Microbiology: Growth Media, Batch Systems & Control

• Aim: To prepare media where micro-organisms can grow. Divided into 2 main groups...
  • Chemically defined/synthetic media - known ingredients and concentration.
  • Complex media - complex, undefined material - rich in vitamins & other nutrients - precise chemical composition undefined and varies on supplier (eg: Yeast extract).

• Various synthetic and complex media commerially available. 
• Preparation = addition of defined mass to specific volume & autoclaving for 15 mins @ 121C @ 100kPa.
• Suitable environment needed - right temp, pH, atmospheric oxygen, osmotic pressure.
• Can be prepared namely in 3 forms...

• Liquid medium (nutrient broth) - fermentation, biochemical studies, biomass determinations - micro-orgs. not visable to naked eye - growth conditions supply nutrients, temp etc. 3 methods detecting microbial growth:
  • 1. Turbidity/opaqueness - cloudy
  • 2. Pellicle formation - mass of cells floats on surface.
  • 3. Sediment formation - cells deposit at bottom.
  • 4. Slime production - cells dont separate - covers sides.

• Solid media:
  • Examintation of colony formation properties, isolation, storage of pure cultures, biochemical reactions.
  • Any liquid medium + agar.
    • Agar - unbranched polysaccharide - cell membranes of red algae - melts at 95 and solidifies at 42 - no nutrient properties - not hydrolysed by many orgs.
  • Prepared as slant, stab, plate - petri dish.
  • Microbes will grow, divide until form a colony - visable on surface. 

• Semi-solid media:
  • fairly soft - reduced amounts of agar.
  • mainly used for testing motility, oxygen requirements of organisms.

• Micro-organisms - reproduce by budding (yeasts) or binary fission (bacteria)
• Microbial growth = increase in cell numbers NOT size - growth is exponential
• Cell numbers expressed as 'colony forming units' (CFU) on solid media - turbidity (less accurate) on liquid media.
• Bactera - grow in batch systems = closed system with limited nutrients - typically follows a growth curve - Lag, log, stationary and death phases...

LAG PHASE

• 1st phase - right after inoculation of culture into medium - little/no cell division takes place.
• Length of lag phase depends on type of org, culture conditions etc. Essential enzymes, cofactors and ATP need to be produced before growth can commence.
  • Longer lag phase - older cells or cells under stress (need longer time to recover)

EXPONENTIAL/LOG PHASE

• Most experiments done during this phase.
• Phase where microbes grow + divide at max rate.
• Growth depends on medium and how well cells adapt to new environment (temp, pH etc)
• Growth rate is constant - ie: cells dividing at regular intervals
• Max cell concentration at end of log growth varies depending on type of microbe and environ. factors.

STATIONARY PHASE

• Antibiotics harvested at this point
• Population growth ceases - curve reaches a plateau
• Some cells still active but no net increase due to many factors (depletion of o2, nutrients etc)

DEATH PHASE

• Detrimental envrion. changes - nutrient deprivation and toxin build up results in log death/decline.

Temperature, Irradiation, Desiccation & Filtration

Temperature

• Heat treatment most common.
• Moist heat - denatures and inactivates proteins (enzymes) and disrupts cell membranes.
• Dry heat - causes oxidation of cell constituents.
• 3 types of treatments used:
  • Pasteurization
    • Reduces number/type of microbes - short term solution - remaining microbes begin growing again. (eg: Milk)
    • LTLT - Low temp, long time
    • HTST - High temp, short time
    • UHT - Ultra high temperature.
  • Sterilization
    • Temps of 121+ used to destroy bacterial spores
    • Labs use autoclaving (industrial type pressure cooker) - combining steam + pressure
    • 121 degrees, 15 min, 2 bar.

• • Tyndallisation
    • Breaks heat-labile chemical bonds by heating with flowing steam at 100 degrees for 30 min on 3 consecutive days.
    • Does not destroy dormant spores but does destroy any that for during 3 day period.
• Glassware and medical instruments - dry heat 170C for 2 hours in oven
• Heat labile products - vitamins, plastics, spices - sterilized by irradtion or filtration.
• Bacteristatic = extremely low temps used to slow/prevent microbial growth.
• Bactericidal = Product not permanently sterilized. (freezer, fridge etc)
• Rapid freezing less damaging than slow freezing there used to keep microbes viable over many years.

Irradiation:

• Used to permanently damage living organisms by altering their nucleic acids.
• UV light - dimerization of thymine residues in DNA - leading to cell death - Low penetating power so best used to sterilize surfaces.
• Ionising radiation - X-rays, gamma rays - highly penetrating - breaks sugar-phosphate backbone of DNA - sterilizes surgical instruments and heat sensitive substances (spices)
• Bacterial endospores are resistant to irradation. 

Desiccation

• Induces hypertonic environment by dehydrating or increasing sugar/salt content - inhibits microbes (not completely eliminating them)
• Many foodstuffs preserved by drying or addition of salt/sugars.
• Freeze-drying done at very low temps - ensures food not damaged by drying process.
• Biltong, dried fruit, jams...

Filtration

• Sensitive substances - vitamins, antibiotics, protein solutions - sterilized using a filter - very small pore size that will retain mostmicrobes.
• Heat sensitive substances are filtered.
• Problem: viruses get through.