The 24th was originally organized as "The Iowa Temperance Regiment".

According to the source below, it was after the Battle of Champion's Hill that they held a religious service. The singing of "Old Hundred" takes place the next day, when they were not a part of the pursuit and fighting at Big Black.

From “Iowa and The Rebellion” by Lurton Dunham Ingersoll
Page 176
(This section covers the battle which the author refers to as “Champion Hills”.)

“The Twenty-fourth Iowa,” says a correspondent of the Cincinnati Commercial, writing from the field of Battle, “is called a Methodist regiment. The colonel and several of the captains are Methodist preachers, and a majority of the soldiers are members of the Methodist church. They did some of the best fighting of the day, yesterday. They went into the battle full of enthusiasm, and not one of them flinched during the engagement. Their major was wounded late in the day. He walked from the field, and on his way to the hospital captured a stalwart confederate, and compelled him to carry him on his back to the Provost-Marshal’s head-quarters. It was a laughable sight to see Major Wright riding his captive into camp. The casualty list of the Methodists is very large, and shows they stood up to the work like true soldiers. On returning from the battle-field in the evening they held a religious meeting, at which the exercises were very impressive. As I write they are filling the woods with ‘Old Hundred.’ “