Mini Project 2

Projects with bacteria are fun and easy! Use this simple procedure as a basis for designing your own experiments or science fair projects. Ideas for other projects are listed at the end. Bacteria are one-celled, or unicellular, microorganisms. They are different from plant and animal cells because they don't have a distinct, membrane-enclosed nucleus containing genetic material. Instead, their DNA floats in a tangle inside the cell. Individual bacteria can only be seen with a microscope, but they reproduce so rapidly that they often form colonies that we can see. Bacteria reproduce when one cell splits into two cells through a process called binary fission. Fission occurs rapidly in as little as 20 minutes. Under perfect conditions a single bacterium could grow into over one billion bacteria in only 10 hours! (It's a good thing natural conditions are rarely perfect, or the earth would be buried in bacteria!) Growing and testing bacteria is a fun any-time project or a great science fair project. Bacteria are everywhere, and since they reproduce rapidly they are easy to study with just a few simple materials. All you need are some petri dishes, agar, and sterile swabs or an inoculating needle. Agar is a gelatinous medium that provides nutrients and a stable, controlled environment for bacteria growth. Most bacteria will grow well using nutrient agar, but some more fastidious bacteria (those with more complex nutrient requirements like Bacillus stearothermophilus,Branhamella catarrhalis, and Bacillus coagulans) prefer tryptic soy agar. You also need a source for bacteria, and this is not hard to find! You can swab your mouth or skin, pets, soil, or household surfaces like the kitchen sink or toilet bowl. If you want to study a particular type of bacteria, you can also purchase live cultures. Adult supervision is recommended when working with bacteria. Growing bacteria is a safe activity if you keep the lids closed and always wash your hands after handling the dishes. Preparing Culture Dishes Before you can grow bacteria, you'll need to prepare sterile culture dishes. A 125ml bottle of nutrient agar contains enough to fill about 10 petri dishes. 1.

Melt the agar in either a hot water bath or a microwave. The bottles will get very hot, so use tongs or a potholder to pick them up. Water Bath Method - Loosen the agar bottle cap, but do not remove it completely. Place the bottle in hot water at 170-190 ºF until all of the agar is liquid. Microwave Method - Remove the bottle cap and microwave on high for 30 seconds. Swirl the bottle to distribute the heat. Repeat in 15-20 second increments until the first signs of boiling appear and all of the agar is liquid.

2. Let the agar cool to 110-120 ºF (when the bottle still feels warm but not too hot to touch) before pouring into petri dishes.

3. Slide open the cover of the petri dish just enough to pour agar into the dish. Pour enough agar to cover 1/2 to 2/3 of the bottom of the dish (about 10-13ml). Don't let the bottle mouth touch the dish. Cover the dish immediately to prevent contamination and tilt it back and forth gently until the agar coats the entire bottom of the dish. (Fill as many dishes as you have agar for: you can store extras upside down in the refrigerator until you're ready to use them.)

4. Let the petri dishes stand one hour for the agar to solidify before using them. Preparing Sensitivity Squares One method for testing the antibacterial effectiveness of a substance is to use "sensitivity squares." Cut small squares of blotter paper (or other absorbent paper) and then soak them in whatever substance you want to test: iodine, ethyl alcohol, antibacterial soap, antiseptics, garlic, etc. Use clean tweezers to handle the squares so you don't contaminate them. Label them with permanent ink, soak them in the chosen substance, and blot the excess liquid with a paper towel.

Setting Up an Experiment Each experiment should have a control dish that shows bacteria growth under normal conditions and one or more test dishes in which you change certain variables and examine the results. Examples of variables to test are temperature or the presence of antiseptics. How do these affect bacteria growth?

1. Using a sterile swab or an inoculating needle (heat-sterilized in a flame), collect bacteria from your chosen source (e.g., kitchen sink, inside of your cheek, or live culture). Remove the cover of a petri dish and lightly rub the swab across the surface of the agar in a zigzag pattern. (You may want to turn the dish a quarter turn and zigzag again for maximum coverage.) Cover the dish again immediately. Repeat with a new swab for the other dishes you are using in your experiment. 2.

