Bacteriology lecture 5 Flashcards
what is the rate of microbial death is affected by?
- Microbial characteristics/susceptibilities
- Environmental influences
- Number of Microbes (Proportional death rates)
- Time required for sterilisation
Sterilisation
The killing or removal of all microorganisms in a material or object
Disinfection:
Bactericide: Agent that kills bacteria (though not spores)
Reduction in the number of pathogenic microorganisms 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 destroy microbes or inhibit their growth
Bacteriostatic agent
An agent that inhibits the growth of bacteria
Bactericide
Agent that kills bacteria (though not spores)
I deal qualities for selecting a disinfectant
- Fast-acting
- Non-toxic
- Non-damaging to material
- Wide spectrum
- Easy to prepare/stable
- Inexpensive
- Odour
Effects on Proteins
Denaturation of Protein:
- Permanent/Temporary
- hydrolysis by acids/alkalis
- oxidation by H2O2, KMnO4, halogens
- alkylating agents (e.g. some dyes) etc.
Effects on Membranes
- Denaturation of Protein Component
- Disruption of Lipids:
- Surfactants (Alcohols, detergents, quats)
- Wetting agents
- Indirect effect
Effects on Other Cell Components
- Nucleic Acids: Damage from heat, radiation, chemicals
- Energy-producing systems: E.g. Fermentation inhibition
by lactic acid or propionic acid - Cell walls: Dyes (e.g. crystal violet) can interfere with cell wall formation
Control methods can be
- Chemical
- Physical (e.g. heat or irradiation)
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 materials
- 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
Many Physical Agents used for food preservation and preparation Includes:
- Heat (Various Methods)
- Refrigeration
- Desiccation
- Irradiation
- Filtration
Physical Agents: Heat
- Cheapest, most effective and widely used control
- Denatures enzymes
- Suitable for materials undamaged by heat
- Various approaches:
- Dryheat
- Moistheat
- Pasteurisation
Dry heat
- Used to sterilise metal objects and glassware
- Flame: Innoculating loops, flasks/tube mouths
- Dry heat sterilisation for moisture-sensitive materials:
1) 170 °C for 1 h
2) 160 °C for 2 h
3) 121°Cfor16h
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
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°C140°C 74°C (5 sec) Can be stored at RT
Lower temperatures
- Refrigeration: (2 – 8 °C) Slows growth based on reduction in enzyme kinetics
- Freezing: (-20 °C) Slows metabolic activity to prevent food spoilage but does not kill organisms
Dessication
- Water absence inhibits enzymatic activity
- Usually bacteriostatic, but may be bactericidal depending on the species
- Used to preserve some foods
- Freeze-drying: (Lyophilisation)
- Freeze-dried coffee
- Used to preserve bacterial cultures
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
Filtration
- Passage of liquid/gas through small pores; traps in a size- specific 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
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
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
what is used when
Evaluating A Disinfectant?
- Phenol coefficient:
- Filter Paper Method:
- Use-Dilution Test:
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 steps
- 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
What methods are used in
Measuring sensitivity to an antimicrobial agent?
- Disc-diffusion (Kirby-Bauer) method
- E (epsilometer) test
- The dilution method
Disc-diffusion (Kirby-Bauer) 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
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
- Can combine with second test to determine if bacteriostatic or bacteriocidal (minimum bactericidal concentration; MBC)
