APPENDIX A Media and Reagents

Pauline K. w. Yu, M.S.

The use of appropriate and dependable media is integral to the 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), (BBL 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 and 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 . 1

Campylobacter Media a. C-O 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. It contains no antibiotics, and is, therefore, not useful for the isolation of Campylobacter from feces; it is use• ful for culturing nonnally sterile fluids or for subculture work. b. C-1 Agar C-o agar containing final concentrations of each of the following antibiotics: 25 U/ml Novobiocin 5 f'g/ml Colistin 1 f'g/ml Cephalothin 5 f'g/ml c. C-2 Agar c-o agar containing final concentrations of each of the following antibiotics: Vancomycin 10 f'g/ml Trimethoprim 5 f'g/ml Polymyxin B 1 f'g/ml Cephalothin 5 f'g/ml Stock antibiotic solutions, with the exception of bacitracin, are prepared at 1000 times their final concentrations; bacitracin is prepared at 40 times its final concentration. All stock solutions are stored frozen at -20°C. Thawed solutions are added to C-O agar which has been allowed to cool to 50°C. C-1 and C-2 agars are used for isolating Campylobacter from feces, as well as from sites or tissues likely to harbor fecal flora.

Carbohydrate Fermentation Broth, Andrade's a. Broth Base Peptone 10 g Meat extract 3g NaCI 5g Andrade's indicator 10ml Distilled water 1000ml Final pH 7.6. Mix dry ingredients in water and boil to dissolve. Adjust pH to 7.25, then add Andrade's indicator. Dispense in tubes with inverted Durham tubes and sterilize at 121°C for 15 min. Final pH should be 7.6. Carbohydrates are generally prepared as 10% (w/v) aqueous solutions; less soluble carbohydrate (e.g., dulcitol) is prepared as a 5% solution. I. Media for Aerobic and Facultatively Anaerobic Bacteria 753

Stock solutions are filter sterilized to prevent possible degradation due to autoclaving. They are added to the broth base to achieve a final concentra• tion of 1 %. Dulcitol is added to yield a final concentration of O.S%. See p. 207 for use of the medium. b. Andrade's Indicator Acid fuchsin o.Sg NaOH,IM 16ml Distilled water l00ml Dissolve acid fuchsin in distilled water and add NaOH. H the fuchsin is not sufficiently decolorized after several hours, add an additional 1 or 2 ml of the alkali. The amount of NaOH which should be used depends on the dye content of different lots of acid fuchsin. Once decolorization is achieved, let indicator solution stand overnight. Decant off supernatant and discard the sediment. Andrade's indicator improves somewhat on aging and should be prepared 6 months ahead of its antiCipated use. The indicator is used in the proportion of 10 ml per liter of medium.

Carbohydrate Fermentation Broth, Phenol Red Base Trypticase peptone 10 g NaCI 5g Phenol red 0.018 g Distilled water 1000mi Final pH 7.4. Dissolve IS g of the dehydrated medium in 1000 ml of distilled water. Dispense in S ml amounts in screw-capped tubes. Sterilize at 116 to 118°C for IS min. Carbohydrates are incorporated to a final concentration of 1 % . See p. 168 for use of the medium.

Carbohydrate Utilization Media for Neisseria a. Serum-Free Medium5 (1) Agar Base GC medium base 36g Distilled water 970ml Dissolve by boiling, and then add 20 ml of the supplement mixture: ( a) L-Glutamine Ig Distilled water 90ml (b) Fe(NOsh·9H20 O.05g Distilled water 10ml Final pH 7.6. 754 Appendix A: Media and Reagents

(2) Phenol Red Stock Solution Phenol red 0.2g NaOH,O.IM 5.70ml Distilled water 94.30ml Dissolve dye in NaOH and dilute with water. Add 10 ml of 0.2% phenol red stock solution to the agar base. Distribute in 90 ml volumes in screw• capped bottles. Sterilize at 121°C for 10 min. Allow to cool to 50°C and add 10 ml of appropriate filter sterilized 10% (wIv) carbohydrate solution to each bottle. Mix well and dispense 3 ml aliquots into sterile small screw• capped vials and slant. See p. 162 for use of the medium. b. Cystine Trypticase Agar (CTA) CTA basal medium 28.5 g Distilled water lOOO ml Final pH 7.3. Heat to dissolve. Dispense 90 ml amounts in screw-capped bottles. Auto• clave at 121°C for 15 min. Cool to 50°C and aseptically add 10 ml of a 10% (wIv) filter-sterilized appropriate carbohydrate solution. Mix well and dis• pense 5 ml aliquots into small screw-capped vials and allow to harden. See p. 162 for use of the medium.

Cetrimide Medium (Pseudosel Agar) Gelysate peptone 20 g MgCb 1.4 g K ZS04 10 g Agar 13.6 g Cetrimide ( cetyltrimethylammonium bromide) 0.3g Distilled water lOOOml Final pH 7.2. Dissolve 45.3 g of dehydrated medium in 1000 ml of distilled water by heat• ing. Dispense 6 ml into tubes. Sterilize at 121°C for 15 min. Allow agar to solidify in slant position. Cetrimide medium is inoculated lightly, with a straight wire, from a young agar slant culture and incubated at 35°C for 7 days. Growth of the organism on this medium is interpreted as a positive result. Over 95% of strains of and Pseudomonas fluores• cens are cetrimide-tolerant, as are 85 to 90% of Pseudomonas putida and 70 to 75% of Pseudomonas cepacia and Pseudomonas pseudoalcaligenes. Most strains of other species of Pseudomonas are cetrimide-intolerant. I. Media for Aerobic and Facultatively Anaerobic Bacteria 755

Charcoal Agar12 (Difco Laboratories) Beef heart, infusion from 500g Peptone 10 g NaCl 5g Soluble starch 109 Yeast extract 3.5 g Activated charcoal (Norit SG) 4g Agar 18 g Distilled water 1000 ml Final pH 7.3. Suspend 62.5 g of dehydrated medium in 1000 ml of distilled water. Heat to boiling with frequent stirring. Dispense 21.5 ml of agar into screw-capped tubes. Sterilize at 121°C for 15 min. Cool and store tubes at 4°C. When needed, melt one tube of charcoal agar and aseptically add 2.5 ml of defibrinated sheep blood and 1 ml of cephalexin (1000 p.g/ ml ). Mix well and pour content into petri dish. The charcoal neutralizes substances, such as fatty acids and peroxides, that are toxic to Bordetella pertussis, and the cephalexin selectively inhibits the growth of bacteria indigenous to the oropharynx.

Charcoal Yeast Extract (CYE) Agar3 Yeast extract 10 g Activated charcoal (Norit A) 2g L-cysteine HCI· H 20 0.4 g Ferric pyrophosphate, soluble O.25g Agar 17 g Distilled water 1000ml Final pH 6.9. Dissolve all ingredients except L-cysteine HCl and soluble ferric pyrophos• phate in water by boiling. Sterilize at 121°C for 15 min. Allow to equilibrate to 50°C in a water bath. Prepare fresh solutions of L-cysteine HCI and soluble ferric pyrophos• phate as described for Feeley-Gorman agar (p. 759). Adjust pH to 6.9 with 1 M KOH. Pour 20 ml per petri dish, swirling medium between pouring to keep charcoal suspended. This medium is suitable for culture of the . It is a more sensitive medium than Feeley--Gorman agar but does not provide the distinctive brown soluble pigment obtainable on that medium.

Chocolate Agar GC agar base Peptone (Polypeptone or Proteose no. 3) 15g 756 Appendix A: Media and Reagents

Cornstarch 19 K2HP04 4g KH2P04 19 NaCI 5g Agar 109 Distilled water 1000mi Final pH 7.2. Prepare double strength GC agar base by adding 7.2 g to 100 ml of distilled water. Mix well and heat to boiling for 1 min with frequent shaking. Sterilize at 121°C for 15 min. Cool agar to 50°c' Add 2 ml of IsoVitaleX (BBL Microbiology Systems) or 2 ml of supplement B (Difco Laboratories) that has been warmed to room temperature and 100 ml of 2% hemoglobin solu• tion that is maintained at 50°c' Mix well and pour plates. is recommended as a primary plating medium for spinal fluids, eye cultures, gonococcal cultures, and any other specimens which may contain fastidious organisms because it supplies the special growth require• ments (X and V factors) of H aemophilus influenzae and, when incubated in CO2, of and . With pro• longed incubation (approximately 7 days), the medium is suitable for isolation of LegioneUa pneumophila from normally sterile body fluids and lung tissue.

Citrate Agar (Simmons) NaCI 5g MgS04 0.2g (NH4 )H2P04 19 K2HP04 19 Sodium citrate 2g Agar 15g Bromthymol blue 0.08g Distilled water 1000 ml Final pH 6.9. Dissolve, dispense in tubes, and sterilize at 121°C for 15 min. Allow agar to solidify in a slant position. See p. 193 for use of the medium.

Columbia Colistin-Nalidixic Acid (CNA) Agar Polypeptone peptone 10 g Biosate peptone 10 g Myosate peptone 3g Cornstarch 19 NaCI 5g Agar 13.5 g I. Media for Aerobic and Facultatively Anaerobic Bacteria 757

Colistin 10mg Nalidixic acid 15mg Distilled water 1000 ml Final pH 7.3. Dissolve 42.5 g of the dehydrated medium in 1000 ml of distilled water with heating and frequent stirring. Boil for 1 min. Sterilize at 121°C for 15 min. Let the medium cool to 50°C, add 50 ml of sterile defibrinated sheep blood. Mix well, and aseptically distribute into petri dishes. CNA agar is a selective medium for the isolation of staphylococci and streptococci. It inhibits the growth of most gram-negative bacilli; however, it is useful as a selective medium for the isolation of Haerrwphilus vaginalis (Corynebacterium vaginale) from genital materiaJ.7

Cystine Tellurite Blood Agar Mueller tellurite base Casamino acids, technical 20g Casein 5g KH2P04 0.3 g MgS04 0.1 g L-Tryptophan 0.05g Agar 20g Distilled water 1000ml Dissolve by boiling 22.5 g of Mueller tellurite base in 500 ml of distilled water. Sterilize at 121°C for 15 min and allow to cool to 50°C. Add 25 ml of sterile, defibrinated rabbit blood, 75 ml of 0.3% sterile potassium tellurite solution, and 22 mg of L-cystine. Swirl the medium while pouring it into petri dishes to keep the cystine suspended. Final pH of the medium is 7.4. This medium is used for the isolation of Corynebacterium diphtheriae. Tellurite inhibits the growth of bacteria indigenous to the oropharynx and imparts the gray or black color characteristic of C. diphtheriae.

Decarboxylase Medium (Moeller) Thiotone peptone 5g Beef extract 5g Bromcresol purple 15 mgo Cresol red 5mg

" Commercially available dehydrated broth base powder contains 10 mg; an additional 5 mg is added to intensify color change in positive reaction. Dissolve 0.25 g of dye in 46.25 ml of 0.1 M NaOH. Add 53.75 ml of distilled water. Use 2 ml for each liter of medium. 758 Appendix A: Media and Reagents

Glucose 0.5g Pyridoxal 5mg Agar 3 gt Distilled water lO00ml Final pH 6.0. The medium is divided into four 250 ml portions, one of which is tubed for control purposes without the addition of any amino acid. To the remaining portions of base medium are added L-Iysine dihydrochloride, L-arginine monohydrochloride, and L-ornithine dihydrochloride, respectively, to a final concentration of 1 %. If DL amino acids are used, they are added to a final concentration of 2%. The pH of the ornithine portion should be readjusted after the addition and prior to sterilization. These media are tubed in 6 ml amounts 16 X 150 mm tubes, properly labeled or color-coded, and sterilized at 121°C for 10 min. On occasion, a small amount of precipitate may be seen in the ornithine medium; however, this does not interfere with the reaction. See p. 195 for use of the medium.

DNase Test Agar a. For Differentiating Gram-Negative Bacilli Deoxyribonucleic acid (DNA) 2g Phytone peptone 5g Trypticase peptone 15g NaCI 5g Agar 15g Toluidine blue 0 (0.25% aqueous) 40ml Distilled water 960ml Final pH 7.3. Suspend 42 g of the powder in 960 ml of distilled water. Add toluidine blue a and mix thoroughly. Heat with frequent agitation and boil for 1 min. Sterilize at 121°C for 15 min. Cool and pour into petri dishes. See p. 203 for use of the medium. b. For Differentiating Staphylococci Prepare agar as in a. above except omit toluidine blue o. See p. 139 for use of the medium.

Eosin-Methylene Blue (EMB) Agar, Levine Peptone (Bacto or Gelysate) 10 g Lactose 10 g

t 3 g of agar is added to each liter of broth medium to prepare a semisolid medium. I. Media for Aerobic and Facultatively Anaerobic Bacteria 759

K2HP04 2g Agar 15 g Eosin Y 0.4 g Methylene blue 0.065 g Distilled water 1000ml Final pH 7.1. Sterilize medium at 121°C for 15 min. Agitate medium frequently while pouring plates. This differential medium supports the growth of most gram-negative bacilli while inhibiting many of the gram-positive bacteria. It is used both in primary plating and for subculturing (Tables 3-3, 3-4, 3-6, 3-7, 3-8, and 3-9).

Esculin Agar Esculin Ig Ferric citrate 0.5g Heart infusion agar 40 g Distilled water 1000 ml Final pH 7.0. Sterilize medium at 121°C for 15 min and allow agar to harden in slant position. Esculin is hydrolyzed to glucose and esculetin. The latter combines with ferric ion in the medium to form a black complex. The following species of hydrolyze esculin: Klebsiella pneumoniae, Enterobacter aero genes, and Serratia marcescens. Esculin is not hydrolyzed by Escherichia coli, EdwardsieUa, , Arizona, Citro• bacter freundii, Proteus, Morganella, or Providencia. Esculin may be hy• drolyzed by Pseudomonas pseudomallei, Pseudomonas cepacia, Pseudo• monas putrefaciens, and Pseudomonas maltophilia, but is not hydrolyzed by other species of Pseudomonas.

Feeley-Gorman (F-G Agar) 4 Mueller-Hinton agar 38 g L-Cysteine HCI· H 20 0.4 g Ferric pyrophosphate, soluble (Mallinckrodt) O.25g Distilled water l000ml Final pH 6.9. Prepare 1000 ml of sterile Mueller-Hinton agar. Allow it to equilibrate to 50°C in a water bath. Separately dissolve 0.4 g of L-cysteine HCI in 10 ml of distilled water and 0.25 g of soluble ferric pyrophosphate in 10 ml dis• tilled water. Filter sterilize each solution and add separately to the Mueller• Hinton agar, starting with the L-cysteine HCI. Mix well, adjust pH to 6.9 to 7.0 with 1 M HCI if necessary and pour plates. 760 Appendix A: Media and Reagents

Soluble ferric pyrophosphate must be kept dry and stored in the dark at room temperature. It should be discarded when the granules change from green to yellow or brown. The solution can be warmed in a 50DC water bath to facilitate complete dissolving of particles, but should never be heated over 60DC. As with L-cysteine HCI, only a freshly prepared solution should be used for preparing new batches of F-G medium. This medium is suitable for the isolation of Legionella pneumophila from normally sterile body fluids and lung tissue. Colonies produce a distinctive browning of the surrounding medium in 3 to 4 days, and a yellow fluores• cence is seen within and around the colonies under long wave (366 nm) ultraviolet light after the plate has been incubated for 4 to 5 days.

Fletcher's Medium Peptone 0.3g Beef extract 0.2g NaCI 0.5g Agar 1.5g Distilled water 920ml Final pH 7.9. Dissolve 0.25 g of dehydrated medium in 92 ml of distilled water by heating to boiling. Sterilize at 121 DC for 15 min. Cool the medium to 56DC and add 8 ml of reconstituted Leptospira enrichment (Difco Laboratories, cat. no. 0452-60). Mix well and aseptically dispense 7 ml amounts in screw-capped tubes. Inactivate all the tubes the following day at 56DC for 1 hr. This medium is used for the isolation, cultivation, and maintenance of Leptospira.

Gelatin Medium Beef extract 5g Gelatin 120 g NaCI 5g Distilled water lOOOml Add beef extract and NaCI to water and stir solution rapidly while gradually adding gelatin. Place suspension in double boiler and heat with occasional stirring for 20 to 30 min or until gelatin has completely dissolved and medium becomes clear. Dispense into tubes in 6 ml amounts and autoclave at 121 DC for 15 min. See p. 206 for use of the medium.

Glucose Cysteine Agar Pancreatic digest of heart muscle 3g Papaic digest of soymeal 10 g I. Media for Aerobic and Facultatively Anaerobic Bacteria 761

NaCI 5g L-Cysteine hydrochloride 19 Glucose 25g Agar 14g Thiamine 0.05mg Distilled water 1000ml Final pH 6.8. Suspend 58 g of dehydrated medium in 1000 ml of distilled water. Heat with constant stirring and boil for 1 min. Sterilize at 118 to 121°C for 20 min. Cool the medium to 50°C, and aseptically add 50 ml of defibrinated rabbit blood. Also add 1 ml each of penicillin (100,000 U Iml), polymyxin B sulfate (100,000 U Iml), and cycloheximide (100 ""g/ml). Mix well and pour into plates. This medium is used for the recovery of tularensis from con• taminated specimens.

GN (Gram-Negative) Broth Glucose 19 n-Mannitol 2g Sodium citrate 5g Sodium deoxycholate 0.5 g K2HP04 4g KH2P04 1.5g NaCI 5g Tryptose 20g Distilled water lO00ml Final pH 7.0. Dissolve ingredients by heat. Dispense in tubes and sterilize at 116°C for 15 min. This is an enrichment medium for the isolation of Salmonella and Shigella and is recommended by many for the isolation of Shigella (Table 3-9). It is inhibitory to gram-positive organisms; coliforms are usually inhibited up to 6 hr.

Heart Infusion Blood Agar Beef heart, infusion from 500g Tryptose 109 NaCl 5g Agar 15 g Distilled water 1000 ml Final pH 7.4. 762 Appendix A: Media and Reagents

Suspend 40 g of dehydrated medium in 1000 ml of distilled water. Heat to boiling to dissolve completely. Sterilize at 121°C for 15 min. Cool agar to 50°C and aseptically add 10 ml of defibrinated sheep blood. Mix well and pour plates. This medium supports the growth of Brucella, many strains of fungi, and other fastidious microorganisms.

Heart Infusion Broth This is similar to heart infusion agar with omission of the agar.

Hektoen Enteric (HE) Agar Lactose 12g Sucrose 12g Salicin 2g Bile salts no. 3 9g Proteose peptone 12g Beef extract 3g NaCl 5g Sodium thiosulfate 5g Ferric ammonium citrate 1.5 g Agar 14 g Thymol blue 0.065 g Acid fuchsin 0.1 g Distilled water 1000ml Final pH 7.5. Suspend 76 g of dehydrated medium in 1000 mi of water and boil until it has completely dissolved. Do not autoclave this medium. Cool and pour plates. This selective medium is used for the isolation and identification of enteric (Tables 3-8 and 3-9).

Indole Broth Peptone (Bacto or Trypticase ) 15 g NaCI 5g Distilled water lO00ml Final pH 7.1. Dispense 2 ml into 13 X 100 nun screw-capped tubes. Sterilize at 121°C for 15 min. See p. 197 for use of the medium. I. Media for Aerobic and Facultatively Anaerobic Bacteria 763

Lactose Agar (10 %) Phenol red broth base 15g Lactose 100g Agar 20g Distilled water 1000ml Final pH 7.3. Dissolve phenol red broth base in 200 ml of distilled water and filter sterilize. Add solution, agar, and lactose to 800 ml of distilled water. Heat with frequent stirring and boil for one minute to dissolve completely. Adjust pH to 7.9 with 1 M NaOH. Dispense 5 ml aliquots into screw-capped tubes and autoclave at 110°C for 8 min. Final pH of medium should be 7.3. Let medium solidify in a slant position. See p. 220 for use of the medium.

Loeffler Medium Veal infusion broth 25g Glucose 10 g Distilled water 1000mi Add dry ingredients to water. Heat to dissolve and bring to a boil. Dispense 50 ml amounts in screw-capped bottles. Add five pieces of CaCOa chips to each bottle and sterilize at 12PC for 15 min. Cool broth to 50°C and add 150 ml of sterile horse serum to each bottle. Mix well and aseptically dis• pense 5 ml amounts into sterile 16 X 125 mm screw-capped tubes. Slant tubes in a metal rack and inspissate medium at 87°C for 30 min in a sterilizer with an isothermal control.

Lysine-Iron Agar Peptone (Bacto or Gelysate) 5g Yeast extract 3g Glucose Ig L-Lysine 10 g Ferric ammonium citrate 0.5g Sodium thiosulfate 0.04g Bromcresol purple 0.02g Agar 15 g Distilled water 1000mi Final pH 6.7. Dispense into screw-capped tubes and sterilize at 121°C for 15 min. Tubes should be slanted to obtain a deep butt and a short slant. See p. 192 for use of the medium. 764 Appendix A: Media and Reagents

MacConkey Agar Peptone (Bacto or Gelysate) 17 g Peptone (Proteose or Polypeptone) 3g Lactose 109 Bile salts 1.5 g NaCI 5g Neutral red 0.03g Crystal violet 0.001 g Agar 13.5 g Distilled water lOOOml Final pH 7.1. Sterilize at 121°C for 15 min. Lactose-fermenting colonies are red, and may be surrounded by an opaque zone of precipitated bile. This is due to the action of acids on the bile salts and the subsequent absorption of neutral red. Colonies of non• fermenters are colorless and transparent. This differential medium supports the growth of most gram-negative bacilli and may be used interchangeably with eosin-methylene blue agar.

Malonate Broth, Ewing Modified Yeast extract 19 (NH4 )2S04 2g K2 HP04 0.6g KH~04 0.4 g NaCI 2g Sodium malonate 3g Glucose O.25g Bromthymol blue 0.025 g Distilled water lOOOml Final pH 6.7. Sterilize at 121°C for 15 min. Dispense 1 ml in 13 X 100 mm screw-capped tubes. See p. 205 for use of the medium.

Mannitol Fermentation Agar Trypticase 10 g Yeast extract 1.5 g Sodium chloride 5g D-mannitol 109 Agar 15 g Bromcresol purple 0.015 g Distilled water lOOOml Final pH 7.1. I. Media for Aerobic and Facultatively Anaerobic Bacteria 765

Heat to dissolve and dispense in screw-capped tubes to a height of 8 cm. Autoclave at 121°C for 15 min. See p. 138 for use of the medium.

McBride Medium Peptone (Biosate or Tryptose) 109 Beef extract 3g NaCI 5g Glycine anhydride 10 g Lithium chloride 0.5 g Phenylethanol 2.5 g Agar 15 g Distilled water 1000ml Final pH 7.S. Mix dry ingredients in 1000 ml of distilled water. Bring to a boil to dissolve completely. Sterilize at 121°C for 15 min.

Motility Medium Casitone 10 g Yeast extract 3g NaCI 5g Agar 3g Distilled water 1000ml Final pH 7.2. Mix dry ingredients in water, heat to a boil. Dispense 6 ml per tube. Sterilize at 121°C for 15 min. See p. 174 for use of the medium.

MR-VP Broth Peptone (Buffered or Polypeptone ) 7g Glucose 5g K 2HP04 5g Distilled water 1000ml Final pH 6.9. Sterilize at 121°C for 15 min. Dispense in 0.5 ml amounts in 13 X 100 mm screw-capped tubes. Although several modifications of the Clark and Lubs formula are avail• able, the one given above has been used satisfactorily in this laboratory. See p. 199 for use of the medium.

