Study Guide Questions

Question 1

What were some of the features of the lab room that are typical for working with level 1 biological agents? [7]

What would be needed to make it suitable for working with level 2 biological agents? [7]

Answer 1

Biosafety Level 1 & 2 [7]:
* Doors kept closed
* Hazard warning signs
* Hand washing sink
* Waste materials segregated
* Lab surfaces must be cleaned & decontaminated
* PPE worn as needed
* Supervisors must have general microbiological training

Biosafety Level 2 [7]:
* Access is more limited
* Biosafety manual
* Sharps precautions (e.g., needles, glass)
* Gloves
* Biological safety cabinet
* Face protection (if needed)
* Autoclave available

Question 2

You have been asked to make some Vegetable Broth agar plates. What could you do? [4]

Answer 2

• Follow a recipe for the media
• Add the ingredient(s) to water
• Autoclave to sterilize and melt agar
• Dispense when cool

Question 3

Describe the structure of a Gram-positive bacterial cell.

Answer 3

• Thick peptidoglycan cell wall and inner cytoplasmic cell membrane
• Also note, compared to Eukaryotes (e.g., yeast):
  
  • Much smaller
  • Lack a membrane bound nucleus
  • Lack mitochondria
  • DNA is circular, smaller in size
  • May contain plasmids

Question 4

Describe the structure of a Gram-negative bacterial cell.

Answer 4

• Outer membrane, thin peptidoglycan cell wall, and inner cytoplasmic membrane
• Also note, compared to Eukaryotes (e.g., yeast):
  
  • Much smaller
  • Lack a membrane bound nucleus
  • Lack mitochondria
  • DNA is circular, smaller in size
  • May contain plasmids

Question 5

You have been handed a tube that contains bacteria. You don’t know if it is a pure or mixed culture.

Suggest two methods that you could do that would allow you to say with some confidence that the tube contains a mixed culture.

Answer 5

• Streak plate: Using aseptic technique, spread bacteria from the tube onto a solid agar medium in a petri dish in pattern that gradually dilutes the sample; after incubation, if more than one colony morphology is present, that suggests the tube contains a mixed culture.
• Gram-stain: Stain a sample with crystal violet and iodine, then decolorize with alcohol and counterstain with safranin; if more than one microscopic morphology is present, that suggests the tube contains a mixed culture.

Question 6

Students were preparing streaking plates to get isolated colonies. Despite many attempts, each plate had a few lines of thick confluent bacterial growth.

What could the students do
differently that might help them get isolated colonies? [4]

Answer 6

1. Reduce the amount of inoculum: use less bacteria; use a sterile swab to transfer a small amount of bacteria; prepare serial dilutions
2. Increase the streaking area: use a larger petri dish to help allow further dilution of bateria
3. Change the streaking pattern: try a different pattern that provides more dilution of the bacteria, like the quadrant method, or the T-streak method
4. Check the incubation conditions: if the plates are incubated too long, or at too high a temperature this can result in overgrowth of the bacteria

Question 7

Why are isolated colonies important? [3]

Answer 7

1. To ensure purity: important for accurate identification and characterization of bacteria
2. Study of individual characteristics: allow for study of properties of the bacteria such as colony morphology, pigmentation, and hemolysis; can help differentiate betweeen strains
3. Subculturing: provides a pure source for future experimentation (e.g., antibiotic susceptibility testing, genetic analysis); important for ensuring accurate and reproducible results.

Question 8

You been given a MacConkey agar plate and told that it is useful for determining whether a
bacterium ferments lactose. You decide to streak half of the plate with a bacterium that does
not ferment lactose, and the other half that does.

What might you expect to see and why do you see this?

Answer 8

• On the half of the plate streaked with the non-lactose fermenting bacterium, you may see colorless or transparent colonies. This is because the bacterium is unable to ferment lactose and therefore does not produce acid. As a result, the pH of the medium remains neutral or alkaline and the neutral red indicator does not change color.
• On the half of the plate streaked with the lactose-fermenting bacterium, you may see pink or red colonies. This is because the bacterium is able to ferment lactose and produces acid as a result. The acid lowers the pH of the medium and causes the neutral red indicator to turn pink or red.

