Study Guide Questions Flashcards
(37 cards)
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]
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
You have been asked to make some Vegetable Broth agar plates. What could you do? [4]
- Follow a recipe for the media
- Add the ingredient(s) to water
- Autoclave to sterilize and melt agar
- Dispense when cool
Describe the structure of a Gram-positive bacterial cell.
- 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
Describe the structure of a Gram-negative bacterial cell.
- 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
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.
- 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.
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]
- Reduce the amount of inoculum: use less bacteria; use a sterile swab to transfer a small amount of bacteria; prepare serial dilutions
- Increase the streaking area: use a larger petri dish to help allow further dilution of bateria
- Change the streaking pattern: try a different pattern that provides more dilution of the bacteria, like the quadrant method, or the T-streak method
- 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
Why are isolated colonies important? [3]
- To ensure purity: important for accurate identification and characterization of bacteria
- Study of individual characteristics: allow for study of properties of the bacteria such as colony morphology, pigmentation, and hemolysis; can help differentiate betweeen strains
- Subculturing: provides a pure source for future experimentation (e.g., antibiotic susceptibility testing, genetic analysis); important for ensuring accurate and reproducible results.
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?
- 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.
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?
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.
How does the Gram staining process work? [4]
- Flood the slide with Crystal violet: crystal violet is positively charged and binds to negatively charged cell wall structures.
- Flood the slide with iodine: iodine binds to CV molecules (via non-covalent bonds) and forms insoluble complexes in the peptidoglycan and inner membrane
- 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
- Counterstain with safranin: safranin is a positively charged stain that binds to the negatively charged cell wall structures in the Gram-negative bacteria
What would you see under a microscope if you forgot to the crystal violet in your gram staining procedure?
Both gram (+) and gram (-) will be pink due to counterstain at the last step
What would you see under a microscope if you forgot to apply iodine during your gram staining procedure?
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.
What would you see under a microscope if you forgot to apply decolorizer during your gram staining procedure?
Gram (+) and (-) will look purple
Decolorization is an important step that differentiates between bacteria.
What would you see under a microscope if you forgot to apply the counterstain during your gram staining procedure?
- Gram (+) purple
- Gram (-) colourless
What would you see under a microscope if you forgot to the crystal violet in your gram staining procedure?
Both gram (+) and gram (-) will not be stained, and both will be pink due to counterstain at the last step
What are the four phases of a culture growth, and what are the cells usually doing in these phases?
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
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?
- 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.
Why do restriction enzymes only cut DNA plasmids one or a few times?
- 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.
How would you make a gel that contains 6% agarose and 12% agarose?
- 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.
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?
- 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.
Distinguish between food intoxication and food infection.
- 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)
How is ELISA similar/different from a lateral flow device? When would you use one over the other?
- 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.
How is FISH similar/different from PCR? When would you use one over the other?
- 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.
Provide four examples of how microbes are involved in food production or food preservation.
- 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.
