RO 5 Flashcards
What is the rate of microbial death affected by?
Microbial characteristics/susceptibilities
*Environmental influences such as:
- Number of Microbes (Proportional death rates)
- Time required for sterilisation
Sterilisation
The killing or removal of all microorganisms in a material or object
Disinfection
Reduction in the number of pathogenic microogranisms on surfaces/objects to the point where they pose no danger of disease
Antiseptic
Chemical agent that is safe to use externally on living tissue to destry microbes or inhibit their growth
Bacteriostatic Agent
An agent that inhibits the growth of bacteria
Bactericide
Agent that kills bacteria (though not spores)
What are the ideal qualities for selecting a disinfectant?
Fast-acting
Non-toxic
Non-damaging to material
Wide spectrum
Easy to prepare/stable
Inexpensive
Odour
Mechanisms of Action - Effects on Protein
Denaturation of Protein:
- Permanent/Temporary
- hydrolysis by acids/alkalis
- oxidation by H2O2, KMnO4, halogens
- alkylating agents (e.g. some dyes) etc.
Denaturation of Protein (Bacteriocidal)
Active Protein - Inactive Protein - No reconfiguration; permanentely denatured
Denaturation of Protein - Bacteriostatic
Active Protein - Inactive Protein - Reconfiguration, temporarily denatured
Mechanisms of Action - Effects on Membranes
Denaturationof Protein Component
Disruption of Lipids
- Surfactants (Alcohols, detergents, quats)
- Wetting agents
- Indirect effect
Mechanisms of Action for Nucleic Acids
Nucleic Acids: Damage from heat, radiation, chemicals
Mechanisms of Action for Energy-producing systems
E.g. Fermentation inhibition by lactic acid or propionic acid
Mechanism of Action - Cell Walls
Dyes (e.g. crystal violet) can interfere with cell wall formation
Control Methods can be…?
Chemical or Physical
Chemical Antimicrobial Agents - Soaps and Detergents
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
Chemical Antimicrobial Agents - Phenols
Phenol and phenol derivatives (phenolics):
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
Chemical Antimicrobial Agents - Halogens
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
Chemical Antimicrobial Agents - Alcohols
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)
Chemical Antimicrobial Agents - Heavy Metals and their compounds
Selenium, Mercury, Copper, and Silver
Very effective in small quantities (oligodynamic action)
Selenium sulphide: Kills fungi; Anti-dandruff shampoo
Silver wound dressings; Calamine lotion
Chemical AntiMicrobial Control - Oxidising Agents
Disrupt disulfide bonds - hydrogen peroxide is used to clean puncture wounds, potassium permangenate to disinfect instruments.
Chemical Antimicrobial Agents - Alkalating Agents
Disrupt structure of proteins and nucleic acids.
Fomehaldehyde is used to inactivate viruses without destroying antigenic properties, glutaraldehyde to sterilize equipmentn
Chemical Microbial Agents - Dyes
May interfere with replicatoin or block cell wall syntheisis
Acridine is used to clean wounds, crystal violet to treat some protozoan and fungal infections.
Name the Physical Agents used as control methods
Heat (Various Methods)
Refrigeration
Desiccation
Irradiation
Filtration
Heat
Cheapest, most effective and widely used control
Denatures enzymes
Suitable for materials undamaged by heat
Various approaches:
Dry heat
Moist heat
Pasteurisation
Dry Heat
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
Moist Heat
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
Pasteurisation
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
Physical Agents - Lower temperatures
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.
Physical Agents - Dessication
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.
Physical Agents - Radiation. Name the 3 main types
Ultraviolet (UV) light
Ionising Radiation
Microwave Radiation
Ultraviolet (UV) light
(40 – 390 nm) Works best at 200nm;
Used for sterilising surfaces
Damages DNA and proteins; DNA repair can aid survival
Ionising radiation
: X-rays and gamma rays (< 40 nm) – dislodge electrons
from atoms, creating ions and radicals.
Radicals kill by interacting with DNA, proteins, lipids etc.
Microwave radiation
(1mm – 1m) Acts on water molecules which release
heat – not effective on spores
Physical agent-filtration
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
What pore sizes of membrane filters are used most often?
0.45-0.22um
Physical Agents
Osmotic Pressure
High salt/sugar concentrations draw water from cells
Bacteriostatic – metabolism cannot progress
Food preservation: Curing, pickling, jams etc
Sound
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
Evaluating A Disinfectant
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
Phenol coefficient:
- Comparison to the ‘original’ disinfectant
- Some Disadvantages
Use-dilution Tests:
Test bacteria coated onto
carrier rings
Incubated in test solutions
(time based on product)
Cultured in broth to detect
surviving microbes
- Filter Paper Method:
Efficacy of a Chemical Agent applied to a filter paper disc
In vitro results may not translate in practice
Measuring sensitivity to an antimicrobial agent
(antibiotic susceptibility testing)
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
E (epsilometer) test
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
The dilution method:
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)