Key ingredients: crystal violet/bile salts (inhibits Gram-positive bacteria); lactose/neutral red (bacteria that ferment lactose produce acid which changes the pH and changes the colour of the indicator)

Question 9

All culture media are selective to some extent – there is no universal growth media that all
bacteria to grow.

Why is something like “Tryptic Soy Agar” usually considered as a general-purpose media?

Answer 9

Tryptic Soy Agar (TSA) is considered a general-purpose medium because it contains a variety of nutrients that support the growth of a wide range of bacteria. TSA contains enzymatic digests of casein and soybean meal, which provide amino acids, peptides, and other nitrogenous compounds that bacteria can use for growth. It also contains glucose as a source of energy and sodium chloride to maintain osmotic balance. However, it is not suitable for all bacteria and some fastidious bacteria may require additional growth factors or nutrients.

Question 10

How does the Gram staining process work? [4]

Answer 10

1. Flood the slide with Crystal violet: crystal violet is positively charged and binds to negatively charged cell wall structures.
2. Flood the slide with iodine: iodine binds to CV molecules (via non-covalent bonds) and forms insoluble complexes in the peptidoglycan and inner membrane
3. Decolorize slide with ethanol: alcohol dissolves the outer membrane of Gram-negative and disrupts the thin peptidoglycan layer so that the Crystal-iodine complexes escape; CV-I complexes in Gram-positive cells are retained
4. Counterstain with safranin: safranin is a positively charged stain that binds to the negatively charged cell wall structures in the Gram-negative bacteria

Question 11

What would you see under a microscope if you forgot to the crystal violet in your gram staining procedure?

Answer 11

Both gram (+) and gram (-) will be pink due to counterstain at the last step

Question 12

What would you see under a microscope if you forgot to apply iodine during your gram staining procedure?

Answer 12

Gram (+) wouldn’t be able to retain purple colour and will look pink, just like gram (-) due to counterstain

Without iodine, the crystal violet would not be fixed to the cell wall and could be removed during the decolorization process.

Question 13

What would you see under a microscope if you forgot to apply decolorizer during your gram staining procedure?

Answer 13

Gram (+) and (-) will look purple

Decolorization is an important step that differentiates between bacteria.

Question 14

What would you see under a microscope if you forgot to apply the counterstain during your gram staining procedure?

Answer 14

• Gram (+) purple
• Gram (-) colourless

Question 15

What would you see under a microscope if you forgot to the crystal violet in your gram staining procedure?

Answer 15

Both gram (+) and gram (-) will not be stained, and both will be pink due to counterstain at the last step

Question 16

What are the four phases of a culture growth, and what are the cells usually doing in these phases?

Answer 16

Lag: adaptation to new media and/or growth conditions; gearing up to take advantage of nutrients
Exponential: cells are growing, dividing, cell numbers (concentration) are increasing
Stationary: cells are adapting to poor environmental conditions, low nutrients, high waste
Death: cells are dying

The doubling time depends on bacteria and growth conditions. E.g., E. coli doubling time is ~20 minutes in rich media at 37C.

Question 17

How does the alkaline lysis process for plasmid isolation work?

What would happen if you
forgot to use the NaOH-SDS solution when trying to isolate plasmids?

Answer 17

• The lysis buffer contains (1) sodium hydroxide (NaOH), (2) SDS.
• SDS solubilizes the cell membrane
• NaOH helps break down the cell wall, but more importantly it disrupts the hydrogen bonding between DNA bases, converting dsDNA to ssDNA (plasmids are not denatured)
• Solution is neutralized by addition of KOAc (chromosomal DNA and proteins precipitate out (plasmids remain in solution; centrifuged out in supernatant)
• Plasmid DNA then precipitated by additional of ethanol; centrifuged; suspended in a buffer.
• If you forgot to use the NaOH-SDS solution when trying to isolate plasmids, you would not lyse the cells and release the DNA.

Note: In lab we used the column elution method, not ethanol.

Question 18

Why do restriction enzymes only cut DNA plasmids one or a few times?

Answer 18

• They recognize specific target sequences and cut DNA at or near those sequences.
• Each RE recognizes one or a few target sequences.
• Plasmids are small, circular pieces of DNA that can be cut by RE.
• The number of times a plasmid is cut by a RE depends on the number of target sequences present on the plasmid.
• If a plasmid only has one or a few target sequences for a particular RE, it will only be cut one or a few times.

