Past Exams (2014-2016) Flashcards
Compare nutrient agar (NA) and potato dextrose agar (PDA).
Nutrient Agar (NA):
- pH: 6.8
- Ingredients: agar, water, beef/yeast extract, peptone, NaCl
- For cultivating: Bacteria and fungi
Potato Dextrose Agar (PDA):
- pH: 5.6
- Ingredients: agar, water, potato extract, dextrose
- For cultivating: Fungi
- Can also be acidified to further inhibit bacterial growth.
Explain the gram staining procedure.
- Cells from a fresh, pure culture (< 24 hrs old) are transferred (smeared on) to a glass slide and allowed to dry.
- Culture slides are then gently heated to fix the bacteria in place.
- STAIN with CRYSTAL VIOLET for 30-60 seconds
- Rinse the slide gently with RO water
- STAIN with IODINE (a mordant (fixes the stain) for at least 1 minute
- Rinse the slide gently with RO water
- Rinse with alcohol until it *just* begins to run clear
- Rinse with RO water
- COUNTERSTAIN with SAFRANIN for 30-60 secs
- Rinse with RO water
- Blot dry
- Examine
*** Gram positive bacteria will appear purple and gram negative bacteria will appear red.
Imagine that you have prepared a gram stain of a confirmed pure culture of a bacterium. You see clumps of purple (or blue) cells and scattered red (or pink) cells on the slide.
Is the bacterium Gram-positive or Gram-negative? Explain your answer.
(*What do you think the correct interpretation of this Gram stain is and why?)
The bacteria are most likely gram negative.
When assessing gram-stained bacteria, assessments are made on individual, well-dispersed cells. Clumps of cells are excluded from analysis because being in a clump can cause problems with taking up and/or rinsing off dyes, leading to false positives (e.g. cells that remain violet because the alcohol was unable to penetrate and wash out the excess dye from the clump, leaving the cells unable to accept the safranin dye). For this reason, the blue/violet clumps would be ignored, and the scattered (individual) red/pink cells would be used for evaluating the gram-stain reaction.
You have been asked to identify a bacterium isolated from a plant, animal, food, or soil sample. Outline THREE (3) approaches that you might use, including in your answer at least one growth dependent and one nucleic-acid based approach. Note for each approach if it would be useful for the identification of Gram-negative or Gram-positive bacteria, or both.
Morphological analysis, looking at: (both gram positive and negative)
- Shape
- Size
- Arrangement
- Motility
- Number and position of flagella
- Presence/absence of endospores
Metabolic test kit
- Mostly for gram-negative bacteria
- e.g. Microbact™ which shows various colour reactions based on a pH change brought about by metabolism of the substrate (12-24 diff substrates).
Chemical composition test, e.g. FAME (analysis of Fatty Acid Methyl Esters)
- Provides a summary of the types and proportions of fatty acids in membranes
- variation exists in chain length, saturation, rings, branches, hydroxyl groups
Antibiotic resistance (growth dependent)
Pathogenicity/symbiosis (growth dependent)
GC ratio (guanine : cytosine) in DNA
- variable in bacteria, among and within groups
- usefull in determining unrelatedness; i.e. whether two samples are related or not
Nuceic acid probes
- to detect specific sequence
- ssDNA with sequence unique to organism in question and that is labelled with a reporter molecule is added to lysed DNA (so it too is single stranded) and allowed to reanneal. The unbound probes are washed off, and the amount of hybridisation is measured using the radioactive/fluorescent reporter.
PCR (Polymerase Chain Reaction)
DNA sequencing
- useful for determining relatedness
You have a pure culture of a Gram-negative, rod-shaped, motile*, obligately anaerobic*, endospore-forming* bacterium.
- Explain what’s meant by the starred* terms, and discuss the advantages they may confer.
- Choose TWO (2) of the following approaches that might help to identify this organism (to genus or species). Explain how each of these approaches works and any limitations that might apply.
(i) Metabolic tests (e.g. Microbact, Biolog)
(ii) Analysis of Fatty acid methyl esters (FAME)
(iii) Physiological characteristics
Motile:
- Able to move around using flagella, fimbriae, and/or pili
- Advantages:
- Can move around to exploit more (and more desirable) resources
- Escape predation
- Be a better predator
Obligately anaerobic:
- Can only survive in anaerobic (oxygen poor) conditions
- Whether this is an advantage or disadvantage depends on the environment the bacteria is living in
- if in the gut, or underwater, its an advantage
- it would be more of an advantage to be facultatively anaerobic, meaning it could switch to aerobic respiration if in the presence of oxygen.
Endospore-forming:
- Endospores are basically a bacteria’s ultimate survival strategy
- They’re non-sexual survival structures (not spores) formed inside a parent cell
- Occur in bacteria within the Firmicutes phylum
- Have a thick cell wall to protect them
- Very resistant/tough/long-living
- Means they can withstand unfavourable conditions while they wait for optimal conditions to thrive
Metabolic tests (e.g. Microbact, Biolog)
Analysis of Fatty Acid Methyl Esters (FAME)
Physiological characteristics
Microbial successions are common in the production of fermented foods. Some successions involve both bacteria and fungi (filamentous fungi and or yeasts).
Discuss microbial successions and the factors which influence the microbial community during production of fermented foods. In your answer provide at least one example which included both bacteria and fungi.
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Explain what is meant by microbial succession in the context of food, fodder (e.g. silage) and beverages, using two examples to illustrate the principles and processes involved in transforming the starting materials into a desirable product.
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Lactic acid bacteria often occur as part of a succession of microorganisms.
Describe one such succession and explain the changes in dominant microorganism(s) over time and the factors that drive those changes.
Explain why lactic acid bacteria are important in the production of food and fodder (such as silage).
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Outline the process of making compost aerobically from plant-derived materials in terms of the microbial successions that may occur, and dismiss the environmental and nutritional features that influence those successions.
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Choose TWO (2) of the following approaches that may be used to extend the shelf life of food. Describe how they work AND explain their influence on the eventual spoilage of food:
(i) canning/heat/high sugar or salt content
(ii) chemical preservatives
(iii) irradiation
(iv) fermentation
(v) pH
XX
Briefly describe C. botulinum, noting the features that make this microorganism difficult to control, and discuss the strategies for food preservation that might be implemented specifically to minimize the risk of botulism.
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Discuss the role of microorganisms in the fixation of N, as free-living organisms and in symbiotic (or mutualistic) relationship(s) with plants.
XX
Briefly describe one of the following ecosystems in terms of the microbial succession and the change in environmental factors (e.g. nutrients, oxygen availability, temperature, pH) over time, then explain how spoilage can occur as a result of disruption of these factors:
(a) compost
(b) silage
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Write short notes on methods for establishing pure cultures.
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Write short notes on sterilization by filtration vs autoclave
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