Label one dish "Control." Then in your test dish, use tweezers to add the sensitivity squares that have been soaked in a substance you wish to test for antibacterial properties. It's a good idea to add a plain square of blotter paper to see if the paper by itself has any effect on bacteria growth. For best results, use multiple test dishes and control the variables so the conditions are identical for each dish: bacteria collected from the same place, exposed to the same amount of antibacterial substance, stored at the same temperature, etc. The more tests you perform, the more data you will collect, and the more confident you can be about your conclusions.

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Place all the dishes in a dark, room-temperature place like a closet. (Unless you are testing them for reaction to different temperatures, of course.)

Wait 3-7 days and examine the bacteria growth in the dishes, without removing the lids. You will see multiple round dots of growth; these are bacteria colonies. Depending on where you collected your bacteria samples, you may have several types of bacteria (and even some mold!) growing in your dishes. Different types of colonies will have different colors and textures. If you have a compound or stereo microscope, try looking at the colonies up close to see more of the differences. Compare the amount of bacteria in the control dish to the amount in the test dishes. Next, compare the amount of bacteria growth around each paper square. Which one has bacteria growing closest to it? Which one has the least amount of bacteria growing near it? If you did more than one test dish, are the results similar in all the test dishes? If not, what variables do you think might have caused the results to be different? How does this affect your conclusions? Disposing of Bacteria Cultures Remember to use care when experimenting with bacteria. The kind of bacteria you're using may be types that are normally present in your house, but you are culturing them in greater numbers than usual, and this can be hazardous. When you are finished with your experiment, pour a little bleach into each petri dish, seal the dishes in a plastic bag, and throw them away. More Bacteria Experiment Ideas Here are some other project ideas for you to try on your own or use as a basis for a bacteria science fair project:

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Household cleaners. Which household cleaners work best against bacteria? Try swabbing a surface in your home, like the kitchen sink or a toilet, and then use sensitivity squares to test different cleaners such as Lysol, bleach, Windex, etc.

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Natural substances. Test to see if garlic really has antibacterial properties. What about tea tree oil, or red pepper, or curry?

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Mouthwash. Swab your teeth and gums and see how well toothpaste or mouthwash work against the plaque-causing bacteria on your teeth.

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Antibiotics. Use an antibiotic disc set to see what different antibiotics can do against bacteria. For a more advanced project, learn how gram staining relates to the use of antibiotics.

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Have you heard people say that dogs' mouths are cleaner than humans'? Design an experiment to test whether this is really true!

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Some band-aids are advertised as being antibacterial. Test to see if they really work better than regular band-aids at inhibiting bacteria.

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Is it safe to keep refilling a water bottle without washing it? Test a sample of water from the bottom of a water bottle that has been used for a couple days and compare it to a sample from a freshly-opened, clean water bottle. You can also test to see if a bottle gets more bacteria in it if you drink with your mouth or with a straw.

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Do bacteria grow in your shoes? Is there a difference in bacteria growth between fabric shoes and leather? Do foot powders work to cut down on bacteria?

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Do bacteria grow on your toothbrush? What are some ways you could try to keep it clean? Mouthwash? Hot water?

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Some people recommend getting new mascara every six weeks because bacteria can grow in the tube. Test this by comparing bacteria growth from old mascara and new, unused mascara. You can also test how much bacteria is on other kinds of makeup.

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What about good bacteria? Try making some homemade yogurt with this kind!

is site has the information to enable any young Pasteur to conduct excellent microbiology experiments at home with materials found in any kitchen. Farmers, gardeners, and amateurs will find useful methods and information on these pages for science projects, family health, gardening, farming, animal care, and food preservation. Inexpensive kits of Cultures and supplies are available. First-time visitors please click here for information moved from this page to make downloading faster. For fast browsing, Bookmark this page, then use your Back Button to return to this index as you finish looking at any page of this site.

Start Your Microbiology Science Project Here, Today! B000 - Index page giving general safety and background information for your microbiology project. This page will help you get an outstanding bacteria project going. Also visit our yeast, fungi, andbacteriophage web sites which are in early construction. •

B001 - Letter to the Beginning Student - Read before trying any of these experiments.

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B002 - Letter to Parents - This letter discusses the safety risks of bacteria projects.

