Bacterial cultivation, preservation and inactivation
Bacterial growth
Considerations for bacterial nutrition and physical and chemical factors
Microbial preservation
Inactivating microorganisms
Binary fission
4 growth phases
Lag -> Exponential growth -> Stationary -> Decline
Lag phase: After innoculation, metabolism to get ready to divide
Exponential phase: Binary fission=exponential increase
Stationary Phase: Essential nutrients depleted / Toxic metabolic products
Decline phase: Old cells die followed by young
Enumeration
Microscopic counting
Colony counting
Opacity/turbidity
From fixed smear, arbitrary number of fields counted
Doesnt account for viability of cell
Make 10 fold serial dilutions, spread fixed volume on each plate and incubate
Count colonies on plates. Viable organisms in suspension=CFU
Opacity tubes. Compare to McFarland Standards
Spectrometry/Optical density
Quick, but no info if viable
Growth requires raw materials: some form of carbon
Autotrophs vs. heterotrophs
Autotrophs use CO2 to make own compounds
Heterotrophs use pre-formed organic compounds
Nutrition
Need lots of C and N
Peptones provide C&N and essential nutrients (P,S,Ca2+, etc)
Phosphates (nucleic acids)
Sulphur (amino acid production)
Extracellular molecules collect nutrients
Siderophores, hemolysins collect iron
Extracellular enzymes
Semi-starvation state: slower metabolism+smaller size
Sporulation and "resting cells"
Very low metabolic rate
Change shape, develop think coat
Endospores form within cells, v. resistant
Spores are for survival, triggered by low nutrients
Culture medium
Defined vs complex
Defined: known amounts of known chemicals
Selective and differential
Complex: hyrolysates, extracts, etc
Selective media limits growth of unwanted microbes
Differential allows differentiation between different microbes
A medium can be both
Physical and chemical growth reuirements
Highly diverse in types of conditions can grow in
Growth influences by temp, pH, moisture, atmospheric composition, osmotic pressure
Oxygen requirements
Aerobes
Microaerophiles
Anaerobes
Require oxygen but only small amounts
Obligate anaerobes
Aerotolerant anaerobes
Facultative anaerobes (can grow with or without O2)
Why
Vaccine production
Research and teaching
How
Freezing in liquid nitrogen
Dessication (freeze drying then stored as ampoules in dark)
Inactivation useful terms
Asepsis
Antimicrobial chemicals
Sepsis
Aseptic technique
Prevent microbial contamination of cultures (lab) or wounds (clinical)
Microbial contamination
Absence of significant contamination
Disinfectant (for inanimate objects)
Antiseptic (for living tissue)
Expected to destroy pathogens but not to achieve sterilization
Disinfection
Sterilisation
Antisepsis
Bacteriostatic
Bactericidal
Kill bacteria
Inhibits bacterial reproduction
Destruction of microorganisms by direct exposure to chemical or physical agents
Destroy all microbial life
Chemicals applied to body surfaces to destroy/inhibit pathogens
Microbe types and life cycle phases different susceptibilities to controls
Organic matter (e.g. Faeces in dairy footbath)
Ability heat/chemical to kill microbes
Exposure time and working concentration
Physical methods
Chemical methods
Refrigeration
Freezing
Boiling
Vaccum packing
Pasteurization (HTST/Flash or UHT)
Thermal death point, thermal death time and decimal reduction time
Moist heat more effective than dry heat
Filtration
Radiation (Gamma or UV)
Increase osmotic pressure (using salts/sugars) -> plasmolysis
Acidification
Sulfur dioxide
Disinfectants
Surfactants
Phenols an phenolics
Acid-anionic detergens
Cationic detergents
Halogens
Alcohols
Chlorine
Iodine
Heavy metals
Oligodynamic action -> denature proteins by altering disulphide bonds
Silver
Mercury
Copper