Procedures for Gen Bio II Lab

Bacteria and gram Staining-->Bacteria Susceptibility to Antibiotics ANSWERS

-Gram-Positive bacteria are more sensitive to antibiotics - E. Coil(Gram negative): most power antibiotic was neomycin and least was erythromycin and penicillin -S. aureus(gram positive): most powerful was ampicillin and least was neomycin

Biuret Lab--> Melamine

For example, since 2004 melamine has been found as a contaminant in various food products imported from China: in pet foods, baby formula, protein powders. In fact many pets in the U.S. died after eating tainted food. Melamine was added by manufacturers because it produces a positive reaction like protein does in the Kjeldahl Test, which is used routinely to measure the amount of protein in foods. Melamine is not protein, is not digestible, and damages kidneys. But since it is cheaper than protein, it has been added by unscrupulous manufacturers to make the food product appear to have a higher protein content.

Acids, Bases, pH, and Buffers--> The Phosphate Buffer System HCL

HCL(Acid) - Take a 50 mL beaker from the Containers shelf and place it onto the workbench. Add 10 mL of 0.5 M hydrochloric acid (HCl) to the beaker. Take a dropper from the Containers shelf and place it onto the workbench. test tube 1 and add two drops Test #1: 5 mL water --> negative pH change (more acidic) Test #2: 5ml of 1 Sodium Dihydrogen Phosphate( NaH2PO4)---> negative pH change Test#3: 5ml of 0.1 M Sodium Hydrogen Phosphate( Na2HPO4) --> negative pH change Test #4: 2.5ml of NaH2PO4 and 2.5ml of Na2HPO4--> negative pH change but it was effected the least

Biuret Lab--> calibration

The blank solution today is more than just water (e.g. we have KCl and biuret reagent in the samples too) This amount of light absorption would be the same in all of your tubes since they will all have the same solvent and the same solutes except for the added protein (albumin). Therefore, this calibration/standardization sets a baseline of light transmitted equally through all the solutions unless something else is in any solution to absorb more light. That "something else", of course, is the colored complex of biuret reagent and albumin which is present in different concentrations in all of your solutions except #1.

Permeability Lab--> Observation of diffusion of solutes.

1. Agar is a polysaccharide; when a dilute aqueous solution of agar is heated to boiling and then allowed to cool, the solution gels (like gelatin prepared at home) into a semisolid form. The gel layer in the dish is 1% agar (the solute) and 99% water, but it's not fluid as any other typical dilute solution would be. 2.You see that two "wells'' have been cut into the agar; their facing edges are 1.5 cm apart. From an eyedropper bottle carefully put 4-5 drops of bromophenol blue dye into one well and (from the other eyedropper bottle) 4-5 drops of HCl solution into the other well. Handle both solutions carefully; wear gloves. Don't overfill either well; keep the acid and dye inside the wells, not on the agar surface. 3. When you see this dye edge flattening, use a dissecting microscope to observe that flattening edge. Bromphenol blue is a pH indicator dye; it changes color from blue (purplish) to yellow as the pH falls below 4. Under the microscope, is that bluish dye edge really flat? 4. You're looking at the zone where the protons from the acid well are colliding with the dye molecules. Since the protons cause a color change of the dye molecules, it's easy to see where that zone is

Biuret Lab--> Error

1. Always remain alert to potential sources of error in your work and do what you can to minimize error. 2. How carefully did you do the pipetting?...any error there? 3. Did you take care to be sure that each solution in its test tube was thoroughly mixed? You wouldn't get proper color development if the solutions weren't thoroughly mixed. 4. Were you and your partner equally careful? Maybe the problem is traceable to one factor in the procedure and you notice that your partner did that particular step. 5. Did you standardize the colorimeter properly? 6. Did you rinse the colorimeter tube (cuvette) thoroughly between samples? 7. If a plotted point seemed "way off," did you recheck the calculations? and the accuracy of your plot? and the proper setup of scale divisions on your graph?