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B003 - What is microbiology? What are bacteria, archeobacteria, viruses, fungi, prions?

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B004 - Introduction to bacteria: their food and growth conditons; their place in bioworld

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B005 - BacteriaStudyList - mail list for K-12 students, parents, farmers, home makers, et al.

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B006 - Believe it or Not. Surprising, but true facts about bacteria. INCOMPLETE

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B007 - Bacteria: Friends or Foes? INCOMPLETE

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- plasmids, genetics, phages, (introductory info every beginner should know for safety).

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B??? - More basic information about bacteria

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- more beneficial bacteria

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- some harmful bacteria

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books, magazines about bacteria

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short history of microbiology

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how bacteria got their names

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links to bacteria sites

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into to growth requirements of bacteria.

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B017 - Suggested gifts for your young Pasteur--some are free.

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bacteria on and in our bodies - could we live without them?

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B018 - Reports on bacteria projects completed by K-12 students.

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B019 - Sample Student Research Proposal - planning your project before you begin

B020 - Start your own Home Microbiology Lab by reading this index page. Introductory experiments, media, tools, and methods for beginners using foods and items found at home--few or no purchases required. B020a prints a work plan for starting your bacteria project. •

B021 - An easy non-sterile first experiment. Can yeast use corn syrup, sugar, or starch?

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B022 - Make E-Broth from ground beef on the kitchen stove - Easy-meat broth medium.

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B023 - Dispense your media as stabs, slants, plates, deeps: aerobic and anaerobic media

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B024 - Sterilize and store your media. Use pressure cooker or steamer; store dust-free

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B025 - Make a loop and streak gelatin or agar plates to isolate pure colonies. Methods.

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B026 - Start your own pure cultures collection. Make chart of bacterial species traits.

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B027 - More meat-based media: liver, egg, bouillon cubes, gelatin, heart infusion, brain

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B028 - Milk media. Many bacteria grow better in milk than in meat broth. A B

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B029 - Make media from potato, carrot, tomato, rice, hay, turnip; slices, plugs, and liquid

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B030 - Stock Cultures Media - as used at Indiana Biolab. <== Study this important page.

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B031 - Chemolithotropic media: rocks and sulfur; mud jars. For bacteria that eat rocks.

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B032 - Simple diagnostic media which can be made at home. Gas tubes, sugars used.

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B033 - Growth conditions; Aerobic vs anaerobic bacteria; media, incubation, and methods.

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B034 - Introduction to enrichment and selective media for growing specific bacteria.

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B037 - Reuse plastic plates and trays. Experiments using bacterial lawns. Reuse & recycle.

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B038 - How to count bacteria. Sub pages on statistics, equipment, simple to complex

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B039 - Summary of tools, equipment, and media for your Home Micro Lab. Incomplete

B040 - Index page to Standard Methods used in Microbiology: This chapter shows you how to perform the techniques used in colleges, hospitals, and professional laboratories. The amateur microbiologist may not have access to this equipment, but these pages help him design or select substitutes so that he is able to complete many additional experiments. •

B041 - What are agar, tryptone, yeast extract, and the other ingredients used in media?

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B0?? - Preparing professional media from tryptone, yeast extract, agar grades, origin of

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B0?? - Formula for professional media

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B042 - Formula for diagnostic media - carbohydrate media

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B0?? - Formulae for bacteriological stains

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B0?? - Streaking plates to isolate bacteria and obtain pure cultures

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B0?? - Making slide mounts and using microscope

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B0?? - Building and maintaining a collection of bacteria generally considered safe.

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B0?? - ?building equipment for advanced work, laminar flow hoods, safety cabinets.

