Kligler Iron Agar

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282 Section III H - K 6.Submerge a KL antitoxin strip or equivalent beneath the agar prior to solidification. 7.Test samples of the finished product for performance using stable, typical control cultures. Procedure Process the specimen according to accepted practices.

4 Inocu- late the complete medium by streaking a loopful of a 24-hour culture in a single line across the plate perpendicular to (right angle to) the antitoxin strip. (Do not touch the actual strip itself.) As many as eight cultures may be tested on a single plate. 5 Place test isolates about 1 cm apart.

Also inoculate a toxigenic (positive control) and a nontoxigenic (negative con- trol) C. diphtheriae strain approximately 1 cm on either side of the test isolates. 5 Incubate the inverted plates under CO 2 at 37 ° C for 72 hours.

Examine at 24-, 48- and 72-hour intervals. Expected Results Toxigenic (virulent) cultures of C. diphtheriae will show fine lines of precipitation at approximately 45 ° angles from the culture streak.

This line forms where toxin (from the bacteria) combines with antitoxin from the strip. Primary precipitin lines form an arc of identity with the precipitin line produced by an adjacent positive control strain. 6 Nontoxigenic strains of C.

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will show no lines of precipitation. Limitations of the Procedure 1.False-positive reactions may be seen after 24 hours as weak bands near the antitoxin strip. These can be recognized when compared with the positive control. 7 2. Corynebacterium ulcerans and C. pseudotuberculosis may also produce lines of toxin-antitoxin. 8 References 1.Elek. 1948. Br. Med. J. 1 :493. 2.King, Frobisher and Parsons. 1949. Am. J. Public Health 39 :1314. 3.Hermann, Moore and Parsons. 1958. Am. J. Clin. Pathol. 29 :181. 4.Funke and Bernard. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 5.MacFaddin.1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins, Baltimore, Md. 6.Washington. 1981. Laboratory procedures in clinical microbiology. Springer-Verlag, New York, N.Y. 7.Lennette, Balows, Hausler and Truant (ed.). 1980. Manual of clinical microbiology, 3rd ed. Ameri- can Society for Microbiology, Washington, D.C. 8.Branson. 1972. Methods in clinical bacteriology. Charles C. Thomas, Springfield, Ill. Availability Difco " KL Virulence Agar Cat. No.212192Dehydrated 3 500 g Difco " KL Virulence Enrichment Cat. No.298610Tube 3 12 × 20 mL* BBL " Taxo " KL Antitoxin Strips Cat. No.231740Vial 3 12 strips* BBL " Tellurite Solution 1% Cat. No.211917Tube 3 20 mL *Store at 2-8 ° C. Kligler Iron Agar Intended Use Kligler Iron Agar is used for the differentiation of members of the Enterobacteriaceae on the basis of their ability to ferment dextrose and lactose and to liberate sulfides. Summary and Explanation In 1911, Russell described a new double sugar tube medium for the isolation of typhoid bacilli from urine and feces. 1 Six years later, Kligler developed a simple lead acetate medium for the differentiation of the typhoid-paratyphoid group. 2 Subse- quently, Kligler evaluated culture media used in the isolation and differentiation of typhoid, dysentery and allied bacilli and endorsed Russell 9s medium. 3 Bailey and Lacey substituted phe- nol red for the Andrade indicator previously used as a pH indicator. 4 The current formulation of Kligler Iron Agar combines fea- tures of Kligler 9s lead acetate medium with those of Russell 9s double sugar agar. Principles of the Procedure Kligler Iron Agar, in addition to casein and meat peptones, contains lactose and dextrose which enable the differentiation of species of enteric bacilli due to color changes of the phenol red pH indicator in response to the acid produced during the fermentation of these sugars. The dextrose concentration is only 10% of the lactose concentration. The combination of ferric ammonium citrate and sodium thiosulfate enables the detection of hydrogen sulfide production. Lactose nonfermenters (e.g., Salmonella and Shigella ) initially produce a yellow slant due to acid produced by the fermenta- tion of the small amount of dextrose. When the dextrose sup- ply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline (red slant) due to