Endospores And Control Of Microbial Growth

Question 1

What is an endospore?

Answer 1

Dormant and inert bacterial structures
▪ Produced only by two Gram+ Genera: Bacillus and Clostridium

Question 2

What are endospores speciality?

Answer 2

• Mechanism of survival to harsh environment that would be lethal for the bacteria
  ▪ nutrients depletion,
  ▪ environmental stresses (extreme temperature, pH),
  ▪ chemical stresses (antibiotics, disinfectants), etc
• Extraordinarily resistant to desiccation, heat, chemicals, and radiation
• Endospores are able to survive for long periods → centuries/thousands of years

Question 3

What is sporulation? + what it entails

Answer 3

*endospore formation
*germatitve stress + environmental stress = sporulation to make endospore

Question 4

What is the endospore structure (core)?

Answer 4

An endospore has an ovoid shape and a multi-layered structure, containing:
➢ a core (dehydrated and inactive), with:
▪ DNA, ribosomes, essential proteins and calcium
dipicolinate (responsible for dehydration)

Question 5

What is the endospore structure outside of the core?

Answer 5

➢ Surrounded by 3-4 different coating layers:
▪ Core walls: innermost layer
▪ Cortex: made of peptidoglycan
▪ Spore coat: thick and impermeable protective
layer to chemicals/antibiotics
▪ Exosporium: thin covering, not always present

Question 6

What is germination and what it entails?

Answer 6

• Process of an endospore that revert back to a vegetative cell very rapidly (even after
  decades), through removal of the stress inducer
  ▪ It also requires an activation step (usually heating to damage the spore coat),
  which is reversible
  ▪ Divided in 3 sequential steps, activation, initiation and outgrowth
  ▪ During germination, the core releases calcium dipicolinate becoming is hydrated,
  spore layers are disassembled, and the bacterial metabolism is restored

Question 7

What are endospores very resistant to?

Answer 7

Extraordinary resistance to desiccation, heat, chemicals, and
radiation in comparison to the vegetative bacteria
* This makes endospores difficult to eliminate
▪ Highly resistant to the disinfection procedures

Question 8

Why is endospore resistance to chemicals bad?

Answer 8

Issues in clinical settings or aseptic environments (e.g. pharmaceutical
preparation)
▪ Increasing the risk of healthcare-associated infections (HAIs)

Question 9

What is sterilisation?

Answer 9

Completely eliminate any microbial life (including endospores)

Question 10

What is disinfection?

Answer 10

Reduces microbial load to the point where they no longer cause
diseases. Not active against bacterial endospores.
▪Disinfectant: agents applied on inanimate objects/surfaces (toxic to human tissue)
▪Antiseptic: agents applied on living tissue

Question 11

What is sanitisation?

Answer 11

reduces microbe on items to safe levels by public health standards

Question 12

What is antimicrobial therapy?

Answer 12

use of drugs to prevent or treat infections to
SELECTIVELY inhibit growth of microorganisms minimising host tissue toxicity

Question 13

What are the agents to control microbial death?

Answer 13

Effectiveness of the control agents depends on:
* Time of exposure
* Microbial characteristics
* Number of microbes
* Environment (organic matter, temperature, biofilms)

Question 14

What are the mechanisms to microbial death?

Answer 14

• Alternation of membrane permeability: Disrupts the integrity of microbial cells
• Protein denaturation: disrupts enzyme function and cellular processes
• Damage to nucleic acids: Prevents replication and proper cellular function

Question 15

What are some physical treatments to control microbial growth?

Answer 15

• Temperature
  ▪ Heat (moist and dry)
  ▪ Cold temperatures
• Filtration
• Radiations
• Desiccation

Question 16

What are some chemical treatments to control microbial growth?

Answer 16

• Alcohols
• Halogens
  ▪ Iodine
  ▪ Chlorine
• Phenolics
• Aldehydes
• Quaternary ammonium compounds
• Sterilizing gases
• Heavy metals

Question 17

What is moist heat?

Answer 17

hot water, boiling water, or steam between 60°C and 135°C (autoclave,
pasteurisation). Kills microbes by denaturation their proteins
*more effective

Question 18

What is dry heat?

Answer 18

hot air or a flame (Bunsen Burner, incineration), which ranges from 160°C
to thousands of degrees. Kills microbes by dehydrating the cell & protein denaturation

Question 19

What is the autoclave + properties?