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B0?? - working with strict anaerobic such as rumen bacteria and methane producers

B060 - Experiments on Growth Requirements of Bacteria. Effect of environmental factors on the growth of bacteria. Performing these experiments will help you understand bacteria and how to protect your food from spoilage and poisoning by bacteria . Salt, sugar, pH, oxygen level, heat, and other agents are all used to protect and preserve foods. In these experiments you will aloso learn how different genera and species of bacteria differ in their response to environmental conditions. •

B061 - Oxygen requirements of bacteria - INCOMPLETE

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B062 - Optimum temperature for some common bacteria: 4C, room temp, 37C, 55C

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B063 - Osmotic pressure (salt) of medium and bacteia - INCOMPLETE

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B06? - pH effect on bacterial growth. Table: species, comments

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B064 - Nutritional requirements of bacteria

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B065 - Carbohydrates used by bacteria - diagnostic assays Henry page 232

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B0?? - Carbon sources used by bacteria

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B0?? - Nitrogen sources used by bacteria

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B000 - Disinfectants action on bacteria

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B000 - Killing of bacteria by UV, heat, and agents is a logarithmic function; killing curves.

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B000 - chemo- and organo- tropic nutrition

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B000 - Bacteriophages (viruses that attack bacteria). Also see phage site.

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B000 - Bacteriocins that kill bacteria

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Growing bacteria on minimal media; streak 10 species on a minimal plate.

B090 - Beginner's Guide to Classification of the Bacteria. The section introduces the beginner to the many types of bacteria and will help students pick an interesting group for a science project. See B400 for a complete classification of the bacteria. •

B095 - Distinctive well-defined Genera of Bacteria which the good student should know.

B100 - Food Bacteriology: Experiments with the bacteria you eat every day INCOMPLETE •

B10? - Making foods by selective fermentations of cabbage, cucumbers, peanut, sorghum, tea fungus, drinks, soybean, breads, cover most primitive fermented foods. `

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B10? - Improvement of primitive fermented foods by pure culture methods

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B120 - NEW - index to milk fermentations, LAB, olives, etc

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isolation of pure cultures of bacteria from human foods by students at home.

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B121 - Isolating and using pure cultures of bacteria in production of fermented foods.

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B122 - Isolation of Brevibacterium linens from Limburger Cheese at home by a beginner.

B200 - Isolation of bacteria from nature based on their traits. •

B203 - Isolation and characteristics of Vibrio phosphoreum, glows brightly.

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Eventually dozens of bacteria will be covered in detail in this section.

B400 - The Bergey Classification of Bacteria •

The Cyanobacteria (blue-green alga) - will not be covered in this site this year.

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The Bacteria (the classical bacteria) - will be covered as time permits - see next line.

The Bergey Classification of Bacteria into 19 parts. Some familiar genera are listed. •

Phototrophic Bacteria: Rhodospirillum - Rhodopseudomonas - Chromatium

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Gliding Bacteria: Myxococcus - Beggiatoa - Simonsiella - Leucothrix

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Sheathed Bacteria: Sphaerotilus - Leptothrix

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Budding / Appendaged Bacteria: Caulobacter - Gallionella

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Spirochetes: Spirochaeta - Treponema - Borrelia

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Spiral and Curved Bacteria: Spirillum - Auqaspirillum - Oceanospirillum Bdellovibrio

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Gram-negative Aerobic Rods and Cocci: Pseudomonas - Xanthanomonas - Zoogloea Gluconobacter - Azotobacter - Rhizobium - Agrobacterium - Halobacterium Acetobacter

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Gram-Negative Facultative Anaerobic Rods: Escherichia - Citrobacter - Salmonella Shigella - Klebsiella - Enterobacter - Serratia - Proteus - Yersinia - Erwinia - Vibrio Aeromonas - Zymomonas - Chromobacterium - Flavobacterium -

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Gram-negative anaerobes: Bacteriodes - Fusobacterium - Desulfovibrio - Succinimonas

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Gram-Negative cocci: Nisseria - Branhamella - Acinetobacter - Paracoccus

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Gram-negative anaerobic cocci: Veillonella - Acidaminococcus

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Gram-Negative Chemolithotrophic: Nitrobacter - Thiobacillus - Siderocapsa

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Methane producing:

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Gram-Positive Cocci: Micrococcus - Staphylococcus - Streptococcus - Leuconostoc Pediococcus - Aerococcus - Peptococcus - Ruminococcus - Sarcina

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Endospore-forming Rods and cocci: Bacillus - Clostridium - Sporosarcina

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Gram-positive, non-sporing rods: Lactobacillus - Listeria - Erysipelothrix Caryophanon

