Your First Microbiology Experiment

The first microbiologists did not have culture tubes, fancy equipment, or special supplies. Just like them, we can accomplish much with the same kitchen foods they used. If you have culture tubes, use them, but otherwise you can conduct this experiment using a drinking glass or small bottles or any other useful container. One advantage of culture tubes is that it costs less to fill them, but the materials for this experiment are cheap.

Our first experiment is very inexpensive. We will try to culture (grow) baker's yeast (from the grocery store) on various carbohydrates learn what it needs to grow. Before a microbiologist can do much with a microbe, he must find a way to grow it. As we work with baker's yeast and then other microbes, you will learn what is needed to grow microbes and what containers will be useful. Thus you can begin building up supplies and equipment for your home microbiolgy lab.

Safety Now. Don't begin this experiment until you have read my general safety page and the special safety notes for these experiments at the bottom of this page. Become a student who always works carefully and safely.

Expt 1.   Growing a Baker's Yeast Culture

Growing a yeast culture is simple and easy, yet it introduces many concepts you need to understand if you want to conduct a serious microbiology project. This experiment will demostrate some of the biochemical principles used to identify bacteria and yeast species. You can find everything you need at home or in the grocery store and that is the first reason we are beginning with bakers yeast, Saccharomyces cerevisiae, but another reason is that since we eat yeast, we know it is safe for beginners to work with. This simple experiment can be expanded into many more complex experiments and become an outstanding science project.

The standard steps for growing a microbe are

• study the literature (books, journals, and web) to learn what food, light, temperature, and other conditions it needs for growth, or just make a guess from your knowledge of its habitat.
• prepare the culture medium (the liquid or agar mixture for growing it)
• put the medium in tubes, plates, or other container
• sterilize or pasteurize the containers of medium
• store the sterile containers of medium under suitable conditions
• inoculate the sterile medium with the microbe
• incubate the culture containers under suitable conditions: temperature, light if needed
• observe the growth, production of gas, and other details
• record results for future use and to plan future experiments.

To search the web for information useful for this project, I would try these keywords: yeast, baker's yeast, baking yeast, "Saccharomyces cerevisiae", culture, yeast culture,culture medium, bread making, agar, microbiology, science project, home science project, science fair, zymology, enzymes, glucose, sucrose, starch, hydrolysis, slime flux, etc.

Preparation of the Medium.

Medium is the name for the food we grow microbes in. The medium must contain everything our yeast needs.

• First we need to figure out what this yeast needs. Bakers use flour, table sugar, and water to make bread. In nature, yeasts are found in flower nectars, in slime fluxes (sap running out of tree wounds; such sap is alive with yeasts and insects that like sugars and yeasts).
• Recall that living organisms need a carbon source, an energy source, a nitrogen source, minerals, and may need vitamins. Carbon and energy source is often a single compound such as glucose (corn syrup). If in doubt, always try corn syrup.
• We will try corn syrup and water in this experiment. We will hope the water has enough minerals to supply the yeast's needs. After all, home bakers do not add any minerals.
• This mixture may not have enough nitrogen for the yeast to grow. If the yeast does not grow we could try meat broth, peptone, or another source of amino acids. The dry power yeast probably contains some items the yeast needs. Very likely the manufactuer adds them to insure the home baker is successful. However, we are not using flour and flour does contain proteins, minerals, vitamins, and starch.
• It tapwater does not have enough minerals, we could try water from a mud puddle or soil water made by mixing soil with water and using the clear supernatant water after the mud has settled. Such soil extract will contain lots of bacteria and should be sterilized first.
• How much corn syrup shall we use? Microbiologists commonly use 10 grams of the carbon and energy source per liter (1.1 quart). Corn syrup is mostly glucose which is a sugar most microbes can use. Recalling that a liter is 1000 mL or 1000 cubic centimeters, we will add 10 mL of corn syrup to a liter of water. Your ruler is marked in centimeters. So we want to add a slug of corn syrup about 1 cm x 1 cm x 10 cm long. You can estimate this volume easily and you need not be exact. A level tablespoon of corn syrup is probably about 10 to 13 grams of glucose content.
• Corn syrup and water may be an adequate medium, but dry yeast has some minerals and perhaps some nitrogen matter; lets try it and see if the yeast will grow. Read the label on your syrup. Much syrup sold today contains corn syrup and high fructose syrup. Fructose is sweeter than glucose and some microbes can use both sugars. Later we will study microbes which can't grow in the presence of sugars and other organic matter.

