Lecture 3 - The two sides of bacterial endospores Flashcards

1
Q

If nutrient depletion occurs or a physical stress occurs then the bacteria can respond in one of three ways:

A

Cell death

Go into a protective dormancy which is going into a metabolically less active state which may also eventually lead to cell death

Some bacteria are able to form endospores which are a survival structure

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2
Q

Most endospores dorm in

A

gram positive rods, commonly microbes in the soil as they are subjected to stresses all the time

Examples = Bacillus anthracis (anthrax), clostridium perfringens (gangrene), c.tetani (tetanus), c.botulinum (food poisioning) - these are medically significant bacteria that form endospores

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3
Q

Endospores allow..

A

for a dormant state during times of stress, they form within the cell

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4
Q

Endospore formation outcome

A

It is not a reproductive process we are just having one cell give rise to one endospore in this metabolically dormant survival state - survival not replication

Process …
Endospores - layers of protective coverings around the dormant genetic material - formation of this takes around 8 hours - germination of endospores takes about 15 minutes
If there are stresses going on in the bacterial environment during this time, this bacterial cell might still die but this is worth the risk

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5
Q

Formation process

A

Vegetative cell dividing by binary fission, genetic material i.e. DNA replicated

Septum forms diving the cell asymmetrically - smaller side will become the endospore - larger part becomes mother cell which helps build protective layers before dying

Larger compartment engulfs smaller compartment which forms the forespore (precursor)

Content and coat formed, dehydration

Lysis of cell and release of the endospore

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6
Q

Structure of the endospore

A

Structure of the endospore - all of the layers mean that the endospore can sit in harsh conditions and handle the environment until germination conditions become optimum

(inner most layers) Core, inner membrane, germ cell wall/core wall = contain normal cell structures such as DNA and ribosomes but it is metabolically inactive, very low water content
- Called the germ cell wall as it contains the peptidoglycan that will form the wall of the vegetative cell that grows out of the spore following germination

Cortex, outer membrane = made up of peptidoglycan but it is less cross linked than that of vegetative cells
Cortex may occupy as much as half the spore volume
Outer membrane is a phospholipid bilayer

Coat = composed of several protein layers and can be very thick, impermeable and it is responsible for the endospore’s resistance to chemicals, UV rays etc.

Exosporium = thin outer layer mainly lipids, carbohydrates, and proteins which acts like a shell that gives the final protection, made up of glycoproteins

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7
Q

(inner most layers) core, inner membrane, germ cell wall/core wall

A

(inner most layers) Core, inner membrane, germ cell wall/core wall = contain normal cell structures such as DNA and ribosomes but it is metabolically inactive, very low water content
- Called the germ cell wall as it contains the peptidoglycan that will form the wall of the vegetative cell that grows out of the spore following germination

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8
Q

Cortex, outer membrane

A

Cortex, outer membrane = made up of peptidoglycan but it is less cross linked than that of vegetative cells
Cortex may occupy as much as half the spore volume
Outer membrane is a phospholipid bilayer

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9
Q

Coat

A

Coat = composed of several protein layers and can be very thick, impermeable and it is responsible for the endospore’s resistance to chemicals, UV rays etc.

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10
Q

Exosporium

A

Exosporium = thin outer layer mainly lipids, carbohydrates, and proteins which acts like a shell that gives the final protection, made up of glycoproteins

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11
Q

What determines resistance in the endospore

A

Physical
Coat (thick layers)
Inner membrane (impermeable)

Chemical (core)
Low water content (15%, compared to >80% in vegetative cells) - Low water content in core of endospore makes the core a gel like texture and this immobilises the proteins and nucleic acids until they are needed for sporilation

Lower pH
Slightly lower than a vegetative cell

High content of dipicolinic acid and Ca2+ - Combine to form a lattice structure and the lattice structure complexes between the bases of the DNA and stabilises it. This cross linking structure in the core represents about 10% of the dry weight of the endospore

Small acid-soluble proteins
These bind to the DNA and when they bind they alter the conformation of the DNA slightly and protect it from damage

Chemical (cortex)
Decreased peptidoglycan cross linking index = 2.9% in endospore (33% in vegetative cells)
This modified peptidoglycan wall is required for the maintenance of spore coat dehydration, and the accompanying metabolic dormancy and heat resistance - it is one more layer, giving the total effect of a very resistant structure

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12
Q

Endospore germination

A

Activation - Preparing cells for germination
Germination - Swells and takes on water, breaks the spore coat which causes a loss of resistance for all the stresses in the environment but also an increase in metabolic activity in the bacteria
Outgrowth - Spore emerges from broken coat and develops into an active bacterial cell that can undergo binary fission

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13
Q

Endospores are the …

A

benchmark for physical and chemical control mechanisms

if endospores are killed then other microbes have also been killed

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14
Q

Why do we need to control microbial growth?

