Lab Packet

This topic submitted by eli, Mike, Mike, Alijandra, Shirley at 6:25 AM on 10/17/02. Additions were last made on Wednesday, December 10, 2008. Section: Cummins

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

Bacterial Resistance To
Anti-Microbial Agents

A Research Proposal By:

Shirley Wang
Michael Durisen
Alejandra Miller
Eli Balkin
Michael

Dr. Hays Cummins
Natural Systems
Fall 2002
WCP 121-A

Abstract

Bacteria play a key role in the process of evolution. The first documented life to appear on earth was bacteria. Life has needed it to survive. In humans, there are numbers of different types of bacteria in our stomachs that allow us to digest our food. There is however, bacteria that cause illness and even death. This is why in addition to our immune system; we have created anti-bacterial agents to kill the bacteria. We split these agents into two groups, name brand and generic brand.
This experiment is based on the question, which type of anti-bacterial agent kills bacteria better, name brand or generic brand. The experiment hopes to show a significant difference between the killing effects of each brand of anti-bacterial solution. The hypothesis is that the name brands will kill the bacteria quicker and more effectively than the generic brand.

Introduction

Bacteria have played an important role in the existence of life on this planate. In fact, it was bacteria that were the first life to appear on the earth around 2.5 billion years ago. Ever since then, bacteria have been essential to the survival of all other living things. Almost every animal and plant depends on bacteria to live. There are countless bacteria responsible for the digestion of food that allows creatures to absorb nutrients, and sometimes, the bacteria themselves are the food. Of course not all bacteria are good to have around. Some bacteria cause harmful diseases that hamper the existence of other beings, and sometimes prove to be fatal to the infected individual. Many creatures have their own defenses to fight these invaders that can be quite affective. Humans, though, besides having natural defenses, have created their own agents to fight the growth of unwanted bacteria. There are many different kinds of products that have been created to kill off bacteria; some made by large companies, and other that are generic. Most all of them, however, claim to be better than the competing company. They even conduct tests that supposedly prove their superiority.
There are many anti-bacterial cleaners that boast of their fantastic ability to kill the bacteria hat grows in the environment around the house. In the vast array of cleaners, there are those that are offered and advertised from big companies, or Òname brandsÓ. Then there are those cleaners that claim to perform the same job, except they lack the fancy labels and marketing techniques, better known as Ògeneric brandsÓ. Is it worth it to pay the few extra dollars to buy the name brands, or are generic brands just as good? It is this question that we will be examining. We are curious to determine whether there is a significant difference in the effectiveness of the name brand anti-bacterial agents as compared to the generic brands. We believe that the name brand cleaners will be more effective in killing bacteria than the generic kinds. According to how thorough and how fast the anti-bacterial agents work, we believe that there will be a noticeable advantage to the use of name brand products. We predict that the name brands will be more affective in kill the bacteria than the other brands. By conducting this experiment, we are hoping to show that differences in quality exist between cleaning brands. It is always interesting to look at well-known products and a less expensive unknown brand and wonder whether or not there is actually a difference in quality. Hopefully in this experiment it can be determined if a difference exists between how well the generic brands work as compared to name brands.

