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 megaterium (grown on nutrient agar for 2 days and resuspended in liquid medium)
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.
In each class, three of the following disinfectants will be used:
benzalkonium chloride bleach (NaOCl) ethanol (70%)
o-phenyl phenol listerine formaldehyde
How are each of these classified? What is the primary mode of action for each one?
Each lab bench will form a group (some of you can move if needed), and each group will be assigned 1bacterium and three disinfectants. You may divide into 3 subgroups if you wish. 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 large group 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, 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.
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 2 days 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, much like you did in the heat treatment experiment.
For this experiment, you will need two more tubes with cultures of the same bacterium. You will also need a sterile 96 well microplate, a micropipettor set for 390 l and sterile tips, and a nutrient solution (TSB with Thiazolyl Blue added). To prepare for the experiment, pipet 390 l of TSB into each well of any 3 rows in the plate. Divide up the work anyway you like.
To begin the experiment, pipet 10 l of culture into well#1. This is your positive control, cells before any disinfectant treatment. When you are ready to start timing, add 2.8 ml of disinfectant to a culture and immediately mix well AND start timing. Have a 10 l micropipet with a sterile tip ready. At each of the times listed in the chart, remove a 10 l sample and put it in the corresponding well of your row. At the same time using another culture of the same bacterium, use a different disinfectant and a different micropipet and tip, and put samples in a different row. You may divide up the work anyway that seems to work for you.
The sampling regimen is listed in the following table:
well# / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12Time (min) / 0 / 1 / 2 / 5 / 10 / 15 / 20 / 25 / 30 / 40 / 50 / 60
Special care needs to be taken that each sample goes into the correct well. Try to keep the plate covered whenever possible, a difficult task with so many samples being added.
Why are we doing the experiment this way? Examine the next figure. Bacteria subjected to heat or disinfectants under constant conditions die the same way that they grow: exponentially. Theoretically, a single surviving bacterium is enough to multiply and make a culture medium turbid. If there is at least 1 bacterium left alive in that 10 l that you add to the well (about 100 bacteria per ml), growth will occur and the medium will become turbid. Fewer bacteria than that, and the well will remain clear. Thus, we will be looking to see how long a disinfectant treatment is needed for the bacterial population to drop below 100 bacteria/ml. The more effective that concentration of the disinfectant is (and the more sensitive the bacterium is), the sooner the population will drop below that threshold and we will see no growth in the wells.
To try and make it easier to determine whether growth has occurred, we have added a substance called Thiazolyl Blue Tetrazolium to the TSB supporting bacterial growth in the wells. This is a colorless compound that detects metabolic activity. During dehydrogenation reactions, electrons are passed to the tetrazolium dye, reducing it, and changing it from colorless to blue. As long as there are metabolically active bacteria in the well, the dye should turn blue. If no living bacteria are present, there will be no color change. This will make it easier to determine whether growth has occurred in the wells or not. This figure shows examples of some outcomes. The bacteria in row A grow in all the wells and were never killed by the disinfectant. By contrast, they were killed more quickly be another disinfectant in row D.
We will collect the results from this experiment during the following lab period. If it goes well, you will be provided with questions with which to analyze the results and understand the principles involved.