Mueller-Hinton Agar Beef, infusion from 300g Peptone (Acidicase or Bacto-Casamino Acids, 17.5 g Technical) 766 Appendix A: Media and Reagents

Starch 1.5 g Agar 17 g Distilled water 1000 ml Final pH 7.4. Mix the medium thoroughly in distilled water, with heat and frequent agita• tion. Boil for approximately 1 min and dispense. Sterilize by autoclaving at 121°C for not more than 15 min. This medium, which was designed for the primary isolation of Neisseria gonorrhoeae and Neisseria meningitidis, is most frequently used for the de• termination of antimicrobial susceptibility by the disk or agar-dilution method. Because it does not inhibit drug action, susceptibility tests of sul• fonamide and trimethoprim should be determined on this medium.

Mueller-Hinton Broth This is similar to Mueller-Hinton agar in formulation, with the omission of agar. It is used primarily for antimicrobial susceptibility and should be supplemented with 50 mg of Ca and 25 mg of Mg per liter of medium.

Nitrate Broth Nutrient broth base Sg KNOa 19 Distilled water 1000ml Final pH 7.0. Stir until dissolved. Bring to a boil. Dispense 2 ml amounts in 13 X 100 mm screw-capped tubes. Sterilize at 121°C for 15 min. See p. 221 for use of the medium.

Nutrient Agar Beef extract 3g Peptone (Bacto or Gelysate ) 5g Agar 15 g Distilled water 1000ml Final pH 6.S. Dissolve dry ingredients in water with heat and frequent agitation. Dispense and sterilize at 121°C for 15 min.

Nutrient Broth This is similar to with the omission of agar. I. Media for Aerobic and Facultatively Anaerobic Bacteria 767

Oxidation-Fermentation (O-F) Medium (Hugh and Leifson) Peptone 2g NaCI 5g KzHP04 0.3g Agar 2.5g Bromthymol blue 0.03g Distilled water 1000ml Final pH 7.1. Distribute basal medium in bottles and sterilize at 121°C for 15 min. Cool medium to 50°C and add appropriate IDter-sterilized carbohydrate solutions to give a final concentration of 1 %. Mix well and aseptically dispense 5 ml amounts into 16 X 125 mm screw-capped tubes. See p. 218 for use of the medium.

Phenylalanine Agar Yeast extract 3g DL-Phenylalanine 2g ( or L-phenylalanine) (1 g) Na2HP04 Ig NaCI 5g Agar 12g Distilled water l000ml Final pH 7.3. Dispense and sterilize at 121°C for 10 min. Allow to solidify as a long slant. See p. 202 for use of the medium.

Phenylethyl Alcohol (PEA) Blood Agar Trypticase peptone 15 g Phytone peptone 5g NaCI 5g Phenylethyl alcohol 2.5 g Agar 15 g Distilled water 1000 ml Final pH 7.3. Sterilize at 121°C for 15 min and cool to 50°C. Aseptically add 50 ml of defibrinated sheep blood, mix well, and pour into petri dishes. PEA is used for the selective isolation of gram-positive cocci, either from clinical speci• mens or by subculture of primary plating media. Pseudomonas aeruginosa, however, is not inhibited. Hemolysis cannot be reliably determined on this medium; therefore, streptococci should be subcultured onto sheep blood agar. 768 Appendix A: Media and Reagents

Phosphate-Buffered Saline (PBS), pH 7.2, for Legionella pneumophila

0.15 M KH2P04 stock solution KH2P04 20.41 g Distilled water lOooml

0.15 M NazHP04 stock solution Na2HP04, anhydrous 21.29 g Distilled water lOooml PBS, pH 7.2 KHzP04 stock solution 24ml NazHP04 stock solution 76ml NaCI 1.7 g Distilled water 100ml Dispense 5 ml amounts into screw-capped tubes and sterilize at 121°C for 15 min. This is a diluent used for preparing a suspension of lung tissue for isola• tion of Legionella pneumophila.

Pigment Production Media Pseudomonas agar P or Tech agar Peptone (Bacto or Gelysate) 20g Glycerol (c.P.) 10ml MgClz 1.4 g K2S04 10 g Agar 13.6 g Distilled water lOooml Final pH 7.2. Pseudomonas agar F or Flo agar Peptone (Proteose no. 3 or Polypeptone) 20g K2HP04 1.5g MgS04·7H20 1.5g Agar 14 g Distilled water lOOOml Final pH 7.2. Tube and sterilize at 121°C for 15 min. Slant tubes so as to obtain a deep butt. See p. 223 for use of these media.

Rogosa SL Agar Tryptone 109 Yeast extract 5g I. Media for Aerobic and Facultatively Anaerobic Bacteria 769

Glucose 109 Arabinose 5g Sucrose 5g Sodium acetate 15g Ammonium citrate 2g KH2P04 6g MgS04·7H20 0.57g MnS0 4 0.12g FeS04 0.03g Sorbitan monooleate 19 Agar 15 g Distilled water lOOOml Final pH 5.4. Add dry ingredients to distilled water and heat to boiling to dissolve the solids completely. Add 1.32 m1 of glacial acetic acid and continue boiling for 2 to 3 min. Do not autoclave. Cool to 50°C and pour plates. This selective medium is useful in the cultivation and presumptive iden• tification of lactobacilli.

Salmonella-Shigella (55) Agar Beef extract 5g Peptone (Polypeptone or Proteose) 5g Lactose 109 Bile salts 8.5 g Sodium citrate 8.5g Sodium thiosulfate 8.5 g Ferric citrate 1.0 g Agar 13.5g Brilliant green O.33mg Neutralred 0.025 g Distilled water lOOOml Final pH 7.0. Heat to boiling for 2 to 3 min to dissolve completely. Do not autoclave. Dispense medium into petri dishes after it has cooled to 50°C. SS agar is an inhibitory medium used for the isolation of Salmonella, Shigella, and Yersinia enterocolitica (Tables 3-8 and 3-9).

Selenite Broth Peptone (Tryptone or Polypeptone) 5g Lactose 4g Na~P04 109 770 Appendix A: Media and Reagents

Sodium acid selenite 4g Distilled water 1000mi Final pH 7.0. Dissolve ingredients in water and bring to a boil. Boil for 1 min. Do not auto• clave. Dispense 5 ml into sterile screw-capped tubes. Store tubes at 4°C until use. This is an enrichment medium for the isolation of Salmonella and Shigella (Table 3-9). Selenite and GN broths provide roughly comparable isolations of Shigella when subcultured onto a variety of plating media; however iso• lations of Salmonella tend to be greater from selenite broth.!

Sodium Bicarbonate Agar Soybean-casein digest agar 90ml NaHC03,7% aqueous solution lOml

Add 10 ml of filter-sterilized NaHC03 solution to 90 ml of sterile soybean• casein digest agar that is maintained at 50°C. Mix well and pour into petri dishes. This medium is used to enhance the capsule production by anthracis.

Sodium Chloride Broth, Modified2 Heart infusion broth (dehydrated) 25g NaCI 60 g Glucose 109 Bromcresol purple, 1.6% in 95% ethyl alcohol Iml Distilled water 1000mi Dispense in 5 ml aliquots in screw-capped tubes. Autoclave at 121°C for 15 min. See p. 150 for use of this medium.

Soybean-Casein Digest Agar (Tryptic Soy, Difco Laboratories, or Trypticase Soy, BBL Microbiology Systems) Pancreatic digest of casein 15 g Papaic digest of soy meal 5g NaCI 5g Agar 15 g Distilled water 1000mi Final pH 7.S. Sterilize at 121°C for 15 min. I. Media for Aerobic and Facultatively Anaerobic Bacteria 771

Soybean-Casein Digest Broth (Tryptic Soy, Difco Laboratories, or Trypticase Soy, BBL Microbiology Systems) Pancreatic digest of casein 17 g Papaic digest of soy meal 3g NaCI 5g K 2HP04 2.5g Glucose 2.5g Distilled water lOOOml Final pH 7.3. Dissolve, dispense in tubes, and sterilize at 121°C for 15 min. This medium is a general purpose nutrient broth that is particularly useful for growing streptococci. It is also suitable for blood cultures and supports the growth of a variety of aerobic, facultatively anaerobic, and anaerobic bacteria.

Starch Agarll Beef extract 19 Proteose peptone 109 NaCI 5g Corn starch 109 Agar 15 g Bromcresol purple, 0.25% in 95% ethyl alcohol 6ml Distilled water lOOOml Final pH 6.S. Suspend ingredients in distilled water, heat to boiling with constant stirring. Boil for 1 min to dissolve completely. Sterilize at 121°C for 15 min. Cool agar to 50°C and dispense into petri dishes. This medium is used for the identification of vaginalis. Acid production from starch is denoted by color change of the indicator from purple to yellow.

Thayer-Martin Medium, Modified9,lo,14 GC agar base ( double strength) 100ml Hemoglobin, 2% aqueous 100ml Iso VitaleX enrichment (BBL Microbiology Systems) 2ml or Supplement B (Difco Laboratories) Antibiotics, to contain final concentration of vancomycin 3 Itg/ml colistin 7.5 Itg/ml 772 Appendix A: Media and Reagents

nystatin, or 12.5 U/ml anisomycin 20 p.g/ml trimethoprim lactate 5 p.g/ml Glucose 2g Add hemoglobin solution, supplement, and antibiotics to sterile GC agar base that is maintained at 50°C. Mix well and pour plates. This medium or one of its modifications is recommended for the selective isolation of Neisseria gonorrhoeae and Neisseria meningitidis from genital and/ or respiratory tract sources.

Thioglycollate-135C (BBL Microbiology Systems) Trypticase peptone 17 g Phytone peptone 3g Glucose 6g NaCl 2.5g Sodium thioglycollate 0.5g Agar 0.7 g L-Cystine 0.25g Na2SOa 0.1 g Final pH 7.0. Dispense 10 ml in screw-capped test tubes and sterilize at 121°C for 15 min. This thioglycollate medium does not contain an Eh indicator. It is able to support the growth of a wide variety of aerobic and anaerobic bacteria and is highly recommended for use as a general utility broth in the clinical laboratory.

Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar Yeast extract 5g Polypeptone peptone 10 g Sodium citrate 10 g Sodium thiosulfate 109 Oxgall 5g Sodium cholate 3g Sucrose 20g NaCl 10 g Ferric citrate 19 Bromthymol blue 0.04g Thymol blue 0.04g Agar 14g Distilled water 1000 ml Final pH 8.6. Dissolve dehydrated medium in distilled water by heating to boiling for 1 min; do not autoclave. Cool to 50°C, and pour plates. I. Media for Aerobic and Facultatively Anaerobic Bacteria 773

This medium is recommended for the selective isolation of Vibrio cholerae and Vibrio parahaemolyticus (Tables 3-8 and 3-9).

Todd-Hewitt Broth Beef heart, infusion from 500g Neopeptone 20g NaCl 2g Na2C03 2g Na2HP04 0.4 g Glucose 2g Final pH 7.8. Bring ingredients to a slow boil and boil for 15 min. Dispense in tubes, and autoclave at 121°C for 15 min. This is an enriched medium for the cultivation of streptococci. An addi• tional 8 g/liter of glucose can be added to enhance growth of streptococci for antigen preparation in Lancefield's grouping (p. 254). Cysteine, 1 g/liter, can also be added to the basal medium for susceptibility testing of streptococci in broth.

Toxigenicity Test Agar Bacto-KL Virulence Agar (Difco Laboratories) 37.5 g Distilled water 1000ml Suspend the dehydrated medium in distilled water and dissolve by boiling. Dispense molten agar in 10 ml amounts in screw-capped tubes and sterilize at 121°C for 15 min. When ready for use, supplement each 10 ml of cooled (50°C) basal medium with 2 m1 of Bacto-KL Virulence Enrichment and 0.5 ml of filter sterilized potassium tellurite, 1% solution. Mix and pour into petri dishes (10 mljdish). This medium is used for the in vitro toxigenicity test of Corynebacterium diphtheriae (p. 169).

Triple Sugar-Iron Agar (TSIA) Beef extract 3g Yeast extract 3g Peptone (Bacto) 15 g Peptone (Proteose) 5g Lactose 109 Sucrose 109 Glucose 19 FeS0 4 0.2g NaCI 5g 774 Appendix A: Media and Reagents

Sodium thiosulfate 0.3g Agar 15 g Phenol red 0.024 g Distilled water 1000mi Final pH 7.4. Sterilize at 121°C for 15 min. The medium is slanted with a deep butt. See p. 190 for use of the medium.

Urea Agar (Christensen) a. Base Medium

Peptone (Bacto or Gelysate) 19 NaCI 5g Glucose 19 KH2P04 2g Phenol red 0.012 g Urea 20g Distilled water 100ml b. Agar Agar 15g Distilled water 900ml c. Complete Medium Dissolve 29 g of the base medium in 100 ml of distilled water. Adjust pH to 6.7--6.8. Filter sterilize. Dissolve 15 g of agar in 900 ml of distilled water. Sterilize at 121°C for 15 min. Cool to 50°C and aseptically add the base medium. Mix well and dispense into screw-capped tubes. Slant tubes with a deep butt. Final pH of the complete medium should be 6.8 to 6.9. See p. 194 for use of the medium.

Veal Infusion Broth Veal heart, infusion from 500g Proteose peptone no. 3 109 NaCI . 5g Distilled water 1000ml Final pH 7.4. I. Media for Aerobic and Facultatively Anaerobic Bacteria 775

Dispense 5 ml aliquots into screw-capped tubes. Autoclave at 121°C for 15 min. This medium is used to grow fastidious bacteria.

"W" (Wisconsin) Mediums.15 To 200 ml of sterile heart infusion agar maintained at 50°C, aseptically add the following antibiotics: Actidione: 2 ml of 10 mglml stock solution; final concentration = 100 p.g/ml of agar. Bacitracin: 1 ml of 5000 U Iml stock solution; final concentration = 25 U Iml of agar. Polymyxin B. 1.2 ml of 1000 U Iml stock solution; final concentration = B U/ml of agar. Add 10 ml sterile sheep blood aseptically, mix well, and pour plates. This is a selective medium for isolation of Brucella from contaminated surgical specimens.

Xylose-Lysine-Deoxycholate Agar (XLD) Yeast extract 3g L-Lysine 5g Xylose 3.75g Lactose 7.5 g Sucrose 7.5 g NaCI 5g Phenol red 0.08g Agar 15 g Distilled water 1000ml Heat mixture to boiling to dissolve the ingredients. Sterilize at 121°C for 15 min, cool to approximately BO°C, and aseptically add 20 ml of sterile solution containing: Sodium thiosulfate 34g Ferric ammonium citrate 4g Distilled water 100ml Mix well, and then add 25 ml of 10% sterile solution of sodium deoxycholate per liter. Mix well and adjust pH to B.9. The commercially available "completed" XLD agar includes all the above ingredients. This moderately selective medium is used for the isolation of enteric pathogens, especially Shigella (Tables 3-8 and 3-9). 776 Appendix A: Media and Reagents

II. Media and Reagents for Anaerobic Bacteria

Anaerobic Media Supplements a. Hemin Stock Solution. 5 mg/ml Hemin 0.5g NaOH,lM 10ml Dissolve hemin in NaOH and adjust volume to 100 ml with distilled water. Store solution at 4°C. Add 1 ml to 1000 ml of medium to give 5 ",g/ml, prior to autoclaving. b. Vitamin Kl Stock Solution for Solid Media. 10 mg/ml Vitamin Kl 0.2 g Ethyl alcohol, 95% 20ml Store solution in a sterile brown bottle at 4°C. Add 1 ml to 1000 ml of sterile agar to give 10 ",g/ml. c. Vitamin Kl Stock Solution for liquid Media. 0.1 mg/ml Vitamin Kl for solid media (10 mg/ml) 1 ml Distilled water 100mi Store solution in a brown bottle at 4°C. Add 1 ml to 1000 ml of liquid medium to give 0.1 ",g/ml, prior to autoclaving.

Bile-Deoxycholate a. Broth Thioglycollate-135C 30g Oxgall 20g Sodium deoxycholate 19 Hemin (5 mg/ml) 1ml Vitamin Kl (0.1 mg/ml) 1ml Distilled water 1000ml Dissolve ingredients by heating and bring to a boil. Dispense 5 ml amounts into screw-capped tubes. Sterilize at 121°C for 15 min. This medium is used for the identification of Bacteroides. Alternatively, oxgall-impregnated paper disks may be used (p. 331). b. Disk Oxgall 10 g Distilled water lOml II. Media and Reagents for Anaerobic Bacteria 777

Dissolve and sterilize at 121 DC for 15 min. Also autoclave 6 rom paper disks in a glass petri dish at 121 DC for 30 min in a dry cycle. Dry disks with dish cover ajar under a hood. Saturate each disk with one drop of oxgall solution and let dry overnight. Store disks with desiccant at 4DC.

Brucella Blood Agar Brucella agar base Trypticase peptone 109 Thiotone peptone 109 Glucose Ig Yeast autolysate 2g NaCI 5g NaHSOa 0.1 g Agar 15g Distilled water 1000 ml Final pH 7.0. Suspend 43 g of dehydrated medium in distilled water and add 1 ml of hemin (5 mg/ml). Heat with agitation and boil for I min. Sterilize at 121 DC for 15 min. Let agar cool to 50DC and add 50 ml of defibrinated rabbit or sheep blood and 1 ml of vitamin Kl (10 mg/ml). Mix well and pour into petri dishes. This medium is used for the primary isolation and subculturing of anaerobic bacteria.

Carbohydrate Fermentation Base for Anaerobic Bacteria Base medium Thioglycollate medium without dextrose or indicator 24g Yeast extract 2g Hemin (5 mg/ml) Iml Bromthymol blue, I % solution Iml Distilled water 1000ml Final pH 7.0. Dissolve ingredients by heating. Bring to a boil. Dispense 10 ml in screw• capped tubes. Sterilize at 121 DC for 15 min. Cool medium to 50DC and aseptically add I ml of a 10% (5% for arabinose and trehalose) filter sterilized carbohydrate solution to give a final concentration of 1 % (or 0.5% ). The carbohydrate stock solution contains 1 p,g of vitamin Kl per ml so that the final concentration of vitamin Kl in fermentation broth is 0.1 p,g/ml. Bromthymol blue indicator Bromthymol blue Ig NaOH,IM 20ml Distilled water 80ml 778 Appendix A: Media and Reagents

Dissolve indicator in alkali and add distilled water. See p. 334 for use of the medium.

Chopped Meat Medium Ground beef (fat-free) 500 g NaOH,lM 25ml Distilled water 1000ml Mix ingredients and bring to a boil. Cool and refrigerate overnight. Skim off any remaining fat. Filter through two layers of gauze and spread out meat particles to dry. Restore volume of broth to 1000 ml with distilled water and add: Trypticase peptone 30g Yeast extract Sg K2HP04 Sg L-Cysteine hydrochloride· H 20 O.S g Hemin (S mg/ml) 1ml Agar IS g Heat to boiling, adjust pH between 7.4 and 7.8. Dispense 6 ml of medium into screw-capped tubes and add meat particles so that the final volume in each tube is approximately 8 ml. Sterilize at 121°C for 15 min. Let tubes cool in a slant position. This medium is used to induce spore production in clostridia.

Cycloserine, Cefoxitin, Fructose, Egg Yolk Agar (CCFA) 6 Egg yolk fructose agar base Proteose peptone no. 2 40g Na2HP04 Sg KH2P04 19 NaCI 2g MgS04, anhydrous 0.1 g Fructose 6g Agar 20 g Neutral red, 1 % solution in ethyl alcohol 3ml Distilled water 1000ml Final pH 7.28. Dispense 100 ml amounts in screw-capped bottle. Sterilize at 121°C for 15 min. Store at 4°C until needed. Melt basal medium and maintain at SO°C. Aseptically add the following to each bottle: Cycloserine base, final concentration of SOO Itg/ml Cefoxitin base, final concentration of 16 Itg/ml 5 ml egg yolk (50% suspension in saline) II. Media and Reagents for Anaerobic Bacteria 779

Mix well and dispense 20 ml per plate. This is a selective medium for Clo8- tridium difJicile (Table 3-9; p. 355).

Egg Yolk Agar Trypticase peptone 40g NazHP04 5g NaCI 2g MgS04, 5% aqueous 0.2ml Glucose 2g Agar 25g Distilled water lOOOml Dissolve ingredients by heating. Bring to a boil. Distribute into two 1 liter flasks. Adjust pH to 7.3 to 7.4. Sterilize at 121°C for 15 min. Cool medium to 60°C. Soak two eggs in 95% ethyl alcohol for 30 to 45 min. With sterilized forceps, make an opening at one end of the egg. Pour out egg white and remove remaining traces with forceps. Beat egg yolk with forceps and transfer one egg yolk to each flask after flaming egg shell. Mix well and pour plates. See p. 354 for use of the medium.

Esculin Broth Heart infusion broth 25g Esculin Ig Agar Ig Hemin (5 mg/ml) Iml Vitamin Kl (0.1 mg/ml) 1 ml Distilled water lOOOml Final pH 7.0. Dissolve ingredients by heating. Bring to a boil. Dispense 5 ml amounts into screw-capped tubes. Sterilize at 121°C for 15 min. See p. 333 for use of the medium.

Gentamicin-Vancomycin Laked Blood Agar Brain heart infusion agar base Calf brain, infusion from 200g Beef heart, infusion from 250g Polypeptone 109 Glucose 2g NaCI 5g Na2HP04 2.5 g Agar 15 g Distilled water lOOOml 780 Appendix A: Media and Reagents

Suspend 52 g of dehydrated medium in 1000 ml distilled water. Add 1 ml of hemin (5 mg/ml) and 5 ml of gentamicin (10 mg/ml). Heat to boiling to dissolve completely. Adjust pH to 7.6. Sterilize at 121°C for 15 min. Cool medium to 50°C and add the following: 0.75 ml of vancomycin (10 mg/ml) 1 ml of vitamin Kl (10 mg/ml) 50 ml of laked rabbit blood" Mix well and pour into petri dishes. This is a selective medium for the isolation of anaerobic gram-negative bacilli (Table 3-4).

Indole-Nitrate Broth Indole-nitrite medium Trypticase peptone 20g Na2HP04 2g Glucose Ig Agar Ig KNOs Ig Distilled water 1000 ml Final pH 7.2. Suspend 25 g of dehydrated medium in water. Add 1 ml of hemin (5 mg/ml) and 1 ml of vitamin Kl (0.1 mg/ml). Stir to dissolve. Bring to a boil. Dis• pense 2 ml amounts into screw-capped tubes. Sterilize at 121°C for 15 min. See p. 332 for use of the medium.

Motility Medium for Anaerobic Bacteria Same as for aerobic bacteria (p. 765).

Peptone Yeast Glucose (PYG) Medium Medium Peptone 109 Yeast extract 10 g Glucose 10 g Resazurin [one tablet (Allied Chemical, cat. no. 506) in 44 ml distilled water] 12ml Salt solution 40ml Hemin (5 mg/ml) 1m} L-Cysteine hydrochloride· H 20 0.5 g

.. Laked blood is prepared by freezing blood overnight and then thawing. II. Media and Reagents for Anaerobic Bacteria 781

Vitamin Kl (0.1 mg/ml) 1ml Distilled water 1000ml Salt solution CaCb, anhydrous 0.2g MgS04, anhydrous 0.2 g K2HP04 1.0 g KH~04 1.0 g NaHCOs 109 NaCI 2g

Dissolve CaCl2 and MgS04 in 300 ml of water. Add 500 ml of water and rest of the salt. Stir until solids have totally dissolved and add more water to give a total volume of 1 liter. Store at 4°C. H salts precipitate out during refrigeration, warm solution at 50°C in water bath before use. Suspend ingredients in water except L-cysteine hydrochloride and vitamin K1 . Heat to boiling to dissolve completely. Cool medium to 45°C. Add L-cysteine hydrochloride and vitamin Kl solution. When dissolved, adjust pH to 6.8 with 0.1 M NaOH. Dispense 5 ml amounts into -serum bottles ( 20 ml size, Wheaton Scientific) and reduce medium inside anaerobic glove• box. Apply stoppers (flange type, gray butyl, 200 mm, Wheaton Scientific) and secure with crimped three piece aluminum seals (20 mm, Wheaton Scientific). Sterilize at 121°C for 15 min. This medium is used to prepare broth cultures of anaerobic bacteria for GLC (p. 336).

