Clinical Bacteriology MT 6320 1st Term PDF

[HANDOUT] ORDER ENTEROBACTERELES ORDER ENTEROBACTERELES CULTURE MEDIUM EOSIN METHYLENE BLUE MACCONKEY AGAR HEKTOEN ENTERIC AGAR PUR...

[HANDOUT] ORDER ENTEROBACTERELES ORDER ENTEROBACTERELES CULTURE MEDIUM EOSIN METHYLENE BLUE MACCONKEY AGAR HEKTOEN ENTERIC AGAR PURPOSE Gram Negative Enterics MEDIA TYPE Selective & Differential Bile Salts Bile Salts INHIBITORS Eosin Crystal Violet Sodium Citrate Methylene Blue pH INDICATORS Neutral Red Bromthymol Blue H2S INDICATOR & Ferric Ammonium Citrate w/ Sodium Thiosulfate SOURCE OF SULFURE Lactose CARBOHYDRATE Lactose Sucrose FERMENTED Salicin COLOR OF FERMENTER Pink – Purple Red Carrot Orange COLOR OF Colorless Yellow-Colorless Blue to Green NONFERMENTER AUTOCLAVE Yes No Pink-purple w/ green E. coli Shigella dysenteriae Blue-green w/o black center metallic sheen Pink-purple mucoid w/ or Enterobacter Salmonella typhi Blue-green w/ black center ADDTL INFO w/o dark centers (fish-eye) ALL ENTERICS grow in this medium Carrot orange w/ black Large pink-purple mucoid LF H2S (+) center Klebsiella colonies w/ or w/o dark Carrot orange w/o black center LF H2S (-) center; usu PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  TRANS: ORDER ENTEROBACTERELES Normal flora; except Y. enterocolitica (sucrose fermenter) (true pathogen) CULTURE MEDIUM SALMONELLA SHIGELLA AGAR XYLOSE LYSINE DESOXYCHOLATE AGAR BISMUTH SULFITE AGAR PURPOSE Differentiates Salmonella from Shigella Gram Negative Enterics For Salmonella typhi MEDIA TYPE Highly Selective & Differential Selective & Differential Selective Bile Salts Desoxycholate Bile Salts Brilliant Green INHIBITORS Brilliant Green Citrate Bismuth Sulfite Sodium Citrate pH INDICATORS Neutral Red Phenol Red H2S INDICATOR & Ferric Citrate w/ Sodium Thiosulfate Ferric Ammonium Citrate w/ Sodium Thiosulfate Ferrous sulfate w/ Peptone & Sulfite SOURCE OF SULFURE CARBOHYDRATE Lactose, Sucrose Lactose Glucose FERMENTED Xylose COLOR OF FERMENTER Red Yellow Black w/ metallic sheen COLOR OF Yellow Red NONFERMENTER AUTOCLAVE No S. arizonae Lysine added as a mechanism to differentiate Red w/ black center (LF) Salmonella NXF LDC (-) & H2S (-) Shigella S. typhi (NLF Yellow w/ black center XF LDC (+) & H2S Red colonies ADDTL INFO Uninhibited organisms appear dark green (+) Shigella XF LDC (-) H2S (-) XF LDC (+) H2S (+) Red w/ black center Shigella Salmonella Red w/o black center sonei (LF) XF LDC (-) H2S (+) Citrobacter PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  TRANS: ORDER ENTEROBACTERELES VIBRIONACEAE (Vibrio) THIOSULFATE CITRATE BLUE SALTS SUCROSE AGAR Vibrio species PURPOSE CARBOHYDRATE FERMENTED Sucrose Gr (-) Non Enteric MEDIA TYPE Selective & Differential COLOR OF FERMENTER Yellow H2S INDICATOR & SOURCE OF None COLOR OF NONFERMENTER Green SULFURE pH INDICATORS Bromthymol Blue AUTOCLAVE No SF V. cholerae, V. alginolyticus Thiosulfate Citrate INHIBITORS ADDTL INFO NSF V. parahaemolyticus Bile Salts High pH Variable V. vulnificus BIOCHEMICAL TESTS MEDIA TRIPLE SUGAR IRON AGAR LYSINE IRON AGAR 10 parts (1%) Lactose Lysine 10 parts (1%) Sucrose COMPOSITION Peptone 1 part (0.1%) Glucose Glucose (0.1%) 2% Peptone (abundant) pH INDICATOR Phenol Red Bromcresol Purple Ferrous Sulfate Ferric NH4 Citrate H2S INDICATOR Sodium Thiosulfate Sodium Thiosulfate GAS PROD’N Yes Variable / None Detection if Gr (-) rod is able to ferment glucose and lactose or sucrose Ability to deaminate lysine, decarboxylate lysine H2S production H2S production (weak) Gas production Deamination : Aerobically Decarboxylation : Anaerobically PRINCIPLE Useful to ID Salmonella, Proteus, Providencia, Morganella 1. Whole medium turns yellow in 8-12 hrs Lysine decarboxylation > Prod’n of cadaverine making butt purple 2. Slant becomes red (oxidation of fermentation products) Deamination > Acetoacetic acid product > Burgundy red in the presence of H2S 3. If lactose / sucrose are fermented, overcomes the oxidation indicator & coenzyme FMN (alkalinity) of slant making the slant yellow PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  TRANS: ORDER ENTEROBACTERELES UNINOCULATED Red Purple Yellow ACIDIC Yellow GLc (Fermentation Purple ALKALINE Red Red K/K Escherichia E.coli DA (-) / LDC (+) Klebsiella Enterobacter aerogenes A/AG or A/A GLc Fermenter Enterobacter Klebsiella H2S (-) Serratia; C. diversus Serratia & Plesiomonas R/A DA (+) / LDC (-) Proteus Salmonella arizonae A/AG+ or A/A Gic Fermenter Providencia* Citrobacter freundii H2S (-) Morganella* K/K (+) Pseudomonas* DA (-) / LDC (+) K/K Salmonella Alcaligenes* GLc Fermenter Edwardsiella H2S (+) RESULT K/A Shigella sonnei A/A Y. enterocolitica E.sakazakii Shigella; Plesiomonas DA (-) K/A E.cloacae Providencia LDC (-) Y. enterocolitica GLc Fermenter Shigella Morganella H2S (+) K/AG or K/A Citrobacter koseri Providencia Salmonella typhi K/A+ K/A+ Edwardiella DA (-) LDC (-) Citrobacter freundii Salmonella GLc Fermenter S. paratyphii K/AG+ or K/A+ Proteus H2S (+) Inoculate: Stab-Streak ADDTL INFO H2S prod’n doesn’t occur in Proteus Incubate & Read w/in: 18-24 hrs PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  BIOCHEMICAL TESTS TESTS OXIDASE TEST UREASE NITRATE RED’N SIM Oxidase Rgt Nutrient Broth w/ 0.1% KNO3 Kovac’s Method Christensen’s Urea Agar Sulfide Infole Motility Agar MEDIUM Rgt : N,N-dimethyl-alpha- 0.5 – 1% Tetramethyl-P- Stuart’s Urea Broth Kovac’s Rgt (PDAB) napthylamine & sulfanilic acid Phenylenediamine (diHCl) pH INDICATOR Phenol Red Semi-solid medium Detects: Determines if organism is capable of Detects presence of Ability to reduce nitrate to nitrite 1. H2S prod’n (sodium thiosulfate & PRINCIPLE hydrolyzing urea into alkaline end cytochrome C and further to nitrogen gas peptonized iron) products 2. Indole protein 3. Motility UNINOCULATED Yellow Clear / Yellow Yellow Rapid : Red AGAR throughout medium H2S (+) : Blackening Late : Red in slant Indole : Red ring RESULT Purple Red Motility : Growth away from Rapid : Red inoculation BROTH Late / Weak : Pink- Orange H2S, Indole, Mot Proteus, Providencia, Proteus vulgaris RAPID (+) Morganella REPRESENTATIVE H2S (-), In (+), Plesiomonas Most Enterics E. coli ORGANISM Motile Citrobacter, Klebsiella, LATE Enterobacter, Yersinia, H2S (-), In (-), K. pneumoniae Serratia NonMot S. sonnei