microbial ecology Flashcards
less than __% of microbes have been grown under lab conditions
5%
list some reasons why a bacterium may be unculturable (5)
microbe is slow growing, growth requirements are unmet, bacteria may require the activities of other bacteria, the microbe is present in low numbers, the bacteria may be in a VBNC state
how do you overcome a slow growing microbe
incubate samples for longer periods of time (weeks-months)
how do you overcome the unmet growth requirements of a microbe
make use of diffusion chambers
how do diffusion chambers work
a sample is inoculated into the chamber and the chamber is placed back into the natural environment. Allows for diffusion of environmental materials into the chamber while restricting the movement of cells
what activities of other bacteria might some bacteria require
growth promoting factors, may require the activity that alters the environment, siderophores
how do you overcome a bacteria needing the activity of other bacteria to grow (3)
co-culture the sample with other bacteria, use diffusion chambers, grow in a conditioned spent medium
list 2 approached you can take when the microbe is present in low numbers
reduce the diversity of the sample by dilution (dilution to extinction technique) or do the enrichment culture technique
describe the dilution to extinction technique
collect a sample from the environment of choice, look under a microscope to assess numbers, perform multiple dilutions on multiple samples until there are 1-10 cells per tube. Incubate cultures at the appropriate temp, then look for evidence of growth
what is the enrichment culture technique good for
isolating specific microbes that may be present in low numbers within a natural community. It’s used to encourage the growth of the desired microbe while inhibiting the growth of others
give 2 examples where you would use the enrichment culture technique to isolate a desired bacterium
- to isolate a thermophile, modify the physical conditions (temp)
- to isolate a nitrogen-fixing bacteria, modify the nutrient content of the medium
what does VBNC stand for
viable but non culturable
list 2 techniques for determining the number of bacteria in an environmental location of interest
most probable number (MPN) technique
staining technique
what type of bacteria can MPN be used on
only on culturable bacteria
T or F: MPN can be used on all types of bacteria; culturable, non culturable
false; can only be used on culturable bacteria
what types of samples can MPN be used on (2)
natural samples (food and water), and enrichment cultures
describe the MPN procedure
perform a tenfold dilution on the sample. 1 ml of each dilution is placed into triplicate tubes of growth medium. The tubes are incubated and examined for growth via turbidity
list 3 assumptions of the MPN technique
- bacteria are distributed randomly within the sample
- the bacteria are separate (don’t grow in clumps or chains)
- detectable growth will be observed even if one organism is inoculated
in the MPN technique, what steps do you do after you observe growth in the tubes (ie, how do you assess the numbers once you have the growth data)
choose the first dilution in which there was no growth (all 3 tubes of that dilution must have no growth). use the two dilutions prior to include in the set. Indicate the number of positives from each set, then consult an MPN table based on the numbers to get the MPN value. multiply the MPN value by the middle set from the dilution (reciprocal) to get number of bacteria per ml of sample
what type of cells can the staining technique be used for
VBNC or dead cells
T or F: the staining technique cannot be used to count VBNC cells or dead cells
false; it can! (unlike MPN technique for counting)
describe how the staining technique works
uses a fluorescent nucleic acid stain to count the total number of microbes present
list 2 stains used in the staining technique
DAPI and acridine orange
what do DAPI and acridine orange stain
both RNA and DNA
after the staining technique, how does one determine if the cells are living or dead? (as they all stain the same color)
use the LIVE/DEAD BacLight Bacteria Viability Procedure
how does the LIVE/DEAD BacLight Bacteria Viability Procedure work
makes use of 2 stains: 1 is membrane permeable green fluorescent nucleic acid stain, the other is a membrane impermeable fluorescent stain (propidium iodide). The stains are applied in a 1:1 mix and viewed using the fluorescent microscope
after the LIVE/DEAD BacLight Bacteria Viability Procedure, what color are living cells? dead cells? VBNC cells? why?
live cells are green b/c green is membrane permeable. dead cells are red b/c red is membrane impermeable and dead cells have a damaged membrane. VBNC cells are also green
list 4 techniques used to determine the diversity of a sample
FISH, sequencing analysis, PCR followed by denaturing gradient gel electrophoresis, phylochip
what level of diversity does FISH allow us to determine
allows us to identify different genera (genuses) of microbes present within a sample
outline the procedure of FISH
fix the sample, permeabilize the fixed cells, incubate the sample with ss oligonucleotides that are specific to a group of microbes of interest (called probes). Probes are fluorescently labeled. Wash off any unbound probe and then view under the fluorescent microscope
after doing FISH, what do you do if you also want to quantify the bacteria
make use of a flow cytometer
what two procedures can be done during sequencing analysis
shotgun metagenomics or PCR
describe shotgun metagenomics
sequencing all of the DNA that’s isolated from an environment using next generation sequencing, and comparing that info to a database to determine the microbes present
what level of diversity does shotgun metagenomics show
phylum
describe PCR
extract DNA from a sample and use PCR primers that target a specific gene from a microbial group of interest
what is qPCR and what is it used for
quantitative PCR, used to determine the number of specific groups of microbes present
T or F: PCR for microbial diversity is very accurate
false; there are some issues with its accuracy
describe how PCR may yield inaccurate results
some rRNA genes of microbes are more readily amplified than others. This may be due to GC content, degradation, or ease of lysis
what is a phylotype
a taxon identified based upon nucleic acid sequencing only
which technique for diversity identified phylotypes
sequencing analysis (PCR)
in PCR, give an example of a primer that can be used
16SrRNA
why does PCR followed by denaturing gradient gel electrophoresis allow the isolation of bacteria
all bacteria have the same conserved regions of the 16S rRNA, so by using this in PCR, only bacteria will be isolated (primers targeted the conserved regions only)
after PCR, describe how the bacteria will act when loaded onto an agarose gel
the bands will be the same size, because bacteria only differ within their variable regions, and this is NOT what the primers were targeting
describe what occurs when samples are loaded onto the DGGE
fragments will migrate until they become denatured. This results in a series of bands = a DNA fingerprint for that microbial communuty
based on the results of the denaturing gradient gel, how can we assess microbial diversity
high % GC = fragment will not be denatured, so it will migrate further down the gel
list 2 applications of PCR followed by DGGE
- can use to compare one microbial community with another (ie water vs soil)
- can look at how a microbial community changes over time
describe the phylochip method for determining diversity within a sample
the chip contains ss-16SrDNA sequences from thousands of bacteria. Isolate the DNA from the environmental sample and fluorescently label it. Apply it to the chip. If there’s a match, hybridization will occur. Labels are detected by laser, and then the data is computer analyzed. This gives info on which bacteria are present in the environment
other than numbers and diversity, what might ecologists want to study from a sample?
