382Add all ingredients except Mg SO4 to 1 liter distilled water. Heat to boiling with stirring. Add Mg SO4 and adjust pH. Dispense 8 ml portions into 16 × 150 mm tubes. Autoclave 15 min at 121°C. Incline tubes to obtain 5 cm slant. Final pH, 6.7 M3. Acid Broth Proteose peptone 5 g Yeast extract 5 g Dextrose 5 g K2HPO4 4 g Distilled water l liter Dissolve ingredients and dispense 12-15 ml portions into 20 × 150 mm tubes. Autoclave 15 min at 121°C. Final pH, 5.0. M4. AE Sporulation Medium, Modified (for C. perfringens) (AE base is available commercially) Polypeptone 10.0 g Yeast extract 10.0 g Na2HPO4 4.36 g KH2PO4 0.25 g Ammonium acetate1.5 g Mg SO4·7H2O 0.2 g Distilled water 1 liter Dissolve ingredients and adjust to pH 7.5 ± 0.1, using 2 M sodium carbonate. Dispense 15 ml into 20 × 150-mm screw-cap tubes and sterilize by autoclaving for 15 min at 121°C. After sterilization, add
3830.6 ml of sterilized 10% raffinose and 0.2 ml each of filter-sterilized 0.66 M sodium carbonate and 0.32% cobalt chloride (CoCl2·6H2O) dropwise to each tube. Check pH of one or two tubes; it should be 7.8 ± 0.1. Just before use, steam medium for 10 min; after cooling, add 0.2 ml of filter-sterilized 1.5% sodium ascorbate (prepared daily) to each tube. M5. Alkaline Peptone Agar Peptone 10 g NaCl 20 g Agar 15 g Distilled water 1 liter Boil to dissolve ingredients. Adjust pH so that value after sterilization is 8.5 ± 0.2. Autoclave 15 min at 121°C. Solidify agar in tubes as slants. M6. Alkaline Peptone Salt Broth (APS) Peptone 10 g NaCl 30 g Distilled water 1 liter Dissolve ingredients. Adjust pH so that value after sterilization is 8.5 ± 0.2. Dispense 10 ml into tubes. Autoclave 10 min at 121°C. M7. Alkaline Peptone Water Peptone 10 g NaCl 10 g Distilled water l liter
384Adjust pH so that value after sterilization is 8.5 ± 0.2. Dispense into screw-cap tubes. Autoclave 10 min at 121°C. M8. Anaerobe Agar Base Trypticase (tryptic) soy agar 40 g Agar 5 g Yeast extract 5 g L-Cysteine (dissolved in 5 ml 1 N NaOH) 0.4 g Distilled water l liter Heat with agitation to dissolve agar. Adjust pH to 7.5 ± 0.2. Autoclave 15 min at 121°C. Cool to 50°C. Hemin solution. Suspend 1 g hemin in 100 ml distilled water. Autoclave 15 min at 121°C. Refrigerate at 4°C. Vitamin K1 solution. Dissolve 1 g vitamin K1 (Sigma Chemical Co., St. Louis, MO) in 100 ml 95% ethanol. Solution may require 2-3 days with intermittent shaking to dissolve. Refrigerate at 4°C. Final medium. To 1 liter base add 0.5 ml hemin solution and 1 ml Vitamin K1 solution. Mix and pour 20 ml portions into 15 × 100 mm petri dishes. Medium must be reduced before inoculation by 24 h anaerobic incubation in anaerobic glove box or GasPak jar. M9. Anaerobic Egg Yolk Agar Agar base Yeast extract 5 g
385Tryptone 5 g Proteose peptone 20 g NaCl 5 g Agar 20 g Distilled water 1 liter Autoclave 15 min at 121°C. Adjust pH to 7.0 ± 0.2. 2 Fresh Eggs Treatment of eggs. Wash 2 fresh eggs with stiff brush and drain. Soak eggs in 70% ethanol for 1 h. Crack eggs aseptically. Retain yolks. Drain contents of yolk sacs into sterile stoppered graduate and discard sacs. Add yolk to equal volume of sterile 0.85% saline. Invert graduate several times to mix. Egg yolk emulsion (50%) is available commercially. Preparation of medium. To 1 liter melted medium (48-50°C) add 80 ml yolk-saline mixture (available from Difco as Bacto Egg Yolk Enrichment 50%), and mix. Pour plates immediately. After solidification dry 2-3 days at ambient temperature or at 35°C for 24 h. Check plates for contamination before use. After drying, plates may be stored for a short period in refrigerator.
386M10. Antibiotic Medium No. 1 (Agar Medium A) Gelatone or gelysate 6 g Tryptone or trypticase 4 g Yeast extract 3 g Beef extract 1.5 g Dextrose 1 g Agar 15 g Distilled water 1 liter Autoclave 15 min at 121°C. Final pH, 6.5-6.6. Commercially available in dehydrated form as Difco Penassay Seed Agar or BBL Seed Agar. M11. Antibiotic Medium No. 4 (Agar Medium B) Tryptone or trypticase 6 g Yeast extract 3 g Beef extract 1.5 g Dextrose 1 g Agar 15 g Distilled water 1 liter Autoclave 15 min at 121°C. Final pH, 6.5-6.6. Commercially available in dehydrated form as Difco Yeast Beef Agar or BBL Yeast Beef Agar. M12. Arginine-Glucose Slant (AGS) Peptone 5 g Yeast extract 3 g
387Tryptone 10 g NaCl 20 g Glucose 1 g L-Arginine (hydrochloride) 5 g Ferric ammonium citrate 0.5 g Sodium thiosulfate 0.3 g Bromocresol purple 0.02 g Agar 13.5 g Distilled water 1 liter Suspend ingredients in distilled water and boil to dissolve. Dispense into tubes (for 13 × 100 mm tubes use 5 ml). Autoclave 10-12 min at 121°C. After sterilization, solidify as slants. Final pH, 6.8-7.0. M13. Bile Esculin Agar Beef extract 3 g Peptone 5 g Esculin 1 g Oxgall 40 g Ferric citrate 0.5 g Agar 15 g Distilled water 1 liter Heat with agitation to dissolve. Dispense into tubes, autoclave 15 min at 121°C, and slant until solidified. Final pH, 6.6 ± 0.2. M13Bismuth Sulfite Agar (Wilson and Blair) Polypeptone (or peptone)10 g
