Every time you manipulate your hive you run the risk of killing the queen, she could run between the frames just as you are pushing them back together, or you could drop her off the frame. Or she could just walk off, if you work like me I set a frame on end leaning against the hive when working the hive. I give a good look to try and see that bee with the long body but she can be hard to spot.
Being queenless is one reason a hive can go from gentle to hot, but being molested by a skunk or racoon at night will make them aggressive as well.
The Busy Bee teaches two lessons: One is not to be idle and the other is not to get stung.