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Culture Media Purpose Inhibiting Substance Sugar/supplement indicator Bacterial Growth A.Simple / General Nutrient agar for cultivation of bacteria...

Culture Media Purpose Inhibiting Substance Sugar/supplement indicator Bacterial Growth A.Simple / General Nutrient agar for cultivation of bacteria none none none growth Nutrient broth for cultivation of bacteria none none none turbidity B.Enriched / Enrichment Blood Agar for detection of hemolysis and cultivation of fastidious none 5-7 % blood none Alpha, Beta, Gamma organism Hemolysis Chocolate Agar For use in the isolation and cultivation of fastidious none Heated 5-7% human none Growth microorganisms, blood or fresh 10% particularly Haemophilus and Neisseria species sheep’s blood Selenite F broth for isolation of Salmonella selenium lactose none turbidity Tetrathionate broth for isolation of Salmonella Tetrathionate, bile salt none turbidity none Staphylococci Enrichment broth selective and enrichement.coag.+ tellurite 6.5%NaCl, mannitol none Staphylococci- black colonies Azide Dextrose broth for cultivation of common aerobe ascorbic acid dextrose none turbidity Thioglycollate broth for cultivation of aerobic anaerobic and - - - - microaerophilic organism Enriched Isolation, cultivation and identification of a wide variety of Sodium thioglycollate Casein enzymatic Methylene Blue Obligate aerobes Thioglycollate broth obligate anaerobic bacteria hydrolysate and Microaerophiles Facultative papaic digest of or aerotolerant anaerobes soyabean meal Trypticase Soy broth Used for subculturing various bacteria for primary agar NaCl glucose Phosphoric acid - plates Regan Lowe Charcoal Agar for the isolation of Bordetella pertusis Cefalexin/Cephalexin Defibrinated Horse None B. pertusis – small, gray, and Amphotericin B blood and Beef shiny convex, smooth and Extract raised with a pearl-like somewhat mercury droplet appearance New York City (NYC) medium is primarily designed for the isolation of Vancomycin Lysed horse blood, none - pathogenic Neisseria. It also supports the growth of genital Colistin horse serum, mycoplasmas (Mycoplasma hominis and Ureaplasma Nystatin peptone urealyticum). Trimethoprim cornstarch transparent medium useful in the diagnosis of gonorrhea glucose and in the recognition of active or asymptomatic mycoplasma infections. TODD HEWIT BROTH Production of antigenic streptococcal hemolysis. Gentamicin and - - nalidixic Boric Acid broth For the detection and presumptive identification of Boric acid Escherichia coli on the basis of this organism to grow at 43°c and form gas in the presence of boric acid Brain Heart infusion Agar/broth medium rich in nutrients, suitable for the cultivation of none infusion from calf none Colonies ( agar ) several fastidious strains of bacteria, fungi, and yeasts. brain Turbidity ( broth) used for the cultivation of a wide variety of fastidious microorganisms such as streptococci, meningococci and pneumococci. C. Differential/Selective. - Mac Conkey Agar isolation and differentiation of Gram(-) enteric bacteria crystal violet/bile salt lactose neutral red LF- pink /red NLF- white/yellow Eosin Methylene blue Agar isolation and differentiation of Gram (-) bacteria eosin Y, methylene blue Lactose ,sucrose none E.coli- blue or black greenish-metallic sheen other LF-gray-brown NLF-colorless/ transparent amber Mannitol Salt Agar isolation of pathogenic staphylococci 7.5% NaCl mannitol phenol red pathogenic Staphylococci- yellow zone non-pathogenic cocci-red /purple zone Azide Blood Agar for isolation of streptococci sodium azide none none growth Baird-Parker Agar