Colorimetry lab--> Graph

1. An absorption spectrum plots absorbance (A), not % transmittance, on the y-axis, as a function of wavelength on the x-axis. 2. Specifically, A = log (1 / T), where T is the decimal equivalent of %T. Example: if %T = 50%, then T = 0.5 and (1 / T) = 2.0, and A = 0.301. Make sure you understand this algebraic relationship.

Permeability Lab--> Error

1. Both ends of each bag must be tied so as to be leak proof. 2. Handle the membranes carefully to avoid causing small punctures. 3. Keep the dialysis tubing moist and pliable as you work with it; it may crack if it dries out. 4. Pour the solutions into the dialysis bags carefully to avoid spills and loss of the solutions. 5. Note that as you prepare the bags, one by one, they will go into the water in the cups at different clock times. So, your weighings of each bag at the 30 minute intervals must be staggered accordingly. Plan ahead so that you will weigh each bag at the proper times. 6. Keep the balance pan clean and dry. Drops of liquid and dried sucrose on the balance pan will add mass to your readings; that creates error. 7. When you remove the bags from the water in the cups to weigh them, be sure you blot them well, especially at the tied ends, to remove water adhering to the outer surface. Any liquid adhering to the outside of a bag will add mass to your reading; that's error. You are interested in changes in weight of the solutions inside the bags.

Bacteria and gram Staining-->Staining

1. Create crystal violet-iodine complexes by adding Gram's iodine from the Materials shelf to the slide. You should see the middle of the slide turn a reddish-brown color. 2. Take a water bottle from the Materials shelf and place it onto the bacterial slide. The water will wash excess iodine solution from the slide. 3. Decolorize the cells by adding 95% ethanol from the Materials shelf onto the middle of the slide 4. Take a water bottle from the Materials shelf and place it onto the bacterial slide. The water will wash excess ethanol from the slide. 5. Add the counterstain to the slide by taking the safranin from the Materials shelf and placing it onto the slide. You should see the middle of the slide turn pink 6. Take a water bottle from the Materials shelf and place it onto the bacterial slide. The water will wash off the excess stain from the slide

Permeability Lab--> Preparation of the membrane bags (dialysis bags).

1. Label the 7 cups #1 - #7. Fill each one with water almost to the top. Later as you place a dialysis bag in each cup, you need to have enough water in each cup to completely immerse each bag. 2. Into each cup put one of the flat pieces of dialysis tubing (the membrane); let them soak for about 5 minutes while you read ahead here to remind yourself of what you're about to do. 3. For each piece of tubing fold over one end about 3/8 inch and tie securely with doubled thread. See B & C in the previous drawing. This is awkward for a single pair of hands, so work with your partner to be sure that the folded end is tied securely. 4. Once you've begun you must watch the time carefully so that you measure the weight of each bag at the proper time. You will use the graduated cylinder to dispense 15 mL of various fluids into the 6 bags; bags #2 and 3# will receive the 15 mL solutions that you've already prepared in the 2 test tubes. 5. Carefully blot dry the bag's surface, especially the tied ends where water may get trapped in the folded membrane. Then weigh the bag to the nearest 0.1 g, put the bag into its cup of water, and mark the time (time zero). 6. You will need to record the weight of each bag again at 30 minutes (then put it back into its cup) and at 60 minutes. To do that, dry each bag's surface before weighing, and be sure to keep the balance pan clean and dry

Mitosis and Meiosis lab --> Relative Time Spent in Mitosis

1. Select one side of the root tip. Scan 5 columns across and 20 cells down within each column. Identify the phase that each cell is in, to the best of your understanding. Use initials for the phases: 2. Determine the number of cells in each stage of mitosis by counting every incidence of its corresponding letter. For example, add all letter I's to determine the number of cells in interphase, and so on for each stage. Record this information. -------------------------------------- RESULTS: Interphase: 80%-90% Mitosis: 10-20% Prophase: 13% Metaphase: 1% Anaphase and Telophase: 3%

