Study Guide

Question 1

• Name a bacterial species that you have encountered in this course that can be found in each of the following environments.
• Give its cell shape (coccus, bacillus) and Gram reaction.
• Is it a lactose fermenter? (Yes, No, or Don’t Know)

Question 2

After performing a serial dilution of Pseudomonas aeruginosa, you plate 0.1 ml of a 10^‑5 dilution. After incubating for 24 hours at 37 C, you count 112 colonies.

• A. How many CFU per ml in the 10‑3 dilution tube?
• B. How many CFU per ml in the original culture?
• C. This species gives you an oxidase-positive reaction on a dry slide. What does that tell you about its electron transport system?
• D. Is this species a psychrophile, mesophile, or thermophile?
• E. What kind of compound does this bacterium secrete that gives it a green color when you grow it at 37oC?

Question 3

If you wanted to achieve a final dilution of 5 x 10^-3 in a test tube, how might you best set this up while keeping dilution tubes to a minimum and using between 1.0 ml and 0.1 ml transfer volumes?

Question 4

If the gradation marks on your ocular micrometer were found equal to 1.5 μm in length and you were measuring a Gram (+) rod that was 30 marks by 12 marks:

• what are the actual dimensions of this bacterium?
• Is this a reasonable finding for any of the species we observed in our course?

Question 5

Describe exactly how you would combine the use of a spectrophotometer and dilution plating to make a standard curve estimating CFUs/unit OD.

Question 6

For EACH of the selective and differential media you’ve encountered, discuss the chemical reactions taking place as well as the putative identity of the bacteria producing those reactions.

Question 7

Questions A-F below pertain to gram staining of a Streptococcus spp

• A. What color are the cells after the first staining step and what primary stain is used?
• B. What is the color of the cells after the application of the mordant and what is the mordant?
• C. What is the color of the cells after the application of the decolorizer and what is used to decolorize?
• D. What is the color of the cells after the second staining step and what secondary stain is used?
• E. What would be the final result if you decolorized too much?
• F. What would be the final result if you mistakenly used the secondary stain first and the primary stain second?
• G. What would be the final result if you mistakenly decolorized too long?
• H. What would be the final result if you Gram stained old cells from a plate that was stored in the refrigerator for a week?

Question 8

Questions A-F below pertain to gram staining of a Klebsiella spp

• A. What color are the cells after the first staining step and what primary stain is used?
• B. What is the color of the cells after the application of the mordant and what is the mordant?
• C. What is the color of the cells after the application of the decolorizer and what is used to decolorize?
• D. What is the color of the cells after the second staining step and what secondary stain is used?
• E. What would be the final result if you decolorized too much?
• F. What would be the final result if you mistakenly used the secondary stain first and the primary stain second?
• G. What would be the final result if you mistakenly decolorized too long?
• H. What would be the final result if you Gram stained old cells from a plate that was stored in the refrigerator for a week?

Question 9

• What staining technique have we used that requires the use of Maneval’s Solution?
• Is it a cationic or anionic stain?
• Is it attracted to or repelled by the surface of most cells?

Question 10

What is a negative stain and what bacterial cell structure can it be used to visualize?

Question 11

You have an unknown sample of cells that are rod-shaped and stains Gram positive, and appears to have small clear-looking spots in each cell.

• What might these clear spots be?
• What staining technique might you perform to verify your suspicions?

Question 12

• Give an example of a complex medium that you have used in class.
• How does this kind of medium differ from a defined medium?

Question 13

• Autoclaving of normal microbiological culture medium should be done for at least _____ minutes at a minimum of _____ pounds per square inch of pressure and _____ degrees Celsius.

Question 14

• How do you avoid the following foibles of media-making:
  
  • boiling over the medium in the autoclave?
  • getting agar plates full of bubbles?

Question 15

Why do you always add agar powder AFTER you dissolve all other ingredients, check the pH of your medium, and bring it to its total volume?

Question 16

What technique could you use to sterilize a heat-labile vitamin solution?

Question 17

• What technique would you use to enumerate bacterial density, yielding an answer in CFU/ml?
• What technique would you use to enumerate bacteria density, yielding an answer in cells/ml?
• Why might you get different numbers from an identical culture, when counting CFU/mL and cells/mL?

Question 18

• When measuring the optical density of a bacterial culture, you must have a __________________________ in order to interpolate the OD to actual cells/mL or CFU/mL.

Question 19

When measuring optical density of a bacterial culture: what wavelength would you use if the cells were suspended in:

• Luria broth (LB), which is yellow in color?
• Phosphate buffer, which is clear?
• Nutrient broth, which is light brown?

Question 20

When counting CFUs on a normal sized Petri plate, you want to choose plates that have fewer than ___ colonies but more than ___ colonies.

Question 21

Question 22

What compound can be used to “scrub” oxygen from medium by reducing it to H2O?

Question 23

In medium containing the dye resazurin, what does a pink color indicate?

Question 24

• How would you differentiate between these microorganisms? Use your knowledge of biochemical reactions, including growth on selective or differential media.
  
  • Be able to name specific tests, and the biochemical reactions that differentiate the two species.
    
    1. A. Enterobacter aerogenes and Escherichia coli
    2. B. Staphylococcus aureus and Staphylococcus epidermidis
    3. C. Pseudomonas aeruginosa and Escherichia coli

Question 25

Local water supplies can be contaminated by fecal matter from humans or other animals. Three important bacterial pathogens transmitted by contaminated water include E. coli, Salmonella sp. and Shigella sp. * How would you differentiate among these organisms using a single test?