Prokaryotes are the most common type of bacteria, they are generally single-celled organisms, although some occur as aggregates, colonies or simple multi-cellular forms. The three most common prokaryotic shapes are spherical (cocci), rod-shaped (bacilli), and helical forms. Nearly all prokaryotes have external cell walls, which protect and shape the cell and prevent osmotic bursting. Many species secrete sticky substances that form capsule, and some have surface appendages called pili outside of the call wall. Both structures help the cells adhere to one another, and some pili are specialized for conjugation. Motile bacteria propel themselves by flagella, use flagella-like filaments positioned inside the cell wall, called spirochetes. Prokaryotic cells are not compartmentalized by endomembranes. However, invaginations of the plasma membrane may provide internal membrane surface for specialized functions. The prokaryotic genome consists of a single circular DNA molecule in a nucleoid region unbounded by a membrane. Many species also possess smaller separate rings of DNA called plasmids, which code for special metabolic pathways and resistance to antibiotics. Bacteria reproduce asexually by binary fission Genetic variation occurs in prokaryotes through mutation and gene transfer by transformation (genes are take up from the surrounding environment), conjugation (genes are transferred directly from one prokaryote to another), or viral transduction (genes are transferred between prokaryotes by viruses).A short generation span enables prokaryotic populations to adapt very rapidly to environmental changes. This is very important to the continuing success of prokaryotes. All major types of nutrition and metabolism evolved among prokaryotes. Prokaryotes are the most metabolically diverse organisms on Earth. Most bacteria are chemoheterotrophs, which require organic molecules as a source of both energy and organic carbon. Several groups of bacteria metabolize nitrogen compounds unavailable to other organisms. By doing so, these prokaryotes play critical roles in the cycling of nitrogen in the environment. The ability or inability to survive in the presence of oxygen also reflects variation metabolism.
Prokaryotes, along with fungi, are decomposers that recycle chemical elements in ecosystems. Some prokaryotes live with other species in symbiotic relationships of mutualism, commensalisms or parasitism. Some parasitic prokaryotes are pathogenic, causing disease in the host by invading tissues or poisoning with endotoxins or exotoxins. Bacteria have been put to work in laboratories, Sewage treatment plants, and the food and drug industry. One especially exciting development has been the use of prokaryotes in recombinant DNA technology

ÒDisinfectants and biocides are a chemically diverse group of agents which are generally considered to exhibit poor selective toxicity.Ó All anti-bacterial operate using one of three types of action: physical, chemical, or ionic. Regardless of the type of action used the objective is the same: the destruction of bacteria. Disinfectants go about this in several ways. Disruption of the transmembrane motive force by preventing the accumulation of a transmembrane force, this type of biocide prevents the cell from carrying any material in to out of the cell. Inhibition of respiration or anabolic rations, this method prevents the cell form caring out chemical processes necessary for bringing oxygen into the cell and carbon-dioxide out, or vice versa. This method also prevents the cell form bringing in and food and energy products. Disruption of replication; this type of action is often used in conjunction with other methods. However, it can be quite effective on its own. By preventing the cell from dividing or replicating, you can assure that the generation being exposed to the agent will be the last generation of that organism in a given sample. Loss of Membrane integrity/cellular leakage. By causing the membrane to decompose, chemical agents can effectively destroy a cell. Breaching the cellular membrane allows the Òintracellular constituentsÓ to leak out; additionally it allows foreign materials to enter the cell. This is one of the most common methods of bacterial destruction, however because of the membrane breakdown that occurs, bacterial DNA if often left behind. This can pose a serious threat and lead to bacterial resistance to antibiotics. The process of lysis is one of the most effective in the disinfectant arsenal; however it is also one of the slowest acting. Lysis causes a dis-coagulation of the material inside the cell without breaching the cell membrane. This kills the organism and prevents the DNA from becoming exposed. Coagulation of intracellular material is the opposite process of lysis. Just as lysis causes a thinning of intracellular fluids and structures, coagulation results in the opposite effect. The fluids within the cell become semi-solid in nature and prevent any of the natural process from occurring and thus destroy the cell. In the most extreme cases of coagulation. The cell hardens to such a point that is solidifies in a state of seeming biostasis with all of the structures imprisoned within the cell.

There are many anti-bacterial cleaners that boast of their fantastic
ability to kill the bacteria that grows in the environment around the
house. In the vast array of cleaners, there are those that are offered
and advertised from big companies, or Òname brandsÓ. Then there are
those cleaners that claim to perform the same job, except they lack the
fancy labels and marketing techniques, better known as Ògeneric
brandsÓ. Is it worth it to pay the few extra dollars to buy the name
brands, or are generic brands just as good? It is this question that
we will be examining. We are curious to determine whether there is a
significant difference in the effectiveness of the name brand
anti-bacterial agents as compared to the generic brands. We believe
that the name brand cleaners will be more effective in killing bacteria
than the generic kinds. According to how thorough and how fast the
anti-bacterial agents work, we believe that there will be a noticeable
advantage to the use of name brand products. We predict that the name
brands will kill the bacteria faster, and be more affective than the
other brands. By conducting this experiment, we are hoping to show
that differences in quality exist between cleaning brands. It is
always interesting to look at well-known products and a less expensive
unknown brand and wonder whether or not there is actually a difference
in quality. Hopefully in this experiment it can be determined if a
difference exists between how well the generic brands work as compared
to name brands.

