1.4 Microbiology Flashcards
What are the 3 PHYSICAL differences between bacteria that we look at? One of the physical features, depending on the structure, can impact the _____ techniques/methods used to identify the bacteria.
a) Size
b) Shape
c) Cell wall structure - this physical feature depending on the cell wall structure can impact staining methods used to identify the bacteria.
Give 2 GENETIC differences that we look at between bacteria… The last point has to do with the type of proteins found on their surface.
a) Varying metabolic features
b) Antigenetic properties - i.e. the type of proteins found on the surface of the bacteria - those proteins are called ANTIGENS.
A genus (a taxonomic category that’s found above ‘species’ & below ‘family’) can have 1 of 3 main SHAPES that can be used to identify/categorise the bacteria. The name of the genus can indicate that shape. What are the three types of genus names we look at and give the name of the shape.
a)BACILLUS - Rod shaped bacteria
b)COCCUS - Spherical shaped bacteria
c)SPIRILLIUM - Corkscrew-shaped/Spiral shaped
Apart from the shape itself allowing us to categorise bacteria, we can also identify bacteria from the way they group/cluster with each other (bacteria of the same kind).
There are 3 types of ways bacteria spherically shaped cluster/organise themselves. Identify the names and whether they organise in chains or large clusters.
a) StreptoCOCCUS - They tend to group in CHAINS. The spherical shaped bacteria form chains.
b) StraphyloCOCCUS - They tend to group in CLUSTERS. The spherical shaped bacteria cluster around each other.
c) DiploCOCCUS - Bacteria are in PAIRS.
Another method of bacteria identification is the ‘Gram Stain Reaction’. What is the ‘Gram Stain Reaction’?
It’s another method of bacteria identification microbiologists use to distinguish between Gram +VE bacteria & Gram -VE bacteria.
Following on from the last card. Why do these two types of bacteria (Gram +VE and Gram -VE) require different types of staining techniques? Simple answer.
Because it’s dependent on the chemical composition of the bacteria’s cell wall.
What colour do Gram -VE bacteria appear compared to Gram +VE? Why is this? Think about the types of stains used…
Gram +VE bacteria appear a purple/violet colour due to the stain used, ‘crystal violet’.
Gram -VE bacteria appear a red colour due to the COUNTER stain used, ‘red safranine’.
Why can the crystal violet stain bind more effieciently to the cell wall of Gram +VE bacteria compared to Gram -VE? Think about the layer that Gram -VE bacteria have that Gram +VE don’t.
Because Gram +VE bacteria lack an outer lipopolysaccharide layer that’s found in Gram -VE bacteria.
What is the significance of the outer lipopolysaccharide layer found only in Gram -VE bacteria - i.e. It does what against lysozyme enzymes & penecillin? It does what against crystal violet dye?
The cell wall of Gram -VE bacteria is supplemented by a lipopolysaccharide layer that PROTECTS the bacteria from lysosome enzymes and penecillin as well as excluding the crystal violet dye!
If the Gram +VE bacteria lack a supplementary lipopolysaccaride layer what does this make Gram +VE bacteria succeptible to? 2 things that the lipopolysaccharide layer protects against in Gram -VE bacteria…
They’re succeptible to PENECILLIN & LYSOZYME ENZYMES.
What is a lysozyme enzyme? Why are they membrane bound in our cells? Why are they essentially a threat to bacteria?
Lysosyme enzymes are DIGESTIVE enzymes and are membrane bound to protect surrounding organelles. They’re a threat to bacteria because the enzyme HYDROLYSES the bonds holding the peptidoglycan layer together.
What does penecillin do to the cell wall’s (what does it prevent ?) of Gram +VE bacteria? Penecillin ____ the cell wall of Gram +VE bacteria. Because Penecillin _____ the cell wall it makes the bacterial cell suceptible to LYSIS/BURST due to water uptake…
Penecillin PREVENTS the bonds inter-linking the peptidoglycan molecules. Penecillin WEAKENS the cell wall of Gram +VE bacteria.
Because the cell wall is weakened by penecillin the uptake of water can BURST the cell wall - cell can’t hold itself together due to weakening of cell wall…
If the lipopolysaccharide layer in Gram -VE bacteria protects the bacteria from PENECILLIN how do we protect against Gram -VE bacteria?
We use another type of antibiotic that impacts the bacteria’s ability to synthesise proteins.
If we use the antibiotic that impact’s the Gram -VE bacteria’s ability to synthesise proteins won’t that impact the ukaryotic cell’s ability to also synthesise proteins?
NO, because the machinery responsible for protein synthesis in eukaryotes is different from bacteria!
a) What is the name of the couter stain used for Gram -VE bacteria?
b) What is the name of the stain used initially that will lead to a purple colour to appear on Gram +VE bacteria?
a) Red Safranine
b) Crystal Violet
What do we use to wash off the stain?
Alcohol - it’s a decoulouriser. Removes the colour from Gram -VE bacteria making them colourless. Can’t do this to Gram +VE bacteria though - purple remains…
What do we use to help the crystal violet stain bind to the peptidoglycan strongly?
IODINE - it’s a MORDANT.
MOVING ON FROM CLASSIFICATIO»_space;>
Bacteria undergo what to reproduce? NOT ‘mitosis’… Define that term…
Bacteria UNDERGO ‘Binary fission’.
Binary fission is the replication of the circular DNA and plasmids and is the division of the cytoplasm forming two IDENTICAL daughter cells.
What 5 things do bacteria/microorganisms require for growth? The next few cards will look at testing you on each…
a) Nutrients
b) Growth Factors (i.e. vitamins and mineral salts such as Na+)
c) Temperature
d) pH
e) Oxygen
Bacteria require NUTRIENTS that can be supplied in a ______.
