Lecture 5 Methods in Microbial Ecology and Biotechnology from cell counting to nucleic acids based methods Flashcards
What did Antoni van Leeuwenhoek discover?
Discovered bacteria and is now called the father of microbiology
What is Kochs postulates?
- The suspected pathogen must be present in all cases of disease and absent from healthy animals
- The pathogen must be grown in pure culture
- Cells from a pure culture of the pathogen must cause disease in a healthy animal
- The pathogen must be reisolated and shown to be the same as the original
What is the Winogradsky column? And what can it show?
Organic matter is incubated with light and supplements and an environmental gradient is created, oxygen decreases and sulphide increases. This gradient creates specific niches for bacteria
What is standard plate counting? How do you work it out?
A method for counting microbial abundance. To work it out (=total number of colonies x dilution factor)
How many colonies should you have on a standard plate counts?
30-300
What is the colony counting technique for marine samples and what is its equation?
Membrane filter techniques
CFU/ml = CFU x dilution factor/volume
What is light microscopy used for and how can you help improve them?
See how many microbes are present directly, very useful as you dont have to grow them, but a stain can help improve contrast and differentiate properties
What is the limit of compound microbes in light microscopy?
That a magnification of 1000x is required to resolve objects of 0.2μm, which is the size of the smallest microbes so cannot differentiate their properties
How does fluorescence microscopy work?
- Cells are made to fluoresce by illuminating them from above with light of a single colour
- Filters are used so that only fluorescent light is seen, thus, cells appear to glow in a dark background
Why is fluorescence microscopy considered easier than light microscopy?
Because it has a better contrast to visualize the bacteria
Name three benefits of fluorescence microscopy
- Use of natural fluorescence within cells
- Use of compound specific stains
- Enable in situ cell counts of individual cells instead of colonies, and independent of culturability
Give two reasons why electron microscopes are good?
- Highest resolution
- Highest magnification
What can a transmission electron microscope do?
See through cells to review intracellular structures
What can scanning electron microscopes do?
Examine surfaces
What is the fastest way to count cells?
Flow cytometrey
What are the three steps of flow cytometry?
- The individual cells flow through a narrow tube
- Lasers shine through and a detector detects natural optical properties
- It also detects laser-stimulated fluorescence
Why is flow cytometry so good in comparison to epifluorescence microscopy?
It has a much higher throughput
What does fluorescence in situ hybridisation (FISH) allow for?
Allows for the visualisation and quantification of different microbes
How is SSU rRNA used in FISH?
Used to pick a stretch unique to the organisms and attach a fluorescent dye fixed on a probe, which goes through the cell membrane
How does FISH work?
- A fluorochrome is attached to specific DNA
- A specific temperature is used to combine the complementary bases so they fluorescent labelled oligonucleotides hybridize
- The excess probes are washed
- Then a microscope or flow cytometer is used to identify the microbes
How does CAtalysed Reporter Deposition (CARD-FISH) differ to FISH?
Horseradish peroxidase probes (HRP) are used instead of the fluorochrome probes
What are the two steps of CARD-FISH?
- HRP is hybridized with 16S rRNA
- Fluorescent tyramide is added and multiple molecules are activated by a single HRP, giving a much stronger fluorescence signal
What is the advantage of CARD-FISH?
- Actively growing cells with many rRNA signals present
- Further amplification
- Allows for the detection of small or even rare microbes within a mixed community
Where are these from?
FISH or CARD-FISH