6. Factors Influencing The Microbial Growth Flashcards
What affects microbial growth?
Oxygen levels, temperature, pH, osmolarity(water availability) ionic concentration
When it comes to the relationship between oxygen and bacteria, how can bacteria be differentiated?
1)Obligate aerobic bacteria(aerobes) =growth only in the presence of O2(atmospheric)
2)Obligate anaerobic bacteria(anaerobes)=growth only in absence of O2
3)Miceoaerophilic bacteria(microaerophiles)= recquire only limited O2(<atmospheric)
And we have:
4) Facultative and aerotolerant anaerobic bacteria=tolerate or are indifferent to O2
What’s is sodium thiogycollate used for when it comes to cultivation?
It’s reducing agent used to slow down oxygen diffusion into the tube
How are ROS formed and what they are
Reactive oxygen species(ROS) or free radicals are very toxic , since they are able to react and demage cellular macromolecules, particularly DNA
ROS are made as by-products during respiration( reduction of molecular oxygen to H2O)
Give examples of ROS
O2^-(superoxide); H2O2(hydrogen peroxide) OH•
What enzymes eliminate toxic intermediates that form during the reduction of molecular oxygen to water
1) catalase
2)peroxidase
3)superoxide dismutase
4)superoxide dismutase/catalase in combination
5)superoxide reductase
How is culture medium modified to eliminate oxygen
1) adding reducing agents: cysteine, ascorbic acid, thio-glicolic acid. There is also the need to add an indicator of redox reactions, generally resazurin
2)Boiling the liquid media: after the boiling addition of an oil layer or vaseline ensures maintenance of the anoxic enviroment
What is CO2 used for in bacteria
Synthesis of some cellular components( fatty acids). CO2 can favour the synthesis of virulence factors(toxins) and capsule
How can optimal [CO2] be obtained
1)Gas pack
2) CO2 thermostats , where is CO2 insufflated
How can we classify microbs based on temperature they prefer
(From smaller to higher temp)
Psychrophiles
Mesophiles
Thermophiles
Hyperthermophiles
Relationship between pH and bacteria
pH range is species-specific and range for optimal growth of bacteria is fairly narrow. Optimal range must be determined empirically
Classification of basteria based on pH
Neutrophiles: pH: 5.5-8
Acidohphiles: they can be
-obligate: pH:3
-facultative: pH:3-7
Alkaliphiles: pH: 8-11.5
At what pH do bacterial enzymes function and how is that pH kept constant
Around pH=7. Protonic pump ATPase and Na+/H+ exchanger maintain pH at constant levels(internal buffering)
Why is osmotic pressure and presence of solutes less important
Because of ability of the bacteria to regulate their internal osmolarity and ionic strength. But it might be important for some mo that recquire high salt or sugar concentrations
What are osmophiles
Bacteria that recquire high concentrationa of sugars
Why is evaporation of water detrimental for microbial growth
1) less water is available for essential metabolic processes
2) loss of water can lead to a consequent increase of mediun solutes and therefore–> increase osmotic pressure–> osmotic shock–> cell death by osmotic lysis
What are 2 types of measuring growth by determining or monitoring
- Cell concentration; number of cell unit per volume; direct technique
- Biomass conc.;weight of cells per unit of volume; indirect technique
Are 2 methods of measuring growth by determining/monitoring equivalent?
No
How to measure cell number
- By machine: electronic cell counter
- Total counting of the cell number (pro: rapid,inexpensive method ; con: inability to disciminate dead from viable cells, not suitable for diluted samples or w small bacteria)
- Viable cell counting based on plating methods,only for cells that are able to form colonies(no filamentous bacteria) (pro: very sensitive method; con: some bacteria grow into biofilm and a lot of them can’t be cultured there; “great plate count anomaly”)
How to measure microbial mass
Measure by:
-microbial dry weight proportional to microbial mass
-cell components(DNA,RNA,proteins)
-turbidity(spectrophotometer: optical density,O.D.)
What’s impedometry method based on
Measurment of the variations in electric conducibility in the culture medium
Explain impedometry method
Microbial growth leads to modifications of the impendance of the medium due to bacterial growth, that involves the transformation of large macromolecules to low-molecular weight, charged molecules.
