Do a 300 words minimum lab summar.

50

Effectiveness of disinfectants/antiseptics & antibiotics as bacteriacidal &/or bacteriastatic agents:

Kirby Bauer zone of inhibition testing

We use chemical preparations called disinfectants and antiseptics in our everyday lives to kill or inhibit the growth of microorganisms. Chemicals that kill microbes are bacteriacidal. Those that do not kill microbes but keep them from growing are bacteriastatic. Disinfectants are chemicals that are normally applied to non-living surfaces such as benches, carts, tables, etc. They can be bacteriacidal or bacteriastatic. Examples of disinfectants include sodium hypochlorite (bleach), ammonia, and chemicals used in hospitals such as Staphene, Vesphene, and Amphyl. Antiseptics are chemicals that are safe to apply to living tissue. They are less harsh that disinfectants, therefore, they are usually only bacteriastatic. Examples of antiseptics include ethanol, detergents, iodine and H2O2.

Some disinfectants & antiseptics are less effective against some types of microorganisms than others. For instance, oxidizing agents such as bleach and peroxide are less effective against pigmented organisms. Organic chemicals such as phenols are less effective against microbes capable of catabolizing unusual organics, such as species of the genus Pseudomonas. We can easily test the effectiveness of various disinfectants and antiseptics against any culturable microorganism using a simple zone-of-inhibition plate assay. SEE IMAGE “Novobiocin sensitivity for gamma hemolytic Staphylococci: left S. saprophyticus, right S. epidermidis.”

To do this, place 0.1mL (~2 drops) of a broth culture on a plate. Dip a "hockey stick" spreader in EtOH and flame it as instructed. Touch the stick to the agar to cool it. Now, use the stick to spread the culture as evenly as possible over the surface of the plate, leaving no part untouched. While you are at it, spread a second plate for the next experiment. Set the plates on the bench right-side-up for a few minutes until most of the moisture has soaked into the plate.

Performing the test: Decide which disinfectants & antiseptics that you want to test. I suggest using no more than 5. Once the plates are dry, use forceps to pick up a filter paper disc, and touch the disc to one of the disinfectants or antiseptics to saturate the disc. Now touch the saturated disc to the edge of the container to drain-off any excess fluid. Gently lay the disc on the agar surface in the plate, and lightly press down to secure the disc on top of the agar. Repeat for each disinfectant & antiseptic that you wish to test. The discs must be spaced evenly on the surface of the plate and not too close

to the edge. Let the plate sit upright for a few minutes before inverting and placing

in the incubator. Incubate for 24-48hrs. at 37oC. Measure the size of the

zones-of-inhibition, and compare the values for each disinfectant and antiseptic.

These values indicate the “Minimum Inhibitory Concentration” (MIC) of the

compounds tested. How do they compare? Possible problems with this method

include the fact that the zones are not always symmetrical (not perfectly round), so

do your best when measuring. Also, if your disc was excessively saturated, the fluid

will run or spread on the surface of the plate, thus skewing your data.

Antibiotics are chemicals produced by certain species of microorganisms to kill or inhibit the growth of other microorganisms. Why? It is all about competition. Antibiotic-producing microbes include filamentous fungi (such as the genus Penicillium which produces penicillin), species of the bacterial genus Bacillus (producing bacitracin & polymyxin), and most prolifically, species of the filamentous bacterial genus Streptomyces (which produces many compounds including erythromycin, tetracycline and many others). Antibiotics are classified based upon their mechanism-of-action, meaning the way the antibiotic kills or prevents the growth of the microorganism. Mechanisms-of action of antibiotics include preventing cell wall synthesis, membrane synthesis, replication, transcription, translation, folic acid synthesis, etc. For this reason antibiotics can be characterized by their spectrum (or range of effectiveness), meaning which groups of microbes are they particularly effective against. For example, those antibiotics that affect cell wall synthesis are more effective against Gram positive bacteria. Those antibiotics that affect virtually all bacteria fairly equally by virtue of their mode-of-action are referred to as “broad spectrum” antibiotics, as opposed to “narrow spectrum” antibiotics which affect a particular, well-defined group of species. “Moderate spectrum” drugs are somewhere in between. NOTE the table of antibiotics listed on the following page.

Strains of pathogenic bacteria that reside in hospital patients and cause nosocomial (hospital born) infections are notorious for antibiotic resistance due to their constant exposure to antibiotics. Methicillin resistant Staphylococcus aureus (MRSA) is a notable example. These organisms are a major problem, especially for immune suppressed patients. Many hospitals use some type of MIC test on a regular basis to quantitate the degree of resistance of their resident bacterial strains to particular antibiotics, and to monitor changes in this resistance over time. This information is critical when determine minimum-effective-doses of antibiotics for patients.

The Kirby Bauer test is much more precise and quantitative. The Kirby Bauer test is conducted under very consistent conditions in regard to media (Mueller Hinton agar), growth temperature, humidity, pH, time of incubation, etc. This way, the results are reproducible and the results from different testing episodes are comparable. The Kirby Bauer test can be conducted as a zone-of-inhibition plate assay just as we did above, although more involved methods are usually used in medical labs. For the plate assay, spread the organism on the plate and place discs soaked with antibiotic rather than disinfectant on the plate. Incubate and measure any zone of inhibition. Compare the results to determine which antibiotics are effective against which organisms. The effect of a combination of antibiotics can also be tested. As above, a gradient of antibiotic will be present (less and less as you go further from the disc) due to diffusion of antibiotic out of the disc. Some methods allow the technician to accurately measure the concentration of antibiotic at any point in the zone-of-inhibition.

Performing the test: We can use a simple Kirby Bauer zone-of-inhibition plate assay in this lab using TSA agar to differentiate between our Gamma hemolytic Staphylococcus species (S. epidermidis vs S. saprophyticus) based upon their level of susceptibility / resistance to the antibiotic “Novobiocin.” Using the same method as described herein, spread a TSA plate with a TSB broth culture (incubated ~24hrs) of either S. epidermidis or S. saprophyticus. Apply a pre-soaked disc of novobiocin and incubate for 24 – 48hrs. Examine the zone-of-inhibition for diameter and relative clarity. S. epidermidis is more susceptible to this antibiotic than S. saprophyticus. SEE IMAGE 62.

Diameter of zone Relative clarity of zone

S. epidermidis: ~ 2.5cm clear

S. saprophyticus ~ 1 cm cloudy

 Don’t forget the table of antibiotics on the next page! 

* +++/- indicates a narrow or moderate spectrum with particular effectiveness against Gram+ bacteria

+/- indicates a broad spectrum antibiotic

+/--- indicates a narrow or moderate spectrum with particular effectiveness against Gram- bacteria