Microilgeal| Ctr Matbods 22 cataton at ‘Applies ab Manual, ‘nueabes gh Ein list thatthe agar shakes have been liquefied for you prior to lab time.) Label the six tubes with the organisms assigned to you (one organism per tube), your initials, and as- signment number, Note: Handle the tubes gently to avoid taking on ‘any unwanted oxygen into the media. If the tubes of FTM are pink in the upper 30%, they must be oiled a few minutes to drive off the oxygen, then ‘cooled t0 inoculate. 3. Heavily inoculate each of the TGYA shake tubes 4, Inoculate each of the FTM tubes with the appro. priate organisms. 5. Streak your three organisms on the plate of anaer- ‘obie agar in the manner shown in figure 22.4, Note that only three straight-line streaks, well separated, are made. Place the Petri plate (in- verted) in a cannister with the plates of other stu- dents that is to go into the GasPak jar 6. Once all the students’ plates are in cannisters, place the cannisters and tubes into the ja. 7. To activate and seal the GasPak jar, proceed as follows: a, Peel apart the fol at one end of a GasPak indi- cator strip and pull it halfway down. The indi- ccator will tur blue on exposure to the ait. Place the indicator strip in the jar so that the wick is visible. b, Cut off the comer of each of three GasPak gas generator envelopes with a pair of scissors Place them in the ja in an upright position, Tenens Compas Cultivation of Anaerober + Exercise 22 ¢. Pipette 10 ml of tap or distilled water into the ‘open comer of each envelope. Avoid forcing the pipette into the envelope. 4. Place the inner section of the lid on the jar, making certain it is centered on top of the jar Do not use grease or other sealant on the iim of the jar since the O-ring gasket provides an effective seal when pressed down on a clean surface. . Unscrew the thumbscrew of the outer lid until the exposed end is completely withdrawn into the threaded hole. Unless this is done, it will be impossible to engage the lugs of the jar with the outer f. Place the outer lid on the jar directly over the inner lid and rotate the lid slightly to allow ito ‘drop in place. Now rotate the lid firmly to en- ‘gage the lugs. The lid may be rotated in either direction. 8 Tighten the thumbscrew by tuning clockwise. If the outer lid raises up, the lugs are not prop- cetly engaged. 8. Place the jar in a 37° C incubator. After 2 or 3 hhours check the jar to note if the indicator strip has lostits blue color. If decolorization has not oc- ‘curred, replace the palladium pellets and repeat the entire process, 9, Incubate the tubes and plates for 24 to 48 hours. SECOND PERIOD (Culture Evaluations and Spore Staining) Remove the lid from the GasPak jar. If vacuum holds the inner lid firmly in place, break the vacuum by slid ing the lid to the edge. When tansporting the plates ©. sporogenes Figure 22.4 Three organisms are streaked on agar plate as straight-line streaks. 9