Materials and Method:

Materials:
-Agar plates
-10 Petri dishes
-Swabs
-Controlled Environment for Incubation
-Filter paper
-Electrical Tape
-Pipettes
-Test tubes
-Distilled water
-Antibacterial agents

Methods
Most of these materials will be obtained from the Microbiology department, and the anti-bacterial agents will be purchased at a local convenience store. To begin with, the bacteria need a medium in which to grow. For this we will use 10 Petri dishes that contain a medium of Agar, which will serve as food for the bacteria to grow on. In order to obtain the bacteria from the environment for culturing, we will use sterilized swabs to collect samples. To grow the bacteria once it is collected, we require an incubator so that the bacteria can be kept in a controlled environment for optimal growth. In order to apply the antibacterial agents to the bacterial contaminated Petri dishes, we need filter paper, pipettes, test tubes, and distilled water. The filter paper will be soaked and then used to apply to the Petri dishes. The test tubes and distilled water and the pipettes will be needed in order to create different concentrated solutions of the antibacterial agents.
In order to test the effectiveness of the cleaning agents, the bacteria will first need to be collected and cultured. We will be collecting the bacteria using the sterile swabs to swab door handles and other implements in the restroom facilities of Peabody. The contaminated swabs will then be smeared onto a Petri dish containing Agar. This Petri dish will then be incubated in a controlled environment for about 24 hours until the bacteria is given a chance to grow. Since there are many bacteria in the environment, the sample will no doubt be a lawn of many different types of bacterium. In order to select one of the colonies to test the antibacterial agents, we will then swab a specific colony from the original Petri dish, and then smear it on new Petri dishes.
Onto this second Petri dish, we will be adding the antibacterial agent. To do this, we will soak the filter paper in three different concentrated solutions of each agent. Three soaked filter papers will then be placed onto the swabbed second dishes, two pares of three per dish with a control filter paper soaked in distilled water in the middle. These second dishes will then be incubated for 24 hours. After this time, the dishes will be observed, and the diameter of the areas where no bacteria grew will be measured, and then the area will be calculated. This procedure will be repeated until enough acceptable data is gathered to compare the different anti-bacterial agents.
In order to dilute the anti-bacterial agents for the use in the experiment, first we will need to distill water. To do this we will take tap water and boil it, then let it cool. This will be repeated three times in order to insure that any microorganisms in the tap water will be killed off. We will have 3mL of concentrated anti-bacterial solution in one test tube. One milliliter of the concentrated solution will then be added to another test tube containing 2mL of distilled water creating a 1 to 2 solution. A third solution will be created by taking 1mL of the 1 to 2 solution and adding it to another test tube containing 2mL of distilled water creating a 1 to 3 solution. These solutions will then be used to soak the filter paper to introduce it to our bacteria cultures in the fashion described above.
To try and discern which agent is more a more affective anti-bacterial agent, we will be comparing the surface areas where no bacteria grew around the filter paper. This will tell us which is more affective in hindering the growth of bacteria. We will also be comparing the results of the different dilutions of the agents to see how strong the agents are compared to one another. These results will then be compared in Stat View in order to see if there is a significant difference in the effectiveness of the agents.