Phenyl ethyl Alcohol (PEA) Agar for Anaerobic Bacteria PEA medium 42.5g Hemin (5 mg/ml) 1ml Distilled water 1000ml Final pH 7.3. Sterilize at 121°C for 15 min. Cool medium to 50°C and add 1 ml of vitamin Kl (10 mg/ml) and 50 ml of sheep blood. This medium is used as a primary isolation medium for anaerobic bacteria (Table 3-4).

Schaedler Broth Trypticase peptone 5.6g Phytone peptone 19 Polypeptone peptone 5g NaCI 1.7 g K~P04 0.82g Glucose 5.82g 782 Appendix A: Media and Reagents

Yeast extract 5g Tris-(hydroxymethyl ) -aminomethane 3g Hemin 0.01 g L-Cysteine hydrochloride· H 20 0.4 g Distilled water 1000ml Final pH 7.6. Suspend 28.4 g of dehydrated medium in 1000 ml of water. Add 1 ml of vitamin Kl solution (0.1 mg/ml). Mix and heat with frequent agitation. Boil for 1 min. Dispense 5 ml amounts in screw-capped tubes. Sterilize at 121°C for 15 min. This medium is used for susceptibility testing of anaerobic bacteria by the microbroth dilution method (p. 361).

Starch Broth Medium Soluble starch 5g Peptone yeast glucose (PYG) medium 1000ml Prepare medium as for PYG medium (p. 780) with added starch. Dispense 10 ml amount into screw-capped tubes and sterilize at 121°C for 15 min. See p. 351 for use of the medium.

Supplemented Thioglycollate for Anaerobic Bacteria Medium Thiogl ycollate-135C 30g Hemin (5 mg/ml) Iml Vitamin Kl (0.1 mg/ml) Iml Distilled water 1000ml Final pH 7.0. Dissolve by boiling. Dispense 10 ml amounts into screw-capped tubes. Add one CaCOa chip to each tube. Sterilize at 121°C for 15 min. Before use, boil thioglycollate tubes for 10 min. When cooled, add 1.5 ml of Fildes extract• NaHCOa mixture.

Fildes extract-NaHCOa mixture NaHCOa 6g Distilled water 600ml Bacto Fildes extract 300ml Dissolve 6 g of NaHCOs in 100 ml distilled water and filter sterilize. Asepti• cally add solution to 500 ml of sterile water, and 300 ml of Fildes enrich• ment. Addition of 1.5 ml of this mixture to 10 ml of supplemented thio• glycollate medium will result in a final concentration of 5% of Fildes extract and 1 mg of NaHCOa per ml of medium. III. Media and Reagents for Mycobacteria and Nocardia 783

This medium is used for the primary isolation (Table 3-4) and sub• culturing of anaerobic bacteria.

Thiogel Medium Trypticase peptone 17 g Phytone peptone 3g Glucose 6g NaCI 2.5g Sodium thioglycollate 0.5g Agar 0.7 g L-Cysteine hydrochloride· H20 0.25 g Na2SOa 0.1 g Gelatin 50g Distilled water lOOOml Final pH 7.0. Suspend 80 g of dehydrated medium in water. Add 1 ml of hemin (5 mg/ml) and 1 ml of vitamin Kl solution (0.1 mg/ml). Heat to dissolve and bring to a boil. Dispense 8 ml amounts into screw-capped tubes. Sterilize at 121°C for 15 min. See p. 348 for use of the medium.

Transport Vial for Anaerobic Bacteria Peptone 109 Yeast extract 109 Resazurin 12ml Salt solution 40ml Hemin (5 mg/ml) 1ml L-Cysteine hydrochloride· H20 0.5 g Vitamin Kl (0.1 mg/ml) 1ml Distilled water lOOOml Final pH 6.8. Prepare and bottle as for PYG medium (p. 780), in aliquots of 1 ml per vial. This vial is used for transporting specimens for anaerobic culture (p. 23).

III. Media and Reagents for Mycobacteria and Nocardia

Bennett's Agar Yeast extract 19 Beef extract 19 784 Appendix A: Media and Reagents

N -Z amine A (Sheffield Chemical) 2g Glucose 10 g Agar 15 g Distilled water lOOOml Final pH 7.3. Dissolve by boiling. Sterilize at 121°C for 15 min. Dispense 35 ml amounts into petri dishes. This medium is useful as an isolation medium to demon• strate the typical morphology of Nocardia and Streptomyces.

Casein Agar Skim milk (dehydrated or instant nonfat milk) 50 g Agar 109 Distilled water l000ml Dissolve skim milk in 500 ml distilled water by heating. Do not boil. Dis• solve 10 g of agar in 500 ml distilled water and heat to boiling. Sterilize solutions separately at 121°C for 15 min. Cool solutions to 45°C, mix and pour 20 ml into petri dishes. See p. 399 for use of this medium.

Decontamination and Digestion Solutions Bromocresol Purple Indicator Bromocresol purple 1.2g Ethyl alcohol, 95% 50ml Distilled water 50ml Dissolve bromocresol purple in alcohol, then add water. Sodium Hydroxide Solution (2% w /w) NaOH 40.8g Distilled water 2000ml Hydrochloric Acid Solution HCI 165ml Bromocresol purple 9ml Distilled water 1835 ml Add acid to water, mix, then add indicator solution. These reagents are used for the digestion and decontamination of speci• mens before culturing for mycobacteria (Table 3-6).

Lowenstein Medium Base Asparagine 3.6 g KH2P04 2.4 g Magnesium sulfate O.24g III. Media and Reagents for Mycobacteria and Nocardia 785

Magnesium citrate O.6g Potato Hour 30.0g Malachite green 0.4 g Distilled water 1000mi This is the basal medium used in Lowenstein-Jensen and Lowenstein• J ensen-Gruft media.

Lowenstein-Jensen Medium Lowenstein medium base 37.2g Glycerol 12ml Homogenized eggs (fresh) 1000mi Distilled water 588ml Dissolve the Lowenstein medium base and glycerol in distilled water by boiling. Sterilize at 121°C for 15 min. Cool medium to 60°C. Add homogen• ized eggs and mix gently to obtain a uniform mixture. Dispense in sterile screw-capped tubes. Do not slant. Coagulate the medium in an inspissator, water bath, or autoclave at 85°C for 45 min. Store tubes in refrigerator until used. See p. 386 for use of this medium in the catalase test. By adding 5% NaCI, the medium can be used to test NaCI tolerance of mycobacteria (p. 387). The medium is also used for isolation of mycobacteria (Tables 3-4,3-6).

Lowenstein-Jensen-Gruft Medium This is Lowenstein-Jensen medium to which has been added: Penicillin 50 U /ml, final concentration Nalidixic acid 35 JLg/ml, final concentration Ribonucleic acid 0.05 JLg/ml, final concentration This medium is used for isolation of mycobacteria (Tables 3-4, 3-6, 3-7).

Middlebrook 7H9 Broth Ammonium sulfate 0.5g L-Glutamic acid (sodium salt) 0.5g Sodium citrate 0.1 g Pyridoxine 0.001 g Biotin 0.0005 g Disodium phosphate 2.5 g Monopotassium phosphate 1.0g Ferric ammonium citrate 0.04g 786 Appendix A: Media and Reagents

Magnesium suHate 0.05 g Calcium chloride 0.0005 g Zinc sulfate 0.001 g Copper sulfate 0.001 g Dissolve 4.7 g of dehydrated powder in 900 ml of distilled water, or 900 ml distilled water containing 0.5 g Tween-80 or 2 ml of glycerol if desired. Glycerol and Tween-80 should not be used together. Distribute in 180 ml amounts and sterilize at 121°C for 10 min. Aseptically add 20 ml of Middle• brook ADC enrichment to 180 ml of sterile medium maintained at 45°C and dispense into screwcapped tubes. This is a basal medium used for the arylsuHatase (p. 384), tellurite (p. 389), niacin (p. 390), and cycloserine susceptibility tests (p. 393).

Middlebrook 7H10 Agar (Difco) Ammonium sulfate 0.5 g Monopotassium phosphate 1.5 g Dipotassium phosphate 1.5 g Sodium citrate 0.4 g Magnesium sulfate 0.025 g Calcium chloride 0.0005 g Zinc sulfate 0.001 g Copper sulfate 0.001 g L-Glutamic acid (sodium salt) 0.5g Ferric ammonium sulfate 0.04g Pyridoxine hydrochloride 0.001 g Biotin 0.0005g Malachite green 0.00025 g Agar 15 g Distilled water lO00ml Final pH 6.6. Dissolve 19 g of dehydrated medium in 1000 ml distilled water containing 0.5% glycerol by boiling. Distribute in 200 ml amounts in dark bottles and sterilize at 121°C for 10 min. Aseptically add 20 ml Middlebrook OADC enrichment to each bottle of medium maintained at 50° to 55°C. Dispense 25 ml of medium into plates or 6 to 7 ml into 16 X 125 mm screw-capped tubes. Keep prepared medium in the dark before and after inoculation. This medium is used for the isolation of mycobacteria (Tables 3-4, 3-6, and 3-7).

Middlebrook 7H11 Agar This is with 0.1% of enzymatic casein hy• drolysate. It is used for the isolation of mycobacteria (Tables 3-4, 3-6, and 3-7). IV. Media for Fungi 787

Middlebrook Selective S7H11 Agar This is Middlebrook 7H10 agar to which has been added: PolymyxinB 20 p,g/ml, final concentration Trimethoprim lactate 20 p,g/ml, final concentration Carbenicillin 50 p,g/ml, final concentration This medium is used for the selective isolation of mycobacteria from contaminated clinical material (Tables 3-4, 3-6, and 3-7).

Tyrosine Agar Nutrient agar 23g Tyrosine 5g Distilled water 1000ml Dissolve nutrient agar in 900 ml of water by boiling. Suspend tyrosine in 100 ml of water. Heat to dissolve. Add tyrosine solution to the nutrient agar. Adjust pH to 7.0. Sterilize at 121°C for 15 min. Dispense 25 to 30 ml amounts in petri dishes, swirling flask to distribute crystals evenly. See p. 399 for use of this medium.

Xanthine Agar Nutrient agar 23g Xanthine 4g Distilled water 1000 ml Dissolve nutrient agar in 900 ml of water by boiling. Suspend xanthine in 100 ml of water and dissolve by heating. Add xanthine solution to nutrient agar. Adjust pH to 7.0. Sterilize at 121°C for 15 min. Dispense into petri dishes, swirling flask to distribute crystals evenly. See p. 399 for use of this medium.

IV. Media for Fungi Brain Heart Infusion Agar (BHIA) Infusion from calf brains 200g Infusion from beef heart 250g Proteose peptone 109 Glucose 2g NaCI 5g Na2HP04 2.5 g Agar 15 g Distilled water 1000ml Final pH 7.4. 788 Appendix A: Media and Reagents

Dissolve 52 g of the dehydrated medium in 1000 ml of distilled water by boiling. Sterilize at 121°C for 15 min. Pour 35 ml amount into petri dishes. This medium is used for the isolation and subculture of fungi and Nocardia (Tables 3-4, 3-6, and 3-7). BHIA-3 Brain heart infusion agar 52g Gentamicin 5mg Chloramphenicol (solvent: 2m195% ethanol) 16mg Distilled water 1000ml BHIA-4 Brain heart infusion agar 52g Gentamicin 5mg Chloramphenicol 16mg Cycloheximide (solvent: 5 ml acetone) 500mg Distilled water 1000ml Prepare both media similarly to brain heart infusion agar. Cool to 50°C and aseptically add defibrinated sheep blood to a final concentration of 10%. BHIA-3 and BHIA-4 are used for the selective isolation of fungi, exclud• ing dermatophytes (Tables 3-4, 3-6, and 3-7).

Brain Heart Infusion (BHI) Biphasic Medium (Castaneda) Bottle Brain heart infusion agar (BBL Microbiology Systems) 52g Agar 3.5g Distilled water 1000ml Brain heart infusion broth (Difco Laboratories) 37g Distillated water 1000ml Prepare as described for Brucella biphasic medium bottle (p. 750) with• out added CO2 • This biphasic medium is used for the isolation of fungi from blood (Table 3-2). The use of BHI agar from BBL Microbiology Systems and BHI broth from Difco Laboratories eliminates the necessity of incorporating an anti• coagulant in the culture medium.

Cornmeal agar Yellow cornmeal 62.5g Distilled water 1000ml Agar (Difco Laboratories) 19 g Heat the cornmeal and water mixture in a water bath for one hour at 52°C and filter. Dilute the filtrate with distilled water to a final volume of 1500 m!. IV. Media for Fungi 789

Add 19 g of agar and sterilize at 121°C for 15 min. Dispense into sterile plastic petri dishes (15 ml/plate). See p. 428 for use of this medium.

Cottonseed Conversion Medium Glucose 20g Agar 109 Pharmamedia (Traders Protein Division, 20g Fort Worth, TX) Distilled water 1000ml Suspend dry ingredients in water. Heat to dissolve and bring to a boil. Dispense into sterile screw-capped tubes. Sterilize at 121°C for 15 min. Let medium cool in a slant position. See p. 471 for use of the medium.

Czapek Solution Agar Sucrose 30g Sodium nitrate 2g K:JIP04 Ig MgS04 0.5g KCI 0.5g FeS04 0.01 g Agar 15 g Distilled water 1000ml Final pH 7.3. Suspend 49 g of dehydrated medium in 1000 ml of water. Dissolve by boil• ing. Sterilize at 121°C for 15 min. Dispense 35 ml amounts into petri dishes. See p. 439 for use of the medium.

Cystine Heart Agar Cystine heart agar base (BBL) Beef heart, infusion from 500g Polypeptone peptone 109 Glucose 10 g NaCI 5g L-Cystine 19 Agar 15 g Distilled water lOOOml Final pH 6.8. Suspend 25.5 g of dehydrated medium in 250 ml of water to prepare a double strength base. Heat with frequent agitation and boil for one minute. Sterilize at 121°C for 15 min. Cool the basal medium to 50°C. Aseptically 790 Appendix A: Media and Reagents add 250 ml of 2% hemoglobin solution that is maintained at 50°C. Mix well and dispense into screw-capped tubes. Let medium harden in slant position. This medium may be used for in vitro conversion of dimorphic hyaline molds (p. 471).

Inhibitory Mold Agar (lMA) Tryptone 3g Beef extract 2g Yeast extract 5g Glucose 5g Starch (soluble) 2g Dextrin 19 Chloramphenicol 0.125 g SaltA 10ml SaltC 20ml Agar 17 g Distilled water 970ml SaltA NaH2P04 25g Na2HP04 25g H 20 250ml SaltC MgS04·7H20 10 g FeS04·7H20 0.5 g NaCI 0.5g MnS04·H20 1.2g H 20 250ml Add one or two drops of concentrated HCI to salt C to solubilize com• ponents. Suspend dry ingredients, except chloramphenicol, in water. Heat to dis• solve and bring to a boil. Dissolve chloramphenicol in 2 ml of 95% of ethyl alcohol and add to the medium. Adjust pH to 6.7. Sterilize at 121°C for 15 min. Dispense 35 ml amounts into petri dishes. This medium is used for isolation and subculturing of fungi (Tables 3-4, 3-6, and 3-7). Chloramphenicol should not be incorporated in the medium when culturing spinal fluid.

Mycosel Agar (SSl Microbiology Systems) Phytone peptone 109 Glucose 10 g Cycloheximide 0.40 g Chloramphenicol 0.05 g IV. Media for Fungi 791

Agar 15g Distilled water 1000mi Final pH 6.5. Suspend 36 g of dehydrated medium in 1000 ml of distilled water. Heat to dissolve and bring to a boil. Sterilize at 118 D e for 15 min. This medium is used for the isolation of dermatophytes (p. 115).

Potato Dextrose Agar Potato, infusion from 200g Glucose 20g Agar 15g Distilled water l000ml Final pH 5.6. Suspend 39 g of dehydrated medium in 1000 ml of water. Bring to a boil to dissolve completely. Sterilize at 121 DC for 15 min. Dispense into petri dishes. This medium is used for demonstrating pigment production by Tricho• phyton rubrum.

Rice Grain Medium Polished white rice (without added vitamins) 8g Distilled water 25ml Sterilize at 121 DC for 15 min. Microsporum audouinii, unlike other Microsporum species, is unable to grow on this medium.

Sabouraud Dextrose Agar (Emmons' modification) Glucose 20g Neopeptone 109 Agar 17 g Distilled water 1000ml Final pH 6.8-7.0. Dissolve ingredients by heating and bring to a boil. Sterilize at 121 DC for 15 min. Dispense 35 ml amounts into petri dishes. This medium is used for isolation, subculturing, and identification of filamentous fungi (Tables 3-4, 3-6, and 3-7).

Trichophyton Agars (Difco Laboratories) Bacto-Trichophyton Agar 1 Bacto-vitamin free casamino acids 2.5g 792 Appendix A: Media and Reagents

Bacto-dextrose 40g Monopotassium phosphate 1.8 g Magnesium sulfate 0.1 g Bacto-agar 15 g Distilled water 1000 rnl Bacto-trichophyton agar 2 through 7 are composed of 59 g of trichophyton agar 1 with the following supplements per liter: Trichophyton Agar 2 Inositol 50 mg Trichophyton Agar 3 Inositol 50mg Thiamine 200 p.g Trichophyton Agar 4 Thiamine 200 p.g Trichophyton Agar 5 Nicotinic acid 2mg Trichophyton Agar 6 Ammonium nitrate 1.5 g Trichophyton Agar 7 Ammonium nitrate 1.5 g OT histidine 3mg Prepare each medium according to the manufacturers' instructions. Sterilize at 121°C for 12 min. Dispense into petri dishes. See p. 460 for use of these media.

Yeast Extract Agar Yeast extract Ig Buffer 2rnl Agar 20g Distilled water 1000 ml Heat to dissolve and sterilize at 121°C for 15 min. Dispense 35 ml amounts into petri dishes. Buffer Na2HP04 40g KH2P04 60 g Distilled water 400ml

Dissolve Na2HP04 in 300 ml distilled H 20; then add KH2P04• The pH is 6.0. If necessary adjust with 1 M HCI or NaOH. Adjust the volume to 400 ml with distilled H 20 and store at 4°C. Final pH should be 6.0. V. Media, Reagents, and Buffers 793

This medium is used for the identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis.

V. Media, Reagents, and Buffers for Chlamydiae, Mycoplasmas, and Viruses

A. Media

General Considerations The work should be done in the clean hood area where the chances of air• borne bacterial or mycotic contamination of the medium are minimal. All constituents are equilibrated to room temperature. The contents of the flask are mixed well after addition of each solution. Constituted medium is trans• ferred to sterile 100 ml milk dilution bottles which are labeled with the name of the medium and the dates of preparation and expiration. Medium is stored at 4°C for a maximum of 6 months except when stated otherwise.

Chlamydia Medium See Table A-I for preparation.

Chlamydia Transport Medium (2 SP) 1. Phosphate buffer a. NaHzP04·H20 0.552 g Distilled water 200ml

Table A-i. Preparation of Chlamydia Medium"

Component Volume (mil

Distilled H20 600 Eagle's dry medium 10x 100 Sodium bicarbonate, 7.5% 7.5 Glucose 20 Glutamine, 200 mM, 100X solution 10 Gentamicinb 0.2 Hepes buffer, 1 Mb 20 Fetal bovine serum 100 Distilled water to make 1000

• Expiration date is 6 months . • See Solutions (p. 797-805). 794 Appendix A: Media and Reagents b. Na2HP04 1.42g Distilled water 500ml Mix 29 ml of a. with 73 ml of b. Sterilize by filtration. Final pH 7.2.

2. Sucrose-phosphate solution Sucrose 6.84 g Phosphate buffer 102 ml ~~o ~~ Add filter-sterilized sucrose to phosphate buffer with nystatin. Adjust pH to 7.2 with 1 N NaOH. Dispense in 1.8 ml aliquots and store at -20°C. No other antibiotics are included when 2 SP is used for transport of both Ureaplasma and Chlamydia; when used for Chlamydia only, 2 SP medium should include gentamicin (50 mg/100 ml medium). Expiration date when frozen is 1 year.

Chlamydia Storage Medium (4 SP) This medium is identical to 2 SP medium except that 13.68 g of sucrose and 0.2 ml of 0.5% phenol solution are added to the phosphate buffer.

Large Colony Mycoplasma Media 1. Unsupplemented media a. Agar PPLO agar (Difco Laboratories) 35 g Distilled water 1000ml Heat to dissolve the agar and dispense 80 ml amounts into 100 ml bottles. Sterilize at 121°C for 15 min. Store at room temperature. b. Broth PPLO broth without crystal violet ( Difco Laboratories) 21 g Distilled water 1000ml Mix and distribute 80 amounts into 100 ml bottles. Sterilize at 121°C for 15 min. Store at room temperature.

2. Supplemented (complete) medium See Table A-2 for preparation. Diphasic medium is prepared by adding 3 ml amounts of PPLO agar to 16 X 125 mm screw-capped tubes, slanting the

o See Solutions, pp. 797-805. V. Media, Reagents, and Buffers 795

Table A·2. Preparation of Complete Mycoplasma Medium

Volum (ml) Agar Diphasic Final concentration Component Plates broth per ml of medium

PPLO agar 70 PPLO broth 70 Horse serum 20 20 Yeast extract 10 10 Penicillin Ga 1 1 1000 U Thallium acetatea 0.5 0.5 500 ftg Amphotericin Ba 0.5 0.5 5 ftg Glucosea 2 10 mg Phenol reda 1 20 ftg

• See Solutions (pp. 797-805) . tubes at a 45° angle until the agar has solidified, and then adding 3 ml of the Complete medium (Table A-2) to each tube.

3. Mycoplasma medium with arginine See Table A-3 for preparation. Dispense in 10 ml amounts and store at _20°C.

Ureaplasma Media 1. U-9 basal broth Tripticase soy broth powder (BBL Microbiology Sys• 0.75g terns) or Tryptic digest broth powder (Dilco Laboratories)

Table A·3. Mycoplasma Medium with Arginine

Component Amount

PPLO broth 70.0 ml Horse serum 20.0 ml Yeast extracta 10.0 ml Amphotericin Ba 0.5 ml Thallium acetatea 0.5ml Penicillin Ga 1.0 ml Glucosea 2.0ml Phenol reda 1.0 ml Arginine (Calbiochem) 200.0 mg

• See Solutions (pp. 797-805). 796 Appendix A: Media and Reagents

NaCI 0.5g KH2P04 0.02g Distilled water 100ml Disolve ingredients and adjust pH to 5.5 with 2 N HCI. Sterilize at 121°C for 15 min.

2. U-9 complete medium Sterile U -9 basal broth 95ml Unheated normal horse serum 4ml Urea solution '" 0.5ml Phenol red solution'" O.lml Penicillin G '" 1ml Final pH 6.0.

3. -Mycoplasma agar (A-3 agar base) Trypticase soy broth powder (BBL Microbiology 30g Systems) Distilled water 1000mi Adjust to pH 5.5 with 4 N HCl, then add: Agar Ionagar No.2 (Oxoid) 7.5g Dissolve the agar by bringing the solution to a boil three times. Dispense in 170 ml aliquots in 250 ml screwcapped bottles and sterilize at 121°C for 15 min.

4. Ureaplasma urealyticum-Mycoplasma complete (A-3) agar A-3 agar base (melted, 50°C) 140ml Horse serum, with 0.5% urea 40ml Yeast extract, pH 5.5 20ml Penicillin G '" 2ml Phenol red'" OAml Dissolve and dispense 10 to 15 ml amounts to 60 X 15 mm plates. Allow to stand at room temperature overnight, then store wrapped (Saran wrap) at 4°C for a maximum of 2 weeks.

