BACTERIA. BIOCHEMICAL TESTS 28 



BACTERIA. MEDIA 



is a key to identification of bacteria, is 

 often useful. 



Motility, agglutination, lysis under 

 influence of bacteriophage, ingestion by 

 leucocytes and many other phenomena 

 can best be observed by examination of 

 living bacteria by direct illumination or 

 in the darkfield. Smears, usually fixed 

 by heat, are, however, most often used. 

 A choice must be made from many well 

 known stains including : Anilin Gentian 

 Violet, Loeffler's Methylene Blue, 

 Giemsa, Gram and Carbol Fuchsin. 

 Others are best listed under the particu- 

 lar structures to be demonstrated 

 Spores, Flagella, Capsules. In some 

 cases search for bacteria in Milk, Soil, 

 Cheese, Sputum, etc. is indicated. 

 When bacteria are so few in number that 

 they may be missed, or large numbers 

 are required separated from the tissues 

 for chemical analysis, Concentration 

 methods may be useful. Accurate 

 localization of bacteria requires their 

 study in sections. See Giemsa's stain, 

 Gram-Weigert stain, Goodpasture's 

 stain (!MacCallum's modification), Mal- 

 lory's Phloxine-Methylene blue and 

 Acid Fast Bacilli. The darkfield 

 examination of stained preparations is 

 said to be an advantage (Goosemann, 

 C, J. Lab. and Clin. Med., 1935-36, 

 21, 421-424). Appearance when viewed 

 at high magnification with electron 

 microscope (Mudd, S., Polevitsky, K., 

 and Anderson, T. F., Arch. Path., 1942, 

 34, 199-207). See Fluorescence micros- 

 copy, Negative Strains, Dead bacteria, 

 Tubercle bacilli, Leprosy bacilli, Mito- 

 chondria and Bacteria in same cells, 

 Rickettsia, Gonococcus, Diphtheria Ba- 

 cilli, Bacterium Tularense, Bacterium 

 Monocytogenes. 

 Bacteria. Biochemical Tests. Given in 

 greater detail by H. R. Livesay in 

 Simmons and Gentzkow, 387-389. 



1. Indicators of pH. Incorporate in 

 basic culture of medium measured 

 amounts of 0.02% aq. phenol red, 0.04% 

 aq. bromcresol purple, or 0.1% aq. 

 bromthymol blue. Their pH ranges 

 and colors are given under Hydrogen 

 Ion Indicators. 



2. Indoltest. Use Bohme's reagents. 

 To 5 day culture in 1% aq. peptone add 

 1 cc. ether, shake and settle. Let 1 cc. 

 of following run down inside tube : p- 

 dimethylaminobenzaldehyde, 4 gm.; 

 95% ethyl alcohol, 380 cc; cone, hydro- 

 chloric acid, 80 cc. If after 1 min. no 

 color develops add 1 cc. sat. aq. po- 

 tassium persulf ate . Positive , pale pink 

 to deep magenta. 



3. Ilosvay's Nitrate reduction. To 

 5 day culture at 37°C. in broth + 0.1% 

 HNO3 add 1 cc. of following solution. 



Dissolve 1 gm. a-naphthylamine in 22 

 cc. aq. dest. Filter and add 180 cc. of 

 dilute acetic acid (sp. gr. 1.04). Then 

 1 cc. of sulfanilic acid (0.5 gm. in 150 cc. 

 dilute acetic acid). Positive, pink, 

 red or maroon; negative, no color. 



4. Ammonia. To 5 day peptone 

 water culture add 0.5 cc. Nessler's Re- 

 agent. Positive, brown; negative, faint 

 yellow. 



5. Hydrogen sulfide. Inoculate or- 

 ganisms on lead acetate agar made by 

 sterilizing extract broth containing 4% 

 peptone + 2.5% agar and equal volume 

 0.1% aq. basic lead acetate. Positive, 

 brown or black; negative, no color. 



6. Reductase. To a 24 hr. broth cul- 

 ture add 1 drop 1% aq. methylene blue. 

 Incubate at 37°C. Positive, complete 

 decolorization; weakly positive, green 

 color; negative, no decolorization. 



7. Catalase. Pour 1 cc. HjOj over 

 24 hr. agar slant culture incubated at 

 37''C. holding tube on incline. Posi- 

 tive, gas bubbles; negative, none. 



8. Methyl red. To 4 day culture in 

 glucose phosphate medium at 37°C. 

 add 5 drops 0.04% methyl red in 60% 

 alcohol. Positive, red; negative, yel- 

 low. 



9. Voges-Proskauer. To 4 day cul- 

 ture in glucose phosphate medium at 

 37°C. add 5 cc. 10% aq. KOH. After 

 18-24 hrs. positive, pink fluorescence; 

 negative, no color. 



10. Oxidase. To surface of colony 

 add loop full or 1-2 cc. fresh 1% aq. 

 dimethylparaphenylenediamine hydro- 

 chloride. Positive, color change from 

 pink to maroon to black. 



Bacteria Flagella. Electron microscopic 

 technique reveals the ultrastructure of 

 bacterial flagella as composite struc- 

 tures made up of a central coiled trypsin 

 resistant filament and a peripheral 

 sheath probably nonresistant (De 

 Robertis, E. and Franchi, C. M., Exp. 

 Cell Res., 1951, 2, 295-298). 



Bacteria. Media. The following are brief 

 summaries of culture media as described 

 by H. R. Livesay in Simmons and 

 Gentzkow, 388-403. 



(Glucose phosphate. Witte or Difco 

 proteose peptone, 0.5 gm.; K2HPO4, 0.5 

 gm.; glucose, 0.5 gm.; aq. dest., 100 

 cc; pH 7.5.) 



Meat extract broth (routine). Add 

 to 1000 cc aq. dest., beef extract, 3 gm. ; 

 peptone, 10 gm.; sodium chloride, 5 gm. 

 Dissolve by stirring with heat (water 

 bath 65°C.). Make up weight loss with 

 aq. dest. and make pH 7.2-7.4. Boil 

 over flame, cool to 25°C., again make 

 up weight loss, clarify and check pH. 

 Place in flasks or tubes, autoclave 15 

 lbs., 15 min. 