isolation and enumeration of coagulase (+) staphylococci sodium tellurite none none S.aureus-black sorrunded by a clear zone Brilliant Green Bile Agar For isolation of Salmonella other than S. typhi. Not Bile salts and Crystal Lactose Neutral red Pink or Purple colonies recommended for Salmonella. violet CIN (Cefsulodine Irgasan Isolation of Yersinia and Aeromonas (Y. Enterocolitica) Sodium desoxycholate, Cefsulodine, Irgasan Mannitol Deep red center surrounded Novobiocin) Agar Crystal Violet, Irgasan by translucent outer zone "bull's eye" SS (Streptococcal Selective) Agar Isolation of Streptococcus pyogenes and agalactiae Coliston, Nalixidic acid Peptone Sheep blood Translucent/opaque colonies surrounded by zones of beta hemolysis. Cysteine Tellurite Blood Agar for isolation of Corynebacteriumdiphtheriae Potassium Tellurite 5% sterile sheep Cysteine Corynebacteriumdiphtheriae (CTBA) blood forms black colonies with brown halos CefoperazoneVancomycinAmpoth for isolation of Campylobacter spp CefoperazoneVancomy 5% defibrinated - Campylobacter jejuni ericin Media(CVA) cin sheep blood will appear as small, mucoid Ampothericin colonies, usually grayish in coloration, flat with irregular edges, and non-hemolytic at 24h Hemin, L-cysteine, For the cultivation of anaerobic microorganisms from Schaedler Agar Neomycin 5% blood for Phenol Red White or Yellow contaminated specimens anaerobes Skirrow agar Isolation of Campylobacters Gram(-)Polymxin B Vancomycin, - Translucent, white colonies Gram(+)Vancomycin Polymyxin B to spreading, flat, Trimethoprim transparent growth - Buffered Charcoal Yeast Extract for the primary isolation of Leigonella Activated charcoal, L-cysteine, Bromo cresol purple Grayish white or blue green, (BCYE) alpha ketoglutarate FericPyrophospate and Bromothymol “ground glass” appearance blue on plate CAMPY Blood agar Selective for Campylobacter spp. Trimethoprim, 10% sheep blood small, mucoid colonies, Vancomycin, agar, peptone, Triphenyltetrazolium usually grayish in coloration Cephalothin dextrose chloride Fletcher medium isolation and cultivation of Leptospira spp. high NaCl concentration 5-fluororacil-peptone, none rabbit serum-none- peptone, beef extract, Leptospira growth (ringed rabbit serum area disk 1-3cm below the surface of medium) VOGEL-JOHNSON AGAR Selective for Staphylococci Tellurite Manitol Phenol red Black/yellow zone- Staphylococcus Differentiation of Manitol and Tellurite-positive Staphylococci Potassium Tellurite Solution Black colonies with a yellow Detection of Staphylococcus aureus from heavily zone. contaminated foods and clinical specimen Dipotassium Phosphate Exhibit gray-black colonies without a yellow zone. EUGON AGAR For the isolation of Gram-positive organisms from clinical Sodium chloride and non-clinical specimens L-Cystine and Glucose 5% hors e blood Isolation of streptococci Sodiumsulfite Isolating, cultivating and determining haemolytic reactions Peptones of fastidious pathogens medium used for count and isolation of mesophilic bacteria For cultivation of a variety of organisms. Well suited for large scale vaccine production. Salmonella-Shigella Agar for the selective isolation and differentiation of pathogenic bile salts, brilliant green lactose neutral red colonies with black enteric bacilli, especially and citrates. centers(Salmonella spp.), those belonging to the genus Salmonella. This medium is pink to red not recommended for the primary isolation of Shigella. colonies(Shigellaspp.) Xylose lysine deoxcholate agar Isolation of enteric pathogens , especially salmonella and brilliant green dye Yeast extract Phenol red -Non fermenting shigella species