check the tubes for thickening of the plasma or the formation of long fibrin threads hourly for the next 4 h. Tubes that have remained liquid can be incubated overnight but must be checked within 24 h of their initial inoculation as coagulated tubes may revert to a liquid after 24 h. A solid clot or a loose clot suspended in the plasma is considered coagulase positive. Sketch the appearance of your tubes and indicate for each whether it is coagulase negative or positive. After incubation, would you expect your uninoculated control tube to appear more like the tube containing Staphylococcus aureus or the tube containing Staphylococcus epidermidis After missing a day of lab, you return to examine the coagulase tube you had inoculated two days previously. The tube is completely liquid, but your lab partner says that he looked at it the previous day and the plasma had coagulated. Understand the use of the citrate test to detect if citrate has been utilized and to aid in the identification of bacterial species. Simmons citrate medium, for example, contains citrate as its only source of carbon and ammonium phosphate as its only source of nitrogen. Organisms able to utilize citrate as their sole carbon source and ammonium phosphate as their sole source of nitrogen will produce alkaline products (carbonates and bicarbonates) as products of citrate catabolism. Use a needle to make a single streak up the slant of the medium to inoculate each tube with the appropriate organism. Tubes displaying neither growth nor color change may be reincubated up to a total of seven days. Tube on the left is positive (note both growth and color change) while the tube on the right is negative. It is recommended that Simmons citrate agar be inoculated with a very small amount of bacteria because a heavy inoculation could deposit small amounts of general-purpose medium onto the Simmons citrate agar, causing a false positive result. Understand the use of the malonate test to detect if malonate has been utilized and to aid in the identification of bacterial species. Bacteria that utilize malonate grow in the medium, producing alkaline end products. Bromthymol blue is included as a pH indicator and will turn from green to blue under alkaline conditions, which is interpreted as a positive reaction. This inhibition will result in the death of the cell unless the bacteria being tested can utilize an alternative pathway to metabolize malonate, resulting in continued bacterial growth. In most cases, this type of medium has a defined formula that includes only one form of an essential nutrient. Malonate medium, by contrast, is an undefined medium, containing small amounts of both yeast extract and glucose, meaning several different carbon sources are available. The large amount of malonate in the medium competitively inhibits a key enzyme in the Krebs cycle that catalyzes the conversion of succinate to fumarate. In most organisms, malonate binds to the enzyme succinate dehydrogenase, preventing the succinate to fumarate reaction and thereby halting the Krebs cycle and eventually killing the cell. Malonate utilization produces alkaline end products, driving the pH of the medium higher and causing the bromthymol blue pH indicator to change from green to blue. Yeast extract and glucose in the medium promote the growth of a wide array of organisms, many of which may produce alkaline end products, but these are negated by a phosphate buffering system that resists small changes in the pH of the medium. Organisms that can ferment glucose may produce acid end products, which can be seen as a slight yellowing of the medium. A blue color throughout the medium indicates that malonate can be utilized as the sole source of carbon, a positive reaction (right). A green color indicates that malonate could not be used as the sole source of carbon and is interpreted as a negative reaction (left). What do you think would be the result if a broth was made that contained only 3 mg malonate/L Understand the use of the decarboxylation test to detect the presence of a decarboxylase enzyme to aid in the identification of bacterial species. Also included in the medium are pyridoxal, a cofactor needed by amino acid decarboxylase enzymes; glucose, a fermentable carbohydrate; and bromcresol purple, a pH indicator. After inoculation, the medium is overlaid with sterile mineral oil to prevent the entry of oxygen and is incubated for up to a week. Initially, fermentation of glucose, if it occurs, will produce acid end products, driving the pH downward and causing the medium to take on a yellow appearance. The base decarboxylation medium is supplemented with a single amino acid as a substrate along with a pH indicator, bromcresol purple. Decarboxylation of the amino acid in the medium produces alkaline end products, causing the medium to turn purple, which is interpreted as a positive reaction. Decarboxylase enzymes remove the carboxyl group from an amino acid, producing an amine and carbon dioxide. Regardless of the specific amino acid being decarboxylated, pyridoxyl phosphate is a cofactor in the reaction. What makes the twenty or so amino acids different from one another is the "R" group, with the chemical nature of this group conferring very specific properties upon the amino acid. Carbon dioxide is also produced during the course of the reaction, but it is far more difficult to test for. In the microbiology lab, the three most useful decarboxylation