Materials and Reagents

1. Gram-negative bacteria (the protocol is developed for Escherichia coli, but it also works on other species)
   
2. Formaldehyde (FA) (Sigma-Aldrich, catalog number: 47608 )
   
3. Glutaraldehyde (GA) (Merck KGaA, catalog number: 1-04239-0250 )
   
4. Tween-20 (Sigma-Aldrich, catalog number: P9416-100ml )
   
5. Triton X-100 (Merck KGaA, catalog number: 1.08643.1000 )
   
6. EDTA (Sigma-Aldrich, catalog number: ED255 )
   
7. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
   Note: The lysozyme is dissolved at 100 μg/ml in the PBS pH 7.2 with 5 mM EDTA ready to use 1 ml aliquots and stored at -20 °C. After using it the leftover is discarded.
   
8. Blocking reagents (F. Hoffmann-La Roche, catalog number: 1096176 ))
   
9. Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, catalog number: 711-165-152 )
   Note: Minimal cross-reaction to Bovine, Chicken, Goat, Guinea, Pig, Syrian Hamster, Horse, Human, Mouse, Rat and Sheep serum proteins. The in buffer freeze-dried Cy3 labeled secondary antibodies are dissolved in H₂O to a final concentration of 1.5 mg/ml and aliquoted as 20 μl samples. Once thawed the secondary antibodies are stored at 4 °C. After one month take a new sample from the -20 °C.
   
10. PBS buffer (pH 7.2) (see Recipes)
    

Equipment

1. Shaking incubator to grow bacteria
   
2. 500 μl or 1 ml tubes (Eppendorf)
   
3. 50 ml Tubes (Greiner Bio-One GmbH, catalog number: 227261 ) (Alternative Sorval SS34 tubes)
   
4. Eppendorf centrifuge 5804 R (Alternative Sorval centrifuge for SS32 rotor)
   
5. Eppendorf centrifuge (cooled)
   
6. Shaking incubation block for eppendorf tubes
   

Procedure