Study Questions Your name (initials), date, name of organism, name of medium

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1 Safety and Introduction 1. What information must you include when labeling your culture plates? Your name (initials), date, name of organism, name of medium 2. Where on an agar plate do you write the label? The label is written on the base (the half with the medium). Written directly on the plastic using a permanent marker. 3. Where is each of the following types of wastes disposed? a. slide with bacterial smear Slides made in lab are disposed of in the beaker with 10% bleach solution b. slide with wet mount Slides made in lab are disposed of in the beaker with 10% bleach solution c. used cotton swab Items contaminated with biologicals are disposed of in an autoclave bag d. used Petri dishes Items contaminated with biologicals are disposed of in an autoclave bag e. cotton swab wrapper If uncontaminated (as it should be), regular trash bin; if contaminated (as it shouldn t be), autoclave bag 4. Why are plates incubated upside down? If placed lid side up, condensation can form on the lid. This condensation could fall back onto the surface of the agar. This position further prevents contamination. 5. Where in lab are microscopes stored? Microscopes are stored in the assigned cabinet at the end of the bench. 6. When should you wash your hands in lab? Hands should be washed after entering, before leaving, and at any time you think it is necessary. 7. What needs to be done to your bench at the beginning of and end of class? The bench needs to be disinfected. 8. What do you use to write a label on an agar plate? Plates are labeled with a permanent marker. 9. What does ubiquitous mean? It means that microbes are everywhere.

2 Safety and Introduction 10. What tools should be found in your equipment tray? Inoculating loop, inoculating needle, clothespins, ruler, forceps, bibulous paper, pen, pencil, permanent marker, wax pencil 11. Is the use of tablets or laptops allowed during lab? No. Laptops and other items are a potential means of transporting bacteria outside the lab. 12. What protective equipment are you required to wear while staining? Gloves and goggles are required to be worn while staining slides and when working with liquid cultures. 13. Where are backpacks and personal items stored while in lab? All personal items are placed in the storage cubicles for the duration of the lab period

3 Microscopy 1. In what position should the stage be when putting a new slide on the microscope? The stage should be fully lowered. 2. What lens should be clicked into position with a new slide? The 4X objective should be clicked in place. 3. What is the correct sequence of steps to focus on a slide with the 40X objective lens? 1) With the 4X objective in position, and the stage fully lowered, place the slide on the stage. 2) Use the coarse focus adj. knob to move the stage all the way up. 3) While looking through the oculars, use the coarse focus adj. knob to lower the stage until the object is in focus. 4) Rotate nosepiece to the 10X objective 5) Use the fine focus adj. knob, if needed, to bring object into focus 6) Rotate nosepiece to the 40X objective 7) Use the fine focus adj. knob, if needed, to bring object into focus 4. To use the oil immersion lens, when should oil be added to a slide? Oil is added to the slide after focusing with the 40X objective, but before placing the oil immersion lens into position. 5. What is the total magnification when using the 100X objective and a 10X ocular lens? The total magnification is 1000X 6. What items are used to clean the oil immersion lens? Lens paper and lens cleaner 7. With what objectives lenses is the coarse focus knob used? The coarse focus knob is used ONLY with the 4X (scanning) objective.

4 Classification 1. For each of the following families, indicate the shape and Gram-stain reaction for the bacteria found in that family. a. Bacillaceae Bacilli, Gram-positive b. Clostridiaceae Bacilli, Gram-positive c. Enterobacteriaceae Bacilli, Gram-negative d. Spirochaetaceae Spirochetes, Gram-negative e. Streptococcaceae Cocci, Gram-positive 2. Name the disease caused by each of the bacteria listed below. a. Bacillus anthracis - Anthrax b. Borrelia burgdorferii Lyme disease c. Clostridium botulinum - Botulism d. Clostridium tetani - Tetanus e. Corynebacterium diphtheria - Diphtheria f. Shigella dysenteriae Shigellosis, Dysentery g. Staphylococcus aureus Impetigo, Carbuncles, Boils, Scalded Skin Syndrome, Furuncles and more h. Streptococcus pyogenes Strep throat, necrotizing fasciitis i. Treponema pallidum - Syphilis j. Yersinia pestis Bubonic plague, pneumonic plague 3. When writing a scientific name in your lab manual, how is it formatted to indicate it is a scientific name? When hand-written, scientific names must be underlined.