reactions are those involving lysine, ornithine, and arginine. Lysine decarboxylase removes the carboxyl group from lysine, producing the amine cadaverine and carbon dioxide. Ornithine decarboxylase removes the carboxyl group from ornithine, producing the amine putrescine and carbon dioxide. Arginine decarboxylation is a multistep process that eventually produces the amine putrescine and urea. Which gas is produced during decarboxylation of amino acids in decarboxylase media A purple color throughout the medium indicates that the amino acid in the medium was decarboxylated, a positive reaction. A yellow/orange color indicates that decarboxylation did not occur and is interpreted as a negative reaction. For each set of decarboxylase tubes, label one tube with the name of each microorganism, and label the final tube "control. What color would a positive reaction produce if neutral red was used as a pH indicator instead of bromcresol purple Understand the use of the phenylalanine deami nase test to detect the presence of the enzyme phenylalanine deaminase and how this result can be used to aid in the identification of bacterial species. The addition of a ferric chloride-containing reagent to the medium after incubation imparts a deep green color to the medium if deamination has taken place. The phenylalanine deaminase test is commonly used to differentiate members of the family Enterobacteriaceae. Detection of the decarboxylation reaction was discussed in Exercise 74 while recognition of deamination is covered here. Because amino acid degradation is an enzyme-catalyzed event that produces specific end products, the ability of a bacterial species to deaminate a particular amino acid can be used as a clue to its identification. The deamination of phenylalanine is commonly used to differentiate organisms in the laboratory. Phenylalanine agar contains yeast extract for growth and phenylalanine as a substrate for the enzyme phenylalanine deaminase. After incubation, a reagent containing ferric chloride is added to the surface of the medium. Benson phenylpyruvic acid to produce a green color that is interpreted as a positive reaction. The phenylalanine deaminase test is commonly used to differentiate the genera Proteus, Providencia, and Morganella, all of which test positive, from other members of the family Enterobacteriaceae, all of which test negative. After incubation of phenylalanine deaminase agar, what rea gent must be added to the medium to visualize the results A positive result in a phenylalanine deaminase test is recognized by the production of a color. Use a needle to inoculate each tube with the appropriate organism by streaking the surface of the slant twice. Along with phenylpyruvic acid, ammonia is produced as a result of phenylalanine deamination. Could you think of a way in which the production of ammonia could be used as an indication of a positive reaction Understand the use of the bile esculin test to differentiate between bacteria based on their ability to hydrolyze esculin in the presence of bile and to aid in the identification of bacterial species. Blackening of the medium begins in the slant and proceeds downward into the butt of the tube. Medically important organisms that give positive reactions on the 76 Bile esculin agar is a medium with both selective and differential properties that is used to distinguish enterococci and Streptococcus bovis from other streptococci. Oxgall (bile) accounts for the selective properties of the medium, inhibiting the growth of Gramnegative bacteria and most Gram-positive bacteria. Hydrolysis of esculin leads to the formation of a deep brown to black color in the medium, which is indicative of a positive reaction. Esculin is an example of a glycoside in which the sugar glucose is bound to esculetin. Although many organisms can hydrolyze esculin under acidic conditions, only group D streptococci and enterococci can do this when bile is present in the medium. Label one tube with the name of each microorganism, and label the final tube "control. Use a loop to inoculate each tube with the appropriate organism by streaking the surface of the slant. The bile esculin test is particularly useful in the identification of certain groups of a. How would you interpret a bile esculin test in which 70% of the medium has turned black If more than one-half of the tube is darkened, the reaction should be considered positive.

This may be seen as a positive characteristic because the mechanotransduction cues womens health 3 month workout plan purchase online premarin. Occasionally menstruation kidney pain buy cheap premarin 0.625mg line, this is also desired to maintain a dedifferentiated phenotype in cultured cells pregnancy resource center purchase genuine premarin line. For example menopause kills marriages discount premarin 0.625 mg free shipping, polyethylene glycol diacrylate hydrogels (with compressive moduli tuned between 2 and 70 kPa) are bioinert and are thus ideal to encapsulate cancer stem cells [39] women's health clinic in oregon city purchase premarin 0.625mg mastercard. In addition pregnancy x ray 0.625mg premarin free shipping, synthetic polymers have more standardized properties that will not vary from lot to lot because they do not need to be organically harvested. Although Cancer mechanobiology: interaction of biomaterials with cancer cells 465 the mechanical properties will be better quantified and replicated, cells can only be