BACTERIA 



33 



BACTERIA. MEDIA 



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 

 Simnions 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 persulfate. 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. H2O2 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. 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. 



Meat extract broth (for water anal- 

 ysis). As above, using beef extract, 

 3 gm.; peptone, 5 gm.; aq. dest. 1000 

 cc. pH 6.4-7. 



Meat extract agar (routine). Dis- 

 solve 20-30 gms. powdered agar in 

 1000 cc. meat extract broth stirring 

 over flame and titrate to pH 7.4. Cool 

 to 50°C., add stirred eggs, heat gently 

 till egg material is firmly coagulated. 

 Remove coagulum with fine wire mesh 

 strainer, filter through cotton, make 

 up filtrate to original weight with aq. 

 dest. and make pH 7.2-7.4. Tubes or 

 flasks. Autoclave 15 lbs., 15 min. 



Meat extract agar (for water anal- 

 ysis). Add 15 gm. best quality agar 

 to 1000 cc of above meat extract agar 

 and make pH 6.4-7. 



Meat infusion broth. Mix 500 gms. 

 ground fat-free beef, or veal round, in 

 1000 cc. aq. dest in ice box 18-24 hrs. 

 Heat over small flame in Arnold steri- 

 lizer, 1 hr., add 5 gm. sodium chloride 

 and 10 gm. peptone. Dissolve our 

 flame, filter, add aq. dest. to 1000 cc, 



