7.8 ENTEROBACTERIACEAE Flashcards

Question

ENZYMATIC TESTS

Answer 1

 Enzymes are the “driving force” of bacterial metabolism  Genetically encoded (the bacterium DNA tells the cell to make the enzyme or not)  Can detect a single enzyme or a complete pathway that utilizes several enzymes

Answer 2

Single Enzyme Tests * Usually quick and easy * Cannot usually ID to species level but can be used to determine next step in ID scheme * i.e. Catalase Metabolic Pathway Tests * Tests the metabolic pathway that an organism takes * Tests for the end-products made by the pathway * May involve several enzymes

Answer 3

 Carbohydrate (CHO) and protein substrates  Oxidative process requires oxygen, fermentation does not  We look for the acid by-products in presence or absence of oxygen through a change in colour (pH indicator)

Answer 4

 We look for enzymes that deaminate or decarboxylate amino acids  i.e. lysine, ornithine, arginine, phenylalanine

Answer 5

 We can test the ability of an organism to grow in the presence of a single nutrient or carbon source

Answer 6

 Single substrate utilization  Tests ability of an organism to utilize sodium citrate as its only source of carbon (and energy) and ammonium salt as its only source of nitrogen  pH indicator – bromthymol blue  Positive: growth (blue)  Negative: no growth (green)

Answer 7

 Tests for metabolic pathways via amino acid degradation  The enzyme decarboxylase can cleave a carboxyl group from an amino acid  This cleaving results in production of CO2 and an amine (alkaline end product)  pH indicator changes colour due to alkaline end product  Decarboxylation is an anaerobic process and requires an acid environment for activation

Answer 8

 Tube contains:  Glucose  pH indicator (bromcresol purple)  Amino acid (lysine or ornithine)  We cover the tubes with oil to create an anaerobic environment

Answer 9

 Glucose in the tube is fermented by all Enterobacteriaceae  The fermentation creates the acidic environment necessary for the decarboxylation to occur  Because of the fermentation, the bromcresol purple will turn from purple to yellow a few hours after inoculation  The acid environment created allows for the decarboxylation to occur  The cleaving of the carboxyl group results in an alkaline pH due to the amine produced  The alkaline environment created causes the bromcresol purple to convert back to purple  Must be controlled by testing basic medium that contains only glucose and the pH indicator  NO amino acid present  Should see a yellow colour in the tube if organism has fermented glucose  If control tube is purple, test results are NOT reliable Positive: purple Negative: yellow

Answer 10

 Single enzyme test  Tests the ability of the organism to hydrolyze DNA via deoxyribonuclease (Dnase)  Agar plate contains polymerized DNA attached to methyl green complex  If DNA is hydrolyzed, methyl green is released for the complex  Green colour around the colony fades  Positive: clear halo around colony  Negative: no clear halo  Positive controls: S. aureus, Serratia marcescens, Moraxella catarrhalis

Answer 11

 Determines an organism’s ability to produce indole from tryptophan  Upon addition of Kovac’s reagent (p-dimethylaminobenzaldehyde), tube will show a bright pink ring Tryptophan (tyrprophanase) Indolepyruvic acid (deaminiation) Indole + Pyruvic acid

Answer 12

 Single enzyme test  Determines an organism’s ability to produce indole from tryptophan via tryptophanase  Breakdown products are pyruvic acid, ammonia and indole  Indole reacts with 1% p-dimethylaminocinnamaldehyde → blue-green colour  Must be performed from BA (or other media with tryptophan)  Saturate filter paper with Spot Indole reagent  Add unknown organism with stick or loop  Read colour within 20-30 seconds  Positive: blue-green  Negative: no colour change

Answer 13

 Determines if an organism possess a flagellum (or flagella)  Tube test contains semi-solid agar (<0.4%) to allow for movement  Touch one colony and stab part way down with a straight wire  Incubate at 35-37°C for 18-24 hours  Positive: growth spreading out from stab line  Negative: no growth away from stab lin

Answer 14

 Combines three tests into one semi-solid media tube:  Motility  Indole  Lysine decarboxylase  Usually contains bromcresol purple indicator  Touch one colony and stab part way down with a straight wire  Incubate at 35-37°C for 18-24 hours  Add Kovac’s reagent after reading motility and lysine to visualize indole result