388Beef extract 5 g Dextrose 5 g Na2HPO4 (anhydrous) 4 g FeSO4 (anhydrous) 0.3 g Bismuth sulfite (indicator) 8 g Brilliant green 0.025 g Agar 20 g Distilled water 1 liter Mix thoroughly and heat with agitation. Boil about 1 min to obtain uniform suspension. (Precipitate will not dissolve.) Cool to 45-50°C. Suspend precipitate by gentle agitation, and pour 20 ml portions into sterile 15 × 100 mm petri dishes. Let plates dry about 2 h with lids partially removed; then close plates. Final pH, 7.7 ± 0.2 DO NOT AUTOCLAVE. Prepare plates on day before streaking and store in dark. Selectivity decreases in 48 h. M14. Blood Agar Tryptone 15 g Phytone or soytone 5 g NaCl 5 g Agar 15 g Distilled water 1 liter Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Cool to 50°C. Add 5 ml defibrinated sheep red blood cells to 100 ml melted agar. Mix and pour 20 ml portions into sterile 15 × 100 mm petri dishes. Final pH of base, 7.3 ± 0.2. Tryptic soy agar, tryptic soy
389agar blood base, or trypticase soy agar [soybean-casein digest agar (M152)] may be used as the basal medium. Commercially available sheep blood agar plates are satisfactory. For Vibrio hollisae, add NaCl to a final concentration of 1%. M15. Blood Agar Base (Infusion Agar) Heart muscle, infusion from 375 g Thiotone 10 g NaCl 5 g Agar 15 g Distilled water 1 liter Heat gently to dissolve. Autoclave 20 min at 121°C. Final pH, 7.3 ± 0.2. Commercially available dehydrated heart infusion agars may be used. M16. Blood Agar Base #2 (Difco) Proteose peptone15 g Liver digest 2.5 g Yeast extract 5 g NaCl 5 g Agar 12 g Distilled Water 1 liter Autoclave at 121°C for 15 min. For blood agar, reduce water to 950 ml. Add 50 ml defibrinated (whole or lysed) horse blood and FBP (4 ml to agar + blood) after autoclaving and cooling to 48°C. Final pH, 7.4 ± 0.2.
390M17. Brain Heart Infusion (BHI) Agar (0.7%) (for staphylococcal enterotoxin) Prepare a suitable quantity of brain heart infusion broth. Adjust pH to 5.3 with 1 N HCl. Add agar to give 0.7% concentration. Dissolve by minimal boiling. Dispense 25 ml portions into 25 × 200 mm test tubes. Autoclave 10 min at 121°C. M18. Brain Heart Infusion (BHI) Broth and Agar Formulations used by selected manufacturers are represented below; these media are normally available as pre-mixed dry powder. Medium 1 Calf brain, infusion from 200 g Beef heart, infusion from 250 g Proteose peptone (Difco) or polypeptone (Bioquest) 10 g NaCl 5 g Na2HPO4 * 2.5 g Dextrose 2.0 g Distilled water 1 liter Medium 2 Brain heart-infusion 6.0 g Peptic digest of animal tissue 6.0 g NaCl 5.0 g Dextrose 3.0 g Pancreatic digest of gelatin 14.5 g
391Na2HPO4 2.5 g Distilled water 1 liter Dissolve ingredients of Medium 1 in distilled water with gentle heat. Suspend ingredients of Medium 2 in distilled water and boil for 1 min to completely dissolve. For both Medium 1 and Medium 2, dispense broth into bottles or tubes for storage. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. Commercially available BHI is acceptable. To prepare brain heart infusion agar, add 15 g agar to 1 liter BHI broth. Heat to dissolve agar before dispensing into bottles or flasks. Autoclave 15 min at 121°C. For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%. M19. Brilliant Green Lactose Bile Broth Peptone 10 g Lactose 10 g Oxgall 20 g Brilliant green 0.0133 g Distilled water 1 liter Dissolve peptone and lactose in 500 ml distilled water. Add 20 g dehydrated oxgall dissolved in 200 ml distilled water. The pH of this solution should be 7.0-7.5. Mix and add water to make 975 ml. Adjust pH to 7.4. Add 13.3 ml 0.1% aqueous brilliant green in distilled water. Add distilled water to make 1 liter. Dispense into
392fermentation tubes, making certain that fluid level covers inverted vials. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.1. M20. Bromcresol Purple Broth Base Peptone 10 g Beef extract 3 g NaCl 5 g Bromcresol purple 0.04 g Distilled water 1 liter Dispense 2.5 ml portions of base solution into 13 × 100 mm test tubes containing inverted 6 × 50 mm fermentation tubes. Autoclave 10 min at 121°C. Final pH, 7.0 ± 0.2. Sterilize stock solutions of carbohydrates (50% w/v) separately by autoclaving or, preferably, by filtration (0.2 µm pore size). Add 0.278 ± 0.002 ml stock carbohydrate solution to 2.5 ml basal medium to give 5% w/v final carbohydrate concentration. For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%. M21. Bromcresol Purple Dextrose Broth (BCP) Dextrose 10 g Beef extract 3 g Peptone 5 g Bromcresol purple (1.6% in ethanol) 2 ml Distilled water 1 liter
