ZIRC cryopreservation (cryoprotectant): bacteria samples

We used the ZIRC protocol for cryopreserving zebrafish sperm with only a few modifications.¹⁴ Before freezing the bacterial samples, two McFarland standards No. 1 and No. 3 (3 × 10⁸ and 9 × 10⁸ bacterial colony-forming units [CFU]/mL, respectively) and a spectrophotometer were used to estimate the density of the cultured broths. We aimed for an absorbance between 0.2 and 0.4 nm, which is estimated to be about 3 × 10⁸–6 × 10⁸ CFU/mL. The bacterial broths were then diluted in 1 × phosphate-buffered saline (PBS) by using a 1:10 serial dilution.

ZIRC cryopreserves 20 μL samples, taken from solutions consisting of 5 μL of sperm, 1 μL E400, and 15 μL Raffinose freezing medium (RMMB). E400 is a high-potassium, buffered salt solution that has an osmolality of 400 mmol/kg that is used after the sperm from the zebrafish have been collected to keep urine from activating the sperm cells.¹⁴ E400 consists of 130 mM KCl, 50 mM NaCl, 2 mM CaCl₂, 1 mM MgSO₄, 10 mM d-(+)-Glucose, and 30 mM HEPES-KOH (7.9).¹⁴ RMMB is the cryopreservant and consists of 20% (w/v) d-(+)-Raffinose pentahydrate (R7630; Sigma), 2.5% (w/v) Difco Skim Milk (No. 232100; Difco), 6.67% (v/v) methanol (Acetone-free, Absolute, Certified ACS Reagent Grade, A412; Fisher Scientific), and 30 mM Bicine-NaOH (pH 8.0).¹⁴ The same ratio of solutions and sample volume were used in this experiment except that the bacteria took the place of the sperm.

A total of 90 μL of RMMB solution was added to a sterile 2 mL cryogenic vial (No. 430659; Corning). To that same vial, 6 μL of E400 and 30 μL of the 10⁴ CFU/mL bacterial concentration were then added and mixed by pipetting. Then, 20 μL of this mixture was transferred into cryo-vials in triplicate. These cryo-vials were then capped and placed onto another empty cryo-vial without a cap, and both were then placed into 15 mL Polypropylene conical tubes (No. 352096; Falcon, Corning). These 15 mL tubes were then capped and placed into a container filled with powdered-dry ice until the caps were flush with the surface of the ice. The samples remained in the powdered-dry ice for at least 20 min. This configuration yields a freeze rate of about 20°C/min. After 20 min, each sample was removed from the conical tubes, placed on a cryogenic cane, and quickly placed in a liquid nitrogen (LN₂) dewar. This same procedure was also done using the starting broth, which had the high bacterial concentration (10⁸ CFU/mL).

• 1:10 serial dilution of starting broth (10⁸ CFU/mL), using 1 × PBS.

• Ratio of solutions used in the ZIRC cryopreservation method:

o 5 μL sperm (in our case bacteria) +1 μL E400 (sperm extender) +15 μL RMMB (Raffinose freezing medium)

• Prepared the following solution:

o Low concentration samples (10⁴ CFU/mL):

□ 30 μL of bacteria (10⁴ CFU/mL) +6 μL E400 + 90 μL RMMB

□ 20 μL of the earlier described solution was pipetted into 2 mL cryogenic vials (done in triplicate).

o High concentration samples (10⁸ CFU/mL):

□ 30 μL of bacteria (10⁸ CFU/mL) +6 μL E400 + 90 μL RMMB

□ 20 μL of the earlier described solution was pipetted into 2 mL cryogenic vials (done in triplicate).

• Final volume frozen = 20 μL/sample

• Final freezing temperature = −196°C