I dont have any experience about this but i think culture on broth media at the first step is best way.

good luck

Hi Kamini, first you should separate BAL eukaryotic cells from bacteria with a low speed spin (3000 rpm for 10 min).

BAL has a lot of neutrophils that kill bacteria and you should separate them ASAP.

After that you can either plate the sample and see what grows or treat the sample with trypsin (0.01 mg/ml for 20 min) and do a high speed spin (~15,000 rpm for 30 min), quench the trypsin with 10% FBS and then plate the pellet.

Trypsin will get rid of any antibodies that are bound to the bugs, but personally I think neutrophils/macrophages in your BAL are the culprit.

Steingrimur Stefansson

Thank you so much for your replay this will be very useful.

Just few clarification

1) When you say "treat" the sample, does that mean resuspending the pellet in trypsin and then tossing the supernatant. How many washes should I do with trypsin?

2) What does "quenching" trypsin in FBS mean?

3) What should my final resuspension be in, since I want liquid sample for my study.

Do you have any other suggestion to remove neutrophils/macrophages

Thanks again for your help,

Kamini

The low speed spin will pellet the eukaryotic cells but not free bacteria. The free bacteria will be in the sup.

Adding trypsin to the sup. should remove any bound Abs from the bacterial surface. Adding FBS (fetal bovine serum) ~10% will inhibit (or quench if you like) all of the trypsin. Serum is full of trypsin inhibitors.

A high speed spin should pellet the bacteria and you can resuspend that pellet in anything you want.

Removing intact neutrophils/macrophages by centrifugation without lysing them would probably be the best option. These guys are full of anti bacterial agents.

Steingrimur Stefansson

very nice answer

Steingrimur Stefansson

Does this protocol make sense?

1) Spin BAL sample

2) Add Trypsin to Sup. and let the solution sit at RT for 20min

3) Add 10% FBS to Sup+Trysin solution

4) Spin at high speed

5) Remove sup and add anything to pellet for resuspension

Sorry this is my first time working with BAL samples and so I'm not sure where to start. Again thanks for all your help!

Looks good to me. Just remember that you can skip the trypsin step after the low speed spin as a control.

Any Abs binding to the bugs are there for the macrophage cleanup.

Steingrimur Stefansson

Hello,

Do you also have suggestion on how I can remove debris from BAL and ETA samples. This way I would be easily identify the pathogenic organisms.

A low speed spin should be good enough to remove debris from the samples.

Steingrimur Stefansson

I tried washing and spinning in saline and L-His, but the debris still remains. I was also doing multiple spins even up to 6 spins. Any other thoughts.

Thanks

Hi, you could try passing the sample through a 0.45 micron disk syringe filter.

Dear Joshi,

If we have a sputum sample, firstly certain concentration of sodium hydroxide must be added to remove contaminant at equal volume of sputum in clean centrifuge tube. After that, DTT (di-thio-thretol) will be added and centrifugation at high speed centrifugation at 14000-16000 rpm at mini-speed high speed centrifuge must be achieved. Hope to be benefit. For more details please read my attached manuscript published recently in this year in a journal affiliated to Clarivate Analytics.

I am ready for any queries and help. Regard

Mushtak T. S. Al-Ouqaili

what% of H2O2 and DTT should I add? I don't want to kill off any pathogen in the sample. Also after centrifugation would the bacteria remain in the supernatant or pellet?