5 Bacterial Cell Morphology 1. What are the most common shapes of bacterial cells? The two most common are coccus and bacillus. Other typical shapes include vibrio, spirillum and spirochete. 2. How can you differentiate between coccus and bacillus shape? Cocci are spherical; bacilli are longer than they are wide (rod-shaped) 3. What are the common arrangements of bacterial cells? The two most common cellular arrangements are strepto- (chains) and staphylo- (grapelike clusters). Other arrangements that may be seen are diplo- (pairs), tetrads (groups of four in a square) and sarcinae (groups of eight in a cube) 4. Do all bacterial cells have an arrangement? No. In fact, the lack of a cellular arrangement is more common than an arrangement. 5. What cellular arrangements are seen in cocci? Cocci may be found in strepto-, staphylo-, diplo-, tetrads and sarcinae. 6. What cellular arrangements are seen in bacilli? Bacilli may be found in strepto- and diplo- 7. Draw the cell shapes and arrangements for each of the following: a. staphylococcus grape-like clusters of cocci b. streptococcus chains of cocci c. diplobacillus pairs of bacilli d. streptobacillus chains of bacilli 8. What is the difference between the terms bacillus and Bacillus? The term bacillus is a bacterial shape; Bacillus is a genus of bacteria. 9. Can you positively identify a bacterial species using cell morphology alone? No.

6 Aseptic Technique 1. What is the purpose of using aseptic technique? Aseptic technique is used to prevent contamination of the bacterial cultures and media used in lab. 2. The incinerator is used to sterilize what microbiology tools? The incinerator is used to sterilize the inoculating loop and the inoculating needle 3. How are forceps disinfected for use in lab? The tips of the forceps are dipped in alcohol. Heat from the incinerator is used solely to increase the rate of evaporation of the alcohol from the forceps. 4. Why is the lab bench disinfected before and after performing lab exercises? The bench is disinfected before conducting exercises to decrease the number of background microbes that could contaminate media while working. The bench is disinfected after to prevent spread of bacteria that were used in lab. 5. Why must you wash your hands before exiting the lab? To prevent spread of bacteria used in lab. Hygiene is an important step in preventing opportunistic infections and the transmission of microbes. 6. What steps are taken to minimize the risk of contamination of broths in test tubes? Tubes are only opened when necessary; caps are held in the hand and never set down on the bench or other surface. The wire of inoculating loops is fully sterilized before performing any culture transfers. 7. Why should test tube caps never be placed on the bench? The benchtop is always a potential source of contamination (even after disinfection) 8. What steps are taken to minimize the risk of contamination of agar in a petri dish? Plates are only opened as needed and closed immediately when not in use. Plates are incubated and stored upside down. We do not breathe on plates when they are opened.

7 Culture Transfer and Streak Isolation 1. What is the purpose of the streak isolation technique? The streak isolation technique is used to produce individual colonies. Individual colonies represent pure culture; individual colonies are needed to observe colony morphology. 2. Explain why an individual bacterial colony is considered a pure culture. A bacterial colony represents a population of cells descended from one or a few colony forming units. As such, all the cells in a colony are clones of one another. 3. Why must you sterilize your loop in between quadrant streaks? The loop is sterilized to remove remaining bacteria from the loop picked up during streaking (and to maintain sterility). 4. What are the steps in the streak isolation technique? 1) Sterilize a loop. Touch the loop to a sterile area of the plate. Transfer a small amount of bacteria from the source plate onto quadrant 1 of the streak plate using a zig-zag motion. 2) The loop is sterilized. 3) The sterile loop is used to subculture and streak cells from quadrant 1 onto quadrant 2 in a zig-zag motion 4) The loop is sterilized. 5) The sterile loop is used to subculture and streak cells from quadrant 2 onto quadrant 3 in a zig-zag motion 6) The loop is sterilized. 7) The sterile loop is used to subculture and streak cells from quadrant 3 onto quadrant 4 in a zig-zag motion 8) The loop is sterilized. 5. Can you positively identify a bacterium based on colony morphology alone? No. 6. What is the most common color of bacterial colonies? Most bacterial colonies are off-white. Additionally, they are generally round, convex, glossy and opaque.

