Biochemical Tests Chart PDF

**Principle** **Reagents or materials** **+/- reaction** **Other** **Oxygen Use & Tolerance** Catalase Test Used to ID microbes that produce **catalase** Catalase is an enzyme found in **aerobes**, **microaerophiles** and **facultative anaerobes** Hydrogen peroxide H~2~O~2~ \+ = bubbles b...

**Principle** **Reagents or materials** **+/- reaction** **Other** **Oxygen Use & Tolerance** Catalase Test Used to ID microbes that produce **catalase** Catalase is an enzyme found in **aerobes**, **microaerophiles** and **facultative anaerobes** Hydrogen peroxide H~2~O~2~ \+ = bubbles bubbles mean gas \- = no bubbles Oxidase Used to ID microbes that make the respiratory enzyme cytochrome C oxidase Reducing agent is added directly to the bacterial growth on solid media If the reducing agent becomes oxidized a purple color is obs. which means that cytochrome C oxidase is present and a pos result No color change = neg result. Thioglycolate Medium The broth supports the growth of most types of bacteria, and contains glucose, amino acids, and sodium **thioglycolate**, a chemical that reduces (transfers electrons to) oxygen within the broth. **Resazurin** is used as an indicator that turns pink in the presence of oxygen but is colorless in the absence of oxygen Gas- Pak These jars are used to establish a completely **anaerobic** environment. A Gas Pak is added to generate H~2~ and carbon dioxide. O~2~ combines with H~2~, resulting in the removal of oxygen gas and production of water within the jar. Gas pak and jar Utilized BHI media Brain heart infusion media contains reducing substances. Basically, helps to drive off oxygen. Growth indicates anaerobic microbe Candle Jar candle burns in a sealed container; oxygen is depleted to extremely low levels while carbon dioxide levels increase. Growth indicates **microaerophile** +-------------+-------------+-------------+-------------+-------------+ | | **Principle | **Reagents/ | **+/- | **Other** | | | ** | materials** | reaction** | | +=============+=============+=============+=============+=============+ | **Oxygen | | | | | | Use and | | | | | | Tolerance | | | | | | cont'd** | | | | | +-------------+-------------+-------------+-------------+-------------+ | O-F Test | Oxidation-F | Contains | A color | Stab | | | ermentation | Glucose, | change to | | | | test uses | | yellow | Note the | | | semi-solid | pH | indicates | addition of | | | media used | indicator | the | mineral oil | | | to | Bromothymol | production | to make it | | | determine | Blue, which | of acid. | an anerobic | | | whether a | is yellow | | env. | | | bacterium | at pH 6.0 | **O tube | | | | uses | or lower | yellow = | | | | oxidative | and green | oxidative** | | | | or | at pH 7.1 | | | | | fermentativ | or higher. | **F tube is | | | | e | | yellow = | | | | carbohydrat | Mineral oil | Fermentativ | | | | e | = Anerobic | e** | | | | metabolism | and | | | | | or is | Fermentativ | **No change | | | | unable to | e | = inert** | | | | metabolize | | | | | | the source | | | | | | carbohydrat | | | | | | e. | | | | +-------------+-------------+-------------+-------------+-------------+ | **Carbohydr | | | | | | ate | | | | | | Metabolism* | | | | | | * | | | | | +-------------+-------------+-------------+-------------+-------------+ | Phenol Red | If the | A variety | Acid | | | Broth sugar | bacteria | of | produced = | | | **fermentat | ferment the | different | color | | | ion** | sugar the | sugars can | change to | | | test (11a) | broth will | be tested, | yellow (+) | | | | change from | and the | result | | | | red to | test is | | | | | yellow | performed | Base | | | | | the same | produced = | | | | Gas | each time. | color | | | | production | Examples of | change to | | | | is | sugars | dark pink | | | | secondary | include | | | | | | glucose, | | | | | | mannitol, | | | | | | lactose, | | | | | | xylose, | | | | | | etc. Phenol | | | | | | Red turns | | | | | | yellow at a | | | | | | pH below | | | | | | 7.0 and | | | | | | darker pink | | | | | | above