Mitosis and Meiosis lab--> Mitosis in Onion Root Cells

1. Take a microscope from the Instruments shelf and place it onto the workbench. 2. Take an onion root slide from the Containers shelf and place it onto the microscope stage. Use different objectives and focus until you can clearly see many cells in one view. 3. Find each phase of Mitosis

Bacteria and gram Staining--> Heat-Fixation of the Bacterial Slide

1. Take a nichrome wire rod and an S. aureus culture tube from the Containers shelf and place them onto the workbench. 2. Take a Bunsen burner from the Instruments shelf and place it onto the workbench. 3. Place the nichrome wire rod onto the Bunsen burner to sterilize it. Insert the nichrome wire rod into the S. aureus culture tube. 4. Place the slide on the Bunsen burner. The slide will automatically pass back and forth through the flame. Return the slide to the staining pan.

Bacteria and gram Staining-->Bacteria Susceptibility to Antibiotics

1. Take a nutrient agar plate from the Containers shelf and place it onto the workbench. 2. Take the bacterial culture tube that you determined to be Gram-positive in Experiment 1 3. Place the micropipette onto the culture tube. Withdraw 100 µL of bacterial culture. 4. Move the micropipette onto the plate and dispense all 100 µL. 6. Empty the culture tube in the waste bin and then place it and the micropipette in the sink. 7. Sterilize your spreader by completing the following steps: ○ Take a 250-mL beaker from the Containers shelf and place it onto the bench. ○ Add 200 mL of 70% isopropanol from the Materials shelf to the beaker. ○ Take an L-shaped spreader from the Instruments shelf and place it into the beaker ○ Move the L-shaped spreader to the burner to ignite the isopropanol. Once the isopropanol burns off, the L-shaped spreader is fully sterilized. 9. Move the spreader onto the nutrient agar plate. You should observe the lid lifting and the instrument moving to spread the bacteria evenly. Drag the spreader and the Bunsen burner back to the Instruments shelf. 10. Fill the antibiotic dispenser with the following antibiotics: ampicillin, erythromycin, neomycin, and penicillin. 11. Run the incubator on these settings: ○ 24 hours ○ 35 °C 12. Observe the nutrient agar plate by placing it in the groove at the base of the projection magnifier. ------------------------------------ REPEAT FOR GRAM NEGATIVE

Acids, Bases, pH, and Buffers--> Buffering Capacity of a Phosphate

1. Take a small test tube from the Containers shelf and place it onto the workbench. 2. Add 2.5 mL of 0.1 M sodium hydrogen phosphate (Na2HPO4) and 2.5 mL of 0.1 M sodium dihydrogen phosphate (NaH2PO4) to the test tube. 3. Take a pH meter from the Instruments shelf and place it in the test tube. Record the pH of the phosphate buffer solution.(7.21) 4. Take a 50 mL beaker from the Containers shelf and place it onto the workbench. 5. Add 20 mL of 0.5 M hydrochloric acid (HCl) to the beaker. 6. Take a dropper from the Containers shelf and place it onto the workbench. 7. Place the dropper into the beaker of hydrochloric acid. You should observe the dropper filling with hydrochloric acid. 8. Add one drop of hydrochloric acid to the test tube. 9. Record the pH and total number of drops. 10.Repeat steps 8 and 9 until the pH falls below 3 or until you have dispensed a total of 15 drops, whichever is reached first. Make sure that you record the pH after each drop is added. ---------------------------------------- 1. Repeat the procedure outlined in Part 1, steps 1-10, using 0.5 M sodium hydroxide (NaOH) in your beaker. Add the base until the pH rises above 12 or until you have dispensed a total of 15 drops, whichever is reached first.

Mitosis and Meiosis Lab--> Mitosis in a Whitefish Blastula

1. Take a whitefish blastula slide from the Containers shelf and place it onto the microscope stage. 2. Find at least one cell in this fish embryo to represent each phase: ------------------------------ The cells of a blastula, or developing embryo, divide rapidly, which makes this whitefish slide useful for viewing the different stages of mitosis.

Permeability lab--> Preparation of the sucrose solutions.

