Micro Labs 7-13

25 July 2022
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question
LAB 7 What was the population density of the original sample? What would have happened if you had inoculated an agar plate with 1mL of the original sample?
answer
8.75x10^4 CFU/mL was the original population density. If the sample would have been inoculated an agar plate then we would not be able to distinguish the colonies from one another. The only growth that would have been visible would have been a thick covering of bacterial growth.
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LAB 7 Did all the bacterial colonies on the countable plate(s) have a similar appearance? If not, how would you explain this?
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They were all similar but not identical because of the different dilution strengths
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LAB 7 You have performed a serial dilution of an unknown sample and counted 73 CFU on a countable plate that was marked as a 10 ^-4 dilution and you use 0.1mL to inoculate the plate. What is the population density of the original sample?
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8.78x10^4
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LAB 7 List at least one advantage and one disadvantage to the direct plate counting method following serial dilution for determining bacterial concentration
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One advantage of direct plate counting method is it limits the number of cells transferred to a reasonable number to be able to count. One disadvantage is if there are too many there could easily be a counting error.
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LAB 7 Compare and contrast direct plate counts for bacterial and viral populations
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Direct count gives an estimated total of bacteria in the sample and form colonies. Whereas viruses do not form colonies on plates and are a lot smaller in size. They are similar because neither of them have a nucleus, they both sometimes have benefits to us, they are virulent.
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LAB 7 What are some sources of error in the serial dilution/direct plate counting method?
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Some sources of error include inaccurate transferring will cause less accurate dispensing. Also the higher the dilution is when doing variable counts, the more common it is for an error to be made when estimating the count.
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LAB 8 EX 1 Why was cow manure used as a potential source of starch degrading bacteria?
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Cow manure supports maximum protease production
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LAB 8 EX 1 What are some other potential sources for starch degrading bacteria?
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Soil
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LAB 8 EX 1 What component makes starch agar selective for starch degrading bacteria?
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Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism.
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LAB 8 EX 1 Why were each of the following steps performed in this experiment: Serial dilution? Growth on the nutrient agar plates? Streak on the starch agar plates?
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The serial dilution is performed to result in concentration curves with a logarithmic scale The growth is to identify certain bacteria. The streak is used to obtain individual colonies.
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LAB 8 EX 2 What substance in MSA confers selectivity? Why?
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NaCl. NaCl is inhibatory to most bacteria
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LAB 8 EX 2 What substance makes MSA differential? Why?
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MSA is differential beause it contains Mannitol. Mannitol fermentation is indicated by a change in the colour of the phenol red indicator which aids in the differentiation of Staphylococcal species.
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LAB 8 EX 2 What purpose does phenol red serve in MSA?
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Indicates if mannitol fermentation has taken place by changing color as the pH changes.
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LAB 8 EX 2 Why are the Nutrient agar plates used in this experiment?
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To isolate specific bacteria
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LAB 8 EX 5 What ingredient(s) makes MacConkey agar selective for Gram-negative bacteria?
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The crystal violet and bile salts inhibit the growth of gram-positive organisms which allows for the selection and isolation of gram-negative bacteria.
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LAB 8 EX 5 What types of bacteria are inhibited on MacConkey agar?
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Bile salts and crystal violet inhibit the growth of Gram positive organisms.
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LAB 8 EX 5 What ingredient(s) makes MacConkey agar differential?
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It can differentiate those bacteria that have the ability to ferment lactose.
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LAB 8 EX 5 Why is an indicator dye used in MacConkey agar?
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The bile salts and crystal violet select for Gram-negative bacteria by inhibiting the growth of Gram-positive organisms.
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LAB 8 EX 5 What are some potentially pathogenic bacteria that are lactose fermenters that will grow on MacConkey agar?
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Salmonella, Citrobacter, Morganella
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LAB 8 EX 5 What are some potentially pathogenic bacteria that do not ferment lactose that will grow on MacConkey agar?
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Acinetobacter, Aeromonas, Alcaligenes, Eikenella
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LAB 8 EX 5 How would you verify that the colonies that grew on a MacConkey agar plate were Gram-negative?
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Stain them and examine them under a microscope.
