Growth of Bacteria Flashcards
Describe how media can be used to cultivate and isolate bacteria
Agar plates are used with various types of media
Minimal media
Nutrient media
Enriched media
selective media
indicator medium
Selective indicator media
Transport media
Describe minimal media
A basic salts based growth media often with a single carbon source.
Describe nutrient Media
A medium with all the basic (general) requirements for growth but without specific supplements. (Often meat/yeast extract so a complex mix of peptides, ions and biochemical intermediates & salt)
Describe enriched media
Nutrient media with additional organism specific supplements.
E.g. Selenite medium to enrich for Salmonella
Describe selective media
A nutrient medium with the basic requirements for growth and added supplements that allows one bacterial type to grow more than another.
E.g. Specific antibiotics that allow isolation of slow growing bacteria
Describe indicator medium
A medium with indicators which react to specific bacteria.
E.g. Lactose fermentation: E.coli (Lac+) & Salmonella (Lac-).
Describe selective indicator media
A combination of selective and indicator media
E.g. MacConkey agar (s: bile salts, i: Lactose ferment)
Describe transport medium
A medium designed to protect the organisms in you sample on the way to the lab.
E.g. swabs
How to spread bacteria onto agar?
Spread bacteria on a plate until they are physically separated.
They then grow up to form individual colonies.
Each colony you see on a plate has grown from a individual bacteria.
If you have a mix of bacteria and spread them out you can separate the different bacteria.
How does bacteria grow?
Grow by Binary fission
Colonies form on plates as piles of bacteria Motile bacteria don’t tend to form colonies as they move quickly & spread out thinly Bacteria have Generation times aka doubling times = length of time required for a single bacterial cell to divide into two daughter cells If bacteria double every 20-30min then colonies arise in 12-24 hours Some slower bacteria can take months
Once the bacteria has been culture and colonies formed, what happens next?
Tests can be done on the bacterial isolates:
Speciation through Molecular tests Biochemical tests Antibiotic sensitivity Typing (various methods) to determine epidemiology of infections
How can bacteria be preserved for long term epidemiology?
Sub culturing (labour intensive & allows bacteria to change).
Freeze drying (lyophilisation).
Freezing with cryoprotection (DMS or Glycerol)
Describe different methods by which you can measure bacterial growth
Direct counting by microscope.
You cannot distinguish viable from non-viable cells.
Colony counting (viable counts, below all basically variants of this).
Serial dilutions spread on surface of plate.
Mix bacteria with agar & pour plate.
Miles-Misra method.
Serial dilution steps
Membrane filtration.
Absorbance in liquid culture.
Measurement of absorbance of liquid culture at 600nm.
Outline the Miles Misra Method
Diluted in log steps
Set volume is plated
The lower the dilution the fewer the amount of bacteria and so when the liquid dries the bacteria form separate colonies
The colonies from the 50µl (5 x 10µl) of the 10-4 dilution are counted
Using this it is then calculated up to how many would be in 1 ml.
The number are expressed using colony forming units / ml (CFU/ml)
These are a viable count for the bacteria in the original sample
Outline the absorbance in liquid culture method
Increase in optical density over time.
Generation (time for one full round of division)
Growth rate (µ) how much doubling per hour.
Can measure by optical density
Describe the basic growth characteristics of bacteria
Lag Phase
Bacteria adjust to the new medium & begin to metabolise & grow
Sensitive
Exponential Phase
Growth rate is determines
Bacteria grow at their best in this phase
More resistant
Stationary Phase
Bacteria reach limit on resources within the media
Maximum population has been reached
(where growth rate is = death rate)
Bacteriostatic antibiotics wont be as effective here as they prevent growth from
occurring
Decline / Death Phase
Medium exhausted of components for bacteria growth
New bacteria cannot grow and death rate remains the same
Measurement of death rate Drop Logs used to represent impact To be aware that 90% / 99% drop doesnt always mean there is no bacteria left E.g a 99.9% drop of (109) means (107) still remains
Define the terms anaerobic, aerobic, facultative anaerobe and microaerophilic
anaerobic ‘cannot grow in presence of oxygen’
Aerobic ‘require oxygen to grow’
Facultative Anaerobe ‘bacteria that can grow with or without oxygen present’
Microaerophilic ‘microbes which require very little oxygen to grow’
What would the clinical relevance in knowing whether bacteria can grow in the presence of oxygen be?
A deep wound will be colonised by different bacteria based on oxygen gradient.