'" See Solutions, pp. 797-805. V. Media, Reagents, and Buffers 797

Media for Viruses 1. Growth and maintenance media

The constituents of these media are given in Table A-4. All constituents should be at room temperature. Dispense final formulations into 100 ml bottles and store at 4°C for a maximum of 6 months except when stated otherwise.

2. Serum-free medium for extraction of swabs

Eagle's serum-free maintenance medium for MK BO ml (TableA-4) Penicillin-streptomycin solution'" 10 ml Nystatin s-olution'" 10 ml

The final medium is transferred to 7.0 ml (2 dram) vials in 2 ml amounts and stored at -20°C.

B. Solutions

1. Agarose Solution Agarose (Marine Colloids, Inc. ) 1.5g Barbital buffer, 0.05 M 200ml

Dissolve the agarose in buffer by heating with continual stirring on a hot plate. Dispense in 6 ml amounts into 16 X 125 mm screw-capped tubes.

2. Alsever's Solution Glucose 2.05g NaCI 0.42 g Sodium citrate O.BOg Citric acid 0.055 g Distilled water to make 100ml Sterilize at 121°C for 15 min or by filtration. Also available commercially. Expiration date is 3 years in unopened containers and 6 months in opened containers. Sheep red blood cells (10% packed cell volume) are suspended in solution for one week prior to use in complement fixation test.

.. See Solutions, pp. 797-805. UI CCI

"C» "C (I) :::I Co x' Table A-4. Growth and Maintenance Media for Virus Isolation ?:" s::: Eagle's basal growth Eagle's Eagle's serum-free (I) Co medium (BME) for maintenance medium maintenance iii' Q) Component MRC-5 Cells for MRC-5 Cells medium for MK :::I Co :c Sterile distilled water 700 ml 1500 ml (I) Q) Earle's 10X (no NaHCOa) cc (I) Eagle's dry medium 10x a 100 ml 200 ml Sodium bicarbonate, 7.5% 7.5ml 1 ml 35ml ~ Tris buffer, pH 7.6, 1 Mb 1 ml/100 ml of BME 20 ml Penicillin (10,000 U/ml)- streptomycin (10,000 ftg/mlP 10 ml 20 ml Gentamicin (10 ftg/ml)b 0.2ml 0.4ml Nystatin (10,000 U/mlp 10 ml Fetal bovine serum 100 ml

a Basal medium, with L-glutamine, without NaHCOa (Biological, Inc.). • See Solutions, pp. 797-805. v. Media, Reagents, and Buffers 799

3. Antibiotics The antibiotic solutions described below are stable for one year when stored at -20°C; however, storage of antibiotic powders or solutions should not exceed the expiration date specified by the manufacturer. a. Amphotericin B Add 50 ml sterile distilled water to a vial containing 50 mg of the powdered antibiotic. Mix, adjust the volume to 50 ml, and transfer to vials in 0.6-ml and 2.6-ml portions, and store at -20°C after wrapping in foil. b. Cycloheximide Add 50 ml sterile distilled water to a vial containing 10 mg of the crystalline antibiotic to yield a final concentration of 200 fLg/ml. The solution is dis• pensed into 1 dram vials in 1.2 ml portions at -20°C. Add 1 ml to 100 ml of Eagle's BME (Table A-4) for a final concentratioll of 2 fLg/ml. c. Gentamicin Add 1 ml of 50 mg/ml solution in vial per 5,000 ml of Eagle's BME for a final concentration of 10 fLg/ml. d. Nystatin Add 5 to 10 ml of sterile distilled water to a vial containing 500,000 units of the antibiotic. Mix, adjust the volume to 50 ml, transfer to vials in 5.2 and 10.2 ml portions, and store at 4°C after wrapping in foil. e. Penicillin G Prepare a stock solution of potassium penicillin G, USP (Calbiochem) by dissolving 0.63 gm of the powdered antibiotic in 10 ml of sterile distilled water. The final concentration of the penicillin G is 100,000 units/mI, based on the specific activity determined by the manufacturer. The antibiotic solution is distributed in vials and stored at -20°C. f. Penicillin-Streptomycin Solution Prepare a stock solution by dissolving the appropriate amounts of penicillin G and dihydrostreptomycin sulfate in about 70 ml of distilled water. From the specific activity of the antibiotic (determined by the manufacturer), calculate the amounts to use so that the final concentration of penicillin is 800 Appendix A: Media and Reagents

10,000 units/ml and of streptomycin, 10,000 p.g/ml. Adjust volume to 200 ml with distilled water. Sterilize the solution by filtration through a 0.45 p'm membrane (Millipore) filter, transfer to vials in 10.2 ml amounts, and store at -20°C. g. Streptomycin and Vancomycin Prepare a stock solution containing 1 mg/ml of each antibiotic, based on the specific activity determined by the manufacturer. The solution is dis• pensed in I-dram vials in 0.5 and 1.0 ml portions and stored at _20°C. The solution is used to treat contaminated specimens by adding 0.1 mljO.5 ml of specimen.

4. Buffers a. Dextrose-gelatin-veronal buffer 5,5'-Diethylbarbituric acid 0.58 g Gelatin 0.6g N a 5,5' -diethylbarbiturate (sodium barbital) 0.38 g CaCl2 (anhydrous) 0.02g MgS04 ·7H20 0.12g NaCl 8.5 g Glucose 10 g Bovine albumin 2g Dissolve the sodium barbital in 200 ml of distilled water by gentle heating. Then add, sequentially, dissolved gelatin and the other ingredients. Adjust volume to 1000 ml and reheat prior to sterilization by filtration. The buffer can be also obtained from commercial sources. The expiration date is 3 years for the buffer in unopened containers. When opened, the expiration date is 6 months. b. HEPES.1 M This solution is obtained from commercial sources and should be prepared for use on the basis of 20 mljliter of medium (20 mM/ml final concentration in medium). c. HEPES Saline-Albumin-Gelatin Buffer (HSAG) • 1 M. pH 6.2 This solution is obtained from commercial sources. V. Media, Reagents, and Buffers 801 d. Phosphate-Buffered Saline (PBS), pH 7.2 (1) NaH2P04·H20 15.75 g Distilled water 228m! (2) Na2HP04 54.8g Distilled water 772ml Mix (1) and (2). Add distilled water to make 2000 ml. Sterilize by filtration. (3) Working PBS, pH 7.2. Mix 40 ml of the buffer with 100 ml of normal saline and then make to 1000 ml with normal saline. Final pH should be 7.2. Sterilize by filtration. e. Tris, 1 M, for Eagle's medium (Table A-4) Tris- (hydroxymethyl) -aminomethane 24.3 g HCI,2N 50ml Dissolve the Tris in approximately 50 ml of distilled water. With constant stirring, add 2 N HCI so that the pH is 7.6. Adjust the volume to 200 ml with distilled water, sterilize at 121°C for 15 min. Final pH should be 7.6. Expiration date is 3 years for the buffer in unopened containers. When opened, the expiration date is 6 months. This buffer is available commer• cially. f. Tris buffer, 0.2 M, for Counterimmunoelectrophoresis See p. 270. Stock solutions: ( 1) Tris- (hydroxymethyl) -aminomethane 24.2g Distilled water to make 1000ml (2) 0.2 M HCI Prepare buffer, pH 8.0, by adding 50 ml of (1) to 26.8 ml of (2). Add distilled water to make 200 m!. g. Veronal Buffer for Complement Fixation Test

(1) MgCI2-CaCI2 Solution

MgCb·6H20 20.3 g CaCh·2H20 4.4 g Distilled water l00ml Mix and store at 5°C. 802 Appendix A: Media and Reagents

(2) Stock Buffer Solution

~a ~g Na-5,5'-diethylbarbiturate 10.19 g Distilled water 1500 ml Mix by swirling until completely dissolved. Add 34.58 ml 1 N HCI and mix. Add 5.0 ml of MgCb-CaCI2 solution (1). Add distilled water to make 2000 ml.

(3) Diluted Buffer Solution Prepare 1: 5 dilution by adding 1 ml of solution (2) to 4 ml of distilled water. The pH should be between 7.3 and 7.4; if not, discard, and prepare fresh solution. Store at 5°C. h. Calcium Chloride for Rubella Test Store 1 M solution (Flow Laboratories) at 25°C until ready for use. Ex• piration date is as specified by the manufacturer.

5. Calcium-Saline Solution for Receptor Destroying Enzyme

CaCb·H20 19 NaCI 9g H 3B03 1.203 g NazB407' H 20 .052 g Distilled water to make 1000 ml Mix the compounds in water in order listed. If used immediately, this solu• tion need not be sterilized. If the solution is to be stored longer than 1 week, sterilize by autoclaving or filtering. Store at 4°C. Expiration date is 1 year.

6. Complement a. For Complement Fixation Serology Thaw diluent and maintain at 4°C. Store the lyophilized complement at 4°C until ready to use. When rehydrating complement, place the diluent, lyophilized complement, and 1 dram vials in a pan of crushed ice. Add 20 ml of the diluent with a syringe to the lyophilized complement and mix using a Vortex mixer. Dispense 0.4 to 0.5 ml aliquots into the 1 dram vials and store at temperatures below -20°C. Expiration date is as specified by the manufacturer. b. For Epstein-Barr Virus Serology Human blood (40-50 ml) is obtained and allowed to clot for about 2 hr at room temperature. The serum fraction is separated by centrifugation at v. Media. Reagents. and Buffers 803

300 g for 10 min. The serum is dispensed into 1 dram vials in aliquots of 0.1 ml and frozen at -70°e.

7. Dextran Sulfate for Rubella Test Store as a 5% solution (Flow Laboratories) at 4°e. Expiration date is as specified by the manufacturer.

8. Gelatin, 0.125% Gelatin Ig Distilled water to make 800ml Add gelatin to 100 ml water, boil, cool to 25°C, and add remaining water. Store at 4°e. Expiration date is one week.

9. Glucose for Chlamydia medium (Table A-1) Glucose 27g Distilled water l00ml Dissolve glucose in 75 ml water and then add remaining water. Sterilize by filtration and store at _20°e. Expiration date is 1 year.

10. D-Glucose for Mycoplasma medium D-Glucose 50g Distilled water 100mi Dissolve glucose in water and store at -20°e. Expiration date is 1 year.

11. Heparin Solution Heparin (159 U fmg) 0.1 g Alsever's solution 100ml Mix heparin in Aisever's solution. Sterilize by filtration. Dispense in 2 mi amounts in 1 dram vials. Store at 4°e. Expiration date is 1 year.

12. Kaolin Kaolin 100 g HCI, IN 400mI Mix kaolin in HCI for 1 hr. Filter through Buchner funnel. Wash filter paper with distilled water until no trace of acid is detected in the filtrate. Dry the 804 Appendix A: Media and Reagents kaolin overnight at room temperature, then grind to a fine powder with a mortar and pestle. Expiration date is 1 year. For use in the hemagglutination test: Kaolin, acid-washed 25g Borate-saline buffer, pH 9.0 100ml Stir the kaolin in buffer. Determine the pH. If less than 8.5, centrifuge at 250 g for 5 min, discard the supernatant, resuspend the kaolin in buffer, and redetermine the pH. If the pH is still less than 8.5, repeat the procedure until it is 8.5. Store at 4°C and recheck the pH prior to use. Expiration date is 1 year.

13. Phenol Red

Media Large Trypsin-EDTA colony solutions Mycoplasma Ureaplasma pH 3 medium

Phenol red powder 50mg 100 mg (Allied Chemical Corp.) Distilled water 25ml 10 ml Phenol red solution, 0.5% Commercially (Flow Laboratories) prepared

14. Potassium Chloride KCI 0.7455 g Distilled water 100ml Sterilize by filtration.

15. Receptor Destroying Enzyme (ROE) Reconstitute RDE with 5 ml of distilled water. Dispense in 1 ml amounts and store at -20°C. Expiration date is 1 year.

16. Thallium Acetate Thallium acetate (Fisher Scientific Co. ) 10 g Distilled water 100ml Mix the thallium acetate in water. Sterilize by filtration and store at -20°C. Thallium acetate is extremely toxic and solutions must be transferred with mechanical pipettes. Expiration date is 1 year. v. Media. Reagents. and Buffers 805

17. Trypsin-EDTA Solution Disodium ethylenediaminetetraacetate 50mg (EDTA or Versene) Trypsin 50mg Sodium bicarbonate, 7.5% 1ml Tris buffer, 1 M 1.6ml Penicillin-streptomycin solution 1ml Nystatin solution 0.5ml lOX special Earle's BSS for trypsin-EDTA solution 10ml Phenol red, 0.5 % 0.2ml Dissolve and add distilled water to make 100 m!. Sterilize by filtration. Distribute into vials in 3 and 5 ml amounts and store at -20°C. Expiration date is 1 year.

18. Tryptose Phosphate Broth Tryptose phosphate broth, dehydrated 29.5g ( Difco Laboratories) Distilled water 1000ml Dissolve the powder in water and distribute in 100 ml bottles. Sterilize at 121 ° for 15 min and store at room temperature.

19. Urea solution for Ureaplasma media Urea 3g Distilled water 30ml Dissolve urea in water. Sterilize by filtration. Dispense in 0.7 ml amounts and store at -20°C.

20. Yeast Extract Yeast ( Fleishman's) 250 g Distilled water 1000 ml Gently boil the suspension of yeast in water for 3 to 5 min. Centrifuge at 250 g for 15 min and filter the supernatant through Whatman no. 1 filter paper. Adjust the pH to 8.0 with 1 N NaOH and to pH 5.5 with 2 N HCl for large colony Mycoplasma and Ureaplasma media, respectively. Sterilize at 121°C for 15 min. Centrifuge at 250 g for 15 min and pour supernatant into 100 ml bottles. Store at -20°C. 806 Appendix A: Media and Reagents

References 1. Dunn, C., and Martin, W. J. Comparison of media for isolation of salmonellae and shigellae from fecal specimens. Appl. M icrobiol. 22: 17, 1971. 2. Facklam, R. R., and Moody, M. D. Presumptive identification of group D streptococci: The bile-esculin test. Appl. Microbiol. 20:245, 1970. 3. Feeley, J. c., Gibson, R. J., Gorman, G. W., Langford, N. c., Rasheed, J. K., Mackel, D. C., and Baine, W. B. Charcoal-yeast extract agar: Primary isola• tion medium for Legionella pneurrwphila. ]. Clin. Microbiol. 10:437, 1979. 4. Feeley, J. C., Gorman, G. W., Weaver, R. E., Mackel, D. C., and Smith, H. W. Primary isolation medium for Legionnaires' disease bacterium. ]. Clin. Micro• biol. 8:320, 1978. 5. Flynn, J., and Waitkins, S. A. A serum-free medium for testing fermentation reactions in Neisseria gonorrhoeae. J. Clin. Pathol. 25:525, 1972. 6. George, W. L., Sutter, V. L., Citron, D., and Finegold, S. M. Selective and differential medium for isolation of difJicile. ]. CUn. Microbiol. 9:214, 1979. 7. Golberg, R. L., and Washington, J. A., II. Comparison of isolation of Hae• rrwphilus vaginalis (Corynebacterium vaginale) from peptone-starch-dextrose agar and Columbia colistin-nalidixic acid agar. ]. Clin. Microbiol. 4:245, 1976. 8. Kuzdas, C. D., and Morse, E. V. A selective medium for the isolation of brucellae from contaminated materials. ]. Bacteriol. 66:502, 1953. 9. Martin, J. E., Armstrong, J. H., and Smith, P. B. New system for cultivation of Neisseria gonorrhoeae. Appl. Microbiol. 27:802, 1974. 10. Martin, J. E., Jr., and Lewis, J. S. Anisomycin: Improved antimycotic activity in modified Thayer-Martin medium. Public Health Rep. 35:53, 1977. 11. Smith, R. F. New medium for isolation of Corynebacterium vaginale from genital specimens. Hlth. Lab. Sci. 12:219, 1975. 12. Sutcliffe, E. M., and Abbott, J. D. Selective medium for the isolation of Bordetella pertussis and parapertussis. ]. Clin. Pathol. 25:732, 1972. 13. Swan, A. The use of a bile-aesculin medium and of Maxted's technique of Lancefield grouping in the identification of enterococci (group D strepto• cocci).]. Clin. Pathol. 7:160,1954. 14. Thayer, J. D., and Martin, J. E., Jr. An improved medium selective for culti• vation of N. gonorrhoeae and N. meningitidis. Public Health Rep. 81:559, 1966. 15. Weed, L. A. Use of a selective medium for isolation of Brucella from con• taminated surgical specimens. Am. J. CUn. Pathol. 27:482, 1957. APPENDIXB Quality Control

fohn P. Anhalt, Ph.D., M.D.

I. Overview

A. General Considerations

The quality control program in Clinical Microbiology at the Mayo Clinic relies heavily on performance testing and good communication between technologists, laboratory directors, and the clinical staff to ensure accurate and meaningful results. To this end, we have implemented a systematic pro• gram with reasonable limits for routine characterizations and operational checks. Good communication serves not only to detect when the inevitable errors occur, but also to decrease the likelihood, we hope, that an error will lead to an adverse result for the patient. Laboratory problems that can lead to errors are of several types, and the actions taken in response should be appropriate for the problem and the medical significance of the resulting error. For example, the underlying cause of a rare or sporadic problem is difficult to determine. Unless the error resulting from such a problem is highly significant from the medical stand• point, the value derived may not justify the costs and time required to solve the problem. In contrast, a systematic or frequent problem is relatively easily solved and usually requires a change in procedure or implementation of additional control procedures. Continuing education of laboratory personnel, including laboratory direc• tors, is an integral part of quality control. Procedures can become outdated

807 808 Appendix 8: Quality Control or gradually changed, and education helps to ensure use of optimal proce• dures, awareness of problems that can occur, and performance of proce• dures according to design. Laboratory safety and the working environment (Chapter 13) also are important factors that can affect performance.

B. Laboratory Manual

A complete and updated laboratory manual is the starting point for accurate laboratory results. Each new technologist is provided with a copy of the manual and is expected to become thoroughly familiar with its contents. Any revisions of procedures are entered, dated, and signed by the laboratory director in a master copy of the manual located in a central area within the laboratory. Technologists are asked to record revisions in their personal copies of the manual. Yearly, the manual is reviewed in detail in a series of meetings with all technologists. Following this review, a new edition of the manual is printed and distributed to all technologists. To prevent older editions from being used, technologists must exchange old copies for new editions. c. Education

New technologists are instructed in the methods for proper collection, iden• tification, and transport of specimens, aseptic technique, inoculation of ap• propriate media, and laboratory safety and policies. They are given a rotat• ing assignment in the laboratory, during which time they work under the immediate and close supervision of a senior or "lead" technologist until their performance is considered to be sufficiently reliable and accurate to allow more independent work. Technologists do not usually assume full responsi• bilities, including on-call work, until they have been in training for 6 months. Weekly meetings of all technologists are held to discuss and to announce new or revised procedures and policies. These announcements are then posted in the laboratory for 1 week. Daily laboratory rounds are held at which senior or lead technologists, postdoctoral trainees, and the staff micro• biologists, pathologists, and infectious disease clinicians discuss interesting or unusual cases and all cases involving isolation of an organism from nor• mally sterile body fluids or tissues. In addition, at various times during the year, lecture series are presented by the staff for technologists. Lastly, attendance and active participation through scientific presenta• tions at national and local meetings and in workshops is expected of senior and lead technologists. I. Overview 809

D. Proficiency Testing

1. Internal Simulated or real clinical specimens are submitted to the laboratory using a conventional request form bearing the name and registration number of a deceased patient. Isolation and identification techniques are tested with simulated specimens containing stock organisms. Serological procedures and antimicrobial assays are tested by using serum remaining from previous tests that has been stored at -60°C, and results are compared with those obtained initially. At least once every 6 months, a proficiency specimen is submitted for each serological procedure. A variety of safeguards are used to ensure that the technologist doing the testing does not know a particular specimen is for proficiency testing and to protect against confusion caused by inadvertent reporting of results outside of the laboratory. The results from these internal proficiency specimens are used as a check on adequacy of procedures and as an indicator for additional technologist training. Causes of problems are investigated, and after appropriate action (e.g., a change in procedure or additional training), a similar specimen is resubmitted to test whether improvement has occurred.

2. External The laboratory participates in or is a referee for various state and federal programs, including those of the College of American Pathologists (7400 North Skokie Boulevard, Skokie, IL 60077) and the Center for Disease Con• trol, Atlanta, GA 30333.

E. Records and Review

All results from proficiency tests, instrument function tests, major items of instrument maintenance, and performance tests of media and reagents are recorded and reviewed monthly by the quality control technologist and staff director of quality control. Results that indicate a failure in procedures, media, reagents, or equipment are recorded in a separate log along with the remedial action taken. Each entry in the log is initialed and dated by the director of quality control. 810 Appendix B: Quality Control

II. Media and Reagents

A. General Considerations

1. Selection Media and reagents should be selected to provide efficient recovery and rapid identification of a wide variety of organisms. Where appropriate, selec• tive media should be used for isolation of common pathogens; however, these media should be used together with nonselective media, because the selective media may inhibit some pathogens. Media and reagents should be purchased from reliable sources that, on request, can provide information regarding quality control of their products. It is also desirable to have alter• native sources readily identified in case an unexpected problem with supply develops.

2. Preparation Media and reagents must be prepared according to directions of the manu• facturer or from well-documented sources. Dehydrated media should be reconstituted in distilled water or deionized water of proved chemical and microbiological quality. Apparently minor variations in procedure (e.g., the order of addition and mixing of components) can have effects on the per• formance of media. It is essential, therefore, that procedures be monitored to ensure that the intended directions are followed. Sterilization of media must be controlled to ensure sterility without degradation of labile con• stituents such as carbohydrates. The pH of each batch of media must be checked to ensure that it is within the proper range. The pH of broth and agar media changes during autoclaving and is, therefore, usually checked after autoclaving rather than before. The media must be equilibrated to room temperature before pH can be measured. The pH of agar media can be measured using a surface electrode. Alternatively, a small portion of the molten agar can be placed in a beaker and allowed to solidify around a standard pH electrode, or the pH electrode can be immersed in solid media that has been minced with a few drops of water. In any case, the electrode must be supported securely to avoid unreliable readings resulting from pressure on the glass tip. The pH of media can usually be adjusted to the proper range by adding dilute NaOH or Hel solutions.

