The first observations taken showed growth only on the first control, which was broth and bacteria with water added. Control #1 featured puntiform and circular colonies across the entire agar surface. Neither the Clorox nor the Envirocide plate had visible colonies. Bacti-Stat had an opaque layer that covered the agar surface, so we could not determine whether or not growth had occurred. Coverage, Lysol, and Enviroquat did not appear to have growth, but upon further examination, we noticed tiny specks in all three plates that could have been either bubbles or potential colonies. Because the presence of too much disinfectant might inhibit growth rather than killing the bacteria completely, we regrew the potential microorganisms on another streak plate. Control #2 had brown particles on the surface of the agar, but these particles clearly were not bacteria colonies. There was an opaque, whitish condensation on the inner surfaces of the petri dish lids of plates that had contained (lie liquid disinfectants, but this was probably not pertinent to the experiment.
New observations, taken three days after first observations (see Appendix A):
The original Bacti-Stat, Clorox, Enviroquat, Envirocide, Lysol, and Coverage plates remained the same; none had bacterial growth. On the other hand, control #1 had undergone a considerable increase in bacterial growth, and the entire dish was literally overrun with circular and puntiform colonies of yellow and white bacteria. There was no bacterial growth was detected on the regrowth plate inoculated from the BactiStat, Coverage, Lysol, and Enviroquat plates, except for two incidents of possible contamination: there was one white circular colony on the very edge of the Bacti-Stat area, and another small white circular colony on the edge of the Enviroquat area. Oddly enough, the Bacti-Stat and Enviroquat areas were across the petri dish from each other, and the contamination occurred on opposite ends of the agar plate. On the second control plate, there was one white circular colony on the peripheral area of the agar, but otherwise, no bacterial growth was present. Contamination can be explained by the various sources of error mentioned in the following discussion.
Discussion.
Under the conditions that were present in this experiment, all cleaners tested were effective. Even though Lysol targeted only "household germs," the bacteria used in our experiment came from the classroom, so there was probably no need for the extra bactericidal action of the other hospital-approved agents. Almost all of the disinfectants definitely contained either ethanol or sodium hypochlorite as their main bactericidal ingredient (see Appendix B for more details), and the soap contained the active ingredient triclosan. Certain concentrations of ethanol will effectively kill bacteria and other pathogens when allowed to work for the proper amount of time [2] (see Appendix C for specific figures); 52.7% ethanol in Enviroquat was the lowest concentration among the disinfectants that used alcohol as an antimicrobial agent, and, as shown in Appendix C, 24 hours would clearly be sufficient time for even Enviroquat to do its job. Hence, it was no surprise that the cleaners containing ethanol were all effective. Sodium hypochlorite also has a bactericidal effect, which can be attributed primarily to its release of hypochlorous acid, HOCI; however, it will disintegrate over time, and its effectiveness is influenced by factors such as pH and temperature 121. Finally, triclosan is commonly used in antibacterial soaps: besides being the active ingredient found in Bacti-Stat, it is also present in certain commercial brands of antibacterial soap, like Jergens and Softsoap.
The results we obtained seemed to disprove our hypothesis that not all disinfectants are created equal. However, our conclusion was not a definite one. For one, the antibacterial soap and disinfectants used in the experiment are generally lathered on the hands and rinsed off quickly, or sprayed on to a surface and wiped off almost immediately. Already, our experiment differed from normal circumstances in two ways: method of application, and duration of time for cleaner to work. First of all, we added the disinfectant to the bacteria's food source, the broth, so the bacteria were essentially ingesting the disinfectant. Normally, the cleaner is applied to a surface on which bacteria arc dwelling, but the bacteria arc not fed the disinfectant, as in our experiment. Second of all, we allowed the cleaners to work for 24 hours. Even assuming that they all remained potent for the entire time, this would stray from normal conditions, in which the cleaner is applied and then almost immediately removed. (10% bleach is allowed 15 minutes to do its job.) Moreover, bleach disintegrates fairly rapidly after usage, breaking down almost entirely to salt water. Thus, if the other disinfectants remained potent, they might have had an advantage over the bleach because they had 24 hours to work, while bleach had only the short time before it broke down. Of course, the disinfectants are probably designed to kill bacteria almost immediately (otherwise, people would be instructed to let the cleaner sit on the surface for a period of time before wiping it off). However, because we are testing the disinfectants and their claims, we cannot make the assumption that it is 100% effective immediately, upon application.
Therefore, a possible variation of this experiment would be to spray the disinfectants on, like they were meant to be applied. Several bacterial lawns would be grown, and disinfectants sprayed directly on to the bacteria. As in real life, the cleaners would be given only a few minutes to take effect. The bacteria from those plate would then be transferred aseptically and streaked on to new sterile agar plates, and allowed to regrow, away from the presence of excess disinfectant (as in the original plate). The results might differ if the bacteria are not directly fed the disinfectants: perhaps only the top layer of bacteria will be killed, for example, and the bacteria underneath will not be harmed by the disinfectant. Another possibility would be to place a disc soaked in disinfectant in the center of the bacterial lawn, and look for a zone of inhibition [3]. These methods would both avoid the rather awkward necessity of pipetting solutions containing bacteria (either alive or dead) on to agar plates, it was a bit difficult to pipet its rapidly as we ideally should have, so we risked contaminating our-"sterile" plates while the lid was lifted and we were attempting to eject the liquid in the pipet.
Another variation on our experiment would be to test bleach under different temperature and pH conditions to see how those factors affect the bleach's effectiveness. Also, we could observe what happens if (lie bleach is more, or less, concentrated than 10%, and whether or not the time needed changes with concentration, pH, or temperature.