A

Without it we would be subjected to more widespread outbreaks of disease, contamination, pollution and spoilage

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15
Q

Death of microbes

A

loss of ability to multiply under any known conditions

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16
Q

Sterilisation

A

complete removal or destruction of all microorganisms (includes spores too) from inanimate objects. It is brought about by the use of physical or chemical methods. Non-selective

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17
Q

Disinfection

A

(removal of infection) destruction or removal of microorganisms (but not bacterial endospores) on inanimate objects by physical or chemical means

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18
Q

Disinfectants

A

agent’s, usually chemical used to carry out disinfection and normally only used on inanimate objects. A disinfectant does not necessarily sterilise an object because viable spores and a few microorganisms may remain

19
Q

Antisepsis

A

chemicals applied to body surfaces to destroy or inhibit growth of microorganisms within host tissues

20
Q

Antiseptics

A

chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth and they also reduce the total microbial population

21
Q

Bactericidal vs bacteriostatic

A

Kills vs inhibits

22
Q

Sanitisation

A

the microbial population is reduced to levels that are considered safe by public health standards

23
Q

Physical control method of heat

A

Heat treatment is still one of the most useful methods of microbial control because it is fast, reliable and inexpensive relative to other methods and it does not introduce potentially toxic substances into the material that is being tested

24
Q

Moist heat

A

Moist heat is much more effective than dry heat since moist heat uses denaturisation of vital molecules in a bacterial cell such as the nucleic acids and proteins therefore some enzymes are going to be affected
Destroys cells and viruses by degrading nucleic acids and denaturing enzymes and other essential proteins, it also disrupts cells membranes

25
Q

Autoclave

A

Moist heat

In the autoclave is pure steam - this creates a high pressure which increases the boiling point  of water so the steam is hotter than usual 
121 degrees celsius of pure steam (this is important as it is the temperature that kills endospores ) and this temperature is held in the autoclave for 15 minutes 
Most vegetative, actively growing cells are killed at about 65 degrees, bacterial endospores with their layers and layers of protection require temperatures above 100 degrees to kill them 
We can autoclave anything that is not heat sensitive or releases any toxic chemicals 
Pure steam (i.e. no air) is crucial 
15 pounds per square inch (15 psi) (103.5 kPa) steam pressure and 121 degrees for 15 minutes
26
Q

Boiling water

A

Moist heat

100 degrees for 15 minutes
This is not sterilisation - it will remove vegetative cells but it will not affect the bacterial endospores therefore has limited applications

27
Q

Pasteurisation

A

Moist heat

Quality control - less than 5 x 10^5 cells/ml = these are lactobacilli which are good for the gut
Batch = 63 degrees for 30 minutes
Flash = 71 degrees for 15 seconds
Removes bacteria from the milk and also gives it a longer keeping time
Plate heat exchanger used = uses metal plate to transfer heat between two fluids so the plates produce a very large surface area which means that the hear can be transferred effectively, can be used to rapidly heat or cool, part of pasturisation is to also cool the fluid
Ultra-high-temperature (UHT) milk = 140 degrees for at least one second
Added implications for after pasteurisation, so it is not sterile product so it needs to be aseptically transferred into a sterile container and sealed and once this is achieved the milk can be stored for much longer and without refrigeration

28
Q

Batch pasteurisation

A

63 degrees for 30 minutes

29
Q

Flash pasteurisation

A

71 degrees for 15 seconds

Removes bacteria from the milk and also gives it a longer keeping time
Plate heat exchanger used = uses metal plate to transfer heat between two fluids so the plates produce a very large surface area which means that the hear can be transferred effectively, can be used to rapidly heat or cool, part of pasturisation is to also cool the fluid

30
Q

Ultra-high-temperature (UHT) milk

A

140 degrees for at least one second
Added implications for after pasteurisation, so it is not sterile product so it needs to be aseptically transferred into a sterile container and sealed and once this is achieved the milk can be stored for much longer and without refrigeration

31
Q

Dry heat

A

oxidises bacterial component - slower process - higher temp and longer time

32
Q

Flaming and incineration

A

Sterilisation method

Extreme temp oxidises material

33
Q

Hot air oven

A

160 degrees for 2 hours

Glassware, metal

34
Q

Freezing

A

Some bacteria may survive, not absolute therefore it is not a sterilisation process

35
Q

Why is moist heat more effective than dry heat?

A

Moist heat is much more effective than dry heat since moist heat uses denaturalisation of vital molecules in a bacterial cell such as the nucleic acids and proteins therefore some enzymes are going to be affected

36
Q

temp, time and effectiveness - boiling water

A

100 degrees
15 minutes
kills cells but does not kill endospores

37
Q

temp, time and effectiveness - steam under pressure (autoclave)

A

121 degrees
15 minutes
Kills cells and kill endospores

38
Q

temp, time and effectiveness - hot air oven

A

160 degrees
2 hours
kills cells and kills endospores

39
Q

temp, time and effectiveness - batch pasteurisation

A

63 degrees
30 minutes
kills some cells but does not kill endospores

40
Q

temp, time and effectiveness - flash pasteurisation

A

71 degrees
15 seconds
Kills some cells but does not kill endospores

41
Q

temp, time and effectiveness - UHT pasteurisation

A

140 degrees
1 second
Kills cells and kills endospores

42
Q

temp, time and effectiveness - flaming/incineration

A

kills cells and endospores

43
Q

temp, time and effectiveness - freezing

A

kills some cells but does not kill endospores