3. Sterility Tests To avoid confusing results, the sterility of media must be assured. A variety of methods can be used to accomplish this goal depending on the nature of the medium and its intended use. Generally, the incidence of contamination II. Media and Reagents 811 is greater in media to which a heat-labile substance (e.g., sugars or blood) is added or which are dispensed into tubes or plates after autoclaving than in media that are terminally autoclaved. Fot most media, incubation of each tube or plate to detect contamination before use is impractical, and only a representative sample of each batch can be tested. The size of the sample that should be tested depends on the maximum incidence of contamination that is acceptable. For example, if the sample consists of 300 tubes or plates from a very large batch, one would expect to detect, based on sampling statistics,4 an incidence of contamination of 1% with about 95% certainty. Our experience with commercially prepared media supports this prediction 0. P. Anhalt, unpublished data). One manufacturer that supplies media to the Mayo Clinic tests 10% of each lot of 4000 to 5000 plates (i.e., a sample of 400 to 500 plates), and discards the entire lot if a single plate is contami• nated. After shipment without refrigeration to our laboratory, each plate is visually inspected, and plates showing contamination are discarded. (Credit is received for discarded plates and the remainder of each lot is used.) Over a period of 6 months, we detected contamination in an average of 0.4% of 165,000 sheep blood agar plates examined and the incidence never exceeded 1.5% per lot of 5000 plates. These findings are in accord with the sensitivity expected for the sample size tested by the manufacturer. Obviously, attempt• ing to detect 1 or 2 % contamination is impractical for clinical laboratories unless each tube, bottle, or plate is tested. Testing a sample of 5 or 10 plates or tubes from a batch has been recommended1 •2; however, this sample size would be expected only to detect reliably a contamination rate of 25 to 40%. In practice, such high rates of contamination are not found except when there has been a gross failure of normal aseptic procedures. When such failures occur, contamination is usually detected during routine per• formance testing or by casual inspection of the media before inoculation and does not require separate sterility testing per se. In part because of the requirements for inspection and accreditation (failure to check all media for sterility is a phase II deficiency in the inspection and accreditation program of the College of American Pathologists3 ) and in part to detect failure of aseptic procedures, we have continued to test sterility by incubation of a single, uninoculated sample from each batch of media prepared in-house. These tests are performed at the same time as performance tests, and the period and conditions of incubation are the same as those used for the per• formance tests. Because of the importance of ruling out low-level contami• nation in some media, such as biphasic blood culture bottles prepared in-house, each bottle is incubated for 48 to 72 hr before use, and bottles show• ing contamination are discarded. For most commercially prepared media, sterility tests involve either incubation of a single sample at the same time as performance tests or careful inspection of each plate or tube prior to use. In the latter case, which is used principally for primary isolation media, media are inspected also for drying, discoloration, or other evidence of 812 Appendix B: Quality Control deterioration. Only those tubes or plates that fail the inspection are dis• carded and the remainder of the lot or shipment is used, unless there is evidence that the problem is characteristic of the entire lot. Commercially prepared blood culture bottles are not checked for sterility prior to use, be• cause experience has shown the rate of contamination in these bottles to be low. Instead, a daily record of the isolation of presumed contaminants (Bacillus sp., Propionibacterium acnes, Corynebacterium sp., Staphylo• coccus epidermidis, Aerococcus viridans, and gram-negative, nonfermenters other than Pseudomonas sp. present in single cultures) is maintained. An incidence of these isolates greater than 2% is considered evidence of a problem with contamination that must be investigated. Finally, media may contain nonviable organisms that may, nevertheless, cause confusion when a Gram-stained smear is performed in the course of normal procedures" Broth media and growth supplements that are used to culture normally sterile fluids (Schaedler broth, transport media, thioglycollate broth, nutrient broth, and rabbit serum) are checked, therefore, by Gram-stained smear before use to ensure that nonviable organisms are not present.

4. Dates All media and reagents are dated when received in the laboratory and when prepared or first put into use. All media and many reagents should also have an expiration date. The expiration date provided by the manufacturer must be followed. When an expiration date is not prOvided, published guide• lines1,5 should be consulted. For most media and working solutions of reagents, an expiration date 6 months following preparation is a useful guide• line. However, because dehydration is the principal determinant of expira• tion for most media, it is frequently desirable to adjust preparation schedules to allow media to be used within 3 months. Simple chemical solutions (e.g., acid-base indicators) and stock buffer solutions can often be stored up to 3 years if there is no evidence of microbial growth and performance is satisfactory. Pure chemicals (e.g., salts) and dehydrated media are gen• erally stable and have unlimited shelf life unless otherwise specified by the manufacturer. To minimize space requirements and inventory, it is usually desirable to purchase quantities of media that can be used within 3 years. Sterile materials used in collection and processing of specimens, such as glass tubes, glass tubes with needles, and sand for grinding tissues, should have an expiration date of 3 to 6 months. These materials are not discarded, but are simply resterilized at their expiration date.

5. Storage Media and reagents should be stored according to directions of the manu• facturer. In most instances, a cool, dry area is satisfactory for storage of dehydrated media and reagents. Prepared media should be stored in sealed II. Media and Reagents 813 tubes or bottles or by wrapping plates in plastic. In general, prepared media should be stored at 2 to BOC, which greatly reduces evaporation of water from plates and tubes with loosely fitting caps. In tightly sealed containers, however, many media may be stored up to 6 months at room temperature.! Media should be allowed to equilibrate to room temperature prior to inocu• lation. Stocks of media and reagents should be rotated so that older supplies are used first.

6. Performance Testing Media and reagents should be tested using controls of known positive and negative reactivity. Specific guidelines for performance tests are given in the following sections as appropriate for each division of Clinical Micro• biology. The frequency of testing varies with the stability of the medium or reagent and the necessity in some tests to have a positive and a negative control for comparison. In general, each batch or lot of media or reagents must be checked for performance. If one lot is received in separate ship• ments, each shipment is checked. Because the aim of performance testing is to identify whether an entire lot or batch is satisfactory, a minimum number of plates (each plate can be divided into sections) or tubes of media should be used to accomplish the required tests. If a medium or reagent does not perform satisfactorily, the entire lot or batch should be discarded. The manu• facturer should be notified and consulted before action is taken with com• mercial materials, because it may be necessary to return them to receive credit.

B. Bacteriology

1. Maintenance of Stock Cultures a. Aerobic and Facultatively Anaerobic Bacteria Two vials of all isolates used for performance testing are stored frozen. Fol• lowing isolation on blood agar, suspend the bacteria in sterile, defibrinated sheep blood that contains no preservatives or anticoagulants. Place two to three drops in a Y:2 dram, screw-cap vial, and store at -60°C or lower. Vials should be thoroughly cleaned and sterilized. To recover bacteria, remove a vial from the freezer and allow the contents to thaw. Streak a loopful or a drop of the suspension on blood agar or other suitable, nonselective medium and incubate overnight at 35°C. The frozen suspensions can be stored indefinitely and are not adversely affected by alternate freezing and thawing. When bacteria in one vial are no longer viable, the second vial is used and a replacement vial is prepared. Frequently used isolates are maintained on plain agar slants (subculture 814 Appendix B: Quality Control every 6 months) or on appropriate agar plates (subculture every 2 weeks). Neisseria and Haemophilus may not survive repeated subculturing and are routinely stored frozen. b. Anaerobic Bacteria To store anaerobic bacteria for long periods, add 10 drops of a turbid broth culture to 0.5 ml of double-strength skim milk medium. Dispense into 1/2 dram vials and store at -60°C or lower. Frequently used strains are main• tained in supplemented thioglycollate broth and are subcultured weekly to fresh broth. Because these cultures often become contaminated with other bacteria, they are subcultured monthly to sheep blood agar, and fresh broth cultures are made from isolated colonies.

2. Stock Organisms Required for Testing Media and Reagents Tables B-1 and B-2 list stock organisms that are used for performance test• ing of media and reagents. Most of these organisms are clinical isolates obtained at the Mayo Clinic, although other isolates with appropriate bio• chemical reactions should be satisfactory. In particular, isolates from the American Type Culture Collection should be used for susceptibility testing ( Section 4.4 ) .

3. Performance Testing of Media and Reagents Tables B-3 through B-9 list the organisms and expected reactions for per• formance testing of media and reagents used in bacteriology. Each batch of

Table B·1. Stock Cultures Used for Testing of Media and Reagents Used in Anaerobic Bacteriology

Organism

Bacteroides fragi/is subsp. vulgatus Bacteroides melaninogenicus Bacteroides thetaiotaomicron Bifidobacterium eriksonii Clostridium sp. (urea positive) Clostridium sporogenes Escherichia coli Fusobacterium nucleatum anaerobius Peptococcus prevotii VeiJIonella parvula II. Media and Reagents 815 medium prepared in-house and each lot of prepared medium purchased is checked before being used for diagnostic purposes. For all media, each test also includes incubation of an uninoculated tube or plate to determine sterility. These samples are incubated 24 hr at 35°C for routine bacteriology and 48 hr at 35°C in an anaerobic chamber for anaerobic bacteriology. For anaerobic bacteria, biochemical tests are read after 48 hr or good growth has appeared, whichever is later. For other bacteria, results are read gen• erally after 24 hr of incubation.

Table B·2. Stock Cultures Used for Testing of Media and Reagents Used for Aerobic and Facultatively Anaerobic Bacteria Organism Organism

Acinetobacter calcoaceticus Neisseria meningitidis. group Y Aeromonas hydrophila Plesiomonas shigelloides Alcaligenes sp. Proteus mirabilis Bacteroides fragilis Proteus vulgaris a Bordetella pertussis Providencia rettgeri Branhamella catarrhalis Pseudomonas aeruginosa Brucella suis Pseudomonas cepacia Campylobacter fetus Salmonella enteritidis. group B Candida albicans Sarcina lutea (ATCC 9341) Citrobacter freundii Serratia marcescens Corynebacterium diphtheriae Shigella flexneri Edwardsiella tarda aureus (ATCC 25923) Enterobacter aerogenes Staphylococcus epidermidis (ATCC 27626) Escherichia coli group A Group II·F Streptococcus group B Streptococcus group D Haemophilus parainfluenzae Haemophilus vaginalis Streptococcus durans Klebsiella pneumoniae Streptococcus equinus sp. Str6ptococcus faecalis Legionella pneumophila Leptospira sp. Streptococcus sanguis I Neisseria sp. (sucrose positive) Streptococcus sangUis II Neisseria gonorrhoeae Streptococcus. viridans group Neisseria lactamica Vibrio parahaemolyticus Neisseria meningitidis. group B Yersinia enterocolitica

a Selected to produce both H.S and DNase. 816 Appendix B: Quality Control

Table 8-3_ Performance Tests for Media and Reagents Used for Anaerobic Bacteria

Medium or reagent Test or test organism Acceptable result

Carbohydrate fermentationsG Arabinose No color change ( - ) Bacteroides fragilis Yellow (+) subsp. vulgatus Cellobiose Veillonella parvula Bifidobacterium eriksonii + Fructose Veillonella parvula B. fragilis subsp. vulgatus + Glucose Veillonella parvula B. fragilis subsp. vulgatus + Inositol Veillonella parvula Clostridium perfringens + Lactose Veillon ella parvula B. fragilis subsp. vulgatus + Maltose Veillon ella parvula B. fragilis subsp. vulgatus + Mannitol Veillonella parvula Bifidobacterium eriksonii + Mannose Veillonella parvula B. fragilis subsp. vulgatus + Raffinose Veillon ella parvula B. fragilis subsp. vulgatus + Rhamnose Veillonella parvula B. fragilis subsp. vulgatus + Sorbitol Veillonella parvula Bifidobacterium eriksonii + Starch Veillon ella parvula B. fragilis subsp. vulgatus + Sucrose Veillon ella parvula B. fragilis subsp. vulgatus + Trehalose Veillon ella parvula Bacteroides thetaiota• + omicron Xylose Veillone;/Ia parvula B. fragilis subsp. vulgatus + Chopped meat slant Clostridium sporogenes Spores after 72 hr

G These tests apply equally to prepared media in tubes or to the Minitek system (BBl Microbiology Systems) . Each batch of tubes is tested. Each shipment (single lot numbers) of Minitek supplies is tested. Minitek disks are checked for sterility by incubating in uninoculated broth. II. Media and Reagents 817

Table B·3 (continued)

Medium or reagent Test or test organism Acceptable result

Clostridium Clostridium perfringens Inhibition of lecithinase perfringens antitoxin type A Disk identification B. fragiJis Fusobac· tests for Bac· subsp. terium teroides fragiJis b vulgatus nuc/eatum Bile Growth No growth Colistin R «14mm) S r"'''14 mm) Erythromycin S S Kanamycin R S Penicillin R S Rifampin S S Vancomycin R R orS Egg yolk agar Clostridium perfringens Lecithinas.s production (opacity around growth) Clostridium sporogenes Lipase production ("waxy" colonies) Esculin brotha Fusobacterium nucleatum No color (add ferric ammo· B. fragiJis subsp. vulgatus Black nium citrate or Minitek disk) Indole/nitrate brotha Fusobacterium nuc/eatum Indole positive Nitrate negative Veillon ella parvula Indole negative Nitrate positive Kovacs' or Erhlich's VeiIJonella parvula No color reagentsa Fusobacterium nuc/eatum Red

Nitrate A and B reo Fusobacterium nuc/eatum Nitrate negative agentsa VeiIJonella parvula Nitrate positive Oxgall Fusobacterium nucleatum No growth B. fragiJis subsp. vulgatus Stimulated growth Peptone-yeast broths Indicator (vent) Color change GLC Record amount of succinic and acetic acids present Phenyl ethyl alcohol Escherichia coli Inhibited agar Peptococcus prevotii growth

b Test a complete set of disks weekly. 818 Appendix B: Quality Control

Table B-3 (continued)

Medium or reagent Test or test organism Acceptable result

Rabbit blood agar B. fragi/is subsp. vulgatus Growth within 48 hr Clostridium perfringens Hemolysis with double zone Peptococcus prevotii Growth within 48 hr Rabbit blood agar B. fragi/is subsp. vulgatus Growth (laked) with genta• Peptococcus prevotii No growth micin and vanco• Bacteroides melanino• Black pigment within 3 to mycin (RGV) genicus 5 days Sheep blood agar Bacteroides fragi/is Growth within 48 hr (Brucella agar subsp. vulgatus base) Clostridium perfringens Hemolysis with double zone Peptococcus prevotii Growth within 48 hr Supplemented thio• All quality control Growth within 48 hr glycollate broth organisms Thiogel Clostridium perfringens Not solidified after refrig• eration B. fragilis subsp. vulgatus Solidified after refrigera• tion Transport vial Pigment No color Introduce air Pink Peptostreptococcus Survival for 3 hr at room anaerobius temperature" Fusobacterium nuc1eatum Survival for 3 hr at room temperature" Urease'" Clostridium perfringens No color Clostridium sp. (urease Purple positive) X-ray film B. fragi/is subsp. vulgatus No removal of emulsion Clostridium perfringens Removal of emulsion

• Prepare inoculum by diluting an overnight thioglycollate broth culture to a density equal to that of a No. 0.5 McFarland standard with brain heart infusion broth. Further dilute 1:10 with the same broth. Mix a 1 }Llloopful of the diluted suspension in 1.0 ml of lactated Ringers solution and inject the whole amount into a vial (final inoculum approximately 0.5 X 10' CFU/ml). After 3 hr, remove 0.1 ml and inoculate onto sheep blood agar (Brucella agar base). Incubate for 48 hr under anaerobic conditions to de• termine survival. II. Media and Reagents 819

Table 8-4. Performance Tests for Media Used for the Isolation of Aerobic and and Facultatively Anaerobic Bacteria

Medium Test organism Acceptable result

Biphasic Brucella blood Brucella suis Growth culture bottles Neisseria gonorrhoeae Growth (Trypticase soy broth)'" Brucella agarb Brucella suis Growth Campylobacter media Campylobacter fetus Growth Escherichia coli No growth Chocolate blood agarb Haemophifus influenzae Growth Neisseria gonorrhoeae Growth Charcoal agarb Bordetella pertussis Growth Colistin-nalidixic acid Escherichia coli No growth agar (CNA)b Streptococcus, group D Growth Haemophifus vaginalis Growth Cystine tellurite agarb Corynebacterium diphtheriae Growth Eosinmethylene blue Shigella f1exneri Growth (no color) (EMB) agar Escherichia coli Growth (green metallic sheen) Feeley-Gorman (F-G) or Legionella pneumophifa cysteine yeast extract Growth (CYE) agar Hektoen enteric (HE) Salmonella enteritidis, group B Green colonies agar with black centers Shigella f1exneri Green colonies Escherichia coli Yellow colonies Leptospira medium Leptospira Growth (Fletcher) Loeffler agar Corynebacterium diphtheriae Growth MacConkey agar Escherichia coli Pink colonies Yersinia enterocolitica Growth (no color) Mueller-Hinton broth Neisseria meningitidis Growth Rabbit serum" Bacteroides fragilis Growth Rogosa medium Lactobacillus Growth Salmonella-Shigella Salmonella enteritidis, group B Growth (no color) (88) agar Shigella f1exneri Growth (no color) Yersinia enterocolitica Growth (no color) Escherichia coli Inhibition (pink colonies)

• Prepare a suspension of organisms to a density equal to that of a No. 0.5 to 1.0 McFarland standard. Add 0.5 ml of a 1: 10' dilution of this suspension. • Prepare a suspension of organisms to a density equal to that of a No. 0.5 to 1.0 McFarland standard and dilute 1:10 twice with broth. Apply 1 ILl of diluted suspension to test medium using a calibrated loop. e Add 1 ml to each of two tubes of thioglycollate broth. Incubate one tube for steril• ity. Inoculate the other tube with the test organism, incubate, and check for growth. 820 Appendix B: Quality Control

Table B-4 (continued)

Medium Test organism Acceptable result

Selenite broth Salmonella enteritidis, group B Growth Escherichia coli Inhibition Sheep blood agarb Listeria monocytogenes Growth with .B-hemolysis Streptococcus, group A Growth with .B-hemolysis Str.eptococcus, viridans groujl Growth with a-hemolysis Streptococcus, group D Growth without hemolysis Supplement Cd Haemophilus influenzae Growth Thiosulfate citrate bile Vibrio parahaemolyticus Green colonies salts sucrose (TCBS) agar Thioglycollate broth Bacteroides fragilis Growth Thayer-Martin mediumb Escherichia coli No growth Staphylococcus epidermidis No growth Neisseria gonorrhoeae Growth JEMBEC platesb Escherichia coli No growth Staphylococcus epidermidis No growth Candida albicans No growth Neisseria gonorrhoeae Growth blood Bacteroides fragilis Growth culture bottlesa Klebsiella pneumoniae Growth Pseudomonas aeruginosa Growth Streptococcus, viridans group Growth

• Filter and add 0.3 ml to a tube of Mueller-Hinton broth.

Table B·S. Performance Tests for Media Used for the Identification of Aerobic and Facultatively Anaerobic Bacteria

Medium Test organism Acceptable resulta Acetate agar Escherichia coli + Edwardsiella tarda Bile-esculin agar Streptococcus, group D Growth with blacken• ing of area around colonies within 24 hr Streptococcus, viridans No color change group (usually no growth) Campylobacter media Brucella broth Campylobacter fetus Growth Nitrate C. fetus +

a Reactions: +, positive reaction as appropriate (e.g., acid production); -, negative or no reaction. II. Media and Reagents 821

Table 8-5 (continued)

Medium Test organism Acceptable resulta

Glycine C. fetus Growth Cysteine C. fetus Growth with black- ening of lead acetate strip Carbohydrate fermen- tation broth, Andrade's Adonitol Enterobacter aerogenes + Citrobacter freundii Arabinose Citrobacter freundii + Edwardsiella tarda Dulcitol Citrobacter freundii + Enterobacter aerogenes Fructose Citrobacter freundii + Pseudomonas aeruginosa Glucose Citrobacter freundii + Pseudomonas aeruginosa Inositol Enterobacter aerogenes + Citrobacter freundii Lactose Citrobacter freundii + Providencia rettgeri Maltose Citrobacter freundii + Providencia rettgeri Mannitol Citrobacter freundii + Edwardsiella tarda Raffinose Citrobacter freundii + Edwardsiella tarda Rhamnose Citrobacter freundii + Edwardsiella tarda Salicin Enterobacter aerogenes + Citrobacter freundii Sorbitol Citrobacter freundii + Edwardsiella tarda Sucrose Citrobacter freundii + Edwardsiella tarda Trehalose Citrobacter freundii + Edwardsiella tarda Xylose Citrobacter freundii + Edwardsiella tarda Carbohydrate utiliza- tion media for NeiSSeria Glucose Neisseria gonorrhoeae + Branhamella catarrhalis Lactose Neisseria lactamica + Neisseria meningitidis 822 Appendix B: Quality Control

Table 8·5 (continued)

Medium Test organism Acceptable result<> Maltose Neisseria meningitidis + Neisseria gonorrhoeae Sucrose Neisseria sp. (sucrose + positive) Neisseria gonorrhoeae Cetrimide Pseudomonas cepacia Growth Acinetobacter calcoaceticus No growth Citrate agar Citrobacter freundii + Edwardsiella tarda Decarboxylase media Arginine Aeromonas hydrophila + Edwardsiella tarda Lysine Edwardsiella tarda + Citrobacter freundii Ornithine Edwardsiella tarda + Klebsiella pneumoniae DNase agar Enterobacter aerogenes Growth with no pink zone Serratia marcescens Growth with pink zone Esculin Enterobacter aerogenes + Pseudomonas cepacia Gelatin Serratia marcescens + Enterobacter aerogenes Indole broth Providencia rettgeri + Enterobacter aerogenes 10% lactose Pseudomonas cepacia + Acinetobacter calcoaceticus (L1A) Citrobacter freundii Alkaline slant/ acid

butt, H2 S Edwardsiella tarda Alkaline slant/alkaline

butt, H2S Providencia rettgeri Red slant/ acid butt,

no H2S MacConkey agar Pseudomonas cepacia Growth Group IJ·F No growth Malonate broth Enterobacter aerogenes + Edwardsiella tarda Methyl red-Voges Citrobacter freundii MR, +; VP,- Proskauer (MR·VP) Enterobacter aerogenes MR, -; VP, + broth Motility agar Edwardsiella tarda + Klebsiella pneumoniae Moti I ity-indole- Edwardsiella tarda M, +; I, +; 0, + ornithine (MIO) agar Klebsiella pneumoniae M, -; I, -; 0,- Nitrate broth Pseudomonas cepacia + Acinetobacter calcoaceticus II. Media and Reagents 823

Table 8-5 (continued)

Medium Test organism Acceptable resulta

Oxidation-fermenta• tion (OF) media Fructose Pseudomonas cepacia + Acinetobacter calcoaceticus Glucose Pseudomonas cepacia + Acinetobacter calcoaceticus lactose Pseudomonas cepacia + Acinetobacter calcoaceticus Maltose Pseudomonas cepacia + Acinetobacter calcoaceticus Mannitol Pseudomonas cepacia + Acinetobacter calcoaceticus Sucrose Pseudomonas cepacia + Acinetobacter calcoaceticus Xylose Pseudomonas cepacia + Acinetobacter calcoaceticus Phenylalanine deami• Providencia rettgeri + nase (PAD) agar Enterobacter aerogenes Pigment F and P agar Pseudomonas aeruginosa + Pseudomonas cepacia Porphyrin test broth Haemophilus parainfluenzae Red fluorescence (8-aminolevul inic Haemophilus influenzae No fluorescence acid substrate) Salmonella-Shigella Pseudomonas aeruginosa Growth (SS) agar Acinetobacter calcoaceticus No growth

Starch agar Haemophilus vaginalis + Streptococcus, viridans group Triple sugar iron agar Citrobacter freundii Acid slant/ acid butt,

(TSIA) gas, H2S Pseudomonas cepacia Alkaline slant! alkaline butt Providencia rettgeri Alkaline slant/ acid

butt, no H2S Urea agar Providencia rettgeri 4+ Edwardsiella tarda Veal infusion broth Streptococcus pneumoniae Growth and lysis with 4% Dreft 824 Appendix B: Quality Control

Table B·6. Performance Tests and Acceptable Profiles for Media Used with the Replica Agar Plating Method (Repliscan, Cathra International) of Gram-Negative Rod Identification

Test organism b Pseudomonas aeruginosa Escherichia coli Proteus Medium" (Mayo strain) (ATCC 25922) vulgaris Citrate + Lysine decarboxylase + + Ornithine decarboxylase + or- + Urease + DNase + Colistin + Cephalothin + + H2S + Bile-esculine Arginine decarboxylase (dihydrolase) + + or- Glucose + + Lactose + Sucrose + Mannitol + Inositole Arabinose + • Media are listed in the order in which results are entered. • Acceptable profiles: Pseudomonas aeruginosa, E240 or C240; Escherichia coli, 6035 or 6075; Proteus vulgaris, 1F28. Reactions: +, positive reaction as appropriate; -, no reaction or not growth as appropriate . • Each batch of inositol or bile-esculin medium is tested with Klebsiella pneu• moniae for a positive reaction.