1. Weigh 34.2 g of sucrose (molecular weight 342 daltons) on the balance and Carefully pour the sucrose into the 100 mL volumetric flask 2. Gradually add water and swirl the flask until all of the sucrose is dissolved. The final volume of the solution must be 100 mL 3. This is a 1 M (1 molar) sucrose solution; it's also 34.2%, weight-per-volume (w/v). That means that in each 1 mL volume of the solution there is 0.342 g of sucrose. 4. Label the 2 large test tubes #2 and #3. Use the graduated cylinder to measure 5 mL of this 1 M sucrose solution and 10 mL water into test tube #2. Then measure 10 mL of the 1 M sucrose solution and 5 mL water into test tube #3

Permeability Lab--> Osmosis and diffusion rate in solutions

1. You will prepare a series of "dialysis bags" (7)Some of them will illustrate osmosis, and some will illustrate the effect of molecular weight (size) of molecules on diffusion rate. 2. The membrane and its pores. Today you will use an artificial membrane, not a unit membrane as found in living systems 3. However, since it is similar in some important ways, we can use it as a model membrane as long as we recognize its limitations. The membrane is called dialysis tubing 4. The cut-off value of this membrane is about 12,000 daltons. That means that molecules smaller than that can pass through the membrane's pores, but larger ones would not pass through 5. the water molecules would move down the water gradient from where their concentration was greater (the pure water in this case) into the solution that had a lower water concentration. 6. that osmotic pressure depends on the total number of solute molecules (or ions) dissolved per unit volume, not the types of solutes. This is an example of a colligative property--> Thus, a 0.1 M glucose solution, a 0.1 M sucrose solution, and a 0.1 M glycerol solution would have the same O.P. value, since each contains the same number of solute molecules per unit of volume. However, molecules that dissociate or ionize in water, such as NaCl becoming Na+ and Cl- ions, contribute more than one osmotically active particle per molecule. 7. The net movement of water (by diffusion) into a dialysis bag could be measured simply by weighing the bag at various times. Water has mass, 1.0 g/mL. The weight gain of a bag reveals the amount of water that enters. 8. What's more, the rate of osmosis is affected by the difference in water concentrations on opposite sides of the membrane. If the water gradient across the membrane is more steep, then net movement of water will be faster.

Biuret Lab--> procedure for albumin

1. You will use BSA, bovine serum albumin, which is the protein extracted from cow's blood, as a representative protein today, but others could be used as well. BSA is the reference protein in this work. (The substance to be used is albumin, which refers to a collection of proteins found in blood, for example. Keep in mind that albumin is only one type of protein and that the procedure you will learn now can be modified for use in measuring concentrations of many types of organic molecules in solutions, not just proteins.) 2. When you add the biuret reagent solution (it happens to be blue) to these protein solutions of known concentration, you will see different degrees of color development (shades of purple) - the greater the protein concentration, the deeper the purple color 3. that is, it will quantify the depth of color in each tube. You will record absorbance values for each solution. You will then generate a graph, absorbance versus concentration of albumin. This is done using the Vernier Spectral Analysis program.

Colorimetry Lab--> Steps to Calibrate

1. make a blank by adding solvent (in this case: water) to a plastic cuvette until it is between 70%-80% full (do not fill beyond 80%... excess solution could spill out into the instrument). If necessary, wipe outside of cuvette with tissue so no liquid will get into the spectrometer. 2.Vernier Spectral Analysis program that you have installed on your computer. A Spectral Analysis window will open prompting you to select from several options... under Absorbance, click on "vs. Wavelength (Full Spectrum)" 3. When prompted, insert a blank cuvette into the cuvette holder on the top face of the SpectroVis Plus **IMPORTANT: always place cuvette so that the clear walls (i.e. no ridges) are in line with the light path, which is from right-to-left in this instrument 4.see in the program window and empty graph of Absorbance vs. Wavelength, and an empty table Data Set 1 on the right. The instrument has been calibrated/standardized, and is ready to read samples