Tube your medium and sterilize it.

• Normally we put our medium in culture tubes, plug them with cotton, and then sterilize them. We will omit the sterilization step for this experiment. This site has an entire page on sterilization. In this experiment we are using so much yeast that the yeast will overwhelm the contaminants. In normal bread making we do everything so fast the contaminants do not have time to grow and poison us. Besides the yeast probably makes substances which interfere with the growth of other organisms.
• It would be nice to use a gas collection tube in this experiment. If you can find a small bottle or small culture tube, fill it with the syrup-water- yeast mixture and invert it in one of your culture containers with the medium. If the gas tube contains a bubble try again. The gas tube will collect gas if your growing yeast makes any gas. If you know some chemistry, you could try to identify any gas produced. [Hydrogen explodes, methane explodes, carbon dioxide and nitrogen extinguish flames by failure to support combustion].

Inoculate your culture(s).

• Inoculate means to introduce a microbe into a tube of medium.
• You can inoculate your tube or tubes with a few bits of the dry baking yeast you got at the grocery store. Save the remainder of the package for future experiments.
• A better way to inoculate tubes is to use the same amount of inoculum in each tube. Therefore, I suggest you dissolve a little dry yeast in water and add one drop of the suspension to each tube of medium. Limit the amount of yeast added so the culture is not cloudy. We like for cultures to be as clear as possible at the beginning so it will be easier to see the cloudiness as the yeast cells begin to grow.
• This will not be a pure culture of yeast because the water, containers, and your fingers carried various fungi and bacteria. Since yeast grows rapidly and outnumbers the contaminants, the contaminants will not have much effect. That is why bakers do not sterilize their medium. Bakers just try to keep things clean as possible. However, due to the contaminants the bread will become moldy after a few days. Commercial bread contains calcium propionate to retard growth of molds. Look at any bread label; what is the additive (hint it gives cheddar type cheeses some of their sharp flavor).
• Do not inoculate all your tubes, save some as controls. Incubate the controls beside the experimental tubes to determine whether control tubes remain unchanged. Since we did not sterilize our medium, the control tubes will likely get turbid soon and eventually rot.
• Any leftover syrup-water will rot. Therefore, you might as well discard it right away.

Incubate your yeast cultures

• Place them in any warm place to grow.
• If you wish place some tubes at different temperatures, such as, in refrigerator, hottest place you can find, and other temperatures in between. Suggested temperatures are 4C (refrigerator), 25C (room temperature), 35C (very hot room), 15C (very cold room)
• Mark your tubes so no adult or child drinks them. The sugar water is harmless, but the contaminating microbes may not be safe. It would be a good idea to place them in labeled box. Absolutely keep all your experiments out of the reach of childern!

Record the results

Examine your tubes for growth at daily or 12 hour intervals. Discard any tubes that get moldy, wash them carefully so you do not inhale any mold spores. If any tube breaks, do not get cut by the contaminanted glass. If you get cut, wash the wound with clean soap and water and flood with alcohol.