passive reactors when unmodified synthetic polymers are used for mechanotransduction assays [44]. However, it is not a physiologically relevant representation of disease progression, which must allow cells to modify the mechanics of their surroundings. If defined natural polymers are utilized instead, it is important to remember that their configuration will drastically affect the biological activity with the cultured cells. For example, many hydrogels are made from methylating hyaluronic acid to allow a cross-linked network to form. These receptors are often critical determinants of tumor growth and disease progression in situ, which can no longer be recapitulated in vitro despite using the same biopolymer [58]. Similarly, many systems will have hydrogels dosed with soluble fibronectin or adsorb fibronectin onto culturing surfaces. However, surface adsorption and soluble dosing do not expose the cryptic binding domain of functionalized fibronectin, Fn-3 [59]. Therefore although the natural biomaterials may be present in the system, without the correct active form, the cultured cells will not biologically interact with the system in physiologically relevant ways. Lastly, it must be stated that not all natural polymers induce biological changes in cultured cells. Although agarose is a natural marine polysaccharide, it is bioinert to mammalian cells because they are not equipped with the proper receptors for this interaction [60]. Finally, the fabrication of each proposed material into the desired platform must be considered. Each potential functionalization must consider the base polymer and the flow of the experimental design. For example, if an acellular hydrogel is cross-linked, washed, and then seeded with cells, a wide variety of cross-linking agents may be used. However, if a 4D system is desired, the potential cross-linking agent must be carefully considered because many are cytotoxic. If 3D printing is the desired fabrication technique, additional mechanical property requirements will be needed of the material. For example, ideal bioinks are shear thinning so that high resolution features can be printed using large-gauge needles. Conversely, if intricate platforms are to be cast, the material of the mold must allow easy demolding of the device, and the material of the device itself must not swell or shrink drastically when gelled. Similarly, most hydrogels will swell 466 Biomaterials for Cancer Therapeutics when surrounded by the cell-culture media, which may affect the final density and topography of the mechanotransduction platform. Especially when comparing two hydrogels of varied stiffnesses, the change in cross-linking can affect the swelling ratio, adding additional variables into any given experiment. Lastly, the degradation rate of the chosen polymer must be applicable for the culturing duration of a given study. Taken together, the intrinsic mechanical properties, biological properties, and requirements for fabrication together create a comprehensive framework from which potential materials can be evaluated when designing mechanotransduction platforms. This has allowed researchers to study disease progression beyond 2D-based cancer cell genetics. The stiffness, diffusion rate, and overarching physical dimensions of a culturing space drastically affect cancer cell biology, from simple proliferation rates to complex drug sensitivity. However, it is also important to remember that cancer cells originate in situ from healthy tissue. To truly mimic this physiological disease progression, cultured cells must be able to interact with and reorganize an existing microenvironment. In addition, it is important to remember that the mechanical features of a material are interconnected at both the microand macroscopic scales. For example, increasing cross-link densities for a 4D collagen hydrogel will increase its stiffness. However, it will also likely reduce pore size, which will affect the diffusion of biochemical signals and inhibit cell migration. When designing experiments, it is thus important to match the mechanical properties of a system and the cell phenotype with the disease stage of interest. Within the material selection, considering what type of forces are most relevant. The interplay between cancer progression and changing mechanical properties already drives several diagnostic techniques, such as manual palpitation and ultrasound elastography [62]. As this field grows, advanced mechanotransduction-based therapeutics show great promise in cancer treatment. Cancer mechanobiology: interaction of biomaterials with cancer cells 467 References [1] F. Naba, Overview of the matrisome-an inventory of extracellular matrix constituents and functions, Cold Spring Harb. West, Modeling the tumor extracellular matrix: tissue engineering tools repurposed towards new frontiers in cancer biology, J. Ruggiero, the collagen superfamily: from the extracellular matrix to the cell membrane, Pathol. Yurchenco, Basement membranes: cell scaffoldings and signaling platforms, Cold Spring Harb. Weaver, Mechanics, malignancy, and metastasis: the force journey of a tumor cell, Cancer Metastasis Rev. Weinberg, New insights into the mechanisms of epithelialmesenchymal transition and implications for cancer, Nat. Chen, Cancer associated fibroblasts: an essential role in the tumor microenvironment, Oncol. Bai, Molecular characterization of circulating tumor cells-from bench to bedside, Semin. Yang, Forcing