the activity of microbes
organisms that are defined by their physiological activity are called a ____
guild
what is a microbial community
a population of microbes that share a common habitat
list 3 methods ecologists use to study the activity of a sample
stable isotope probing (SIP), metatranscriptomics, metaproteomics
how does standard isotope probing allow us to study the activity of microbes
can be used to look at what microbes are taking up an element of interest, and thus which ones are metabolically active in the community
what type of elements does SIP utilize
isotopes of elements
what is an isotope
an element that differs in the number of neutrons
what domain do methanogens belong to
archaea
what is methanogenesis
an anaerobic respiration that produces methane as the final product of metabolism
are methanogens anaerobic or aerobic
anaerobic
what do methanogens use for the terminal electron acceptor
CO2
where does methanogenesis take place in the world
freshwater sediments, bogs, swamps, waterlogged soils
how can SIP be used to determine if organisms are carrying out methanogenesis?
set up a microcosm to mimic the natural conditions of the tested environment, pump in 13CO2, gaseous 13CH4 was collected. RNA extracted from the soil, separate 12C-containing RNA from 13C-containing RNA. Identify the archaea based upon the 13C-containing rRNA that was present
what does metatranscriptomics allow us to do
allows us to see which genes are being actively transcribed in a community (and thus have an active gene product)
what method is used in metatranscriptomics
RNA-Sequencing
describe the procedure of RNA-Sequencing in metatranscriptomics
collect a sample and isolate the mRNA. Convert mRNA to cDNA using reverse-transcriptase, then analyze that by NGS. Info is compared to a database, and then obtain all of the genes that are being transcribed at the time of sampling
in metatranscriptomics, what might one want to determine instead of which genes are being transcribed
whether or not a specific process is occuring
in metatranscriptomics, how can one determine if a specific process is occuring?
look at the presence of a specific mRNA that encodes for a protein that takes place in the process of interest
describe the procedure of determining if organisms are carrying out a specific process in a natural habitat (metatranscriptomics)
isolate the mRNA from the sample and convert to cDNA. Carry out PCR using primers that target a specific gene involved in the process, then run on a gel. If there is a product, then organisms carrying out that process were active at the time of sampling
what is the role of metaproteomics?
look at which proteins are present in the environment at the time of sampling
what procedure does metaproteomics utilize
2D-PAGE
what is 2D-PAGE
form of gel electrophoresis that separates proteins based upon charge and molecular weight
describe the procedure of metaproteomics using 2D-PAGE
proteins are isolated from the sample and radiolabeled, then separated via 2D electrophoresis = a visual representation of proteins in the sample. Cut a protein spot from the gel, digest, then analyze by Mass Spectroscopy to identify the protein
what does it mean for a cell to be viable but not culturable
it’s alive but unable to be cultured in the lab
why might a cell/microbe be VBNC
could be due to pollutants that negatively affect the cell
how are VBNC cells different to dead cells
unlike dead cells, they preserve their cellular integrity, their membranes are not injured, they retain genomic/plasmid DNA, they exhibit metabolic/respiratory activities, have high ATP levels, and may perform transcription/gene expression
how are VBNC cells different to normal viable culturable cells
nutrient transport + metabolic/respiratory activities are reduced, modification of cell wall/membrane + composition + morphology + gene expression
advantages to the VBNC state? (3)
the state is an adaptive strategy to survive harsh environments, cells in this state are able to be resuscitated, and the state allows for modification of certain cellular properties which may be beneficial for the cell/microbe
T or F: it is believed that the majority of microorganisms in nature are in a VBNC state
true
list 2 methods used to resuscitate VBNC cells
siderophores, resuscitation promoting factors
describe siderophores in VBNC resuscitation (2)
they promote cell division and they act as growth factors which reduces the lag phase
describe resuscitation promoting factors in VBNC resuscitation (3)
stimulate cell growth, reduces the lag phase, helps dormant cells revert from a VBNC state
primary use of VBNC cells?
play an important role in the neutralization of harmful environmental pollutants
list 2 ways in which VBNC cells can neutralize harmful pollutants
bioremediation and biodegradation
how does bioremediation work
utilizes the metabolic pathways of microorganisms
which is better in neutralizing harmful pollutants: bioremediation or biodegradation? why?
bioremediation; biodegradation is less efficient
