Principles of sterilization and disinfection:

sterilization: refers to the killing or removal of all organisms in any material or on any object, i.e. free of life of every kind

disinfectant: refers to the reduction in the number of pathogenic organisms on objects or in materials so that the remaining organisms no longer pose a disease threat.

Typically, disinfectants are applied to inanimate objects (too toxic for living tissues), whereas antiseptics are applied to living tissues.

The total number of organisms present when disinfection is begun determines the length of time required to eliminate them; the fewer the number at the begining, the shorter the time to eliminate them.

Potency of Chemical Agents:
The potency, or the effectiveness, of chemical antimicrobial therapy, is affected by time, temperature, pH, and concentration. Consequently, depending upon the interaction of each of the above parameters, antimicrobial agents may be either:

bacteriocidal: kills all bacteria except spores (generally higher concentrations of the agent)

bacteriostatic: growth inhibiting (generally lower concentrations of the agent; note, bacterial growth resumes upon removal of the agent)

Mechanisms of action of chemical agents:

1. Reactions that effect proteins: as much of the cell is composed of proteins, then agents that damage protein inactivate the cell. Alteration of protein structure is called denaturation.
2. Reactions that effect membranes: membranes are composed of lipids and can be disrupted by substances that dissolve the lipids components (called surfactants, which include alcohols, detergents, and quaternary ammonium compounds).
3. Reactions that damage DNA: such agents include ionizing radiation, ultraviolet light, and DNA-reactive chemicals (e.g. alkylating reagents and chemicals that modify the bases in the DNA). Types of Antimicrobial Agents:

a) Physical agents

i) Heat - dealt with previously.
ii) Radiation - ionizing (e.g. gamma radiation) and ultraviolet - currently a prefered method for sterilization of food. However, its application has stirred up considerable debate on the potential harm that may arise from irradiating food.

b) Chemical agents

i) Alcohols - toxic to cells at high concentrations, due to denaturing proteins. Generally, a 70% solution of either ethyl or isopropyl alcohol will disinfect a surface (e.g. when one gets an injection).
ii) Phenol - phenolic derivatives disrupt cell membranes, denature proteins and inactivate enzymes. Amphyl or lysol (something you may use in lab) destroys vegatative forms of bacteria and fungi, and also inactivates viruses.
iii) Heavy metals - include selenium, mercury, copper, and silver; merthiolate tinctures. Silver nitrate was once put into babies eyes following vaginal births to prevent the possibility of gonococcal infection (nowadays pregnant women are screened prior to delivery). Again application of heavy metal solutions do not inactivate spores.
iv) Oxidising agents - include hydrogen peroxide, iodine, chlorine, and bleaches. Work by oxidising sulfyhryl groups in proteins.
v) Alkylating agents - e.g 37% formaldehyde solutions and ethylene oxide (the most effective disinfectant available for dry surfaces.
vi) Detergents - Composed of a long hydrophobic side-chain as well as a charged polar head group. Two kinds of detergent depending on the charged head group, either anionic (negatively charged) or cationic (positively charged). Act by disrupting cell membranes.

Microbial Growth
Definition: orderly increase of all chemical components.

Cell division (see Figure 5:1)
Cell division in bacteria, unlike eucaryotes, usually occurs by binary fission. In binary fission a cell a cell duplicates and divides into two cells. The daughter cell becomes complete when the septum is complete. Unlike eucaryotes, bacterial cells generally do not have a cell cycle. Some microorganisms duplicate through budding e.g. yeast.

Growth Curve (see Figure 5:2)
In a similar fashion to cell death, plotting the log number of bacteria (N) vs time gives a straight line. However, due to some complicated math, the slope equals the rate constant (µ)/2.303. The incept on the Y-axis equals logN0.

Growth Phases: (see Figure 5:3)

a) lag phase: when cells are transferred to fresh culture medium they need time to adapt, especially if they are transferred from a sttionary phase culture. This ia variable in duration, but ingeneral is directly related to the duration of the preceeding stationary phase.

b) exponential phase: cells are in steady state - i.e. they are doubling their biomass at regular intervals - known as the doubling time. Here new material in being synthesized at a constant rate and this continues until one of two things happens: i) one or more nutrients within the medium are exhausted; or, ii) some metabolic product becomes toxic for the culture. For aerobic organisms the nutrient that generally becomes limiting is O2, due to the fact that the rate of diffusion of O2 into the culture medium cannot match the amount needed for the growth of the bacteria.

c) stationary phase: once the exhaustion of nutrients or accumulation of toxic products has reached a certain critical level growth ceases. However, cells continue to turnover with approximately an equal number dying as new ones are being formed. NB: total viable count remains stable.

d) death phase: as cells are held in a non-growing state for long enough they begin to die. Number of factors contribute to this: an important factor is the depletion of cellular energy reserves. The death rate eventually reaches steady state (i.e. straight line). Once the majority of cells in the culture have die, death is reduced drastically, and a few cells remain viable and may persist for months or even years (persistence depends on the bacterium; E. coli persist for a long time; gonococci die very quickly).

Generally, growth is either measured as:

1) Cell concentration: viable cell count - plating known dilutions of of a culture and counting the bacteria. However, done usually by turbidity in a spectrophotometer (see Figure 5:4) - method of choice. Here a beam of light is passed through a bacterial suspesion and the bacteria within that suspension scatter the light, i.e. the reduction in the amount of light transmitted is a direct relection on cell density. NB: This is only of use if plotted against a standard curve. However, if doing by turbidity then the stage in the growth cycle is crucial, i.e. there would be a profound difference if when comparing two cultures of the same bacterium and one was in exponential growth and the other was in stationary phase.

2) Biomass density: in theory biomass can be determined directly by measuring the dry weight of a microbial culture. However, more convenients during balanced growth to measure any cellular component over time e.g. increases in DNA etc.

Note: an increase in mass need not necessarily indicate growth as the organism could be simply increasing their content of storage products such as glycogen or b-hydroxybutyrate. However, during balanced growth, a doubling of the biomass is accompanied by a doubling of all other measuable properties of the population e.g. protein, RNA, DNA, and intracellular water. In other words: cultures undergoing balanced growth maintain a constant chemical composition.

 

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