Table B·7. Performance Tests for Reagents Used for the Identification of Aerobic and Facultatively Anaerobic Bacteria

Reagent or item Test organism Acceptable result

Sodium deoxycholate, Streptococcus, viridans No lysis 10% solution group Streptococcus pneumoniae Lysis Ferric chloride Providencia rettgeri Blue green color on reagent (FeCl s) phenylalanine deami• nase agar (PAD) Enterobacter aerogenes No color change on PAD Escherichia coli Pink to red rod shaped organism Purple to blue coccal shaped organism II. Media and Reagents 825

Table B·7 (continued)

Reagent or item Test organism Acceptable result

Hydrogen peroxide Plesiomonas shigelfoides Weak production of gas

(H 20 2 ) solution, 3% Streptococcus pneumoniae No gas Indole reagent Providencia rettgeri Red in MR-VP broth (Kovacs') Enterobacter aerogenes No color change in MR·VP broth Methyl red indicator Citrobacter freundii Red color in MR-VP broth Enterobacter aerogenes No color change in MR·VP broth Nitrate reagents Pseudomonas cepacia Positive Acinetobacter calco- Negative aceticus ONPG test disks Neisseria lactamica Yellow color Neisseria gonorrhoeae No color Oxidase reagent and Aeromonas hydrophila Blue color oxidase strips Escherichia coli No color change Phenylalanine Proteus mirabilis Brown to black color deaminase strips Escherichia coli No color change Spot indole reagent Escherichia coli Pink color Proteus mirabilis No color change Rabbit plasma (slide Staphylococcus aureus Agglutination and and tube coagulase coagulation tests) Staphylococcus epider- No agglutination and midis coagulation Voges-Proskauer Enterobacter aerogenes Red color reagents Citrobacter freundii No color change

Table B·8. Performance Tests for Media Used to Identify Streptococci

Medium Test organism Acceptable result

Carbohydrate fer• mentation brotha Lactose Streptococcus faecalis Yellow color Streptococcus equinus No color change Mannitol Streptococcus faecalis Yellow color Streptococcus sanguis I No color change Raffinose Streptococcus bovis Yellow color Streptococcus faecalis No color change Sorbitol Streptococcus faecalis Yellow color Streptococcus bovis No color change

a Heart infusion broth base with bromcresol purple indicator and final carbohydrate concentration of 1 %. 826 Appendix B: Quality Control

Table B·8 (continued)

Medium Test organism Acceptable result

Sucrose Streptococcus faecalis Yellow color Streptococcus durans No color change Hippurate hydrolysis Streptococcus, group B Color with ninhydrin (hydrolysis) Streptococcus, group A No color with nino hydrin 1% inulin Streptococcus sanguis I Yellow color Streptococcus,group D No color change Litmus milk Inspection Not decolorized or coagulated Streptococcus, group D Acid with clot Streptococcus equinus No color change NaCI 6.5% broth Streptococcus faecalis Growth Streptococcus, viridans group No growth Potassium tellurite Streptococcus faecalis Growth agar Streptococcus, viridans group No growth Starch agar Streptococcus bovis Hydrolysis of starch Streptococcus faecalis No hydrolysis Sucrose agar Streptococcus, viridans group Growth Sucrose broth, 5% Streptococcus sanguis II Growth Streptococcus mutans Crystalline deposit at 48 hr Streptococcus sanguis I Gel effect Tetrazolium agar Streptococcus faecalis Brick-red centered colonies Streptococcus bovis No growth or clear colonies Todd-Hewitt broth with glucose Streptococcus, group A Growth Todd-Hewitt broth without glucose Streptococcus, viridans group Growth

Table B·9. Performance Tests for Fluorescent Antibody and Serological Tests Used in Bacteriology

Itema Test organism or material Acceptable result

Fluorescent conjugates Streptococcus, group A Streptococcus, group A Fluorescence Streptococcus, group C No fluorescence Streptococcus, group G No fluorescence Staphylococcus aureus No fluorescence

a All turbid antisera are filtered or centrifuged to remove denatured proteins or con• taminating organisms. II. Media and Reagents 827

Table B·9 (continued)

Item'" Test organism or material Acceptable result

Leptospira Homologous Leptospira Fluorescence strain Normal rabbit globulin No fluorescence Listeria monocytogenes Listeria types 1 to 4 Fluorescence Normal rabbit globulin No fluorescence Neisseria gonorrhoeae Neisseria gonorrhoeae Fluorescence Boiled suspension of En- No fluorescence terobacter cloacae Neisseria meningitidis Neisseria meningitidis Fluorescence Normal rabbit serum No fluorescence Streptococcus pneu- Streptococcus pneumoniae Fluorescence moniae Normal rabbit globulin No fluorescence Haemophilus influenzae, Haemophilus influenzae, Fluorescence type b type b Normal rabbit globulin No fluorescence Brucella Homologous Brucella strain Fluorescence Normal rabbit globulin No fluorescence Bordetella pertussis Bordetella pertussis Fluorescence Normal rabbit globulin No fluorescence Legionella pneumophila Positive smear Fluorescence (direct) Negative control No fluorescence Legionella pneumophila Positive control serum Positive at ::"'1 :512 (indirect) Negative control Negative Streptococcal antisera Homologous strain PreCipitate Antisera for counterimmu- noe I ectrophores i s Haemophilus influenzae, H. influenzae, type b an- PreCipitin line type b tigen, 0.04 p.g/ml Streptococcus pneumo- S. pneumoniae, type 3 an- Precipitin line niae polyvalent tigen, 0.1 p.g/ml Neisseria meningitidis, N. meningitidis, group A Precipitin line group A antigen, 0.1 p.g/ml Neisseria meningitidis, N. meningitidis, group B Precipitin line group B culture supernatant, 1 :2 dilution Neisseria meningitidis, N. meningitidis. group C Precipitin line group C antigen, 0.05 p.g/ml Neisseria meningitidis, N. meningitidis, group Y Precipitin line polyvalent groups X-Z control dilution Streptococcus, group B Streptococcus, group B an- Precipitin line tigen (Difco) 828 Appendix B: Quality Control

4. Performance Testing of Reagents and Sera Used for Antimicrobial Susceptibility Tests and Assays

Horse Serum

Purpose: To detect antimicrobial activity and microbial contamination of horse serum. Each lot number of horse serum is tested for antimicrobial activity and each bottle is tested for sterility.

Procedure: 1. Saturate a paper disk, 12.7 mm in diameter (Schleicher and Schuell, cat. no. 740E), with the serum to be tested. 2. Place the saturated disk on a penicillin assay plate containing Sarcina lutea ATCC 9341 as the test organism (Section 12.1). 3. Incubate the plate for 18 hr at 30°C. 4. Repeat the above procedure using a gentamicin assay plate containing Staphylococcus epidermidis ATCC 27626 as the test organism (Table 12.1-6). Incubate this plate for 4 hr at 35°C. 5. Check both plates for zones of inhibition. 6. To test for sterility, remove 1 ml from each bottle of serum as it is to be used and inoculate it into 15 ml of thioglycollate broth. Incubate the broth for 3 days at 35°C. 7. If zones of inhibition are present, or if growth is detected in the thio• glycollate broth, either discard the entire lot or bottle or return to the company for credit. 8. Record results and action taken, if any.

Human Serum

Purpose: To test human serum for the presence of hepatitis B antigen (HB.Ag), antimicrobial activity, sterility, and the presence of inhibitors or inactivators of penicillins or cephalosporins.

Procedure: 1. Test each lot number for HB.Ag by an approved, third generation test. 2. Test each lot number for antimicrobial activity and each bottle for steril• ity as described above for horse serum. II. Media and Reagents 829

3. To test for inhibitors or inactivators of penicillins or cephalosporins, prepare the assay reference concentration of penicillin and cephalothin (Table 12.1-4) in the serum to be tested. Assay these solutions according to the appropriate method (Section 12.1). If there is a loss in activity of either the penicillin or cephalothin, discard the entire lot or return to the company for credit.

CaCl2 Assay Plates

Purpose: To test for complete inhibition of amino glycoside activity. Each batch of plates is tested.

Procedure: 1. Impregnate a paper disk, 6.25 mm in diameter (Schleicher and Schuell, cat. no. 740E) with 20 pl of a solution containing 100 ILg/ml of strepto• mycin. Impregnate a second disk with a solution containing 20 ILg/ml of gentamicin. 2. Apply both disks to a penicillin assay plate containing CaCl2 (Table 12.1-6). 3. Incubate for 16 hr at 30°C and examine for zones of inhibition. 4. If any zone of inhibition is present, discard the entire hatch of agar. 5. Record results.

,B-Lactamases

Purpose: To test for sterility and for penicillinase and cephalosporinase activities. Test each lot of enzyme for activity and each bottle for sterility.

Procedure: 1. Mix 1.6 ml of a solution containing 100 ILg/ml of penicillin G in water or buffer with 0.4 ml of a penicillinase solution containing 10 X 106 kinetic (Kersey) units per ml (BBL Microbiology Systems, cat. no. 11898). 2. Incubate the mixture for 20 min at 35°C. 3. Impregnate a paper disk, 6.25 mm in diameter, with 20 ILl of the mixture and place on a penicillin assay plate (Table 12.1-6). 4. Incubate the plate for 16 hr at 30°C. 5. After incubation, check for a zone of inhibition. 830 Appendix B: Quality Control

6. To test for sterility, inoculate 15 ml of thioglycollate broth with 1 ml of penicillinase solution. Incubate for 3 days at 35°C and check for growth. 7. If microbial growth or a zone of inhibition is detected, discard or return the penicillinase to the company for credit. 8. The broad spectrum of ,B-Iactamase from 569/H9 (p. 699) is tested for sterility and activity against cephalothin in an analo• gous manner to the test for penicillinase. Mix 0.4 ml of the ,B-Iactamase, which contains 10 IU Iml of ,B-Iactamase II and 100 IU Iml of ,B-Iactamase I, with 1.6 ml of cephalothin at 100 fLg/ml. Incubate for 20 min at 35°C and check for residual antimicrobial activity as described above. To test sterility, inoculate 0.1 ml of the ,B-Iactamase into 15 ml of thioglycollate broth and incubate for 3 days at 35°C.

C. Mycology and Mycobacteriology

1. Maintenance of Stock Cultures and Serum Controls Organisms are stored as suspensions in sterile water, on agar slants, or frozen. The water suspensions are contained in tubes sealed with paraffin and are stored at room temperature. This method has been shown to be reliable for storing fungi. Our experience with mycobacteria is limited to only 1 year, during which time no problems have occurred; however, a back-up method should still be used. Organisms are sto~ed frozen at -50°C or lower in a mixture of equal parts of horse serum and Trypticase soy broth (BBL Microbiology Systems). Serum controls are stored at _50°C or lower.

2. Stock Organisms Required for Testing Media and Reagents Stock cultures required for performance testing of media and reagents used in mycology are listed in Table B-I0.

Table 8-10. Stock Cultures Used for Testing Media and Reagents Used in Mycology

Organism Organism

Aspergillus fumigatus Cryptococcus neoformans Aspergillus versicolor Klebsiella pneumoniae Candida albicans Nocardia asteroides Candida glabrata Saccharomyces cerevisiae Candida parapsilosis Staphylococcus aureus Candida pseudotropicalis Streptomyces sp. Candida tropicalis Trichophyton mentagrophytes Cryptococcus albidus Trichophyton rubrum Cryptococcus laurentii Trichophyton tonsurans II. Media and Reagents 831

3. Performance Testing of Media and Reagents Tables B-ll through B-13 list the organisms and expected reactions for per• formance tests of media and reagents used in mycology and mycobacteriol• ogy. Generally, each batch or lot of media or reagents used in mycology is tested before use. Sheep serum, which is used for detecting germ tube formation, is tested daily with each use. For reagents used in mycobac• teriology, a positive and negative control (Table B-13) is tested with each clinical specimen. Positive and negative controls for serological tests in mycology are also done concurrently with each specimen or batch of specimens.

D. Parasitology

Performance tests for reagents and stains used in parasitology are described in Table B-14. A culture of Entamoeba histolytica is maintained on a char-

Table 8-11. Performance Tests for Media and Reagents Used for the Isolation and Identification of Aerobic Actinomycetes and Fungi

Medium or reagent Test organism Acceptable result

Acid-fast stain Nocardia asteroides Acid-fast positive Streptomyces sp. Acid-fast negative Amphotericin B Candida albicans (Sveen) No growth at 0.4 p.g/ml Ascospore agar Saccharomyces cerevisiae Ascospore formation Candida albicans No ascospore forma- tion Bennett's medium Nocardia asteroides Growth Streptomyces sp. Growth Bird seed agar Cryptococcus neoformans Growth with brown pigment Cryptococcus albidus Growth with no pig- ment Brain heart infusion agar Nocardia asteroides Growth Biphasic brain heart Candida albicans Growth infusion blood culture medium Brain heart infusion agar Candida albicans Growth with blood and anti- Cryptococcus neotormans Growth (except on biotics (chlorampheni- agar with cyclo- col, gentamicin, with heximide) or without cyclohexi- mide)a Aspergillus fumigatus Growth (may be only partially inhibited with cycloheximide) 832 Appendix B: Quality Control

Table 8-11 (continued)

Medium or reagent Test organism Acceptable result

Klebsiella pneumoniae No growth Staphylococcus aureus No growth Carbohydrate assimila• Candida albicans Glucose assimilation tion base agar (yeast positive nitrogen base agar) Candida albicans lactose assimilation negative Carbohydrate assimila• tion disks Glucose Cryptococcus laurentii Positive Candida glabrata Positive Inositol Cryptococcus laurentii Positive Candida glabrata Negative lactose Cryptococcus laurentii Positive Candida glabrata Negative Maltose Cryptococcus laurentii Positive Candida glabrata Negative Raffinose Cryptococcus laurentii Positive Candida glabrata Negative Sucrose Cryptococcus laurentii Positive Candida glabrata Negative Trehalose Cryptococcus laurentii Positive Candida glabrata Positive Candida pseudotropicalis Negative Carbohydrate fermenta• tion (yeast fermen• tation broth base) Glucose Candida tropicalis Acid and gas produc• tion Cryptococcus neoformans No gas production lactose Candida tropicalis No gas production Candida pseudotropicalis Acid and gas produc• tion Maltose Candida tropicalis Acid and gas produc• tion Candida pseudotropicalis No gas production Sucrose Candida tropicalis Acid and gas produc• tion Cryptococcus neoformans No gas production Casein agar Nocardia asteroides No hydrolysis Streptomyces sp. Hydrolysis Cornmeal agar Trichophyton rubrum Growth Cornmeal Tween-80 agar Candida albicans Chlamydospore pro• duction and hyphae II. Media and Reagents 833

Table 8·11 (continued)

Medium or reagent Test organism Acceptable result

Czapek agar Aspergillus fumigatus Conidial heads typical for Aspergillus fumi• gatus Flucytosine Saccharomyces cerevisiae No growth at 2.5 (ATCC 9763) p.g/ml Inhibitory mold agar Candida albicans Growth (plate)1J Cryptococcus neoformans Growth Aspergillus fumigatus Growth Klebsiella pneumoniae No growth Staphylococcus aureus No growth Inhibitory mold agar Aspergillus fumigatus Growth at 45°C (slant) Aspergillus versicolor No growth at 45°C Mycobiotic agarlJ Cryptococcus neoformans No growth Staphylococcus aureus No growth Trichophyton rubrum Growth Nigrosin Cryptococcus neoformans Capsules Candida albicans No capsules Nitrate broth Cryptococcus albidus Nitrate positive Cryptococcus neoformans Nitrate negative Trichophyton rubrum Red pigment Trichophyton mentagro• No pigment phytes Sabouraud's dextrose Candida albicans Growth aga .... Cryptococcus neoformans Growth Aspergillus fumigatus Growth Nocardia asteroides Growth Sheep serum Candida albicans Germ tube formation Candida tropicalis No germ tube forma· tion Trichophyton agars # 1, Trichophyton tonsurans #1, #2: 1+ growth #2, #3, #4 #3, #4: 4+ growth Trichophyton rubrum Growth on all four media Tyrosine agar Nocardia asteroides No hydrolysis Streptomyces sp. Hydrolysis Urea R broth Cryptococcus neoformans Pink within 4 hr (positive) Candida albicans No color change in 4 hr Xanthine agar Nocardia asteroides No hydrolysis Streptomyces sp. Hydrolysis

G Prepare a suspension of organisms to a density equal to that of a No. 0.5 to 1.0 McFarland standard and dilute 1: 10 twice. Apply 1 1'1 of diluted suspension to test medium using a calibrated loop. Inoculate media with Trichophyton rubrum, Nocardia asteroides, and Aspergillus fumigatus without dilution in the usual manner. 834 Appendix B: Quality Control

Table 8-12. Performance Tests and Acceptable Profiles for the API 20C Yeast Test Strip

Test organisma Candida Candida Cryptococcus Test parapsilosis glabrata laurentii Glucose (GLU) + + + Glycerol (GlY) + 2-Keto-D-gluconate (2KG) + + Arabinose (ARA) + + Xylose (XYl) + + Adonitol (ADO) + + Xylitol (XLT) + Galactose (GAL) + + Inositol (INa) + Sorbitol (SOR) + + or- Methyl-D-glucoside (MDG) + + or- N-Acetyl-D-glucosamine (NAG) + + Cellobiose (CEl) + lactose (lAC) + Maltose (MAL) + + Sucrose (SAC) + + Trehalose (TRE) + + + Melezitose (MlZ) + + Raffinose (RAF) +

4 Acceptable or usual profile numbers: Candida parapsilosis. 6-756-171; Candida glabrata. 2-000-040; Cryptococcus laurentii. 2-775-773 (usual). coal agar slant containing rice starch and is transferred weekly. Clinical specimens that contain protozoa are fixed in formalin or polyvinyl alcohol (PVA) and are stored in a refrigerator until needed. Blood smears from known cases of malaria usually are not available to test the performance of the Giemsa stain. This stain, which is obtained commercially prepared, is tested using a routine thin film blood smear.

E. Virology

Performance tests for media and cell cultures used to isolate or identify viruses, Mycoplasma, and Chlamydia are described in Table B-I5. Generally, these tests are performed with each new batch or lot of media or cell cul• tures. The test for Chlamydia isolation with McCoy cells is performed with each run, and tests with MRC-5 and primary rhesus monkey kidney cells for virus isolation are performed monthly. Known positive and negative control serums are run concurrently with each serological test. II. Media and Reagents 835

Table 8-13. Performance Tests for Media and Reagents Used for the Isolation and Identification of Mycobacteria

Medium. reagent. or test Test organism Acceptable result

Arysulfatase M. avium-intracellulare Negative M. fortuitum Positive (pink to red color) Catalase M. tuberculosis H37RA Negative M. fortuitum Positive (bubble produc- tion) Fluorochrome stain Streptomyces sp. No fluorescence M. tuberculosis H37RA Fluorescence Kinyoun acid-fast stain Streptomyces sp. Blue branching or bacilli forms M. tuberculosis H37RA Red bacilli Light exposure M. avium-intracellulare No pigment production M. kansasii Pigment production (yellow to orange) Lowenstein medium M. tuberculosis H37RA Growth Lowenstein-Gruft M. tuberculosis H37RA M. tuberculosis H37RA Growth Middlebrook S7H11 M. tuberculosis H37RA Growth Escherichia coli No growth Pseudomonas aeruginosa No growth Middlebrook 7H9 M. avium-intracellulare Homogeneous growth medium M. tuberculosis H37RA Rough growth with clumping of cells Middlebrook 7H10 agar M. tuberculosis H37RA Growth Niacin M. avium-intracellulare Negative M. tuberculosis H37RA Positive (yellow pig- ment) Nitrate reduction M. avium-intracellulare Negative M. kansasii Positive Sodium chloride toler- M. avium-intracellulare Growth of <50 colonies ance test M. fortuitum Growth of >50 colonies Tellurite reduction M. kansasii Negative M. avium-intracellulare Dark pigment Thiophene-2-carboxylic M. tuberculosis H37RA Growth at ~1 p.g/ml acid hydrazide (TCH) M. bovis No growth at ~1 p.g/ml susceptibility Tween-80 hydrolysis M. avium-intracellulare Negative M. kansasii Positive Urease M. avium-intracellulare Negative M. kansasii Positive (pink color) 836 Appendix B: Quality Control

Table 8-14. Performance Tests for Reagents and Stains Used in Parasitology

Stain or reagent Test or test organism Acceptable result

Eosin-saline Entamoeba histolytica Background staining with viable solution (trophozoites) ameba Giemsa stain Blood smear Clear and distinct staining of white blood cells and red blood cells Iodine solution Formalin-treated spec i- Cyst nuclei should stain and be- men with protozoan come readily visible cysts Polyvinyl alcohol Physical appearance Colorless and fluid (PVA) fixative Entamoeba histolytica Good preservation (trophozoites) Trichrome stain PVA-fixed specimen Nuclear characteristics clearly positive for ameba stained

Table 8-15. Performance Tests for Media and Cell Cultures Used for the Isola• tion of Chlamydiae, Mycoplasmas, and Viruses

Medium or cell culture Test or test organism Acceptable result

Chlamydia isolation Eagles MEM Sterility No growth (Chlamydia) McCoy's cells Mycoplasma contamination No growth Chlamydia trachomatis Growth Mycoplasma isolation Large colony agar Sterility (incubate 1 week No growth at 36°C) Growth Ureaplasma urealyti• Sterility No growth cum agar Ureaplasma urealyticum Growth Virus isolation: Eagles BME (virus) Sterility No growth MRC-5 Mycoplasma contamination No growth Herpes simplex virus Characteristic cyto• pathic effects Primary rhesus mon• Parainfluenza virus Characteristic cyto• key (primary pathic effects and hem• cynomolgus monkey) adsorption kidney Adenovirus Characteristic cyto• pathic effects Hemadsorption (not in• No hemadsorption fected) III. Equipment 837

III. Equipment

A. General Considerations

A complete quality control program for laboratory equipment must include preventi ve maintenance and performance checks on a regular schedule. Records must be maintained to enable one to determine readily when and by whom equipment was checked last and whether performance was satisfac• tory. The records also provide maintenance and performance histories that are often useful in solving problems when they arise. Each person using a piece of equipment must be familiar with how to determine that perform• ance is satisfactory. For major equipment, e.g., chromatographs and spectro• photometers, the records should also include an operator's manual, a service manual with a list of replacement parts, calibration procedures, serial num• ber and other identifying numbers, date installed, date of next scheduled service, and a telephone number to call for service.

B. Incubators

Incubator temperature must be checked and recorded daily, and acceptable ranges established. If CO2 is added, the concentration should be checked every other day. A Fyrite analyzer (Bacharach Instrument Co.) can be used to measure the CO2 concentration. A record should also be made of the cylinder pressure and date of installation to help predict when replacement will be necessary and to detect abnormally high use, which may be caused by leaks. Incubators should be cleaned periodically (e.g., quarterly), and servicing of circulation fans and humidity controls should be included in maintenance procedures.

C. Refrigerators and Freezers

Temperatures must be checked and recorded daily and acceptable ranges established. Refrigerators and freezers should be cleaned periodically. If specimens are being stored, there should be an established procedure for decontamination. Freezers should not be self-defrosting.

D. Autoclaves

Autoclaves must have accurate temperature and pressure gauges for control of sterilization procedures. Performance should be checked weekly with 838 Appendix B: Quality Control spore strips or ampules (Kilit, BBL Microbiology Systems) and results recorded. Heat-sensitive tape should be used with every run.

E. Hot-Air Ovens

Operating temperatures should be checked weekly to confirm the function of the thermostat control.

F. Water Baths and Heating Blocks

Water baths must be kept scrupulously clean. Before routine use, the tem• perature of a heating block should be measured in each corner and in the center to ensure that there are no hot spots. Temperature should be moni• tored daily.

G. Thermometers

All new thermometers used for critical temperature measurements (e.g., refrigerators, incubators, water baths, heating blocks) must be calibrated against a reference thermometer that has calibration traceable to a National Bureau of Standards reference. The deviation from the reference ther• mometer should be recorded and generally should be within -I- 1°C. Cali• bration should be repeated yearly. A common source of error, often un• detected, is separation of the mercury column. To correct this, the mercury column should be retracted fully into the bulb by placing the thermometer in an ice-salt or Dry Ice-acetone bath. Calibration should be repeated after this procedure.

