Question 1

The colonies of interest in this case are the blue-green presumptive Shigella colonies. However, growth on hektoen enteric agar is not sufficient for definitive identification. Further tests are needed. First, the lab technicians need to prepare larger numbers of Shigella from your patient's stool sample, and then they need to ensure that they are culturing a single species. The methods used to do this are ________, followed by ________. 
A) streak plating; pour plates
B) enrichment culture; Gram staining
C) Gram staining; enrichment culture
D) Gram staining; capsule staining
E) enrichment culture; streak plating

Accepted Answer

Enrichment culture is the method used to obtain larger numbers of Shigella from the patient's stool sample, and streak plating is used to ensure that a single species is being cultured. This is because Shigella is a fastidious organism and requires enrichment to grow in quantities that can be used in further tests. Streak plating allows for the separation of individual colonies, which can then be tested for further identification. Gram staining is not necessary for culturing a single species, and pour plates and capsule staining are not mentioned in this case.

Question 2

Thayer-Martin agar typically contains a combination of antibiotics: vancomycin, colistin, nystatin, and trimethoprim. Please select the True statement.
A) The antibiotics in Thayer-Martin agar do not kill Gram-negative cells.
B) The antibiotics in the Thayer-Martin medium lyse the sheep RBCs.
C) Neisseria gonorrhoeae is not sensitive to the included antibiotics.
D) Fastidious bacteria are never impacted by common antibiotics.
E) Sheep blood cell in a growth medium protects bacteria from the impact of antibiotics.

Accepted Answer

Thayer-Martin agar is specifically designed to isolate Neisseria gonorrhoeae, which is not inhibited by the antibiotics (vancomycin, colistin, nystatin, and trimethoprim) in the medium. These antibiotics are chosen to suppress the growth of other bacteria, allowing N. gonorrhoeae to grow.

Question 3

A halophilic, aerobic mesophile has a generation time of 20 minutes. You place 1 × 10⁴ of these organisms on an appropriate solid medium, and incubate the plate for 100 minutes. Select the False statement.
A) The growth medium contains NaCl.
B) The organism will grow at 40ᵒC. 
C) After incubation, a colony on the plate contains 3.2 × 10⁵ cells.
D) After incubation, a colony on the plate contains 5 × 10⁴ cells.
E) The organism produces superoxide dismutase and catalase.

Accepted Answer

According to the given generation time of the organism, the number of cells after 100 minutes can be calculated using the equation Nf = Ni x 2^(t/g), where Nf is the final number of cells, Ni is the initial number of cells, t is the incubation time, and g is the generation time. Plugging in the values, we get Nf = 1 × 10⁴ x 2^(100/20) = 3.2 × 10⁵ cells. Therefore, statement C is true and statement D is false. Statements A, B, and E are not contradicted by the information given.

Question 4

The lab technicians culture the sample on Thayer-Martin agar. This complex medium contains 5% sheep blood and a combination of antibiotics. This medium is 
A) differential.
B) selective AND differential.
C) selective.
D) aerobic.
E) chemically defined.

Accepted Answer

Thayer-Martin agar is selective because it contains antibiotics that inhibit the growth of unwanted bacteria, allowing only the growth of Neisseria species and a few other organisms.

Question 5

You are determining the number of colony forming units (CFUs) for Susan, who has a urinary tract infection (UTI). You added 1 mL of Susan's urine sample to 99 mL of diluent, and spread 0.1 mL of that dilution on a culture medium. After appropriate incubation, you count 143 colonies on the plate. What was the concentration of bacteria in the original urine sample? 
A) 1.43 cells/ mL
B) 1.43 × 10² cells/ mL
C) 1.43 × 10³ cells/ mL
D) 1.43 × 10⁵ cells/ mL
E) Cannot be determined

Accepted Answer

The original sample was diluted by a factor of 100 (1 mL in 99 mL), and then 0.1 mL of this dilution was plated. The dilution factor for the plated sample is 1000 (100 for the dilution and 10 for the 0.1 mL plated). Therefore, the concentration in the original sample is 143 colonies × 1000 = 1.43 × 10⁵ cells/mL.