Acids, Bases, pH, and Buffers--> examples of pH Indicators

1. phenolphthalein--> Low pH: colorless; color change interval: 8.3-10; High pH: pink, magenta 2. methyl orange--> Low pH: red; 3.1-4.4; high pH: yellow 3. bromothymol blue--> low pH: yellow; 6.0-7.6; high pH: blue 4. bromocresol green--> low pH: yellow; 3.8-5.4; high pH: blue 5. phenol red--> low pH: yellow; 6.8-8.4; high pH: red

Colorimetry Lab--> collect data from riboflavin's absorption spectrum

1. record the absorbance value at wavelengths from 380 nm to 950 nm 2. Transfer riboflavin solution (0.017 mg/mL) so that it fills a second cuvette to 70-80% 3. Since the absorbance is negligible at wavelengths above 600 nm(yellow), we have chosen to focus on the data points between 380-580 nm. 4. it has 2 peaks and wavelengths can be found between 300-550nm Maximum measured for our lab: 446.9

Biuret Lab--> Solutions in test tubes

8. 1. Set up and number the 8 large test tubes in the rack: #1-#8, left to right. 2. Refer to the Biuret Setup Table for the volumes of the 5 solutions to be dispensed (pipetted) into the 8 test tubes. 3. As you pipette the various solutions into each test tube, CAREFULLY swirl each tube to ensure that the materials are uniformly mixed in the tube. The color-producing reaction of the reagents with the albumin protein won't occur properly unless the solutions in the test tubes are thoroughly mixed. 4. First dispense the albumin stock solution into all tubes that are supposed to get it. (Note that #1, #7, and #8 do not get this solution.) 5. Second, dispense the two albumin 'unknown' solutions into the two tubes that are supposed to get those. 6. Next, dispense the 0.5 M KCl solution into all tubes that are supposed to get it. Since the albumin itself was prepared in 0.5 M KCl solution, this addition of KCl is meant to keep the total volume the same in each of the tubes #2-#6 (5 mL for the sum of the first 2 columns in the setup table). 7. Finally, add the biuret reagent to the tubes. The NaOH (sodium hydroxide) in this is caustic and poisonous and can burn the skin and eyes 8. Let tubes stand undisturbed 20 minutes for full color development. Once formed the color will be stable for the rest of the lab period.

Permeability lab--> the bags

Bag #1- 15 mL water Bag #2- 5 mL of 1 M sucrose + 10 mL water (test tube #2 contents already prepared) Bag #3- 10 mL of 1 M sucrose + 5 mL water (test tube #3 contents already prepared). Bag #4- 15 mL of the 1 M sucrose solution (already prepared by you in the volumetric flask) Bag #5- 15 mL of the 1 M glucose solution (in small bottle, as is) Bag #6- 15 mL of the 1 M glycerol solution (in small bottle, as is) Bag #7- 15 mL of the 1 M sucrose solution containing methylene blue dye (on front bench or side bench). Handle carefully; this dye will stick to paper, fabrics, skin. Wear gloves.

Permeability Lab--> Kidney

For example, some chemical waste molecules in the bloodstream will pass through membranes in the kidney to become urinary wastes, while other (beneficial) molecules such as proteins remain in the bloodstream because they are too large to pass through the same membranes. A kidney dialysis machine performs a similar function though not nearly as well as it happens in a kidney.

Bacteria and gram Staining-->Observing Stained Bacteria with a Microscope

Gram-positive bacteria: thick cell walls composed primarily of a carbohydrate known as peptidoglycan( keeps primary stain= crystal violet and looks purple) Gram-negative bacteria: thinner peptidoglycan cell wall and an outer membrane made of lipopolysaccharides(DOES NOT KEEP crystal violet, uses secondary stain safranin and looks pink)

Biuret Lab-->general

In general, since different organic molecules absorb different wavelengths to different degrees, the molecules might be distinguished from each other based on the differences in their absorption spectra (plural of spectrum). That is useful in identifying molecules. --------------------------------------- Colorimetric procedures are routinely used for detecting and measuring a wide variety of substances in blood, urine, water samples, and other fluids of interest to biologists, chemists, doctors, and others

