Expo 6 (Roel or Ravikant Only)!!!!!

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EXPERIMENT 6: Microbes Everywhere

Read the entire experiment and organize time, materials, and work space before beginning. Remember to review the safety sections and wear goggles when working with chemicals.

Part 1 takes 3 to 6 days to incubate. Allow 15-20 minutes per slide for Part 2. Objective: To demonstrate the growth and characteristics of microorganisms. Materials: Student Provides: Paper towels Timer or clock

Distilled water Clear tape Candle, matches, or lighter

From LabPaq: Safety goggles

Clinical face mask Disposable gloves Test tube clamp Thermometer Wax marker pencil Microscope slides, 5 Field microscope Hand magnifying lens Metal spatula

Staining tray

Experiment Bag: Sterile slant tubes of agar, 6 Sterile swabs, 6 Gram Stain Kit: Crystal violet solution

Gram Iodine stain Safranine stain Decolorizer

Discussions and review: Microbes are microorganisms such as bacteria, molds, and yeasts. They are around us all the time, and no matter how hard we try to clean and disinfect our environment, they always come back. Some microbes are carried in the air and then multiply when they find a hospitable surface. Aerobic microbes need oxygen to live, anaerobic microbes do not. Microbes tend to be found more often in some places than in others. Most microbes, especially those found around the home, grow best in warm areas like the kitchen and bathroom and the warm folds of the human body. They also grow best where there are ample nutrients for them such as body oils or food residue and dirt that accumulate from frequent handling and insufficient cleaning.

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Since microbes can cause infectious diseases, it is important to know where they are most often found and to work to limit their numbers. In this laboratory exercise you will discover and compare the types of microbes found in your personal environment. PROCEDURES: Part 1 - Finding Microbes You will need: 6 sterile agar slant tubes, 6 sterile swabs, safe, warm incubation area. Note: Cultures need 3 to 6 days incubation period. Plan accordingly. Because this experiment involves culturing microorganisms from a human environment, it is possible that unknown microbes may become incorporated into the sample. Any culture that may contain an unknown organism should be treated as potentially pathogenic. Therefore, be certain to wear the gloves and mask provided when handling the cultures to protect yourself from unintended exposure. Handle the cultures carefully and maintain an organized, clutter free work space to prevent spills. As with all materials in your LabPaq, use and store the cultures out of the reach of children and pets. DO NOT CULTURE YOUR OWN THROAT OR OTHER BODY FLUIDS AS THEY MAY CONTAIN BACTERIA THAT WHEN CULTURED COULD INFECT SOMEONE ELSE. 1. Consider where might be the best conditions in your home for microbes to thrive.

Make a list and prioritize it based upon the above information about conditions favorable to microbe growth. Some places to consider are:

electric light switches door knobs residue around a bathtub or shower inside the end of a water faucet exhaust hood over the kitchen range drain pan under the refrigerator inside the garbage disposal (be sure to turn it off first!) anywhere around the kitchen where food might spill

2. Select six sources from your prioritized list that are from different areas of your

home. Set up a data table similar to the one shown below to number your microbes and record information and observations about them for Part 1 and Part 2 activities.

Location Of

Microbes Colony Growth Notes .... Gram Stain Notes ......... # Description Temp Shape Color Size Type Shape Color Size Type Observations 1 2 3 6

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3. Select a place where you can incubate cultures of the microbes you will collect. Chose a warm spot where the cultures will not be disturbed and cannot be reached by small children and pets. An ideal site is often the top of a hot water heater. If you choose to use your hot water heater, first place a multi-folded bath towel on top of the heater surface, and then place the incubation tubes on the towel. The towel will keep the tubes from rolling off plus provide insulation to keep them from overheating.

Electrical equipment tends to generate heat and makes another potential incubation site. Consider areas around electronics that run most of the time such as a transformer, computer, or stereo. (As far as we know noise does not bother microbes.) There are usually lots of warm spots around a kitchen. However, if you incubate cultures in your kitchen, NEVER open the tubes around where food is prepared or eaten. Rather, the culture tubes should be sealed when you take them into the kitchen and not opened until you leave the kitchen.

4. Mark the six sterile slant tubes of agar with a wax marker pencil, numbered 1

through 6. These numbers will correspond to the microbes you will collect from the sources you numbered 1 through 6 in Step B above. Do not open the agar incubation tubes until you are ready to place specimens inside them.

