The Real Truth About Bathroom Bacteria!

This topic submitted by Chris Jones, James Diewald, Paul Georgeadis, Michelle Cisar (cubbiesseven@yahoo.com) at 1:59 AM on 12/10/02. Additions were last made on Wednesday, December 10, 2008. Section: Cummins

Natural Systems 1 Fall, 2002 -Western Program-Miami University

Bathroom Bacteria




Introduction:


As Western campus students, what are potential bacterial risks we may come in contact with on a daily basis, and how can we minimize these risks.

The purpose of this experiment is to collect and identify various forms of bacteria found on Western campus.

The bathrooms of Peabody are suspected to have the highest concentration of bacteria as well as the widest variety due to that fact that it has the largest number of students and faculty living and working there. It is believed that Mary Lyon holds the least bacteria because it is the smallest Western dorm, thus there are less people to transmit bacteria.

Bacteria we expect to find include E-Coli, Staphylococcus Aureus, Streptococcus, Campylobacter, and Salmonella. We also expect standard household cleaners such as Lysol to be sufficient disinfectants.

Bacteria can be found everywhere. Though most people prefer to ignore this fact, we would like you to be aware of its presence and the possible effects that go along with them. The truth is that most bacteria are relatively harmless. However, there are exceptions; many of the harmful bacteria can cause death. This is why the presence of bacteria around us should be made known, so that we can protect ourselves and others.

Bacteria can be clearly defined as: Microorganisms that lack a nucleus and have a cell wall composed of peptidoglycan, a protein-sugar molecule. They are the most common organisms on Earth and are intimately connected to the lives of all organisms. (Bacteria, Encarta Online) Diagram 1 shows the basic structure of a typical bacterium cell. We all have been taught the basics about bacteria in lower level sciences, so the point to focus on in our experiment is that they are found everywhere.

The basic needs of bacteria are closely related to higher forms of life. They require an energy source, a carbon soure, and a nitrogen source in order to fill their nutritional needs, the majority of bacteria are tolerant to the presence or absence of free oxygen in their environment, though few are not. Temperature and moisture levels also play a huge role in the survival of bacteria. For the kinds of bacteria we will study (bacteria parasitic to man and warm-blooded animals) the natural growing environment is between 25 and 40 degrees C. Since over 80% by weight of a bacterial cell consists of water, moisture is essential for growth. However, some species (ie. Staphylococci) are able to survive weeks or months in dry areas. Finally, bacteria grow best in darkness because ultra violet rays are lethal regardless of whether they are natural or artificial.

We should not be hasty to say bacteria are all bad. Bacteria are important parts of the lives of many organisms. For example, probiotics have recently been used in childhood gastrointestinal disorders for effective prevention and treatment of such conditions as diarrhea, irritable bowel syndrome, and inflammatory disorders. These “good” bacteria can displace potential pathogens in the intestines, deter growth of negative bacteria, lower the pH in the colon, and stimulate immune antibodies against viruses. (Journal of Pediatric Gastroenterology and Nutrition. 1998;27:323-332)

Sanitary conditions in public places have always been a major problem, especially bathrooms! Health departments are continually checking the cleanliness and safety of these bacteria breeding grounds to prevent the spread of sickness and disease. In a research experiment conducted by J. Barker and S.F. Bloomfield of the Pharmaceutical Science Institute, it was found that in four out of six bathrooms tested, Salmonella bacteria existed in close proximity to toilets. This experiment was pursued after a report of an attack of salmonellosis in the home of one of the test bathrooms. In recent studies done in England, it was found that infectious intestinal diseases, such as the Salmonella found in Barker and Bloomfield’s research, occurs in one our of five people each year. (Wheeler et al. 1999) Also, in reports done by the Communicable Disease Surveillance Center, it was calculated that 136 unreported cases in the community causing considerable morbidity. On a more local level, a group of natural systems students researched the bacteria of Peabody Hall bathrooms in 2001. These young scientists took samples from boys and girls bathrooms and compared which had a greater amount of bacteria. We will be using similar techniques and our ideas are similar, but we will further their studies by identifying types of bacteria we sample and by covering a broader area.

Our research project can relate some larger questions also pertaining to the world outside Miami. Are public bathrooms clean enough? How do we know that disinfectants really work? How do we know Western Campus’ cleaning staff does a thorough job of ridding our bathrooms of harmful bacteria? Hopefully, our research can help answer some of these questions or at least spark someone else to attempt to answer them.


