Lecture 1-Midterm Flashcards
Distinguish between culture-dependent and culture independent techniques of studying bacteria (give examples of each)
Culture-dependent (ex. streaking a plate) - relies on growing the bacteria in culture before hand
Culture-independent (ex. microscopy) - doesn’t matter if the bacteria or alive or dead, they must simply be there
What is “the great plate count anomaly”?
The idea that after serial dilution, we still end up with only 10-1000x of what the original cell count was - a very different result than what is seen under the microscope
What does it mean to say that microbial research is usually treated as a “black box”?
It means that we have no idea what bacteria or how many bacteria perform a process, but instead just measure the reactions themselves (ex. nitrogen fixation) in controlled environments
IT is thought that ____% - ____% of bacteria present in a sample can be cultivated
0.1% - 1%
Sewage is generally ____% cultivatable bacteria whereas soil is more like ____%
30%, 0.01%-0.1%
What are some advantages of disadvantages of culture dependent analysis of bacteria?
Advantages:
Can create pure culture
Can do genetic/biochemical characterization
After isolating species, we can observe rate of biochemical reactions
Disadvantages:
Most of the organisms present cannot be observed in any way
What are some advantages and disadvantages of culture independent analysis of bacteria?
Advantages: Easy and cheap Many media (Selective, or general purpose - there is no universal medium) Disadvantages: Harder to isolate organisms
How do nutrient concentrations in culture media compare to that of the natural environment?
Concentration in culture media is thousands of times that of the natural environment
Distinguish between oligotrophs and Copiotrophs
Oligotrophs - like to live in low nutrient environments (won’t grow in culture)
Copiotrophs - Like to live in high nutrient or polluted environments
What lab media can be used to isolate oligotrophs
Dilute versions of other lab media.
What are different reasons why bacteria may not grow on standard growth media?
May have very low growth rate (months to years)
May be oligotrophic
What is an “MPN”? How does one used this?
Most probable number - statistical technique used to quantify bacteria.
- Large bottles of varying dilutions are prepared from a sample
- Grow 3mL of each sample in 3-5 separate tubes (and a control)
- Observe tubes for turbidity.
- Results are plugged into a table based on turbidity
- Most probable number of bacteria present is calculated
How does the use of MPNs allow scientists to obtain pure cultures?
At a certain dilution it is highly likely that only 1 bacterium was inoculated into the tube - so if you streak that it results in a pure culture
Why is it that bacterial colonies observed on agar after 6 weeks are the same as those observed at 4 weeks?
Because bacteria are dormant a lot of the time, usually under nutrient stress. Randomly, bacteria will break dormancy and appear as a colony.
Why is it that pyruvate or rare sugars are better energy sources in growth media than glucose?
Glucose breakdown results in more toxic by-products that are harmful to oligotrophic bacteria.
TRUE OR FALSE: Oligotrophic or nutrient-starved bacteria are more susceptible to toxic by-products of cellular respiration
TRUE
IF one wants to grow oligotrophic bacteria, what is the best mixture of substances
Small (miniscule) amounts of carbohydrates, organic acids, hydrocarbons, amines
There are ______ to ________ of different species of bacteria in one gram of soil
Thousands to potentially Millions
How did scientists find a way to get bacteria to produce “unlimited” antibiotics?
Using two hydrophobic plastic layers covered in wells leading to holes slightly smaller than a single bacteria. Between them is a dialysis membrane.
The plastic is added to water and the space between the two layers (containing the dialysis membrane) mimics the atmosphere. Each well should maintain very few bacteria coming from a single bacterial source (in the hole)
Observations are made using a microtiter reader, observing which wells have bacteria.
These bacteria had 40-50% cultivatability.
Why is it that a single cell per volume will grow better than multiple cells?
It just does. We have no idea why,
What is a FACS machine and how does it work?
Fluorescence activated cell sorting machine.