pH | | | | | | 7.0. | | | +-------------+-------------+-------------+-------------+-------------+ | **Amino | | | | | | Acid | | | | | | Metabolism* | | | | | | * | | | | | +-------------+-------------+-------------+-------------+-------------+ | Decarboxyla | detection | detected | \+ dark | | | tion | of the | using | purple | | | | enzyme | **Bromocres | | | | (11b) | decarboxyla | ol | \- no | | | | se | Purple**, | change or | | | | | which is | yellow | | | | Decarboxyla | **yellow** | | | | | tion, | at pH 5.2 | | | | | the removal | and | | | | | of the | **purple** | | | | | carboxyl | at pH 6.8. | | | | | group from | After | | | | | an amino | inoculation | | | | | acid | , | | | | | (**lysine** | a layer of | | | | | , | mineral oil | | | | | **arginine* | or melted | | | | | * | Vaseline is | | | | | or | added to | | | | | **ornithine | prevent | | | | | **), | oxidative | | | | | yields | deamination | | | | | alkaline |. | | | | | which is a | | | | | | base! | | | | +-------------+-------------+-------------+-------------+-------------+ | Phenylalani | Testing for | **Ferric | Green = a | | | ne | phenylalani | chloride is | positive | | | deamination | ne | added | result. A | | | | deaminase | following | negative | | | (11b) | | incubation* | result is | | | | which | * | indicated | | |  | amino group | with | color after | | | | from the | **phenylpyr | addition of | | | | amino acid | uvic** | ferric | | | | phenylalani | acid (if | chloride. | | | | ne | present) to | | | | | to produce | form a | | | | | phenylpyruv | green | | | | | ic | precipitate | | | | | acid and |. | | | | | ammonium. | | | | +-------------+-------------+-------------+-------------+-------------+ +-------------+-------------+-------------+-------------+-------------+ | | **Principle | **Reagents/ | **+/- | **Other** | | | ** | materials** | reaction** | | +=============+=============+=============+=============+=============+ | **Anaerobic | | | | | | Respiration | | | | | | Tests** | | | | | +-------------+-------------+-------------+-------------+-------------+ | Nitrate | Do bacteria | Add | If after | | | Reduction | reduce | **sulfanili | the drops | | | Tests (11c) | Nitrate to | c | are add you | | | | Nitrate | acid** and | get a red | | | | | **dimethyl | result it | | | | Testing for | -- α -- | means | | | | the enzyme | napthylamin | Nitrite is | | | | that does | e** | there and | | | | this | to the | the enzyme | | | | nitrase | tube. | is present | | | | | | | | | | | If there is | (+) Result | | | | | no color | | | | | | change then | If pink | | | | | there are | after zinc | | | | | two | is added | | | | | possible | then | | | | | reasons why | nitrate was | | | | | | not | | | | | 1. bacteria | reduced. | | | | | did reduce | | | | | | the nitrate | (-) result | | | | | | | | | | | 2\. the | No color | | | | | bacteria | change | | | | | further | means | | | | | reduced | nitrite was | | | | | the | reduced | | | | | nitrite | further. | | | | | to other | | | | | | compounds | (+) result | | | | | like | | | | | | nitrogen | | | | | | gas. | | | | | | | | | | | | To | | | | | | differentia | | | | | | te | | | | | | between the | | | | | | two add | | | | | | **zinc**. | | | | | | If nitrate | | | | | | is there | | | | | | then zinc | | | | | | can take | | | | | | the | | | | | | electrons | | | | | | and the | | | | | | broth will | | | | | | turn pink | | | +-------------+-------------+-------------+-------------+-------------+ | **Hydrolyti | | | | | | c | | | | | | Enzyme | | | | | | Tests** | | | | | +-------------+-------------+-------------+-------------+-------------+ | Bile | The medium | Ferric | Following | | | Esculin | is prepared | citrate | incubation, | | | (11d) | as a slant | | a color | | | | and | | change to | | | | contains | | dark brown | | | | bile (a | | is | | | | salt, to | | indicative | | | | select for | | of a | | | | bacteria | | positive | | | | that are | | result. | | | | salt | | | | | | tolerant), | | | | | | esculin (a | | | | | | nutrient | | | | | | whose | | | | | | metabolic | | | | | | products | | | | | | can be | | | | | | detected) | | | | | | and ferric | | | | | | citrate | | | | | | (reacts | | | | | | with the | | | | | | product of | | | | | | esculin | | | | | | hydrolysis, | | | | | | esculetin, | | | | | | producing a | | | | | | dark brown | | | | | | precipitate | | | | | | ). | | | | +-------------+-------------+-------------+-------------+-------------+ | Starch | Detecting | Iodine | Halo + | | | Hydrolysis | for the | | | | | | presence of | | No halo - | | | (11d) | amylase | | | | +-------------+-------------+-------------+-------------+-------------+ | Urea (11d) | Do they use | Urea disk | Pink = + | | | | the carbon | which | for | | | | in urea. | contains | ammonium | | | | Testing for | urea and | which is a | | | | urease the | phenol red | base | | | | enzyme that | | | | | | breaks down | | Yellow = - | | | | urea into | | | | | | ammonium | | | | | | and CO~2~ | | | | +-------------+-------------+-------------+-------------+-------------+ +-------------+-------------+-------------+-------------+-------------+ | | **Principle | **Reagents/ | **+/- | **Other** | | | ** | materials** | reaction** | | +=============+=============+=============+=============+=============+ | **Hydrolyti | | | | | | c | | | | | | Enzyme | | | | | | Tests** | | | | | +-------------+-------------+-------------+-------------+-------------+ | ONPG | Used to | ONPG media | If | | | | determine | and ONPG | B-galactosi | | | Stands for | if the | disk | dase | | | O-Nitrophen | microbe | | is present | | | yl | produces | | it | | | -B-D | | | hydrolyzes | | | galactopyra | the enzyme | | ONPG | | | noside | B-galactosi | | resulting | | | (11d) | dase | | in | | | | need to | | | | | | hydrolyze | | Β-D | | | | lactose | | galactopyra | | | | into | | noside | | | | glucose and | | + | | | | B-galactose | | | | | | | | o-Nitrophen | | | | | | ol | | | | | | byproduct | | | | | | produces | | | | | | bright | | | | | | yellow | | | | | | color | | +-------------+-------------+-------------+-------------+-------------+ | Casein | | Litmus milk | Pink color | 7--14-day | | (11d) | | contains | indicates | incubation | | | | casein | acid | | | | | (cloudy | production | | | | | appearance) | from sugar | | | | | , | fermentatio | | | | | lactose, | n | | | | | and the | | | | | | **pH | Presence of | | | | | indicator | solid curd | | | | | litmus** | indicates | | | | | | casein | | | | | | protein | | | | | | coagulation | | | | | | | | | | | | Clear & | | | | | | dark | | | | | | blue-purple | | | | | | media | | | | | | indicates | | | | | | casein | | | | | | hydrolysis | | +-------------+-------------+-------------+-------------+-------------+ | Gelatin | determine | | \+ result: | | | (11d) | if bacteria | | liquefied | | | | produce the | | media after | | | | exoenzyme | | cooling | | | | gelatinase, | | | | | | which | | ─ result: | | | | hydrolyzes | | solid media | | | | the protein | | after | | | | gelatin | | cooling | | +-------------+-------------+-------------+-------------+-------------+ | **IMViC | | | | | | Tests** | | | | | +-------------+-------------+-------------+-------------+-------------+ | Indole | Can the | Kovacs | Pink ring | | | (11e) | bacteria | | indicates | | | | metabolize | | that indole | | | | the amino | | is present | | | | acid | | and | | | | tryptophan | | therefore | | | | using the | | the microbe | | | | enzyme | | has | | | | tryptophana | | tryptophana | | | | se? | | se | | | | | | | | | | | | No ring = - | | | | | | result | | +-------------+-------------+-------------+-------------+-------------+ | MRVP (11e) | MRVP tubes | | | A few drops | | | contain | | | (3-4) of | | | glucose (a | | | the | | | fermentable | | | indicator | | | sugar), | | | methyl red | | | peptone (a | | | are added | | | protein | | | to ONE of | | | source), | | | the | | | and | | | reaction | | | dipotassium | | | tubes. | | | phosphate | | | Methyl red | | | (a buffer | | | is red at | | | to resist | | | pH 