through tumor metastasis: the interplay between tissue rigidity and epithelial-mesenchymal transition, Trends Cell Biol. Md Akil, Classification, processing and application of hydrogels: a review, Mater. Cooper, the development and causes of cancer, the Cell: A Molecular Approach, second ed. Lin, Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells, Acta Biomater. Lipke, Polymeric biomaterials for in vitro cancer tissue engineering and drug testing applications, Tissue Eng. Jordan, Targeting hyaluronic acid family for cancer chemoprevention and therapy, Adv. Zia, Glycoproteins functionalized natural and synthetic polymers for prospective biomedical applications: a review, Int. Haier, Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids, Am. These cell types are particularly important with regard to nanoparticle interactions with the immune system and therefore will be discussed in greater detail. These immune cells are produced in primary lymphoid organs (fetal liver, bone marrow, and thymus) and mature in secondary lymphoid organs (spleen and lymph nodes) [1]. It is within these secondary lymphoid organs that a significant amount of antigen presentation and cell activation occurs. Macroscale biomaterials can be engineered to mimic these secondary lymphoid organs to achieve an optimal environment for cell stimulation. A primary role of these cells is to serve as sentinels that collect and process foreign and pathogenic molecules and particulates to generate appropriate immune responses, often stimulating controlled inflammation. This broad functional definition, devoid of ontogeny, underscores the variety of different specialized tasks that tissue-specific macrophages perform. Macrophages are critical for tissue maintenance and regularly phagocytose senescent and apoptotic cells. Alveolar macrophages utilize these receptors to actively phagocytose and clear bacteria and debris from the lungs [5]. In contrast, gut resident macrophages demonstrate significantly reduced inflammatory cytokine production, even after phagocytosis of bacteria [7]. This tissue-dependent response demonstrates that a focus on delivering to a particular cell type, with no regard to tissue or organ, is not an effective strategy for macrophage immunostimulation. Aside from the highly diverse category of tissue-resident macrophages, there also exists a group of macrophages that typically appear at the site of tissue damage or infection. These macrophages are derived from Ly-6Chi monocytes and typically exist somewhere on a spectrum of polarization between two states, termed "M1" and "M2. Both types of macrophages can be pathogenic, and it is generally important to note that macrophages can switch polarizations if needed. In fact, macrophages display surprising plasticity, a trait that can make them potentially very interesting targets for immunomodulatory treatments [10]. This ability to evade immune destruction was recently added to the list of eight essential hallmarks of cancer and is thus considered a vital component of cancer progression and development [23]. Myeloid cells play a critical role in host defense against infection as well as in wound healing, and dysregulation of this latter role within solid tumors contributes significantly to cancer immune evasion. Myeloid cells secrete an array of growth factors, cytokines, and proteinases that induce the formation of new tissue, influence tissue remodeling, and suppress immune responses that were initially intended to combat infection [24]. This process shares numerous mechanisms with wound healing, which is a biological process that has received decades of attention from the materials community. As a result, prior strategies and materials developed to induce and regulate wound repair have been applied for controlled immunostimulation in the treatment of cancer. Tissue engineering scaffolds, and in particular hydrogels, have emerged as key tools for wound regeneration and have therefore been applied to modulate immune dysregulation observed in cancer. Both antigen and adjuvant can be localized in these polymeric matrices, often creating what is essentially an artificial lymph node for controlled immunostimulation. Furthermore, hydrogel scaffolds have long been used for controlled and sustained drug delivery, which is advantageous for the release of antigen and adjuvant in a dynamic or triggered fashion. Here, we describe several seminal and more recent tissue engineering scaffolds and hydrogels that have been employed for immunostimulation during cancer therapy. Such scaffolds form naturally following tissue injury, resulting in a controlled display of antigen and adjuvant as well as a dynamic environment to direct cell function during the course of wound healing. Recruited inflammatory cells and local diffusion from neighboring tissue both increase concentrations of cytokines and chemokines within the fibrin scaffold, which can be retained by diverse motifs within fibrin itself as well as by sites of glycosylation. Cytokines are factors responsible for immune cell activation, differentiation, and proliferation, while chemokines direct cell migration. In addition, the mechanics of the fibrin gel can promote specific cell differentiation and activity. Mimicry of these processes has resulted in fibrin being employed extensively for tissue engineering applications as well as for cancer Immunostimulatory materials 477 therapy. Immunomodulatory factors