Abstract:


As a continuation of our experiment and as a means of statistical analysis we have obtained 16 additional agar plates, which we will use to culture and observe bacteria before and after cleaning times. Through this data we will be able to draw conclusions in the effectiveness of the janitorial staff of Peabody Hall, bacteria hotspots in our bathrooms, and safety issues.

We expect the door handle and the shower in Peabody’s 3 south bathroom to be the highest in bacteria contact due to frequency of contact and moisture conditions in which bacteria thrive. Furthermore, the sink will probably have the lowest bacteria levels because people using the sink often use antibacterial agents while washing their hands and these agents may come to coat various areas of the sink.


Procedures and Materials:


In order to conduct our experiment we again contacted the microbiology department and were able to obtain 16 agar plates. We also used cotton swabs, a peminant marker to mark the agar dishes, and tape to seal the agar after cleaning.

In terms of procedures, we took two sets of samples on each of two days. One of these sample sets was taken before cleaning and another directly after. Each sample set contained on sample from each of the following places;

Two sample points will also be selected; one of these being the outgoing door handle and the other will be randomly selected by use of a number generator from the following list.


1) The Toilet Flush Handle, Seat, and Outgoing Handle
2) The Outgoing Door Handle of the Bathroom
3) The Shower Floor
4) The Sink Handle and Basin.

The plating method we custom designed to be identical for each plate. It consisted of on single stroke around the edge of the agar and an X drawn across it. (See Pictures) These samples were then sealed and placed in a warm, dark place for three days to grow. At the end of the growth period the samples were removed and observed.

Visual descriptions, total percent coverage, and percent coverage by species were noted. To calculate the percent coverage for both species and total coverage, we took digital photos at a set distance and inserted them into Photoshop. Using the grid feature in the program we were able to zoom into the plates and calculate an accurate percentage.

Time Line:



-Sept. 30, 2002: Gather Materials
-Oct. 2, 2002: First Collection Date
-Oct. 6, 2002: Second Collection Date
-Oct. 7-14, 2002: Observation/Recording Days
-Oct. 15-16, 2002: Disposal and Testing
-Oct. 22-25, 2002: Data Analysis
-Oct. 30-Nov. 9, 2002: Backup Procedure Repeat Dates


-Sept. 30, 2002: On this date we plan to gather the following materials:


Materials:
-Latex Gloves
-Recording Instruments
-Masking Tape
-Cellophane Tape
-Q-Tips
-Petri Dishes (20)
-Agara gel culture medium based on a seaweed extract, widely used for growing microorganisms in laboratories
-Microscope
-Slides
-Cover Slips
-Methyl-violet Stain
-Iodine solution (2 g. iodine, 10 ml. sodium hydroxide, 90 ml. Distilled Water)
-Acetone
-fuchsin stain
-Sterile Loop
-Bunsen Burner

The majority of this equipment will be available to us from Dr. Anne Morrishooke, the Chair of Microbiology at Miami University. The research group will purchase remaining Supplies.

-Oct. 2, 2002: Due to cleaning schedules, we have selected 12:00pm on Wed. Oct. 2nd to be a time where bacteria levels are the lowest. At this time we will collect samples by the process described below.


The Bathroom Table below assigns a number to each bathroom on Western campus that receives moderate traffic. (If there are two bathrooms on a floor, bathroom A represents north/west and B represents south/east) Using a random number generator, 10 of these 15 will be selected for sampling.

See Table 1


The first sample point will be swabbed with one side of the Q-Tip and the second with the other side. Both samples will then be applied to opposite sides of a Petri Dish by a standard plating method illustrated below. The Petri Dish will than be placed in an incubator until it is ready for analysis.

-Oct. 6, 2002: Due to cleaning schedules, we have selected 6:00pm on Sun. Oct. 6th to be a time where bacteria levels are the highest. We will follow the same collection process as above.

-Oct. 7-14, 2002: On or about the date listed we intend to analyze the bacteria that have accumulated on our Petri dishes. We will take a digital photo of each dish and describe its physical characteristics in the table below. We will then utilize a microscope and Gram's Staining Method to identify the bacteria. (See Gram's Staining Method Below) Drawings of the bacteria may also be included.