oxidation of the acids. The reversion does not occur in the anaerobic environ- ment in the butt, which remains acid (yellow butt). Lactose fermenters produce yellow slants and butts because enough acid is produced in the slant to maintain an acid pH under aerobic conditions. Organisms incapable of fermenting either carbohydrate produce red slants and butts. Hydrogen sulfide production is evidenced by a black color either throughout the butt, or in a ring formation near the top of the butt. Gas production (aerogenic reaction) is detected as individual bubbles or by splitting or displacement of the agar. KL Virulence Agar, cont. 283 H - K User Quality Control Identity Specifications BBL " Kligler Iron Agar Dehydrated Appearance:Fine, homogeneous, free of extraneous material. Solution:5.2% solution, soluble in purified water upon boiling. Solution is medium to dark, orange to red, with or without a tint of brown, clear to slightly hazy. Prepared Appearance:Medium to dark, orange to red, with or without a tint of brown, clear to slightly hazy. Reaction of 5.2% Solution at 25 ° C:pH 7.4 ± 0.2 Cultural Response BBL " Kligler Iron Agar Prepare the medium per label directions. Stab inoculate with fresh cultures and incubate at 35 ± 2 ° C for 24 hours. Formula BBL " Kligler Iron Agar Approximate Formula* Per Liter Pancreatic Digest of Casein......................................10.0g Peptic Digest of Animal Tissue.................................10.0g Lactose.....................................................................10.0g Dextrose.....................................................................1.0g Sodium Chloride........................................................5.0g Ferric Ammonium Citrate...........................................0.5g Sodium Thiosulfate....................................................0.5g Agar.........................................................................15.0g Phenol Red...............................................................25.0mg *Adjusted and/or supplemented as required to meet performance criteria. Directions for Preparation from Dehydrated Product 1.Suspend 52 g of the powder in 1 L of purified water. Mix thoroughly. 2.Heat with frequent agitation and boil for 1 minute to com- pletely dissolve the powder. 3.Dispense and autoclave at 121 ° C for 15 minutes. 4.Cool in a slanted position such that deep butts are formed. For best results, the medium should be used on the date of preparation or melted and resolidified before use. 5.Test samples of the finished product for performance using stable, typical control cultures. Procedure To inoculate, carefully touch the center of an isolated colony on an enteric plated medium with a cool, sterile needle, stab into the medium in the butt of the tube, and then streak back and forth along the surface of the slant. Several colonies from each primary plate should be studied separately, since mixed infections may occur. Incubate tubes with loosened caps for 18-24 hours at 35 ± 2 ° C in an aerobic atmosphere. To enhance the alkaline condition in the slant, free exchange of air must be permitted through the use of a loose closure. If the tube is tightly closed, an acid reaction (caused solely by dextrose fermentation) will also involve the slant. Expected Results After incubation, record the reaction in the slant and butt, noting gas formation and hydrogen sulfide production. Typical reactions produced by members of the Enterobacteri- aceae (majority of the species in the particular genus) are presented in the following table. 5 Uninoculated Tube ORGANISMATCC " RECOVERYSLANTBUTTH 2 S Escherichia coli 25922GoodAcidAcid with gas 3 Morganella morganii 8019GoodAlkalineAcid with 3 or without gas Pseudomonas aeruginosa 27853GoodAlkalineAlkaline 3 without gas Salmonella choleraesuis subsp. choleraesuis serotype Typhi19430GoodAlkalineAcid without gas+ Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium14028GoodAlkalineAcid with gas+ Shigella flexneri 12022GoodAlkalineAcid without gas 3 Kligler Iron Agar, cont. Echerichia coli ATCC " 25922 Morganella morganii ATCC " 8019 Salmonella typhimurium ATCC " 14028 284 Section III H - K Intended Use Koser Citrate Medium is used for differentiating Escherichia coli from Enterobacter aerogenes based on citrate utilization. Summary and Explanation In 1923, the work of Koser demonstrated that coli-aerogenes bacteria could be differentiated by their use of certain salts of organic acids. 