could be retained within these gels for a slow release and modulation of locally recruited macrophages to induce antitumor T-cell responses that prevent postsurgery tumor recurrence. Key design parameters for engineering such scaffolds include the combination and rate of delivery of multiple cytokines and chemokines, the type and concentration of cell adhesion molecules, and the overall 3D structure of the microenvironment. Natural fibrinogen or collagen matrices have also been used since they already contain important cell adhesion motifs within their protein fibers. Several attempts have been made to better mimic the 3D structures of secondary lymphoid organs in order to better control or model elicited immune responses. In this section, we have seen that tissue-engineered constructs can be used for immunomodulation by applying methods of controlled release that were initially developed for tissue healing and regeneration. Tailoring matrices to present antigens, adjuvants, cytokines, and chemokines in a fashion that mimics natural microenvironments can allow precise control over cellular migration and the immune response. Some discrepancy exists regarding the terminology employed to describe nanotechnology and related nanomaterials. Regulatory and governmental agencies have defined nanomaterials as those materials that exhibit one or more dimensions between 1 and 100 nm [36], which has reached general agreement with the material sciences community [37]. But, researchers whose focus is geared toward the life sciences tend to more broadly define nanomaterials as those materials exhibiting one dimension that is less than 1000 nm [37,38]. Size, shape, charge, and surface chemistry are important variables to consider when designing nanomaterials for the modulation of inflammatory cells. The composition and bioactivity of this coating is dependent on the characteristics of the material surface and influence the charge, hydrodynamic size, and aggregation behavior of the nanoparticle [49]. Often, the proteins that comprise this corona undergo conformational changes that result in the presentation of novel epitopes, altering how the protein interacts with immunoglobulins and various immune cell populations [50].

How is the length of treatment connected to the fact that mycobacterial species tend to grow very slowly Whether analyzing drinking water xanthelasma menopause buy premarin pills in toronto, orange juice menopause youngest age 0.625 mg premarin, or buffers used to dilute pharmaceutical products women's health clinic boca raton purchase premarin with visa, the goal is the same pregnancy symptoms week by week 0.625mg premarin visa, ensuring that living microbes in a sample are below a set limit womens health website discount premarin 0.625mg amex. During the collection process breast cancer network of strength premarin 0.625mg on-line, however, it is inoculated with the normal microbiota of the cow. Because milk has such a rich composition, being laden with water, proteins, fats, and hormones, microbial growth is a common occurrence. The main safeguard against bacterial infections acquired from milk is pasteurization. In this process, heat is used to kill many bacterial species, resulting in a product that, while not sterile, has a reduced microbial load. This decrease in bacterial diversity and number results in a product that is safer to drink and has a longer shelf life. Dairies that sell raw milk rely on more stringent standards of cow health as well as rigorous collection standards. Despite these efforts, raw milk remains a relatively common source of Salmonella and Escherichia coli outbreaks. For both raw and pasteurized milk, the Food and Drug Administration has set limits on the number of both coliforms and total bacteria that may be present in milk before and after processing and/or pasteurization. Technically speaking, coliform bacteria are Gramnegative rods that ferment the sugar lactose, producing acid and gas. The name itself refers to the fact that these bacteria look and act like a "form" of E. Because MacConkey agar selects only Gram-negative bacteria for growth, red colonies can presumptively be considered coliforms (Gram-negative lactose fermenters) while white or clear colonies are considered noncoliforms. One commonly used medium, MacConkey agar, contains lactose, along with a pH indicator that turns bacterial colonies pink if the pH of the medium drops, as would happen if a coliform bacterium were fermenting lactose. Bacterial counts in milk are often determined using a viable plate count, both as part of routine testing and whenever milk products are thought to be the source of a disease cluster. In this method, a sample of milk to be counted is serially diluted to produce several samples with decreasing cell densities. Aliquots of the dilutions are then plated onto media, and the colonies produced are counted after incubation. Each dilution is represented by a dilution factor, a term that corresponds to the amount of the original sample still present in the current sample. For example, if 1 ml of a sample is diluted into 9 ml of diluent (the fluid used for dilution), so that the original sample makes up 1/10 of the current volume, then the dilution factor is 10. If 1 ml were diluted in 99 ml, the dilution factor would be 100 because the sample would be 1/100 as concentrated, 127 128 Case Study Exercise 12 Viable Plate Count and so on. The use of dilution