preparation of bacteria for SEM - fast method for bacteria preparation (Sep/05/2008 )

Hello everybody,

I'd like to know if there is an efficient and fast way for the preparation of bacteria for scanning electron microscope,

I have one method but it takes long time;

1. centrifugation of bacterial broth
2. washing the pellet with phosphate buffer saline for 3 times
3. adding 0.25% gluteraldehyde (in Na-phosphate, pH 7.2)
4. incubation at room temperature for 30 minutes
5. then overnight incubation
6. washing with Na- phosphate buffer for 3 times
7. collection of the pellet by centrifugation
8. dehydrolysis the sample by different ethanol volumes starting; 30%, 50%, 70%, 80%, 90% and 100% and for each ethanol volume incubate for 10 minutes
9.incubation in 100% ethanol for 1 hour
10. preparation of SEM stub by applying the adhesive tap and then adding the bacterial sampl on the tap

if somebody knows better way pls let me know, and if i want to scann treated bacteria do i have to follow the same protocol


appreciate your help

ok fine, since no body answered me I guess I will search bymyself to find the best protocol for bacterial sample preparation

thanks

Scheme for routine bacterial preparation for SEM
CARE – use in fume cupboard only, with eye protection and gloves

Bacteria in broth
↓
Drop on filter membrane (0.45 micron or 0.2 micron), air dry
↓
Fix in 2.5% glutaraldehyde in PBS buffer, 45 min, 1 h
↓
3×15 min rinses in PBS
↓
Refix in 1% OsO4 in PBS buffer, 1h
↓
Series dehydration with ethanol, 10 min each rinse
(30%, 50%, 70%, 80%, 100% Ethanol)
↓
Leave sample in absolute ethanol (15 min approx)
↓
Critical-point dry
↓
Attach to stub
↓
Sputter coat with gold
↓
SEM

Hope this would help

Nal