Bacteriostatic vs Bactericidal: The Effect of Disinfectants on Bacteria
Introduction
Many chemicals have a harmful effect on bacteria. By inactivating proteins that then must be replaced or by poking holes in cell membranes and allowing substances to leak out (or in), chemicals can slow the growth of bacterial cells, or in high enough concentrations, kill them. The terms bacteriostatic and bactericidal, respectively, describe these chemicals, and these can be used as disinfectants or antiseptics. Although many chemicals can be either bacteriostatic or bactericidal, depending on their concentration, we can manipulate the experimental setting to observe either type of reaction.
Not all microbes are affected equally by the same chemicals. Viruses with a lipid envelope around them are inactivated by disinfectants that dissolve lipids, whereas other viruses are not. Bacteria that have converted to endospores are highly resistant to many chemicals, and bacteria that have waxes in their cell walls are more difficult to kill as well. Gram negative bacteria have an outer membrane that prevents access of some chemicals to critical molecules within the cell.
In this set of experiments, the following questions will be addressed:
1. How resistant are various bacteria to disinfectants?
2. How and why might brief exposure to disinfectants differ from growth in the presence of disinfectant?
The bacteria to be used in this experiment are as follows:
Bacillus thuringiensis (grown on nutrient agar at 37 ºC and resuspended in liquid medium)
Bacillus thuringiensis (grown in TSB)
Staphylococcus aureus
Pseudomonas aeruginosa
These bacteria have been selected because of their differences. What are they?
Each culture will be adjusted to the same Optical Density reading so that the starting concentrations of bacteria are similar.
Three disinfectants will be used:
bleach (NaOCl), Amphyl (o-phenyl phenol), and generic “Listerine”
How are each of these classified? What is the primary mode of action for each one?
Students will work in pairs with each pair be assigned 1bacterium and all three disinfectants. You will be conducting two different kinds of experiments. In one experiment, you will test your 3 disinfectants on your bacterium to determine how bacteriostatic the disinfectants are. In a second experiment, you will use your bacterium and test how bactericidal the disinfectants are.
Procedure
Testing the bacteriostasic effect.
Each pair will need a TSA plate, a cotton swab, and a culture of the bacterium to be tested. First, make a lawn on a TSA plate by dipping a sterile cotton swab once into the culture, squeezing out the excess culture against the inside of the tube, then swabbing the entire surface of the agar plate, turning the plate several times while swabbing from rim to rim. Upon incubation, an even growth of bacteria would cover the entire agar surface. Keep this bacterial culture, because you will need it for the experiment described later. Next, using sterile forceps, pick up a small, sterile filter disk and dip it halfway into a one of the 3 disinfectant solutions. In this way, disinfectant will wick up the disk and dampen it all. If you submerge the entire disk it will be too wet. Place the disk on your lawn about 2 cm from the edge of the plate. Repeat this with two more disinfectants, evenly spacing the 3 disks in a triangle around the plate. Be sure you label your plate in some way so you will remember which disk is which.
As the bacteria grow to form the lawn, some may fail to grow where the disinfectant concentration is high, and zones of inhibition will be produced. Notice that you are testing the ability of your bacterium to grow in the presence of different concentrations (the gradient around the disk) of each disinfectant. You will measure the size of the zones of inhibition on your plate after a day of incubation.
Measuring the bactericidal effect.
For the second experiment, you will use the same bacterium and 3 disinfectants, but the procedure will be more involved. In this experiment, you will test the killing ability of the disinfectant, and thus the ability of the bacterium to survive treatment with the disinfectant. The bacteria will not be growing in the presence of the disinfectant. This is a timed experiment in which you will combine bacteria with disinfectant and take periodic samples to test for survival. For this experiment, you will need two more tubes with cultures of the same bacterium, 15 cotton swabs, and 3 agar plates. Each plate should be divided into 5 pie shaped wedges by drawing on the bottom of the plate. The sections should be labeled 0, 0.5, 1, 2, 5 minutes. Each plate will be used to test a different disinfectant.
To begin the experiment, take a control sample. Insert a sterile swab into a culture, squeeze the swab against the inside wall of the tube to remove excess culture, and inoculate the zero time section of a plate, making sure to stay inside the lines you have drawn. Take the first disinfectant and pipet out 2.5 ml and add it to the culture tube. Immediately mix it thoroughly and start timing. Your disinfectants are 1:5 dilutions of a concentrate, and there is 2.5 ml of culture in your tube, so the final concentration of disinfectant at the start of the experiment is 1:10. Thirty seconds after adding the disinfectant, use a swab to sample as before and apply the culture to the 0.5 min section of the plate, staying within the lines. Repeat for the 1, 2, and 5 minute samples. Any disinfectant present in your samples will diffuse throughout the 25 ml of agar and thus be diluted to harmless levels.
Repeat this process with the other two cultures and the other two disinfectants on the other two plates. After incubating these plates for a day, we will score the relative growth in each section on the plate to determine how well the bacteria survived the short term disinfectant treatment. Do you think the bacteria being tested will show the same types of results in the disk test and in the tube test?