8 Bacterial Smears and Simple Staining 1. What information regarding cell morphology can a simple stain provide? Simple stains provide information only for shape and cellular arrangement. 2. What information regarding cell morphology is not provided by a simple stain? Simple stains cannot determine if a cell is Gram-positive or Gram-negative, nor if endospores or other cellular features are present. 3. A lab mate performs a simple stain with crystal violet. They observe that all the cells are purple and conclude that their unknown bacterium is Gram-positive. Is she correct? Why or why not? No. A Gram-stain was not performed. With a simple stain, all bacteria will have the color of the stain used, regardless of the cell wall structure. 4. What is the purpose of air-drying a smear prior to heat-fixing? Air-drying a smear is used to partially dehydrate the cells. This prevents the cells from lysing during heat fixing, retaining the shape of the cells. 5. What is the purpose of heat-fixing a smear? Heat-fixing further dehydrates the cells in a smear and strengthens their bond to the microscope slide. In addition, the heat fix will kills the cells important when making smears of pathogens. 6. You conclude that your unknown bacterium is staphylococcus with respect to arrangement and shape. Your lab partner disagrees, noting that the bacteria are overcrowded on the smear. What went wrong during smear production, and why is it that you cannot draw a conclusion regarding arrangement in this case? When cells are overcrowded (too dense) on a smear, they will appear to be clustered and will lead to an incorrect conclusion that they have a staphylo- arrangement. In order to determine if an arrangement exists, you must have a single layer of cells with open background (areas of the slide without cells) around them.

9 The Gram Stain 1. Explain how the Gram stain works. In your discussion, include how Gram-negative and Grampositive bacteria react with the reagents of the Gram stain. The Gram stain works because the reagents react differently with the components of Gram-positive and Gram-negative cell walls. Crystal violet is retained within the thick peptidoglycan of Gram-positive cells, while crystal violet is removed from Gram-negative cells by the decolorizer. The decolorizer damages the outer membrane of Gram-negative bacteria, which results in Gram-negative bacteria appearing colorless after the decolorizer step. Gram-positive bacteria retain the crystal violet when the decolorizing step is done properly. Safranin is used to stain the colorless Gram-negative cells pink, while Gram-positive cells remain purple after the counterstain is added. 2. Complete the table below with regard to the reagents in the Gram stain. Role Reagent Function Primary Stain Crystal Violet Stains all bacteria purple Mordant Decolorizer Counterstain Gram s Iodine Alcohol/Acetone Safranin Forms a complex with crystal violet. This complex is more likely to become trapped in the peptidoglycan of Gram-positive cells Damages outer membrane of Gram-negative bacteria, removing the crystal violet; results in Gram-negatives becoming colorless Dehydrates peptidoglycan of Gram-positives, trapping crystal violet-iodine complex Stains Gram-negative bacteria pink. Gram-positives remain purple from crystal violet. 3. What information regarding cell morphology is provided by a Gram stain? The Gram stain provides information regarding cell wall structure (Gram-negative or Gram-positive), cell shape and cellular arrangement. 4. You perform a Gram stain on a known Gram-positive culture. Your results indicate that the cells are Gram-negative. What may have happened during the procedure? It is most likely that the smear was over-decolorized, removing the crystal violet from the Gram-positive bacteria

10 The Gram Stain 6. Can you definitively identify a bacterium using only the information given by a Gram stain? No. 7. You perform a Gram stain on a known Gram-negative culture. Your results indicate that the cells are Gram-positive. What may have happened during the procedure? It is most likely that the smear was under-decolorized, resulting in a failure to remove the crystal violet from the Gram-negative bacteria, causing them to be purple in color

11 Endospore Stain 1. What is an endospore? A dormant, metabolically inactive bacterial cell that is resistant to harsh environmental conditions such as dehydration, freezing, UV light and heat. 2. What is the function of an endospore? Endospores serve to protect the DNA and the cell during suboptimal environment conditions when vegetative growth would be inhibited. 3. What reagents are used as the primary stain, decolorizer and counterstain in the endospore stain? Primary stain malachite green Decolorizer water Counterstain - safranin 4. What are the purposes of the primary stain, decolorizer and counterstain in the endospore stain? Primary stain stains the endospore coat Decolorizer removes primary stain from vegetative cells Safranin stains vegetative cells pink 5. Would you expect a Gram-negative bacterium to produce endospores? No. Sporulation (endospore production) is a feature that is found only in certain Families of Gram-positive bacteria. 6. What Bacterial Families are known have species that are known to produce endospores? Bacterial Families that produce endospores include the Bacillaceae, Clostridiaceae and Paenibacillaceae 7. What modification to heat-fixing is done prior to the endospore stain? The heat-fix step is extended to two minutes 8. What is the purpose of this modification? The extended heat-fix is used to soften and damage the endospore coat so that the primary stain works more effectively 9. Assume you have prepared an endospore stain of an organism that is known to produce endospores, but no endospores are observed. How could you explain this observation? It is most likely that the culture has yet to enter the growth phase where endospores are produced. This usually happens if the culture is not old enough.

