6. Factors Influencing The Microbial Growth

Question 1

What affects microbial growth?

Answer 1

Oxygen levels, temperature, pH, osmolarity(water availability) ionic concentration

Question 2

When it comes to the relationship between oxygen and bacteria, how can bacteria be differentiated?

Answer 2

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

Question 3

What’s is sodium thiogycollate used for when it comes to cultivation?

Answer 3

It’s reducing agent used to slow down oxygen diffusion into the tube

Question 4

How are ROS formed and what they are

Answer 4

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)

Question 5

Give examples of ROS

Answer 5

O2^-(superoxide); H2O2(hydrogen peroxide) OH•

Question 6

What enzymes eliminate toxic intermediates that form during the reduction of molecular oxygen to water

Answer 6

1) catalase
2)peroxidase
3)superoxide dismutase
4)superoxide dismutase/catalase in combination
5)superoxide reductase

Question 7

How is culture medium modified to eliminate oxygen

Answer 7

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

Question 8

What is CO2 used for in bacteria

Answer 8

Synthesis of some cellular components( fatty acids). CO2 can favour the synthesis of virulence factors(toxins) and capsule

Question 9

How can optimal [CO2] be obtained

Answer 9

1)Gas pack
2) CO2 thermostats , where is CO2 insufflated

Question 10

How can we classify microbs based on temperature they prefer

Answer 10

(From smaller to higher temp)
Psychrophiles
Mesophiles
Thermophiles
Hyperthermophiles

Question 11

Relationship between pH and bacteria

Answer 11

pH range is species-specific and range for optimal growth of bacteria is fairly narrow. Optimal range must be determined empirically

Question 12

Classification of basteria based on pH

Answer 12

Neutrophiles: pH: 5.5-8
Acidohphiles: they can be
-obligate: pH:3
-facultative: pH:3-7
Alkaliphiles: pH: 8-11.5

Question 13

At what pH do bacterial enzymes function and how is that pH kept constant

Answer 13

Around pH=7. Protonic pump ATPase and Na+/H+ exchanger maintain pH at constant levels(internal buffering)

Question 14

Why is osmotic pressure and presence of solutes less important

Answer 14

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

Question 15

What are osmophiles

Answer 15

Bacteria that recquire high concentrationa of sugars

Question 16

Why is evaporation of water detrimental for microbial growth

Answer 16

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

Question 17

What are 2 types of measuring growth by determining or monitoring

Answer 17

1. Cell concentration; number of cell unit per volume; direct technique
2. Biomass conc.;weight of cells per unit of volume; indirect technique

Question 18

Are 2 methods of measuring growth by determining/monitoring equivalent?

Answer 18

No

Question 19

How to measure cell number

Answer 19

1. By machine: electronic cell counter
2. 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)
3. 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”)

Question 20

How to measure microbial mass

Answer 20

Measure by:
-microbial dry weight proportional to microbial mass
-cell components(DNA,RNA,proteins)
-turbidity(spectrophotometer: optical density,O.D.)

Question 21

What’s impedometry method based on

Answer 21

Measurment of the variations in electric conducibility in the culture medium

Question 22

Explain impedometry method

Answer 22

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

Question 23

What are some indirect methods for measuring microbial mass

Answer 23

Nutrient consumtion
Formation of metabolic product
ATP content( [ATP]/mass unit is cont.over time)

Question 24

What’s biofilm

Answer 24

Highly organized 3D structure where bacteria are embedded in a self-produced conplex matrix, made of extracellular polymeric substrates(EPS)

Question 25

What are steps of formation of biofilm

Answer 25

1. Planktonic bacteria=free-swimming bacteria can reversibly attach to different types of surfaces
2. Auto-aggregation-bacteria travel from reversible to irreversible attachement due to EPS production
3. Establishment-easy development of biofilm architecture into stable supercellular structure
4. 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

Question 26

On what bacteria can turbiometric measurments be applied

Answer 26

Planctonic bacteria

Question 27

What are phases of bacterial growth curve

Answer 27

•Lag phase
•Log phase
•Stationary phase
•Death phase

Question 28

What’s lag phase

Answer 28

Latency(lag) phase-cell increase in volume but not in number; bacteria adaptation to the new enviroment. Basically: metabolic activity without division

Question 29

What’s log phase

Answer 29

Exponential/logaritmic (log) phase- cell devide with duplication(doubling) time that depends on the strain and enviromental factors. It has balanced growth rate

Question 30

What’s stationary phase

Answer 30

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

Question 31

What’s death phase

Answer 31

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

Question 32

What’s innoculum

Answer 32

Population of mo or cells that is introduced in the fermentation medium or any other suitable medium

Question 33

What factors determine length of lag phase

Answer 33

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

Question 34

What is lag phase happening

Answer 34

•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

Question 35

What’s phase of adaptation

Answer 35

Bacteria need to adapt to the new growth conditions so they:
⬆️cell size
⬆️synthesis of specific enzymes for new metabolites
⬆️synthesis of RNA

Question 36

When is there no lag phase

Answer 36

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

Question 37

What is the goal of microbial growth(selection of optimal culture media)

Answer 37

The creation of optimal conditions for the growth of mo object of study

Question 38

What’s the procedure for microbial growth(selection of optimal culture medium)

Answer 38

-plate-seeding on suitable growth media(normal,enriched…)
-incubation(aerobiosis,anaerobiosis,microaerophilia)
-isolation
-identification

Question 39

What’s common approach of identification represented by

Answer 39

Determination of the nutritional and metabolic properties of mo

Question 40

What properties do identification test measure

Answer 40

Enzymatic properties and ability of isolated clone to grow and survive in the presence of certain inhibitors

Question 41

Why are enzymatic assays useful

Answer 41

Bc they allow us to measure activity of a single enzyme or of the comolete metabolic cycle (with involvement of several enzymes)

Question 42

What’s phage typing

Answer 42

Test used for interpreting lytic action of the phages-phage types

Question 43

What’s bacitracin test

Answer 43

Sensitivity test

Question 44

What’s optochine test

Answer 44

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

Question 45

What’s DNase test

Answer 45

•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

Question 46

What’s motility test

Answer 46

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

Question 47

Can we use biochemical activity for identification of mo

Answer 47

Yes but only if it’s a pure culture