393Dissolve ingredients in distilled water. Dispense 12-15 ml into tubes. Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2 M22. Egg Yolk Emulsion, 50% Wash fresh eggs with a stiff brush and drain. Soak eggs 1 h in 70% ethanol. Drain ethanol. Crack eggs aseptically and discard whites. Remove egg yolks with sterile syringe or wide-mouth pipet. Place yolks in sterile container and mix aseptically with equal volume of sterile 0.85% saline. Store at 4°C until use. Egg yolk emulsion (50%) is available commercially. M23. Heart Infusion (HI) Broth and Agar (HIA) (for Vibrio) Beef heart, infusion from 500 g 1 liter Tryptose 10 g NaCl 5 g Dissolve ingredients and dispense into tubes. For halophilic Vibrio spp. add an extra 15 g NaCl/liter. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. For heart infusion agar, add 15 g agar/L and boil to dissolve before dispensing and sterilizing.Commercially available from Difco. M24. Kligler Iron Agar Polypeptone peptone 20 g Lactose 20 g Dextrose 1 g NaCl 5 g Ferric ammonium citrate 0.5 g
394Sodium thiosulfate 0.5 g Agar 15 g Phenol red 0.025 g Distilled water 1 liter Heat with agitation to dissolve. Dispense into 13 × 100 mm screw-cap tubes and autoclave 15 min at 121°C. Cool and slant to form deep butts. Final pH, 7.4 ± 0.2.Commercially available from Difco, BBL, and Oxoid.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%. M25. Lysine Decarboxylase Broth (Falkow) (for Salmonella) Gelysate or peptone 5 g Yeast extract 3 g Glucose 1 g L-Lysine 5 g Bromcresol purple 0.02 g Distilled water 1 liter Heat until dissolved. Dispense 5 ml portions into 16 × 125 mm screw-cap tubes. Autoclave loosely capped tubes 15 min at 121°C. Screw the caps on tightly for storage and after inoculation. Final pH, 6.8 ± 0.2. For halophilic Vibrio spp., add NaCl to a final concentration of 2-3%. M26. Lysine Decarboxylase (LDC) Medium (for Gram-negative nonfermentative bacteria) L-Lysine HCl 0.5 g
395Dextrose 0.5 g KH2PO4 0.5 g Distilled water 100 ml Dissolve ingredients. Adjust pH to 4.6 ± 0.2. Autoclave 15 min at 121°C. Aseptically dispense 1 ml portions to sterile 13 × 100 mm tubes. M27. Lysine Iron Agar (Edwards and Fife) Gelysate or peptone 5 g Yeast extract 3 g Glucose 1 g L-Lysine hydrochloride 10 g Ferric ammonium citrate 0.5 g Sodium thiosulfate (anhydrous) 0.04 g Bromcresol purple 0.02 g Agar 15 g Distilled water 1 liter Heat to dissolve ingredients. Dispense 4 ml portions into 13 × 100 mm screw-cap tubes. Autoclave 12 min at 121°C. Let solidify in slanted position to form 4 cm butts and 2.5 cm slants. Final pH, 6.7 ± 0.2. M28. MacConkey Agar Proteose peptone or polypeptone 3 g Peptone or gelysate17 g Lactose 10 g
396Bile salts No. 3 or bile salts mixture 1.5 g NaCl 5 g Neutral red 0.03 g Crystal violet 0.001 g Agar 13.5 g Distilled water 1 liter Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121°C, cool to 45-50°C, and pour 20 ml portions into sterile 15 × 100 mm petri dishes. Dry at room temperature with lids closed. DO NOT USE WET PLATES. Final pH, 7.1 ± 0.2. M29. Motility-Indole-Ornithine (MIO) Medium Yeast extract 3 g Peptone 10 g Tryptone 10 g L-Ornithine HCl 5 g Dextrose 1 g Agar 2 g Bromcresol purple 0.02 g Distilled water 1 liter Dispense 4 ml portions into 13 × 100 mm tubes. Autoclave 15 min at 121°C. Final pH, 6.5 ± 0.2. M30. Motility Medium (for B. cereus) Trypticase 10 g
397Yeast extract 2.5 g Dextrose 5 g Na2HPO4 2.5 g Agar 3 g Distilled water 1 liter Heat with agitation to dissolve agar. Dispense 100 ml portions to 170 ml bottles. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. Cool to 50°C. Aseptically dispense 2 ml portions to sterile 13 × 100 mm tubes. Store at room temperature 2 days before use. M31. Motility-Nitrate Medium, Buffered (for C. perfringens) Beef extract 3 g Peptone (Difco) 5 g KNO3 1 g Na2HPO3 2.5 g Agar 3 g Galactose 5 g Glycerin (reagent grade) 5 ml Distilled water 1 liter Dissolve all ingredients except agar. Adjust pH to 7.3 ± 0.1. Add agar and heat to dissolve. Dispense 11 ml portions into 16 × 150 mm tubes. Autoclave 15 min at 121°C. If not used within 4 h, heat 10 min in boiling water or flowing steam. Chill in cold water.
398M32. Motility Test Medium (Semisolid) Beef extract 3 g Peptone or gelysate10 g NaCl 5 g Agar 4 g Distilled water 1 liter Heat with agitation and boil 1-2 min to dissolve agar. Dispense 8 ml portions into 16 × 150 screw-cap tubes.Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. For Salmonella: Dispense 20 ml portions into 20 × 150 mm screw-cap tubes, replacing caps loosely. Autoclave 15min at 121°C. Cool to 45°C after autoclaving. Tighten caps, and refrigerate at 5-8°C. To use, remelt in boiling water or flowing steam, and cool to 45°C. Aseptically dispense 20 ml portions into sterile 15 × 100 mm petri plates. Cover plates and let solidify. Use same day as prepared. Final pH, 7.4 ± 0.2.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%. M33. Nitrate Broth Beef extract 3 g Peptone 5 g KNO3 (nitrite-free) 1 g Distilled water 1 liter Dissolve ingredients. Dispense 5 ml portions into 16 × 125 mm tubes. Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2.
