FISH Protocoal to fix bacteria

① Fresh samples are collected and fixed as soon as possible;

② Prepare 4% PFA solution 20 mL;
a. Take 13 mL deionized water in a beaker;
b. Heat this beaker 2min @ 60ºC with stirring;
c. Pour 0.8g PFA into this beaker;
d. Add a few drops of 2M NaOH to help dissolve PFA until the PFA dissolved
entirely;
e. Put the beaker into ice to make the temperature of the beaker fall;
f. Add 6.6mL 3×PBS;
g. Add a few drops of 2M HCl to adjust pH to 7.2 (not strict);
h. Filtrate the solution through a 0.22μm filter;
i. Move biomass in 2 mL tube and add 1 mL of 4% PFA solution in the tube;
j. Store it at 4℃ and use it during 24h.
③ Fixation:
a. Gram Positive cells: mix equal volumes of sludge and 96% ethanol in a test tube.
Keep the solution in the freezer (-20ºC) for 2-3 hours;
b. Gram negative cells: mix equal volumes of sludge with cold 8% PFA, Keep the
solution in the freezer (-20ºC) for 2-3 hours;
c. Take 1mL activated sludge into a 2ml EP tube and centrifugalize:
12000rpm×5min;
d. Discard supernatant and wash with PBS buffer two times;
e. Suspend the sludge pellets with 300l of PBS buffer;
f. Add 900μl 4% PFA into the tube, biomass: 4% PFA=1:3. Mix well;
g. Ice bath the tube at 4℃ in refrigerator for 3-24h;
h. Centrifugalize: 12000rpm×5min and discard all supernatant, then add 1ml PBS
and shake the tube until the sludge resuspending;
i. Centrifugalize: 12000rpm×5min and discard all supernatant, then add 0.5ml
PBS and 0.5ml ethanol, namely PBS: ethanol=1:1, then shake the tube until the
sludge resuspending. The volume of added PBS and ethanol is depends on the
biomass concentration;
j. Store the sample at -20℃ and use it during 6 months;
④ Fix the sample on slide
a. Disperse samples intermittently with a ultrasonic processor at 20W for
5~10min at the first time; and from the second time on, disperse the samples
intermittently at 20W for 30s before fix sample on slide.
b. Spread 3μl of one sample onto a well (diameter of a well is 9mm) which is on a
slide (every slide has 12 wells).
c. Desiccate the samples at room temperature.
⑤ Cryosectioning
a. Separate PFA and biomass by centrifugation (3500 g for 8 min);
b. Dissolve the pellet in 5mL cold 1:1 PBS/EtOH. Separate by centrifugation (3500
g for 8 min). Repeat this step once more;
c. Resuspend the pellet after the last step in 1-5 ml cold 1:1 PBS/EtOH and mix,
the fixed sample can be kept in the freezer (-20ºC) for several months;
d. The sample can typically be applied directly to the slide to maintain spatial
resolution within the flocs, or else after gentle homogenization by gently rubbing
two glass slides with 20µL sample against each other;
e. Turn on cryotome a night before cutting and fix temperature at 30ºC;
f. Distance is fixed at 10nm and thin slices are carried out on a cryotome at (-30ºC)
and slices are immediately placed on a slide (room temperature), where they
melt. The slices are allowed to dry on the bench for 3 hour;
⑥ Hybridization
a. The slide with fixed samples is dehydrated sequentially in 50%, 80%, and 98%
of ethanol/H2O, and then put the slide vertically on a filter paper.
b. Preparation of hybridization buffer and washing buffer. The two buffers need
be prepared every experiment. (See another protocol);
c. Prepare 2 mL of hybridization buffer for desired percentage of formamide, see
composition of the hybridization buffer add SDS at end;
d. Transfer 8 µL of hybridization buffer onto the slide within a small area or into
each well;
e. Add 1 µL of each gene probe and mix carefully (avoid contact with the sample)
with hybridization buffer;
f. Place the slide horizontally into a 50 mL polyethylene tube with a piece of tissue
wetted with 1 – 2 mL of hybridization buffer, biomass should be on the other
side of the tissue;
g. Place the tube in the hybridization oven (46ºC) for at least 2 – 3 hours;
h. During hybridization prepare the washing buffer in a 50 mL polyethylene tube,
 see composition of the washing buffer corresponding to the formamide
concentration applied during hybridization;
i. Preheat the washing buffer in a 48ºC water bath;
j. Gently rinse the slide by pouring a few millilitres of the preheated washing
buffer over the sample (must be carried out in the fume hood to avoid toxic
formamide fumes);
k. Transfer the slide to the 50 mL polyethylene tube with the remaining preheated
washing buffer and incubate for 15 min at 48ºC (water bath);
l. Remove the slides from the washing buffer and dip in cold dH2O. Let the slide
air-dry;
m. Add DAPI solution (1µg/mL in distilled water) to cover the sample, and stain
for 15 min at 4ºC in dark;
n. Rinse with plenty of dH2O and let the slide air dry;
o. Please note that DAPI is staining DNA only, for other counterstaining methods;