Lecture 2 - Bacterial nutrition Flashcards
Culture media
a solid or liquid preparation use to grow, transport and store micro-organisms. To be effective the medium must contain all the nutrients the microorganism requires for growth
Defined media
Chemically defined, know the exact composition, know every component and the amount of every component as every component is added individually
Made with specific chemicals - specific sugars, amino acids, vitamins
Less common
Grows most chemoautotrophs and photoautotrophs (photoautotrophs use CO2 as a carbon source and light as an energy source therefore they can be grown on media containing sodium carbonate or bicarbonate (sources of CO2), nitrate or ammonia as a nitrogen source, sulphate, phosphate and other minerals)
Complex media
Do not not the exact composition, not chemically pure
Use complex sources of nutrients - glucose (source of energy and carbon), beef extract, yeast extract (both the beef and yeast extract supply water soluble acids that are rich in nitrogen and carbon), peptones ( digestive protein material)
Most common
Grows most chemoheterotrophic organsims
Transport media
Should maintain the viability of all organisms in the specimen without cell division - Want specimen to arrive at the lab in exactly the same condition as it was taken from the patient
Contains only buffers and salts (there for protective purposes)
Lacks carbon, nitrogen and organic growth factors (since you do not want any microbial growth happening)
The two words to describe transport media….
temporary storage
Enriched media
Contains general nutrient supplements (e.g. serum or yeast extract), to provide precursor metabolites
Made to accommodate lots of bacteria even the fastidious (fussy) ones
Would use when you are doing a bit of a survey about what microbes are present
Nutrient supplements to help grow a wide range of bacteria
Good for when you do not know what you are growing
Enriched media two words
generally encouraging
Enrichment broths
Encourage the growth of one particular organism, put things in it that will get a specific microbe to grow
Gives a competitive edge to the desired microbe, which then becomes the dominant species ( not hidden anymore by the other microbes)
Commonly used in the clinical laboratory - to culture human samples since humans have lots of microbes and the ones we are investigating are often pathogens which can be present in low numbers
Enrichment broth example
Salmonella
Low concentration of salmonella in faecal specimen so an enrichment broth is used to promote salmonella growth so that it can be detected
Tetrathionate broth holds the normal flora in the lag phase of growth whilst promoting logarithmic growth of salmonella as it has the tetrathionate reductase enzyme
Concentration of pathogens is usually low, concentration of intestinal normal microflora is high
Enrichment broth two words
Specifically encouraging
Selective media
Encourages the growth of some organisms while specifically inhibiting the growth of others
Used in clinical samples
Selective media 2 words
Specifically inhibiting
Mannitol salt agar
Differential and selective
Selective
7.5% sodium chloride - inhibits all except staphylococci therefore it is selective so staphylococci
Selective ingredient which is 7.5% sodium chloride + general nutrients to support microbial growth
Inhibits all gram negative, inhibits most gram positive excluding staphylococci (gram positive)
Differential Mannitol is a carbohydrate so microbes that can ferment will react with this and produce acid which results in a pH change shown by the phenol red Phenol red (pH indicator) Mannitol fermented = acid Mannitol fermenters (S. Aureus) change the agar colour from pink to yellow = pathogens Non-mannitol fermenters (S.epidermidis) agar colour not changed (remains pink) S. Aureus (pure culture) - can tolerate the selective ingredient and so it grows and it also is a mannitol fermenter so the pH indicator picks up on this and there is a colour change from pink to yellow S. Epidermis - can grow since it can tolerate the selective ingredient which is the 7.5% sodium chloride but it cannot ferment the mannitol which is the differential ingredient therefore it remains pink in colour
Hektoen agar
Differential and selective, has 7 different ingredients
selective
Bile salts (selective ingredient) + general nutrients
Inhibits gram-positive and some (non-enteric) gram negative bacteria
Selective for gram-negative enteric (gut) bacteria
Differential
3 carbohydrates = lactose, sucrose and salicin
2 pH indicators = acid fuchsin, bromothymol blue - which allow us to visualise if any carbohydrates are being utilised
Carbohydrate (s) fermented => acid (yellow/pink)
Fermenters e.g. E. coli will turn the agar yellow/pink
Nonfermenters e.g. shigella and salmonella (pathogens) colony colour is blue/green
Sodium thiosulphate and ferric ammonium citrate work together
FAC is a source of iron which allows the production of hydrogen sulphate from sodium thiosulphate, H2S production gives black colonies or black centres
H2S creates black colonies int he middle of salmonella (blue/green with black centres) but not shigella (blue/green) therefore can be differentiated from other members of the enterobacteriaceae family (yellow/pink)