Gram's Staining Method

Staining is essential to identify bacteria because of their clear protoplasm, which is difficult to see without it. Before staining, one must fix a sample to a slide using a sample taken from the culture. We will use a sterilized loop to extract a sample from our cultures. The loop will then be spread over the surface of a new slide. The loop will then be resterilized and the slide carefully dried. We will then fix the slide by quickly passing it through the flame of a Bunsen burner three times. The stain (Methyl-violet) will then be applied to the slide with a dropper and after five minutes the stain will be washed off with an iodine solution. We will allow the iodine to act for two minutes and drain off the excess iodine with acetone for no more than five seconds. The slide will then be immediately rinsed with water and a fuchian stain will be applied. Finally the slide will be washed with water and blotted dry.

-Oct. 15-16, 2002: After completing our observations, we will use Lysol disinfectant spray on our dishes and record its effects from a visual standpoint only and an approximate effectiveness will be established.

-Oct. 22-25, 2002: Data Analysis

-Oct. 30-Nov. 9, 2002: These dates are available for a repeat of testing if our results are not adequate.


Results:



This graph shows a basic overview of our experiment results. Here, we can see the differences in percent coverage before and after cleaning, at each individual location in the bathroom. The toilet is relatively clean when comparing it to the other locations in the bathroom. We can see that cleaning did not effect the toilet and shower nearly as much as it did the sink and door.

Before Cleaning vs. After Cleaning


From this Stat View bar graph we can see the effects of cleaning the bathroom sink and door. Though the P-value is not great enough to show a significant difference between the two, we can see that there is still a difference. Before cleaning, the percent coverage by species was around 10-13%. After cleaning on both days, the percent coverage by species went down to nearly 0%! If we had more time to conduct our experiment, we could have swabbed more Petri dishes and therefore had more data numbers to allow the P- value to rise.

Cleaning Effectiveness on a Whole


From this Stat View graph we can see the overall effects of cleaning bathrooms by looking at before and after percent coverage of all our Petri dishes. Before cleaning the percent coverage of all the Petri dishes was about 10%. After cleaning, the percent coverage of all the Petri dishes was about 5%. The bacteria in the bathroom were eliminated by about 50% overall. So, cleaning is effective to a point, but not as effective as we like to think it is. We would have liked to see percent coverage of bacteria to be reduced to 0% coverage.

Cleaning Effectiveness By Location


From this Stat View graph we can see the change in percent coverage by species for each location in the bathroom. The toilet had very little percent coverage of bacteria before cleaning, but it barely changed after it was cleaned. The shower, as we can see, had a very high percent coverage of bacteria before cleaning, and also, did not really change after being cleaned. We can determine that the cleaning techniques used on the toilet and shower are not effective since there was hardly any change in percent coverage before and after cleaning. On the other hand, we can see the cleaning techniques used on the sink and door are effective since there is a great change in percent coverage of bacteria before and after cleaning.

Bacteria Levels by Location


Here, we can view the average percent coverage of before and after cleaning by specific bathroom locations. We can determine that the main “home” of bacteria at any given time is in the shower. The warm, moist location is the main breeding ground for bacteria in the bathroom. We can also see that, interestingly enough, the toilet lacks bacteria by percent coverage more than any other location in the bathroom we tested. The sink is found to have a slightly higher percent coverage of bacteria than the door. This is probably due to the moisture and “dirty” hands that come in contact with the sink. The door doesn’t receive as much because most people wash their hands before touching the handle, though, when someone does not, their bacteria spreads to those who touch the door with clean hands.

Bacteria Levels by Location (continued)


We can see the individual location results of cleaning very well shown in this Stat View graph. The toilet shows little change in percent coverage as well as the shower. The sink and door show great changes between their before and after percent coverage of bacteria. Again, we can determine that the cleaning techniques of the toilet and shower are not nearly as effective as the techniques used on the sink and door in dealing with percent coverage of bacteria.

Bacteria Species levels found Before and After Cleaning


This Stat View graph shows the differences in percent coverage by species before and after cleaning. It does not take into consideration the location in the bathroom. We can determine how effective cleaning was just looking at the species of bacteria.
Staphylococcus was reduces from about 10% to 7.5% coverage. Salmonella had little change at all; it stayed at about 5% coverage. Campylobactor went from a whopping 22% coverage before cleaning, to about 3% coverage after cleaning. Streptococcus changed from 12% to 8% coverage after cleaning. And E-coli actually increased from 1% to 2% after being cleaned. From this, we can gather that Peabody’s bathroom cleaning methods are effective on Staphylococcus, Campylobactor, and Streptococcus, however, it did not effect Salmonella at all, and increased the percent coverage of E-coli, though only by less than 1%.