xylose/lactose/sucrose appear as red colonies -CHANGES ITS COLOR TO YELLOW Columbia CAN agar For isolation of Gram positive micro organism from clinical nalidixic acid and 5% sheep blood none Greenish beta hemolytic and nonclinical specimen colimycin reaction Bile Esculin Azide Agar with For cultivation of vancomycin-resistant enterococci from Azide None Ferric Ammonium Enterococcus faecium, vancomycin clinical and surveillance specimens Citrate Enterococcus faecalis Phenylethyl Alcohol Agar For cultivation and selective isolation of gram + bacteria, Phenylethyl Alcohol 5% Sheep blood None Staphylococcus specifically gram + cocci with mixed flora. aureus Mac Conkey Sorbitol Agar Selection and Differentiation of E. coli 0157:H7 in stool Crystal violet and bile D-sorbitol Neutral red Sorbitol Fermenting strain of specimen salts E.coli – Pink-red colonies E.coli possessing beta-D- glucuronidase – Blue colonies E.coli possessing beta-D- glucuronidase and Sorbitol Fermenting – Purple colonies Regan Lowe Charcoal Agar for the isolation of Bordetella pertusis Cefalexin/Cephalexin Defibrinated Horse None B. pertusis – small, gray, and Amphotericin B blood and Beef shiny convex, smooth and Extract raised with a pearl-like somewhat mercury droplet appearance MIDDLEBROOK 7H11 AGAR For selective isolation and cultivation of pathogenic -malacjite green - Casein hydrolysate none Colorless to pale tan to light mycobacteria fromspecimens potentially contaminated with carbenicillin - tan-green, slightly hazy to bacteria and funji trimetophrim -lactate - hazy. Mycobacterium Light polymixins yellowish green, very slightly to slightly opalescent BUFFERED CHARCOAL- YEAST recommended for use in the cultivation and primary isolation contains anisomycin, bromcresol purple Legionella spp. present a EXTRACT with antibiotics of Legionella spp. in water and other samples suspected of colistin, and and bromothymol white to gray coloration. harboring the bacteria. vancomycin. blue, They may also have blue, Anisomycin inhibits the pink, purple, maroon, growth of fungi. Gram- greenish-yellow or dark red negative organisms are pigmentation that fades, inhibited by colistin. becoming whiter and more Gram-positive filamentous with age organisms are inhibited by vancomycin D.Special Cyclosporine Cefoxitin Fructose CCFA is primarily utilized for the selective isolation and CCFA is selective for Agar (CCFA) identification of Clostridium difficile, a bacterium usually Clostridium difficile, which linked with antibiotic-related diarrhea and Cyclosporine and Fructose Neutral red may thrive on this medium pseudomembranous colitis in humans. Cefoxitin while preventing the growth of many other bacteria. The medium promotes the formation of characteristic colonies of C. difficile, aiding in its isolation and identification. Promotes the growth of Pseudomonas aeruginosa, Cetrimide Agar Primarily used for the selective isolation and identification of which produces a green Pseudomonas aeruginosa due to its inhibition of other pigment on cetrimide agar. Cetrimide Pancreatic digest of Neutral Red pH Cetrimide is the selective bacterial species. (Cetyltrimethylammoniu gelatin indicator agent and inhibits most m bromide) bacteria by acting as a detergent Growth of almost all Czapek Agar For cultivation of fungi and chlamydospore production by saprophytic Aspergilli with Candida albicans. Streptomycin and Sucrose Slightly neutral characteristic mycelia and Aureomycin candida formation. For cultivation, maintenance Dorset Egg Medium and transport of pure cultures of mycobacteria A non selective medium well suited for maintenance of pure Malachite green