Differential media
Contains indicators that visually distinguish between organisms - usually a colour change
Can give a preliminary or preemptive result
e.g. mannitol salt agar and Hektoen agar
Differential media two words
visually distinguishing
Blood agar
Enriched = blood Differential = blood Bacterial enzymes (haemolysins) act on the animal cytoplasmic membrane (red blood cell membrane and it forms a pore that results in the lysis of the RBC) Observe by streaking organism onto blood agar, once bacterial growth has begun the haemolysin is release and lyses the surrounding the red blood cells, creating a zone of haemolysis Types of haemolysis Alpha = partial lysis of RBC —> green discolouration Beta = complete lysis of RBC —> complete clear zone Gamma = non-haemolytic
Blood agar two words
enriched and differential
Batch culture
Closed system
Nothing is added or removed during the growth of a culture
Displays the growth curve
Continuous culture
Not a closed system, continuously adding nutrients in and removing waste
Known as chemostats
Similar to the human digestive system
Want to keep bacteria in the exponential phase because they may produce components that are useful to us
Rate of growth determined by the rate of addition of fresh medium into the culture flask
Turbidostat
Second type of continuous culture system and it has a photo cell that measures the turbidity (defined as the amount of light scattered) of the culture in the growth vessel
Chemostat
Most simple of the continuous batch culture devices
Constructed so that the rate at which a sterile medium is fed into the culture vessel is the same as the rate at which the medium containing microorganisms is removed
Primary metabolite
Essential for growth of the microbe
Produced during the logarithmic/exponential phase
Mirrors growth because the primary metabolites are formed in the exponential phase of growth
Such as amino acids, nucleotides and fermentation products such as ethanol and organic acids
Secondary metabolite
Not essential for growth e.g. antibiotics - But very useful if you are antibiotic producing microbe because you have a competitive advantage and can kill off other microbes that might be in the environment around you
Accumulate during stationary phase (accumulate when nutrients become limited or waste products accumulate following the active growth phase) - Most antibiotics fall into this category - If we wanted to harvest these we would set up a chemostat differently so that it would keep the bacteria in the stationary phase
Four phases of the microbial growth
Lag
log/exponential
stationary
death
what has the characteristic growth curve?
The theoretical growth of a bacterial batch culture (liquid), incubated under ideal conditions has a characteristic growth curve
Lag phase
The time required to get biosynthetic reactions running, lag between adding the cells to the media to when it actually starts growing
It not a time of inactivity but it is rather cells are synthesising new component which are needed before growth can begin
Log/exponential phase
Cells are actively dividing and nothing is limiting for growth
During this phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium and the environmental conditions
Population is doubling in a constant time interval which is determined by the mean generational time
Slope reflects the rate of division … steep = short mean generation time i.e. the numbers build up quickly
Stationary phase
Total number of surviving cells remains constant
Numbers are static - cells still forming by binary fission are mirrored by cell death
Nutrients used up (nutrient limitation) or waste becomes toxic is what causes this phase
Death phase
Exponentially decreasing, cell death is outnumbering cell growth, cells dying at a constant rate
Death = unable to reproduce
Long-term stationary phase
long-term stationary phase is a highly dynamic period in which the ‘birth’ and ‘death’ rates are balanced. That is, long-term batch cultures have an apparent carrying capacity that can only support a certain number of cells.
Long-term growth experiments reveal that after a period of exponential death some microbes have a long period where the population size remains more or less constant
How to calculate the no. of bacteria during exponential growth
Final number = initial number x 2^n where n= generation number and initial=no. of microbes before growth began
Direct count
Advantage = quick Disadvantage = counts both live and dead cells Haemostatometer = physically count the cells Spectopertrometer = counts cells directly but through the optical density (turbidity)
Viable count
Advantage = only counts live cells
Disadvantage = slow
Dilute culture - plate culture - one cell = one colony on plate which we then count
Gram positive
thick layer of peptidoglycan in bacterial cell wall which traps crystal violet masking the red dye
Gram negative
thin layer of peptidoglycan with inner and outer membranes which means that crystal violet is easily rinsed away, revealing the red dye
Organisms most likely to survive extreme
Archaea
