5 - Environmental Influences & Control of Microbial Growth

Question 1

What are the different types of physical sterilization?

Answer 1

(1) Autoclave sterilization
(2) Liquid filtration
(3) Radiation sterilization

Question 2

What is autoclave sterilization?

Answer 2

Most equipment (especially in the lab) is sterilized using an autoclave. They are put in a chamber and steam is pumped in (moist heat + pressure)

Question 3

What is liquid filtration?

Answer 3

Filter sterilization can be used to sterilize liquids.The solution to be sterilized is passed through a nitrocellulose membrane with pore sizes in the order of submicrons. Pores are no larger than 0.2 microns. This method sterilizes the solution from bacteria, but not necessarily viruses.

Question 4

Does radiation sterilization work on everything?

Answer 4

Not everything gets killed by radiation. For example, Deinococcus radiodurans can’t be killed by radiation

Question 5

How does Deinococcus radiodurans survive radiation?

Answer 5

It has exceptional DNA repair

Question 6

What was the first chemical used to sterilize bacteria?

Answer 6

Phenol which is an antiseptic

Question 7

What is antiseptic surgery?

Answer 7

We use antimicrobial substances on living tissue/skin to reduce the possibility of infection.

Question 8

What is aseptic surgery?

Answer 8

We reduce the risk of infection by making sure that everything that touches the living tissue/skin is sterile, for example surgical equipment. So, we don’t apply chemicals to the living tissue.

Question 9

How do we sterilize plastic equipment used in the hospitals without melting it?

Answer 9

Plastic cannot be placed in an autoclave. We cannot apply radiation or harsh chemicals on plastic either. Plastic equipment is placed in sealed packages with gas permeable membranes. Then they are exposed to gas such as ethylene oxide which will enter the plastic with the gas permeable membrane in order to sterilize the contents.

Question 10

What are we referring to when we say biological sterilization

Answer 10

Antibiotics

Question 11

How was penicillin discovered?

Answer 11

It was discovered by Alexander Fleming when Penicillium notatum (a mould) grew on a petri dish containing Staphylococcus colonies, resulting in the formation of a zone of inhibition due to the antibiotic that was inhibiting the growth of the bacterium.

Question 12

What does penicillin as an antibiotic target?

Answer 12

This antibiotic targets “penicillin binding proteins” (PBP) that have a crucial role in cell wall synthesis. PBP catalyze the cleavage of the terminal D-Ala to link several NAMs together. Penicillins have a \beta-lactam ring

Question 13

How would you grow low Pi cultures to study toxin growth regulation?

Answer 13

Grow in a chemostat and vary phosphate concentration and see what toxins they make as a result.

Question 14

What is the main starvation response?

Answer 14

ppGpp = alarm-mone = magic spot

If you starve cells when they feel that starvation they make this molecule which alternates RNA polymerase

Question 15

What are the key players during the starvation response?

Answer 15

Ribosomes. They:

• are sensors
• need to be stored during hibernation

Question 16

How can you remove endo-spores from growth media?

Answer 16

Autoclaving

Question 17

What are microplasma?

Answer 17

Bacteria that lack peptidoglycan cell walls. Despite this they are sensitive to penicillin antibiotics. This is because they make peptidoglycan but for another essential function. Make peptidoglycan ring just at the point of dividing then destroy ring and use it for cytokinesis

Question 18

What is the E.coil MazEF toxin-antitoxin system

Answer 18

Is it a type of programmed cell death. Antitoxin is unstable and is passed on to protease. To neutralize toxin you need to constantly make antitoxin. This happens normally in E.coli. In starvation, ribosome makes ppGpp which binds RNA polymerase Lots of genes turn off and the bacteria no longer make the antidote. Lots of cells then die.

Question 19

What are some other examples of toxin/anti-toxin system?

Answer 19

(1) Tuberculosis which survive in granulose
(2) Plasmids - Once you get a plasmid you cannot lose it. If you happen to lose it by dividing unequally, quite often the cell dies

Question 20

What is the optimal temperature for psychrophiles?

Answer 20

9 degrees celsius

Question 21

What is the optimal temperature of mesophiles?

Answer 21

37 degrees celsius

All human pathogens are mesophiles

Question 22

What is the optimal temperature of thermophiles?

Answer 22

68 degrees celsius.

Most are prokaryotes

Question 23

What is the optimal temperature of extreme thermophiles?