Yellow and other fastidious and cultures of Mycobacterium. Glycerol nonfastidious organisms. Lowenstein-Jensen Medium for isolation of Mycobacterium malachite green none none none Deoxychollate Lactose Agar is selective against gram-positive organisms which Sodium deoxychollate Lactose Neutral red are inhibited by optimum concentration of sodium Pink- Lactose fermenters deoxycholate and sodium citrate in the medium. It Colorless- Non lactose helps to differentiate between lactose fermenting and fermenters nonfermenting enteric bacilli. BiGGY agar(Nickerson) sulfite reduction by Candida species glycine, sulfite dex, none C.albicans-brown-black C.tropicalis-dark brown with black centers Bismuth Sulfite Agar isolation of Salnomella typhosa bismuth sulfite Dex none S.tyhosa-black w/metallic brilliant green ferrous sulfate sheen S.gallinarum,S.paratyphi- green other enteric bacilli Dieudonne’s medium Used for cultivation and detection of vibrio cholerae. Penicillins, Glucose, Mannitol, Yellow Microorganism are nutrient Cephalosporins, Sucrose, Lactose broths or lysogeny broth Carboxypenicillins, medium. Liquid media are Diaminopyrimidines often mixed with agar poured via a sterile media to solidify. Bordet-Gengou Medium isolation and detection of B.pertussis penicillin 1% glycerol,potato, none B.pertussis- pearl or 20-50 % blood mercury like droplets Chapman Agar Selective for Staphylococci NaCl Mannitol bromthymol blue after add'n of bromthymol yellow clear zone after add'n amm.SO4 Dubos Oleic Agar A diagnostic medium used for the isolation and susceptibility Dextrose Phenol Red DOA primarily supports the testing of Mycobacterium spp., particularly Mycobacterium Oleic acid and Bovine growth of mycobacteria, tuberculosis. serum albumin which are slow-growing and acid-fast bacteria. Edward Hayflick Agar A selective medium used primarily for the cultivation and Horse serum Phenol Red Phenol red alters the isolation of Mycoplasma spp. and Ureaplasma spp. Penicillin medium's color to either yellow or purple, serving as an indicator of mycoplasma growth. Egg yolk Agar An enriched, nonselective, and differential medium used for Support good growth of the presumptive differentiation of Clostridium spp. and other Natural lipid content none Bromthymol blue obligate anaerobes found in obligate anaerobes. clinical infections; support typical lecithinase reaction produced by some Clostridium spp., and a lipase reaction produced by some Fusobacterium and Clostridium species. Ellinghausen, McCullough, Nurtures an environment Johnson and Harris (EMJH) suitable for the growth of medium Enrichment medium for the isolation, cultivation and sulfamethoxazole, Albumin and Straw colored or Leptospira spp. maintenance of Leptospira spp. For performing MAT or trimethoprim, polysorbate 80 amber A healthy culture in EMJH is Microscopic Agglutination Test amphotericin, straw coloured, and when fosfomycin and 5- swirled gently appears fluorouracil (STAFF) almost like a plume of smoke. Fletcher’s semi solid medium Used for the cultivation of Leptospira Peptic digest of animal 5-fluorouracil- None Ringed area (disk) 1-3 cm tissue, beef extract, peptone below the surface of the NaCl, agar, medium.  