APPENDIX A Media and Reagents Pauline K. w. Yu, M.S. The use of appropriate and dependable media is integral to the isolation and identification of microorganisms. Unfortunately, comparative data docu­ menting the relative efficacy or value of media designed for similar purposes are often lacking. Moreover, one cannot presume identity in composition of a given generic product which is manufactured by several companies because each may supplement the generic products with components, often of a proprietary nature and not specified in the product's labeling. Finally, the actual production of similar products may vary among manufacturers to a sufficient extent to affect their performance. For all of these reasons, therefore, product selection for the laboratory should not be strictly based on cost considerations and should certainly not be based on promotional materials. Evaluations that have been published in the scientific literature should be consulted when available. Alternatively, the prospective buyer should consult a recognized authority in the field. It is seldom necessary for the laboratory to prepare media using basic components since these are usually available combined in dehydrated form from commercial sources; however, knowledge of a medium's basic compo­ nents is helpful in understanding how the medium works and what might be wrong when it does not work. Hence, the components have been listed for each medium included in this chapter. All dehydrated media must be prepared exactly according to the manu­ facturers' directions. Any deviation from these directions may adversely affect or significantly alter a medium's performance. Containers of media should be dated on receipt and when opened, and the media should never be used beyond expiration dates specified by the manufacturers or recom­ mended by quality control programs. 747 748 Appendix A: Media and Reagents The media included in this chapter were selected primarily on the basis of efficacy and reliability in dse by the authors. This listing is by no means an exhaustive compilation of currently available media. Nearly all the media are available in dehydrated form from commercial sources. Specific brands or commercial sources are cited only when their use is deemed important to the medium's performance. Also included in the chapter are reagents. Once again, those listed are those in use in this laboratory. I. Media for Aerobic and Facultatively Anaerobic Bacteria Acetate Agar NaCI 5g MgS04 0.2g (NH4) H2P04 Ig K2HP04 Ig Sodium acetate 2g Agar 15 g Bromthymol blue 0.08g Distilled water 1000ml Final pH 6.7. This medium is prepared in a similar manner as Simmons' citrate agar. See p. 204 for use of the medium. Bile-Esculin Agar2•13 Bile-esculin agar base (Difco Laboratories) Beef extract 3g Peptone 5g Oxgall 40g Ferric citrate 0.5 g Agar 15 g Distilled water 1000mi Final pH 6.6. Esculin Ig Suspend 63 g of base medium and 1 g of esculin in 1000 ml of distilled water. Heat to boiling to dissolve completely. Dispense into screw-capped tubes and sterilize at 121 DC for 15 min. Let agar solidify in a slant position. See p. 149 for use of the medium. I. Media for Aerobic and Facultatively Anaerobic Bacteria 749 Bismuth Sulfite Agar (Modified Wilson and Blair Medium) Beef extract 5g Peptone (Bacto or Polypeptone) 10 g Glucose 5g Na2HP04 4g Ferrous sulfate 0.3g Bismuth sulfite indicator 8g Brilliant green O.025g Agar 20g Distilled water 1000mi Final pH 7.5. Suspend 52 g of dehydrated medium in 1000 ml of distilled water and heat to boiling to dissolve completely. Agitate the flask thoroughly to evenly dispense the characteristic precipitate present in the medium; pour into plates. Plates should be used the same day they are prepared. See p. 114 for use of the medium. Blood Agar Prepare 1000 ml of blood agar base medium-soybean-casein digest [Tryptic Soy Agar (Difco Laboratiories), Trypticase Soy Agar (BBL Microbiology Systems), etc.]. Adjust pH to 7.3. Sterilize at 121°C for 15 min. Cool to 50°C, and aseptically add 50 ml of sterile defibrinated blood. Mix and pour plates. This plating medium supports the growth of most medically significant bacteria. It is used for primary plating and for subculturing of colonies and is especially useful for detecting hemolytic activity of bacteria. With certain exceptions, sheep blood is recommended for general use, mainly because colonies of ,a-hemolytic streptococci show characteristic clear zones on this blood medium. In addition, colonies of streptococci that are neither typically a- nor ,a-hemolytic on human blood agar appear as typical a-hemolytic colonies after 18 to 24 hr of incubation. One advantage of sheep blood is its inhibitory effect