You take absorbance readings on a spectrophotometer across a 6-hour culture of E. coli cells growing in tryptic soy broth (TSB). Your absorbance readings clearly indicate a lag phase, a log phase, and a stationary phase. You come back in and take readings at 8, 10, 12, 14, and 16 hours, but the absorbance number remains the same. Shouldn't it start coming down as the closed batch culture enters death phase? What's the most likely thing that is happening?
A) Something is likely wrong with the spectrophotometer and it isn't measuring the correct values. Perhaps something is on the detector, making it register Falsely high absorbance numbers.
B) A spectrophotometer can measure absorbance as an indicator of cell number, but can't discriminate between a live cell and a dead one. The presence of dead cells keeps the absorbance high even into the death phase.
C) E. coli just grows slowly, and has a long generation time. After 16 hours the cells are still in stationary phase, so the cell numbers are expectedly constant.
D) Perhaps there's a big smudge of something on the tube that is blocking some of the light. This would lead to an elevated reading for every timepoint after the smudge was placed on the tube.
E) There is no point in measuring the absorbance of the culture because the cells are bacterial. It would make more sense to measure would the transmission value.

Question

You take absorbance readings on a spectrophotometer across a 6-hour culture of E. coli cells growing in tryptic soy broth (TSB). Your absorbance readings clearly indicate a lag phase, a log phase, and a stationary phase. You come back in and take readings at 8, 10, 12, 14, and 16 hours, but the absorbance number remains the same. Shouldn't it start coming down as the closed batch culture enters death phase? What's the most likely thing that is happening?
A) Something is likely wrong with the spectrophotometer and it isn't measuring the correct values. Perhaps something is on the detector, making it register Falsely high absorbance numbers.
B) A spectrophotometer can measure absorbance as an indicator of cell number, but can't discriminate between a live cell and a dead one. The presence of dead cells keeps the absorbance high even into the death phase.
C) E. coli just grows slowly, and has a long generation time. After 16 hours the cells are still in stationary phase, so the cell numbers are expectedly constant. 
D) Perhaps there's a big smudge of something on the tube that is blocking some of the light. This would lead to an elevated reading for every timepoint after the smudge was placed on the tube.
E) There is no point in measuring the absorbance of the culture because the cells are bacterial. It would make more sense to measure would the transmission value.

Quizplus · Accepted Answer

Spectrophotometers measure absorbance as an indicator of cell number, but they cannot discriminate between live and dead cells. Therefore, the presence of dead cells keeps the absorbance high even into the death phase, explaining the consistent readings after 6 hours.

You Take Absorbance Readings on a Spectrophotometer Across a 6-Hour