12 Motility 1. What structure is commonly used by bacteria for motility? Flagella 2. Are all bacteria motile? No.

13 Bacterial Enumeration 1. What are the individual and overall dilution factors for each of the tubes in the image below? Individual dilution factors: A. 1/100 B. 1/100 C. 1/10 D. 1/10 E. 1/2 Overall dilution factors A. 1/100 B. 1/10000 C. 1/ D. 1/ E. 1/ If plate F has 134 colonies, what is the CFU/mL of the original sample in the flask? 1.34 x 10 8 CFU/mL

14 Bacterial Enumeration 2. You have performed a serial dilution followed by spread plating 0.1 ml of sample from each tube. The data appear below. Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Dilution Factor of Tube Number of Colonies TNTC TNTC TNTC Which plate would you use to determine the CFU/ml? Plate 4 What is the CFU/ml of the original sample? 4.25 x 10 7 CFU/mL 3. Why do we use plates with between colonies in enumeration? Plates with more than 300 colonies are difficult to count, and plates with less than 30 colonies give statistically unreliable numbers of colonies to count. 4. Why is the term aerobic plate count most appropriate for the enumeration method used in lab? In lab, the plates are incubated aerobically; therefore, only organisms that can grow under aerobic conditions would grow and be counted. 5. What are the four phases in the standard bacterial growth curve? Lag, log (exponential), stationary, death (decline) 6. What is a colony forming unit (CFU)? A colony forming unit is a live (viable) cell or small group of cells that gives rise to a colony. Arrangement, capsules and other cellular features have an effect on how bacteria are spread and deposited on the surface of a plate. 7. Which would you expect to have more colonies after spread-plating, a 0.1 ml of a 1/10 dilution from a sample with 2500 CFU/mL, or 0.1 ml of a 1/100 dilution from a sample with 2500 CFU/mL? 0.1 ml of a 1/10 dilution would be expected to have more colonies.

15 Bacterial Enumeration 8. Calculate the CFU/mL for each of the following scenarios. a. 150 colonies on a plate from a 0.1 ml sample from a tube with an overall dilution factor of 1/ x 10 4 CFU/mL b. 34 colonies on a plate from a 0.1 ml sample from a tube with an overall dilution factor of 1/ x 10 5 CFU/mL c. 88 colonies on a plate from a 0.1 ml sample from a tube with an overall dilution factor of 1/ x 10 6 CFU/mL

16 MAC, MSA and PEA 1. You have a suspected gram-positive culture. Would it be more appropriate to grow it on MacConkey agar or PEA agar to confirm these results? It would be better to grow it on PEA agar; growth would confirm it was Gram-positive. PEA selects for Gram-positive bacteria. 2. In addition to selecting for halophilic bacteria, MSA can detect the fermentation of mannitol. What color will the plate turn if mannitol is fermented? MSA will turn yellow if mannitol is fermented. 3. You discover that an unidentified bacterium you have been given is Gram-negative. Would you use MAC, PEA or MSA to determine if the bacterium ferments lactose? You would use MAC to determine if a Gram-negative can ferment lactose. 4. If an organism does not grow on MacConkey, what conclusions can you draw regarding its ability to ferment lactose? You cannot draw any conclusions regarding lactose fermentation. If an organism does not grow on MacConkey, there is no opportunity for fermentation to occur. 5. When using selective media, why must a culture also be grown on TSA? You must always use TSA as a positive control for growth. No growth on a selective plate could be due to plating a non-viable culture. Without a positive control for growth, you cannot determine if the bacterial culture was selected against. 6. What are the selective reagents in PEA, MAC and MSA? PEA 2.5% phenylethyl alcohol MAC bile salts, crystal violet MSA 7.5% NaCl 7. What are the differential reagents in MAC and MSA? MAC lactose, neutral red MSA mannitol, phenol red

17 KOH 1. Why does KOH become gooey in the presence of Gram-negative cells? The Gram-negative cells lyse, releasing proteins, DNA and other molecules that then denature in the presence of KOH. 2. What is denaturation? Loss or change in secondary, tertiary and quaternary structure in proteins and other molecules. 3. As a beginning microbiologist, which do you think is more reliable, a Gram stain or the KOH test? The KOH test is more reliable. One is less likely to make mistakes in the KOH test than in the Gram stain.