Machine sends tiny tiny blobs of liquid which may or may not contain bacteria past a laser beam. This excitational laser beam induces fluorescence in bacteria. If beads fluoresce, they are sent down a tube into one container, and if they do not fluoresce they are sent to another, Sorting can be further differentiated by telling the machine to only take beads containing more than one bacterium, or to sort them into 0 bacteria, 1 bacterium, or more than one.
Micrococcus sp. contain _______ which results in the production of a peptide that may resuscitate gram positive cells. How can this be used in experimental conditions?
Resuscitation factor.
It is produced by stationary cells and pumped into the external environment. This can be extracted by removing cells from the system. This solution full of resuscitation factor can be added to dormant gram positive bacteria (ex. other micrococcus) to get them to grow
What is “co-cultivation”?
Some bacteria will only grow in the presence of another type of bacteria, but we don’t know why.
One thing we do is streak colonies out in lines, then streak the sample across the lines (crosshatch).
Sometimes at the intersection of two lines the bacteria will be found.
Another method is to grow some bacteria suspended in agar, and the others grown on top of the agar on a dialysis membrane
Is direct microscopic counting a culture dependent or culture independent technique?
Culture independent
What are the advantages and disadvtanges of Microscopy as an analytical method?
Advantanges: inexpensive and easy
Disadvantages: Hard to tell what is bacteria vs eukarya or inorganic water droplets or rock fragments, it’s fucking boring, can’t tell the difference between live or dead cells
What is phase contrast microscopy?
Get better contrast between cells and background
Why would one prepare soil and water samples for phase contrast microscopy?
Add dye to water samples, but soils samples require both dilution and dye, run through filter (usually polycarbonate)
What is “epifluorescent” microscopy? How does it relate to the black stain found on polycarbonate filters?
Microscope with light bending substance is used to induce fluorescence in bacteria on black-stained filters. This results in a black background but bright bacteria.
What are some dyes used in fluorescence microscopy? What are some advantages and disadvantanges?
Acridine orange (stains both protein and DNA - usually different colours - may stain things that are not bacteria) DAPI or Hoechst 33258 (used more commonly - stains DNA more specifically)
What are some benefits of fluorescence microscopy?
May see bacteria that are too small to be seen with regular light microscopes, and usually can pick up far more bacteria than standard plate counts
What are “ultra micro bacteria”?
Bacteria so small that they cannot be seen with standard microscopy. They make up most of the biomass of some ecosystems
What is the formazan dye reduction technique?
Oxidized formazan dye can enter living bacteria and can be reduced inside the cell as part of the ETC. As the dye is reduced it precipitates and a red colour is produced. Once the sample is observed under a standard bright field microscope, red cells are alive and uncoloured cells are dead
What is a LiveBac stain?
A stain that results in live and dead cells appearing as different colours
What is the difference between viable cells and culturable cells? Are viable cells considered alive?
Viable simply means they stain as “live” cells whereas culturable cells can form a colony. Viable cells are often considered alive, but they may be dead, dying, or becoming dormant.
What is meant by the term “VBNC”?
Viable but not culturable - Cells are considered alive but may not divide or cause disease
How is Vibrio cholerae shown to sometimes be VBNC
If a mouse ingests cholera bacteria, they may not get sick, but when they excrete the V. cholerae, those bacteria may now be pathogenic. Something in the mouse activated these VBNC cells
How does TB dormancy result in TB infections late in life?
Early in life someone can be infected with TB, then it can be triggered over 30 years later by some form of stress (ex. absence of tumour necrosis factor)
How can one break dormancy of TB in a lab?
Dormant bacteria can be reactivated by living viable bacteria.
What are indirect/functional methods of bacterial analysis?
Things that measure/count a property or reaction that is proportional to bacterial population (ex. measure O2 output)
How can one measure cell components to observe bacterial ecology?
Usually one can measure DNA or protein (usually Lowry or Bradford methods). The problem is that these methods are not specific to microorganisms
How can one measure DNA to observe bacterial ecology?