Question 6

In addition to the few Shigella colonies that are noted, there are also abundant salmon pink colonies, around which the medium has turned yellow, losing its original blue color. You know that hektoen enteric agar must contain ________. 
A) lactose
B) a pH indicator
C) a weak acid
D) malachite green
E) penicillin

Accepted Answer

Hektoen enteric agar contains a pH indicator that changes color based on the pH of the medium. The original blue color of the medium changes to yellow around colonies that produce acid, indicating fermentation of lactose or other carbohydrates. Therefore, choice A (lactose) is not necessary for the color change, and choices C (a weak acid), D (malachite green), and E (penicillin) are not components of hektoen enteric agar.

Question 7

A physician sends a stool sample to your lab, and wants to know if there are lactose fermenting microbes in the sample. How might you determine if these microbes are present or not from this mixed-microbe specimen?
A) Streak the sample for isolation on Thayer-Martin agar (which contains lactose and particular antibiotics for selectivity).
B) Streak the sample for isolation on a blood agar plate (which contains lactose and red blood cells that enrich the culture for iron).
C) Streak the sample for isolation on a MacConkey agar plate (which contains lactose and a pH indicator that turns pink when acid byproducts are present).
D) Either Thayer-Martin agar or blood agar would work, as long as the plates are incubated in an anaerobic chamber.  
E) None of the answer choices would work-there's no way to reliably determine this feature from the specimen given.

Accepted Answer

Streaking the sample for isolation on a MacConkey agar plate containing lactose and a pH indicator that turns pink when acid byproducts are present provides a selective and differential method for detecting lactose-fermenting microbes. The pH indicator will turn the plate pink if lactose-fermenting microbes are present. Thayer-Martin agar and blood agar plates are not specific for lactose-fermenting microbes. Incubation in an anaerobic chamber is not necessary for the detection of lactose-fermenting microbes.

Question 8

A cluster of cases of legionellosis have occurred in your neighborhood. People with the illness have cough, fever, shortness of breath, chest pain and sometimes diarrhea. The illness is caused by the bacterium Legionella pneumophila, a facultative intracellular parasite that survives in certain ameba. The organism has an absolute requirement for L-cysteine.
-Diagnosis of legionellosis depends partly on culture of the causative agent from medical samples. Typically, the organism is grown on buffered charcoal yeast extract (BCYE) agar. What essential component must this medium have to ensure growth of L. pneumophila?
A) red blood cells
B) chlorine
C) NaCl
D) cysteine
E) All of the answer choices are correct.

Accepted Answer

L. pneumophila has an absolute requirement for L-cysteine, which is an important component for its growth on BCYE agar. Therefore, the essential component that this medium must have to ensure the growth of L. pneumophila is cysteine. Red blood cells, chlorine, and NaCl are not essential components for the growth of L. pneumophila.

Question 9

Clostridium paradoxum grows optimally at 55°C, pH 9.3; it will not grow in the presence of O₂. Please select the True statement about this organism.
A) It is spherical in shape.
B) It is a psychrophile.
C) It is an obligate anaerobe.
D) It is an acidophile.
E) It is an aerotolerant anaerobe.

Accepted Answer

Clostridium paradoxum is unable to grow in the presence of oxygen, making it an obligate anaerobe. It grows optimally at a high temperature and alkaline pH, but no information is given about its shape or ability to tolerate acidity or cold temperatures.

Question 10

If you are asked to make 200 mL of Thayer-Martin agar, who much sheep's blood would you add to the medium? Recall that this complex medium contains 5% sheep blood.
A) 10%
B) 5 mL
C) 10 mL
D) Random quantities
E) 10,000 RBCs

Accepted Answer

To make 200 mL of Thayer-Martin agar, 5% of it must be sheep's blood. Therefore, the calculation goes as follows: 
5% = 5/100 = 1/20
1/20 of 200 mL = 10 mL of sheep's blood. 
Therefore, the correct choice is C) 10 mL.

Question 11

You are in charge of water quality for your city's water treatment plant. Of the methods at your disposal, which will be the most efficient and cheapest method of determining the number of viable bacteria in the water coming out of your plant?
A) Direct counts using a microscope and a counting chamber.
B) Using a Coulter counter machine.
C) Performing serial dilutions and doing spread plate counts.
D) Using membrane filtration followed by placing the membrane in a growth medium for colony counts after incubation.
E) Biochemical analysis of secondary metabolites in the water that are given off by bacteria.