399M34. Nitrate Broth, Enriched (CDC) Infusion broth 25 g KNO3 (nitrite-free) 2 g Distilled water 1 liter Dispense 4 ml portions into 13 × 100 mm tubes with inverted Durham tubes. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2. M35. Oxidative-Fermentative (OF) Test Medium Base Peptone 2 g NaCl 5 g K2HPO4 0.3 g Bromthymol blue 0.03 g Agar 2.5 g Distilled water 1 liter Heat with agitation to dissolve agar. Dispense 3 ml portions into 13 × 100 mm tubes. Autoclave 15 min at 121°C.Cool to 50°C; pH, 7.1. Carbohydrate stock solution. Dissolve 10 g carbohydrate in 90 ml distilled water. Sterilize by filtration through 0.22 µm membrane. Add 0.3 ml stock solution to 2.7 ml base in tube. Mix gently and cool at room temperature.Inoculate tubes in duplicate. Layer one tube with sterile mineral oil. Incubate 48 h at 35°C.For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%. M36. Selenite Cystine Broth Medium 1 (modification of Leifson’s formulation for selenite broth)
400Tryptone or polypeptone 5 g Lactose 4 g Sodium acid selenite (NaHSeO3) 4 g Na2HPO4 10 g L-Cystine 0.01 g Distilled water 1 liter Heat to boiling to dissolve. Dispense 10 ml portions into sterile 16 × 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Final pH, 7.0 ± 0.2. The medium is not sterile. Use same day as prepared. Medium 2 (North-Bartram modification) Polypeptone 5 g Lactose 4 g Sodium acid selenite (NaHSeO3) 4 g Na2HPO4 5.5 g KH2PO4 4.5 g L-Cystine 0.01 g Distilled water 1 liter Heat with agitation to dissolve. Dispense 10 ml portions into sterile 16 × 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Use same day as prepared. M37. Sheep Blood Agar Blood agar base (Oxoid No. 2) 95 ml Sterile sheep blood, defibrinated 5 ml
401Rehydrate and sterilize base as recommended by manufacturer. Agar and blood should both be at 45-46°C before blood is added and plates are poured. Commercial pre-poured sheep blood agar plates may be used. M38. Shigella Broth Base Tryptone 20 g K2HPO4 2 g KH2PO4 2 g NaCl 5 g Glucose 1 g Tween 80 1.5 ml Distilled water 1 liter Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2. Novobiocin solution. Weigh 50 mg novobiocin into 1 liter distilled water. Sterilize by filtration through 0.45 µm membrane. Add 2.5 ml concentrate to 225 ml base. M39. Simmons Citrate Agar Sodium citrate2 g NaCl 5 g K2HPO4 1 g NH4H2PO4 1 g MgSO4 0.2 g Bromthymol blue 0.08 g
402Agar 15 g Distilled water 1 liter Heat gently with occasional agitation. Boil 1-2 min until agar dissolves. Fill 13 × 100 or 16 × 150 mm screw-cap tubes ⅓ full. Autoclave 15 min at 121°C. Before medium solidifies, incline tubes to obtain 4-5 cm slants and 2-3 cm butts. Final pH, 6.8 ± 0.2. M40. Sorbitol-MacConkey Agar Peptone or gelysate 17.0 g Protease peptone No. 3 or polypeptone 3.0 g Sorbitol 10.0 g Bile salts, purified 1.5 g NaCl 5.0 g Agar 13.5 g Neutral red 0.03 g Crystal violet 0.001 g Distilled water 1 liter Dissolve ingredients in distilled water by heating with stirring. Autoclave 15 min at 121°C. Final pH, 7.1 ± 0.2. M41. Sporulation Broth (for C. perfringens) Polypeptone 15 g Yeast extract 3 g Starch, soluble 3 g MgSO4 (anhydrous) 0.1 g Sodium thioglycollate 1 g
403Na2HPO4 11 g Distilled water 1 liter Adjust pH to 7.8 ± 0.1. Dispense 15 ml portions into 20 × 150 mm screw-cap tubes. Autoclave 15 min at 121°C. M42. Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar Yeast extract 5 g NaCl 10 g Peptone 10 g Ferric citrate 1 g Sucrose 20 g Bromthymol blue O.04 g Sodium thiosulfate·5H2O 10 g Thymol blue 0.04 g Sodium citrate·2H2O 10 g Agar 15 g Sodium cholate 3 g Distilled water 1 liter Oxgall 5 g Prepare in flask at least 3 times larger than required volume of medium. Add ingredients to warm distilled water and heat to dissolve. Bring just to boil, and immediately remove from heat. DO NOT AUTOCLAVE. Cool to 50°C and pour into petri dishes. Dry the plates overnight or at 37-45°C before use. M42. Triple Sugar Iron Agar (TSI)
404Medium 1 Medium 2 Polypeptone 20 g Beef extract 3 g NaCl 5 g Yeast extract 3 g Lactose 10 g Peptone 15 g Sucrose 10 g Proteose peptone 5 g Glucose 1 g Glucose 1 g Fe(NH4)2(SO4)2·6H2O 0.2 g Lactose 10 g Na2S2O3 0.2 g Sucrose 10 g Phenol red 0.025 g FeSO4 0.2 g Agar 13 g NaCl 5 g Distilled water 1 liter Na2S2O3 0.3 g Phenol red 0.024 g Agar 12 g Distilled water 1 liter These two media are interchangeable for general use.Suspend ingredients of Medium 1 in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min to dissolve ingredients. Fill 16 × 150 mm tubes ⅓ full and cap or plug to maintain aerobic conditions. Autoclave Medium 1 for 15 min at 118°C. Prepare Medium 2 in the same manner as Medium 1, except autoclave 15 min at 121°C. Before the media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt. Final pH, 7.3 ± 0.2 for Medium 1 and 7.4 ± 0.2 for Medium 2. For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.
405M150. Trypticase Novobiocin (TN) Broth Trypticase soy broth 30 g Bile salts No. 3 1.5 g Dipotassium phosphate 1.5 g Novobiocin 20 mg Distilled water 1 liter Dissolve all ingredients except novobiocin by heating and stirring; autoclave at 121°C for 15 min. Prepare stock solution of novobiocin by adding 20 mg monosodium novobiocin per ml of distilled water. Filter-sterilize. Make fresh stock each time of use, or store frozen at -10°C in the dark (compound is light-sensitive) for not more than 1 month (half-life is several months at 4°C). Add 1 ml stock solution per liter of medium. M43. Trypticase (Tryptic) Soy Agar Trypticase peptone 15 g Phytone peptone 5 g NaCl 5 g Agar 15 g Distilled water 1 liter Heat with agitation to dissolve agar. Boil 1 min. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2. For use with halophilic Vibrio
406M44. Tryptone (Tryptophane) Broth, 1% Tryptone or trypticase 10 g Distilled water 1 liter Dissolve and dispense 5 ml portions into 16 × 125 or 16 × 150 mm test tubes. Autoclave 15 min at 121°C. Final pH, 6.9 ± 0.2. M45. Tryptone Yeast Extract Agar Tryptone 10 g Yeast extract 1 g *Carbohydrate 10 g Bromcresol purple 0.04 g Agar 2 g Distilled water 1 liter Dissolve agar with heat and gentle agitation. Adjust pH to 7.0 ± 0.2. Fill 16 × 125 mm tubes ⅔ full. Autoclave 20 min at 115°C. Before use, steam medium 10-15 min. Solidify by placing tubes in ice water. *Glucose and mannitol are the carbohydrates used for identification of S. aureus. M46. Tryptose Phosphate Broth (TPB)(for cell culture) Tryptose 20 g Dextrose 2 g NaCl 5 g Na2HPO4 2.5 g Distilled water 1 liter