Mycobacterium species, Fildes Enrichment Medium It provides essential nutrients and growth factors that may Malachite green Glycerol None particularly Mycobacterium be lacking in standard culture media, allowing for the growth tuberculosis. and isolation of certain bacteria that might not thrive in a more general-purpose medium. Promotes growth of small, Fetal Bovine Serum Agar w/ Used as a growth supplement for cell culture media α1-antitrypsin, Glucose Phenol red pH fastidious, gram-negative vancomycin containing large amount of nutrients vancomycin indicator rod which is Haemophilus ducreyi Provides a favorable Fraser broth Serves as a selective medium for the growth of Listeria Ferric Ammonium Mannitol or Glucose Contain a pH environment for the growth monocytogenes. It helps in the enrichment and isolation of Citrate, Acriflavine indicator, typically of Listeria monocytogenes this bacterium from various food and clinical samples. Hydrochloride and phenol red. and Listeria spp. Nalidixic acid Gram Negative (GN) Broth A enrichment and selective medium for enterobacteria, with Sodium citrate and Peptone, Mannitol Turbidity Mannitol restrains the a strong inhibitory action against Gram positive bacteria Sodium deoxycholate growth of Proteus and because of its high content of citrate and deoxycholate facilitates the proliferation of Salmonella and Shigella Gram-negative enteric Hektoen Enteric Agar (HEA) A selective and differential medium designed to isolate and Bile Salts and dyes Lactose, sucrose, Bromothymol blue microorganisms differentiate members of the species Salmonella and and salicin and acid fuchsin Shigella from other Enterobacteriaceae M. pneumoniae and M. Friis agar A selective medium for Mycoplasma hyopneumoniae. Bacitracin Horse serum and Phenol Red genitalium are able to grow Bacitracin inhibits the synthesis of the cell wall of Swine serum because they do not accompanying gram-positive bacteria respond to Bacitracin that target cell wall synthesis. Hugh-Leifson OF medium Hugh-Leifson OF medium It is used to determine if gram-negative bacteria metabolize Mineral oils Glucose Yellow carbohydrates oxidatively, by fermentation, or are nonsacchrolytic and therefore have no ability to use the carbohydrate in media. Gilitti- cantoni broth Gilitti- cantoni broth Potassium tellurite (+) Dextrose (glucose) None Giolitti Cantoni broth is used for the isolation and cultivation and lithium chloride (-) of Brucella bacteria, which can cause brucellosis. The purpose of this broth is to provide an environment conducive to the growth of Brucella while inhibiting the growth of other bacteria in clinical samples or research specimens. Lim Broth Used for the selective enrichment of group B streptococci Colistin and nalidixic Glucose None (Streptococcus agalactiae), especially from genital acid specimens Loeffler Coagulated Serum Slant The Loeffler coagulated serum slant is a specialized culture Proteose peptone, medium used in microbiology laboratories for specific dextrose, horse objectives, most notably the identification and cultivation of Potassium tellurite serum Phenol red certain bacteria, most notably Corynebacterium diphtheriae, the bacterium that causes diphtheria. Glycerol Lowenstein-Jensen Medium Asparagine Lowenstein-Jensen (LJ) is the selective medium which is used for the cultivation and isolation of Mycobacterium, Malachite Green Phenol Red specifically Mycobacterium tuberculosis from clinical Potassium Nitrate specimens. MacConkey Agar Bile salts and crystal Lactose Neutral Red Used for the isolation of gram-negative enteric bacteria and violet the differentiation of lactose fermenting from lactose non- fermenting gram-negative bacteria. MacConkey Sorbitol Agar For the selection and differentiation of E. coli O157:H7 in Crystal violet and bile D-Sorbitol Neutral Red stool specimens salts Mannitol Salt Agar Sodium chloride Mannitol Phenol Red Valuable for identifying and isolating Staphylococcus species, particularly Staphylococcus aureus, which is a significant human pathogen. J27 McBride Media A medium recommended for use