Biuret lab--> graph

It is the Beer-Lambert relationship that makes colorimetric tests so useful for quantifying solutes such as proteins in solutions. The Beer-Lambert Law (relationship) says that there exists a concentration range within which there is a direct, linear (straight line) relationship between solute concentration and absorbance -This linear relationship between solute concentration and absorbance is called a standard curve. To produce such a standard curve, you prepare a series of solutions of known protein concentration, subject them to the color reagent, and then measure the absorbance of each solution. Solutions of greater protein concentration would produce more color. the points won't be exactly linear. Therefore, you draw the line of best fit through the set of points; that is the standard curve... you can compare the absorbance of 'unknown' protein solutions with the standard curve to determine the protein concentration of those unknowns.

Acids, Bases, pH, and Buffers--> Introduction to pH Indicators using bromothymol blue

NO pH change for any: Test #1: 10ml water --> turned green Test #2: 10 mL acetone --> turned green Test #3: 10 mL 5 M citric acid--> turned yellow Test #4: 10 mL 5% vinegar--> turned yellow Test #5: 10 mL 4 M ammonia--> blue Test#6: 10 mL diluted bleach--> turned blue

Acids, Bases, pH, and Buffers--> The Phosphate Buffer System NaOH

NaOH(base) -10 mL of 0.5 M sodium hydroxide (NaOH) Test#1: 5ml water--> positive pH change(basic) Test #2: 5ml of 1 Sodium Dihydrogen Phosphate( NaH2PO4)--->positive pH change Test#3: 5ml of 0.1 M Sodium Hydrogen Phosphate( Na2HPO4) --> positive pH change Test #4: 2.5ml of NaH2PO4 and 2.5ml of Na2HPO4--> positive pH change but it was effected the least

Colorimetry lab--> molecule of interest

Riboflavin is a water-soluble vitamin. The yellow solution in one of the tubes in your rack contains riboflavin dissolved in water, 0.017 mg/mL; nothing else is present.

Colorimetry Lab --> Calibration of spectrophotometer

Since the point is to measure the absorption of one particular type of molecule in a solution, it is necessary to correct for (to "subtract" out) light absorption by everything else (the glass of the test tube, the solvent, any color-producing reagents present). To do that you calibrate/standardize the instrument before doing your measurements. ------------------------------------------------- In order to get the most accurate possible data for determining the absorption spectrum of riboflavin, you must calibrate/standardize the colorimeter to correct for light absorption by the glass and the solvent (water in this case). Therefore, you have another tube containing only water, that is, everything except the molecule of interest: riboflavin; this second tube is called the blank. You use the blank to calibrate/standardize the instrument. In effect, you will be "telling the instrument to ignore" light absorption by everything except the molecule of interest, riboflavin.

Biuret Lab--> Biuret and peptide bonds

The small molecule called 'biuret' (also known as carbamylurea) reacts with copper sulfate and NaOH (sodium hydroxide) in an aqueous solution (aqueous = pertaining to water) to produce a purple/violet color. -Although the molecule, biuret, itself, is of little significance, there is a group of very important molecules, the proteins, which react with copper sulfate and NaOH the same way that biuret reacts -proteins are made of many amino acids attached to each other by peptide bonds. Because the arrangement of atoms at every peptide bond is similar to part of the biuret molecule (see structure above and compare with peptide bond), peptide bonds within protein molecules react the same way biuret reacts. That means that proteins in the solution give a positive biuret reaction, with the result that solutions containing proteins turn purple.

Mitosis and Meiosis Lab-->Live Mitosis in Cultured Mammalian Cells

The video showed the same process of the cell dividing. You can clearly see the metaphase, anaphase, and telophase because the chromosomes are lined up in the middle and then separated and pulled to opposite sides and finally pinched off to make 2 cells. However, the interphase and prophase have no clear distinction, so it is hard to tell what phase it is in since it happens so quickly. Once the cell divided and created 2 cells, the cells stayed near each other.