factors, along with careful measurement, allows the accurate determination of cell densities in any liquid. When several dilutions are performed in sequence, a serial dilution is the end result, with each sample being less concentrated than the one before. The most important part of performing a serial dilution is being able to calculate exactly how dilute a sample is when compared to the original. A dilution factor is always found by dividing the volume of the first sample by the volume of the first sample plus the volume of the diluent, and then taking the reciprocal. For example, diluting 1 ml of a milk sample into 99 ml of sterile water results in a 1/100 dilution and a dilution factor of 100. In a serial dilution, the final dilution factor is simply the product of each individual dilution factor. For example, if an undiluted liquid (dilution factor of 1) is diluted into 9 ml of diluent, 1 ml of this sample is diluted into 9 ml of diluent, and 1 ml of this sample is diluted into 9 ml of diluent, then the final dilution factor is (1)(10)(10) (10) = 1000. Finally, the cell density of the original broth is a function of the volume of the sample plated onto the media, the dilution of the sample, and the number of colonies on the plate. Define the following terms using the laboratory exercise or the glossary: Viable For example, if 0. Although we tend to think of a colony arising from a single cell, it is important to remember that for some organisms, single cells are rarely if ever seen. Once milk has gone through the process of pasteurization it is considered sterile. For each of the media in the accompanying table, indicate what type of bacteria is selected for and how lactosefermenting bacteria are differentiated from lactosenonfermenting bacteria. Each student team will be responsible for determining the bacterial count of coliforms and total bacteria from one type of milk, either raw or pasteurized. Inoculate the first plate with undiluted milk (shake the milk again if it has been sitting more than 3 min since last being mixed), the second plate with the 1/10 dilution, and the third plate with the 1/100 dilution. The rod is sterilized by dipping it in ethanol and then passing it through the flame of a Bunsen burner. Place the spreader rod in the beaker, and add enough ethanol to the beaker to cover the lower portion of the rod. Have nearby an empty beaker large enough to place over the small beaker and spreading rod. If the alcohol catches fire, simply place the large beaker over the smaller beaker to extinguish the flame. If a turntable is used, gently spin the turntable while moving the spreader back and forth. In either case, finish by rotating the plate one complete revolution while holding the spreader against the edge of the plate. Label the bottom of all 6 plates and the 3 water blanks with your name and lab time. After making sure the sample container is completely closed, vigorously shake your milk sample for 10 sec. Pipette up and down several times to mix, and transfer 1 ml of the 1/10 dilution to the 1/100 tube. Pipette up and down several times to mix, and transfer 1 ml of the 1/100 dilution to the 1/1000 tube. Pipette the 130 Case Study Exercise 12 Viable Plate Count 1 ml 1 ml 1 ml Milk sample 1/10 dilution 1/100 dilution 1/1000 dilution 0. Use dilution factors to establish the total number of each type of microorganism per milliliter of milk, using only those plates containing between 25 and 250 colonies. Compare the growth between plates inoculated with each type of milk (raw versus pasteurized). Using the morphological appearance of the colonies on each plate, determine the number of different types of colonies present on each plate. Why was the milk used for coliform counting diluted to a lesser extent than the milk used for total bacterial counting In most cases, campylobacteriosis (infection with Campylobacter) causes diarrhea, abdominal cramping, fever, nausea, and vomiting lasting about a week. Rarely, especially when the person involved has a weakened immune system, Campylobacter may spread to the bloodstream and cause a lifethreatening infection. All three persons had consumed raw (unpasteurized) milk from Ropelato Dairy in northern Utah. Both were hospitalized, and the parent died a week later of multisystem organ failure. Among the 98 persons interviewed, 52 reported drinking raw milk from Ropelata Dairy. In addition to ensuring that the dairy follows proper safety procedures, samples of milk are tested to determine the total number of bacteria, number of coliform bacteria, and number of somatic cells they contain. Although Campylobacter is commonly found in feces, it is not a coliform, and tests designed to detect coliforms will not identify its presence. Mostly leukocytes (white blood cells) that are produced in response to infection, a high number of somatic cells is indicative of a sick cow. To be sold in Utah, raw milk may contain no more than 20,000 bacterial cells, 10 coliforms, and 400,000 somatic cells per milliliter. Ropelata Dairy met this standard on their June 1 inspection, as well as two inspections on June 12 and July 13 ordered after the outbreak was recognized. Based on their successful test results, the dairy was allowed to continue selling raw milk. Case Study Exercise 12 Viable Plate Count 133 was obtained, adjusted to a pH of 7. Although many people believe raw milk provides health benefits not available in pasteurized milk, there is no