407Sterilize by filtration through 0.20 µm membrane. M47. Urea Broth Urea 20 g Yeast extract 0.1 g Na2HPO4 9.5 g K2HPO4 9.1 g Phenol red 0.01 g Distilled water 1 liter Dissolve ingredients in distilled water. DO NOT HEAT. Sterilize by filtration through 0.45 µm membrane. Aseptically dispense 1.5-3.0 ml portions to 13 × 100 mm sterile test tubes. Final pH, 6.8 ± 0.2. REAGENTS R1. 4 M Ammonium Acetate Ammonium acetate 308.4 g Distilled water to make 1 liter R2. 0.25 M Ammonium Acetate Ammonium acetate 19.3 g Distilled water to make 1 liter R3. Basic Fuchsin Staining Solution
408Dissolve 0.5 g basic fuchsin dye in 20 ml 95% ethanol. Dilute to 100 ml with distilled water. Filter if necessary with Whatman No. 31 filter paper to remove any undissolved dye. (TB Carbolfuchsin ZN staining solution, available from Difco Laboratories, is satisfactory.) R4. 0.1 M Bicarbonate Buffer (pH 9.6) Na2CO3 1.59 g NaHCO3 2.93 g Distilled water 1 liter Store at room temperature for not more than 2 weeks. R5. Bovine Serum Albumin (BSA) (1 mg/ml) Nuclease-free bovine serum albumin 10 mg Distilled water 10 ml Place 0.5 ml portions into 1.5 ml plastic conical centrifuge tubes. Store frozen. R6. 1% Bovine Serum Albumin in Cholera Toxin ELISA Buffer Bovine serum albumin (BSA)1 g ELISA buffer for (cholera toxin), pH 7.4 100 ml Dissolve BSA in ELISA buffer. Aliquot and store at -20°C. R7. 1% Bovine Serum Albumin in PBS Bovine serum albumin (BSA)1 g Phosphate-buffered saline, pH 7.4 100 ml
409Dissolve BSA in PBS buffer. Aliquot and store at -20°C. R8. Brilliant Green Dye Solution. 1% Brilliant green dye 1 g Distilled water (sterile) 10 ml Dissolve 1 g dye in sterile water. Dilute to 100 ml. Before use, test all batches of dye for toxicity with known positive and negative test microorganisms. R9. Bromcresol Purple Dye Solution. 0.2% Bromcresol purple dye 0.2 g Distilled water (sterile)100 ml Dissolve 0.2 g dye in sterile water and dilute to 100 ml. R10. Bromthymol Blue Indicator. 0.04% Bromthymol blue 0.2 g 0.01 N NaOH 32 ml Dissolve bromthymol blue in NaOH. Dilute to 500 ml with distilled water. R11. Butterfield’s Phosphate-Buffered Dilution Water Stock solution KH2PO4 34 g Distilled water 500 ml
410Adjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15 min at 121°C. Store in refrigerator. Dilution blanks Take 1.25 ml of above stock solution and bring volume to 1 liter with distilled water. Dispense into bottles to 90 or 99 ± 1 ml. Sterilize 15 min at 121°C. R12. Catalase Test Pour 1 ml 3% hydrogen peroxide over growth on slant culture. Gas bubbles indicate positive test. Alternatively, emulsify colony in l drop 3% hydrogen peroxide on glass slide. Immediate bubbling is positive catalase test. If colony is taken from blood agar plate, any carry-over of red blood cells can give false-positive reaction. Rl2a. Chlorine Solution, 200 ppm, Containing Q.1% Sodium Dodecyl Sulfate Commercial bleach (5.25% sodium hypochlorite) 8 ml Distilled water containing 1 g sodium dodecyl sulfate 992 ml Dissolve 1 g sodium dodecyl sulfate in 992 ml distilled water. Add 8 ml commercial bleach and mix well. Make immediately before use. R13. 0.05 M Citric Acid (PH 4.0) Citric acid (monohydrate)10.5 g Double distilled water to make l liter Dissolve citric acid in 900 ml distilled water. Adjust pH to 4.0 with 6 M NaOH and dilute to 1 liter. Store in refrigerator.
411Rl4. Clark’s Flagellar Stain Solution A Basic fuchsin, special 1.2 g 95% ethanol 100 ml Mix and let stand overnight at room temperature. Solution B Tannic acid 3.0 g NaCl 1.5 g Distilled water 200 ml Mix solutions A and B. Adjust pH to 5.0 with 1 N NaOH or 1 N HCl, if necessary. Refrigerate 2-3 days before use. Stain is stable l month at 4°C or may be stored frozen indefinitely (50 ml portions). To use, thaw stain, remix, and store at 4°C. Optimum staining time for each batch varies 5-15 min. To determine staining time (after 2-3 days refrigeration at 4°C), stain a known flagellated organism on 3 or more cleaned slides for various times (e.g., 5, 10, 15 min). Mark best staining time on all containers. IMPORTANT: Stain will not work unless slides are clean. To clean slides, soak them 4 days at room temperature in cleaning solution (either acid dichromate or 3% concentrated HCl in 95% ethanol). Rinse 10 times in fresh tap water and twice in distilled water. Air-dry at room temperature. Store in covered container. Staining Procedure
412To prepare suspension, pick small amount of growth from 18-24 h plate (equivalent to 1 mm colony). Do not pick up agar. Suspend gently in 3 ml distilled water. (Flagella can be knocked off.) Suspension should be faintly opalescent. To prepare slide, pass cleaned slide through blue part of burner flame several times to remove residual dirt. Cool slide, flamed side up, on paper towel. Mark wax line across slide to give area 2.5 × 4.5 cm. Place large loopful of suspension in center of slide adjacent to wax line. Tilt slide, letting drop run down center of slide to end. If drop does not run evenly, slide is dirty. Discard it. Air-dry slide on level surface. R15. Coating Solution for V. vulnificus EIA Phosphate-buffered saline 100 ml Triton X-100 (a polyoxyethylene ether)20 µl Mix Triton X-100 with PBS, pH 7.4. R16. Crystal Violet Stain (for Bacteria) 1. Crystal violet in dilute alcohol Crystal violet (90% dye content) 2 g Ethanol (95%) 20 ml Distilled water 80 ml 2. Ammonium oxalate crystal violet.
413Either solution is generally considered suitable as a simple stain to observe morphology. R17. Disinfectants (for preparation of canned foods for microbiological analysis) 1. Alcoholic solution of iodine Potassium iodide 10 g Iodine 10 g Ethanol (70%) 500 ml 2. Sodium hypochlorite solution *Sodium hypochlorite 5.0-5.25 g Distilled water 100 ml Laundry bleach, which is 5.25% sodium hypochlorite (NaOCl), may be used. R18. Dulbecco’s Phosphate-Buffered Saline (DPBS) NaCl 8.0 g KCl 200 mg Na2HPO4 1.15 g KH2PO4 200 mg CaCl2 100 mg MgCl2·6H2O 100 mg Distilled water 1 liter Dissolve ingredients in water. Sterilize by filtration. Final pH, 7.2.