Question 19

How would you make a gel that contains 6% agarose and 12% agarose?

Answer 19

• Make two separate solutions with the desired % agarose and combine them
• E.g., Dissolve 3 grams of agarose in 50 mL of buffer for the 6% solution. Pour into casting tray and wait until solidified. Then dissolve 6 grams of agarose in 50 mL for 12%, pour into casting tray and wait until solidified.

Question 20

You have a linearized DNA plasmid of about 4000 bp in length. If you were to electrophoresis the DNA fragment in 6% agarose gel or 12% agarose gel, under the same voltage conditions, which gel
would the plasmid move faster and farther in 1 hour? Why does this happen?

Answer 20

• The plasmid would move faster and farther in the 6% agarose gel.
• Agarose gels are used to separate DNA fragments based on their size.
• The % agarose affects the pore size of the matrix; a higher % results in a smaller pore size.
• In a 6% agarose gel, the pore size is larger than in a 12% agarose gel. This means that DNA fragments can move more easily through the gel matrix. As a result, the plasmid would move faster and farther in the 6% agarose gel compared to the 12% agarose gel under the same voltage conditions in 1 hour.

Question 21

Distinguish between food intoxication and food infection.

Answer 21

• Food poisoning/intoxication is a form of foodborne illness due to preformed toxins in food.
• Food infection is a form of foodborne illness due to consumption of pathogens that cause infection when ingested (e.g., they release toxins or cause other detrimental effects only after being eaten)

Question 22

How is ELISA similar/different from a lateral flow device? When would you use one over the other?

Answer 22

• Both are immunoassays that can be used to detet the presence of a target substance in a liquid sample; both rely on use of antibodies to bind the target and produce a detectable signal.
• Key difference: ELISA is used in lab settings and requires multiple steps (including incubation); LFDs are used in the field and provide results quickly (within minutes)
• Another difference: ELISA is more sensitive and specific.
• The choice depends on: desired level of sensitivity and specificity, the need for rapid results, and the availability of lab equipment and trained personnel.

Question 23

How is FISH similar/different from PCR? When would you use one over the other?

Answer 23

• Both are used to detect and analyze nucleic acids.
• FISH detects and localizes specific sequences in cells or tissues involving fluorescently labelled probes that bind to complementary sequences.
• PCR amplifies specific DNA sequences.
• Key difference: FISH can be used to visualize the spatial distribution of nucleic acid sequences within cells or tissues, while PCR can be used to amplified specific DNA sequences for further analysis.
• The choice depends on the research question and experimental design.

Question 24

Provide four examples of how microbes are involved in food production or food preservation.

Answer 24

• Yeasts ferment sugars to ethanol and carbon dioxide in bread production.
• Synergistic starter culture bacteria ferment lactose to lactic acid to thicken milk and produce the distinctive sour taste and mouthfeel of yogurt.
• Mould ferments soybeans in soy sauce production
• Acetic acid bacteria ferment various starter materials (e.g., wine, cider, juice) to produce vinegar.

Question 25

Clostridium botulinum is an anaerobic bacterium, and
sometimes is present in home-canned food. How would you test whether the toxin was present in a can of tuna?

Answer 25

• ELISA
• This method can detecct the presence of specific antigens, such as botulinum toxin, in a sample.
• This test involves the use of antibodies that bind specifically to the toxin and produce a detectable signal.

Question 26

Brucellosis is a disease that humans can acquire from drinking raw milk contaminated with the bacteria Brucella melitensis. As a young microbiologist, your job is to determine whether this particular species of bacteria are present in sample of raw milk. What method would you choose and why?

Answer 26

• Prepare serial dilutions.
• Selective spread plating
• Gram-staining and biochemical tests specific to B. melitensis
• Serological tests

Question 27

What is the problem with the use of antibiotic in livestock production?

Answer 27

• Development of antibiotic-resistant bacteria.
• Routine use means bacteria that are exposed can develop resistance; these can spread to humans through the food chain or through direct contact.
• This can make it more difficult to treat bacterial infections in humans, as antibiotics that were effective may no longer work.
• Can affect other organisms in the environment as well (e.g., beneficial bacterial that play important roles in nutrient cycling)

Question 28

How would you determine how long food should be treated with heat/irradiation to make food “safer” to eat after storage?