• Did the clear medium become cloudy (evidence of multiplying cells)?
• Did any odor develop (Never sniff moldy tubes)?
• Did any sediment develop in the tubes; compared to the uninoculated tubes?
• Did any gas collect in the filled inverted gas tube inside your culture?
• Did growth occur differently at different temperatures?
• Did any growth occur at the hottest temperature. If not move that tube to the temperature that gave the most growth. Does it then grow? Some microbes die at high temperatures. (Of course very high temperatures will kill any microbe, but the killing temperature varies. If spores are produced, they survive much higher temperatures.
• Write down any ideas you have for improving your next experiment.
• As time goes on, growth will slow down and eventually all the cells will die.
• Does this yeast make spores? How can you determine that?

If you wish, you could perform the above experiment using a bit of garden soil (or slime flux) as the inoculum instead of bakers yeast. Since garden soil contains dozens of different species of bacteria and fungi, the results will be different and perhaps useless because soil usually contains bacteria capable of growing at most temperatures and on most foods.

Expt 21B. Growth of Bakers Yeast on Other Sugars.

Biochemists have discovered dozens of sugars and other carbohydrates in nature. Various species of bacteria and yeasts differ in their ability to grow on these carbohydrates. The ability of a strain of bacteria or yeast to grow on certain sugars is one of the main methods for indentifying them to Genus and species. Since fungi have huge genomes and the genetic codes to make enzymes to digest most carbohydrates, sugar digestion is not as useful in for identifying fungi.

• You can repeat Experiment #1 using different carbohydrates. If the yeast grew on corn syrup water medium, try making media from water and each carbohydrate you can find.
• Starch and table sugar are the other two carbohydrates found in most kitchens. Some people may have maltose. Can you make some maltose by using sprouting barley which contains the enzyme maltase?
• Table sugar (sucrose) is a disaccharide made by plants (maple, beet, cane, and others). Plants make sucrose by joining one molecule of glucose to one molecule of fructose. Microbes can't use sucrose unless they have an enzyme capable of splitting sucrose apart to give glucose and fructose.
• Starch is a long molecule composed of hundreds of glucose molecules hooked together to make a long chain-like polymer. Microbes can't use starch unless they have enzymes to break it apart into separated glucose molecules. You probably should boil the starch in water before you use. It will still be long chains, but the boiled solution will be cloudy instead of white.
• Maltose is a disaccharide consisting of two molecules of glucose joined together covalently.
• Lactose is a disaccharide consisting of one molecule of glucose and one molecule of galactose covalently joined. Recall milk contains a trace of glucose but lots of lactose. Does bakers yeast ferment milk. If you do your work carefully using lots of yeast, not just one crumb, the yeast/milk should be safe to taste, provided a qualified cook (your mother?) helps you.
• If you have a interest in becoming a bio-scientist, you could memorize this info about starch, glucose, sucrose, lactose, maltose. This one is easy lactose = glucose + galactose. The ability to recongnize the importance of learning such things will help others know you have aptitude and interest in biochemistry.

Testing the ability of bakers yeast to grow on three carbohydrates.

• Dissolve 10 mL of cornsyrup per liter of tap water. While it is not pure, label it "Glucose Water."
• Dissolve 10 mL of table sugar per liter of tap water and label it "Sucrose Water."
• Dissolve 10 mL of starch per liter of tap water and label it "Starch Water."
• Dissolve 10 mL of dry milk per liter or use skim milk if you have it and label "Milk". In microbiology experiments we prefer skim milk to avoid the little globules (balls) of fat which make the solution turbid.
• Prepare your inoculum by dissolving yeast in tap water. Try a couple matchheads amount in 15 or 20 drops of water--I am guessing.
• I like to do all my experiments in triplicate. Thus, I would set up 3 tubes of Glucose water, 3 tubes of Sucrose water, 3 tubes of Milk, and 3 tubes of Starch water. If you make 3 identical tubses, it sometimes happens that the three tubes do not give identical results. Sometimes it is difficult to find a reason for the difference. If you made only one tube and it had an error, you would be mislead by the false result.
• I would now inoculate each tube by adding the same amount of inoculum. I would add one drop of yeast suspension to each tube.
• I would conduct all experiments using an inverted gas collection tube in each carbohydrate tube if possible. You might not get the same gas result from all carbohydrates.
• This experiment would be more informative if you sterilize your medium. See B02?

