Exercise 1 gram-positive and gram-negative bacteria.The difference between these bacteria is that gram positives are not decolorized when alcohol or acetone is applied to the smear.They thus retain the primary stain and are colored purple when observed microscopically under oil immersion.Gram-negative bacteria,however,lose the primary stain upon decolorization and appear pink or red because of the safranin counterstain. >>>>> 1.To prepare a smear from a broth culture(liquid suspension of bacterial cells),merely spread a loopful of the culture over a 2 cm square area of the microscope slide.Aseptic technique must be used to accomplish this transfer and remember to ALWAYS FLAME LOOP BEFORE AND AFTER USE!! 2.For colonies on agar plate,place a loop(or two)of water on a glass slide Using aseptic technique,gently touch the bacterial surface growth with an inoculating needle or loop and remove a small part of the colony.Emulsify the bacteria on the loop into the drop of water until it just looks cloudy. Flame the loop to incinerate excess bacteria and allow it to cool.Continue to spread the smear with the loop until you obtain an even distribution of organisms over a 2 cm square area and remember to FLAME YOUR LOOP BEFORE PUTTING IT DOWN ON THE BENCH TOP. 3.Allow the smears to air dry(you may speed the drying by holding the slide high above the flame but do not allow them to become hot). 4.Heat fix the dried bacterial smears by passing the glass slides quickly through the flame two or three times. 5.Stain and observe the microscopic morphology and staining characteristics of each microorganism Gram stain Place the slide on the staining rack and stain the films according to the following method: (1)Crystal violet,1-2 minutes (2)Rinse in tap water and blot dry (3)Gram's iodine,1 minute (4)Rinse in tap water and drain excess water (5)Tilt the slide and add acetone-alcohol,drop by drop,until it flows colorlessly (about 5-10 seconds) (6)Rinse in tap water and drain excess water (7)Safranin,30 seconds (8)Rinse in tap water and blot dry (9)Examine with the oil immersion lens and draw the microscopic morphology microscopic morphology file://CWINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise1/exercise1b.htm (3of 6)[8/3/0024:9:54 PM) gram-positive and gram-negative bacteria. The difference between these bacteria is that gram positives are not decolorized when alcohol or acetone is applied to the smear. They thus retain the primary stain and are colored purple when observed microscopically under oil immersion. Gram-negative bacteria, however, lose the primary stain upon decolorization and appear pink or red because of the safranin counterstain. >>>>> 1. To prepare a smear from a broth culture (liquid suspension of bacterial cells), merely spread a loopful of the culture over a 2 cm square area of the microscope slide. Aseptic technique must be used to accomplish this transfer and remember to ALWAYS FLAME LOOP BEFORE AND AFTER USE!! 2. For colonies on agar plate, place a loop (or two) of water on a glass slide. Using aseptic technique, gently touch the bacterial surface growth with an inoculating needle or loop and remove a small part of the colony. Emulsify the bacteria on the loop into the drop of water until it just looks cloudy. Flame the loop to incinerate excess bacteria and allow it to cool. Continue to spread the smear with the loop until you obtain an even distribution of organisms over a 2 cm square area and remember to FLAME YOUR LOOP BEFORE PUTTING IT DOWN ON THE BENCH TOP. 3. Allow the smears to air dry (you may speed the drying by holding the slide high above the flame but do not allow them to become hot). 4. Heat fix the dried bacterial smears by passing the glass slides quickly through the flame two or three times. 5. Stain and observe the microscopic morphology and staining characteristics of each microorganism. Gram stain Place the slide on the staining rack and stain the films according to the following method: (1) Crystal violet, 1-2 minutes (2) Rinse in tap water and blot dry (3) Gram's iodine, 1 minute (4) Rinse in tap water and drain excess water (5) Tilt the slide and add acetone-alcohol, drop by drop, until it flows colorlessly (about 5-10 seconds) (6) Rinse in tap water and drain excess water (7) Safranin, 30 seconds (8) Rinse in tap water and blot dry (9) Examine with the oil immersion lens and draw the microscopic morphology microscopic morphology Exercise 1 file:///C|/WINDOWS/Desktop/clown/Microb Lab Web 2001/Exercise1/exercise1b.htm (3 of 6) [8/31/2002 4:29:54 PM]