Time Table
10/17/02 Ð receive materials from microbiology dept.
10/22/02 Ð collect bacterial samples
10/22/02 Ð 10/23/02 Ð grow original culture
10/24/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
10/24/02 Ð 10/39/02 Ð observation and data collection

10/31/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
10/31/02 Ð 10/39/02 Ð observation and data collection

11//02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
11/7/02 Ð 11/12/02 Ð observation and data collection

11/14/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
11/14/02 Ð 11/19/02 Ð observation and data collection

11/21/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
11/26/02 Ð 10/39/02 Ð observation and data collection

11/28/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
11/28/02 Ð 12/3/02 Ð observation and data collection

12/5/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
12/5/02 Ð 12/10/02 Ð observation and data collection

12/12/02 Ð isolate bacterial species from original culture, on second plate, with anti- bacterial
12/12/02 Ð 12/17/02 Ð observation and data collection

In addition to a data sheet our group has elected to use a data collection form. We believe that this will produce more a more accurate record of our experiment especially considering that many of the individuals collecting data will be our classmates and will not have a full understanding our methods and procedures.

Supervising experimenter:__________________________________________________

Assisting experimenter:____________________________________________________

Date:____________________________________________________________

Time:____________________________________________________________

Plate number:__________________________________________________________

Anti-Bacterial being tested:_________________________________________________

Side A aura diameter_______________(mm)

Side B aura diameter :______________(mm)

Photo taken: YES NO

Photo file name:_________________________________________________________

NOTES:__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

DATA TABLE
Date Time Plate/Side Aura Diameter (mm)
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The following is a list of the sources consulted during the formulation this project. As this project is a continually growing and evolving entity, we fully expect this list to grow and develop as well. Because of the dynamic, multi-dimensional nature of the internet, when ever possible, any text that is directly removed formerly previously published work is linked to a full copy of that work. With the understanding that a citation is used to give credit and to allow the reader to obtain the full text, we have elected to o use the dynamic-link citation system. We have directly connected the reader with the original work without using the standard citation as in intermediary.

Citations and Sources
Sterilizing Internal Parts Using Vapor
Effectiveness of Some Disinfectants
Detection of Bacteria in Food
Disinfectant Testing
Application of Disinfectants
The testing of Disinfectants
Cleaning Microbes
Disinfectants on Surfaces
Future Techniques of Disinfection
Anthrax Toxin
Metacillin Resistance Bacteria
Mechanism of Action of Disinfectants
Efficiency of Disinfectants

University of California, Berkeley. Museum of
Paleontology

High Levels of Colicin Resistance in Escherichia coli
Michael Feldgarden, Margaret A. Riley
Evolution, Vol. 52, No. 5. (Oct., 1998), pp.
1270-1276.

When can Reduced Doses and Pesticide Mixtures Delay
the Build-up of Pesticide Resistance? A Mathematical
Model
C. P. D. Birch, M. W. Shaw
Journal of Applied Ecology, Vol. 34, No. 4. (Aug.,
1997), pp. 1032-1042.

A Consideration of the Evolutionary and Taxonomic
Significance of Some Biochemical, Micromorphological,
and Physiological Characters in the Thallophytes
Richard M. Klein, Arthur Cronquist
Quarterly Review of Biology, Vol. 42, No. 2. (Jun.,
1967), pp. 108-296.

The Evolution of Plasmids Carrying Multiple Resistance
Genes: The Role of Segregation, Transposition, and
Homologous Recombination
Richard Condit, Bruce R. Levin
American Naturalist, Vol. 135, No. 4. (Apr., 1990),
pp. 573-596.

The Influence of Temperature on the Life Processes and
Death of Bacteria
Bettylee Hampil
Quarterly Review of Biology, Vol. 7, No. 2. (Jun.,
1932), pp. 172-196.

The Relation of Adaptability to Adaptation
G. F. Gause
Quarterly Review of Biology, Vol. 17, No. 2. (Jun.,
1942), pp. 99-114.

Evolution in Bacterial Plasmids and Levels of
Selection
William G. Eberhard
Quarterly Review of Biology, Vol. 65, No. 1. (Mar.,
1990), pp. 3-22.

Additionally the following web sites were consulted; however no data from these locations was used.
→University of California, Santa Cruz Biology Department Biosafety Front Page (http://www.biology.ucsc.edu/Safety/index.html)
→Ohio State Department of Biological Science Safety Page
(http://www.biosci.ohio-state.edu/~jsmith/SafetyPage.htm)
→ATTC Cultures Incorporated
(http://www.atcc.org/SearchCatalogs/Bacteria

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