Laboratory evaluation of antimicrobial agents

Definitions:

Biocide - chemical or physical agent which kills viable organisms, both

pathogenic and nonpathogenic

Microbicide - refer specifically to an agent which kills microorganisms

Biocidal , bactericidal , fungicidal and viricidal - an agent with killing

activity against a specific class or classes of organism

Bacteriostatic and fungistatic - inhibit the growth of bacteria or fungi

Tolerance - ability of some bacterial strains to survive but not grow

Minimum inhibitory concentration (MIC) - the lowest concentration of

antimicrobial which will inhibit the visible growth of a microorganism

Minimum bactericidal concentration (MBC) - the lowest concentration of

antimicrobial which will kill the microorganism

MIC and MBC values are recorded in milligrams per litre or per millilitre
(mg/L or mg/ml)

Factors affecting the antimicrobial activity of disinfectants

Concentration of agent
Duration of exposure to the active agent
Temperature
pH

Innate (natural) resistance of microorganisms

Microorganisms adhering to surfaces as biofilms or present within other

cells, may reveal a marked increase in resistance to disinfectants and
biocides.
When evaluating new disinfectants, a suitable range of microorganisms
and environmental conditions must be included in tests.

Microbial density

The larger the number of microorganisms present, the longer it takes a

disinfectant to complete killing of all cells
For instance, using identical test conditions, it has been shown that 10
spores of the anthrax bacillus (Bacillus anthracis) were destroyed in 30
minutes, while it took 3 hours to kill 100 000 (105) spores

Disinfectant concentration and exposure time

-

The more concentrated the disinfectant, the greater its efficacy and the
shorter the time necessary to achieve microbial kill. Considering the length
of the disinfection time, which depends on the potency of the germicide,
also is important.
Items must be exposed to the germicide for the appropriate minimum
contact time. The exact times for disinfecting medical items are somewhat
elusive because of the effect of the aforementioned factors on disinfection
efficacy.

Physical and chemical factors

Temperature
- The activity of most disinfectants increases as the temperature increases.
Increase in temperature causes the disinfectant to degrade and weakens
its germicidal activity and thus might produce a potential health hazard.
pH
- directly influence the survival and rate of growth of the microorganism
under test acid or a base
- The non - ionized molecule is the active state (capable of crossing the cell
membrane/partitioning) and alkaline pHs which favour the formation of
ions of such compounds will decrease the activity
Divalent cations

The presence of divalent cations (e.g.Mg2+,Ca2+),for example in hard

water, has been shown to exert an antagonistic effect on certain biocides
while having an additive effect on the cidal activity of others.
Metal ions such as Mg2+ and Ca2+ may interact with the disinfectant itself
to form insoluble preipitates and also interact with the microbial cell
surface and block disinfectant adsorption sites necessary for activity.

Presence of extraneous organic material

Blood, serum, pus, faeces or soil is known to affect the cidal activity of
many antimicrobial agents

Disinfectants are tested in hard water containing 0.3 g/L bovine albumin
to mimic dirty conditions
Disinfectants should be evaluated under both clean and dirty conditions
Disinfectant whose activities are particularly attenuated in the presence of
organic contaminant include the halogen disinfectants. (e.g. sodium
hypoclorite) where the disinfectant reacts with the organic matter to form
inactive complexes, biguanides, phenolic compounds and QACs.

Evaluation of liquid disinfectants

Evaluation of a disinfectants efficacy was based on its ability to kill

microbes
Capacity- use dilution test- developed by Kelsey, Sykes and Maurer.
Measured the ability of a disinfectant at appropriate concentrations to kill
successive additions of a bacterial culture
number of viable cells remaining after exposure to the disinfectant at
known concentration for a given time interval
Disinfectant tests require determinations of appropriate cidal levels

Antibacterial disinfectant efficacy tests

The most readily accessible and recent guide to the methodology of

possible bactericidal, tuberculocidal, fungicidal and viricidal disinfectant
efficacy tests, is that of Kampf and colleagues (2002)

Tests for bacteriostatic activity

Disc diffusion
These are really modifications of the earlier cup or ditch-plates
procedures where filter-paper discs impregnated with antimicrobial replace
the antimicrobial-filled cups or cells.
Dilution tests
These usually employ liquid media but can be modified to involve solid
media.
It require a number of controls e.g. sterility control growth control, and
the simultaneous testing of a bacterial strain with known MIC to show
that the dilution series is correct.
E-tests
The most convenient and presently accepted method of determining
bacterial MICs.