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Actinomycetes and Related: Corynebacterium - Arthobacter - Brevibacterium Cellumonas - Kurthia - Propionibacterium - Eubacterium - Actinomyces - Archina Bifidiobacterium - Rothia - Mycobacterium - Frankia - Streptosporangia - Nocardia Streptomyces - Streptoverticillium - Micromonospora

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Rickettsias: Rickettsia - Erhlichia - Wollbachia - Bartonella - Chlamydia

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Mycoplasmas: Mycoplasma - Acoleplasma - Thermplasma - Spiroplasma

B800 - Medical bacteriology; move pathogenic discussions here - not ready (incomplete) •

Introduction to bacteria pathogenic to humans- Required reading for safety

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B801 - First micro safety page for beginners - required reading for safety

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B802 - Safety page for High School and Begin College students

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B??? - lists of pathogenic bacteria class 1,2,3, 0=no report of disease

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B0?? - The pathogenic bacteria: disease causing bacteria, plants and animals, microbiol.

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safety

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Rutger's University Medically Important bacteria - very sketchy

B900 - Bacterial genetics INCOMPLETE barely started.

First time Visitors Please Read This You can easily and safely run a bacteria project at home for a few years. Such long-term projects usually win college scholarships for young scientists. These pages were written for the young Pasteur who has an interest in microbes, but has no books, no equipment, no microbes--nothing but a desire to learn. You will find protocols for isolating bacteria from nature, descriptions of species, and useful information not found elsewhere on the Web. With this information you will be able to start you own collection of pure cultures and identify many of them, at least assign them to the correct genus. If you put in that much effort, you will probably learn as much as many students in a good college microbiology course. Likewise elementary and highschool teachers, farmers, gardeners will learn much to aid them. Eventually this site will be 20 times as large as it is today. You do not need a microscope. Toy microscopes are useless for studying bacteria. A microscope good enough to see bacteria costs hundreds of dollars. You would have to pay over $1,000 for a microscope that would be really useful. If you want to see your bacteria, take them to school and use a microscope there. Instead of spending money on a microscope, it is better to concentrate on isolating bacteria from nature and studying their biochemical traits. Someone once told Pasteur

he had called a bacterium a coccus (round) when it was actually rod-shaped. "If you only knew how little difference that makes to me," Pasteur replied. There is no need to go out and buy lots of equipment. Begin with Home Micro Lab by making media from common kitchen foods and isolate some bacteria. Bottles can serve as culture tubes, or your teacher may loan you culture tubes and supplies. If not try a hospital, they throw away lots of items you can use and if autoclaved they are perfectly safe. Before you trouble a hospital for gifts, do a few week's work at home and prove that you are really interested and planning to work hard. Can lids, or flat bottles can serve as petri plates. Most students only have access to plastic petri plates, but is sometimes possible to reuse them. BA.htm - Isolation and study of Bacillus strains CL.htm - Isolation and study of Clostridum strains Some day there will be pages on yeasts and fungi. You may send private e-messages to Dr. Eddleman and he will reply, usually within 24 hours.

Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria MANY crude oil constituents are biodegradable in the presence of oxygen; however, a substantial anaerobic degradation has never been demonstrated1,2. An unusually low content of n-alkanes in oils of certain deposits is commonly attributed to selective utilization of these hydrocarbons by aerobic microorganisms3,4. On the other hand, oil wells and production fluids were shown to harbour anaerobic sulphate-reducing bacteria5–8, but their actual electron donors and carbon sources were unknown. On the basis of nutritional properties of various bacterial isolates it was assumed that fatty acids and H2 are potential electron donors for sulphate reduction in situ5–8. Here we demonstrate that hydrocarbons in crude oil are used directly by sulphate-reducing bacteria growing under strictly anoxic conditions. A moderately thermophilic pure culture selectively utilizesn-alkanes in oil for sulphate reduction to sulphide. In addition, a mesophilic sulphate-reducing enrichment culture is shown to oxidize alkylbenzenes in oil. Thus, sulphate-reducing bacteria utilizing aliphatic and aromatic hydrocarbons as electron donors may present a significant source of sulphide in oil deposits and oil production plants. ,,,,,& Topof page

Abstract To read this article in full you may need to log in, make a payment or gain access through a site license (see right). Bacteria Basics This experiment will get you started with the basics of studying bacteria by growing a bacteria culture using a sample from the cheek cells inside your mouth. Remember to treat every bacterial culture with great caution! Adult supervision recommended. Make a culture dish using the following directions: Prepare agar according to the directions on the label, then pour enough into a petri dish to just cover the bottom. Rotate the dish to obtain even surface coverage. Cover it immediately and let stand until firm. Store upside down in the refrigerator until ready to use.