on the growth of bacteria such as H aemophilus haemo­ lyticus, the colonial morphology and hemolytic properties of which may resemble those of the ,a-hemolytic streptococci. In order to isolate Haemo­ philus spp., 5% sterile fresh rabbit or horse blood may be added to the SallIe base in place of the sheep blood. Rabbit and horse blood provide both factors X and V. Bordet-Gengou (B-G) Agar Base Potato, infusion from 125 g NaCI 5.5g Agar 20g Distilled water 1000mi 750 Appendix A: Media and Reagents Add 10 ml of glycerol to 1000 ml of distilled water. Mix well and add 30 g of dehydrated basal medium. Mix thoroughly and heat with frequent agita­ tion. Boil for 1 min. Sterilize at 121°C for 15 min. Store this sterile "B-G base" at 4°C. Just prior to use, melt the base, cool to 50°C, and add 15% sterile defibrinated sheep or rabbit blood aseptically. Mix well, and pour plates. This medium is used to isolate Bordetella pertussis. Penicillin (0.5 unit/ ml of agar base) can be added prior to dispensing to inhibit the normal flora of the respiratory tract. However, some strains of B. pertussis may be inhibited by penicillin, so a plate without penicillin also should be used. The preferred medium in this laboratory for isolation of B. pertussis is charcoal agar (p. 755). Brucella Biphasic Medium (Castaneda) Bottle Soybean-casein digest agar (Trypticase, BBL Microbiology Systems), 50 ml/bottle Soybean-casein digest broth (Trypticase, BBL Microbiology Systems), 60 ml/bottle The agar base is melted and poured (50 ml/bottle) into 8 oz prescription bottles which are capped with a sponge stopper and sterilized at 121°C for 15 min. As soon as the bottle is cool enough to handle, the sponge stopper is replaced by a sterile red rubber sleeve serum stopper with needle septum. This will maintain the suction in the bottle necessary for the introduction of CO2• The agar is then slanted by placing the bottles on their sides on a level surface and allowing the agar to cool and harden for 24 hr. The broth is then added aseptically (60 ml/bottle) through sterile tubing attached to a sterile needle that is used to puncture the rubber stopper. CO2 is then introduced into the bottle to a concentration of 10%. This bottle is used to recover Brucella and Neisseria from blood. On receipt and daily thereafter, it is examined for the presence of turbidity in the broth and colonies on the agar slant. If these signs of growth are absent, the bottle is tipped onto its side so that the blood broth mixture flows over the agar slant. Other basal media may be used (e.g., brain heart infusion) in the bottle instead of Brucella medium, and sodium polyanetholsulfonate (0.025%) can be added to the broth to facilitate the recovery of other aerobic and facultatively anaerobic bacteria from blood. Brucella Broth Polypeptone peptone 20 g Glucose Ig Yeast extract 2g I. Media for Aerobic and Facultatively Anaerobic Bacteria 751 NaCl 5g NaHSOs 0.1 g Distilled water 1000ml Final pH 7.0. Suspend 28 g of dehydrated medium in 1000 ml of distilled water. Heat with agitation until completely dissolved. Sterilize at 121°C for 15 min. For Campylobacter identification: Add 1.6 g of agar to 1000 ml of Bru­ cella broth. Heat with agitation until completely dissolved. To three 200 ml portions of medium are added potassium nitrate, 0.2 g, glycine, 2 g, and L-cysteine hydrochloride· H20, 0.04 g, respectively. Dispense 10 ml of each medium in screw-capped tubes, and sterilize at 121°C for 15 min. Keep caps on tightly during storage. Buffered Glycerol-Saline Solution (Sachs' Modification) NaCl 4.2g K2HP04 3.1 g KH2P04 1.0 g Phenol red 0.003 g Distilled water 700ml Final pH 7.2. Dissolve 8.3 g of dehydrated material in 700 ml of distilled water. Add 300 ml of glycerol. Dispense in 10 ml amounts in 30 ml screw-capped bottles. Sterilize for 15 min at 121°C. This medium serves as a stool specimen preservative and may be used to transport fecal material. Of various available transport media, buffered glycerol-saline appears to be the best for Shigella. 1 Campylobacter Media a. C-O Agar Brucella agar 43g FeS04·7H20 0.25g Sodium metabisulfite 0.25g Sodium pyruvate 0.25g Hemin (5mg/ml) 1ml Distilled water 1000 ml Final pH 7.0. Mix the ingredients thoroughly in distilled water with heat and frequent agitation. Boil for approximately 1 min. Sterilize at 121°C fo 15 min. Allow to cool to 50°C. Add 50 ml of sterile defibrinated sheep blood and 1 ml of vitamin Kl solution (10,000 ftg/ml). Pour into petri dish. For preparation of hemin and vitamin Kl solution, see p. 776. 752 Appendix A: Media and Reagents This is an enrichment medium containing reagents designed to reduce the toxicity of oxygen for Campylobacter.