ALL ENTERICS are Oxidase (-) Rapid : 2-4 hrs (+) ADDTL INFO except Plesiomonas Late : 24 hrs (+) *Not an Enteric PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  BIOCHEMICAL TESTS TESTS ONPG DNASE GELATINASE DECARBOXYLASE DEAMINASE Moeller’s Decarboxylase Broth containing: Phenylalanine / ONPG Disk or Tablet (ortho- DNAse test medium o 1% Lysine Tryptophan Ager or MEDIUM nitrophenyl-beta-D- containing 0.2% DNA 12% Gelatin o 1% Ornithine Tablets galactopyranoside) 1N HCl Rgt o 1% Arginine 10% Ferric Chloride Sterile Mineral Oil pH INDICATOR Bromcresol Purple Detection of decarboxylase (LDC, ODC, ADH) Determines if it is a late Detects production of Decarboxylases : lactose fermenter or true extracellular DNAse that Attack carboxyl grp of non fermenter hydrolyze DNA specific amino acids to Determine if organism Differentiates Salmonella Heavy inoculum Ability to liquify / gelling form amins & CO2 can oxidatively PRINCIPLE from Citrobacter inoculated to agar of gelatin 1. Lysine >> Cadaverine deaminate phenylalanine Beta-galactosidase Incubate for 18-24 hrs Presence of gelatin (Alk) / tryptophan converts ONPG to Add 1N HCl galactose & o- Unhydrolyzed DNA : Ins 2. Ornithine > Putrescine nitrophenyl to HCl (Alk) 3. Arginine >> Citruline > Ornithine > Putrescine (Alk) UNINOCULATED Colorless (+) Green (PAD) RESULT Yellow w/in 20 mins / 24 hrs Soluble, Clear Zone Gelling / Liquefy Gelatin Purple (+) Brown (Tryptophan Deaminase) K. pneumoniae LDC (+) K. oxytoca ALL LACTOSE E. aerogenes Serratia Proteus REPRESENTATIVE FERMENTERS are (+) Serratia Salmonella Proteus vulgaris & P. mirabilis PAD (+) Providencia ORGANISM Some NLF may be (+) Pantoea Pseudomonas* E. aerogenes Morganella o E. taylorae ODC (+) E. cloacae ADH (+) E. cloacae Decarboxylation Add 10% Ferric Chloride happens in anaerobic after incubation condition and if acidic ADDTL INFO Interpret after refrigeration Table dissolve in 1 mL (initial) sterile water Differentiates Klebsiella Aerobic condition from Enterobacter PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  TEST MUG TEST KCN TEST MEDIUM Substrate : 4-methylumbelliferyl-Beta-d-glucoronide impregnated into a disk KCN Broth Used to presumptively identify E. coli and verotoxin producing E. coli PRINCIPLE Detection of enzyme beta-d-glucoronidase (+) Electric Blue Fluorescence RESULT (-) No Fluorescence Use 366 nm UV light REPRESENTATIVE (+) E. coli (+) Growth, Turbidity ORGANISM (-) Other Enterics – Klebsiella, Verotoxin E. coli ADDTL INFO Can be used for Water testing alongside MPN method PPM, Citrobacter, Klebsiella, Enterobacter, Serratia, Hafnia BIOCHEMICAL TEST: IMVIC TEST VOGUS PROSKAUER (BARRITT’S TEST INDOLE TEST METHYL RED CITRATE UTILIZATION METHOD – GRAM (-) BACILLI) Tryptophan Broth Kovac’s Rgt (para-dimethyl amino benzaldehyde) MRVP Broth (dextrose broth w/ Simmon’s Citrate Agar MEDIUM 2 Methods: peptone) pH Indicator : Bromthymol Ehrlich’s Indole Test for Methyl Red Indicator Rgt Blue anaerobes & nonfermenters Kovac’s Uses mixed acids pathway for Determines if enteric organism PRINCIPLE Detection of tryptophanase fermenting glucose utilizes citrate as sole