Biology Reference
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4. Columbia salt agar (CSA): Columbia broth (as per the manufacturer's instruc-
tions, 35 g/L in ddH 2 O), 2% (w/v) Bacto Agar, and 2% (w/v) NaCl (store at
4 °C).
5. Tryptic soy agar (TSA): As per the manufacturer's instructions 40 g/L in ddH 2 O
or blood agar—TSA with 5% sheep's blood (store agar plates at 4 °C).
6. Phosphate-buffered saline (PBS) without calcium or magnesium: 5.6 m M
Na 2 HPO 4 , 0.94 m M KH 2 PO 4 , pH 7.4, and 155 m M NaCl.
7. Anesthesia: Ketamine 33 mg/kg, Xyla-Ject (xylazine) 2.5 mg/kg, and
Acepromezine Maleate 2.5 mg/kg (see Table 1A ) (make anesthesia fresh for each
use).
8. Small volume (20 μL) disposable pipette tips.
9. Syringes, 1 mL with 27-gauge needles.
10. A spectrometer capable of reading light transmission at 650 nm.
2.2. Treatment
1. Anesthesia: As above (freshly made).
2. Syringes, 1 mL with 27-gauge needles.
3. BD Angiocath flexible FEP polymer catheters, 22-gauge (for delivery of semi-
solid formulations if desired).
4. Syringes, 1 mL.
5. Small volume (20 μL) disposable pipette tips.
2.3. Recovery
1. Ethanol: 70% (v/v) in water (or equivalent for surface sanitation) and 100% for
flaming instruments.
2. PBS + Tween-20: PBS (above), 0.5% (v/v) polyoxyethylene monolaurate (Tween
20) (make fresh for each harvest, Tween 20 is generally sterile as received).
3. TSA + 7.5% NaCl: TSA (above) and 7.5% (w/v) NaCl (store at 4 °C).
Table 1A
Anesthesia Preparation and Dosing by Animal Weight
Anesthesia
Dose (mg/Kg)
Ketamine
33
Xylazine
2.5
Acepromezine
2.5
Preparation of standard anesthesia solution: Ketamine solution at
100 mg/mL, use 20 μL/animal dose; Xyla-Ject (xylazine) solution
at 20 mg/mL, use 7.5 μL/animal dose; acepromezine solution at
10 mg/mL, use 15 μL/animal dose; phosphate-buffered saline, use
157.5 μL/animal dose. Make 50% more animal doses than needed.
 
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