H. pH Meters

Before each use, standardized buffer solutions (pH 4.0 and 7.0) should be used to calibrate the instrument. A standard at pH 10.0 should be used when the instrument is going to be used to measure alkaline solutions. When not in use, place electrodes in saturated KCI or distilled water, de• pending on the recommendation of the manufacturer. To prevent polariza• tion of electrodes, they should never be removed from a solution when the instrument is in the measuring mode. The electrodes should be cleaned monthly. Protein deposits may be removed by alternately dipping electrodes in 0.1 mol/liter NaOH and 0.1 mol/liter HCl followed by a thorough rinse with distilled water. A 5% solution of NaOCI (Clorox) works well for III. Equipment 839 removing deposits of blood. Never place electrodes in an autoclave. When necessary, electrodes can be sterilized with a 5% solution of phenol or NaOCI. Yearly, the instrument should be thoroughly cleaned, and an anti• static solution should be applied to the face of the indicating meter, if appropriate.

I. Centrifuges

The centrifugal speed should be checked annually with an accurate tachometer, and other maintenance appropriate to the type of motor and braking system should be done. Centrifuges that are used for specimens should be disinfected with a 5% solution of phenol or with Amphyl at least weekly.

J. Balances

Balances must be kept clean and level, and should be protected against temperature variations, vibration, and high humidity. Preventive main• tenance should be performed annually and should include a check of ac• curacy using National Bureau of Standards Class S weights.

K. Biological Safety Cabinets

Specific instructions of the manufacturer should be followed in regard to operation and maintenance, including correct air velocity, frequency of and methods for changing filters, and decontamination. Generally, daily dis• infection is recommended.

L. Microscopes

Daily, clean all lenses with lens paper. H the oil immersion lens was used, clean the lens with lens paper that has been lightly moistened with xylene, then wipe dry with a clean piece of lens paper. Preventive maintenance should be performed every 6 months and should include an overall inspec• tion with adjustment and lubrication of all necessary parts.

M. Pipettes and Dilutors

The appropriate pipette for the type of work being performed must be used. For example, a volumetric pipette, rather than a serological one, 840 Appendix B: Quality Control should be used for delivering relatively large volumes accurately. Pipettes of the Eppendorf type should be calibrated monthly by a gravimetric method. Dilutors should be tested by a gravimetric method or other method appropriate to the volume delivered.

References

1. Bartlett, R. C., Allen, V. D., Blazevic, D. J., Dolan, C. T., Dowell, V. R., Gavan, T. L., Inhorn, S. L., Lombard, G. L., Matsen, J. M., Melvin, D. M., Sommers, H. M., Suggs, M. T., and West, B. S. Clinical Microbiology. In Inhorn, S. L. (ed.), Quality Assurance Practices for Health Laboratories. Washington, D.C., American Public Health Association, 1978, p. 871. 2. Blazevic, D. J., Hall, C. T., and Wilson, M. E. Cumitech 3: Practical Quality Control Procedures for the Clinical Microbiology Laboratory. Balows, A., (co• ordinating ed.). Washington, D.C., American Society for Microbiology, 1976. 3. College of American Pathologists Commission on Inspection and Accreditation: Inspection Checklist, Section IV, Microbiology. Skokie, IL, College of American Pathologists, 1978, checklist item no. 04.1220.00. 4. Moroney, M. J. Facts from Figures. Baltimore, Penguin Books, 1951, p. 82. 5. Prier, J. E., Bartola, J. T., and Friedman, H. (eds.). Quality Control in Micro• biology. Baltimore, University Park Press, 1975. Index

A Aerotolerance, subcultures to deter• Accident, procedures following, 731- mine, 319 733 Agar dilution, 291-299 Acetate agar, 748 Agar media for salmonellae, shigellae, Acetate utilization test for Escherichia and vibrios, 114 coli, 204-205 preparation in disk Acid-fast stain{s) diffusion tests, 299 for Actinomycetales {Kinyoun and Agarose solution, 797 Ziehl-Neelsen),377-379 Agglutination tests, 209-210 for Nocardia (modified Kinyoun's) cold, for M. pneumoniae, 522 397, 399 Alsever's solution, 797 of sputum, 73, 74 Amantadine (Symmetril) for influenza Acidimetric test for p-Iactamase, 305- type A infections, 545 306 Amebiasis, cutaneous, 662 Acinetobater, 220 Amikacin, chromatogram of, 711 Actinobacillus actinomycetemcomitans, Aminoglycoside assay, 703-712 differentiation of, 226-227 Amphotericin B susceptibility test for Actinomyces, 356-357 fungal isolate, 483-485 cultures for, 322, 323 Ampicillin assay, 685-691 Actinomycetes, common aerobic, 395- Ampicillin testing against Haemophilus, 405 297 Adenovrrus, 592-597 Anaerobic bacteria, 309-363 neutralization to infectivity, 596 clues to presence of, 315-316 Aerobic and facultatively anaerobic identification of, 323-358 bacteria, 126-241 Bacteroides, various, 338-342 gram-negative bacilli, 179-241 Bacteroides fragilis group, 327- gram-negative cocci, 157-164 335 gram-positive bacilli, 164-179 Bacteroides melaninogenicus gram-positive cocci, 132-157 group, 335-338 Aerococcus viridans, 157 Clostridium, 348-356 Aeromonas, 212-214 cocci, 344-348

841 842 Index

Anaerobic bacteria (continued) selection of, 283--284 Fusobacterium, 342-344 solvents and diluents for, 286-287 non-spare-forming gram-positive, Arthropods and artifacts, procedure for, 356-358 662-664 infections caused by, 313-315 Arylsulfatase production by myco• isolation of, 315--323 bacteria, 384-385 media and reagents for, 776-783 Ascospore production by yeasts, 434- storage of, 814 435 susceptibility testing of, 358-363 Anaerobic bacteriology, workcards for, 319,320-321 B Anaerobic glove box, 318 Babesia, 646-648, 649-656 Anaerobic jar, 316-317 Bacilli Anaerobic media supplements, 776 gram-negative, 179-241; see also Anaerobic transport tube or vial, 21 Gram-negative bacilli Andrade's carbohydrate fermentation gram-positive, 164-179; see also broth, 752-753 Gram-positive bacilli Animal inoculation procedures, 665-666 Bacillus, 177-178 test for Listeria, 175 Bacitracin inhibition test for Group A Antibiotic assays, form for requesting streptococci, 145-146 and reporting, 683 Bacteremia and fungemia specimen Antibiotic disk identification for collection, 24-29 anaerobic bacteria, 329-330 Bacterial isolates, identification of, 275 Antibiotic solutions for viruses, 799- Bactericidal tests, 715--726 800 combination studies, 723-726 Antimicrobial activity, special tests of, minimal bactericidal concentratIon 680-726 (MBC),716-719 antimicrobial assays, 680-712 serum antibacterial titer, 719-723 bioassays,684-702 Bacteroides liquid chromatographic assays, indirect FA test for, 264-265 702-712 species identification of bactericidal, 715--726 B. asaccharolyticus, 335 combination studies, 723-726 B. fragilis, 312, 327-335 MBC, 716-719 B. melaninogenicus, 335-338 serum antibacterial titer, 719-723 B. ruminicola, 339, 342 Antimicrobial susceptibility tests various, 338-342 of aerobic and facultatively anaerobic Bacteriology, quality control in, 813- bacteria, 279-306 830 preparation of solutions for, 285- Baerman digestion technique for larval 288 worms, 666-667 testing and reporting protocol, 284 Beaver egg-counting technique, 667 testing procedures, 288-306 Bennett's agar, 783--784 anaerobic bacteria, 359-363 Bi{idobacterium, 356-357 fungi, 473, 483-488 Bile, procedure for, 663 Mycobacteria, 393-395 Bile-deoxycholate, 776-777 Antimicrobials Bile-esculin agar, 748 concentrations of, 291 Bile-esculin test for streptococci, 149- regression lines for, 290 150 Index 843

Bile solubility test for Streptococcus, C

152-153 CaCl2 assay plates, testing, 829 Bile tolerance for Bacteroides fragilis, Caffeic acid-ferric citrate test of, 426- 330-331 428 (modified Wilson Calcium-saline solution for receptor• and Blair medium), 749 destroying enzyme, 802 Bladder biopsies, 661 California virus, CIE for antibodies to, Bladder washout differentiation 566-568 (Fairley test), 43-45 California virus encephalitis, 559-560 Blood agar, 749 CAMP test for streptococci, 148-149 Blood and tissue parasites, examination Campylobacter, 214-216 for, 646-657 Campylobacter fetus, testing protocol maria and trypanosomes, 649, 656, for, 298 657-659 Campylobacter media, 751-752 malaria, babesia, trypanosomes, Candida microfilariae, 646-648, 649-656 characteristics, 422-423 Blood cultures infections, 413 initial processing for, 91-95 morphology, 418-421 media for, 28 Capnophilic bacteria, 309 procedures for, 94 streptococci, subcultures for, 319, Blood mms, examining, 614, 616 322 Blood or blood products, processing, Capsule stain, 82 123 Carbohydrate analysis of cell wall Bone marrow biopsy, 661 constituents in aerobic actinomy• Bordet-Gengou (B-G) agar base, 749- cetes, 403-405 750 Carbohydrate fermentation base for Bordetella, 234-235 anaerobic bacteria, 777-778 Borreliosis (relapsing fever) specimen Carbohydrate fermentation broth collection, 30 Andrade's, 752-753 Brain abscess, direct microscopic phenol red base, 753 examination of specimen, 71 Carbohydrate fermentation test, 207- Brain heart infusion agar (BRIA), 787- 208 788 for Bacteroides fragilis, 334-335 Brain heart infusion (BRI) biphasic for Corynebacterium, 168-169 medium (Castaneda) bottle, 788 of yeasts, 432-433 Broth-dilution bioassays, 700-702 Carbohydrate utilization by yeasts, 430- Broth dilution "checkerboard" protocol, 432 two-dimensional, 720-721 Carbohydrate utilization media for Broth-disk elution for anaerobes, 360- Neisseria, 753-754 361 Carbohydrate utilization tests to Brucella, 231-232 Brucella biphasic medium (Castaneda) speciate neisseriae, 161-162 bottle, 750 Cardiobacterium Iwminis differentia• Brucella blood agar, 777 tion, 226-227 Brucella broth, 750 Casein agar, 784 Buffers Casoni skin test for Echinococcus, phosphate, 288 667-668 for viruses, 800-802 Castaneda bottle, 750, 788 844 Index

Catalase test CIE (counterimmunoelectrophoresis), for Bacteroides fragilis. 332 268-275 for mycobacteria, 385-386 for California virus antibodies, 566- for staphylococci, 135-136 568 Cell cultures for viruses, 527-531 and FA, overview of, 249-250 Cell wall analysis for diaminopimelic Citrate agar, 756 acid (DAP) in aerobic actinomy• Citrate utilization test for cetes, 401-402 Escherichieae, 193-194 Central nervous system (CNS) citrobacter. 187 direct microscopic examination of Clostridium. 310-311 specimens, 70-71 C. botulinum, isolation of. 356 infections of, 32-34 C. difficile Cerebrospinal fluid (CSF) cytotoxicity assay for, 355-356 CIE procedure for, 268-274 glucose fermentation by, 337 collection of, 33 isolation from feces, 355 Cetrimide medium (Pseudosel agar) , gelatin liquefaction for anaerobic 753-754 gram-positive cocci and, 348 Chancroid disease, direct microscopic identification, 348-356 examination of, 79 of lecithinase-positive, 350 Charcoal agar, 755 lecithinase-producing, 354-355 Charcoal yeast extract (CYE) agar, non-lecithinase-producing, 355 755 CMK (cynomolgus monkey kidney) "Checkerboard" protocol (broth cell cultures, 529-530 dilution), 720-721 CMK cells for detecting adenoviruses, Chemicals. danger from, 731, 735, 743 594-595 Chickenpox, 577-578 CMV (cytomegalovirus), 576-578. Chlamydia medium, glucose for, 803 581-582 Chlamydia psittaci, section through, Coagulase test for Staphylococcus. 137- 497 138 Chlamydia storage medium (4 SP). 794 Cocci Chlamydia transport medium (2 SP), anaerobic 793-794 identification of, 344-348 Chlamydiae, 493-505 taxonomy and description of, 312 cultures for, 495-505 gram-negative, 157-164; see also and mycoplasmas and viruses, media Gram-negative cocci and reagents and buffers for, gram-positive, 133-157; see also 793-805 Gram-positive cocci Chlamydial infections, tests for, 495, Columbia colistin-nalidixic acid (CNA) 498, 499 agar, 756-757 Chloramphenicol testing against Complement for virus serology, 802 Haemophilus. 297 Complement fixation (CF) test for Chloroform test for enveloped and non- LVG or C. psittaci, 502-503 enveloped viruses, 540-541 Complement fixation test (CF) for Chocolate agar, 755-756 M. pneumoniae. 521-522 Chopped meat medium, 778 Conjunctivitis etiology, 58-59 Chromatographic assays, liquid, 702- Conjunctivitis specimen collection, 61 712 Contamination of equipment and Chromogenic cephalosporin test for facilities, 733-735 ,B-Iactamase, 304 Corneal infections, 59-61 Index 845

Cornmeal agar, 788-789 Diarrhea, 55, 574 Corynebacterium, 164, 166-172 Dilution tests for susceptibility, 291- Cottonseed conversion medium, 789 299 Counterimmunoelectrophoresis, see CIE Disinfection in laboratory, 736 Coxsackieviruses A and B, 532 Disk diffusion tests, 299-301 CPE Disk elution tests, 301 identification of virus and, 598, 599- Disk method test for urease production 602 of mycobacteria, 388 from adenovirus, 595 Disk placement, 300 from CMV, 581 Disk-plate bioassays, 691-700 from enteroviruses, 533, 534 DNA-containing viruses, 576-598 from HSV, 579-580 adenovirus, 592-597 from mumps virus, 548 herpesviruses, 576-592 from parainfluenza virus, 547 poxvirus, 597-598 from rhinovirus, 534 DNase (deoxyribonuclease) test, 203- from RSV, 530 204 from SV5, 548 for staphylococcus, 139-l40 from VZ, 581 DNase test agar, 758 Creutzfeldt-Jakob disease, 741 Drainage material, aspirates, and spinal Cryptococcus fluid, procedure for, 663 characteristics, 422-423 Dreft test (sodium dodecylsulfate), for infectious, 414 Streptococcus, 153-154 morphology, 418 Duodenal biopsies (aspirates), 661 Culture media, 675 Dye inhibition test for Brucella, 232- Cutaneous amebiasis, 662 234 Cycloserine, cefoxitin, fructose, egg yolk agar (CCFA), 778-779 E Cylinder-plate bioassay with Sarcina EBV (Epstein-Barr virus), 576-592 lutea ATCC 9341, 685-691 immunofluorescence test for anti• Cynomolgus monkey kidney (CMK) bodies to, 586-592 cells, 529-530 EBV infection, antibody responses to, Cystine heart agar, 789-790 593 Cystine tellurite blood agar, 757 EBV serology, complement for, 802 Cytomegalovirus (CMV), 576-578, Echovirus antiserum pools, 538-539 581, 582 Echoviruses, clinical importance of, Cytotoxicity assay for C. difficile in 532 feces, 355-356 Egg-counting technique Beaver, 667 D Stoll,670 D-glucose for Mycoplasma medium, Egg yolk agar, 779 803 , 226-227 DAP (diaminopimelic acid), 401-402 Electron microscopy (EM) test for Decarboxylase medium, 757-758 RVLA, 574-576 Decontamination and digestion solu- Encephalitis tions,784 direct microscopic examination of Dermatiaceous molds, 472, 474-478 specimen, 71 Dermatophytes, 442, 455-458, 459- etiology of, 33 461, 462 Encephalitis viruses, 559-560 Dextran sulfate for rubella test, 803 Enterobacter, 186 846 Index

Enterobacteriaceae, 180-210 Feces, cultures of, 112-113, 116-119; biochemical reaction patterns of, see also Intestinal parasites; Stool 183-185 examination; Stool specimen differentiation of, 186-190 Feeley-Gorman (F-G) agar, 759-760 diseases with, 181 Filamentous fungi, 436-472 Enterovirus infection and meningitis, Filaria, 649-651, 656,657-659 532 Flagella stain (Leifson), 82-83 Enteroviruses, 533, 534 Fletcher's medium, 760 Eosin-methylene blue (EMB) agar, Flucytosine susceptibility test for fungal Levine, 758-759 isolate, 485--488 Eosin-saline wet preparation, 624- Fluorescence microscope, 250-251 625 Fluorescent antibody procedures, see Epstein-Barr virus, see EBV FA procedures Equipment, quality control and, 837- Formalin-ether concentration, 632, 637 840 FranciseUa, 237 Equipment and facilities, contamination Fungal infections, serodiagnosis of, of,733-735 472-473 Erysipelothrix rhusiopathiae, 176 Fungemia and bacteremia specimen versus Listeria mOTUJcytogenes, 173 collection, 24-29 Escherichieae, 186 Fungi,407-488 Esculin agar, 759 definition of, 407 Esculin broth for anaerobic bacteria, definitive tests for, 429-435 779 filamentous, identification of, 436- Esculin hydrolysis for anaerobic gram• 472 negative bacilli, 333-334 Absidia species, 444 Etiologic agents, shipment and dis• Acremonium species, 447 tribution of, 738-739 Alternaria species, 474 Exoantigen microimmunodiHusion test Aspergillus flavus, 447 for dimorphic molds, 462, 469-472 Aspergillus fumigatus, 448 Exposure, procedures following, 731- Aspergillus niger, 448 733 Aureobasidium pullulans, 474 Chrysosporium species, 449 Cladosporium species, 475 F Coccidioides immitis, 464-465 FA (fluorescent antibody) procedures, Cunninghamella species, 444 250-268 Curvularia species, 475 for Bacteroides jragilis, 264-265, Dreschlera species, 476 327-328 Epicoccum species, 476 for group A streptococci, 254 Epidermophyton floccosum, 455 for Legionella, 259 Exophiala jeanselmei, 477 for other bacteria, 263 Fusarium species, 449 for Yersinia pestis, 257 Geotrichum candidum, 450 and CIE, overview of, 249-250 Gliocladium species, 450 conjugate titration techniques, 251- Histoplasma capsulatum, 465-466 254 Microsporum audouinii, 455 direct, 254-263 Microsporum canis, 456 indirect, 263-268 Microsporum gypseum, 456 Fairley test, 43-45 Mucor species, 445 Fecal transport systems, 21 Nigrospora species, 477 Index 847

Paecilomyces species, 451 Gentamicin, chromatogram of, 710 Paracoccidoides brasiliensis, 466- Gentamicin-vancomycin laked blood 467 agar, 779-780 Penicillium species, 451 Germ tube production by Candida Petriellidium boydii, 452 albicans,429-431 Phialophora verrucosa, 478 Giemsa stain, 83-84 Rhizopus species, 445 Glucose, fermentation of, by Scopulariopsis species, 453 Clostridium difficile, 337 Sepedonium species, 453 for chlamydia medium, 803 Sporothrix schenckii, 467-468 Glucose cysteine agar, 760-761 Syncephalastrum species, 446 Glycerol-saline solution, buffered Trichoderma species, 454 (Sachs' modification), 751 Trichophyton mentagrophytes, 457 Gonorrhea, direct microscopic Trichophyton rubrum, 457 examination of, 79 Trichophyton tonsurans, 458 Gram-negative bacilli, 179-241 Trichophyton verrucosum, 458 Campylobacter, 214-216 Wangiella dermatitidis, 478 DNase test agar for, 758 media for, 787-793 Enterobacteriaceae, 180-210 mycotic infections and, 408-410 miscellaneous, 224-245 preliminary tests for, 424-429 anaerobic nonsporing, 311-312 susceptibility tests of, 473, 482-488 Pseudomonas, 216-224 yeasts, identification of, 411-423 with special growth requirements, Fusobacterium, identification of, 342- 225-240 344 Vibrionaceae,211-214 Gram-negative bacteria, major groups G of, 131 Gas liquid chromatography (GLC) for Gram-negative (GN) broth, 761 anaerobic bacteria, 336-338 Gram-negative cocci, 157-164 Gastroenteritis, 45-55, 574 Gram-positive bacilli, 164-179 direct microscopic examination of, anaerobic non-spore-forming, 356- 79-80 358 etiologic agents of, 46-53 Bacillus, 177-178 specimen collection and transport for Corynebacterium, 164, 166-172 bacteria in, 45, 54-55 differential characteristics among, Gelatin, 0.125%, 803 166-167 Gelatin liquefaction test, 206-207 Erysipelothrix, 176 for anaerobic gram-positive cocci and Lactobacillus, 179 Clostridium, 348 Listeria, 172-175 Gelatin medium, 760 nonsporing,311 Genital tract infections, 56-58 cultures of, 113-115 Gram-positive bacteria, major groups due to HSV, 577 of, 130 female, not sexually transmitted, 57- Gram-positive cocci, 132-157 58 Micrococcus, 140-141 cultures of, 113, 115, 120-121 Staphylococcus, 135-140 prostatitis, 57 Streptococcus, 141-157 sexually transmitted, 56 Gram stain, 84-85 Genitourinary tract, direct microscopic of sputum, 72-73 examination of, 78-79 Group A Streptococcus, 254-256 848 Index

H Human infection, mycobacteria im• Haemophilus,225-231 plicated in, 366-367 H. aphrophilus differentiation, 226- Human serum, testing of, 828-829 227 Hyaline molds species, differential characteristics of, aseptate (zygomycetes), 442, 444- 228 446 testing ampicillin and chlor- dimorphic, 461-472 amphenicol against, 297 monomorphic, septate, 442, 447-454 Hafnia,188 Hanging drop test for motility, 174 Hatching test for schistosomiasis, 668 Heart infusion blood agar, 761-762 Immunofluorescence test Heart infusion broth, 762 for adenovirus-infected cells 596-597 HEK cell cultures for viruses, 530-531 for Chlamydia antibodies, 503-505 for adenoviruses, 595 for EBV antibodies, 586-592 Hektoen enteric (HE) agar, 762 for HSV-infected cells, 583-586 Hemadsorption inhibition test Indole broth, 762 for paramyxovirus isolates, 552-554 Indole nitrate broth, 780 for large colony mycoplasmas, 515- Indole test, 197-199 517 for anaerobic bacteria, 332-333 for ortho- and paramyxovirus in• for Enterobacteriaceae, 197 fection, 549-552 Infection Hemagglutination-inhibition (HAl) bacteremia and fungemia, 26 for influenza virus isolates, 556-558 central nervous system, 35 for rubella virus antibodies, 569-573 fluids, other than CNS, 36 for St. Louis and Western equine gastroenteritis, 47 virus antibodies, 563-566 genital tract, 59 Hemagglutination (HA) test intra abdominal, 59 for myxoviruses, 554-555 lower respiratory, 40 for rubella virus, 568-569 parasitemia, 32 for St. Louis and Western equine spirochetemia, 32 viruses, 560-563 tissue, 65 Heparin solution, 803 upper respiratory, 38 Hepatitis infection, preventing, 741, 744 urinary, 44 Herpesviruses, 576-592 wounds, 37 cytomegalovirus (CMV), 576-578, Infectious material, hazardous, 736-741 581-582 Influenza virus growth characteristics, simplex (HSV), 576-586 546-547 zoster (VZ), 577-578 Influenza virus infections, 544-545 Hippurate hydrolysis for streptococci, Inhibitory mold agar (IMA), 790 146-148 Inocula-replicating apparatus, 296 Hookworm culture, 669-670 Inoculum Horse serum, testing of, 828 preparation of, in disk diffusion tests, HSV (herpes simplex virus), 576-586 300 HSV-infected cells, direct immuno- as susceptibility test variable, 282- fluorescence test for, 583-586 283 Human embryonic kidney cells, see Integumentary infections, 64-66 HEK entries Interpretation, 300-301, 302-303 Index 849