5. To collect smears of your six microbe sources, carefully follow these instructions for

each one. Systematically collect only one microbe source at a time. a. First measure and record the temperature at the collection site.

b. Open the cap of the correspondingly numbered sterile agar slant incubation tube

for the source number to be tested. Let the opened cap rest over the mouth of the tube until you are ready to use it. Your objective is to have the sterile tube ready to accept a smear while at the same time limiting its exposure to contaminants

c. Unwrap a sterile swab and tightly grip its stem in your dominant hand, usually the

right hand. Wipe only the end tip of the swab firmly across a small area of the selected surface. Do not swirl the swab or touch anything else with it. You should perform this and the next step fairly quickly, yet very carefully.

d. Remove the cap of the incubation tube and hold the open tube at eye level in the

hand opposite the one grasping the swab. Very, very carefully insert the swab into the incubation tube and firmly touch the contaminated end of the swab tip to the center of the agar slant tube. Ensure the swab tip contacts nothing but this designated site on the agar.

e. Recap the incubation tube; place the used swab in the trash, and repeat the

above steps a. through d. for the next of your five microbe sources.

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6. Place the six tubes containing microbe sources in the site previously selected for incubation. Make sure everyone in your home knows what the incubation tubes are and where they are located and that they should never be disturbed and especially that they should not be opened. Leave a note stating “Experiment in Progress – Do NOT Disturb” with the tubes while they incubate for three full days.

7. Keep the culture tubes closed! Use the hand magnifying lens to

observe the growths of colonies within the tubes. Note their shape, size, color, and anything else distinguishing. Bacteria will grow in small circular colonies, whereas molds will spread out more and may look fuzzy. Yeasts tend to grow initially in tight, compact colonies and their color is somewhat darker than that of bacteria. Record what you see in the data table. Draw what you see for each. Try to deduce the types of microbes each cultured colony contains: bacteria, mold, or yeast. Some cultures may contain more than one type of microbe.

8. If some of the cultures are not yet well developed let all of the cultures

continue to incubate for up to three additional days until you see visible growth in them all. Record final observations for each tube..

Questions: A. From which samples did you observe the most and least microbial growth? B. Why do you think there were differences in the number and types of microbes at the

different sampling sites? C. Did microbes from warmer or cooler sites multiply faster? Hypothesize why. D. How could the information gained in this experiment be useful in your home?

Part 2 - A Closer Look You will need: safety goggles, disposable gloves, face mask, paper towels, microscope slides, metal spatula, gram stain experiment bag containing crystal violet stain (#1), PVP iodine stain (#2), decolorizer (#3) and safranine stain (#4), staining tray, clean water to rinse slides, candle or matches. Most of the microbes that you have cultured are very small, much smaller than you can see individually with a hand lens and even a 150-power field microscope. However, at 150-power magnification you will be able to see the culture’s structure and possibly some individual microbes. That is why you will now prepare microscope slides and take a closer look at your home’s microbes. To make the microorganisms stand out in relief from the background light it is necessary to stain them. A common way of doing this is with Gram stain, a process using a sequence of stains that divide all microorganisms into two categories, either gram- positive or gram-negative. Gram-positive microorganisms will hold the blue color of

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the first stain in the sequence and will appear blue at the end of the process. Gram- negative microorganisms will lose the blue color of the first stain in the decolorizing step, but will hold the color of a subsequent stain, and will finally appear red in color. Past research has identified certain bacteria or pathogens as gram-positive or gram- negative; thus gram staining is often used in medical laboratories as a first step in diagnosis. The color results from gram staining quickly narrows down the possibilities during the microbe identification process. For example, the Streptobacillus from which we get “strep throat” is a gram-negative microorganism. When the medical laboratory cultures and stains the swab from your throat and the final color of the stain is blue, they know that you do not have strep throat. Important: Wear a clinical face mask, disposable gloves, and safety goggles at all times during this part of the experiment. Some microbes can become airborne and you do not want to breathe them in. Do not touch your face, especially your mouth and eyes, during this part of the lab exercise. At the end of the experiment, place the face mask and gloves in the trash and wash your hands and equipment thoroughly. It is wise to perform this experiment in an area where food is not prepared or eaten. While most of the cultures will be harmless, it is always possible to produce one that is contagious, so don’t take chances! Put on your mask, gloves, and goggles! 1. Select four of the six cultured microbes from which to prepare microscope slides.

