BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES 65 



Agar itself is a good desiccant when dry. Weighings should be made promptly after 

 cooling and done rapidly. An efficient desiccant is essential. 



Solubility, cold. Dissolve 1.5 g of solids (1.87 g of agar of 80 per cent of total solids, 

 for example) in 100 ml of H2O by autoclaving at 120°C for 20 min in 250-ml tared 

 flask. Remove flask at 99-100°C, swirl thoroughly to mix, and make up to 100 g net. 

 Take 15.2-15.5 ml of solution into Luer-type syringe. Cap with pinched-off needle. 

 Weigh to 0.1 g. Uncap syringe, and eject contents slowly into 50-mm aluminum-foil 

 dish. Recap Luer, and reweigh. Difference is weight of aliquot (A). After 15 min 

 cut gel freehand with knife into 3- to 4-mm squares and place dish in 3- to 5-mm layer 

 of H2O in ice-cube tray or equivalent. Put tray on 1- to 2-mm cardboard in freezing 

 compartment at —5 to — 10°C. Hold overnight. Transfer dish contents to petri 

 plate, break into squares with single-edged razor blade, and retransfer to 30-ml 

 medium-porosity fritted glass crucible. Wash with four 10-ml portions of H2O at 

 10-15''C, using suction at end of each wash. Stir and gently press flakes with flat- 

 tened rod while washing. Evaporate combined washings in porcelain dish tared to 

 0.1 mg on steam bath. Dry residue 2 hr at 105°C, cool in desiccator, and weigh. 

 Residue is B. 



Per cent cold solubles = -^— j — 

 A 



Hot-water solubles. Dissolve 1.5 g of solids in 100 of ml H2O at 120°C for 20 min. 

 Swirl at 99-100°C. Filter at 80-90°C through fine-porosity 30-ml fritted glass 

 crucible tared to 0.1 mg. Wash flask and crucible three times with 25 ml of 80-90°C 

 H2O. Dry crucible 2 hr at 105°C, and weigh. Net is C. Run a blank to obtain cor- 

 rection for solubility of sintered glass membrane (D). Correction may approach 

 1 mg. 



T> .u . 1 u^ 100[1.5 - {C + D)] 



Per cent hot solubles = ^-= 



1.0 



Gelation temperature. Dissolve 1.5 g of solids in 100 ml of H2O at 120°C for 

 20 min. Swirl at 99-100°C. Cool to 60-70°C, and make to 100 g net. Place 15 ml 

 in 15- by 150-mm test tube, and insert calibrated Weston-type dial thermometer. 

 Insert 2- to 3-mm-OD glass tube with 90° bent tip and 0.5-mm orifice. Run air 

 through tube at }i to 1 bubble per second. Note temperature at which course of 

 rising bubbles becomes impeded. 



Gel melting temperature. Dissolve 2.0 g of solids in 100 ml of H2O at 120°C for 

 20 min. Swirl at 99-100°C. Cool to 60-70°C, and make to 100 g net. Place 10 ml 

 in 15- by 150-mm test tube. Stopper, and support tube at 30° angle. Hold 1 hr at 

 15-25°C. Place tube vertically in 70°C water bath 1 hr. If slant does not collapse, 

 sample passes test. 



Rate of dissolution. Arrange a 500-ml spherical three-necked flask with reflux 

 condenser, sealed agitator with circular, segment paddle, and stopper. Place 200 ml 

 of hot water in flask, start agitator, and so place and adjust a bunsen burner that the 

 flame covers approximately half the wetted portion of the flask and maintains slow but 

 definite ebullition (3 to 5 drops per second). Place 3 g of solids in length of glass 

 tubing small enough to enter neck of flask. Provide a glass-rod-rubber-stopper 

 plunger for tubing. Force sample from tube into flask with plunger in such a manner 

 that particles do not become attached to flask. Restopper flask. Stop heating and 

 agitation after 10 min. If no undissolved agar remains in liquid, sample passes test. 



Sol turbidity. Dissolve 2.0 g of solids in 100 ml of H2O at 120°C for 20 min. Swirl 

 at 99-100°C. Cool to 60-70°C, and make to 100 g net. Stopper, and hold at 45- 

 47°C for 3 hr. Simultaneously hold the glassware of a Jackson Coleman or Hellige 



