1. You add 0.1 ml of undiluted cell culture to 9.9 ml of sterile broth. What is the dilution factor? 2. Transfer 1.0 ml and 0.1 ml of your dilution from step 1 onto separate plates (A and B). Report the final dilution factors of the plates on Table 1. 3. You take 0.1 ml of the cell suspension you made in question 1 and add it to 9.9 ml of sterile broth. By how much did you dilute it at this step? What is the total dilution of the original culture after this second step? 4. [Transfer 1.0 ml and 0.1 ml of your dilution from question 3 onto separate plates (C and D). Report the final dilution factors of the plates on Table 1 5. You take 1 ml of the cell suspension you made in question 3 and add it to 9 ml of sterile broth. What is the total dilution of the original sample after this third step? 6. Transfer 1.0 ml and 0.1 ml of the suspension you made in question 5 onto separate plates (E and F). Report the final dilution factors of the plates on Table 1 After 24 hours of incubation you count the number of colonies on Plates A – F (see Table 1). Calculate the original density of the starting culture in colony forming units (CFU) Plate | Final Dilution Factor | Number of Colonies | CFU of original culture A | | TNTC | B | | TNTC | C | | 267 | D | | 58 | E | | 69 |
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The first dilution is 0.1 ml of cell culture in 9.9 ml of broth. This is a 1:100 dilution (0.1 ml in 10 ml total volume). Show more…
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The following set of questions goes through the steps you would follow to do a viable count. You would do serial dilutions of the culture and plate an aliquot of each dilution on nutrient agar. Some terminology: a 1:10 dilution = 1/10 dilution = 1x10^-1 dilution; a 1:100 dilution = 1/100 dilution = 1x10^-2 dilution, and so on. a) A bacterial culture contains 2 x 10^6 cells/ml. A 1:10 dilution of the culture is made by taking 0.1 ml out of the culture and adding it to a test tube that contains 0.9 ml of diluent. How many cells are present in the 0.1 ml sample taken from the culture? (units: # of cells) What is the new concentration of cells after you add the 0.1 ml sample to the tube with 0.9 ml of diluent? (units: cells/ml) You remove 0.1 ml from the 1:10 dilution and spread it on nutrient agar. How many colony forming units (CFU) do you expect to grow on the plate? b) A 1:100 dilution of the culture is made by taking 0.1 ml out of the 1:10 dilution made in (a) and adding it to a test tube that contains 0.9 ml of diluent. (1/10 * 1/10 = 1/100 or 1:100) How many cells are present in the 0.1 ml sample taken from the 1:10 dilution from (a)? What is the new concentration of cells after you add the 0.1 ml sample to the tube with 0.9 ml of diluent? (units: cells/ml) You remove 0.1 ml from the 1:100 dilution and spread it on nutrient agar. How many colony forming units (CFU) do you expect to grow on the plate? c) A 1:1000 dilution of the culture is made by taking 0.1 ml out of the 1:100 dilution made in (b) and adding it to a test tube that contains 0.9 ml of diluent. (1/10 * 1/10 * 1/10 = 1/1000 or 1:1000) How many cells are present in the 0.1 ml sample taken from the 1:100 dilution from (b)? What is the new concentration of cells after you add the 0.1 ml sample to the tube with 0.9 ml of diluent? (units: cells/ml) You remove 0.1 ml from the 1:1000 dilution and spread it on nutrient agar. How many colony forming units (CFU) do you expect to grow on the plate?
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Practice Dilution Questions If you add 0.1 mL of a 10^-1 dilution to 9.9 mL of water, what is the final dilution? You count 87 cfu on a 10^-2 dilution plate after plating 0.25 mL. Calculate the cfu/mL of the original sample. 3x10^0 cfu/mL of soil was suspended in 990 mL of distilled water. From this, serial dilutions are made by aliquoting 10 mL into 90 mL water blanks. If the original cfu/mL of soil is 3x10^0, how many colonies are expected if 1 mL is spread from the last dilution? You have a sample of bacteria at 10^-10 cfu/mL and remove 0.5 mL into 4.5 mL of water. One mL is removed from that dilution tube into 9 mL of water, and after mixing, 1 mL is removed into 99 mL of water. Next, 1 mL is serially diluted three times. If you plate 0.1 mL, how many cfu do you expect? Given a bacterial suspension serially diluted 1 mL into 99 mL three times and 0.1 mL of the third dilution plated, resulting in 47 colonies. What is the cfu/mL of the original sample? 3x10^0 cfu/g soil mixed with 99 mL of water. After mixing, 1 mL of the suspension is diluted in 98 mL of water and 0.1 mL is spread on an agar plate. If 56 colonies grew after incubation, calculate the original cfu/g soil.
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Purification of proteins for use as biopharmaceuticals is often accomplished by ion exchange chromatography, in which a process fluid passes through a column packed with small resin beads whose ionic surface charge causes them to adsorb some stream components more strongly than others. An ion-exchange run takes place in two steps: (1) the load step, in which the process stream flows through the column and the target protein (the product) and some undesired impurities are adsorbed onto the resin; and (2) the elution step, during which another fluid passes through the column and desorbs the impurities and the protein from the resin. The elution fluid consists of an aqueous solution of a solute known as Tris diluted with an $\mathrm{NaCl}$ solution, with the NaCl-to-Tris ratio starting at 0 and steadily increasing with time. The impurities desorb into the fluid when the NaCl concentration is low, and the effluent is collected in a waste vessel. As the NaCl concentration increases, the target protein desorbs. When analysis of the effluent reveals the presence of the target protein, the flow is switched to the product collection vessel, and the effluent is collected until no more product is detected in the effluent. The collected product is then subjected to additional process steps to further isolate the protein, and the column is cleaned for reuse. Consider an elution step in which solutions of 1 M $\mathrm{NaCl}$ (solution A) and 50 mM Tris (solution B) are mixed and fed to a loaded ion-exchange column. The system is programmed to keep the total volumetric flow rate $\left(\dot{V}_{\Lambda}+\dot{V}_{B}\right)$ into the column constant at 120 Lh while linearly increasing the volume fraction of solution A in the feed from $0 \%$ to $20 \%$ over a period of 33.6 minutes, at which point the elution is declared to be complete. The flowchart is shown below: (a) Calculate $V_{\mathrm{t}}(\mathrm{L}),$ the total amount of solution fed to the column. (b) Derive an equation for the volumetric flow rate of solution $A, \dot{V}_{A}(t),$ assuming that the densities of both fluids are the same. Use the calculated value to determine calculate $V_{\mathrm{A}}(\mathrm{L}),$ the total volume of that solution fed to the column, and $m_{\mathrm{A}}(\mathrm{g} \mathrm{NaCl})$, the total mass of $\mathrm{NaCl}$ fed. Then determine $\dot{V}_{B}(t)$ and $V_{\mathrm{B}}(\mathrm{L})$ and $n_{\mathrm{B}}(\text { mol } \text { Tris) }, \text { the total volume of solution } \mathrm{B}$ and total moles of Tris fed, respectively. (Hint: Once you've done the calculations for solution A, those for B should be trivial.) (c) Suppose in one run product is detected in the effluent at the same time impurities are detected $-$ that is, product protein starts desorbing earlier than in previous runs. List up to five possible causes of the problem.
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