Answer 28

• Estimate the decimal reduction time in lab using pure cultures.
• Treat the food with a process equivalent to 12 x D.

Note the y-axis is in log scale. The D-value is the time to reduce the population size by 1 log, to 10% of its initial size.

Question 29

What is the D-Value?

Answer 29

• The time to reduce the population size by 1 log, to 10% of its initial size.

Question 30

For the following examples of food, what method of food preservation would you consider
best? Your goal is to have the food safe to eat up to one year after treatment (the food
nutritional quality is another matter – not relevant here).

The methods available include heat sterilization, irradiation, pasteurization and salting.

Why did you pick the method for each food group?

Foods: Flour, rice, fresh apples (whole), fresh tomatoes (whole), ground cinnamon, eggs, green beans and ground chicken.

Answer 30

• Flour: heat sterilization; heat to high temp for short time
• Rice: heat sterilization; heat to high temp for short time
• Fresh apples (whole): Irradiation
• Fresh tomatoes (whole): irradiation
• Ground cinnamon: heat sterilization or irradiation
• Eggs: Pasteurization; heat the eggs to a specific temp for specific length of time to kill harmful bacteria without cooking the egg
• Green beans: heat sterilization or irradiation; blanching in water/steam or expose to ionizing radiation
• Ground chicken: heat sterilization; cook to internal temp of 165F to ensure harmful bacteria are killed

Irradiation does not noticeably change the taste, texture, or appearance of food.

Question 31

Antibiotics are sometimes used in the maintenance of dairy cattle that are then milked, and the resulting milk is then sent to a processing plant. What are the methods that you could use to ensure that the milk is a reasonable quality (i.e., the bacteria present are below the required threshold)?

Answer 31

• Bacterial testing: Milk samples can be tested for the presence of bacteria using methods such as standard plate counts or coliform counts. These tests can provide an indication of the overall bacterial load in the milk and can help to identify any potential contamination issues.
• Antibiotic residue testing: Milk samples can be tested for the presence of antibiotic residues using methods such as microbial inhibition assays or immunoassays. These tests can detect the presence of antibiotics in milk and can help to ensure that milk from treated animals is not sold for human consumption until the antibiotic residues have cleared.
• On-farm hygiene practices: Good hygiene practices on the farm can help to reduce the risk of bacterial contamination of milk. This includes practices such as regular cleaning and sanitizing of milking equipment, proper storage and handling of milk, and good animal husbandry practices.

Question 32

What were some of the features of the lab room that are typical for working with level 3 biological agents? [7]

What would be needed to make it suitable for working with level 4 biological agents? [7]

Answer 32

Biosafety Level 3 [3]:
* Physical separation from access corridors
* Negative airflow
* Exhausted air is not recirculated

Biosafety Level 3 & 4 [3]:
* All requirements of BSL1&2
* Controlled access (e.g., keycard)
* Decontamination of all lab clothing before laundering

Biosafety Level 4 [6]:
* Separate building or isolated zone
* Dedicated supply/exhaust, vacuum, decontamination systems
* All material is decontaminated on exit from facility
* Clothing changed before entering
* Full-body, air-supplied, positive pressure personnel suit
* Shower on exit

Question 33

You have been asked to make some saline dilution blanks. What could you do? [4]

Answer 33

• Follow a recipe for the media
• Add the ingredient(s) to water
• Dispense into tubes
• Autoclave

Question 34

Describe the lag phase.

Answer 34

• Cells do not divide
• Cells are adjusting to culture conditions and preparing for cell division.

Question 35

Describe the exponential (a.k.a. log) phase.

Answer 35

• Cells actively proliferate
• Cell density increases exponentially

Question 36

Describe the stationary phase.

Answer 36

• Growth reaches a plateau as the number of cells dying equals the number of dividing cells.
• Due to growth-limiting factors such as depletion of essential nutrient and/or formation of an inhibitory metabolite or waste product.

Question 37

Describe the death phase.

Answer 37

• Charaterized by an exponential decrease in the number of cells.