Question 26

**MSA medium** * When using mannitol salt agar (MSA) plates, what biochemical process can Staphylococcus spp. do that might turn the medium yellow? * Which Staphylococcus species give this reaction? * Why do most bacteria NOT grow on MSA? * What aspect of MSA makes it a selective medium, and against which bacteria does it select? * What aspect of MSA makes it a differential medium, and among which bacteria does it differentiate?

Question 27

**MacConkey’s agar:** * When using MacConkey’s agar plates, which organisms produce the characteristic “brick red” color? * What is the cause of this reaction? * What aspect of MacConkey’s agar makes it a selective medium, and against which bacteria does it select? * What aspect of MacConkey’s medium makes it a differential medium, and against which bacteria does it differentiate?

Question 28

**Sheep blood agar:** * Which bacteria can produce greenish zones on sheep blood agar? * What is the term used to describe production of these zones? * Which bacteria can produce clearing zones on sheep blood agar? * What is the term used to describe production of these zones, and what virulence factor contributes to their production?

Question 29

**Eosin methylene blue agar:** * When using Eosin methylene blue (EMB) plates, which organism produces the characteristic “green metallic sheen”? * What is the cause of this reaction?

Question 30

**Catalase, coagulase, and oxidase:** * Which Gram positive bacterial genera (which we worked with in this course) are usually catalase-positive? * What reaction is occurring during a catalase-positive test? * Which test, catalase, coagulase, or oxidase can differentiate between pathogenic and nonpathogenic Staphylococcus? * What reaction is occurring during a positive oxidase test? * What reaction is occurring during a positive coagulase test? * If you are testing an unknown and find it to be a Gram negative rod, which test would help you decide whether it is an enteric: catalase test, coagulase test, or oxidase test?

Question 31

How would you determine whether a Gram negative, facultative, oxidase negative rod might be Proteus? Name two separate tests.

Question 32

Which of the following are enteric bacteria? * a. Escherichia * b. Enterobacter * c. Enterococcus * d. Shigella * e. Salmonella * f. Klebsiella

Question 33

Which of the following are coliform? What characteristic makes coliform bacteria a subdivision within the Enterobacteriaceae? * a. Escherichia * b. Enterobacter * c. Enterococcus * d. Shigella * e. Salmonella * f. Pseudomonas

Question 34

Specifically, what group of bacteria can be identified with the RapID 20E kit AND what are their three defining characteristics?

Question 35

Match the reagents in the right column with the appropriate biochemical test in the left column (indicate by using each letter only once).

Question 36

What are the three sugars added to TSI agar?

Question 37

Upon observing a red over yellow (e.g., K/A) TSI agar slant, which of the three sugars have been fermented? If you were to observe a blackened butt, what would this indicate?

Question 38

What enzyme is present in bacteria that give a positive urease test?

Question 39

What cellular component is present in bacteria that give a positive citrate test?

Question 40

* Competent bacterial cells are generated in the laboratory by repeated washing of logarithmic-phase cells with ice-cold \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_. * They are transformed by electroporation, which makes the cell membrane porous, or by heat shock. What is the purpose of keeping cells+DNA on ice for a few minutes prior to heat shock? * The process of heat-shock makes the cell membrane more _____________ so that DNA can enter.

Question 41

Why did you culture your heat-shocked, transformed cells for 45 minutes in unselective medium before plating on agar containing ampicillin?

Question 42

What was the role of arabinose in the LB+amp+arab plates?

Question 43

Green fluorescent protein was observed in your cells if they were: * a. living * b. dead * c. induced to metabolize arabinose * d. undergoing catabolite suppression * e. induced by UV light * f. exposed to UV light

Question 44

You transformed 100 uL of cells with 12.5 ng of the plasmid pGLO. You added 900 uLs of Luria broth to the transformation reaction, and incubated for one hour at 37oC. Then, you plated 100 uL onto LB + amp + ara and incubated at 37 C overnight. The next day you observed 48 colonies on your plate. * What was your transformation efficiency (in CFU/ug DNA)?

Question 45

* What controls should always be included in a transformation experiment? * Describe the purpose of each – what false negative or positive results would each control detect?

Question 46

* Describe how you would test foodstuffs for bacterial growth. * If you discovered bacteria on a food sample, how could you further test to determine whether it is a potential food poisoning organism? * Which of the organisms we studied this term could cause food poisoning? * How would they look under the microscope? * Could you further identify them with a special staining technique? * Selective or differential media? * Enyzmatic tests?

Question 47

* What do the terms “enteric”, “coliform”, and “fecal coliform” mean? * Draw a Venn diagram depicting these groups in relationship to one another.

Question 48

Why is E. coli a good indicator for fecal contamination?

Question 49

* What enzymes must be present in the bacteria of a water sample for the Colilert test to turn out positive for fecal coliforms? * Which types of organisms have each of these enzymes?

Question 50

Why did you place the filter right-side up on m-Endo agar prior to incubation, rather than upside-down?

Question 51

* What enzyme specific to E. coli causes both Colilert and NA-MUG plates to fluoresce? * What indicator compound must be cleaved to release the fluorescent dye?

Question 52

What is the difference between a bacteriocidal compound and a bacteriostatic compound?

Question 53

Which would you use on your countertop: disinfectant or antiseptic?

Question 54

Which would you use on your hands: disinfectant or antiseptic?