484 METHODS AND FORMULAS DS 23.215-DS 23.216 



23.215 Ryo 1937 11796, 14:218 



STOCK solutions: I. water 100, phenol 2.5, tannic acid 10, potassium alum 4, II. sat. ale. 



sole. {circ. 10%) crystal violet 

 WORKING solution: stock I 100, stock II 10 

 method: [heat-fixed smear] -^ stain, 3-5 mins. — » tliorough wash -^ dry 



23.215 Sclavo test. 1904 Besson Besson 1904, 172 



REAGENTS required: A. 1% tannin in 50% ale; B. 5% phosphotungstic acid; C. DS 



23.211 Ehrlich 1882 

 method: [heat-fixed smears] -^ A, 1 min. —* rinse — > 5, 1 min. — ♦ rinse — > C, heated to 

 steaming, 3-5 mins. 



23.215 Shunk 1920 11056, 5:181 



reagents required: A. ADS 12.2 Shunk 1920; B. 95% ale. 80, aniline 20; C. DS 11.44 



Lofiier 1890 90, 95%, ale. 8, aniline 2 

 method: [heat-fixed smear] — > A, on slide —> B, added to A on slide, about 10% of quan- 

 tity of A used, 15 sees. — > drain — » C, 15 sees. — > water, wash — > dry 



23.215 Trenkmann 1890 23684, 8:385 



REAGENTS REQUIRED: A. ADS 12.2 Trenkmann (1904); B. sat. aq. sol. iodine; C. DS 



23.211 Ehrlich 1882 

 method: [heat-fixed smear] — > water — > A, 6-8 hrs. -^ wash —* B, 1 hr. -^ water -+ C, 



30 mins. -^ wash — > balsam, via usual reagents 



23.215 Tribondeau, Fichet, and Dubreuil 1916 6630, 79:710 



STOCK solutions: I. water 100, potassium alum 3.5, tannic acid 3; II. 1% crystal violet 

 PREPARATION OF STOCK I: Mix ingredients. Autoclave, 20 lbs. for 30 minutes. Cool. Filter. 

 WORKING solution: stock I 100, stock II 10 



method: [heat-fixed smear] -^ stain, heated to boiling, 30 sees. — > wash —* dry 

 note: If background too granular, decrease proportion of stock II. If flagella not 

 stained, increase proportion of stock II. A detailed description of the use of this tech- 

 nique is given under 23.20 above. 



23.215 Wright 1928 see DS 23.215 Pittfield 1910 



23.215 Yokata 1924 6630, 90:1303 



REAGENTS REQUIRED: A. ADS 12.2 Yokata 1924; B. sat. sol. (circ. 3.5) aniline 100, 



magenta 0.03 

 method: [heat-dried smear] -^ A, raised to boiling, 30 sees. — > water, wash — > B, on 



slide, raised to boiling 2 or 3 times -^ water, wash -^ dry 



23.215 Zettnow 1891 see MS 33.1 Zettnow 1891 



23.215 Zikes 1930 see DS 23.215 Lofiier 1890 (note) 



£3 £16 Spore Stains 



The reagents here given are intended not so much to stain bacterial spores in their 

 free condition as to differentiate a spore within a bacterium, in order that its spore- 

 forming nature may be determined for diagnostic purposes. Tlie majority of these 

 methods depend on differentiating a first stain with an acid solution, and then ap])ly- 

 ing a counterstain to bring into contrast the main body of the bacteria. Other methods 

 depend upon the selective affinity of malachite green or light green for the spore itself, 

 and thus permit the direct coloration of the spore without subsequent differentiation. 

 Probably the surest and simplest method is that of Muzzarelli 1931, though the selec- 

 tion from the methods given is largely a matter of opinion. 



23.216 Abbott test. 1920 Stitt Stitt 1920, 55 

 REAGENTS REQUIRED: A. DS 11.44 Lofflcr 1890; B. 2% nitric acid; C. 1% eosin 

 method: [lieat-fixed smears] — » /I, on slide, raised to boihng 3 or 4 times, 1 min. -^ B, 



till colorless -^ water, wash — » C, 15 sees. — > water, wash — > dry 

 result: spores blue on yellow. 



