Week 3 - Bacterial Growth, Sterilization, & Disinfection

Bacterial growth

• Increase in number of cells
• Reproduce by binary fission (2 daughter cells)
• Daughter cells of equal size
• Cells may or may not separate after division
• Produce characteristic morphology

Chromosome replication

• Chromosome replication initiates cell division
• DNA replicated at origin of replication (oriC)
• Proceeds in both directions until terminus (ter)
• Rapidly growing cells can replicate on the new chromosomes even before division

Measurement of bacterial growth

In liquid media:

• Tubidity (absorbance of light)
• Count particles
• Viable counts
  In solid media:
• Serial dilution

Generation time

• Each time bacteria divide the population doubles
• Time taken for doubling to occur = generation/doubling time
• Characteristic to each bacteria (under ideal conditions)
• Greatly affected by culture conditions

Bacterial growth rates

• Microorganisms grow very rapidly (E. coli = 23 mins)
• Microorganisms grow slower in vivo (E. coli = 12hr in gut)

Requirements for bacterial growth

• Physical requirements: Temp, pH, Osmotic pressure, moisture
• Gaseous requirements: Oxygen, Carbon dioxide
• Nutrient requirements: Carbon, Nitrogen, Sulphur, Phosphorus, Trace elements, Vitamins & growth factors

Effects of temp on bacterial growth

• Bacteria can grow over a range of temps
• All bact. have optimum growth temps
• Most pathogenic bact. are mesophile (optimum = 37C)
• Temps above op are more deleterious than lower

Effects of pH on bacterial growth

• Can grow over a range
• All bact. have op growth pH
• Most pathogenic bact. are neutrophiles (op = 7)
• Products of bact. metabolism usually acidic
• Some bact. grow at pH 2(stomach acid)

Effects of oxygen on bacterial growth

• Obligate aerobes (need O2)
• Obligate anaerobes (need no O2)
• Facultative anaerobes (prefer O2 but can grow without)
• Aerotolerant anaerobes (grow with or without no problem)
• Microaerophiles (super strict, 1-10% O2)

Nutrient requirements

• Pathogenic bact. are chemoheterotrophs

• Require: C, N, S, P, K, Mg, Ca, & trace elements

• Some require growth factors (NAD or NADP)

Solid media

• Agar is added at 1-3% to solidify media
• Media commonly dispensed into petri dishes (plates)
• Bacteria grow as colonies
• Each colony grew from one cell
• Allows determination of different bacteria present and to isolate single colonies for pure culture

Enriched media

Supplemented w/ nutrients to support the growth of bacteria

• Blood agar
• Chocolate agar (heated blood agar)
• Dorset egg media
• Lowenstein Jensen media

Reading a blood agar

Bacteria break down RBC (lysis) for nutrients

• Alpha hemolysis: partial breakdown of RBC, greenish/brownish discoloration
• Beta hemolysis: complete lysis of RBC, clear zone around colonies
• Gamma hemolysis: no hemolysis or change in agar

Selective media

Inhibit the growth of certain bacteria while allowing others to grow

• Inhibitors: chemicals or dyes to suppress unwanted bacteria
• Differential agents: indicators (lactose, pH) to distinguish between bacteria based on metabolic activities

Cultivation of fungi

• Chemoheterotrophs, similar requirements to bacteria
• Nutrient-rich media
• Temp: 22-30C
• pH: 5.6
• Moisture: moist environments
• Incubation time: 3-7 days

The diagnostic process - sample to diagnosis

1. Patient (clinical diagnosis)
2. Sample collection
3. Microscopy (staining)
4. Culture
5. Sensitivities (to help determine treatment)

Types of cleaning (killing bacteria)

• Sanitizing (reduces number of bact.)
• Disinfecting (killing or inactivating most/all bact.)
• Sterilizing (guarantee killing/inactivating all bact.)

Methods of sterilization

• Physical: heat, radiation, filtration
• Chemical: gas, liquid

Heat sterilization

• Dry heat (hot air oven, 160C)
• Moist heat (boiling or autoclaving) - most effective, quicker, kills spores

Pasteurization of milk

• Low-temp, long-time: 63C for 30mins (traditional method, no longer used/effective)
• High-temp, short-time: 72C for 15-20 secs (modern method)
• Ultra-high temp: 135C for 2-5 secs (dairy products w/ long shelf life)

Irradiation sterilization

• Ultraviolet: poor penetration, only kills surface level, causes formation of pyrimidine dimers = error in DNA replication
• Ionizing: x-ray & gamma rays, used w/ water, forms toxic O2 metabolites, highly oxidizing agents kill microorganisms

Chemical disinfection - liquids

• Bleach (sodium hypochlorite), very effective against broad range
• Hydrogen peroxide (H2O2), surface disinfection, wound care, damages bact. proteins & membranes
• Alcohols (ethanol, isopropanol), skin disinfection & surfaces in healthcare, denatures proteins & disrupts membranes
• Quats (ammonium compounds), surface disinfection, effective against bact. but not against spores

Chemical disinfection - gases

• Ethylene oxide (EtO), sterilizes equipment, attacks bact. & spores, used in low-temps
• Ozone (O3), disinfect air, water, and surfaces, disrupts cell walls & internal structure
• Chlorine dioxide (ClO2), disinfect air & surfaces, attacks bact. spores & vegetative bact.

Materials that require sterilization

• Microbiological and tissue culture
• Medicines
• Surgical, optical, & electrical instruments
• Reagents
• Equipment or contains in which any above are handled
• Infected material

Factors affecting the action of disinfection

• Period of exposure
• Concentration
• Temp
• pH
• Organic matter
• Number of bacteria present