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Microbiology Biochemical Tests

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									                                                                Microbiology: Biochemical Tests

  Test              Medium            Substrate              Product(s)                     Reagent                                       Result
Amylase         starch agar        starch            glucose, maltose, dextrins   iodine added after incubation      + = colorless zone around organism after iodine
Caseinase       skim milk agar     casein            amino acids, peptides                                           + = clear zone around organism

DNase           DNase agar         ds DNA            nucleotides                     methyl green in agar (bound     + = colorless zone around organism
                                                                                     to DNA)
Lipase          spirit blue agar   lipid             glycerol, fatty acids           spirit blue in agar             + = blue/clear zone around organisms, blue ppt.
                                   (triglyceride)                                                                    in organisms

                                                                     Selective and Differential Media
            Test                     Medium         Substrate           Selective           pH                           Result                         + organisms
                                                                       Ingredient        indicator
EMB agar – selective for           Eosin            Lactose        Eosin +                           Growth = gm -;
gm - & differential for            Methylene                       Methylene Blue                    Dye accumulation (color) in organism
lactose fermentation               Blue (EMB)                                                        = lactose fermentation:
                                   agar                                                                         Metallic green                         E. coli
                                                                                                                Fish eyes (pink with purple center)    E. aerogenes
MacConkey’s agar –                 MacConkey’s      Lactose        Crystal violet +    Neutral red   Growth = gm -;                                    E. coli;
selective for gm - &               agar                            bile salts          in agar       Pink ppt. in organism = lactose fermentation      E. aerogenes
differential for lactose
Mannitol Salt Agar                 MSA              Mannitol       7.5% NaCl           Phenol red     Growth = salt tolerant (staphylococci)           S. epidermidis
(MSA) – selective for staph                                                            in agar                                                         & S. aureus;
& differential for mannitol                                                                           Acidic (yellow) media = mannitol fermentation    S. aureus

         Test                       Media               Substrate         Product(s)          Reagents               pH indicator     + appearance       + organisms
Indole                    Tryptic Soy Broth (TSB)      Tryptophan        Indole         Kovac’s layered on top                        Red at surface    E. coli
Methyl Red                MRVP broth                   Glucose           Organic acids Methyl red                  (Methyl red )      Red throughout    E. coli
Voges-Proskauer           MRVP broth                   Glucose           Non-acids      VP-A & VP-B (Barritt’s)                       Red throughout    E. aerogenes
Citrate Utilization       Simmons’ citrate agar        Citrate           Na2CO3                                    Bromthymol         Royal blue        E. aerogenes
                                                                                                                   blue in agar
                                                                        TSI (Triple sugar iron)
    Test              Media          Substrate          Product(s)          Reagent       pH indicator                + appearance                     + organisms
H2S Gas           TSI agar        Cysteine             H2S               Iron in media                   Black ppt. when H2S reacts with iron        S. typhimurium
                  (stab & drag)                                                                          to form iron sulfide                        P. vulgaris
Carbohydrate      TSI agar        Glucose, lactose,    Organic acids                    Phenol red in    Media: yellow = acidic (A);
Fermentation      (stab & drag)   sucrose                                               agar                    red = alkaline (K);
                                                                                                         Record reactions as “slant”/”butt”:
                                                                                                         K/A=glucose only,
                                                                                                         A/A=sucrose &/or lactose +/-glucose
                                                                                                         K/K=none fermented
                                                                                                         Black butt = acidic

                                                               Carbohydrate Fermentation Series
            Test                             Media                         Substrate            Product(s)            pH indicator                 + appearance
Carbohydrate Fermentation              Carbohydrate broth       Glucose, lactose, or sucrose   Organic acids        Phenol red in broth       Acidic (yellow) media
Gas Production                         with Durham tubes                                           CO2                                        Bubble in Durham tube

         Test                     Media                 Substrate         Product(s)          pH indicator                  + appearance               + organisms
Urease                  (Christensen’s) urea agar     Urea              Ammonia, CO2      Phenol red in agar     pink (alkaline) media                 Proteus
Litmus                  Litmus milk                   Casein, lactose   Various           Litmus in media        pink = acid; sugar fermentation
                                                                                                                 hard curd = acid
                                                                                                                 soft curd = rennin clots casein
                                                                                                                 blue = alkaline; casein breakdown
                                                                                                                 brown = further casein breakdown
                                                                                                                 white = litmus reduction

    Test                 Substrate                    Product(s)                             Reagent                  + appearance                 + organisms
  Catalase        Hydrogen peroxide        Water, oxygen                                                          Bubbles                      Staphylococci
 Coagulase        Fibrinogen               Clumping factor-fibrinogen complex       Staphyloside latex beads      Blue ppt. (clumping)         S. aureus
(Cytochrome)      Cytochrome c             Oxidized cytochrome c                    Phenylenediamine              Blue color                   P. aeruginosa
Bile Solubility                                                                     Tris buffer + deoxycholate    Clearing (lysis of cells)    S. pneumoniae
  Test            Media                    Substrate                            Results                           + appearance                 + organisms
*Alpha     Blood agar (5% SB)        RBCs & hemoglobin (Hb)       Hemolysis; partial digestion of Hb       Green/brown zone               Strep. pneumoniae
                                                                                                           around/beneath organisms      E.(S) faecalis
                                                                                                                                          Staph. epidermidis
**Beta     Blood agar (5% SB)        RBCs & hemoglobin (Hb)       Hemolysis; complete digestion of Hb      Clear zone around organisms    Strep. pyogenes,
                                                                                                                                          E.(S) faecium
                                                                                                                                          Staph. aureus
Gamma      Blood agar (5% SB)        RBCs & hemoglobin (Hb)       No hemolysis                             No clearing around
*Alpha-hemolytic Strep differentiated by optochin sensitivity (S. pneumoniae)
**Beta- hemolytic Strep differentiated by bacitracin sensitivity (S. pyogenes)

       Test              Media         Substrate        Product(s)               Reagent                                        Appearance
Nitrate Reduction    Nitrate broth    Nitrate          Nitrite;         Nitrate A & B, zinc        1. Add nitrate A & B:
                                                       N2/ammonia       added after incubation     Red color = nitrate reductase
                                                                                                   2. If colorless after nitrate A & B add zinc:
                                                                                                   Still colorless after zinc = both nitrate and nitrite reductase
                                                                                                   If red after zinc = neither nitrate or nitrite reductase

                                             Nitrate reductase                                         Nitrite reductase
               Nitrate (NO3)                                                  Nitrite (NO2)                                       N2 (gas) /NH3 (ammonia)
               Nitrate A & B               reduces nitrate to nitrite         Nitrate A & B                                              Nitrate A & B
            + nitrate = colorless                                             + nitrite = red                              + N2 (gas) or NH3 (ammonia) = colorless

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