If we were merely repeating our experiment, there would still be several necessary changes. We made an error in not filtering the bouillon broth after it cooled and before it was autoclaved, because the broth had residue and chunks of fat floating in it that inhibited our observations of whether or not bacteria was present in the solution after we had inoculated and incubated it. Nutrient broth, being clearer, might be a better choice of broth; however, bouillon broth should be fine if it is properly filtered.
We also incubated our bacteria at different temperatures throughout the experiment: at first, the bacteria was incubated at 37' C, which probably encouraged the bacteria found on skin more than the bacteria found on inanimate surfaces because it is closer to body temperature than to room temperature. Later, when regrowing the bacteria front our culture tubes, we incubated the plates at 320 C because 37' C had melted our agar. However, this might have encouraged the other kind of bacteria (from the environment, rather than from the body) to grow. Obviously, this does not make much sense, because we encouraged the growth of one kind of bacteria. tested that bacteria, and then regrew it in conditions suitable for another kind of bacteria. It is possible that we saw two types of bacteria on control #1 (yellow bacteria and white bacteria) because we had two types growing: skin bacteria and bacteria from the environment, Hence, incubation temperature should be kept constant (32' C might be the safer option because, among other reasons, it did not harm the agar).
We chose to dilute the disinfectants because we wanted to see how effectively bleach would work at 5% strength. Therefore, the disinfectants were all twice as dilute as usual. We initially thought that all of our cleaners were successful in killing the bacteria because we had too high a ratio of disinfectant to bacteria present. However, upon further thought, we realized that perhaps concentration might be more important to the effectiveness of the cleaners than the ratio of bacteria to disinfectant. If so, our results would seem to indicate that at half-strength, the cleaners were already cffective, so full-strength would be, if anything, even better. It would be interesting to perform a follow-up experiment to determine whether it is the ratio of disinfectant to bacteria that matters, or if it the concentration of disinfectant is the only important factor. To do this, we would vary the amount of bacteria in several culture tubes, but add in the same amount of a single disinfectant. For example, 1 mL of broth and bacteria, 2 mL sterile broth, and 1 mL disinfectant might be added to one culture tube, then. 3 mL broth and bacteria and 1 mL disinfectant might be added to another tube, and the two results compared.
The antibacterial soap was included in our variables because we wanted to test if there was a difference between the effects of disinfectants and the effects of soaps. However, the soap was considerably thicker in consistency than the other variables. and therefore, was difficult to pipet and to transfer. Moreover. it formed a layer on the agar plate that obscured any possible bacterial growth, so we had to transfer it to another streak plate and regrow it a third time. Diluting it would have still been a problem, because the other disinfectants were half as concentrated as usual, but the soap would have been even less concentrated.
Another question that occurred to us after performing the experiment was that if certain concentrations of ethanol are needed in order to kill bacteria, why was it that the cleaners still worked at half their normal concentrations? We thought that perhaps the ratio of bacteria to cleaner had some influence, and that maybe we had put in much more cleaner per bacterium than we normally might have. Again, we would need to perform the aforementioned alternate experiment that attempts to determine whether it is the concentration of the disinfectant itself. or the ratio of bacteria to disinfectant, that determines the results.
An interesting experiment would be to see whether the bacteria develop an immunity to the disinfectants being used on them. This would be important based solely on its applications in everyday life: maybe the disinfectants used in households and even in hospitals stop working after a certain period of time, and we do not realize it. To do this experiment, we would probably have to add very small, dilute amounts of cleaner to the bacteria, perhaps killing some but letting some remain alive. These bacteria would be regrown, and then disinfectant would be added once again. Eventually, perhaps the bacteria would become invulnerable to the disinfectant.
Finally, further experimentation can be done to test the effectiveness of other disinfectants. Possible antibacterial agents to look at include peroxides, iodine, silver compounds, and phenolic compounds [2].
Different methods of applying the disinfectant to the bacteria can be experimented with, including spraying it on a bacterial lawn and adding it to the bacterial food source.
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Sterilization: The complete destruction of all forms of microbial life-including cells, vaible spores, and viruses is called Sterilization. This can be achieved by heat, autoclaving, gas (ethylene oxide), various chemicals (such as formaldehyde) and certain levels of radiation with UV or gamma rays.
Disinfection: Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are called disinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.
Sanitization reduces microbial populations to levels considered safe by public health standards. Restaurant glassware, china, and tableware are subject to sanitization to minimize chances of disease transmission from one user to another. This is usually accomplished by high temprature washing followed by a dip in a mild chemical disinfectant.
Microbicidal Agent: is one that kills bacteria, but not endospores of bacteria. A disnfectant that kills fungi is called fungicide and one that kills viruses is called virucide. Germicide is a general term used to refer any agent that destroys any agent that destryos germs or harmful microorganisms.
Microbistatic Agents: is a drug or chemical that inhibits growth and reproduction of microorganisms. A bacteristatic agent is one that inhibits the metabolism and reproduction of bacteria. Microbiostatic agents and processes include desiccation(drying), freezing, concentrated sugar and salt solutions and some microbicidal agents including some antibiotics.
Asepsis: The term sepsis refers to growth of infectious microbes on living tissues or in sewage tanks. Aseptic means that an object or an area is free of pathogens. Thus aseptic techniques are designed to eliminate and exclude all infectious microbes by sterilization of equipment, disinfection of the environment, and cleansing of body tissues with antiseptic. Antiseptic technique was first developed by Lister 1867. He use dilute carbolic acid to clean surgical wounds and equipment.