Accepted Answer

Membrane filtration followed by placing the membrane in a growth medium for colony counts after incubation is both efficient and cost-effective for determining the number of viable bacteria in water. This method concentrates the bacteria present in large volumes of water onto a filter, which is then placed on a growth medium. After incubation, the colonies that grow can be counted, providing a direct measure of the number of viable bacteria in the water sample. This method is preferred for water quality testing because it is specific for viable bacteria, relatively simple, and does not require expensive equipment.

Question 12

Shigella species and Salmonella species are classified as facultative anaerobes. You know that given this information, the bacteria in your patient's fecal culture will grow best
A) in the absence of oxygen.
B) in the presence of oxygen.
C) at low levels of oxygen.
D) in high levels of carbon dioxide.
E) if catalase is added to the medium.

Accepted Answer

Facultative anaerobes, such as Shigella and Salmonella species, can grow in both the presence and absence of oxygen. However, they grow best in the presence of oxygen because they can utilize aerobic respiration, which is more efficient in energy production than anaerobic pathways.

Question 13

Eosin methylene blue agar (EMB) is a selective and differential medium. The dyes in the medium inhibit most Gram-positive organisms. Gram-negative organisms such as the coliforms grow on this medium. Coliforms that are fecal in origin produce colonies with a metallic green color on EMB, while those that are non-fecal produce colorless colonies. You plate a urine sample onto an EMB plate and incubate it. Metallic green colonies develop. From this, you can conclude that 
A) the sample contains bacteria that are Gram-negative AND are non-fecal in origin.
B) the sample contains bacteria that are Gram-negative AND are fecal in origin.
C) the sample contains bacteria that are Gram-positive AND are fecal in origin.
D) the bacteria in the colonies are Gram-positive AND are rod-shaped.
E) normal microbiota in the bladder is fecal in origin AND is aerobic.

Accepted Answer

The metallic green colonies on EMB indicate the presence of Gram-negative bacteria that are fecal in origin.

Question 14

Mrs. Thomas comes to the emergency department where you work, complaining of nausea and vomiting, bloody diarrhea, abdominal discomfort and fever. She tells you that she went out to a picnic the previous day, where barbecued chicken was served. Mrs. Thomas is a vegan, so she ate only salad. You think that she likely has salmonellosis, caused by the bacterium Salmonella enterica. However, the presence of blood in her stool causes you concern, and you want to rule out the possibility that patient has shigellosis, caused by Shigella bacterial species.
-You take a stool sample from Mrs. Thomas, and send it to the lab for analysis. You tell her that the technicians will do a variety of tests to confirm the identity of the organism causing her illness. The first thing that will be done is to inoculate some of her stool sample onto a medium called hektoen enteric (HE) agar. On this medium, Shigella species produce blue-green colonies, while Salmonella produces blue-green colonies with a black center, and most Gram-positive bacteria do not grow at all. You explain to Mrs. Thomas that this type of medium is called a(n) ________ medium.
A) selective
B) differential
C) enrichment
D) selective and differential
E) differential and enrichment

Accepted Answer

Hektoen enteric (HE) agar is both selective and differential. It is selective because it contains bile salts and dyes that inhibit the growth of many Gram-positive bacteria and some Gram-negative bacteria, allowing for the isolation of Gram-negative enteric bacteria. It is differential because it contains indicators that allow for the differentiation of various enteric bacteria based on their ability to ferment lactose or sucrose and produce hydrogen sulfide gas. The distinction between Shigella and Salmonella is based on the production of hydrogen sulfide gas, which causes the center of the colony of Salmonella to turn black.

Question 15

You are culturing a new bacterium in the laboratory. You cannot find any glucose it the lab when you are making growth medium, so you decide to use honey instead. You add twice as much honey than is called for in the medium recipe, because you want to give the organism the best opportunity or growth. To your surprise, the organism does not grow on your modified medium. Select the most likely explanation.
A) The growth medium became hypotonic with the addition of so much honey; the bacteria underwent plasmolysis and died.
B) The growth medium became hypertonic with the addition of so much honey; the bacteria underwent plasmolysis and died.
C) The growth medium became halophilic with the addition of so much honey; the bacteria underwent plasmolysis and died.
D) The growth medium became hypertonic with the addition of so much honey; the bacteria underwent osmotic lysis and died.
E) The growth medium became hypotonic with the addition of so much honey; the bacteria underwent simple diffusion and burst.

