66 MANUAL OF MICROBIOLOGICAL METHODS 



turbidimeter at 45-60 °C. Run turbidity estimation as with waters, using standard 

 notation based on turbidities equivalent to Si02 content in parts per million. 



Threshold gel concentration. Use solution from gel-melting-temperature deter- 

 mination. To duplicate 140-mm lengths of 14.5- to 15.5-mm-ID glass tubing, 

 stoppered at one end, add 17.5-ml portions of 70-90°C H2O, using Luer-type syringe 

 without needle. Immediately add 2.5-ml portions of sample solution with syringe, 

 making 20 ml per tube. Stopper, and mix by inverting six times. Hold tubes 1 hr 

 at 20-25°C, then 1 hr at 0-5°C, and, finally, 1 hr at 20-25°C. Remove top stoppers, 

 and gently add 3-1 ml of H2O to tubes. Taking each tube individually, lay on hori- 

 zontal wooden surface, remove bottom stopper, and induce cylinder of gel to slide 

 from tube by elevating bottom end slightly and moving tube backward as cylinder 

 moves forward relative to tube. Object is to remove tube without motion of gel with 

 respect to wood. If neither cylinder of gel breaks under pull of gravity in 10 sec, 

 sample passes test. 



Protein nitrogen. Use Hen wood & Garey (Hengar) modification of Kjeldahl 

 method with 0.1 g of solids. Not more than 0.32 per cent of N (approximately 2 per 

 cent protein) shall be found after correcting for reagents by running a blank on sucrose. 



Reducing substances as galactose after autoclaving. Dissolve 1 g of solids in 

 100 ml of H2O in 500-ml short-neck spherical flask containing one 3-mm glass bead. 

 Use 120°C for 40 min. Swirl at 99-100°C. Transfer immediately to flame or heater 

 preadjusted to maintain a steady boil. Add 0.2 g of NaOH and 4 mg of methylene 

 blue chloride, dry or as freshly made solution. Close flask with two-hole stopper, one 

 hole of which holds 90° vent tube. Titrate through open stopper hole to blue which 

 is stable 0.5 min. Total titration time 2.5 ± 0.25 min. Use Soxhlet's solutions 

 standardized according to National Bureau of Standards Circular 0440, page 187, 10 ml 

 of solution A plus 10 ml of solution B diluted to 100 ml on day of use. No more 

 titrating solution must be needed by the sample than the quantity necessary to react 

 with 80 mg of Eastman galactose No. 141 dried to constant weight over boiled H2SO4 

 and carried through the same procedure. 



Chlorides as NaCl. Blend 1 g of solids in 200 ml of H2O in Waring blender or 

 equivalent device provided with rheostat or auto transformer speed control. Start 

 slowly to avoid splash. Blend, covered, at top speed until visually textureless. Cut 

 speed to point of splash-free agitation; rinse top and sides into mixture. Add 2 ml of 

 10 per cent K2Cr04, and titrate in blending jar with 0.0171 A^ AgNOs to an end point 

 permanent for 1 min. Subtract the value of a blank run on 200 ml of water. Each 

 milliliter of AgNOs solution represents 0.1 per cent of NaCl. 



Viable spores. Transfer 2-g sample as received to 100 ml of sterile tryptone- 

 glucose-beef extract broth in 6-oz screw-capped prescription bottle, using sterilized 

 vegetable parchment for weighing and transferring. Close bottle tightly, and auto- 

 clave at 115°C for 5 min. Agitate gently at 90-95°C, cool to 50-60°C, add 1 ml of 

 sterile skim milk, and pour entire charge into three petri dishes, glass-covered. Invert 

 when cool, and incubate at 35°C for 36 hr. Divide total colonies visible in Quebec- 

 type counter by 2 to get spores per gram. 



Debris count. Dissolve 1.5 g of solids in 100 ml of H2O at 120°C for 20 min. 

 Swirl at 99-100°C, and immediately pour entire charge into two 90-mm petri dishes. 

 Cover with porous tops, and allow to congeal. Examine plates on Quebec-type 

 colony counter in dim light at standard magnification of 1.5 diam, adjusting lens posi- 

 tion for maximum resolution. Note all objects which a trained microbiologist might 

 mistake for a microbial colony. To avoid the counting of bubbles, examine each 

 object found with a 7.5 X magnifier without moving petri dish or 1.5 X lens. The 

 smaller magnifier is to be used for examination only, not for counting. Count only 

 true debris particles clearly visible at 1.5 X. Run a control with each series of 