Additional suggestions:

• you can use 500 ml or other size soda pop bottles to measure volumes.
• 20 drops of water equal about 1 mL
• begin collecting pop bottles and other useful things for your home microbiology lab.

Expt #21C. Using A Complete Medium

Corn syrup water is not a very good medium because it is low in nitrogen. All living things require amino acids to make their proteins. Some proteins make up muscle or other structures; other proteins are called enzymes. Enzymes are catalysts which reduce the energy level required for reactions. Thus, fire at high temperature burns sugar, but using enzymes some bacteria can burn (oxidize) sugar near the freezing point.

While all organisms require nitrogen, very few can use nitrogen directly from the air. Plants can use inorganic salts called nitrates and some can use ammonium salts. Many bacteria can use ammonium, nitrite, or nitrate salts and some can use nitrogren direct from the air. Many microbes and humans can use only amino acids. Humans can eat proteins and digest them down to amino acids. It is hard to find sources of free amino acids around the home, but there are lots of sources of proteins including egg white, milk, meat, and beans (has other things also).

Since some microbes require amino acids, common microbial media contain proteins, peptides, or amino acids. Peptides are partially digested proteins. We could add milk to our corn syrup medium. Milk alone makes a good medium for some bacteria because it contains proteins and lactose, a sugar. The white color of milk makes it difficult to see the growth of the bacteria unless the bacterium has a strong color. Meat broth is a good way to add proteins, peptides, and amino acids to a medium.

Using these ideas you can try to make a more complete medium for bacteria. By February 1998, this site will have a page devoted to home-made media.

For now you can try some experiments on your own, such as adding soil to milk and incubating at 4C, cold room, normal room, or higher temperatures. Expect some very strong odors. If you screw the lids on tight, there is a risk that gas pressures could build so high that the bottle or tube will burst. Don't overlook the use of plastic bags for cultures, but they may leak.

Expt #21D. Selective Medium

Many kinds of food are made using selective medium and bacterial fermentations. Salt or vinegar is often added to prevent the growth of most kinds of bacteria. Try adding varying amounts of salt to tubes of a good medium for yeast add gas collection tube if you have them, does the salt interfere with growth of the yeast. Some yeasts grow at remarkably high salt levels.

Try adding vinegar in varying amounts to your proven yeast medium. In general, yeasts are noted for growing at acidic pH. If you don't have a pH indicator, you might try red cabbage juice. See URL:
http://wwbbs.otherside.com/PUBLIC/HOMEPAGE/haroldeddleman_303/INDEX.HTM

Here is a cabbage experiment. Slice some cabbage finely. Pack it tightly into 3 small jars. Add dilute vinegar to jar #1. Add dilute salt water to Jar #2. Add nothing to jar #3. Put the lids on loosely and incubate at room temperature or a little cooler. Jar #3 will rot quite quickly. Jars #1 and #2 will rot but less so. For an interesting project, get a canning book and the help of your mother, grandmother, or any experienced canner and try making fermented pickles or sauerkraut using such selective methods. The food is safe to eat only if you follow the directions carefully--it is safer to have an expert cook with canning experience to help you.

During 1998 this site will have pages dealing with selective enrichment methods. The first such page is Isolation of Vibrio phosphoreum using low temperature and seawater to select luminous bacteria on fresh seafish or squid. Fresh fish from inland markets is seldom fresh enough to have the bacterium.

Suggested Further Experiments (incomplete)

Much of this will be used on other pages .

