Growing Bacteria collected from surfaces and observing the results.

Procedures:

1. Collecting and Isolating Specimen

• Collecting Specimen: choose one of the following. (Petri-dish’s cap must be held at all times, never place it on the desktop)

-Get a swab whip a public restroom toilet sit and whip swab gently back on the agar then cover plate immediately

• Store plates upside down in an incubator

-next class record the number of bacterial cultures by sketching

-compare with “control” plate

• DON’T OPEN PLATES or TOUCH THE INSIDE
• Isolating by Aseptic Technique

-examine your bacterial cultures. What you have now is a mixture of different bacterial cultures

-Since accurate studies of a bacterial species are possible only through the use of pure cultures, an incubator from one of the colonies must be physically separated. The method used is a four phase streaking pattern.

-the inoculating loop is used for making transfers and streaking of bacterial cultures

1. Flaming the Loop

• Heat from the middle of the wire and slowly move towards the loop
• Heat the wire until red hot

                              2. Before touching your selected colony, air cool loop or touch loop to another      

                            part of the agar where there is no growth to cool loop.

                          3. Using the sterile loop, streak cultures over one fourth of the surface of the                

                              agar plate, Then flame loop

                      4. Cool loop, pass the cooled the loop three or four times over intial streaked  

                               portion of the plate. Streak it without over lapping the next quadrant.

                     5. Flame loop and allow it to cool

                      6. Pass the loop over the streak portion of the second quadrant two or three times a    

             and then streak materials without over lapping over the third quadrant of the            

        plate

                   7. Repeat step 6 to streak last quadrant

                    8. Flame loop before you put it down. Finished

2. Identification

1. Bacterial Morphology

• To be entered in your data section, not your procedure. Type of margin, elevation, pigmentation, texture, light transmission

2. Gram Stain

• Make a bacterial smear
• Place bacterial smear in tray. Cover the smear with Crystal violet for about two minutes. (Use forceps for this procedure to keep the reagents off your skin)
• Place slide in Gram’s Iodine tray for about 1-2 minutes
• Remove
• Decolorize by washing slide under alcohol
• Cover with Safranin counter stain for two minutes
• Rinse with water blot excess without rubbing smear
• Dry

3. Viewing Bacteria Under Oil Immersion

1. Select a lightly stained area
2. Focus your sample with the 10x objective first (lowest power), then 40x, and finally the oil-immersion, 100x
3. Decrease the amount of light if you are having trouble seeing something
4. Turn the barrel of the microscope so that your smear is halfway the 40x and 100x objectives
5. Place a drop of oil in the middle of your smear
6. Carefully turn the objective until the 100x lens contacts the oil and snaps into position
7. Examine the lens, it should almost touch the slide, the oil should fill the space between the slide and the lens
8. If it touches the slide, carefully back up lens until there is about 1mm between lens and slide. While looking through eye piece use Fine Adjustment knob to move lens up and down. This should help you focus

3. Sensitive’s

Day 1

• Add approximately l ml of broth culture to Petri-dish wit bacterial growth media
• Spread over full surface of agar by rotating and titling Petri dish
• Allow film to dry (15 minutes)
• With sterile forceps or applicator place sensitivity discs on the agar surface
• Discs should be about 1 in apart and no closer than a half a inch from edge of agar
• Incubate

               Day 2

• Measure in millimeters the diameter of the zones of the inhibition zone
• Inhibition zone

-area between edge and outer edge of growth (not including disc)

Materials:

-inoculating loop

-gram stains

(Crystal violet, Gram Iodine)

-Microscope

-Petri-dish

-Agar

-Alcohol burner

-incubator

Data and Observations:

I observed that the bacteria grow plenty considering we didn’t culture it very long, only over night. The bacteria had a bad smell. It wasn’t colorful it was just an off whitish color. It mostly grew only on the spot where I rubbed it. It wasn’t liquidly, it was more of solid. In the sensitivities portion of the experiment the hand sanitizer and the ampillin were the only two things that are resistant to the E-coli. They weren’t close together either.

Analysis & Conclusion:

Since we used the incubator the bacteria grew, or cultured, faster than if we just let it sit out in a room temperature room. Also, the incubator controlled the bacteria’s growth by controlling how much heat the cultured bacteria will get and how often. If I was to do anything different I would let the bacteria sit in a room temperature room, to see how fast bacteria grows on its own. I now know that I shouldn’t put my hands in my mouth so much and that I should wash my hands more often because a lot of common surfaces are infected with nasty bacteria.