scientific evidence to support these claims. According to the Centers for Disease Control, unpasteurized milk is 150 times more likely to cause illness and results in 13 times more hospitalizations than pasteurized dairy products. When asked if raw milk is safe to drink, the Food and Drug Administration, the Centers for Disease Control, and the American Academy of Pediatrics all had the same answer. A common method of ensuring pasteurization of milk is to test the activity of an enzyme called phosphatase. An adequately pasteurized milk sample will have only a very small amount (<1 g/ml) of functional enzyme. Bacteria in milk may also be detected directly, that is, by examining a small quantity of milk under the microscope and counting the number of cells present. Why is a viable cell count a better indicator of the safety of milk (especially pasteurized milk) Food and Drug Administration, the Dangers of Raw Milk: Unpasteurized Milk Can Pose a Serious Health Risk. Properly use an anaerobe jar or similar device for the cultivation of anaerobic bacteria. Cooking especially is used as a means of killing the bacteria that are assumed to be present on most foods. Ingested spores are generally destroyed by acid in the stomach or outcompeted for nutrients in the gastrointestinal tract; put another way, ingestion of small numbers of endospores is generally not a problem. If, however, the spores are given the chance to germinate in food, the vegetative cells produce botulin, the most potent bacterial toxin known. Improper canning of food can lead to viable spores being present in the anaerobic environment created by the canning process. When the cans (or jars) are kept at room temperature, these endospores can germinate and begin to produce botulin. Ingestion of botulin interferes with the release of acetylcholine from motor neurons, resulting in flaccid paralysis (where muscles are unable to contract). Successful culturing of anaerobes requires the creation of an anaerobic environment. One method of doing this is to incubate cultures in an anaerobe jar, a container with a tight-fitting lid that prevents oxygen from entering the jar from the outside environment. The water combines with the chemicals inside the packet to produce hydrogen and carbon dioxide gases. A palladium catalyst, also in the packet, then converts hydrogen and oxygen gas within the jar to water, generating an anaerobic environment. A paper strip impregnated with the dye methylene blue is placed inside the jar as a visual indicator of an anaerobic environment. When water is added to the chemical packet inside the jar, a series of reactions take place that result in oxygen within the jar being converted to water, creating an anaerobic environment. A methylene blue indicator strip, visible in the lower left area of the jar, provides visual assurance that the environment is anaerobic, becoming colorless when no oxygen is available. A second method of culturing anaerobic organisms is to use fluid thioglycollate medium, which permits the growth of a wide variety of bacteria and also allows the determination of the oxygen 135 136 Case Study Exercise 13 Cultivation of Anaerobes High Aerobes Oxygen tension Facultative anaerobes Microaerophiles 5. The highest concentration of oxygen in a tube of fluid thioglycollate medium is found a. This medium, which is a thick broth because of the addition of a small amount of agar, has dissolved oxygen expelled from it during autoclaving. The medium is inoculated with a vertical stab from top to bottom, ensuring that organisms are initially present throughout the media. After incubation, the position of growth within the media indicates the oxygen requirements of the bacterium. What two techniques are commonly used to culture anaerobic bacteria such as Clostridium botulinum Why is it important that fluid thioglycollate medium not be mixed or shaken prior to incubation When no oxygen is present, the methylene blue indicator strip in an anaerobe jar appears. How is the environment within the fluid thioglycollate medium different from that found within the anaerobe jar On the following two plates sketch the appearance of growth you would predict for each type of organism. Place one plate in the anaerobe jar and the other in a sleeve to be incubated at normal oxygen concentration.

Through the process the collision between drug crystals and milling balls results in abrasion and cleavage of the crystals pregnancy in weeks cheap premarin 0.625mg otc, and micro- or nanosizing of the drug [25] womens health 2 colon discount premarin online master card. In piston-gap homogenization womens health yoga safe premarin 0.625 mg, the drug particle suspension is forced through a gap women's health center wooster ohio cheap premarin 0.625 mg. As a result menstrual vaginal discharge premarin 0.625 mg lowest price, particle size reduction is achieved by shear force menstrual yoga poses purchase cheapest premarin, cavitation, and particle collision under high pressure [52,53]. The particle size and other characteristics are determined by the brittleness, hardness, and defect density of initial drug crystals [54,55]. These processes also involve high energy (mechanical and hydrodynamic), leading to loss of crystallinity during size reduction. All of these methods share similar issues of production, often requiring a significant amount of excipients during the process to stabilize drug nanoparticles and assist the