Washing Cell Pellet

Hi Micro, welcome back.


I remember doing this step liberally. Normal saline is 0.9% solution of NaCI, which isn't harmful (relatively).

I'm using 0.2 mM (yep, millimolar) NaCl. I'm currently adding 10 mL at first and since NaCl is also used to adjust the OD, I measure the OD after the wash and add more accordingly to get the OD required. Hope I'm not 'over-washing' if that's a thing.

Nice, it's crucial that you measure the optical density, I was about the mention that

I'm kind of struggling with that because I feel the method I'm using is not scientific if you know what I mean. Because after adding 10 ml wash I'm taking a sample and measuring the stock OD then by simple proportion calculate the amount of sterile saline that needs to be added to get to the desired OD. Is that the way to go? I've read about plotting a calibration curve to get the cell density but I need not know the exact quantity of cells. Measuring the OD as a method of standardization.

I see, for the quantity analysis, have you done any microscopic counting?

I have not. Is it necessary? Because I'm performing extraction (DNA, proteins etc) under different conditions and using the OD as a way to ensure that the same quantity of bacteria are present, each time I run the test and compare the products. Do I need to know the cell density?

No, unless cell density is one of the factors under examination examined. I've attached a protocol for examining density using a spectrophotometer and a link to another website.

Cheers for that! This is what I don't understand though: "Bacterial cell cultures are routinely grown until the absorbance at 600 nm (known as OD600; default setting) reaches approximately 0.4 prior to induction or harvesting". If you are measuring the OD after suspending the cell pellet in say sterile saline, the OD is effected by the quantity added.

Post Merge: 10 years ago

Now I think I understood what they meant by that statement. Since it said before harvesting, I would imagine the spectrophotometer is blanked with the broth used for the culturing and the readings are taken routinely during culturing. I am using a fixed amount of incubation time throughout my experiment and measuring the OD after washing to standardize the quantity of bacteria to make the following test fair. What I found bizarre is that I was using the principle OD1 V1 = OD2 V2 (where OD1 is the first OD reading and V1 is the total volume of the culture in the wash and OD2 is the desired final OD so V2 would be the total volume of suspension so V2-V1 would be the amount to be added to get the OD desired) but say OD1 for a 10 mL suspension read 1.6, 1.6*10 = 16 = total volume. When I added 6 mL to the 10 mL suspension to get a final volume of 16 mL, the OD didn't come to 1. I had to add further 1.8 mL to get 1.03. Cannot figure out why :/

No, it is not affected by the quantity added.


It's not perfect. Those are theoretical numbers, but other factors play a role in how it is read. It's the discrepancy that's bothering you, isn't it?

Yep! Because it seems to be a bit 'trial and error'

I would say to continue doing what you're doing. Best way to learn is through trial-and-error.

The problem is its very time consuming. Sometimes it takes 6 additions to get it right and sometimes it even goes below 1.0 (my desired OD) (even though I add the 'proportionally' correct volume), that I have to centrifuge again and start all over. Imagine doing this for 20 samples :/