Nutrient Medium
This nutirent medium/media can be a liquid called a _____ or in solid form called _____.
a) Liquid from called ‘Nutrient BROTH’
b) Solid form called ‘AGAR’
What do nutrient mediums/media provide bacteria? 3 THINGS…
a) Carbon & an energy source - gluecose
b) Nitrogen to allow the bacteria to synthesise amino acids
c) Water
The SECOND requirement for culturing microorganisms is providing growth factors such as? 2 THINGS…
a) Vitamins
b) Mineral salts - Na+ & PO3 4- etc…
The THIRD requirement is a suitable temperature. Why is this key to culturing microorganisms?
Bacterial chemical reactions (i.e. their metabolism) are controlled/facilitated by enzymes. Thus, these enzymes require certain operating temperatures to ensure they’re working fast/efficiently but also the temp. must NOT be over the OPTIMUM level or the enzymes are prone to DENATURINg/DENATURATION.
The FIFTH requirement is OXYGEN (we’ve skipped pH).
a) What are bacteria who NEED oxygen called?
b) What are bacteria who CAN survive without oxygen called?
c) What are bacteria who CANNOT survive in the presence of oxygen called?
a) Obligate aerobes
b) Facultative aeorbes
c) Obligate anaerobes
There are 5 reasons why many bacteria CANNOT be cultured in the conditions we just described/listed. Name and explain them…
a) Some bacteria are mutualistic (i.e they require the presence of another species in order to thrive).
b) Some are INTRACELLULAR parasites
c) Simply have very SPECIFIC growth requirements
d) Very long generation times
e) Poisoned by media components.
There are 4 ways to DESCRIBE media:
a) Defined - i.e. we know what the ingredients present in the growth medium are…
b) Undefined - i.e. we don’t know what some of the media’s components are because they include yeast extract or beef peptone.
c) A selectve medium - i.e. a medium that allows only certain bacteria to grow. E.g. a selective media that only allows Gram -VE bacteria to grow…
d) A COMPLETE MEDIUM - All the chemicals required to support growth are present…
PRINCIPLES OF ASEPTIC TECHNIQUES»_space;>
Bacteria & fungi are put on a nutrient media/medium which is designed to supply them with the nutritional and physical requirements they require for growth. BUT, to do this it must all be done under ASEPTIC CONDITIONS. Define ‘ASEPTIC CONDITIONS’.
These are laboratory requirements that maintain sterility of the apparatus being used as well as the immediate environment where the culture preperation is taking place. It PREVENTS contamination.
What do we do to the apparatus before & after usage?
We sterilise the apparatus to PREVENT INITIAL contamination.
What do we do to the culture vessel’s neck before using it?
a) We flame the neck of the culture vessel before and after using it.
What do we do to the environment where we are operating/working on growing the culture?
We sterilise the environment using a disinfectant.
Describe the 6 step innoculation process. Include all details.
- Hold the culture bottle/vessel in one hand; open the cap with your little finger. Don’t place the cap down.
- Flame the mouth/neck of the bottle/vessel.
- Pass the innoculaitng LOOP thorugh the flame until red hot. Allow to cool soon after in the AIR - i.e. don’t place it down because that will lead to contamination…
- Lift the lid of the petri dish enough so that the wire can go though.
- Secure petri dish lid with tape.
- Incubate at 25 degree.
Why do we not seal (put tape) the petri dish fully?
Because this will create anerobic conditions that can promote the growth of pathogenic bacteria.
METHODS OF MEASURING GROWTH»_space;>
There are 2 ways the size of a liquid culture can be measured. Directly couting cells & indirectly counting cells (i.e. measuring what’s called _____. For direct couting of cells what are the names of the 2 types of counts (think about live and dead cells).
Measuring what’s called TURBIDITY using a HAEMOCYTOMETER - a cell counting chamber used origionally to quantify RBC.
Viable counts - i.e. counting the number of colonies (live cells)
Total counts - i.e. using a HAEMOCYTOMETER - a cell counting chamber used origionally to quantify RBC. Total counts highlight the no. of dead AND live cells.
We spoke about measuing TURBIDITY (i.e. cloudiness) as an indirect method. When is this technique used mostly? Explain what is done to the sample of bacteria to quantify the no. of bacteria in the sample.
Fieldwork.
To quantify the number of bacteria cells in a given sample we measure the light absorbed by a particular sample, e.g. a sample of river water. The more cloudy the water the less light shall be absorbed indicating a high bacterial count.
A device called a _____ can be used to determine light absorbtion for a given sample. Does this method give you a viable or a total count - explain your answer.
COLORIMETER. A colourimeter provides a TOTAL count for a given sample becuase obviously a colourimeter cannot distinguish between viable (i.e. live cells) and dead cells in the given sample.
Measuirng growth directly is only possible by first carrying out serial ______. This method relies on each bacterial cell forming what to provide a VIABLE count?
Serial DILUTIONS
This method relies on the bacterial cells forming colonies with each other. A colony is a cluster of cells.
Give the formula that allows you to find the ‘dilution factor’.
diliution factor = 1 / conc.
After the serial dilutions are complete 1cm^3 of each diluted sample is then _____? After that it is incubated at what temp. and for how long?
Spread over a large AGAR PLATE then incubated at 25 degrees for 2 days.
What happens if the dilution is too great?
What happens if the dilution is insufficient?
a) There shall be too FEW colonies that appear on each plate for the count to be statistically sound!
b) There shall be overlapping between colonies (i.e. they merge). Leads to an accurate/underestimation of numbers.