production. Additionally, particle size tends to have a broad distribution and low crystallinity is common. The bottomup approach involves phase transition of drug molecules dispersed in the liquid 98 Biomaterials for Cancer Therapeutics state to the final solid state [54,58]. This process avoids using high-energy input which may otherwise lead to degradation of active pharmaceutical ingredients [27]. Only after a nucleus becomes large enough can the internal energy become dominant over the surface energy, ensuring continuous growth. It is possible that a solution can stay supersaturated without forming any nuclei. A narrow size distribution is mostly desired because of Ostwald ripening [48], as well as to achieve predictable and consistent pharmacokinetic performance. Delivering anticancer drugs as carrier-free nanocrystals 99 simultaneously throughout the solution. Furthermore, to generate a vast amount of nuclei and minimize continuous growth, the metastable state needs to be maintained as closely as possible to the metastable limit in the mixing process [60]. In general, upon freezing, the solubility of a drug in solution decreases, creating the supersaturation for crystallization [72]. Freezing rate, degree of freezing, and composition of the solution are controlling factors for the particle size and distribution of resultant crystals. On the other hand, during spray drying, the supersaturation is created by solvent evaporation. However, it is difficult to achieve a uniform particle size distribution through this method, and it is not always possible to find a suitable solvent for the spray drying process [27]. Lastly, there are combination technologies that use both top-down and bottomup techniques. Such a process typically starts with a crystallization step followed by high-energy comminution. In a recent report, ultrasound is used as the high-energy step to reduce the particle size after precipitation [73]. Crystals are, in general, physically stable because of the crystalline state, likely enabling predicable and consistent in vivo performance. This may be due to the anionic nature of the dye that limits the molecules across cell membranes. Furthermore, no significant changes of the intercellular drug concentration were detected as incubation time increased. The amount of the drug found inside cells was significantly increased from about 15% at 0. Accordingly, the drug concentration in the culture media decreased over time from more than 80% to less than 40%. This is not only because organic crystals share similar contrast with the biological background with regard to electron refraction, but also because a drug crystal is constantly dissolving. Li, Exploring intracellular fate of drug nanocrystals with crystal-integrated and environment-sensitive fluorophores, J. This test was aimed to further examine dissolution kinetics of the crystals inside the cells. The results (not shown here) provide additional support for the cellular uptake mechanisms and kinetics [44]. The uptake difference between the two cell types could result from distinct endocytic pathways and cellular detoxification responses. Oral or parenteral delivery requires a drug substance that is readily dissolved and molecularly dispersed with the local aqueous environment. Inside phagocytic cells, nanoparticles likely dissolve slowly in phagolysosomes but additional understanding is lacking. The freely lipophilic drug molecules may permeate through the phagolysosomal membrane and enter the cytoplasm and then exit the cell because of the concentration gradient across the membrane. It is believed that the polymer coating interacts preferentially with water molecules in circulation, forming a hydrophilic, protective shield [91,92]. The aqueous layer then minimizes the recognition of such a delivery system by signaling proteins and immune cells, thus reducing phagocytosis and elimination by the immune system. In vivo results showed significantly improved efficacy and greater reduced toxicity compared with conventional solubilizing formulations, as indicated by changes in the tumor volume, body weight, and survival rate. During the initial 4 days of treatment, all three groups almost completely suppressed tumor growth. The increased growth rate of tumors at the late stage was attributed to the use of only a single injection, and as such, eventual elimination of the drug from the body. The differences among the treatment groups were outstanding, taking into account the experimental error. This change could be even larger if the weight of tumor was taken into consideration as well. In our studies [40,41,43,44], tissue distribution of the drug in mice was measured through tritium-labeling of the drug and scintillation counting. The weights of the removed tumors were measured after the completion of the in vivo assays (C). Delivering anticancer drugs as carrier-free nanocrystals 109 formulation was shown to distribute extensively in all major organs, especially to the heart. However, it is expected that drug concentration can be maintained at an ideal level if multiple doses are given. The underlying support lies in the fact that the intrinsic solubility of a drug is higher than the effective concentration needed to kill cancer cells, even if the drug is poorly soluble. Pharmacokinetics and biodistribution become significantly different from traditional delivery designs. 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