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LAB 8 EX 5 Look up the formulation for MacConkey agar either in a microbiology text book or online. Is this a chemically defined or complex media? Why is that important?
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Complex. It is important because depending on what you are trying to grow and study you want to use a complex media.
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LAB 9 EX 1 oxidase Which bacterial plates contained oxidase positive bacteria? Which plates contained oxidase negative bacteria? Did both of the colonies from each plate show the same color change? Why or why not? How would you interpret this?
answer
Sink only. Shoe, phone and control. No. Different types of bacteria are rubbed on each side of a petri dish. If there is more bacteria put on one side that side is going to grow more. Being in the same petri dish does not mean they will grow the same amount. Using my sink/show petri dish as an example, the sink bacteria oxidized and the shoe bacteria did not.
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LAB 9 EX 1 Which bacterial samples have cytochrome c oxidase as the terminal enzyme of their ETC? Which bacterial samples do not have cytochrome c oxidase as the terminal enzyme of their ETC?
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The sink bacteria. Shoe, phone and control.
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LAB 9 EX 1 Is the control test in this experiment positive or negative control? What was the purpose of a negative control?
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Negative control. A negative control is used because it is known for the start of the experiment that there will not be a reaction in that area. No bacteria should be produced and it will not react in the DrySlide assay.
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LAB 9 EX 1 What is the purpose of a positive control?
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The purpose is to verify that a reaction will occur. This would have been the case if we would have put a bacteria on the control part of the petri dish that we knew would grow and cause a color change in the DrySlide assay.
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LAB 9 EX 1 How are mitochondria in eukaryotic cells similar to bacteria that use aerobic respiration?
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Reaction occur in the mitochondria in both cells and both types of reactions produce ATP.
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LAB 9 EX 2 catalase Which bacterial plates contained catalase positive bacteria?
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sink only
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LAB 9 EX 2 Which plates contained catalase negative bacteria?
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shoe and phone
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LAB 9 EX 2 Did the colonies from each plate show the same reaction? Why or why not? How can you interpret this?
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No. There are separate types of bacteria in each section and they may not grow at the same rate. For example, in my dish with the sink and the shoe, there were no traces of bacteria on the shoe side but a lot of bacteria on the sink side. If the bacteria were to have multiplied and spread due to being in the same dish, it would have given me a positive result on the shoe side which it did not.
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LAB 9 EX 2 Were any controls used in this experiment?
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The control portion of a petri dish was used but we did not test to see if it reacted with the hydrogen peroxide, so there was technically no control in this case.
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LAB 9 EX 2 What reaction would you expect when performing a negative control in the catalase assay?
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I would expect no reaction from a negative control in the catalase assay. No bubble should form.
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LAB 9 EX 2 What reaction would you expect when performing a positive control in the catalase assay? What would it mean if a known catalase positive bacteria did not produce bubble after the addition of the H2O2?
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I would expect a reaction to occur and bubbles to form. It would mean that a different strain of bacteria was used that doesn't react to hydrogen peroxide.
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LAB 9 EX 2 If flavoprotein performs the alternate pathway of transferring an electron directly to oxygen rather than to the next component of the ETC, what toxic molecules are produced? What other effects of bypassing the ETC have on the cell?
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It can produce a superoxide radical which is toxic. You can further damage the mitochondria of a cell using premature aging.
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LAB 10 EX 1 Mold Growth on Bread and Fruit-What are Optimal Conditions for Fungal Growth? Which condition produced the most mold growth?
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bright, sugar
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LAB 10 EX 1 Which condition produced the fastest mold growth?
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bright, sugar
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LAB 10 EX 1 Which condition produced the least mold growth?
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dark, lemon
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LAB 10 EX 1 Which condition produced the slowest mold growth?
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dark, lemon
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LAB 10 EX 1 Based on your results, what conditions are most favorable for mold growth on bread and apples?
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bright, sugar
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LAB 10 EX 1 Would these conditions apply to all fungal growth?
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No. Different conditions produce different results depending on the fungi trying to be grown.
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LAB 10 EX 1 How would changing the type of bread (fresh from the bakery, no preservatives versus prepackaged with preservatives) affect the results?
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Bread with preservatives would take longer to grow mold than bread without preservatives.
