Transfer and Isolation Materials required (per group):

Transcription

1 Aseptic Technique and Culture Transfer - 1 Aspetic transfer The purpose of this laboratory exercise is to develop facility in transferring bacterial cultures from one kind of medium to another using various instruments such as the loop, needle and pipette. An axenic culture may be defined as a population or organisms, all of which are of the same type. If a suspension of mixed cells is spread so thinly over a solid surface of a culture medium that individual cells are widely separated on the surface, then each cell will neg m to grow and soon divide, as will each of its progeny cells. On a solid surface, the migration of cells is not possible or is slowed greatly, thus the progeny or the one original cell will accumulate in a small area and pile up there to form a colony uncontaminated by neighboring cells. A colony mav sometimes be described as a clone, a population in which all of the organisms are the direct descendants of a single ancestor. Such a colony is visible to the naked eye, and may contain from 10 7 to individual cells. Such colonies often require from one to two days to achieve a size large enough to be noted easily. Dispersal of a population into individual cells and subsequent colony growth forms the basis of the streak plate isolation method, first developed by Robert Koch in the 1880s. To spread and separate cells, a sterile inoculating loop is placed in a suspension of mixed cells and is then lightly streaked across the surface of an agar plate. As the plate is streaked, organisms are gradually dislodged from the loop, first in large numbers, but later in smaller and smaller numbers as the process is continued. The pattern of streaking can he arranged to insure a high probability that cells will eventually be deposited individually and widely separated from one another in some region of the plate. There are several equally effective variations in streaking patterns. Streaking a plate for colony isolation will be the subject of a future lab exercise. Aseptic transfer of microorganisms using a needle, loop, and pipette are summarized in figures shown on the next few pages. Practice the transfer of bacteria from slants and broth to slants, broth, and plates, as directed by your instructor. Transfer and Isolation Materials required (per group): tryptic soy agar (TSA) slants plates of TSA tubes tryptic soy broth (TSB) sterile petri dishes sterile 1 ml pipettes sterile water blanks A slant culture, broth cuture and a plate culture will be provided.

2 Aseptic Technique and Culture Transfer - 2 Transfer Procedure: 1. Transfer the bacteria from the slant culture provided to {a) a nutrient agar slant and (b) a tube of nutrient broth. Label the slant and the tube with the name of the organism today's date, and your name. 2. Transfer the bacteria in the broth culture to a nutrient agar slant. Label the slant. 3. Transfer the bacteria from the slant culture to one of the nutrient agar plates, using the streak plate procedure. The instructor will demonstrate the technique. You may make a suspension of the slant culture provided in the 1 ml sterile water blank and then streak the plate as demonstrated. Label the plate. 4. Transfer the bacteria from the broth culture to streak a second nutrient agar plate. 5. Label the plate.

3 Aseptic Technique and Culture Transfer - 3

4 Aseptic Technique and Culture Transfer - 4

5 Aseptic Technique and Culture Transfer - 5

6 Aseptic Technique and Culture Transfer - 6

7 Aseptic Technique and Culture Transfer - 7

8 Aseptic Technique and Culture Transfer - 8

9 Aseptic Technique and Culture Transfer - 9

10 Aseptic Technique and Culture Transfer - 10

11 Aseptic Technique and Culture Transfer - 11

12 Aseptic Technique and Culture Transfer - 12

13 Aseptic Technique and Culture Transfer - 13

14 Aseptic Technique and Culture Transfer - 14 INOCULATION OF BROTH TUBES WITH A PIPETTE

15 Aseptic Technique and Culture Transfer - 15 ALTERNATIVE METHOD OF CULTURE TRANSFER FROM TUBES, The Two-Tube Method. This method can be faster and be a little less prone to contamination from airborne organisms, but it requires practice and mastery of the other methods shown on the previous pages.