3/4/96

Hi All:
I have been asked to examine, using the SEM, some bacteria that have been exposed to a drug solution. The interest is in whether the exposure to the drug solution has altered the bacterial shape and size (mostly size) and thus effect its retention on a filter. I propose to simply filter the bacterial suspension onto a polycarbonate membrane, air dry, mount, sputter coat, and examine. This will supposedly work for the Gram positive microbes but may not work for the Gram negatives (they are said to have a much thinner wall and could shrink). An alternate approach is to heat fix the G- to a glass coverslip, sputter coat, and examine.

Is this approach OK or is there a better way to do this, perhaps using a fixative? I would rather not have to go the route of CPD. Any suggestions will be greatly appreciated!!!

Damian Neuberger
Email: neuberd@baxter.com

I've had excellent results air drying from hexamethylsilizane at room temperature. Peldri will also work if you've got any left. Filtering through a filter works fine.

But what % size change are you looking for? Recall the measuring things, especially biological specimens in the SEM is dicey at best--and can be grossly misleading.

Phil Oshel
oshel@ux1.cso.uiuc.edu

Hello Damian!
No fix and air drying will almost certainly change the shape and size of the bacteria. If you wish to avoid critical point drying you could try fixation followed by a CPD substitute such as HMDS (hexamethyldisilazane), TMS (tetramethylsilane) or Peldri (if available). Alternatively, freeze-drying, cryo-SEM or LVSEM would be preferable to air-drying without fixation.

Robin Cross
eurc@giraffe.ru.ac.za

Dear Damian,
One alternative to CPD is to filter, fix, dehydrate and follow the final 100% ETOH dehydration with 3 baths of 100% HMDS (Hexamethyldisilizane). Polycarbonate membranes handle the solvent very well. They dry in minutes and you can quickly afix them to stubs and sputter coat.

Rosemary Walsh
rw9@email.psu.edu

Get your bacteria to attach to a polylysine coated cover slip. Fix with glut and osmium, dehydrate and dry via HMDS (two changes of hexamethyldisilazane after 100% alcohol, then air dry finishing up with ten minutes in a 60 C oven). Then sputter coat Will give you much better results than heat fixing and air drying.

Greg Erdos
E-mail: gwe@biotech.ufl.edu

[Return to Tips & Tricks Menu]