Biology 250--Bacteriology
Disinfection and Antisepsis (Exercise 24--Handout)

Chemical agents that can be used as disinfectants and antiseptics generally cannot be relied on for sterilization, which implies that all living cells are killed. One chemical agent that is used for sterilization is ethylene oxide, which is used to sterilize plastic-ware in a special chamber that controls temperature and humidity. Generally speaking, a chemical used as a disinfectant can be applied to nonliving materials, and will act on infectious agents (microorganisms). Frequently disinfectants are bactericidal, but not necessarily. Chemicals used as antiseptics can be applied to living surfaces to act on infectious agents (microorganisms), and are often bacteriostatic.

Disinfectants and antiseptics are most useful when a low number of microorganisms are present, and on surfaces that are clean and free of contaminating material, especially protein-containing material. As a general rule, gram positive organisms are often more sensitive than gram negative ones. Mycobacteria, pseudomonads, and spore-formers are less affected by disinfectants than other kinds of microorganisms. Organisms in active, or exponential, growth are most sensitive. Many disinfectants are more effective at warm temperatures than at cold ones. Many disinfectants and antiseptics are inhibited by the presence of organic matter.

The targets of disinfectants and antiseptics are most frequently

  1. the cytoplasmic membrane, in which lipids and proteins are denatured, leading to changes in membrane permeability, and
  2. nucleic acids and proteins, which are denatured as the hydrogen and covalent bonds that stabilize their structure are broken.

This leads to interference with nucleic acid replication and protein synthesis, with consequent damage to enzyme-mediated cell processes.

An ideal disinfectant would have all of the following properties, but of course there is none: rapid action, effective against all microorganisms, penetrating, water-soluble, not inhibited by organic matter, slow to decompose, not damaging to materials, harmless to skin, nontoxic internally, cheap, safe, pleasant to use.

Major groups of disinfectants and antiseptics include the following:

  1. formaldehyde (usually used at 8%, bactericidal, toxic, not used as an antiseptic, better in combination with 70% ethanol, glutaraldehyde less toxic) (available in lab: 8% formaldehyde)
  2. halogens (I- and Cl-containing compounds) (bactericidal or bacteriostatic depending on concentration, used in both antiseptics and disinfectants, inhibited by organic matter, alcohol enhances ability of I to get into cells) (available in lab: 5% chlorine bleach, 0.5% iodine, Betadine, Weladine)
  3. phenolics (chemicals related to phenol, 5-10% phenol bactericidal for most species including mycobacteria, and effective against some viruses, 1% hexachlorophene is bacteriostatic) (available in lab: 5% and 10% phenol)
  4. ethanol (70%, EtOH) or isopropanol (70-90%) (bactericidal for many g+ and g- vegetative bacteria and fungicidal for some fungi, useless for spores and many viruses, enhances activity of other chemicals) (available in lab: 70% ethanol)
  5. heavy metal ions (0.1-0.2% mercury, bacteriostatic for g+) (1% silver nitrate) (also tin, zinc, arsenic, copper) (available in lab: 10% ferric chloride, 0.1 M mercuric chloride, 0.2 M silver nitrate)
  6. quaternary ammonium compounds (organic salts of ammonium chloride, bactericidal for most bacteria except pseudomonads, spores and mycobacteria, inactivated by soaps, detergents and some organic materials) (available in lab: Roccal, Septisol, and Lysol may contain quaternary ammonium compounds, among other agents)
  7. hydrogen peroxide (1.5-3%, catalase neg. g+ only) (available in lab: 3% hydrogen peroxide)

Procedure for Exercise 24: Disinfectants

This procedure replaces exercise 24 in your lab manual. Students will test several disinfectants and antiseptics in a disk diffusion test for their ability to inhibit the growth of various organisms.

Equipment:
(per group) forceps, beaker of alcohol, paper towels, sterile filter paper disks (1 glass petri dish of about 20 per group), sterile swabs (7 per group)
Cultures:
Each group should use one of the following cultures: Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans (or similar species)
Media:
(per group) 7 nutrient agar plates
Reagents:
the ones in the other handout, plus whatever commercial mouthwashes or antiseptics you or the students bring in (it would be especially helpful to have some of the new hand sanitizer stuff....)

Procedure:

  1. Choose one culture to be used by all members of the group.
  2. Each member of the group should prepare 1-2 plates to be used to test 4 reagents each.
  3. Label the plates with name and date. Divide the plate into quadrants and label each quadrant with the reagent to be placed in that quadrant.
  4. To prepare a plate, first swab it in three directions with a sterile swab dipped in the broth culture and drained against the side of the tube. This should produce a continuous lawn of growth after incubation.
  5. Pick up a sterile filter paper disk with sterile forceps (dipped in alcohol and flamed).
  6. Dip the disk in the reagent to be tested, drain it slightly on a paper towel, and place it in the center of the appropriate quadrant of the labeled plate.
  7. Repeat steps 5-6 for each reagent.
  8. Incubate all plates inverted at 37o C for 24-48 hours.
  9. Measure zones of inhibition around disks for each reagent and record results in a table. Class results for all groups will be collected at the next lab period, copied and returned during lecture if necessary.