4.4 and | | | pH changes | | | yellow at | | | and | | | pH 6.2. The | | | therefore | | | broth will | | | prevent | | | turn | | | death of | | | pink-red if | | | the | | | positive | | | microbes | | | for mixed | | | being | | | acid | | | studied). | | | fermentatio | | | | | | n; | | | | | | any other | | | | | | color is | | | | | | negative or | | | | | | inconclusiv | | | | | | e. | | | | | | | | | | | | The second | | | | | | reaction | | | | | | tube is | | | | | | used to | | | | | | test for | | | | | | butanediol | | | | | | production | | | | | | indirectly | | | | | | by testing | | | | | | for | | | | | | acetoin, | | | | | | another | | | | | | product of | | | | | | the same | | | | | | metabolic | | | | | | pathway. To | | | | | | test for | | | | | | the | | | | | | presence of | | | | | | acetoin, | | | | | | first add a | | | | | | few drops | | | | | | of | | | | | | α-naphthol | | | | | | followed by | | | | | | a few drops | | | | | | of | | | | | | potassium | | | | | | hydroxide | | | | | | (KOH). | | | | | | \*Add the | | | | | | reagents in | | | | | | that order. | | | | | | The | | | | | | positive | | | | | | reaction | | | | | | may take up | | | | | | to two | | | | | | hours and | | | | | | during this | | | | | | time the | | | | | | tubes are | | | | | | agitated, | | | | | | using the | | | | | | shaker. If | | | | | | acetoin is | | | | | | present, | | | | | | the | | | | | | reaction | | | | | | tube will | | | | | | slowly turn | | | | | | pinkish | | | | | | red, | | | | | | indicating | | | | | | a positive | | | | | | result. No | | | | | | color | | | | | | change, or | | | | | | any color | | | | | | other than | | | | | | pinkish red | | | | | | indicates a | | | | | | negative | | | | | | result. | | | | | | Examine the | | | | | | color of | | | | | | the tubes | | | | | | every 15 | | | | | | minutes. If | | | | | | the tube | | | | | | color turns | | | | | | red, the | | | | | | test is | | | | | | complete. | | | | | | If | +-------------+-------------+-------------+-------------+-------------+ | Simmons | citrate as | | The medium | Streak and | | Citrate | their sole | | also | stab | | (11e) | source of | | contains | | | | carbon if | | ammonium | | | | they have | | salts. If | | | | the enzyme | | the | | | | citratase | | bacterium | | | | | | utilizes | | | | | | citrate, it | | | | | | is thought | | | | | | to also use | | | | | | the | | | | | | ammonium | | | | | | salts as a | | | | | | nitrogen | | | | | | source, | | | | | | which | | | | | | results in | | | | | | an | | | | | | **alkaline* | | | | | | * | | | | | | pH shift in | | | | | | the media. | | | | | | The | | | | | | indicator | | | | | | is | | | | | | **Bromothym | | | | | | ol | | | | | | Blue**, and | | | | | | the media | | | | | | is | | | | | | initially | | | | | | green, but | | | | | | will turn | | | | | | cobalt blue | | | | | | if the test | | | | | | is positive | | | | | | for citrate | | | | | | utilization | | | | | |. | | +-------------+-------------+-------------+-------------+-------------+ | **Combinati | | | | | | on | | | | | | Tests** | | | | | +-------------+-------------+-------------+-------------+-------------+ | KIA | Do bacteria | Iron will | Yellow | | | | produce | react with | slant: + | | | | hydrogen | sulfur | for lactose | | | | sulfide | which will | fermentatio | | | | (H~2~S) as | produce a | n | | | | it converts | black | | | | | the amino | precipitate | Yellow | | | | acid |. | butt: + for | | | | cysteine | produces | glucose | | | | into | Media | fermentatio | | | | pyruvic | contains | n | | | | acid. | glucose, | | | | | | lactose, | Cracks or | | | | | peptone, | bubbles: + | | | | | and phenol | for gas | | | | | red to | production | | | | | indicate | | | | | | whether | Black | | | | | fermentatio | precipitate | | | | | n | (only if | | | | | of either | acid was | | | | | or both | produced): | | | | | sugars. | + for H2S | | | | | | production | | +-------------+-------------+-------------+-------------+-------------+