Answer 19

Autoclave (moist heat)
* Preferred sterilisation method
* Closed chamber with hot saturated
steam under pressure
▪ 121.5o C for 15 minutes, to sterilise
microbes/endospores (more time for prions)
* Steam must directly contact material

Question 20

What is pasteurisation?

Answer 20

Pasteurisation (moist heat disinfection): Developed by
Pasteur to prevent the spoilage of beverages by reducing
microbes

Question 21

What are the three methods of pasteurisations?

Answer 21

▪ Classic Pasteurisation: 65 o C for 30 minutes
▪ Flash Pasteurisation (HTST): Used today. 72 o C for 15 seconds
▪ Ultra High Temperature Pasteurisation (UHT): 140 o C for 4 sec.
and quickly cooled in a vacuum chamber. It is a sterilising method
* Except for UHT, the other pasteurisation methods do not sterilise

Question 22

Low vs freezing temperature effect on microbial effect?

Answer 22

Low temperatures (< optimal temp.) slow down microbial growth
▪ Reduces metabolic rate of most microbes to stop their
proliferation and toxin production, but often do not kill microbes
▪ Refrigeration temperatures (4-8 o C) are used for material
preservation
* Freezing temperature (<0o C) can be used for long-term storage and
preservation of some bacterial cultures (useful for analysis)
* Placing solutions in glycerol at -70 oC

Question 23

What is filtration + what it entails?

Answer 23

• Removal of microbes from a solution
  by the use of specific pore sizes (<
  microbe size) as a physical barrier
• Used for heat-sensitive drugs (vaccines, enzymes/proteins)
  ▪Pore size to exclude most bacteria: 0.2 - 0.45 μm
  ▪Pore size for viruses: 0.01 μm

Question 24

What is desiccation?

Answer 24

Based on osmotic pressure and water
reduction
* Adding salts and sugars in foods is used to dry food and
reduce water activity in the environment, creating
hypertonic environment

Question 25

Describe what happens in desiccation

Answer 25

• Water leaves the cell by osmosis
• plasma membrane shrinks away from cell wall and
  bacteria reduce their metabolism (dormant`)
• Cell may not die, but usually halts growth.

Question 26

What does UV light do to microbes?

Answer 26

UV light: Damages DNA (breaks or mutations)
▪ poor penetrating power
▪ Used only for surface sterilisation

Question 27

What does ionising radiation do to microbes?

Answer 27

• Ionising radiations (Gamma rays, electron beams and X-rays): inducing
  reactive free radicals that lead to microbial cell death.
  ▪ High penetrating power into objects
  ▪ Used to irradiate heat-sensitive pharmaceuticals and medical supplies

Question 28

what is important to remember about disinfectant agents?

Answer 28

More often employed in disinfection (inanimate
objects/surfaces) and antisepsis (human tissues).
Only a Few chemical agents achieve sterility.

Question 29

what factors affect the efficiency of chemical agents?

Answer 29

▪ The kinds of organisms present
▪ Degree of contamination
▪ Time of exposure
▪ Nature of the material being treated
▪ Concentration of disinfectant

Question 30

What do chemical agents and antiseptics include?

Answer 30

▪ Alcohols (60-80%): Ethanol and Isopropanol
➢ Used as an antiseptic before injections, in hand sanitisers and cosmetics.
➢ Not suitable for open wounds
o No effect on nonenveloped viruses
▪ Iodine: (tincture → iodine+alcohol)
➢ Used as antiseptic in open wounds during surgery → must be in contact for 30 seconds
▪ Chlorine
➢ Used to disinfect drinking water, pools, and sewage
❑ They damage plasma membranes, proteins, lipids, or DNA to reduce microbial content

Question 31

What does glutaraldehyde do?

Answer 31

Sterilising power Used to sterilise
medical equipment

Question 32

What does betapropiolactone do?

Answer 32

Sterilising power
Chemicals that sterilise heat-sensitive
material in a chamber similar to an autoclave
The structures of some frequently used disinfectants and antiseptics.

Question 33

What are the different antimicrobial therapies and what are they targeting?

Answer 33

▪ Antibiotics → bacterial infections
▪ Antiviral drugs → virus infections
▪ Antifungal drugs → fungal infections
▪ Anthelminthic drugs → worm infections (parasites)
▪ Antiprotozoal drugs → protozoan infections (parasites)