in qualitative procedures Beef extract, Phenyl Casein Peptone, None for selective isolation of Listeria monocytogenes from foods ethanol, Glycine Meat Peptone and clinical samples anhydride, Phenyl ethanol, Lithium chloride Vogel-Jonhson Agar Selective for Staphylococci tellurite Mannitol pheno red blackw/yellow zone- Differentiantion of Mannitol & Tellurite-positive Staphylococci S.aureus gray to blackw/out zone- S.epidermidis Streptococcus Enrichment Broth Selective and Enrichement Azide/sulfite - - growth/turbidity low conc'n of crystal violet Kanamycin Esculin Azide Agar Selective for isolation of enterococci esculin,Kanamycin - - olive green to black complex Thayer-Martin Agar for isolation of N. Gonorrhea Vancomysin, Colistin, Sheep Blood none growth and N. meningitidis Nystatin Saboraud’s Dextrose Agar is a type of agar growth medium containing peptones. It is used to cultivate dermatophytes and other types of fungi, and can also grow filamentous bacteria such as Nocardia.... The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth E. Transport medium Cary – Blair Medium To maintain the viability of the organism - Sodium - - Thioglycollate Disodium Hydrogen Phosphate Sodium Chloride Calcium Chloride Agar Stuart medium a semi-solid, non-nutritional transport medium for fastidious, Sodium pathogenic organisms glycerophosphate non-nutrient soft agar gel containing a reducing agent to Sod. Thioglycolate prevent oxidation, and charcoal to neutralize. Calcium Chloride Methylene blue 1% suspension of charcoal Amies medium an improved transport medium, containing charcoal to Same as Stuart medium prolong the viability of pathogenic organisms. containing It is a semisolid media recommended for use in qualitative glycerophosphate w/c procedures for the transport of clinical swab specimens to is replaced with the laboratory. inorganic phosphate It is the modified Stuart’s Transport Medium produced by buffer (Amies)  replacing glycerophosphate with an inorganic phosphate buffer and adding charcoal to the medium. Note: There are several bacteriological media commercially available. You can use this format, if you want to add other media that you encounter in our discussion or provided in the lecture notes. ID TEST REACTION SUBSTRATE/ ENZYME/ END PRODUCT INDICATOR POSITIVE NEGATIVE POSITIVE NEGATIVE Substance Toxin RESULT RESULT CONTROL CONTROL ACETAMIDE Utilization/ Acetamide deaminase Ammonia Bromthymol blue Blue/royal blue Green P. aeruginosa Stenotrophomonas Deamination maltophilia ACETATE Utilization Sodium acetate Source of carbon Shift to alkaline Bromthymol blue Blue Green E.coli Shigella flexneri BACITRARIN TEST Inhibition Zone of inhibition No zone of S. pyogenes S. agalactiae around the disk inihibition BILE ESCULIN AGAR Hydrolysis 40% Bile-inhibitor hydrolase Esculetin Ferric ammonium Blackening of agar No blackening/ E. faecalis S. mitis esculin citrate slant/Dark Brown Light Brown BUTYRATE DISK Hydrolysis bromo-chloro-indolyl Butyrate esterase indoxyl Bromo-chloro indolyl Blue color w/in 5 min No color change M. catarrhalis N. gonorrhoeae TEST butyrate butyrate CAMP TEST Inhibition blood Beta hemolysin Beta lysin reacts with Synergistic action of Enhanced hemolysis- No enhancement S. agalactiae S.pyogenes (CHRISTIE, ATKINS, CAMP Factor or CAMP factor CAMP factor with arrowhead appearance in hemolysis MUNCH, PETERSEN) protein B S.aureus lysin CATALASE Decomposition 3% H2O2 Catalase H2O+O2 H2O2 Evolution of gas No evolution of gas S.aureus S. pyogenes CETRIMIDE Inhibition Cetyltrimethylammoni Pyocyanin, flourescein Cetrimide Green-blue gren No growth P. aeruginosa E.coli um bromide/ Cetrimide