Species Abundance as a Whole


This Stat View graph shows us the change each day in percent coverage of each individual species. Right away, we can see that the levels of E-coli are so small that they don’t even appear on the graph, yet we know they are still there. We can see that Staphylococcus and Salmonella levels are similar, in that before cleaning the first day percent coverage is lower than on the second day before cleaning. Also, we can see for these that percent coverage after cleaning the first day reduced, but did even more so after the second day of cleaning. Campylobactor was extremely high, as well as Streptococcus, before cleaning on the first day, but was greatly reduced, even through the second day, after cleaning on the first day. Overall, we can see that cleaning did reduce the percent coverage of each individual species of bacteria.

In our experiment, after letting the Petri dish cultures mature, we set out to identify the different bacteria types. It was important for us to know what bacteria inhabit the bathrooms we use each day and where they could come from and how they could affect us.

Based on identification by Gram Staining Method and microscopic views of the different bacteria types from our previous experiment, we were able to gain general knowledge of what some common bacteria types in the bathrooms were. We then applied that knowledge, along with new found pictures and descriptions, to our new set of Petri dish bacteria types.

We found five types of bacteria within our new sets of Petri dishes. We then examined how they were transferred, bodily locations of infection, and possible harmful effects on humans.

Staphylococci:

Staphylococci are yellow, spherical cells that grow in irregular clusters. It is known that an estimated 15-20% of the general population carries these bacteria in their noses and throats. Staphylococci infections are the single most common types of infections within hospitals from surgical or wound infections. They can infect hair follicles resulting in painful boils and may spread across the entire head. Staphylococci are also found to infect the bones after tissue infections and may even spread through the bloodstream. They can infect the intestines as well as the urinary tract, which is most likely where the Staphylococci we found came from. Staphylococci can also infect tampon-wearers in a disease called Toxic Shock Syndrome.

Streptococci:

Streptococci are small, white/ semi-transparent, less spherical cells that grow in chains. While they grow they secrete a large amount of harmful toxins and enzymes. The most common types of infection from Streptococci are sore throat. Mucus membranes become red and swollen, lymph nodes enlarge, temperature rises, and white blood cell count increases. This kind of infection is spread through personal contact with infected or healthy carriers. Sore throat Streptococci is probably the source for our bathroom bacterial sample, seeing as it is so prevalent among college students. Also, Streptococci can cause Scarlet Fever which is a skin rash as a result to hypersensitivity to the toxins Streptococci excretes. It is usually confined to the throat and nasopharynx, but can spread to the bloodstream and cause a Streptococci blood infection. From a small percentage of untreated Streptococci infections, comes an illness called Rheumatic Fever. Streptococci antigens are deposited to the joints and heart, which almost always causes permanent damage. Streptococci can cause very small, acute infections or fatal infections such as Rheumatic Fever.

E-coli:

E-coli are smooth, orange, spherical cells. They are part of the normal flora of the intestinal tract, but can adhere and produce a toxin that prohibits protein synthesis. The result of this infection is most likely death. E-coli is also one of the major causes of diarrhea worldwide. This form of E-coli is very common, but rare types of it can cause a severe cholera-like disease that is fatal. Our bathroom E-coli swabs were most likely from a common diarrheal infection.

Salmonella:

Salmonella are larger, tan, rod-shaped cells. Humans usually become contaminated through ingestion of contaminated food (usu. meat) or water. The contamination usually is a result of introducing feces to either of these from any animal excreting Salmonella. One of the most common sources of Salmonella infections comes from pet turtles that are frequent carriers. Gastroenteritis is a common type of Salmonella infection from contaminated food. It involves symptoms such as headache, abdominal pain, nausea, vomiting, and diarrhea.

Campylobactor:

Campylobactor are larger white, rod-shaped cells. Along with E-coli, it is rated one of the most common causes of diarrhea worldwide. Campylobactor inhabits the intestinal tracts of animals and humans. The spread of infection is by fecal-oral route most likely from farm animals, birds, dogs, and poultry.

Pictures of the bacteria reside in our Power Point presentation - click here to download


Discussion:


While we obtained enough data to gather significant results from our experiment, our experiment could have been greatly improved if we had gathered more data. With more data, it would have been possible to gain better, more accurate statistics. There are a variety of ways that we could have gone about gathering this data. The first and most obvious is to simply take more cultures of bacteria. This would have ensured that our statistics were as accurate as possible. We could have also taken cultures at different times. This would have enabled us to obtain better knowledge of how the bacteria fluctuates before and after cleaning. It would have made our graphs more accurate, and we would have been able to calculate the rate at which the bacteria returned to the bathroom after cleaning. We could have questioned the housekeeping staff about the methods and materials used for cleaning. This would have given us an idea of which methods and materials were working and which ones needed to be reconsidered. It also would have given us an idea of how effective the products being used by the staff were.