18 Thioglycollate 1. What oxygen indicator is used in thioglycolate broth? Why is this oxygen indicator used? Resazurin. It is used to indicate that the top portion of the broth contains oxygen. Obligate aerobes will only grow if oxygen is present, and are expected to grow in this oxygen-containing region. 2. What component of thioglycolate broths maintains anaerobic conditions in the lower portion of the broth? Sodium thioglycolate 3. Why is 0.05% agar added to thioglycolate? Agar is added to prevent the mixing of the upper aerobic portion of the medium with the lower anaerobic portion of the medium. 4. Where in a thioglycolate broth tube would you expect to find growth for the following types of bacteria? a. facultative anaerobes Growth will be in both the aerobic and anaerobic portions. Growth in the aerobic portion is generally greater than that in the anaerobic portion. b. obligate aerobes Growth only in the aerobic portion. c. obligate anaerobes Growth only in the anaerobic portion. d. aerotolerant anaerobes Growth will be in both the aerobic and anaerobic portions. Typically there are no differences in the amount of growth seen in the two areas. 5. What type of metabolism is used by obligate aerobes? Aerobic respiration is the only ATP producing strategy used by obligate aerobes. 6. What type of metabolism is used by facultative anaerobes? Facultative anaerobes will use aerobic respiration when oxygen is present; will switch to fermentation when no oxygen is present. 7. What type of metabolism is used by obligate anaerobes? An obligate anaerobe will use either anaerobic respiration or fermentation, but will not use both. 8. What type of metabolism is used by aerotolerant anaerobes? Aerotolerant anaerobes typically use fermentation to make ATP. 9. Would you expect an obligate aerobe to be capable of using fermentation to make ATP? No. Obligate aerobes are incapable of fermentation.

19 Sulfide-Indole-Motility Medium 1. What are the differential reagents in SIM? Sulfide production sodium thiosulfate and ferrous ammonium sulfate Indole production casein peptone (contains tryptophan) Kovac s reagent is not an ingredient of SIM, but must be added after incubation to detect indole production. 2. Can SIM be used to detect motility in obligate aerobes? SIM typically cannot be used to detect motility in obligate aerobes. Obligate aerobes do not typically grow well in agar stabs. 3. Indole is an end product of the catabolism of what amino acid? Tryptophan. 4. Hydrogen sulfide reacts with what ingredient to form ferrous sulfide? Ferrous ammonium sulfate. 5. Why does ferrous sulfide form a precipitate? Ferrous sulfide is insoluble in aqueous solution.

20 Nutrient Gelatin 1. What is the substrate of gelatinase? Gelatin (collagen) 2. How is gelatinase used as a virulence mechanism? Gelatinase breaks down collagen (connective tissue). This mechanism can be used for invasion or nutrient acquisition. 3. Why do you think agar is used to solidify media? Gelatin is liquid at temperatures above 28 C, so cannot be used to produce plates. 4. How do you interpret the nutrient gelatin assay? If gelatinase is present, the medium will be liquid. In some cases, a liquid crater forms on the surface. 5. Why must observations for nutrient gelatin be done at temperatures lower than 28 C? The gelatin will be solid at temperatures below 28 C. At temperatures above 28 C gelatin is liquid and would give a false positive reading. 6. What type of biological molecule is collagen? Collagen is a protein.

21 Oxidase and Catalase 1. What enzyme is detected by the oxidase DrySlide test? Cytochrome oxidase C 2. In what pathway is this enzyme used? Cytochrome oxidase is commonly found in the electron transport system of aerobic respiration. 3. Why is there a 20 second time limit for the oxidase DrySlide test? After 20 seconds, a change to blue could be due to factors other than cytochrome oxidase and should be considered a false positive. 4. What is the function of catalase? Catalase is used by bacteria to detoxify peroxide anions. 5. Why is bubbling used to determine if catalase is present? The catalase reaction release molecular oxygen, which is easily observable as bubbles.

22 Starch Agar 1. What enzymes are used by bacteria to hydrolyze starch? Amylases and glucosidases 2. Why is iodine added to the starch plate? Iodine reacts with starch in the plate making zones of clearing easier to observe. 3. Why does a zone of clearing form around organisms that can hydrolyze starch? The exoenzymes are secreted by the bacteria and digest the starch in the vicinity of the culture. 4. Is starch agar a selective or differential medium or both? Starch agar is differential. 5. What ingredient is added to nutrient agar to make starch agar? Soluble starch.

