Methods To Study Microorganisms Flashcards
How much of all bacteria have been isolated in a pure culture?
0.01 to 1%
What are some methods to study microorganisms and their activity?
Sampling- soil, water, sediments
Sample processing- homogenisation, dilution, concentration
Determination of microbial numbers- direct counts, viable counts
Proxy measures of biomass- cell envelope components
Measurement of microbial activity and processes- isotopic techniques, specific inhibitors to measure metabolic intermediates
Culture independent analysis of microbial communities- 16S RNA based, metagenomics
What is the direct cell count procedure?
Cells counted directly, with a microscope
Uses a modified microscope slide known as a counting chamber- Sedwick-Rafter cell OR Haemocytometer
Gives the highest estimate of cell numbers
Good for large morphologically distinguishable cells
Direct counts often proportional to biomass
May be possible to identify dividing cells and use this to estimate mean growth rate
What are the limitations of direct count procedures?
Cannot distinguish between live and dead cells
Background debris can cause problems
Little information about types of organisms present , activity or physiological type
How are direct count procedures used on fungi?
Known amount of sample mixed with molten agar
Transferred to a microscope slide and a known area is covered in a thin agar film
Cells are stained
Thin agar film examined microscopically- length of mycelium measured using an eyepiece graticule, photograph taken and dimensions measured
Determining mean filament diameter allows bio volume and biomass to be calculated
How are direct count procedures used on bacteria?
Sample fixed and diluted
Stained with fluorescent dye and examined by epifluoresence microscopy
Sample filtered and washed
Filter is viewed
Cells are counted
Can work out cell numbers when we know the dilution factor, filtration area, amount of original sample, area of microscopic field of view, and number of fields of view counted
Automated cell counting is a form of direct count procedures, how does this work?
Using a flow cytometer
Sample containing cells added to fine stream of flowing liquid (sheath fluid)
Fluid expelled through a very fine vibrating needle
Droplets pass by optical detector
Properties of particles can be measured- fluorescence AND light scatter/size
What are the limitations of automated cell counting?
Best for aqueous samples
Particulate material can clog needle
Clumps of cells can underestimate numbers
What are the two main methods of viable count procedures?
Plate counts
MPN counts (most probable number counts)
What are limitations of viable count procedures?
Detects only cells that grow on laboratory growth media
Selective
Gives lower estimates than direct counts
Relies on ability to separate cells into reproductive units- rarely achieved as cells habitually form clumps or filaments . For this reason viable counts often reported as Colony forming units (CFUs)
What is the procedure of plate counts?
Organisms grow on solid agar media
Samples are serially diluted, and spread evenly- dilute samples can be filtered onto a sterile filter and placed on the agar surface. Samples can also be mixed with molten agar before the plate is poured
Plates incubated under suitable conditions and macroscopic colonies arising from viable cells are counted
Numbed in the original sample calculated based on dilution factor and volume of sample spread of plate
What are the two types of media that can be used in plate counts?
Selective media- can be used to enumerate particular types of organism
Diagnostic media- can allow organisms to be identified among non-target organisms by their growth on specific agar medium
What is the procedure of MPN counts?
Sample serially diluted, often conducted in a glass bottle
Inoculated into an appropriate growth medium
Incubated under appropriate conditions
Number of positive scores in replicate sub samples at each dilution can be used to calculate most probable number of organisms in the orginal sample
What are the limitations of MPN counts?
Poor for total counts as selectively imposed by growth media
What are the advantages of MPN counts?
Useful for specific organisms
Useful for lithotrophic organisms with low growth yields
What order of magnitude higher does direct count procedures give in comparison to viable bacterial counts?
1-2 order of magnitude higher
Why are measurements of microbial processes often referred to as potentials?
Not always representative of rates in situ, so often referred to as ‘potentials’
How can microbial processes be measured simply, and what are the limitations?
Using chemical measurements
Measuring changes in the concentration of biologically transformed chemical species can be used to measure processes
Limitations:
Often have limited sensitivity
Measure potentials rather than in situ rates
Linked processes can affect measured rates
How do specific metabolic inhibitors work to measure microbial processes, and what are some examples and limitations ?
Inhibitor added to sample and effect on process is measured
Examples:
Process: Sulfate reduction
Inhibitors: sodium molybdate, fluoracetate
Process: methanogenesis
Inhibitors: bromoethane sulfonic acid
Process: denitrification
Inhibitors: sodium chlorate, acetylene
Limitations:
Inhibitors can effect more than one process, not specific
What are two groups that fall under isotopic techniques?
Radio tracer techniques
Stable isotope techniques
What is an example of a stable isotope technique and how does it work?
Measuring nitrogen cycling can be done using ¹⁵N labeled compounds. e.g ¹⁵NO3(-), ¹⁵NO2(-), ¹⁵NH4(+)
Denitrification and linked nitrification can be measured
Samples incubated in sealed vessel and headspace analysed
Gaseous products measured by mass spectrometry
Dissolved nitrogen species can be biologically or chemically reduced to nitrous oxide or dinitrogen gas and measured by mass spectrometry
How can radio tracer techniques be used to measure primary productivity?
Measuring uptake of ¹⁴CO2/H¹⁴CO3(-)
Samples taken from environment
Known amount of radio tracer added
Sealed bottle incubated in lab or in situ
Samples filtered and washed
Radioactivity in particulate material measured by scintillation counting
Light and dark incubations to take account of inorganic carbon uptake by chemoautotrophs and heterotrophs
How can radio tracer techniques be used to measure bacterial (secondary) productivity, and what is a limitation?
A measure of DNA or protein synthesis rates
Measures uptake of thymidine and leucine
Samples taken from environment
Known amount of labelled nucleoside or amino acid added
Sealed bottle incubated in lab or in situ in the dark
Samples filtered and washed
Nucleic acids and proteins extracted
Limitations:
Not all organisms assimilate exogenous nucleotides or amino acids- underestimates bacterial growth rates
Radioactivity in extract measured by scintillation counting
How can radio tracer techniques be used to measure degradation of specific compounds, and what is a limitation?
Degradation of ¹⁴C labelled carbon compounds- conversion of organic carbon to carbon dioxide can be used to measure degradation rates of specific compounds
Known amount of radio labelled compound added to environmental sample
Incubated in sealed vessel containing CO2 trap- KOH
Samples taken from CO2 trap overtime and radioactivity measured over time
Residual radioactivity can be measured in the sample
Limitations:
May overestimate rates of degradation of aged pollutants
How can radio tracer techniques be used to measure sulfate reduction?
Measurement with ³⁵SO4(2-)
Known amount of ³⁵SO4(2-) added to environmental sample
Anoxic conditions maintained
May be measured with or without electron donors
Reduced sulfur distilled by chromium reduction
Sulfied produced trapped in Zinc acetate
Radioactivity in zinc sulfied measured by scintillation counting
What is positive selection?
Growing the culture on a medium that will allow for the growth of only mutant colonies
What is negative selection?
Used to identify mutants that have lost their ability to perform a certain function that their parents had
What is transduction?
Use of bacteriophages to transfer DNA between cells
Genetic material exchanged via viruses
No physical contact
Promotes drug resistance
Helps genetic mapping and engineering
What is transformation?
Acquisition of exogenous DNA fragments from the environment
Promotes Antibiotics resistance
What is conjugation?
DNA exchanged via pilli
Cell to cell contact required
Transfer is non specific
Drug resistance
What is CRISPR?
Clustered
Regularly
Interspaced
Short
Palindromic
Repeat
Two main components:
Cas9 enzyme which cuts DNA
A guide RNA guides these molecular scissors to the sequence we want to cut