Once the culture dish is prepared, use a sterile cotton swab or inoculating needle andswab the inside of your cheek. Very gently rub the swab over the agar in a few zigzag strokes and replace the lid on the dish. You'll need to let the dish sit in a warm area for 3-7 days before bacteria growth appears. Record the growth each day with a drawing and a written description. The individual bacteria are too tiny to see without a high-power microscope, but you can see bacteria colonies. Distinguish between different types of bacteria by the color and shape of the colonies. When you are done observing your bacteria culture, dispose of it: add 1 tablespoon household bleach, re-cover the dish, and seal in a plastic bag before throwing away. Antibacterial Agents You need two culture dishes for this experiment, in which you'll demonstrate how antibacterial agents (such as antibiotics and household cleaners) affect bacteria growth. Leave the dishes with their lids off in a room-temperature location. Leave the culture dishes exposed for about an hour. While you wait, cut small squares of paper (blotter paper works well), label them with the names of the antibacterials you're going to test (e.g. "L" for Lysol, "A" for alcohol, etc.),, and soak each in a different household chemical that you wish to test for antibacterial properties. If you have time, you might also experiment with natural antibacterial agents, such as tea tree oil or red pepper. Wipe off any excess liquid and use tweezers to set each of the squares on a different spot in one of the culture dishes. The second culture dish is your "control." It will show you what an air bacteria culture looks like without any chemical agents. Store the dishes in a dark place like a closet where they will be undisturbed for a few days. After 3-7 days, take both culture dishes and carefully observe the bacteria growth in each dish, leaving the covers on. The bacteria will be visible in small, colored clusters. Take notes of your observations and make drawings. You could also answer the following questions. In the control culture, How much of the dish is covered with bacteria? In the sensitivity square test culture, Have the bacteria covered this dish to the same extent as the control culture? What effect have each of the chemicals had on the bacteria growth? Did a particular chemical kill the bacteria or just inhibit its growth?

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For further study, you could use an antibiotic disc set and bacteria study kit. See our Bacteria Science Project Guide for more tips on setting up an experiment.

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For a more advanced experiment, try this Gram Staining and Antibiotics project.

Homemade Yogurt - Using Good Bacteria Generally when people think of "bacteria," they think of harmful germs. However, not all forms of bacteria are bad! You can enjoy a tasty product of good bacteria by making a batch of yogurt at home. You'll need to use a starter (available at grocery or health food stores), or else one cup of plain, unflavored yogurt that has live cultures in it. (If it contains live cultures, it will say so on the container.) Slowly heat four cups of milk until it is hot, but not boiling or scalding. The temperature should be around 95-120 degrees to kill some of the harmful bacteria. Cool slightly, until milk is warm, and then add one cup of active yogurt or the starter. Put the mixture in a large bowl (or glass jars) and cover. Make sure that the bowl or jars are sterilized before using by either running them through the dishwasher or washing them with very hot water. There are two different methods for culturing the yogurt mixture: You can put the covered bowl or jars into a clean plastic cooler, and fill the cooler with hot water to just below the top of the culture containers. With this method, you will need to occasionally refill the cooler with hot water, so that the temperature of the yogurt stays consistent. The other method is to wrap the containers in a heating pad and towels, setting the heating pad on low to medium heat. Check the mixture after heating for 3 1/2 to 4 hours. It should be "set up," having a smooth, creamy consistency similar to store-bought yogurt. If the mixture is not set up yet, heat it for another 1-2 hours. When it is the right consistency, add some flavoring €”such as vanilla extract, chocolate syrup, or berries €”and store the yogurt in the refrigerator. It should keep for a couple of weeks. For safety, we suggest that you do not eat any yogurt that has separated or has a non-typical consistency.