Correlation curve is obtained by variation in the conductivity of the medium to microbial growth
What are some indirect methods for measuring microbial mass
Nutrient consumtion
Formation of metabolic product
ATP content( [ATP]/mass unit is cont.over time)
What’s biofilm
Highly organized 3D structure where bacteria are embedded in a self-produced conplex matrix, made of extracellular polymeric substrates(EPS)
What are steps of formation of biofilm
- Planktonic bacteria=free-swimming bacteria can reversibly attach to different types of surfaces
- Auto-aggregation-bacteria travel from reversible to irreversible attachement due to EPS production
- Establishment-easy development of biofilm architecture into stable supercellular structure
-
Growth-late development of micro-colonies. Multiple species and secondary colonization of those species can happen
5.Mature biofilm-characterized by complex 3D structure with antibiotic gradient
On what bacteria can turbiometric measurments be applied
Planctonic bacteria
What are phases of bacterial growth curve
•Lag phase
•Log phase
•Stationary phase
•Death phase
What’s lag phase
Latency(lag) phase-cell increase in volume but not in number; bacteria adaptation to the new enviroment. Basically: metabolic activity without division
What’s log phase
Exponential/logaritmic (log) phase- cell devide with duplication(doubling) time that depends on the strain and enviromental factors. It has balanced growth rate
What’s stationary phase
Stationary phase-cells stop dividing, either due to the depletion of essential nutrients or accumulation of toxic metabolites, or both. There is production of secondary metabolites and activation of sporulation genes
What’s death phase
Death phase- decline and death phase is an exponential function and is a linear reduction of the viable cells number over time; mortality rate increases initially and then reaches a constant level
What’s innoculum
Population of mo or cells that is introduced in the fermentation medium or any other suitable medium
What factors determine length of lag phase
Volume of innoculum, time needed for the recovery from physical demage or shock, time needed for synthesis of enzymes necessary to metabolize the substrates present in culture medium
What is lag phase happening
•the innoculum derives from a culture in stationary phase and is introduced in a culture medium similiar to original (metabolic recovery)
•the innoculum contains demaged cells(demage recovery)
•innoculum comes from a rich medium and it is introduced in a minimal medium
What’s phase of adaptation
Bacteria need to adapt to the new growth conditions so they:
⬆️cell size
⬆️synthesis of specific enzymes for new metabolites
⬆️synthesis of RNA
When is there no lag phase
When innoculum, in exponential phase, is transferred in a culture medium that is the same of the medium of origin and is incubated under the same conditions
What is the goal of microbial growth(selection of optimal culture media)
The creation of optimal conditions for the growth of mo object of study
What’s the procedure for microbial growth(selection of optimal culture medium)
-plate-seeding on suitable growth media(normal,enriched…)
-incubation(aerobiosis,anaerobiosis,microaerophilia)
-isolation
-identification
What’s common approach of identification represented by
Determination of the nutritional and metabolic properties of mo
What properties do identification test measure
Enzymatic properties and ability of isolated clone to grow and survive in the presence of certain inhibitors
Why are enzymatic assays useful
Bc they allow us to measure activity of a single enzyme or of the comolete metabolic cycle (with involvement of several enzymes)
What’s phage typing
Test used for interpreting lytic action of the phages-phage types
What’s bacitracin test
Sensitivity test
What’s optochine test
Allows identification between S.pneumoniae(+,lysis by optichine) and alpha-emolytic streptococci(-,resistant to optochine-induced lysis). Chemotherapeutic agent is present that’s active against streptococcus pneumoniae
What’s DNase test
•plate medium contains DNA,peptides and methyl green
DNA and green dye form blue-green complex at pH=7 5
DNase catalyzes the hydrolysis of DNA in small fragmets that dissociate from the dye=>there is clarification next to the growth region
What’s motility test
Seeding by infixition in semi-solid medium. Motile organisms diffuse from the innulation site in the culture medium while non motile stay at innulation site
Can we use biochemical activity for identification of mo
Yes but only if it’s a pure culture