CITRATE Utilization Sodium citrate& Source of carbon, NH3 (Alkaline) Bromthymol blue Blue Green K. pneumonia E.coli inorganic ammonium source of nitrogen salts COAGULASE Coagulation Citrated plasma Bound coagulase Clumping factor Citrated plasma Clumping No S. aureus S. epidermidis Free coagulase Clotting factor slide Clotting clumping/clotting Y.pertussis S.saphropyticus Citrated plasma S.lugdenensis tube DECARBOXYLASE/ Decarboxylation Lysine, Decarboxylase/ Cadavine, Bromcresol purple Alkaline, purple Acid, yellow K. penumoniae E.cloacae MOELLER’S /hydrolyze ornithine, deaminase putrasine, E.cloacae K.pnuemoniae arginine cittruline E.cloacae K.pneumoniae DNA HYDROLYSIS Hydrolysis DNA DNAse Hydrolyze DNA DNA-methyl green clear zone Absence of clear S. aureus S. epidermidis Toluidine blue zone GELATIN Hydrolysis Gelatine Gelatinase Liquid gelatin Gelatin Liquid at 4C Solid at 4C Proteus vulgaris E. aerogenes Serratia GROWTH AT 42C Growth Good growth No growth P. aeruginosa P. fluorescens Campylobacter jejuni HIPPURATE TEST Hydrolsis Hippuric acid Hippurase Glycine, Benzoic acid Ninhydrin Deep purple color Colorless/yellow or Gardnerella vaginalis, S. pyogenes Streptococcus agalactiae, pink Campylobacter jejuni, Listeria monocytogenes. S. agalactiae INDOLE Utilization Tryptophan Tryptophanase Indole Erlich’s + ether/ Red/pink ring at No color change Kovac’s -E.coli Kovac’s Erlich’s- Elizabeth kingia K. pneumonia PRODUCTION chloroform/xylene junction Meningoseptica Erlich’s Kovac’s CDC groupEO-2 LIA Decarboxylation/ Lysine Decarboxylase Cadaverine Ferric ammonium K/K(alkalines slant/ alkaline butt): no sugar K/K with H2S: Salmonella typhi deamination citrate/flavin fermentation K/A: Shigella flexneri mononucleotide K/A(alkaline slant/ acid butt): acid production R/A: Proteus vulgaris is seen A/A(red slant/ acid butt): red indicates deamination METHYL RED- Fermentation glucose MR:acid pyruvic acid , MR: Methyl red MR: bright red color MR:yellow color, MR E.coli MR: Klebsiella pneumoniae Enterobacter species mixed acid 4.5 pH VOGES PROSKAUER Oxidation VP: Barritts A(alpha- VP: pink to red ring VP: yellow VP: (MRVP) VP: acetoin naphthol 5%, ethanol 95%) Klebsiella species VP: E.coli Barritts B(40% KOH Enterobacter species neutral/acethyl- creatine alhpha naphthol) Hafnia species methylcarbinol Serratia species MICRODASE TEST Oxidation 1%tetramethyl-p- Oxidase Indophenol 1%tetramethyl-p- Blue to purple blue Colorless M. luteus S. aureus (Oxidase Test phenylene diamine phenylene diamine color Or Dihydrochloride Dihydrochloride, white And cytochrome C* cytochrome C MUG TEST Hydrolysis 4-nitrophenyl-β-D- Beta-D- 4-methylumbelli-feryl fluorogenic Electric blue Lack of E.coli P.aeruginosa (4-methylumbelliferyl- glucopyranosiduronic glucoronidase moiety compound under fluorescence fluorescence beta-D-glucuronide) acid (PGUA) long UV light NITRATE TEST Reduction Nitrate (NO3-) Nitrate reductase Nitrite (NO2-) Sulfanilic acid + Red Yellow + zinc Escherichia coli reduces Acinetobacter spp. Alpha naphthylamide NO3 (Nitrate) to NO2 (Nitrite). Pseudomonas aeruginosa reduces NO3 (Nitrate) to N2 (Nitrogen). NITRITE TEST Reduction Nitrite reductase NO, N2O, N2, and NH3 Sulfanilic acid + Alpha naphthylamide No color change after Red colored broth Positive: Proteus mirabilis (ATCC12453): colorless, Negative: Acinetobacter baumannii (ATCC19606): red addition of reagents and no gas gas production colouration, no gas production after 2 min and gas production production O-Nitrophenyl-β-D- Lactose fermenter (ONPG Non lactose fermenter ONPG Hydrolysis O-nitrophenyl-B-D- Beta-galactosidase Galactose, Yellow Colorless