This can affect your treatment options
Describe the impact on growth of different pH ratings
Most standard media is buffered at pH7
During bacteria growth acidic metabolites may be produced
This can lead to growth inhibition over time
Most pathogens are neutrophils (grow within 2 pH units of pH7)
The acidophiles & alkaliphiles found in extreme environments are not
pathogenic
Due to environments existing before first higher organisms developed
Describe the impact on growth of different temperatures
The bacteria that infect or colonise animals are mesophilic (27 degrees)
Bacteria adapted to some animals have slightly different optima.
For example birds average temperature is 42°C and Campylobacter which is commonly found in birds grows best at 42°C. It has co-evolved for this environment.
That said it can also grow OK at mammalian body temperatures and cause disease.
flexibility within a range
At what temperature should bacteria be stored at to slow bacteria growth?
Approx 8°C
Slows bacteria growth but doesnt stop it completely
Allows pathogens to survive and replicate outside/between hosts
Describe the impact on growth of different osmolarities
Bacteria membrane is semi permeable
Accumulate or lose solutes to balance the water potential
Bacteria prefer to maintain a slight positive pressure an inflow of water which is resisted by the cell wall.
What is the clinical relevance of knowing the preferred osmolarity for bacteria?
High salts/sugars can be used to preserve foods
Hypertonic environments draw water out of bacteria
leading to changes in the concentrations of solutes in the cytoplasm
Affects a range of enzymes/cell functions and so the changes disrupt cell physiology.
Describe the core nutritional requirements for bacteria
Where does bacteria obtain its nutrients?
Acquire nutrition from the immediate environment.
That environment may be your patients wound, intestine, blood
Bacteria can be:
Fastidious require specific supplements
Non-fastidious grow from basic chemicals. Most veterinary relevant organisms pathogens are chemoheterotrophs They use organic chemical as sources of energy & carbon
How does knowing where bacteria obtain its nutrients and what it consume clinically relevant?
Part of the host defence is to limit access to certain nutrients (eg iron)
reduced bacterial growth
Bacteria can compete for compounds so one organism can potentially block colonisation/infection by another
Where are carbon and hydrogen sourced and what are they primarily used for?
Organic compounds (for chemoheterotrophs)
In many of the organic compounds in the cell
Where is nitrogen sourced and what is its primary use?
Ammonia, Inorganic nitrogen (nitrates etc), already in organic molecules already containing nitrogen
amino acids, nucleic acids nucleotides, and coenzymes
Where is sulphur sourced and what is its primary use?
In organic phosphates and already in some organic molecules containing phosphorous
Amino acids (cysteine, methionine, glutathione), some coenzymes
Where are potassium, magnesium & calcium sourced and what are their primary uses?
salts
Co-factor
Potassium is also an osmolite
Where is iron sourced and what is its primary use?
salts
Co-factor and component of cytochromes, non-haem iron-proteins
Where are trace elements sourced?
metal ions required by certain cells in such small amounts that it is difficult to detect (measure) them. Not required to add them to most culture media as nutrients as in sufficient levels in other constituents
Outline methods to control and manage pathogens in the environment
Sterilisation:
Sterilisation refers to a process that eliminates (removes) or kills (deactivates) all forms of life (in this case bacteria)
Main methods: heat, chemical, irradiations.
Washing.
Will not sterilize unless you use something that kills the microorganisms. But can remove and reduce risk of contamination or spread of infection.
Disinfection.
Do not assume that all disinfectants kill all pathogens.
Disinfectants may not be compatible with all uses.
Common methods are listed on next few tables make sure you understand what the terms refer to
How does disinfection work?
There are a range of different chemicals used to disinfect.
They damage in a general manner by reacting with different targets such as proteins, nucleic acids
Most effects are to denature cell components can damage/affect animal/human cells.
But prions very tolerant as already denatured so not fully effective or need extreme compounds.
Some are washes can be used on patients,
i.e. Chlorohexidine
Others are caustic or toxic.
i.e. formaldehyde, sodium hypochlorite, so need great care.
Selection must account for a lot of factors (few examples below).
Non tainting for food industry
Effectiveness in environment they will be used in (including temp and contamination type).
Effectiveness against the organism you want to target.
Otline the methods of preserving and limiting bacterial growth
Refrigeration 4°C
Reduces growth rate of many organisms
Freezing
Prevents microbial replication
Boiling
Inactivation of vegetative bacteria and fungi but not endospores
Pasteurization
Inactivation of vegetative bacteria and fungi but not endospores or prions
Autoclaving (heat under pressure to 120°C or more)
Inactivation of all pathogen groups including endospores but not fully
effective against prions.
Acidification
Inhibits many bacteria. Weak acids used in food preservation pickling.
Increased osmotic pressure
Inhibits bacteria multiplication.
Irradiation
Inactivation agents, except Prions.
