8.1: Introduction

Learning Objectives

• Learn about disinfectants and the various factors that need to be considered when choosing a disinfectant.
• Set up an experiment to determine the effectiveness of five different common disinfectants.
• Learn how to determine antibiotic susceptibility using the Kirby-Bauer method.
• Set up an experiment to test for antibiotic production by three different strains of Streptomyces.

The use of chemicals to control microbial growth dates back at least as far as the 1800’s. Tincture of iodine was used as antiseptic during the Civil War, and Joseph Lister established the practice of aseptic surgery using a disinfectant known as carbolic acid (phenol) in the 1860’s. Since that time, many types of disinfectants (agents that are used to eliminate or kill vegetative cells on surfaces) and antiseptics (agents that are used to eliminate or reduce vegetative cells on living tissue) have been used. Although disinfectants and antiseptics may be effective at killing vegetative cells, they do not usually achieve sterilization.

Various factors need to be considered when choosing a disinfectant or antiseptic. It is very important to know which microbes are present to determine what type of disinfectant would work best. It is also important to realize that the effectiveness of a particular disinfectant may be affected by pH, temperature, concentration, and exposure time. Ideal disinfectants should be effective against the particular contaminants present, usable at a low concentration, require a relatively short exposure time, and have a long shelf life. It should also be water soluble, non-toxic to humans and animals, and cost-effective.

The efficacy of a disinfectant or antiseptic can be tested in several ways. One way is to inoculate an agar plate with a lawn of bacteria and add filter paper disks that have been moistened with the disinfectant being tested. This is known as the filter paper disk method, or agar disk diffusion assay. After incubation, plates are observed for the presence of a zone of inhibition (area around a disk where no microbial growth is detected). Generally speaking, the larger the zone, the more effective the disinfectant is against that particular microbe. However, other factors such as the solubility of the test agent and the molecular weight of the disinfectant molecules (which determines the diffusion rate of the disinfectant through the agar) can also affect results.

Figure 8.1.1: Filter paper disk method experiment showing zones of inhibition

The use of antimicrobial agents to treat infections began in the early 1900’s, when Paul Ehrlich developed Salvarsan to treat individuals infected with Treponema pallidum, the spirochete that causes syphilis. In 1928, Alexander Fleming later observed that the Penicillium mold growing on his agar plates could inhibit the growth of bacteria: years later penicillin was purified and used to treat many types of infections. Since this time, many other antimicrobial agents have been used to treat a wide variety of bacterial infections. Antibiotic producers include many types of fungi (Penicillium, Cephalosporium) and bacteria (Bacillus, Streptomyces). In addition, many antimicrobial agents currently used to treat infections are either synthetic (made in a laboratory) or semisynthetic (a modification of a naturally-produced antibiotic). Today there are over 100 different antimicrobials that are used to treat infectious diseases. These include broad-spectrum and narrow-spectrum antimicrobial drugs (see chart below). Narrow-spectrum drugs are more desirable to use whenever possible because they target the pathogen more specifically and do less damage to the normal microbiota; broad-spectrum drugs are used when the cause of the infection is unknown or when other antibiotics are not effective.

Activity spectra of the major classes of antibiotics

	Mycobacteria	Gram Negative	Gram Positive	Chlamydiae	Rickettsiae
Tetracyclines		✓	✓	✓	✓
Streptomycin	✓	✓
Penicillin		✓	✓	✓
Sulfonamides, Quinolones, Cephalosporins		✓	✓
Vancomycin			✓
Polymyxin		✓
Isoniazid	✓

It is important to remember that not all antibiotics are effective at killing all types of bacteria. Bacteria may have intrinsic resistance to a particular antibiotic. For example, gram negative bacteria are intrinsically resistant to vancomycin because the drug cannot penetrate the outer membrane of the gram negative cell wall. Also, the misuse and overuse of antibiotics has led to the evolution of resistance among bacteria by selecting for individual cells within a population that are not affected by the drug. This acquired resistance can occur in several ways, including through transformation, conjugation and mutation. Antibiotic-resistant bacteria have become a major problem of growing concern in health care, as it is often difficult (or impossible) to treat bacterial infections caused by these microbes (for example, multidrug-resistant Staphylococcus aureus, or MRSA). Therefore clinical isolates are often tested for their antibiotic susceptibility in a laboratory setting so that health care providers can choose an appropriate drug to treat a particular infection.

There are several ways to determine antibiotic susceptibility in a laboratory setting—one common test is called the Kirby-Bauer method. This method is similar to the filter paper disk method used to test disinfectants, except that it uses filter paper disks impregnated with a known concentration of an antimicrobial compound. It also uses Mueller-Hinton agar, and is often performed with larger (150 mm) petri dishes that allow for the testing of several antibiotics simultaneously. When performing the Kirby-Bauer method, it is important to measure the size of the zones of inhibition and compare them to a set of standardized values established by the Clinical Laboratory Standards Institute (CLSI).

Antibiotic Production by Soil Bacteria

Streptomyces is a bacterial genus of the order Actinomycetales. These Gram-positive spore-formers closely resemble fungi because of their branched filamentous structure. They are commonly found in soils and are primarily saprophytic, which means that they feed off of decaying matter. Streptomyces are characterized by having complex and abundant gene clusters that code for bioactive secondary metabolites. From a human health perspective they are very valuable, as they produce 2/3 of all the antibiotics of natural origin. Secondary metabolites from Streptomyces have antifungal, antiviral, antitumor, and immunosuppressant activities.

In this lab you will set up experiments to evaluate the effectiveness of several disinfectants and antibiotics. In addition, you will test three strains of Streptomyces for their ability to produce antimicrobial compounds, and determine the effect of these compounds on different types of bacteria.

Key Terms

disinfectant, antiseptic, filter paper disk method (agar disk diffusion assay), zone of inhibition, Kirby-Bauer method, broad-spectrum antimicrobial drugs, narrow-spectrum antimicrobial drugs, susceptibility, resistance, intrinsic resistance, acquired resistance, Streptomyces, saprophytic, secondary metabolites