Measure UV absorbance of DNA using spectrophotometer or use fluorescence. There is sometimes interference from abiotic components.
Why is peptidoglycan a great way to measure bacterial components of a sample? How does one do this?
Because Eukarya and Archaea don’t have peptidoglycan. This is performed by lysing cells and acid hydrolysing peptidoglycan. This is dependent on the proportion of gram negatives and gram positives because they contain different amounts of peptidoglycan
What is the benefit of using lipopolysaccharides to quantify microbes in a sample?
It is specific for gram negatives
Why is the measurement of ATP one of the most sensitive ways to determine live cells in a bacterial sample? How is this performed? What are some disadvantages?
It allows one to measure only living cells and it is highly sensitive.
Performed using “luciferase”, which reacts with O2, luciferin, and ATP to give off small flashes of light.
The ATP must be removed from the cells almost instantly, as it will degrade very quickly.
How are Biolog tests performed?
Using 96 well microtiter plates. In some, you have only a carbon source and in others a nitrogen source. Each well has a pH indicator, and each well is inoculated with the sample. Whether the organisms used the substrate is determined. This allows one to observe the biochemical needs of the whole bacterial community
How are isotopes used in microbial ecology?
Radioactive isotopes can be used to follow compounds through an organism, ecosystem, or sample.
Radioactive isotopes were popularized in the sciences in the _______, after _____
1940s, after World War II
What is an “isotopic ratio”
The idea that many elements have multiple isotopes that have varying abundance. The light isotope is nearly always more abundant
How can isotopic ratios be used in microbial ecology?
One can substitute an element (ex Hydrogen) for an isotope of that element. The ratios of Hydrogen1 to the isotope can be measured to track Hydrogen progress
How can isotopes be used to track Nitrogen?
One can use isotopes of hydrogen to bind to ammonia. Or, radioactive isotopes (Hydrogen3) can be used to “tag” the molecule
TRUE OR FALSE: In bacteria, Carbon is fixed by the Calvin cycle
FALSE, there are many ways bacteria fix carbon
How can one use radioactive Carbon14 to observe carbon fixation?
CO2 tagged with Carbon14.
Carbon14 can be found along the cycle in varying intermediates along the cycle. measurements taken slightly after one another can show the steps of the cycle.
How does one tell the difference between radioactively-tagged bacteria and non-radioactively-tagged bacteria?
Dilute the sample and pour it over X-ray film. Develop the film, and the glowing bacteria are radioactive.
Another way is to filter the bacteria, roll the filter into a tube and use machinery to measure the amount of the radioactive isotope present.
In an experiment a scientist adds 14C tagged glucose to a flask containing bacteria. How do they measure the rate of respiration (CO2 produced/unit weight/unit time)
Put solution in erlenmeyer flask with tube leading to alkaline solution. CO2 should bubble off into solution (CO2 trap) and measured.
What are “dissimilatory processes”?
Molecule is not incorporated into biomass and instead is released as a waste product
Why is dissimilation so important for the Nitrogen cycle in bacteria?
Because N2 in such organisms is reduced to NH3 but not assimilated into the organism. This results in most of our atmospheric N2
Distinguish between DOC and DON
Dissolved organic carbon
Dissolved organic nitrogen
Distinguish between POC and PON
Particulate organic carbon
Particulate organic nitrogen
How does one separate primary and secondary productivity measurements in microbes?
label isotopes that are used in both.
What is the primary reason that we use radioisotopes in microbial ecology?
Techniques to measure radioisotopes are VERY VERY VERY VERY sensitive, which is necessary because often we are working with less than a micromole of substance
____ is the standard laboratory method to quantify the radioactivity of low energy radioisotopes, mostly beta-emitting and alpha-emitting isotopes. The sensitive detection method requires specific cocktails to absorb the energy into detectable light pulses.