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LAB 10 EX 1 How would changing the temperature at which the bags were incubated affect the results?
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Changing the temperature would either produce more mold or inhibit the growth of the mold depending on the conditions and the type of mold attempting to be grown.
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LAB 10 EX 1 How would you test these predictions (hypotheses)?
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By performing the same experiment. However, I would change the conditions (temperature, light, dark, etc.) and recording the difference in growth.
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LAB 10 EX 1 Look up the pH of lemon juice and vinegar. Based on your results and your knowledge of favorable environmental conditions for fungal growth, what can you conclude about the effect of pH on growth? How would making the pH more basic effect the growth?
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A lower pH will inhibit mold growth. A more basic pH would cause considerably more growth.
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LAB 10 EX 1 What is the source of the mold that grew on the samples?
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Mold spores. Most like from the surrounding environment. The bread provides the nutrients, and the water and light help aid the growth.
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LAB 11 EX 1 Assessing the Bacterial Load of Milk with Methylene Blue Which sample took the least amount of time to become white? Why do you think this is so?
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The 4 hour sample. Because it contained living bacteria.
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LAB 11 EX 1 Which sample took the greatest amount of time to become white? Why do you think this is so?
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zero hour. because it contained less bacteria
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LAB 11 EX 1 How does refrigeration affect the amount of bacteria present in milk?
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Refrigeration keeps the milk cold. Making difficult for bacteria to grow and thrive.
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LAB 11 EX 1 How does pasteurization affect the amount of bacteria present in milk?
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Pasteurization kills harmful bacteria. It can also kill some good bacteria. Ultimately reducing the number of bacteria.
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LAB 11 EX 1 What difference, if any, would this assay show if performed using raw (unpasteurized) milk?
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Many differences. More bacteria being present would bring dramatically different results.
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LAB 11 EX 1 What differences, if any, would this assay show if performed using milk that was past its expiration date and had an off-putting odor?
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This would also cause many differences. If the milk was expired it would have more bacteria present. Causing the results of the above experiment to be more rapid in the end.
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LAB 11 EX 2 Yogurt Preparation Make a graph of the pH changes over the course of the experiment. How does the pH change at each step? Why?
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The pH slowly decreases with each step. The decrease is caused by the increase of heat.
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LAB 11 EX 2 What attributes does lactic acid confer to yogurt?
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The lactic acid that is produced from the fermentation of lactose contributes to the sour taste and the characteristic texture of yogurt.
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LAB 11 EX 2 How does the consistency of the milk change during the production of yogurt? What facilitates this change?
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The milk begins to get thick and chunky (a yogurt like texture). The lactic acid that is produced during the fermentation of the lactose.
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LAB 11 EX 2 What are the breakdown products of lactose?
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glucose and galactose
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LAB 12 EX 1 Observe the two sets of plates. Determine the CFU/mL of each solution (Normal and Heated), remembering to use a plate that has between 30-300 colonies (hint: refer to Lab 8: Quantitation ). Is there a difference in the CFU/mL counts for the two treatment conditions? What accounts for this difference? Normal 10%: 150 Normal 1%:100 Normal 0.1%:75 Normal 0.01%:50 Heated 10%: 20 Heated 1%:15 Heated 0.1%:5 Heated 0.01%: 1
answer
The difference was that the heat killed some of the bacteria.
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LAB 12 EX 1 How are the colonies from the two treatment conditions different (e.g., size, color, shape, etc.)?
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The normal colonies were small, yellow, off white, and red. They were shaped like irregular circles. The heated colonies were small to medium in size. All were medium and off white. They were also shaped like irregular circles
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LAB 12 EX 1 What effect does the heating and cooling procedure have on the bacteria present in the soil samples?
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the heat terminated some of the bacteria
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LAB 12 EX 1 What is the source of the bacterial colonies in the heated samples?
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bacillus
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LAB 12 EX 2 What effect does heat treatment have on the number of Gram positive and Gram negative bacteria?
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Gram positive bacteria are able to withstand heat better than gram negative bacteria.
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LAB 12 EX 2 What effect does heat treatment have on the number of positive endospores?
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The heat treatment has a little effect on endospores
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LAB 12 EX 2 What effect does heat treatment have on the number of different bacteria isolated?
