Lab Packet #2 - Bathroom Bacteria

This topic submitted by Chris Jones, Michelle Cisar, James Diewald, & Paul Georgeadis (cubbiesseven@yahoo.com) at 2:11 AM on 10/22/02. Additions were last made on Wednesday, December 10, 2008. Section: Cummins

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

Bathroom Bacteria

Research question:

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.

Purpose:

To collect and identify various forms of bacteria found on Western campus

Predictions:

The bathrooms of Peabody will 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. Mary Lyon will hold the least bacteria because it is the smallest Western dorm, thus there are less people to transmit bacteria.

Hypothesis:

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.

Relevance:

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: ñMicroorganisms 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. (ñBacteriaî, 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 focu 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 source, 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 ñgoodî 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 BloomfieldsÍ 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 CampusÍs 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.

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

Detailed Procedure:

-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

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) Sink Knobs

2) Counter Top

3) Shower Knobs

4) Shower Floor

5) Toilet Flush Lever

6) Toilet Seat

7) Inner Stall Latch

8) Paper Towel Dispenser

9) Sink Bowl

10) Toilet Seat

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.

Class Participation:

In order to get the class to participate in our presentation we will make a Powerpoint slideshow of many different kinds of bacteria. We will have the class try to identify the different kinds of bacteria and we will hand out candy to the people who are the most active in participation, not necessarily whomever has the most correct because we realize it may be a bit difficult.

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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