RO 5

Question 1

What is the rate of microbial death affected by?

Answer 1

Microbial characteristics/susceptibilities
*Environmental influences such as:
- Number of Microbes (Proportional death rates)
- Time required for sterilisation

Question 2

Sterilisation

Answer 2

The killing or removal of all microorganisms in a material or object

Question 3

Disinfection

Answer 3

Reduction in the number of pathogenic microogranisms on surfaces/objects to the point where they pose no danger of disease

Question 4

Antiseptic

Answer 4

Chemical agent that is safe to use externally on living tissue to destry microbes or inhibit their growth

Question 5

Bacteriostatic Agent

Answer 5

An agent that inhibits the growth of bacteria

Question 6

Bactericide

Answer 6

Agent that kills bacteria (though not spores)

Question 7

What are the ideal qualities for selecting a disinfectant?

Answer 7

Fast-acting
Non-toxic
Non-damaging to material
Wide spectrum
Easy to prepare/stable
Inexpensive
Odour

Question 8

Mechanisms of Action - Effects on Protein

Answer 8

Denaturation of Protein:
- Permanent/Temporary
- hydrolysis by acids/alkalis
- oxidation by H2O2, KMnO4, halogens
- alkylating agents (e.g. some dyes) etc.

Question 9

Denaturation of Protein (Bacteriocidal)

Answer 9

Active Protein - Inactive Protein - No reconfiguration; permanentely denatured

Question 10

Denaturation of Protein - Bacteriostatic

Answer 10

Active Protein - Inactive Protein - Reconfiguration, temporarily denatured

Question 11

Mechanisms of Action - Effects on Membranes

Answer 11

Denaturationof Protein Component
Disruption of Lipids
- Surfactants (Alcohols, detergents, quats)
- Wetting agents
- Indirect effect

Question 12

Mechanisms of Action for Nucleic Acids

Answer 12

Nucleic Acids: Damage from heat, radiation, chemicals

Question 13

Mechanisms of Action for Energy-producing systems

Answer 13

E.g. Fermentation inhibition by lactic acid or propionic acid

Question 14

Mechanism of Action - Cell Walls

Answer 14

Dyes (e.g. crystal violet) can interfere with cell wall formation

Question 15

Control Methods can be…?

Answer 15

Chemical or Physical

Question 16

Chemical Antimicrobial Agents - Soaps and Detergents

Answer 16

Remove microbes, oily substances and dirt
Anionic: Clothes laundering, household cleaning agents - less effective
Cationic: Sanitize food utensils (kill some viruses)
Quaternary ammonium compounds (quats)
Mixtures can increase efficacy

Question 17

Chemical Antimicrobial Agents - Phenols

Phenol and phenol derivatives (phenolics):

Answer 17

Denature proteins/enzymes and disrupt membranes
Action not impaired by organic material
Halogen addition can increase effectiveness
E.g. Amphyl and Lysol: retain properties for days,
safe on skin and medical instruments

Question 18

Chemical Antimicrobial Agents - Halogens

Answer 18

Particularly Iodine and Chlorine; alone (I2 or Cl2) or part of
compounds (NaOCl)
Agent used in drinking water and swimming pools (HClO)
Can be inactivated by organic material
Iodophors (Iodine combined with organic molecule): slow release, less irritating , surgical scrubs and skin antiseptic

Question 19

Chemical Antimicrobial Agents - Alcohols

Answer 19

Denature protein when mixed with
water
Dissolve lipids (cell membranes)
Effective against bacteria & fungi, but not endospores unenveloped viruses
Evaporates quickly (low exposure time)
Used as skin antiseptic (isopropanol or ethanol; effective at 60-95% (v/v)

Question 20

Chemical Antimicrobial Agents - Heavy Metals and their compounds

Answer 20

Selenium, Mercury, Copper, and Silver
Very effective in small quantities (oligodynamic action)
Selenium sulphide: Kills fungi; Anti-dandruff shampoo
Silver wound dressings; Calamine lotion

Question 21

Chemical AntiMicrobial Control - Oxidising Agents

Answer 21

Disrupt disulfide bonds - hydrogen peroxide is used to clean puncture wounds, potassium permangenate to disinfect instruments.

Question 22

Chemical Antimicrobial Agents - Alkalating Agents

Answer 22

Disrupt structure of proteins and nucleic acids.
Fomehaldehyde is used to inactivate viruses without destroying antigenic properties, glutaraldehyde to sterilize equipmentn

Question 23

Chemical Microbial Agents - Dyes

Answer 23

May interfere with replicatoin or block cell wall syntheisis
Acridine is used to clean wounds, crystal violet to treat some protozoan and fungal infections.