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The heat treatment lowers the number of bacteria that are isolated.
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LAB 12 EX 2 What conditions favor endospore formation?
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Endospores are mostly found in soil and water and can survive for long periods of time. It is safe to assume endospores can survive a variety of different conditions.
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LAB 12 EX 2b Which treatment condition produced more catalase positive bacteria? What can explain the difference in the number of catalase positive bacteria?
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Heated. The heated contained more bacillus bacteria which are catalase positive.
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LAB 12 EX 2b Write out the reaction that is catalyzed by the enzyme catalase. Why is this enzyme important?
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2H2O2 --> 2H2O + O2 It plays an important role in protecting the cell from oxidation destruction.
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LAB 12 EX 2b What would you conclude if all samples you tested were catalase negative?
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That the heat killed all of the endospore bacteria.
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LAB 12 EX 2b What types of controls could you include in the experiment ensure all components of the system are working properly?
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Be certain the peroxide used was 3% and not expired or old.
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LAB 12 EX 3 Where does carbon come from in a Winogradsky column?
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the shredded newspaper
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LAB 12 EX 3 why is carbon important?
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It starts the process of anaerobiosis
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LAB 12 EX 3 What purpose does calcium sulfate serve in the Winogradsky column
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it serves as an electron acceptor
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LAB 12 EX 3 Why is a hydrogen sulfide gradient generated from bottom to top in a Winogradsky column
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Sulfate-reducing bacteria utilizing fermentation by-products such as alcohols of anaerobes as carbon sources
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LAB 12 EX 3 How is photosynthesis different between cyanobacteria (growing at the top of the column) and green and purple sulfur bacteria (growing near the bottom 1/3 of the column)?
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Photosynthetic cyanobacteria and algae: in water layer; use light for energy, release oxygen gas as by-product, fix carbon dioxide, and produce organic molecules Green sulfur bacteria: belong to the obligate anerobic photoautotrophic bacteria. Purple sulfur bacteria: belong to the proteobacteria capable of photosynthesis
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LAB 13 EX 1 Which DNA bases pair with each other?
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A with T C with G
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LAB 13 EX 1 How is DNA information used to make proteins?
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Enzymes read the information in a DNA molecule and transcribe it into an intermediary molecule also called messenger ribonucleic acid(mRNA). The information contained in the mRNA molecule is translated into the language of amino acids. This language tells the cell's protein-making machinery the exact order in which to link the amino acids to produce a specific protein.
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LAB 13 EX 1 What is the purpose of the following reagents in the experiment: Salt in the DNA extraction solution?
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The salt neutralizes the negative charges on the DNA enabling the DNA strands to stick together. It also causes proteins and carbohydrates to precipitate.
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LAB 13 EX 1 what is the purpose of the following reagent in the experiment: detergent in the DNA extraction solution
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It pulls apart the fats, or lipids as well as the proteins that make up the membranes surrounding the cell and nucleus.
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LAB 13 EX 1 what is the purpose of the following reagent in the experiment: ethanol
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Everything except the DNA will dissolve in ethanol. The ethanol pulls water from the DNA molecule so that it then collapses in on itself and precipitates. The DNA will become visible as white mucous strands that can be spooled with the wooden applicator stick.
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LAB 13 EX 1 What else might be in the ethanol/aqueous interface? How could you eliminate this?
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When you precipitate the solution with cold ethanol 70% you would have a mix of DNA, RNA and some soluble salts. You could eliminate this by straining with cheese cloth so the DNA/RNA forms a pellet and drain the liquid out.
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LAB 13 EX 1 What is the texture and consistency of the DNA?
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thin and wirey
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LAB 13 EX 1 Is the DNA soluble in the aqueous solution or alcohol?
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No
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LAB 13 EX 1 What surprised you about DNA replication and protein synthesis?
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It surprised me that extracting DNA was so easy. I would never have thought it was something I could do.
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LAB 13 EX 2 What is the expected size of the plasmid plus the cut foreign DNA?
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720 bp
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LAB 13 EX 2 What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work?
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Ligase. It reforms the phosphodiester backbone.
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LAB 13 EX 2 How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?
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A gene could be copied by using RF cloning.