414R19. 0.5 M EDTA Na2EDTA 186.12 g Dissolve in 800-900 ml dH2O . Adjust pH to 8.0 with 10 N NaOH. Add distilled water to make 1 liter. R20. EIA (V. vulnificus) Wash Solution NaCl 87.65 g Tween 205.0 ml Dissolve ingredients in 10 liters of deionized water. R21. Ethanol Solution, 70% Ethanol, 95% 700 ml Distilled wateradd to final volume of 950 m. R22. Ferric Chloride. 10% FeCl3 10 g Distilled water 90 ml R23. Formalinized Physiological Saline Solution Formaldehyde solution (36-38%) 6 ml NaCl 8.5 ml Distilled water 1 liter Dissolve 8.5 g NaCl in 1 liter distilled water. Autoclave 15 min at 121°C. Cool to room temperature. Add 6 ml formaldehyde solution. Do not autoclave after addition of formaldehyde.
415R24. Gel Diffusion Agar. 1.2% NaCl 8.5 g Sodium barbital 8.0 g Merthiolate (crystalline) 0.1 g Noble special agar (Difco) 12.0 g Distilled water 1 liter Dissolve NaCl, sodium barbital, and merthiolate in 900 ml distilled water. Adjust pH to 7.4 with 1 N HCl and/or 1 N NaOH. Bring volume to l liter. Add Noble agar. Melt agar mixture in Arnold steamer. Filter in steamer, while hot, through 2 layers of analytical grade filter paper (e.g., No. 588, Schleicher and Schuell or equivalent). Dispense in small (15-25 ml) portions into 4 oz prescription bottles. Do not remelt more than twice. R25. Gel-Phosphate Buffer Gelatin 2 g Na2HPO4 4 g Distilled water 1 liter Use gentle heat to dissolve ingredients. Sterilize 20 min at 121°C. Final pH, 6.2. R26. Giemsa Stain Giemsa powder 1 g Glycerol 66 ml Methanol (absolute) 66 ml Distilled stain in glycerol by heating 1.5-2.0 h at 55-60°C. Add
416methanol. Store stain in tightly stoppered bottle at 22°C for at least 2 weeks. Dilute stock solution with distilled water (1+9) before use. R27. Glycerin-Salt Solution (Buffered) Glycerin (reagent grade) 100 ml K2HPO4 (anhydrous) 12.4 g KH2PO4 (anhydrous) 4 g NaCl 4.2 g Distilled water 900 ml Distilled NaCl and bring volume to 900 ml with water. Add glycerin and phosphates. Adjust pH to 7.2. Autoclave 15 min at 121°C. For double strength (20%) glycerin solution, use 200 ml glycerin and 800 ml distilled water. R28. Gram Stain (commercial staining solutions are satisfactory) Hucker’s crystal violet Solution A Crystal violet (90% dye content) 2 g Ethanol, 95% 20 ml Solution B Ammonium oxalate 0.8 g Distilled water 80 ml Mix solutions A and B. Store 24 h and filter through coarse filter paper.
417Gram’s iodine Iodine 1 g Potassium iodide (KI) 2 g Distilled water 300 ml Place KI in mortar, add iodine, and grind with pestle for 5-10 s. Add 1 ml water and grind; then add 5 ml of water and grind, then 10 ml and grind. Pour this solution into reagent bottle. Rinse mortar and pestle with amount of water needed to bring total volume to 300 ml. Hucker’s counterstain (stock solution) Safranin O (certified) 2.5 Ethanol, 95% 100 ml Working solution: Add 10 ml stock solution to 90 ml distilled water. Staining Procedure (Gram stain)Fix air-dried films of food sample in moderate heat. Stain films 1 min with crystal violet-ammonium oxalate solution. Wash briefly in tap water and drain. Apply Gram’s iodine for 1 min. Wash in tap water and drain. Decolorize with 95% ethanol until blue color is no longer released (about 30 s). Alternatively, flood slides with ethanol, pour off immediately, and reflood with ethanol for 10 s. Wash briefly with water, drain, and apply Hucker’s counterstain (safranin solution) for 10-30 s. Wash briefly with water, drain, blot or air-dry, and examine. R29. Endospore Stain (Schaeffer-Fulton)Solution AMalachite green10 g Distilled water 100 mlFilter to remove undissolved dye.Solution BSafranin O0.25 g Distilled water20 ml
418R30. Hippurate Solution, 1% Dissolve 0.1 g sodium hippurate in 10 ml distilled water. Filter-sterilize and store refrigerated or in 0.4 ml aliquots at -20°C. Commercial preparations are also available. R31. Horseradish Peroxidase (color development solution) Solution A (horseradish) HRP color development reagent 60 mg Ice cold methanol 20 ml Mix to dissolve. Protect from light and prepare fresh. Solution B Ice cold hydrogen peroxide, 30% 60 µl Tris-buffered saline 100 ml Prepare fresh before use. Mix ice cold solution A with room temperature solution B. Use immediately. R32. 1 N Hydrochloric Acid HCl (concentrated) 89 ml Distilled water to make 1 liter R33. Kovacs’ Reagent p-Dimethylaminobenzaldehyde 5 g
419Amyl alcohol (normal only) 75 ml HCl (concentrated) 25 ml Dissolve p-dimethylaminobenzaldehyde in normal amyl alcohol. Slowly add HCl. Store at 4°C. To test for indole, add 0.2-0.3 ml reagent to 5 ml of 24 h bacteria culture in tryptone broth. Dark red color in surface layer is positive test for indole. R34. 0.1 N Lithium Hydroxide Lithium hydroxide (anhydrous) 2.395 g Distilled water 1 liter R35. Lugol’s Iodine Solution Potassium iodide (KI)10 g Iodine 5 g Distilled water 100 ml Dissolve KI in about 20-30 ml of distilled water. Add iodine and heat gently with constant mixing until iodine is dissolved. Dilute to 100 ml with distilled water. Store in amber glass-stoppered bottle in the dark. R36. Methyl Red Indicator Methyl red 0.10 g Ethanol, 95% 300 ml Distilled water to make 500 ml Dissolve methyl red in 300 ml ethanol. Bring volume to 500 ml with distilled water.