Most advanced microbial experiments require the use of solid media for isolation of pure cultures and the use of sterile medium. Pasteur accomplished much using selective methods, but he and others began using sterile medium and then rapid advances began.

move page LINK for moremedia and steiliar;ona

• look for slime flux on tree and inoculate karo water.
• colored milk in discarded milk jugs. Psudomonas
• can reteat exptusing yel dust basteria

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• Mix some corn syrup, or table sugar, or meat broth with tap water (or water from a clear puddle). Use all three if you wish. This will be your medium for the growth of your microbe. It is best if your medium is very clear (free of particles). It is better if it is close to water white so don't use black corn syrup unless that is all you have. Do not use too much sugar because too much sugar will interfere with the growth of your microbe. __ teaspoon of sugar to __ cup of medium is about right.
• The medium will contain lots of bacteria and mold spores. Boiling for a while will kill most of the growing bacteria (vegetative cells) but not all of the spores.
• in each container.
• Put some food in each container

Observe examples of microbial growth around you.

Selective growth of an organism. vingegar, salt.

Notice that when microbes multiply gets cludy, corn syrup in water and inoculate.

sterile toothpicks tostart rotten apples

tyddalize milk 3 days in a row save a conrtol

Safety Rules for Microbiology

Be sure to keep your experiments away from small childern

Work with your parents to plan a place to keep your things so that small kids or unsuspecting adults do not drink or eat your experiments because they thought it was food. This means you should not keep your experiments in the refrigerator or on food shelves. You can keep them in suitable boxes with a sign so they are will marked.

Safety Rules.

Safety First. Do not sniff moldy items. There is a chance that the spores could get into your longs and begin to grow. Certain fungi can grow in our lungs and antibiotics do not help much. Death has a good chance of occuring if you get infected by such fungi. I will write about the fungi later. Actually, one cubic centimeter of air in homes commonly has 5,000 or 8,000 particles of dust and some of these are mold spores including the dangerous ones. Each good deep breath contains about 20 million particles.

Rule #1. Don't waste too much time wishing for equipment you don't have. Spend your time thinking about things you can do with what you have.

I love working with bacteria, yeasts, fungi, protozoa, algae, and bacterial viruses at home. The study of these things is called microbiology. The study of all of them uses approximately the same equipment. Therefore, this series of pages B020 => B024 will concentrate on the supplies and equipment for studying bacteria. Later, I will write pages about the equipment to study the other organisms.

You should read these pages in the order listed.

I might be helpful to read the first four pages before you do anything. The begin some simple experiments and you will learn what you need. Later you may want to buy some supplies, but at first see how much you can do with items from your home, farm, and gifts of discarded items.

• B021 - First do some simple experiments to learn what you need. Working with non sterile equipment.ooker, loop, sterile sticks or paper strips.
• B022 - Making a meat broth medium.
• B023 - Tubing your media
• B024 - Sterlizing and storing your media

These experiments still need lots of rewriting, but the facts are correct. If you were able to sterilize your medium. Save a culture of the yeast for your collection, but it is just as well to wait until we can isolate a culture by streaking in a later experiment.

If you have a pressure cooker, put your liquid medium in a glass jar with the lid on loosely and cook (autoclave) at 15 pounds pressure for 15 minutes. Read the manual for the pressure cooker to learn the setting for 15 pounds and read the safety precautions. Get the owner of the cooker to supervise your work. It is possible for a pressure cooker lid to fly off and injure you. Do not forget to p

In fact, the early humans and all animals and plants are involved with microbes. With each breath, you inhale about 5 million to 20 million dust particles and many of those are microbes.

Bacteria and other microbes are all around us. I am having trouble getting this page started because there are so many things we can do that I do not know what will interest you the most. I will just begin listing some simple experiments and you can do what ever you like.

IF YOUR YEAST DID NOT GROW ON STARCH

• recall that your saliva contains an enzyme called salivary amylase which digests starch. does it digest starch enough to allow yeast to grow on starch.
• recall that acids catalyze hydolysis of starches, proteins and other condensation polymers. Add a drop of acid. battery acid, muractic acid, vinegar. and place the tube in boiling water for a while.
• can you think of other ways to dryrlize starch, such as barley sprouts (malt)