Accepted Answer

Honey is a concentrated solution, so adding twice as much as called for in the recipe would make the growth medium hypertonic. Hypertonic solutions have a higher solute concentration than the bacterial cytoplasm, so water will leave the bacterial cell by osmosis, causing the cell to undergo plasmolysis and die.

Question 16

A cluster of cases of legionellosis have occurred in your neighborhood. People with the illness have cough, fever, shortness of breath, chest pain and sometimes diarrhea. The illness is caused by the bacterium Legionella pneumophila, a facultative intracellular parasite that survives in certain ameba. The organism has an absolute requirement for L-cysteine.
-L. pneumophila is widespread and is found in various water sources, including hot tubs, water cooling towers, swimming pools, grocery store misters, and shower water. Select the False statement  regarding the organism, given this information.
A) It survives in the presence of chlorine.
B) It is a mesophile.
C) It is an obligate anaerobe.
D) It can survive in water pipes.
E) It is a mesophile AND it can survive the presence of chlorine.

Accepted Answer

L. pneumophila is not an obligate anaerobe, as it can grow in the presence of oxygen. It is a facultative intracellular parasite, meaning it can survive and multiply within host cells, but can also grow outside of them. It is also a mesophile, meaning it grows best at moderate temperatures between 25-45°C. However, it can survive in colder temperatures as well. L. pneumophila is known to survive in water pipes, and has been found to grow in biofilms that can form in plumbing systems. While chlorine is commonly used as a disinfectant for water, L. pneumophila has been shown to survive in the presence of low levels of chlorine, especially in biofilm environments. Therefore, statement E is also false; L. pneumophila is a mesophile but it cannot always survive in the presence of chlorine.

Question 17

You take absorbance readings on a spectrophotometer across a 6-hour culture of E. coli cells growing in tryptic soy broth (TSB). Your absorbance readings clearly indicate a lag phase, a log phase, and a stationary phase. You come back in and take readings at 8, 10, 12, 14, and 16 hours, but the absorbance number remains the same. Shouldn't it start coming down as the closed batch culture enters death phase? What's the most likely thing that is happening?
A) Something is likely wrong with the spectrophotometer and it isn't measuring the correct values. Perhaps something is on the detector, making it register Falsely high absorbance numbers.
B) A spectrophotometer can measure absorbance as an indicator of cell number, but can't discriminate between a live cell and a dead one. The presence of dead cells keeps the absorbance high even into the death phase.
C) E. coli just grows slowly, and has a long generation time. After 16 hours the cells are still in stationary phase, so the cell numbers are expectedly constant. 
D) Perhaps there's a big smudge of something on the tube that is blocking some of the light. This would lead to an elevated reading for every timepoint after the smudge was placed on the tube.
E) There is no point in measuring the absorbance of the culture because the cells are bacterial. It would make more sense to measure would the transmission value.

Accepted Answer

Spectrophotometers measure absorbance as an indicator of cell number, but they cannot discriminate between live and dead cells. Therefore, the presence of dead cells keeps the absorbance high even into the death phase, explaining the consistent readings after 6 hours.

Question 18

A cluster of cases of legionellosis have occurred in your neighborhood. People with the illness have cough, fever, shortness of breath, chest pain and sometimes diarrhea. The illness is caused by the bacterium Legionella pneumophila, a facultative intracellular parasite that survives in certain ameba. The organism has an absolute requirement for L-cysteine.
-In some labs, samples containing suspected L. pneumophila are cultured on traditional BCYE medium as well as BCYE medium lacking L-cysteine. How would L. pneumohila be identified in this situation?
A) Compare the two plates. Colonies that grow on traditional BYCE medium but not on BYCE medium lacking cysteine are likely L. pneumophila.
B) Compare the two plates. Colonies that grow on BYCE medium lacking cysteine but not on traditional BYCE medium are likely L. pneumophila.
C) Compare the two plates. Colonies that grow on both traditional BYCE medium and on BYCE medium lacking cysteine are likely L. pneumophila.
D) Compare the two plates. Colonies that do not grow on traditional BYCE medium or on BYCE medium lacking cysteine, but do grow on nutrient agar are likely L. pneumophila.
E) Compare the two plates. Colonies that grow on traditional BYCE medium and are blue, yellow or cyan in color are likely L. pneumophila.