Question 24

Name the Physical Agents used as control methods

Answer 24

Heat (Various Methods)
Refrigeration
Desiccation
Irradiation
Filtration

Question 25

Heat

Answer 25

Cheapest, most effective and widely used control Denatures enzymes Suitable for materials undamaged by heat Various approaches: Dry heat Moist heat Pasteurisation

Question 26

Dry Heat

Answer 26

Used to sterilise metal objects and glassware Flame: Innoculating loops, flasks/tube mouths Dry heat sterilisation for moisture-sensitive materials Longer heating times/temperatues than moist heat

Question 27

Moist Heat

Answer 27

Causes denaturation of proteins and may disrupt membrane lipids-widely used Boiling water can kill most vegetative bacteria and fungi Heating water under pressure (higher temps) in an autoclave: 121 °C for 15-20 min can kill also spores

Question 28

Pasteurisation

Answer 28

Invented by Pasteur; does not achieve sterility Kills pathogenic organisms in raw products (milk etc) - Flash method: 71.6 °C for 15 sec - Holding method: 62.9 °C for 30 min - Ultrahigh temperature processing (UHT): 74°C— 140°C— 74°C (5 sec) Can be stored at RT

Question 29

Physical Agents - Lower temperatures

Answer 29

Refrigeration: (2-8 degrees) Slows growth based on reduction in enzyme kinetics Freezing : (-20 degrees) slows metabolic activity to prevent food spoilage but does not kill organisms.

Question 30

Physical Agents - Dessication

Answer 30

Water absence inhibits enzymatic activity Usually bacteriostatic, but may be bactericidal depending on the species Used to preserve some foods Freeze-drying: Lyphilisation - used to preserve bacterial cultures.

Question 31

Physical Agents - Radiation. Name the 3 main types

Answer 31

Ultraviolet (UV) light Ionising Radiation Microwave Radiation

Question 32

Ultraviolet (UV) light

Answer 32

(40 – 390 nm) Works best at 200nm; Used for sterilising surfaces Damages DNA and proteins; DNA repair can aid survival

Question 33

Ionising radiation

Answer 33

: X-rays and gamma rays (< 40 nm) – dislodge electrons from atoms, creating ions and radicals. Radicals kill by interacting with DNA, proteins, lipids etc.

Question 34

Microwave radiation

Answer 34

(1mm – 1m) Acts on water molecules which release heat – not effective on spores

Question 35

Physical agent-filtration

Answer 35

Passage of liquid/gas through small pores; traps in a sizespecific manner Membrane filters: Specified pore sizes (0.025 – 25 µm) Suitable for heat-sensitive materials (e.g. media supplements, drugs, vitamins) HEPA (High-efficiency particulate air) filters in labs

Question 36

What pore sizes of membrane filters are used most often?

Answer 36

0.45-0.22um

Question 37

Physical Agents Osmotic Pressure

Answer 37

High salt/sugar concentrations draw water from cells Bacteriostatic – metabolism cannot progress Food preservation: Curing, pickling, jams etc

Question 38

Sound

Answer 38

Ultrasonic waves can cause bacteria to cavitate – denatures proteins and disintegrates bacteria Can be used to lyse bacteria to release components for study, but not practical for sterilisation

Question 39

Evaluating A Disinfectant

Answer 39

Phenol coefficient: Filter Paper Method: Use-Dilution Test: Evaluating A Disinfectant Agents that prevent growth at the lowest concentrations are considered the most effective disinfectants

Question 40

Phenol coefficient:

Answer 40

- Comparison to the ‘original’ disinfectant - Some Disadvantages

Question 41

Use-dilution Tests:

Answer 41

Test bacteria coated onto carrier rings Incubated in test solutions (time based on product) Cultured in broth to detect surviving microbes

Question 42

* Filter Paper Method:

Answer 42

Efficacy of a Chemical Agent applied to a filter paper disc In vitro results may not translate in practice

Question 43

Measuring sensitivity to an antimicrobial agent (antibiotic susceptibility testing)

Answer 43

Disc-diffusion (Kirby-Bauer) method: Disc-diffusion method Lawn of target bacteria spread over plate Specific concentrations of antimicrobial agents on filter paper discs Zones of inhibition measured (diameter) Issues due to bacteriostatic vs. bactericidal actions

Question 44

E (epsilometer) test

Answer 44

determines sensitivity to antibiotic and also minimum inhibitory concentrations (MIC) – antimicrobial gradient Gradient of antibiotic concentrations on a strip (high to low) Intersection of zone of inhibition with the strip used to determine the MIC

Question 45

The dilution method:

Answer 45

Bacteria inoculated into serial dilutions of an antimicrobial agent MIC can be determined Measuring sensitivity to an antimicrobial agent (antibiotic susceptibility testing) Can combine with second test to determine if bacteriostatic or bacteriocidal (minimum bactericidal concentration; MBC)