420R37. Methylene Blue Stain (Loeffler’s) Solution A Methylene blue (90% dye content) 0.3 g Ethanol (95%) 30 ml Solution B Diluted potassium hydroxide (0.01%) 100 ml Mix solutions A and B. R38. Nitrite Detection Reagents A. Sulfanilic acid reagent Sulfanilic acid 1 g 5 N acetic acid 125 ml B. N-(l-naphthyl)ethylenediamine reagent N-(l-naphthyl)ethylenediamine dihydrochloride 0.25 g 5 N acetic acid 200 ml C. alpha-Naphthol reagent alpha-Naphthol 1 g 5 N acetic acid 200 ml To prepare 5 N acetic acid, add 28.75 ml glacial acetic acid to 71.25 ml distilled water. Store reagents in glass-stoppered brown bottles. To perform test, add 0.1-0.5 ml each of reagent A and either reagent B or reagent C (as specified in method) to culture grown in liquid or semisolid medium. Development of red-violet color with reagents A and B or orange color with reagents A and C indicates that nitrate has been reduced to nitrite. Since color produced with reagents A
421and B may fade or disappear within a few minutes, record reaction as soon as color appears. If no color develops, test for presence of nitrate by adding small amount of zinc dust. If color develops, nitrate has not been reduced. Nitrate reduction test for enteropathogenic E. coli. To 3 ml of 18-24 h culture in indole-nitrite medium, add 2 drops each of reagents A and B.Red-violet color indicates that nitrate has been reduced to nitrite. Check negative tests by adding small amount of zinc dust; if red-violet color does not appear, nitrate has been reduced. D. Alternative test reagents. 5-Amino-2-naphthylene sulfonic acid (Cleve’s acid) and N,N-dimethyl-1-naphthylamine have been recommended as substitutes for preparation of reagent B. Absolute ethanol may be substituted for acetic acid in reagent C. However, comparative evaluations should be conducted before substitution of these alternative reagents. R39. Oxidase Reagent N,N,N’,N’-Tetramethyl-p-phenylenediamine·2HCl 1 g Distilled water 100 ml This is the preferred reagent. Use freshly prepared. However, reagent can be used up to 7 days if stored in a dark glass bottle under refrigeration.Apply freshly prepared solution directly to young culture (24 h) on either agar plate or slant. Oxidase-positive colonies develop a pink color and progressively turn dark purple. If cultures are to be preserved, complete the transfer from plates to which
422reagent has been added within 3 min, since reagent is toxic to organisms. R40. Peptone Diluent, 0.1% Peptone 1 g Distilled water 1 liter Autoclave 15 min at 121°C. Final pH, 7.0 + 0.2. R41. O.01 M Phosphate-Buffered Saline (pH 7.5)Stock solution (0.1 M) Na2HPO4 (anhydrous) 12.0 g NaH2PO4·H20 2.2 g NaCl 85.0 g Distilled water 1 liter Dissolve ingredients in distilled water and bring volume to 1 liter. Dilute stock solution 1+9 in double distilled water. Mix well. Adjust pH to 7.5 with 0.1 N HCl or 0.1 N NaOH if necessary. R42. 0.02 M Phosphate Saline Buffer (PH 7.3-7.4) Prepare stock solutions of 0.2 M mono- and disodium phosphate in 8.5% salt solutions and dilute 1:10 for preparation of 0.02 M phosphate saline buffer. Stock solution 1 Sodium phosphate dibasic anhydrous Na2HPO4 (anhydrous) (reagent grade) 28.4 g
423NaCl (reagent grade) 85.0 g Distilled water to make 1 liter Stock solution 2 Sodium phosphate monobasic monohydrate NaH2PO4 H2O (monohydrate) (reagent grade)4 227.6 g NaCl (reagent grade) 85.0 g Distilled water to make 1 liter To obtain 0.02 M phosphate-buffered saline (0.85%), make 1:10 dilutions of each stock solution. For example: Stock solution 1 50 ml Stock solution 2 10 ml Distilled water 450 ml Distilled water 90 ml Approximate pH, 8.2 Approximate pH, 5.6 Using pH meter, titer diluted solution 1 to pH 7.3-7.4 by adding about 65 ml of diluted solution 2. Use resulting 0.02 M phosphate saline buffer solution in the lysostaphin susceptibility test on S. aureus. NOTE: Do not titer 0.2 M phosphate buffer to pH 7.3-7.4 and then dilute to 0.02 M strength. This results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2. R43. Physiological Saline Solution 0.85% (Sterile) NaCl 8.5 g Distilled water 1 liter
424Dissolve 8.5 g NaCl in water. Autoclave 15 min at 121°C. Cool to room temperature. R44 Potassium Hydroxide Solution. 40% KOH 40 g Distilled water to make 100 ml R45. Saline Solution. 0.5% (Sterile) NaCl 5 g Distilled water 1 liter Dissolve NaCl in water. Autoclave at 121°C for 15 min. R46. Salts-Phosphate Buffered Saline Solution (Salts-PBS) NaCl 121 g KCl 15.5 g MgCl2 12.7 g CaCl2·2H2O 10.2 g NaH2PO4·H20 2.0 g Na2HPO4·7H20 3.9 g Distilled water 1 liter Adjust pH to 7.4. R47. Slide Preserving Solution Prepare 1% acetic acid solution (10 ml glacial acetic acid, reagent grade + 990 ml distilled water). Add 1 ml glycerin to each 100 ml of solution.
425R48. Sodium Bicarbonate Solution. 10% Sodium bicarbonate 100 g Distilled water to make 1 liter Sterilize by filtration. R49. 0.2 M Sodium Chloride Solution NaCl 11.7 g Distilled water to make 1 liter Dispense in suitable containers. Autoclave 15 min at 121°C. R50. 1 N Sodium Hydroxide Solution NaOH 40 g Distilled water to make 1 liter Use for adjusting pH of culture media. R51. 10 N Sodium Hydroxide Solution NaOH 400 g Distilled water to make 1 liter R52. Standard Saline Citrate (SSC) Solution (20%) NaCl (reagent grade) 175.3 g Sodium citrate 88.2 g Dissolve in 800 ml deionized water and adjust to pH 7 with 10 N NaOH. Bring volume to 1 liter.
