SEM of Filters

12/10/96

Hello,

I have an environmental engineer who is interested at looking at

bacterial samples that have been filtered. He wants to look at the

bacteria on the filters themselves. Does anyone know how to process

such samples? Do I let the filters air-dry or should I fix,

dehydrate, and CPD the filters? Any help would be much appreciated.

Thank you in advance,

Ginger Baker

EM Lab Manager

Dept. APP

250 Veterinary Medicine

Oklahoma State University

Stillwater, OK 74078

(405) 744-6765

FAX: (405) 744-5275

Email: lizard@okway.okstate.edu

Dear Ginger,

Use ESEM ( environmental scanning electron microscope) equipped with a cold

stage going down to 1-2 degrees C. You will be able to use wet filter with a

bacterial deposit on it and to dry carefully water out while in the

microscope. For a short time ~ 5 minutes, you should be able to see and

identify your bacteria without substantial distortion. Alternatively, fix

them with 1% of OsO4 in water while on filters and repeat as above. Use 20

kV and high condenser setting (60-70%) for artefact free observation.

Cheers,

Wis Jablonski OiC EM/X-ray Microanalysis, CSL, University of Tasmania

W.Jablonski@csl.utas.edu.au

Ginger,

Once you filter the suspension of bacteria

through a 0.2um polycarbonate filter, remove

the filter from the housing and place it into

fixative (2.5% glutaraldehyde in a buffer)>

Follow this with:

buffer washes,

an optional post-fixation (osmium tetroxide)

buffer washes

gradient alcohol series (3X100% ETOH)

At this point you have 2 options;

3 washes in HMDS (hexamethyldisilizane)

followed by air-drying

or Critical Point Drying.

Fix the filters to the SEM stub with double-sided

sticky tape, add a drop of silver dag to the edge of

the filter and sputter-coat.

Additional tips---during the intial stage of filtering,

remove the syringe from the filter housing, pull

plunger back, reattach and push a column of air

through the housing---this pushes all of the filtrate

through the filter so that it doesn't drain off when

removing the filter from the Swinney holder.

-------------------the filter housing and filter can be

autoclaved ahead of time and sterile 1 cc. syringes

used to insure a reliable prep.

Best of luck,

Rosemary Walsh

rw9@psu.edu

I would definitely recommend to dehydrate and CPD the filters with

the bacteria. After they are dry, you can cut the filter for suitable size

to be mounted on a SEM-stud, whereafter they have to be coated for conductivity.

Good luck and happy holidays

Jouko

Jouko K. Mäki, Ph.D., Laboratory Manager

University of Turku, Laboratory of Electron Microscopy

Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND

Tel: + 358 2 333 7318 GSM: + 358 40 505 2521 FAX: + 358 2 333 7380

jouko.maki@utu.fi

Microscopy & Analysis (US Edition Issue No. 21, November 1996) ran an

article you might be interested in - "Cryo-preparation of small or lightly

attached biological specimens" by Robinson et al.

Basically, the idea is a variation on cryo-fixation - this normally

involves plunging a specimen into liquid N2. In the case of specimens

similar to yours, the result is that all the particles of interest drop

off! However, if you simply fix the specimen on to a stub at room

temperature and then transfer it to the pre-cooled cryo-stage, freezing is

still relatively rapid. Obviously, you still loose internal specimen detail

because of ice crystal growth but a considerable amount of external detail

is successfully preserved. The authors present a number of good SEM images.

Whether the procedure will work successfully with specimens as small as

bacteria, I'm not sure.

If you want further details, contact Ken Robinson at KRO@pcmail.nerc-bas.ac.uk.

Regards,

Dr L. P. Stoter Technical Editor, MICROSCOPY & ANALYSIS

Technesis

17, Rocks Park Road email: LPS@teknesis.demon.co.uk

Uckfield, E. Sussex Phone: +44 (0)1825 766911

TN22 2AT Fax: +44 (0)1825 766911

United Kingdom

I would try to minimize handling as much as possible to avoid losing any

bacteria. We have resorted to exposure to osmium vapors for an extended

period such as 24-72 hours and then air drying.

G.W. Erdos, Ph.D. Phone: 352-392-1295

Scientific Director,

ICBR Electron Microscopy Core Lab

218 Carr Hall Fax: 352-846-0251

University of Florida E-mail: gwe@biotech.ufl.edu

Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/

Some bacteria can stand airdrying. I suggest trying this with

part of the sample. If the bacteria or other organisms look

deflated you can CPD the remainder.

Dave

DAVID PATTON

D-PATTON@wpg.uwe.ac.uk

Ginger:

I have done similar preps while at UMASS. Are the bacteria already

on the filters? There are several kinds of filters. Some are better

than others for SEM. The best ones to use for SEM are polycarbonate

(Nuclepore type, Bio-Rad also sold some). Other ones are

a torturous path type and it will be hard to find the bacteria on them.

Your best bet if there is a ?? is to look at an unused filter so you know

what the background is. Polycarbonate ones are smooth with round holes in them.

They come in several pore sizes. You will probably want ~.2um for bacteria.

Too many bacteria will clog them up and stop the flow. My guess is these are

some type of water filters. Certainly try to get a control to look at.

By all means fix the samples, osmicate, ethanol dehydrate, CPD and Au/Pd coat

for best results. Fixing should also aid in keeping the bacteria in place.

You can try air drying for comparison but the morphology will

be poor. It depends on what your final purpose is; bacterial counts or

being able to identify types and have nice morphology. I would recommend

running a test batch first to be sure there are no problems. Feel free

to call if you have any ???

Is Bill Chissoe at Stillwater?

Best of luck

Ed Basgall, PhD

Penn State Univ.

Dept. of Chemistry

University Park, PA 16802

Ph: 814-865-0493

edb@chem.psu.edu

Ginger Baker

If the samples haven't already been filtered, then first sputter

coat the filters before use, or use silver filters. Fewer imaging problems

that way.

With the bacteria on the filters, fix, dehydrate, and dry from

hexamethyldisilizane at room temp or 60 C in a fume hood! I usually get

excellent results this way.

(Note: check Crang and Klomperens "Artefacts in Biological EM",

title approximate, about fixing and dehydrating and their effects on

bacterial coats. The slime coat may be important, and it's usually lost.)

Phil

Philip Oshel

Microscopy

Station A

PO Box 5037

Champaign, IL 61825-5037

(217)244-3145 days

(217)355-3145 evenings

oshel@ux1.cso.uiuc.edu

(11) Microscopy & Analysis (US Edition Issue No. 21, November 1996) ran an