Accepted Answer

Legionella pneumophila requires L-cysteine for growth, so it will grow on traditional BCYE medium that contains L-cysteine but not on BCYE medium lacking L-cysteine.

Question 19

Mr. Jones comes to your clinic complaining of genital discharge. He admits that he recently had unprotected sex with an old friend with whom he reconnected after a school reunion. You suspect that he may have gonorrhea. You take a sample of the discharge and send it to the hospital lab for analysis.
-The causative organism of gonorrhea is Neisseria gonorrhoeae, a fastidious, Gram-negative diplococcus. What does fastidious mean in this context?
A) An organism with complex nutritional requirements.
B) An organism that requires oxygen for growth.
C) An organism that survives in the presence of specific antibiotics.
D) An organism that only grows in pure culture.
E) An organism that requires a living host.

Accepted Answer

Fastidious means that the organism has complex nutritional requirements and requires specific nutrients to grow. In the case of N. gonorrhoeae, it requires specific growth factors such as hemin and nicotinamide adenine dinucleotide (NAD) for optimal growth.

Question 20

Mrs. Thomas comes to the emergency department where you work, complaining of nausea and vomiting, bloody diarrhea, abdominal discomfort and fever. She tells you that she went out to a picnic the previous day, where barbecued chicken was served. Mrs. Thomas is a vegan, so she ate only salad. You think that she likely has salmonellosis, caused by the bacterium Salmonella enterica. However, the presence of blood in her stool causes you concern, and you want to rule out the possibility that patient has shigellosis, caused by Shigella bacterial species.
-On HE medium, Shigella species produce blue-green colonies, while Salmonella produces blue-green colonies with a black center. There are two noticeable types of colonies growing on the hektoen enteric (HE) medium in Mrs. Thomas' test. One type of colony is greenish in color. This leads you to conclude that the bacteria in these colonies 
A) is Gram-negative and is likely Shigella.
B) is Gram-negative and is likely Salmonella.
C)  is Gram-positive and is likely Shigella.
D) is Gram-positive and is likely Salmonella.
E) is Gram-neutral and is likely Salmonella.

Accepted Answer

The greenish color of the colonies indicates that the bacteria is producing acid, and HE medium contains lactose, sucrose, and salicin as fermentable carbohydrates which are utilized by their respective bacteria to produce acid, leading to the pH indicator turning green. Shigella species produce blue-green colonies on HE medium, while Salmonella produces blue-green colonies with a black center. Therefore, the greenish colonies suggest that the bacteria is Gram-negative and likely Shigella.

Quiz 4: Dynamics of Microbial Growth

Thayer-Martin agar typically contains a combination of antibiotics: vancomycin, colistin, nystatin, and trimethoprim. Please select the True statement.

A halophilic, aerobic mesophile has a generation time of 20 minutes. You place 1 × 10⁴ of these organisms on an appropriate solid medium, and incubate the plate for 100 minutes. Select the False statement.

The lab technicians culture the sample on Thayer-Martin agar. This complex medium contains 5% sheep blood and a combination of antibiotics. This medium is

In addition to the few Shigella colonies that are noted, there are also abundant salmon pink colonies, around which the medium has turned yellow, losing its original blue color. You know that hektoen enteric agar must contain ________.

A physician sends a stool sample to your lab, and wants to know if there are lactose fermenting microbes in the sample. How might you determine if these microbes are present or not from this mixed-microbe specimen?

Clostridium paradoxum grows optimally at 55°C, pH 9.3; it will not grow in the presence of O₂. Please select the True statement about this organism.

If you are asked to make 200 mL of Thayer-Martin agar, who much sheep's blood would you add to the medium? Recall that this complex medium contains 5% sheep blood.

You are in charge of water quality for your city's water treatment plant. Of the methods at your disposal, which will be the most efficient and cheapest method of determining the number of viable bacteria in the water coming out of your plant?

Shigella species and Salmonella species are classified as facultative anaerobes. You know that given this information, the bacteria in your patient's fecal culture will grow best