4266X SSC NaCl (reagent grade) 52.6 g Sodium citrate 26.5 g Dissolve in 800 ml deionized water and adjust to pH 7 with 10 N NaOH. Bring volume to 1 liter. 3X SSC 6X SSC 500 ml Deionized water 500 ml 2X SSC 6X SSC 333 ml Deionized water 667 ml R53. Tris-Buffered Saline (TBS) (PH 7.5) Tris 2.42 g NaCl 29.24 g Double distilled water to make 1 liter Dissolve ingredients. Adjust pH to 7.5 with HCl and bring volume to 1 liter. R54.Tris-Buffered Saline (TBS), with Gelatin 1% solution Gelatin 1 g TBS, pH 7.5 100 ml
4273% solution Gelatin 3 g TBS, pH 7.5 100 ml Add gelatin to TBS at 40°C. Stir to dissolve. Cool to room temperature before use. R55. Tris-Buffered Saline (TBS)-Tween Tween 2050 F1TBS, pH 7.5 100 ml Dissolve Tween 20 in TBS. R56. Tris-Buffered Saline (TABS), 1% or 3% Gelatin, or Tween 20 Tris 2.42 g NaCl 29.24 g Distilled water 1 liter Dissolve ingredients in distilled water by heating and stirring. Adjust pH to 7.5 with HC1. Autoclave 15 min at 121°C. For 1% and 3% Gelatin-TABS, add 10 g and 30 g gelatin, respectively, to ingredients before autoclaving. Adjust final pH to 7.5 with HCl. For Tween-TABS, add 0.5 ml Tween 20 to ingredient and adjust pH to 7.5 before autoclaving.
428R57. Voges-Proskauer (VP) Test Reagents Solution 1 alpha-Naphthol 5 g Alcohol (absolute)100 ml Solution 2 Potassium hydroxide 40 g Distilled water to make 100 ml Voges-Proskauer (VP) test. Transfer 1 ml of 48 h culture to test tube and add 0.6 ml solution 1 and 0.2 ml solution. Shake after adding each solution. To intensify and speed reaction, add a few creatine crystals to mixture. Let stand at room temperature. Read results 4 h after adding reagents. Development of eosin pink color is positive test. GLOSSARY Asexual reproduction: Reproduction in which sex cells are not involved; as by binary fission or budding. Acute: Having rapid onset, severe symptoms and a short course. Antigen: Foreign substance when gets into the body induces immune response. Antibody: Endogenous glycoprotein, which reacts with antigen. Blood Brain Barrier: Chronic: Of long duration; denoting a disease with slow progression.
429Convulsion: Paroxysms of involuntary muscular contraction and relaxation. Congenital: Present at birth. Dehydration: a condition resulting from loss of excessive body fluid. Disease: Pathological condition of the body that presents with group of clinical symptoms and signs; and abnormal laboratory findings. DNA: A nucleic acid consisting of deoxyribose, phosphoric acid and bases. It is present in chromosomes of the nuclei of cells, is the chemical basisof heredity and the carrier of genetic information for living cells. Endogenous: Produced or originating from with in a cell or organism. Endoplasmic reticulum: Net work of membraneous tubules with in a cell and involved in transport of proteins synthesized on the ribosomes; and synthesis of lipids. Electrolyte: An ionized salt in blood, tissue fluids and cells. Fastidious: Requiring precise nutritional and environmental conditions for growth and survival. Genome: Hematogenous: Through the blood stream. Histone: Positively charged protein that is part of chromatin in eukaryotic cells. Hydrocephalus: Excessive fluid in the brain ventricles. Iatrogenic: Any adverse mental or physical condition induced in a patient through the effects of treatment by a physician or surgeon. Incubation Period: The time interval between exposure and development of disease. Infertility: The inability or diminished ability to produce offspring.
430Lysosome: Cell organelle that is part of the intracellular digestive system. Microscopic: Can not be observed with naked eye. Macroscopic: Can be observed with naked eye. Microscope: Optical instrument that greatly magnifies minute objects. Microorganism: Minute living body not seen with naked eye. Mitochondria: Oval shaped cell organelles that contain the enzymes for aerobic stages of cell respiration and thus the site of ATP synthesis. Microtubule/Microfilament: Tubular structures present in an eukaryotic cell and are important for maintaining rigidity; transporting substances in different directions with in a cell. Nuclear membrane: A membrane enveloping nucleus of a living cell. Nucleolus: Structure in the nucleus of a cell made of DNA, RNA, and protein. It is the site of synthesis of ribosomal RNA(rRNA). Purulent: Full of pus Postulate: A supposition or view, usually self-evident that is assumed with out proof. Pleural effusion: Fluid accumulated in pleural cavity. Primary stain: The dye applied first in differential staining procedures. Counter stain: The dye which stains the micro-organism or part of it after decolorization of the primary stain.
431Mordant: It is a substance which facilitates the reaction of the primary stain with the material to be stained. It combines with the stain and then facilitate the reaction. Basic mordant reacts with acidic stain and acidic mordant react swith basic stain. Decolorizer : It is a chemical added in differential staining procedure to selectively remove the stain from the materials that are not intended to be stained. Pathogen : Organism that causes disease Virulence : Degree of pathogenicity in causing disease which depends on toxin production and invasiveness. Invasiveness : The ability to penetrate in to the tissues, overcome the host defense, multiply and disseminate widely. Toxicity: The capacity to damage the tissues. Opportunistic : Normally harmless organism causing disease during lowered host resistance. Infection: The result of breakdown in the host-parasite relationship and follows when the balance is tipped in favor of the parasite.
432 REFERENCES 1. Monica Cheesbrough. Medical Laboratory Manual for Tropical Countries, Microbiology, volume II, First edition. Tropical Health Technology and Butter Worth-Heinemannith, 1984. 2. Geo.F. Brooks, Janet s. Butel, Staphen A. Morse. Jawetz, Malnick and Adelberg’s Medical Microbiology. 21st edition. Appelton & Langh,1998.
- T.D. Sleight, M.C. Murphy. Notes on Medical bacteriology, 2nd edition. Churchill livingstone, Medical division of Longman group UK limited, 1986. 4. Rajesh Bhatia, Rattan Lal Ichhpujmai, Essentials of Medial Microbiology, 1st edition. Jaypee brothers Medical Publishers Ltd. 1994. 5. Cole and cox(1981). Handbook of Toxic fungi Metabolite Academic press, inc. New York 6. Salle(1981). Fundamental principles of bacteriology, TaTa McGraw – Hill publishing Company Ltd, New Dalhi 7. Mackie and McCartney(1989). Practical medical microbiology 13th edition. Churchill Livingston 8. Bernand D.Davis, Renanto Dulbecco, Herman N.Eisen and Harold S.Ginsberg(1990). Microbiology 4th edition. Lipinocott Company.