Bacteria Lab

Day 1 - Working with Agar

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 resolidify as it cools. Before it cools (and resolidifies) it can be poured into petri dishes. Using sterile technique minimizes the contamination of the agar before you are ready to innoculate it with bacteria from other sources.

You will be provided with a test tube of melted agar and four sterile petri dishes. The top lid is larger and fits over the bottom dish, do not separate the lid from the dish except during pouring, as you will contaminate the dishes.

1. Without opening any of the petri dishes, label the bottom with your name, hour, and a number (#1-4)

2. Open the agar test tube by removing the packing cotton. Be careful, the tubes may be hot as they have been sterilized and autoclaved (heated and pressurized) to remove any bacteria.

3. Heat the lip of the test tube and keep the tube angled for pouring. Avoid raising the tube upright since bacteria in the air will enter the sterile agar.

4. 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 until the dish is 1/3 to 1/5 full and replace the lid.

5. Repeat this process for the next three petri dishes, stacking them one on top of the other. This stacking allows them to cool more evenly and reduces condensation.

6. Return the agar test tubes to the instructor.

7. The agar will resolidify. Turn the dishes upside down, the dishes will be stored and incubated upside down. Until the plates are innoculated, they will be stored in the refrigerator.

Day 2 - Innoculating Your Plates

Instructions

1. Open the sterile swab and moisten with distilled water.

2. Rub swab across the object with all sides of the swab by twisting and turning. Each plate will use a different swab to avoid cross contamination.

3. Open the lid of the plate only when you innoculate and close right after. Innoculate with the cotton swab by gently rubbing it over the surface of the agar. Do not rub hard enough to gouge the agar.

4. Use the scribble method to innoculate yoru plates. Streak then turn plate 1/3, streak then turn plate 1/3, streak.

5. Store petri dishes upside down, they will be placed in an incubator overnight.

6. WASH HANDS!

Day 3 - Analyzing Colonies

Identifying and categorizing different, isolated bacterial colonies based on varied appearance and morphology (form and structure) will permit the selection and transfer of different species from a mixed culture to a sterile medium. When a single bacterial cell is deposited on the surface of a nutrient medium (agar), it begins to divide exponentially. After thousands of cells are formed, a visible mass appears, which is called a COLONY. Each species of bacteria will exhibit characteristic colonies.

Terms

1. Colony Shape and size -- round, irregular, punctiform (tiny)
2. Margin edge -- entire (smooth), undulate (wavy), lobate (lobed)
3. Elevation -- convext, flat, raised
4. Color -- color + opaque, translucent, shiny or dull
5. Texture -- moist or dry (rough)

** Draw each plate, showing how colonies are spread across the agar surface.

**Pick several colonies on your plates and describe them.

Preparation of a Bacterial Smear

Choose 2 colonies from your plates to examine. Make a note of the description of these colonies (using the terms above)

1. Clean a slide using a bleach solution and dry the slide using a cloth.
2. Place a very small amount of distilled water on the slide.
3. Remove a small amount of culture from the agar suface and 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.
4. Burn the bacteria from the loop and allow the loop to cool, use the loop to spread the suspension over the surface of the slide to form a thin film.
5. Allow suspension to aire dry then heat fix it 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. Cover the smear with methylene blue.
7. Allow the dye to remain for approximately 1 minute.
8. Wash the excess stain off the slide by picking the slide up and holding it at an angle over the sing. Gently drop distilled water over the slide to rinse the excess dye off.
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.