Safety Precautions:
1. Always use STERILE TECHNIQUE
2. ASSUME that any bacteria that grows on your plate is harmful and treat as such
3. Always WASH HANDS after you are finished handling plates
1. Always use STERILE TECHNIQUE
2. ASSUME that any bacteria that grows on your plate is harmful and treat as such
3. Always WASH HANDS after you are finished handling plates
A solid source for bacterial growth is essential if organisms are to be isolated. In the laboratories of Robert Koch, gelatin was first used to achieve bacteria colonies. Agar now serves as a more useful material; it will remain solid until heating and then will solidify as it cools. Before it cools (and solidifies) it can be poured into petri dishes. Using sterile technique minimizes the contamination of the agar before you are ready to inoculate it with bacteria from other sources.
1. Without opening any of the petri dishes, use a marking pen to divide the dish into four quadrants and label the sections as shown. TIP: We do not label the lids of dishes because they can easily fall off and be mixed up.
2. Holding the test tube with your left hand, raise the lid of the petri dish with your right (raise only enough to pour the agar). Pour the agar into the plate.
3. The agar will solidify shortly. Turn the dishes upside down, the dishes will be stored and incubated upside down. Until the plates are inoculated, they will be stored in the refrigerator.
| Surface 1 | Any surface in classroom, hall or bathroom. Use sterile cotton swabs. |
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Surface 2 |
Any surface in classroom, hall or bathroom. Use sterile cotton swabs. |
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Hair |
Place a small length of your hair in this quadrant |
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Finger |
Touch the circle marked D with your finger. Then wash your hands and rub your finger with alcohol. Place your “disinfected” finger in circle C |
Analyzing Colonies
Terms
1. Colony Shape and size -- round, irregular, punctiform (tiny)
2. Margin edge -- entire (smooth), undulate (wavy), lobate (lobed)
3. Elevation -- convex, flat, raised
4. Color -- color + opaque, translucent, shiny or dull
5. Texture -- moist or dry (rough)
Choose 2 colonies from your plates to examine. Make a note of the description of these colonies (using the terms above)
1. Place a drop of water on a clean slide.
2. Heat the inoculating loopuntil it glows red. Let it cool then remove a small amount of culture from the agar surface; touch it several times to the drop of water until it just turns cloudy.
More is not better! Too much bacteria and you won't be able to see individual cells
3. Burn the bacteria from the loop and allow the loop to cool, use the loop to spread the suspension (water + bacteria) over the surface of the slide to form a thin film.
4. Allow suspension to air dry. This process will be short if you spread the liquid out in step 4.
5. Hold the slide with a clothespin and then heat fix the bacteria on the slide by passing it over the flame 3-4 times (film side up). Do not overheat the slide, it should feel warm but not hot.
6. Place a drop of methylene blue on the slide over where the “smudge” of bacteria and water is
7. Allow the dye to remain for approximately 1 minute (the bacteria will take up the stain during this time)
8. Wash the excess stain off the slide by picking the slide up and holding it at an angle over the sink. Gently rinse the excess dye off with water.
9. Blot off excess stain and water using filter paper - DO NOT RUB!
10. Examine the slide under the microscope using the high power objective.
You may need to repeat this process a few times in order to get a good slide! Enjoy!
** Draw your bacteria; identify the shape and arrangement of the bacteria.
Microbiologists use a special level of classification to describe eubacteria – it is called the “Division”. Eubacteria are placed in a division depending on the type of cell wall they have: gram (+) walls, no walls, or gram (-) walls. Bacteria are stained so that their cell walls can be observed. A bacterium with a cell wall that has a large amount of the carbohydrate –protein compound, petidoglycan, is classified as gram (+) and stains blueish-purple. Bacteria with a thin peptidoglycan layer are classified as gram (-) and will stain pink.
Reconstitute the Culture:
Follow the directions on the pamphlet that comes with your microlive culture.
Gram Staining
Add one or two drops of the reconstituted culture (remember to wait 30 min) to a clean microscope slide held by a clothespin. Tilt the slide back and forth to spread the mixture then allow it to air dry. HEAT FIX the culture by passing it briefly over a flame.
1. Add one or two drops of a known culture to the slide and follow the steps above to heat fix the culture to your slide. The staining process is different for a gram stain. Alternately, you can take your known culture from bacteria grown on a petri dish (if microlive cultures are not available)
2. Add about 5 drops of crystal violet over the fixed culture. Let stand for one minute.
3. Pour off excess stain and gently rinse the slide in tap water. Keep in mind, the objective is to wash off the stain, not the fixed culture.
4. Add about 5 drops of iodine to the smear. Let stand for one minute.
5. Pour off excess iodine and rinse the slide with water as before.
6. Tip the slide and add several drops of the ethyl alcohol to the upper end. Allow alcohol to flow over the smear. After several seconds, rinse the slide again.
7. Add about 5 drops of the safranin to the slide. Let stand for one minute.
8. Rinse the slide again, drain and blot away excess moisture. Let the slide dry.
Lab Report
1. Title (Something clever)
2. Purpose – What are we trying to learn about here?
3. Topics covered in the lab
4. Conclusions – What did you learn? What would you like to do over or expand upon? This is the area for your own reflections and thoughts.
Must be TYPED. Due Date: _________