Positive): E.coli, Klebsiella spp, (O-NITROPHENYL-B-D- galactopyranoside (ONPG) (ONPG Negative): galactopyranoside o-nitrophenol(yellow) colorless Enterobacter spp produce β- Salmonella spp; Shigella spp; GALACTOPYRANOSIDE) galactosidase and permease Proteus spp; Providencia spp Late lactose fermenter (ONPG and Morganella spp do not Positive): Citrobacter spp, produce β-galactosidase so Arizona spp produce only β- cannot ferment lactose. galactosidase so they slowly ferment lactose. OPTOCHIN Sensitivity Ethylhydrocuprein Ethylhydrocuprein Zone of No zone of S. pneumoniae S. mitis Lysis hydrochloride disk hydrochloride inhibition(14mm/using a inhibition (6 mm, 5 µg) (6 mm, 5 µg) 6 mm disk) OXIDASE Test Oxidation 1%tetramethyl-p- Cytochrome Indophenol 1%tetramethyl-p- Deep purple w/in 10 No color change Positive result: Negative result: No change in Development of a dark color (i.e. no blue color seen) KOVAC’S METHOD phenylene diamine oxidase phenylene diamine seconds purple color within 10 Members of family Dihydrochloride* Dihydrochloride* seconds of inoculation. Enterobacteriaceae like E.coli Neisseria gonorrhoeae, are oxidase negative. Vibrio cholera, Pseudomonas Neisseria spp. Micrococcus Campylobacter Pseudomonas Vibrio Aerumonas PHENYLALANINE Deamination Phenylalanine Deaminase Phenylpyruvic acid 10% ferric chloride Green color develops No color change Proteus vulgaris E.coli DEAMINASE (PAD) on slant on slant Providencia Morganella PYR TEST Hydrolysis L-pyrrolidonyl-B- L-pyrroglutamyl- B-naphthylamide 0.01% N,N,methyl- Bright red color w/in No color change/ Group A Streptococci Group B Streptococci aminocinnamaldehyde (Streptococcus pyogenes), (Streptococcus naphthylamide amino- peptidase 5min orange color Group D Enterococci agalactiae), Streptococcus (Enterococcus mitis, S. bovis, S. equinus, S. faecalis and Enterococcus milleri. faecium), Coagulase negative Staphylococcus species such as S. hemolyticus, S. lugdunensis, S. schleiferi., Enterobacter, Citrobacter, Klebsiella, Yersinia and Serratia, Aerococcus, Gamella, Lactococcus, most Corynebacterium (Arcanobacterium) hemolyticum. SALT TOLERANCE Inhibition 6.5% NaCl 6.5% NaCl Visible turbidity w/ or No turbidity and no Streptococcus mitis test It is used to differentiate Aerococcus species such as Aerococcus species from W/o color change from color change A. viridans and A. urinae can other similar organisms Group D Enterococcus It is used to differentiate non-beta-hemolytic strains of catalase- negative, also grow in 6.5% NaCl, such as Stomatococcus purple to yellow Group B streptococcus enterococci (positive) from non-enterococci (negative). gram-positive cocci (i.e. therefore salt tolerance broth and Helcococcus. Enterococcus and can also be used to Aerococcus) based on differentiate their ability to grow in a 6.5% sodium chloride broth. Starch Hydrolysis Hydrolyis Starch Amylase glucose Lugol’s Iodine clearing black Bacillus subtilis Escherichia coli Differention of Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and members ofEnterococcus spp. Triple Sugar Iron Fermentation Glucose, Hexokinase Acidification, H2S Phenol red & ferrous K/K(ALKALINE SLANT/ ALKALINE BUTT): K/K: Pseudomonas aeruginosa (TSI) Agar sulfate non sugar fermenter lactose, Galactoside K/A(ALKALINE SLANT/ ACID BUTT): glucose K/A, H2s(+): Salmonella typhi permease is fermented only B-D galactosidase A/A(ACID SLANT/ ACID BUTT): glucose, A/A: E.coli sucrose lactose and sucrose are all fermented invertase UREA HYDROLYSIS Hydrolysis Urea Urease Ammonia Phenol red Bright red/ change of No color change( Proteus vulgaris E.coli slant from orange to slant and butt magenta remain orange)