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The song is first introduced when Penny sings it to Sheldon when he has caught a cold.
Penny had to sing "Soft Kitty" to Sheldon again on the pretense that "...Homesick is a type of being sick" since he had to spend the night in Penny's apartment because he locked himself out of his and Leonard's apartment.
When Sheldon was feeling sad about being betrayed by his friends (and not being able to attend Comic Con), Penny starts singing "Soft Kitty" in an attempt to cheer him up, but was interrupted by Sheldon as he said, "That's for when I'm sick!! Sad is not sick."
When Penny was a little lightheaded from the powerful pain medication and a muscle relaxer that the hospital gave her after she dislocated her shoulder from slipping in the bathtub, Penny asked Sheldon to sing "Soft Kitty" to her (according to her, "Injured and drugged is a kind of sick"). After much prompting, the two sing it as a round.
Penny sings "Soft Kitty" to Sheldon, or more accurately, Sheldon's Mobile Virtual Presence Device (Sheldon in robot form) after Sheldon falls down the stairs. Sheldon plays "Soft Kitty" on his recorder (woodwind musical instrument) as a round while Penny sings "Soft Kitty".
Leonard and Penny sing the song as a lullaby to put Sheldon to sleep because he hadn't slept in days.