1 Koser found that the sodium salt of citric acid (sodium citrate) is used as a source of carbon by E. aerogenes and not by E. coli . Biochemical identification schemes for iden- tifying E. coli frequently include Koser citrate. E. coli is an important member of the coliform group of bacte- ria. The coliforms are described as aerobic and facultatively anaerobic gram-negative non-sporeforming bacilli that ferment lactose and form acid and gas at 35 ° C within 48 hours. Proce- dures to detect, enumerate and presumptively identify coliforms are used in testing foods and dairy products. 2-5 Presumptive identification is confirmed by performing biochemical tests that specifically identify E. coli . Principles of the Procedure Koser Citrate Medium is prepared with chemically pure salts and tested to determine that no sources of carbon (other than sodium citrate) or nitrogen (other than ammonium salts) are present. Bacteria that are able to use citrate as their carbon source will grow in the medium and cause turbidity. Formula Difco " Koser Citrate Medium Approximate Formula* Per Liter Sodium Ammonium Phosphate..................................1.5g Monopotassium Phosphate........................................1.0g Magnesium Sulfate....................................................0.2g Sodium Citrate...........................................................3.0g *Adjusted and/or supplemented as required to meet performance criteria. Directions for Preparation from Dehydrated Product 1.Dissolve 5.7 g of the powder in 1 L of purified water. 2.Autoclave at 121 ° C for 15 minutes. 3.Test samples of the finished product for performance using stable, typical control cultures. Procedure 1.Transfer growth from a single colony or a loopful of liquid suspension and inoculate the broth medium. 2.Incubate at 35 ± 2 ° C for 18-24 hours. Expected Results Positive:................Turbidity Negative:..............Clear, no turbidity References 1.Koser. 1923. J. Bacteriol. 8 :493. 2.Marshall (ed.). 1993. Standard methods for the microbiological examination of dairy products, 16th ed. American Public Health Association, Washington, D.C. 3.U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC Inter- national, Gaithersburg, Md. 4.Horwitz (ed.). 2000. Official methods of analysis of AOAC International, 17th ed. AOAC Interna- tional, Gaithersburg, Md. 5.Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods, 4th ed. American Public Health Association, Washington, D.C. Availability Difco " Koser Citrate Medium AOAC BAM COMPF SMD Cat. No.215100Dehydrated 3 500 g Koser Citrate Medium SLANTBUTTGASH 2 S Citrobacter AlkalineAcid++ or 3 Edwardsiella AlkalineAcid++ Escherichia coli AcidAcid+ 3 Enterobacter Acid*Acid+ 3 Morganella AlkalineAcid ± 3 Proteus Alkaline or AcidAcid++ Providencia AlkalineAcid ± 3 Salmonella AlkalineAcid++ Shigella AlkalineAcid 3 3 *May revert to alkaline even though lactose fermented ( E. aerogenes ). References 1.Russell. 1911. J. Med. Res. 25 :217. 2.Kligler. 1917. Am. J. Public Health. 7 :1041. 3.Kligler. 1918. J. Exp. Med. 28 :319. 4.Bailey and Lacy. 1927. J. Bacteriol. 13:183. 5.Ewing. 1986. Edwards and Ewing 9s identification of the Enterobacteriaceae , 4th ed. Elsevier Sci- ence Publishing Co., Inc. New York, N.Y. Availability BBL " Kligler Iron Agar BAM CMPH COMPF ISO MCM7 Cat. No.211317Dehydrated 3 500 g 220896Prepared Slants 3 Pkg. of 10* 220897Prepared Slants 3 Ctn. of 100* *Store at 2-8 ° C. User Quality Control Identity Specifications Difco " Koser Citrate Medium Dehydrated Appearance:White, free-flowing, homogeneous. Solution:0.57% solution, soluble in purified water. Solution is colorless, clear. Prepared Appearance:Colorless, clear. Reaction of 0.57% Solution at 25 ° C:pH 6.7 ± 0.2 Cultural Response Difco " Koser Citrate Medium Prepare the medium per label directions. Inoculate and incubate at 35 ± 2 ° C for 18-24 hours. ORGANISMATCC " INOCULUM CFURECOVERY Enterobacter aerogenes 1304810 3 Good Escherichia coli 2592210 3 Marked to complete inhibition Kligler Iron Agar, cont.

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