Our research and results obtained from this experiment affected our lives in many ways. We learned about the importance of thoroughly cleaning bathrooms, and other frequently used rooms and objects. We learned about the rapid growth of bacteria, and many of its harmful characteristics. We also learned that the majority of bacteria is not harmful to humans, and a great deal of it is actually helpful. Another interesting fact previously unknown to us is the tremendous amount of bacteria that exists on this planet. The amount of bacteria combined outweighs all other living things on earth combined. Probably the most shocking, and at the same time, amusing fact we learned about the bathrooms of Peabody, is that the toilets are actually cleaner than the showers. Based on this fact, we came to the conclusion that we should begin cleaning ourselves in the toilets rather than the showers. This is why the members of our group now wash ourselves behind the privacy of the stall doors instead of the shower curtains.

We learned that there is a large amount of bacteria within the showers, and sadly, most of this bacteria is not destroyed during cleaning. To improve this situation, we could meet with the housekeeping staff and bring this information to their attention. We could then discuss new methods of cleaning that are possibly more effective. Because bacteria thrive in moist environments, a drying technique could be used after cleaning the showers. A time period in which the bathrooms are closed after cleaning could also be utilized to ensure that someone does not take a shower and wash the anti-microbial agents away before they have a chance to do their job. This would be an inconvenience to the students, but would most likely be helpful in the long run because fewer people would get sick from exposure to bacteria. The solution to the bacteria in the showers could be as simple as using a different, or more potent cleaning solution. Whatever the case, the amount of bacteria contained in the showers is somewhat unnerving and some action should be taken against this disease causing creature that flourishes in the same place Peabody students clean themselves.


Sources:



Introduction to the Bacteria
-Basic introduction to bacteria, their effects, and where they exist.

Bacteria Museum
Introductory website dedicated to teaching the basics of bacteria.


Encarta - Bacteria
Encyclopedia definition and pictures.


Britannica - Bacteria
Encyclopedia definition and pictures.


Barker, J. and S.F. Bloomfield. Survival of Salmonella in Bathrooms and Toilets in Bathrooms and Toilets in Homes Following Salmonellosis.
Journal of Applied Microbiology 89, 137-144 (10 March 2000): 8pp. Online. Internet. 23 Sept. 2002
- Bathroom connection to salmonella.


Bell, Chris and Alec Kyriakides. Salmonella. Ames Iowa: Iowa State University Press, 2002
-Taxonomy of Salmonella.


Boln, Brogden, et al. Ed. Virulence Mechanisms of Bacterial Pathogens. 2nd Ed. Washington D.C.: ASM Press, 1995
-Behaviorisms of bacteria in various environments.


Dharan, S., P. Mourouga, P. Copin, P. Bessmer, B. Tschanz, and J.Pittet, ed. Routine disinfection of patients environmental surfaces. Myth or reality? Journal of Hospital Infection 42:2 (1999): 113-117.
-Bacteria prevention: Does it really work?


Gibson, S, ed. Human Health: The Contribution of Microorganisms. London: Springer-Verlag London Limited, 1994
-The effects of bacteria on human health.


Gillies, R.R. and T.C. Dodds. Bacteriology Illustrated. 4th ed. New York: Churchill Livingstone, 1976
-Bacteria basics and Gram's Staining method.


Good Bacteria Importance. Journal of Pediatric Gastroenterology and Nutrition (1998;27:323-332) Online. Internet. 23 Sept. 2002
-Culturing methodology.


Soltys, M.A. Bacteria and Fungi Pathogenic to Man and Animals. Baltimore: The Williams and Wilkins Company, 1963
-Bacteria taxonomy and effects on fauna.


Wilson, Michael, Rob McNab, and Brian Henderson. Bacterial Disease Mechanisms. Cambridge: University Press, 2002.
-Picture credits and taxonomy.


Zacheus, O.M. and P.J. Martikainen. Occurrence of Heterotrophic Bacteria and Fungi in Cold and Hot Water Distribution Systems Using Water of Different Quality. Canadian Journal of Microbiology 41:12 (1995): 1088-1094.
-Western connection/ relevance.


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