source of pH 4.4 or less carbon UNINOCULATED Colorless / Yellow Green RESULT Red Ring Red Coloration Deep Blue E. coli Shigella Proteus vulgarise Serratia Klebsiella Salmonella Citrobacter koseri Hafnia Enterobacter Proteus REPRESENTATIVE ORGANISM E. coli Enterobacter Citrobacter Providencia Providencia rettgeri Klebsiella Providencia Morganella Morganella Pantoea Most Salmonella Citrobacter Edwardsiella Add PDAB, xylene / Add rgt after incubation chloroform after incubation Add rgt after incubation 1st : Add alpha napthol rgt then Ehrlich Test : Add 1 mL xylene MR result is reciprocal of VP shake vigorously ADDTL INFO / chloroform shake, then add Inoculate by Streaking (vice-versa) i.e. if MR (+) then 2nd : Add 40% KOH PDAB VP (+) Gently shake to expose to air Kovac’s : No need to add Oxygen oxidizes acetoin xylene; add PDAB PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  Enterobacter Enterobacter Pantoea TEST E. coli K. pneumoniae K. oxytoca Edwardsiella tarda aerogenes cloacae agglomerans INDOLE + - + - - + MR + - V - - + V VP - + + + + - CITRATE - + + + + - LDC + + + + - - + ODC + - - + + - + ADH - - - - + - - MOTILITY + - - + + + + H2S - - - - - - POS (large amt) TEST K. pneumoniae K. oxytoca K. aerogenes Enterobacter cloacae Pantoea agglomerans LDC + + + - - ODC - - + + - ADH - - - + - TEST P. vulgaris P. mirabilis P. penneri INDOLE + - - MR + + + VP - V - CITRATE V V - ODC - + - DEAMINASE + + - H2S + + - TEST P. rettgeri P. stuartii Morganella morganii INDOLE + + + MR + + + VP - - - CITRATE + + - LDC - - - ODC - - + ADH - - - DEAMINASE + + + PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  TEST Salmonella enteriditis Salmonella typhi Citrobacter freundii C. koseri INDOLE - - - + MR + + + + VP - - - - CITRATE + + - + LDC + + - - ODC + - - + ADH V - V + H2S + + + - TEST Serratia marcescens S. liquefaciens INDOLE - - MR V + VP + + CITRATE + + DNASE + + LDC + + ODC + + ADH - - GELATINASE - + PRODIGIOSIN + + TEST Shigella dysenteriae Shigella flexneri Shigella boydii Shigella sonnei GRP A B C D INDOLE V V V - MR + + + + VP - - - - CITRATE - - - - LDC - - - - ODC - - - + ADH - - - - CATALSE - + + + ONPG - - - + MANNITOL - + + + PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION  TEST Yersinia pestis Yersinia enterocolitica Yersinia pseudotuberculosis Plesiomonas shigelloides INDOLE - V - + MR + + + V VP - - - - CITRATE - - - - MOT37C - - - + MOT25C - + + + UREASE - + + - SUCROSE - + - - ONPG + + + Oxidase (+) NONFERMENTERS P. aeruginosa Acinetibacter baumanni Streptrophomonas maltophilia Burkholderia cepacia TSI RXN K/K LDC - - + + OXIDASE + - - + GLUCOSE + V + + OXIDATION XYLOSE + - + V OF MALTOSE - + - V GROWTH AT 42C Yes Var Yes Var MOTILE Yes No Yes PIGMENT PYOCYANIN V - - - PROD FLUORESCEIN + - - ON MAC Colorless Colorless (purplish) Colorless (bluish) Colorless DNASE - - + - NONFERMENTERS Plesiomonas shigelloides Vibrio Aeromonas OXIDASE + + + INOSITOL + - - O/120 Sensitive Resistant TCBS No Growth Growth No Growth GLUCOSE + + + GELATINASE - + + 6.5% NACL - + - 0% NACL + Mostly (-) + PREPARED BY : SIR ARF; TRANSCRIBED BY : RECCION | 3CMT; FORMATTED : ENCARNACION