Liquid scintillation counting
Why is it that we don’t need to use radioisotopes in science any more?
We now have analyticial techniques that allow us to observe the ratios of stable isotopes of an element
What is GC-IRMS and how can it be used to distinguish between two isotopes?
gas chromatography - ion ratio mass spectroscopy. Allows for very sensitive separation of isotopes of the same element
What is “isotopic discrimination”?
The idea that many biological/chemical processes discriminate against one isotope of an element (preferentially use one - usually the lighter one)
Why is isotopic discrimination a way to detect the presence of life?
The ratios of isotopic elements can be used to determine whether life is present, because isotopic ratios will typically favour the light isotopes in the presence of life. It is possible to look at mineral profiles to find how farm back in time living organisms have been on the planet
The isotopic ratio of sigma13C for atmospheric CO2 is -8 parts per thousand. What is the ratio within plant organic material? What does this mean?
-27 parts per thousand. This means that plants preferentially use Carbon12 (the smaller isotope).
If plants have an isotopic carbon ratio of -27 parts per thousand, what do bacteria have? Are all bacteria the same?
Bacteria that consume plant material will have the same isotopic ratio but other types of bacteria have varying ratios. For example, methanogens have a -40 parts per thousand ratio
TRUE OR FALSE: There are no abiotic processes that discriminate against certain isotopes
FALSE - certain geologic processes have designated isotopic ratios
What is “deep evolution”?
The idea that life can be traced back as far as almost 4.1 billion years ago
Prokaryotes can be found as far back as ______
3.7 billion years ago
Why are molecular detection methods the preferred method of microbial analysis?
Allows for culture-independent detection, counting, and identification of microorganisms, which in turn enables one to compare bacterial profiles within ecological research
Why are molecular methods considered universal to all life?
All cellular organisms contain DNA or RNA
PCR was invented in the year _____
1985
How do people harvest bacteria from thermal ponds?
Tying a microscope slide to a fishing line and dipping it in the ponds for long periods of time before viewing them under a microscope
how was the important bacterial species “Thermus aquaticus” or “Taq” discovered?
By collecting it from a thermal pond in yellowstone national park
Why is Taq such a historically important microorganism?
After the thermophilic bacterium Thermus aquaticus was discovered, scientists realized that it must have its own heat-stable DNA polymerase. This is now used in PCR so we do not have to add new polymerase after each cycle
When executing PCR, we want our primers to bind with any possible bacterium. How do we accomplish this?
We need to find a piece of DNA that is found in every bacterial species in the world today
How can PCR be used to determine the presence/absence of bacteria or the quantity of bacteria in a sample?
Searching for bacteria-specific DNA codes allows us to detect/quantify bacteria
How can PCR be used to identify bacteria? Describe this step by step.
16S rRNA is present in all bacteria and there is some variation between each type of bacteria.
- Take a sample and isolate total DNA
- Isolate all 16SrRNA genes (PCR product) - this produces a “gene” library (a bunch of the same gene but all from different bacteria)
- Insert all those genes into different plasmids
- All the plasmids are inserted organisms (ex. E. coli) - transformation. Each E. coli has a single plasmid in it.
- The E. coli cells are plated on agar, and a bunch of colonies grow, each from one CFU of E. coli - Each colony has the same variant of the gene
- Plasmid DNA can be isolate from one clone - you now have multiple copies of the same gene.
How can PCR be used to detect viruses (specifically RNA viruses)?
Can convert RNA to DNA using reverse transcriptase
How can PCR be used to determine whether a gene is active/expressed?
Get PCR machine to search for mRNA sequence, convert with reverse transcriptase to DNA.
In 1 gram of soil there is approximately ______ species of bacteria present
10 000
Why is rRNA used to determine relatedness of microorganism?
Because all cellular organisms require ribosomes to produce protein (and therefore have ribosomal RNA)
What is the eukaryotic equivalent of 16SrRNA?
18SrRNA