Intestinal parasites, 54-55, 616-646; L see also Stool specimens Laboratory, environmental control in, amebas 742-744 cysts of, 633-635 Laboratory-acquired infections, routes Entamoeba histolytica, 618 of,730 nuclei of, 616 Laboratory safety, 729-745 trophozoites of, 626-627 Laboratory waste, 738 ciliates and coccidia, 630-631 ,8-Lactamase tests for Bacteroides collection and transport for, 54-55 fragilis, 328-329 flagellates ,8-Lactamases cysts of, 636 detection of, 301, 304-306 trophozoites of, 628-629 inactivation of, 699-700 helminth eggs, 620, 638 testing of, 829-830 leukocytes, methylene blue stain for, Lactobacillus, 179 625,632 Lactose agar (10% ), 763 nematode and cestode eggs, 639 Lactose agar test, 220-221 nonparasitic objects in stools, 612- Lecithinase-producing and non- 613 lecithinase-producing Clostridium, nuclear visibility of, in unfixed 354-355 material, 624 Legionella pneumophila protozoa, 615, 617, 619 direct FA test for, 250-263 stool examination for fresh indirect FA test for antibody to, specimens,623-645 265-268 trematode eggs, 640 and Legionella-like bacteria, 237- Intracranial abscess, 33 241 Iodine stain test for C. trachomatis, 498, PBS pH 7.2 for, 768 500-502 Leishmania Iodometric slide test for ,8-lactamase, animal inoculation for, 666 304-305 skin biopsies and, 662 Iron hematoxylin staining technique Leptospirosis specimen collection, 29- (Regaud's), 644-645 30 Listeria, 172-175 Loeffier medium, 763 J Loeffier's methylene blue stain, 85-86 Jejunal or duodenal biopsies (aspirates), Lowenstein-Jensen-Gruft medium, 785 661 Lowenstein-Jensen medium, 386, 785 JEMBEC,22 Lowenstein medium base, 784-785 Lung abscess, etiology, 38 K Lung biopsy, open, 104-105 Kaolin, 803-804 Lymph nodes, procedure for, 663 Kilburn method of Tween-80 Lysine-iron agar, 763 hydrolysis, 383 Lysine iron agar (LIA) test, 192-193 Kinyoun stain, 377-378 modified acid-fast, for Nocardia, 397, 399 M Klebsiella, 93 MacConkey agar, 764 Konno-Runyon method test for niacin Malonate broth, Ewing modified, 764 production, 391 Malonate utilization test, 205-206 850 Index

Manganous chloride-urea test for "Microaerophilic" bacteria, 309 Ureaplasma urealyticum, 519-521 Microbial flora, 1-4 Mannitol fermentation agar, 764-765 Microbroth dilution method for MIC, Mannitol fennentation test for 361-363 Staphylococcus, 138-139 Micrococcaceae, 133, 135-141 MBC (minimum bactericidal concen• Microfilariae, 646-648, 649-656 tration),716-719 Micrometer, ocular, 609-611 McBride Listeria medium, 765 Middlebrook selective S7Hll agar, 787 McCoy's cell cultures, 496-497 , 785-786 chlamydial inclusion bodies in, 500 Middlebrook 7HI0 agar, 786 Measles, 545 Middlebrook 7Hll agar, 786 Measles virus (rubeola), 549 Minimal inhibitory concentration, see Media MIC for aerobic and facultatively Morganella species, 189 anaerobic bacteria, 748-775 Motility medium, 765 for fungi, 787-793 for anaerobic bacteria, 780-781 for viruses, 797-805 Motility test and reagents hanging drop, 174 for aerobic and facultatively ornithine, 195-197 anaerobic bacteria, 747-805 in semisolid agar, 174-175 for anaerobic bacteria, 776-783 MRC-5 cell culture (XI00), 528 for chlamydiae, mycoplasmas, and MR-VP broth, 765 viruses, buffers and, 793-805 Mueller-Hinton agar, 765-766 for mycobacteria and Nocardia, Mueller-Hinton broth, 766 783-787 Mumps, 545 Media sterility tests, 810--812 Mumps virus, 545, 547-548 Meningitis Muscle biopsy of meat products, 661- CIE,268 662 direct microscopic examination of Mycobacteria, 365-395, 396 specimen, 70--71 identification of, 367-393 etiology of, 33 media and reagents for, 783-787 Metamyxoviruses, 543 susceptibility testing of, 393-395, Methyl red test differentiating 396 Escherichia from Klebsielleae, various species of, 368-373, 376-377 199-200 Mycology and mycobacteriology, Methylene blue stain for parasites, quality control in, 830-831 625, 632 Mycoplasma (s), 509-522 MIC (minimal inhibitory concentra• large colony, identification of, 511, tion),280--281 513 acceptable values for control properties of, 512 organisms, 296 "pseudocolonies" resembling, 514 microbroth dilution method for, 361- 363 Mycoplasma medium (a) susceptibility and, 281 with arginine, 795 MIC correlates, zone diameter and, n-glucose for, 803 302-303 large colony, 794-795 MIC guidelines, clinically oriented, 292 Mycoplasma, sp. pneumoniae, Mice, inoculation of, for Coxsackievirus cerological tests for, 521-522 A test, 541-543 Mycosel agar, 790-791 Index 851

Myxoviruses, hemagglutination test Orthomyxoviruses, 543 (HA) for, 554-555 hemadsorption patterns and cytopathic effects with, 551 Oxidase test for Neisseria, Haemophilus, N Pseudomonas, Aeromonas, 159- Neisseria, 157-159 161 carbohydrate utilization media for 753-754 ' Oxidation-fermentation (O-F) medium, 767 carbohydrate utilization tests, 161- Oxidation-fermentation (O-F) test 162 for carbohydrate utilization 218- N. meningitidis, antigen detection 220 ' by eIE, 268-274 N. meningitidis, concentrations of antimicrobials to be tested p against, 298 Pan encephalitis, subacute sclerosing Neufeld Quellung reaction for (SSPE),546 pneumococci, 155-157 Parainfluenza virus, 545, 547 Neutralization of infectivity as test for Paramyxoviruses, 543 viral isolate, 535-539 Niacin production test for M. hemadsorption patterns and tuberculosis, 390-392 cytopathic effects with, 551 Parasitemia specimen collection, 30-32 Niger seed agar, 434 Parasites, 609-675 Nitrate broth, 766 blood and tissue, 646-657 Nitrate reduction test, 221-223 filaria ( e ), 657, 659 anaerobic bacteria, 333 for mycobacteria, 380-382 flagellates, 647-648 for yeasts, 425 malarial, 656 Nocardia, 395, 396 microfilaria ( e), 658, 659 acid-fast stain (modified Kinyoun's) Plasmodium species compared, for, 397, 399 652-653 media and reagents for, 783-787 intestinal, 616-646; see also N. asteroides, 396 Intestinal parasites N. brasiliensis, 396 nonfecal specimens, 660 Nutrient agar, 766 specimens other than feces and Nutrient broth, 766 blood, 657-664 Trichomonas vaginalis in urine of males, 658 o urine, 657-658 Occupational health in laboratory, Parasitic diseases, serology for, 664-665 744-745 Parasitic infection, examination of Ocular infections, 58-62 specimens for, 616-623 direct microscopic examination of, Parasitology, quality control in, 831- 80-81 Ocular micrometer, 609-675 834, 836 Onchocerciasis, 662 Parenteral fluids and medications, 122- ONPG test for ,B-galactosidase, 163- 123 164 Pasteurella, 235-236 Open lung biopsy, 74-78 Pectobacterium, 188 Optochin sensitivity test for Penicillin G assay, 692-699 Streptococcus, 154-155 Penicillinase in blood culture media, 29 852 Index

Perlormance testing Q for media and reagents, 831-836 Quality control, 284-285, 807-840 of reagents and sera, 828-830 in antimicrobial susceptibility, 284- of stock cultures, 814-827 285 for yeast test strip, 834 in bacteriology, 813-830 pH3 test for rhino viruses and of equipment, 837-840 enteroviruses, 539-540 media and reagents, 810-837 Phenol red, 804 in mycology and mycobacteriology, Phenol red base carbohydrate 830-831 fermentation broth, 753 in parasitology, 831-834 fermentation test, 168-169 performance testing of media, Phenylalanine agar, 767 reagents and sera, 814-830 Phenylalanine deaminase test for in virology, 834-836 protease, 202-203 Phenylethyl alcohol (PEA) blood agar, R 767 Receptor-destroying enzyme (RDE), Phenylethyl alcohol (PEA) for 804 anaerobic bacteria, 781 calcium-saline solution for, 802 Phosphate-buffered saline (PBS), pH Rectal biopsies, 661 7.2, for Legionella pneumophila, Reovirus-like agents (RVLA), 537-576 768 Reporting times of microbiological Picornaviruses, 531-543 results, 92-93 Pigment production Respiratory syncytial virus (RSV), 546, for mycobacteria, 379-,380 549, 550 on Niger seed agar for Cryptococcus Respiratory tract infections neoformans, 433-434 direct microscopic examination of for Pseudomonas, 223-224 specimen, 72-78 Pigment production media, 768 lower Plesiomonas, 212, 214 cultures of specimens from, 104- Pneumonia etiology, 37-38 109 Polymyxin, broth-dilution assay of, etiology, 37-38 701-702 specimen collection, 39-42 Porphyrin test for Haemophilus, 229- upper 231 cultures of specimens from, 100- Postmortem microbiology, 63-64 104 Potassium chloride, 804 etiology and specimen collection, Potassium hydroxide (KOH) 35-37 preparation, 86 Rhinovirus, 533-536 wet mount, of sputum, 73,75-78 Rice grain medium, 791 Potato dextrose agar, 791 test for Microsporum audouinii, Poxvirus, 597-598 459-460 PRAS media, 318 RNA-containing viruses, 531-576 Propionibacterium, 356-357 ortho-, para-, and metamyxoviruses, Prostatitis etiology, 57 543-558 Proteus, species of, 189 picornaviruses, 531-543 Providencia, species of, 189 reovirus-like agents (RVLA), 573- Pseudomona~ 216-224 576 Pseudosel agar, 753-754 togaviruses, 558-573 Index 853

Rogosa SL agar, 768--769 Sodium bicarbonate agar, 770 Roudabush fixative, 670 Sodium chloride broth, modified, 770 RSV (respiratory syncytial virus), 546, Sodium chloride tolerance test for 549, 550 mycobacteria, 387 Rubella test, dextran suHate for, 803 Sodium dodecylsuHate (Dreft test), for Rubella viral infection, 560 Streptococcus, 153--154 Rubella virus Sodium polyanetholsulfonate (SPS) in hemagglutination-inhibition (HAl) blood culture media, 28 for antibodies to, 569-573 Solvents and diluents for antimicrobials, hemagglutination (HA) test for, 286-287 568--569 Soybean-casein digest agar, 770 Rubeola (measles virus), 549 Soybean-casein digest broth, 771 RVLA (reovirus-like agents), 537-576 Specimen collection bacteremia and fungemia, 24-29 S gastroenteritis, 45, 54-55 Sabouraud dextrose agar (Emmons' instructions for, 5, 20-23 modification), 791 microbiologic, 6-13 Sal11Wnella mycoplasma, Q-fever, and agglutination test for, 209-210 psittacosicosis, 18-19 differentiation of, 187 spirochetemia, 29-30 Salmonella-8higella (SS) agar, 769 virus, 14-18 Salt tolerance test for enterococci, 150- Spirochetemia specimen collection, 152 29-30 Schaedler broth, 781-782 Spore stain, 86-87 Schistosoma haematobium in urine. Sputum, 663 657-658 direct microscopic examination of, Schistosomiasis, 668 72-73 Scotch tape swab, 659 St. Louis virus, 560 Selenite broth, 769-770 hemagglutination-inhibition (HAl) Septicemia, 24 for antibodies to, 563--566 Serratia, 188 hemagglutination (HA) test for, Serum or urine collection, 33-34 560-563 Sexually transmitted disease (STD) Stains and wet mount preparations, cultures of, 113, 120-121 81-88 direct microscopic examination of, 79 Staphylococcus etiology, 56 DNase test agar for, 758 specimen collection and transport, identification of, 135-140 57-58 Starch agar, 771 specimen transport systems, 22-23 Starch broth medium, 782 Shigella STD, see Sexually transmitted disease agglutination test for, 210 Sterile body fluids differentiation of, 186 cultures of, 95, 98-99 Shingles, 577-578 infections of, 32-35 Skin biopsies and specimens from skin, tissue, wounds, and abscesses, 662 procedures for, 96-97 Smallpox, 598 Sterility tests of media, 810-812 Sodium amylosulfate (SAS) in blood Sterilization in laboratory, 735-736 culture media, 28-29 Stoll egg-counting technique, 670-671 854 Index

Stool examination of mycobacteria, 393-395, 396 for intestinal parasites variables in, 281-284 fixed specimen, 645-656 Swabs, 5, 20 fresh specimen, 623-645 anal, 659 and related procedures, 621-622 serum-free medium for extraction of, Stool specimens, see also Intestinal 797 parasites Swube,659 collecting, 47,57-58,616,618, 621 vaginal, 661 finding parasites in, 611, 614, 615 SWUBE disposable paddle (Falcon processing, 612-646 2012),22 protozoa in, 615, 617 Streak plate methods, 98-99 T Streptococcus, 141-157, 254-256 Tapeworm recovery, 671-672 group A, tests for, 144-146 Tapeworms, diagnostic features of direct FA procedures, 254-256 large, 673 group B, tests for, 146--149 TCH (thiophene-2-carboxylic acid group D, tests for, 149-151 hydrazide) susceptibility test for identification of, 142-157 M. bovis and M. tuberculosis, 392 pneumoniae, tests for, 152-157 TCID (tissue culture infective doses), viridans, 151-152 536-537 Streptomyces, 395, 396 Tellurite reduction for M. ooium• Substrate hydrolysis (casein, xanthine, intracellulare, 389-390 tyrosine) for actinomycetes, 399, Tetrazolium reduction test for large 401 colony Mycoplasma, 517-519 Sucrose or sorbitol in blood culture Thallium acetate, 804 media, 29 Thayer-Martin medium, modified, Sucrose-phosphate solution, 794 771-772 Sudan black B fat stain, 87 Thick and thin films, purpose of, 32 Susceptibility, definitions of, 280-281 Thick film, examining, 654 Susceptibility tests Thin film areas for examination, 655 amphotericin B, for fungal isolate, Thiogel medium, 783 483-485 Thioglycollate, supplemented, for of anaerobic bacteria, 358-363 anaerobic bacteria, 782-783 antimicrobial, 279-306 Thioglycollate 135C, 772 preparation of solutions for, 285- Thiopene-2-carboxylic acid hydrazide 288 (TCH) susceptibility test for selection of antimicrobials for, M. bovis and M. tuberculosis, 392 283-284 Thiosulfate citrate bile salts sucrose antimicrobial method, proportional, (TCBS) agar, 772-773 393-395 Threonine or lactate, conversion for of Chlamydia trachomotis, 505 Fusobacterium, 343-344 dilution tests, 291-299 Tissue culture infective doses (TCID), disk diffusion, 299-301 536--537 disk elution, 301 Tissue specimens, 62-63 Hucytosine for fungal isolate, 485- Tissues, cultures of, 99-100 488 Todd-Hewitt broth, 773 of fungi, 473, 479-482 Togaviruses, 558-573 ,B-lactamase detection, 301, 304-306 Toluidine blue 0 stain, 88 Index 855

Toxigenicity test agar, 773 Urine Toxigenicity tests of Corynebacterium bacterial antigens in, 274-275 diphtheriae, 169-172 clean-voided, 78 Toxoplasma, animal inoculation concentration of, 275 procedure for, 666 cultures of, 109-111 Transgrow, 22 Urine specimen collection Transport vial for anaerobic bacteria, clean-voided midstream, 42-43 783 postmortem, 64 Transtracheal aspirate (TTA), 73-74 or serum collection, 33-34 "Trichinoscope," 661-662 suprapubic aspiration (SPA), 43, 44 Trichomoniasis, 79 Trichophyton agars, 791-792 V and nutritional requirements of Varicella zoster (VZ), 576-578 dermatophytes, 460-461 Veal infusion broth, 774-775 Trichrome staining technique, 642--644 Vibrio, 211-214 of PVA fixed specimens, 645-646 Viral isolates, reporting of, 598-602 Triple sugar-iron agar (TSlA), 773- , 151-152 774 Virology, quality control in, 834-836 test, 190-192 Virus dementia, transmissible, 741 Trypanosomes, 646-659 Virus excretion and antibody response Trypsin-EDTA solution, 805 during rubella, 570 Trypsinization for subculturing MRC-5 Virus isolation (s) cells, 528 frequency of, 599 Tryptose phosphate broth, 805 growth and maintenance media for, Tween-80 hydrolysis of mycobacteria, 798 382-383 Virus neutralization tests, 535-543 Tyrosine agar, 787 Viruses, 525-602 clinical importance of, 526-527 U DNA-containing, see DNA-con• U-9 basal broth, 795-796 taining viruses U-9 complete medium, 796 media for, 797-805 Urea agar, 774 RNA-containing, see RNA-con• Ureaplasma media, 795-796 taining viruses urea solution for, 805 Voges-Proskauer (V-P) test for Ureaplasma urealyticum, 511, 513- Klebsielleae, 201-202 515, 516 Vulvovaginitis, 79 manganous chloride-urea test for, VZ (varicella-zoster), 576-578 519-521 Ureaplasma urealyticum-Mycoplasma w agar (A-3 agar base), 796 "w" (Wisconsin) medium, 775 Ureaplasma urealyticum-mycoplasma Western equine virus, 560 complete (A-3) agar, 796 hemagglutination-inhibition (HAl) Urease production, test for for antibodies to, 563-566 Enterobacteriaceae, 194-195 hemagglutination (HA) test for, mycobacteria, 388-389 560-563 Nocardiae,403-404 Wilson and Blair medium, modified, 749 for Trichophyton species, 459 Worm fixation, 672-675 yeasts, 424-425 Worms, staining of, 670 856 Index

Wound, abscess, and cellulitis, direct in clinical specimens, 411 microscopic examination of speci• definition of, 407 men,7I human infection and, 413-414 Wound infections, 34 identification of, 411-423 microscopic morphological features of, 419, 420-421 x morphology of, 418 Xanthine agar, 787 Yersinia, differentiation within, 190 Xylose-lysine-deoxycholate agar Yersinia pestis, identification of, 257- (XLD),775 258

y Z Yeast extract, 805 Ziehl-Neels en stain, 378-379 Yeast extract agar, 792-793 Zone diameter and MIC correlates, Yeasts 302-303 body sites of, 412 Zygomycetes (aseptate hyaline molds), characteristics of, 212-213 442,444-446 2·1a 2·1b 2·1c

,

2·1d 2·1e 2·1f

Figure 2·1. Gram stained smears (original magnification, 1000x). Cerebrospinal fluid demonstrating (a) faintly staining, pleomorphic gram-negative coccoba• cilli and bacilli, typical of Haemophilus influenzae; (b) gram-negative diplococci typical of Neisseria meningitidis; (c) encapsulated gram-positive diplococci typical of Streptococcus pneumoniae. Cultures of (d) Listeria monocytogenes and (e) Corynebacterium demonstrating angular and palisading arrangement of nonsporulating gram-positive bacilli. Exudate or pus demonstrating (f) round gram-positive cocci in clusters typical of Staphylococcus aureus; (g) gram• positive bacilli, gram-negative bacilli, and cocci suggesting mixed anaerobic bacterial infection; (h) gram-positive cocci in chains typical of group A strep• tococci; and (i) large, nonsporulating gram-positive bacilli resembling Clos• tridium or Bacillus. Blood cultures demonstrating (il highly pleomorphic gram-negative bacilli suggesting bacteremia due to Bacteroides tragilis, (k) slender gram-negative baci II i with tapered ends typical of Fusobacterium nucleatum, and (I) nonsporulating, nonbranching bacilli demonstrating extreme pleomorphism and gram-variability from a patient with lactobacillemia. Pha• ryngeal exudate demonstrating (m) gram-positive cocci typical of streptococci in association with a polymorphonuclear leukocyte demonstrating loss of cytoplasmic and cellular integrity, suggesting group A streptococcal pharyn• gitis. Expectorated sputum from patients with (n) Branhamella catarrhalis and (0) pneumonia. Male urethral exudate demonstrating intracellular, gram-negative diplococci diagnostic of gonorrhea (p). (q) Yeasts with branching hyphae from left ureter. 2-19 2-1h

2-1i 2-1j

2-1k 2-11

2-1m 2-1n 2·10 2·1p

2·1q 2·3a

2·3b 2·4

Figure 2·3. Sinus tract exudate with sulfur granules (a) comprising masses of nonsporulating gram-positive bacilli (b) identified as Actinomyces israelii.

Figure 2·4. Loeffler's methylene blue stain of Corynebacterium diphtheriae demonstrating metachromatic granules (x 1000). 2·8a 2·8b

Figure 2·8. Gram (a) and Sudan black fat (b) stained smears of Legionella pneumophila.

Figure 2·9. Toluidine blue stained impression smear of open lung biopsy demonstrating Pneu• mocystis carinii cysts (x640).

2·9

2·10a 2·10b

Figure 2·10. Gram-stained smears of drops of well-mixed urine specimens: (a) yeasts, pus cells; (b) gram-negative bacilli, no pus cells (x1000).

Figure 2·12. Spore stain of Clostridium sporo• genes.

2·12 Figure 4.2-2. CAMP reac• tion consisting of arrow• head-shaped area of f3- hemolysis at juncture of lines of growth on blood agar of Staphylococcus aureus and group 8 strep• tococci.

a b c

Figure 4.2-3. Reactions in triple sugar iron agar (TSIA) demonstrating: (a) alka• line slant! acid butt (KI A) reaction without gas or H2 S, (b) AI A reaction with gas and no H2 S, (c) AI A reaction with gas and H2 S. Figure 4.2-5. Christensen's urea agar dem• onstrating negative reaction in the first tube on the left and increasing gradations of positive reactions in the next three Figure 4.2-4. Positive (left) tubes. and negative (right) reac• tions on Simmons' citrate agar.

Figure 4.2-6. Decarboxylation of argmme and ornithine (center two tubes) but not of lysine (left) or in the control (right) Figure 4.2-7. Positive (left) semisolid Moeller's medium. Diffuse and negative (right) indole growth extending away from stab line of tests. inoculation reflects the organism's mo• tility. Figure 4.2·8. Positive (left) and Figure 4.2·9. Positive (left) and negative (right) methyl red tests. negative (right) Voges-Proskauer tests.

a

Figure 4.2-10. Positive (left) b and negative (right) phenyl• alanine deaminase tests. Figure 4.3-1. Examples of a positive (4+) and negative ("""'1 +) FA stain using fluorescein• labeled antibody against group A Streptococ• cus: (a) group A Streptococcus; (b) non-group A Streptococcus. •• "'-e ~ • • , • • • • 0 • ., • • • • - Figure 8-3. Fluorescence of intra• Figure 10-21. Immunofluorescence of cytoplasmic inclusion bodies result• MRC-5 cells infected with herpes ing from reaction of antibodies to simplex virus (x 250). C. trachomatis with LGV-1-infected McCoy's cells in the indirect test (x 250). • 'I: •••• t - ~ • Figure 10-24. Immunofluorescence of ~ Epstein-Barr virus-infected HR1 K • cells after reaction with serum con• • taining IgG antibodies to the viral • ~ .. capsid antigen (x 250). ... . ~..'

Figure 10-25. Immunofluorescence of Epstein-Barr virus-infected HR1 K cells after reaction with serum con• taining IgM antibodies to the viral capsid antigen (x 250).

Figure 10-26. Immunofluorescence of Epstein-Barr virus-infected Raji cells after reaction with serum containing antibodies to EBV nuclear antigen (EBNA) (x 400) .