Ideally you should choose the culture with the largest growth plus, assuming they exist, one each of the cultures that appears to be bacteria, yeast, and mold per your earlier identification. In your data table, note the four culture selections next to their corresponding numbers, source names, and suspected types. Label four microscope slides with the corresponding numbers of the four sources selected.

2. As detailed below, successively process the four selected cultures onto separate

microscope slides. These instructions are not complex, but they are long with many steps in this process. Read the instructions and fully understand all the steps before you begin. Complete all work for each slide before going on to the next culture.

a. Light a candle or have a match or lighter ready to ignite.

b. Place a clean microscope slide on a flat surface and arrange it to hang over the

side of the surface so you can later grasp it with the test tube clamp.

c. Open the culture tube containing your selected culture. Carefully insert the LabPaq’s metal spatula into the culture tube and scrape off a small portion of the targeted culture. Recap the culture tube.

d. Grasp the clean microscope slide with the test tube clamp and smear the

removed culture onto the center of the slide.

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e. Sterilize the metal spatula tip by slowly passing it through the flame of a candle, match or lighter flame for five seconds. Lay the spatula on a clean paper towel that can be used to wipe off any soot or other material collected on it.

f. Pass the underside of the smeared

microscope slide back and forth a few inches above the tip of a candle, match, or lighter flame. You want to heat the smear until it is dry, but not burn it. The heat fixes the culture on the slide so it will not wash off during the staining process

g. Hold the slide over the staining tray from the LabPaq as

shown in the photo on the right, place three drops of crystal violet solution (Gram stain #1) on the smear, and let it stand for one minute. Crystal violet is a very strong stain. Do not let it contact anything that can be damaged. If you must set the stained slide down, set it in the tray.

h. After one minute, lightly rinse the stained slide by briefly passing it through a thin

stream of running tap water. Gently shake the excess tap water from the slide into the sink and rinse the excess down the drain. Note: these slides can also be rinsed by dunking them into a glass of clean water several times and then shaking the excess water into the staining tray or onto a paper towel.

i. While holding the slide over the staining tray, place three drops of PVP Iodine

(Gram stain #2) on the smear and let it stand for one minute. Iodine is also a strong stain so do not let it contact anything that can be damaged.

j. After one minute, again lightly rinse the stained slide by

and gently shake off excess tap water.

k. While holding the stained slide over the staining tray, slowly and repeated drop single drops of decolorizer (Gram stain #3) onto the smear, as shown, until the drops running off the slide no longer have any color. Briefly rinse the slide again r and gently shake off the excess tap water.

l. Place three drops of safranine (Gram stain #4) on the smear and let it stand for

30 seconds. Safranine is also a strong stain so do not let it contact anything that can be damaged.

m. After 30 seconds, briefly rinse the smear again and gently shake off the excess

tap water.

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n. Gently blot the unsmeared areas of the slide with a paper towel or tissue, but

NOT the actual smear area. Let the slide air-dry for a minute or two.

o. Note the color of the stained material on the slide. Examine the slide under your microscope. Describe and draw what you see. Do not wash the slide yet. Keep it for now so you can compare it to the other slides.

p. Repeat steps a. through o. above to successively stain and examine slides for

each of the other cultures selected. 3. After you have finished examining all the slides and answered the following

questions, wash the slides well with detergent, rinse them with distilled water, and allow them to air-dry before returning them to your LabPaq. Properly clean all other equipment. Place the sealed incubation tubes and used paper towels in the trash.

Questions: A. Was the structure or arrangement of the colonies of microorganisms different among

what you identified as bacteria versus yeast versus mold? B. Four slides are, of course, a very small sample, but regardless of this limitation, what

can you hypothesize about differences in the microbe growth patterns? Does your hypothesis match the descriptions at the beginning of the experiment?

C. Were you able to see any individual microorganisms? If so what would you guess

they are (e.g., mold, yeast, etc.)? D. Do any of the slides appear to have more than one type of microorganism? Did you

determine this by physical appearance of the culture or by color of stain?