23 Phenol Red Carbohydrate Broths 1. Phenol red turns what color in an acidic solution? Yellow. 2. You have an unknown culture and do not have Gram stain results. You are allowed one medium to determine if it can ferment lactose. Would you use phenol red lactose broth or MacConkey agar? Phenol red lactose broth. MAC will not give any results regarding fermentation if you are working with a Gram-positive organism. 3. You have grown an obligately aerobic culture in phenol red glucose broth, and some of the media has turned yellow. How would you explain this? Obligate aerobes cannot ferment carbohydrates. However, the metabolic pathways found in obligate aerobes can produce acids. It is the presence of these acids that turn the phenol red yellow (the broth will often appear orange in these instances). 4. A culture ferments mannitol and glucose to acid, but not lactose and sucrose. Is this possible? Why? Yes. The genetics of a given bacterium may allow it to produce the enzymes to metabolize some sugars but not all sugars. 5. What type of molecule is used as the final electron acceptor in fermentation? Organic molecules are used as the final electron acceptor in fermentation. 6. You check your fermentation broth and find that the top is red and the bottom is yellow. What can you conclude? The tube has been incubated too long. The organism originally produced acid and the broth was yellow. As bacteria continue to grow, the carbohydrate in the tube will run out and they will metabolize other compounds - this will raise the ph, turning the top of the tube red. 7. What is the purpose of the Durham tube? To capture gases produced via fermentation. 8. What are the end products in each of the following types of bacterial fermentation? a. homolactic fermentation Lactic acid b. heterolactic fermentation Lactic acid, ethanol, CO2 c. mixed acid fermentation Lactic acid, acetic acid, succinic acid, formic acid, ethanol, CO2, H2 d. 2,3 butanediol fermentation 2,3 butanediol, lactic acid, ethanol, CO2, H2

24 Phenol Red Carbohydrate Broths 10. What observations would you expect to see in Phenol Red Glucose Broth for each of the following types of fermentation? a. homolactic fermentation Yellow broth, no bubble in Durham tube b. heterolactic fermentation Yellow broth, bubble in Durham tube c. mixed acid fermentation Yellow broth, bubble in Durham tube d. 2,3 butanediol fermentation Yellow broth, bubble in Durham tube

25 Microbial Control 1. How do you interpret the disk diffusion assay for antibacterial efficacy? Measure the diameter of the zone of inhibition. 2. What would you hypothesize to explain why there may be differences in the usefulness of antibiotics on different species of bacteria? Inherent differences in the physiology and structure of bacteria lead to different reactions to antibiotics. For example, penicillin is not effective against Gram-negative bacteria. 3. What is meant by a broad-spectrum drug? A broad spectrum drug is one that is effective against multiple types of bacteria. Often, broad spectrum drugs have a mode of action that targets common features of many bacteria, such as ribosomal subunits. 4. Is UV effective for control of all bacteria? Explain. No. UV is not effective against endospores. 5. How does UV light affect bacteria? UV light produces thymine dimers. Thymine dimers disrupt the function of DNA.

26 Bacterial Transformation 1. What is transformation? The uptake of DNA molecules from the environment by a bacterial cell. 2. What promoter controls the GFP gene? The AraC promoter. 3. What factors effect transformation of bacteria? These factors include: Amount of bacteria used, stage of bacterial growth, amount of DNA used, use of transformation buffer, temperature of heat shock, length of heat shock, temperature of bacteria when heat-shocked. 4. What is meant by competency? Competency is the ability of a bacterium to undergo transformation. In the laboratory, we induce artificial competency. Some bacteria are naturally competent. 5. What is the purpose of a selective marker? The selective marker allows for the selection of bacteria that contain a plasmid, that is, bacteria that have been transformed. In the pglo exercise, the selective marker is ampicillin resistance. Only bacteria that contain the pglo plasmid will grow on the LB/Amp plates. 6. What is the purpose of the heat shock step? The heat shock creates transient openings in the cell wall that allow the plasmid to diffuse into the cell. 7. Why are cells held on ice prior to heat shock? The cells will acclimate to the reduced temperature, making the heat shock more effective. 8. What was the purpose of the calcium chloride transformation solution? Calcium chloride allows the plasmid to come into proximity to the cells, increasing the efficiency of transformation. 9. What is a promoter? A promoter is a DNA sequence used where RNA polymerase binds to begin transcription.

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