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Quality Control Tests Recommended at Broth Stage: 3.1. Qualitative Tests 3.1.1. Check for freedom from visible contaminants 3.1.2. The pH of the bacterial broth shall normally be between 6.5 and 7.5 3.1.3. Smear and Gram stain 3.1.3.1. Reagents a. Ammonium oxalate crystal violet stain weigh 0.2 g of crystal violet and dissolve in 20 ml of 95 percent ethyl alcohol. Dissolve separately 0.8 g of ammonium oxalate in 80 ml of distilled water. Mix the two solutions and filter through a filter paper. [Act No.6 of 2006] 47 b. Iodine solution Iodine 1.00 g Potassium Iodide 2.00 g Distilled water 300 ml Weigh the ingredients and dissolve in water. Filter through a filter paper. c. Erythrosine Erythrosine 1.00 g Phenol 5.00 g Distilled water 100 ml Weigh the ingredients, dissolve in distilled water and filter through a filter paper. 3.1.3.2. Procedure Prepare a smear on a clean microscope slide, fix over a flame by gentle and intermittent heating, air cool and flood with ammonium oxalate crystal violet stain for 1 min. After removing the excess of ammonium oxalate crystal violet, wash the slide under a gentle stream of running tap water. Flood the slide with iodine solution for half a minute remove excess stain wash with 95 percent ethyl alcohol and finally wash under a gentle stream of running tap water. Flood the slide with erythrosine stain for about 3 min, wash under a gentle stream of running tap water and dry between the folds of a filter paper. Examine the slide under a compound microscope using an oil immersion objective. Note:- A smear prepared from undiluted broth should be free from Gram positive cells. The presence of a few 48 [Act No.6 of 2006] gram positive cells in occasional fields which may be due to dead cells in the medium may be disregarded. 3.1.4. Absence of Growth on Glucose - Peptone Agar The composition of the glucose - peptone agar is as follows: Glucose 10.0 g Peptone 20.0 g Sodium chloride (NaCl) 5.0 Agar (IS 6850) 15.0 Distilled water 1000 ml Bromocresol purple ethyl alcohol solution 10 ml of 1.6 persons pH 7.2 Note:- When a loopful of the broth is streaked into this medium and incubated at 28 +/- 20 C for 24 h, the purple - violet colour of the medium (due to the indicator bromocresol purple) shall not change. If the colour changes to yellow (acidic reaction) or blue (alkaline reaction) the broth is grossly contaminated. Hence, the broth should be rejected. 3.1.5. Streak on yeast Extract mannitol Agar with Congo Red When a loopful of broth culture is streaked to a plate of this medium and incubated at 28 +/- 20 C for 3 to 10 days, it shall show colonies of bacteria with growth characteristics same as that of the pure culture used in the preparation of the broth. Otherwise, the broth should be rejected. [Act No.6 of 2006] 49 3.2. Quantitative Test 3.2.1. Viable or Plate Counts Serially dilute one milliliter of the broth to obtain dilutions of the order of 106 to 109. Plate 0.2 ml aIiquots of the dilutions on YEMA plates and incubate at 28 +/- 20 C for 2 to 6 days, depending on the species of Rhizobium. The counts of viable Rhizobium in the final broth from shake culture or fermentors shall be not less than 108 to 109 cells / ml. Otherwise, the broth should be rejected.