Answer 15

 Permease: allows lactose to enter the bacterial cell  β-galactosidase: breaks down lactose to glucose and galactose  LF possess both enzymes (can ferment quickly)  LLF possess only β-galactosidase (can ferment but just slower)  Determines if an organism possesses β-galactosidase and therefore confirms its ability to ferment lactose  β-galactosidase hydrolyzes the substrate ONPG to orthonitrophenol (yellow)  Positive: yellow  Negative: no colour change

Answer 16

 Single enzyme method  Used most often to differentiate between groups of Gram negative bacteria  Determines if an organism possesses the enzyme cytochrome oxidase  Aerobic process  Substrate: 1% tetramethyl-p-phenylenediamine dihydrochloride  If the organism possesses the enzyme, the substrate is oxidized to indophenol causing a colour change  Positive: dark purple  Negative: no colour change

Answer 17

False negatives * Colonies >24 hours old * Test done from selective medium or medium containing glucose (i.e. MAC) False positives * Iron or chromium loop (use wooden sticks) * Auto-oxidized reagent

Answer 18

Filter Paper * Saturate a filter paper with substrate * Rub organism on and read within 10 seconds Disc or strips * Impregnated with substrate Swab * Add reagent after swabbing colonies

Answer 19

 Amino acid degradation test  Determines an organism’s ability to oxidatively deaminate phenylalanine to phenylpyruvic acid (PPA)  Inoculate by streaking up slant and incubate for 18-24 hours at 35-37°C  PPA is detected with 10% ferric chloride (FeCl3)  Positive: dark green after addition of FeCl3  *All Tribe Proteae are positive  Negative: no change with addition of FeCl3

Answer 20

 Amino acid degradation test  Determines an organism’s ability to deaminate tryptophane to indolepyruvic acid  Indole pyruvic acid turns brown in the presence of 10% ferric chloride (FeCl3)  Positive: brown after addition of FeCl3  Negative: no colour change

Answer 21

* 1 part glucose (0.1%) * 10 parts lactose (1%) * 10 parts sucrose (1%) * Peptone (provides nitrogen) * Phenol red (pH indicator) * Ferric sulfate * Sodium thiosulfat

Answer 22

 Determines if suspect organism can ferment sugars and produce gas and hydrogen sulfide  Inoculation:  Stab to bottom of butt (ANO2)  Streak up surface of slant (O2)  Incubate no longer than 24 hours with cap loosened  All Enterobacteriaceae ferment glucose  After 8-12 hours of incubation, the small amount of glucose will be fermented and acid end products will cause a change in the phenol red indicator from red to yellow in the slant and butt

Answer 23

 After ~12 hours, the pH changes on the slant due to the oxidation of peptone to amines  CO2 and H2O products cause the oxidation of the fermentation products  Slant reverts to red (butt still yellow)

Answer 24

 There is a large volume of acid end products  Any amines produced are neutralized by the large amount of acid  Even after ~12 hours, slant remains yellow

Answer 25

 There will be bubbles or cracks in agar  There may be separation of agar from inner surface of tube

Answer 26

 H2 gas + Na thiosulfate → H2S (colorless)  H2S + ferric sulfate = black precipitate  MUST have an acidic environment for this reaction to occur  Acid environment provided by fermentation of sugars (will only see in butt if glucose is the only sugar fermented)

Answer 27

 Do not incubate >24 hours  Oxidation products will eventually overwhelm acids and tube will revert to red  Be sure to provide atmospheric oxygen to allow for peptone metabolism on slant and gas formation (loose caps)

Answer 28

 Single enzyme test  Determines the ability of an organism to hydrolyze urea by the enzyme urease  Tube contains broth or agar with urea as primary carbon source  Urea → ammonia + CO2 + H2O  Ammonia causes the pH of the medium to increase (alkaline)  Phenol red indicator turns magenta  Inoculate by streaking up the slant of the agar  Incubate for 18-24 hours at 35-37C  Positive: magenta  Negative: light orange or yellow

Answer 29

 Methyl Red Test  Tests for lactic, formic, and acetic acids produced after glucose utilization  We will not be performing this test

Answer 30

 Looking for neutral end products of glucose utilization  Glucose fermentation follows the EMP metabolic pathway resulting in the end product pyruvic acid  Pyruvic acid can further follow the butylene glycol pathway with acetoin as its neutral end product  After the addition of 40% KOH, acetoin is converted to diacetyl  Diacetyl + 𝛼-napthol reagent + guanido group forms a red complex Acetoin (40% KOH) Diacetyl Red (a-napthol) complex

Answer 31

-Positive: red colour after addition of 40% KOH and α-naphthol  Negative: yellow or no colour change

7.8 ENTEROBACTERIACEAE Flashcards

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