Answer 23

95 degrees celsius

They need to have very stable enzymes and structures to allow their growth and survival.

Question 24

Heat-stable DNA polymerases can be isolated from which organisms in order to be used in PCR to amplify DNA?

Answer 24

Both thermophiles and extreme thermophiles

Question 25

What is the rule of 40?

Answer 25

There are no bacteria whose range of growth temperature is more that 40 °C.

Question 26

What is a special feature of bacteria that are human pathogens?

Answer 26

They have a very narrow temperature range. That is why fever is an important immune mechanism to drive these bacteria out.
o Example includes mycobacterium leprae

Question 27

What is an Arrhenius plot?

Answer 27

It displays the growth rate constant on the Y axis and the inverted temperature on the X axis. They are used to analyze the effect of temperature on the rates of chemical reactions.

Question 28

What does the straight line part of an Arrhenius plot represent?

Answer 28

This part is where all reactions are obeying the simple laws of physics

Question 29

When we use an Arrhenius plot to assess growth rate, what do we assume?

Answer 29

That the cells obey biochemical laws. All reactions in the cell are interpreted as being simply biochemical.

Question 30

What happens when temperature is sutiable?

Answer 30

E. coli will grow faster because all the molecules are moving faster. E. coli doesn’t have to adjust anything to grow faster.

Question 31

What temeprature is at the upper edge of the Arrhenius line?

Answer 31

37 degrees celsius

Question 32

What happens to bacteria at high temperatures?

Answer 32

Growth rates will fall because enzymes denature, they don’t fold properly and the membrane becomes loose. In response to these high temperatures, E. coli, just like any other organism, have what we call “a heat shock response”.

Question 33

What are some of the most universally conserved proteins?

Answer 33

Proteins made to counteract the heat stress. Some of these proteins are chaperones.

Question 34

What do chaperones do?

Answer 34

They form a sort of a closed box in which a protein can get in a fold properly

Question 35

What are other heat shock proteins?

Answer 35

Proteases. If a protein is severely damaged, it won’t get into a chaperon, it will get degraded by proteases. The amino acids are then recycled.

Question 36

Why are the genes encoding chapersones and proteases referred to as orthologs?

Answer 36

Because chaperones and proteases are conserved in bacteria and eukaryotes and they perform the same functions in both species

Question 37

What happens to bacteria in low temperatures?

Answer 37

Growth rates fall because of the decrease in membrane fluidity and enzymatic activity. Cellular synthesis of most proteins is inhibited, except for cold-shock-inducible proteins that have unwinding activity. There is a set of specific enzymes that counteracts cold shock responses. Most of these enzymes are helicases that will try to unwind the stiff (rigid) proteins.

Question 38

What property is very important for growth?

Answer 38

Salinity

Question 39

Why does a high concentration of salt preserve food?

Answer 39

because high concentrations of those solutes will absorb water and microbes need water to grow, so microbes will dry out and die.

Question 40

Why does water not passively cross the membrane?

Answer 40

Because it is hydrophilic and hydrophilic substances cannot cross a hydrophobic membrane. Water, therefore, requires a channel.

Question 41

What are aquaporins?

Answer 41

Integral inner-membrane proteins that serve as channels that can quickly move water across membranes (facilitated diffusion) to equilibrate internal and external pressure across membranes

Question 42

What are aquaporin channels lined by?

Answer 42

Hydrophobic residues that speed water through the open pore.

Question 43

Where are aquaporins located in bacteria vs OmpF?

Answer 43

In bacteria, aquaporins are located on the inner membrane of both Gram negatives and Gram positives, whereas OmpF are located on the outer membrane of Gram-negatives.

Question 44

Are aquaporins conserved across bacteria and eukaryotes?

Answer 44

Yes

Question 45

What do OmpF let through vs aquaporins?

Answer 45

OmpF are big enough that they can let water and other molecules, such as antibiotics, pass freely (they from holes). However, in the case of aquaporins, there is a very precise channel (not holes) and only water can go through

Question 46

What are the structural differences between OmpF and aquaporins?

Answer 46

OmpF proteins tend to have the beta-barrel structure (since they are located in the OM), whereas aquaporins are made of alpha-helices (since they are located in the IM).

Question 47

What other apparently spontanous reactions use proteins/enzymes?

Answer 47

CO2 gas/aqueous solubility and O2 gas/aqueous solubility in all three domains.