Chapter 9: Biochemical Testing

Question 1

Stereotyping:

Answer 1

Use of antibodies to detect specific pathogens located on cell surface

Question 2

Molecular biology is based on:

Answer 2

Based on genotype and nuclei acid sequences

Question 3

MALDI - TOF MS based on:

Answer 3

Characterization of microbial proteins

Question 4

Lactose utilization:

Answer 4

Most important carb determination

Can be used to differentiate lactose fermenting vs. Non-lactose fermenters

Question 5

Lactose is:

Answer 5

Glucose and galactose

Question 6

Lactose degradation:

Answer 6

Requires b-galacto side permease (transports lactose through cell wall) and b-galactosidase ( breaks lactose to yield subunits). Afterwards, glucose is available for metabolism

Question 7

Some bacteria lack permease, but still have

Answer 7

B-galactosidase

Question 8

Two pathways of glucose metabolism are

Answer 8

Embolden - myerhof-parnas pathway and entner-dovdoroft pathway

Question 9

Fermentation:

Answer 9

Anaerobically utilized. Glucose to pyruvate that’s oxidized to other acid products and gases
Acid detected by pH indicates. Single acid and double/mixed fermenters.

No oxygen, uses a different inorganic molecule

Question 10

Oxidation:

Answer 10

Aerobic, glucose to pyruvate to CO2

Generally weak acid producers, acid neutralized by alkaline reaction from peptones

Question 11

O/F Basal Media (OFBM) Purpose and Description:

Answer 11

Help classify as oxidizer or fermenter, lower concentration of peptones

• pH indicator is bromothymol blue.
• Uninoculated is green.
• Acid pH is yellow.
• Alkaline pH is blue

Question 12

Triple Sugar Iron Agar (TSI):

Answer 12

• Contains sucrose, glucose, and lactose
  – Lactose and sucrose present in 10:1 ratio to glucose.
  – Kligler iron agar (KIA) is similar but no sucrose.
• Ferrous sulfate and sodium thiosulfate
  – Detect production of hydrogen sulfide (H2S), which gives a black color
• Phenol red
  – pH indicator
• Below 6.8 yellow (acidic)
• Above 6.8 red (alkaline)
• Reaction chambers
  – Slant is aerobic.
  – Butt is anaerobic

Question 13

Reactions on TSI or KIA:

Answer 13

must be read at 18 and 24 hours

read as slant/butt

Question 14

Ortho-Nitrophenyl-β-D-
Galactopyranoside (ONPG) Test Purpose:

Answer 14

To determine if an organism is a dLF (one that lacks the enzyme β-galactoside permease but possesses β-galactosidase) or is a true NLF

Question 15

ONPG test mechanism:

Answer 15

• ONPG is structurally similar to lactose, but ONPG is
  more readily transported through the bacterial
  plasma membrane
• β-Galactosidase hydrolyzes ONPG, a colorless
  compound, into galactose and o-nitrophenol, a
  yellow compound. ONPG remains colorless if the
  organism is a NLF and becomes yellow for a dLF

Question 16

Steps to perform ONPG Test:

Answer 16

– Make a heavy suspension of bacteria in sterile saline
– Add ONPG disk/tablet.
– Incubate at 35° C.
– Positive results usually seen within 6 hours

Question 17

Glucose metabolism:

Answer 17

Occurs via the Embden–Meyerhof pathway, produces several intermediate by-products (pyruvic acid which can then degrade into mixed acids as end products)

Question 18

Enterics take two separate pathways:

Answer 18

mixed acid fermentation and butylene glycol pathway

Question 19

The methyl red (MR) and Voges–Proskauer (VP) test
detect

Answer 19

end products of glucose fermentation

Question 20

MRVP Test Procedure Highlights:

Answer 20

• Inoculate glucose-containing broth.
• Incubate for 3 to 5 days.
• Transfer half of bacterial suspension into a
  clean dry tube for the VP test.
• Remaining half of suspension is for MR test

Question 21

Decarboxylase test:

Answer 21

Test the presence of enzymes capable of removing carboxyl group (COOH)

• Specific for amino acids like lysine, ornithine, arginine

Lysine - lysine decarboxylase - cadaverine + CO2

Ornithine - Ornithine decarboxylase - putrescine + CO

Question 22

Dihydrolase test:

Answer 22

Arginine - arginine dihydrolase - citrulline - ornithine
- putrescine

Question 23

Moeller Decarboxylase Base Medium Description:

Answer 23

Broth medium used to detect decarboxylation (contains glucose, peptones), pH indicators are bromocresol purple and cresol red

– Specific amino acid at a concentration of 1%
– Overlay with oil
– Initial pH of 6.0

Uninoculated is purple; initial fermentation drops pH to 5.5: turns medium yellow

Question 24

Deaminase Test Description and Procedure:

Answer 24

Amino acids can be metabolized by deaminases that remove an amine (NH2) group

Question 25

Phenylalanine deaminase (PAD) test:

Answer 25

– Deamination produces phenylpyruvic acid. – Slant medium – Inoculate slant and incubate – Add 10% ferric chloride * Green indicates positive test (phenylpyruvic acid present).

Question 26

Citrate Utilization:

Answer 26

* Citrate test determines whether an organism can use sodium citrate as a sole carbon source * Use of citrate results in an alkaline pH. * This changes the indicator from a green color to a blue color (Blue is a positive test) * Inoculum must be light. – Dead organisms can be a source of carbon.

Question 27

Deoxyribonuclease (DNase):

Answer 27

Endonucleases produced by bacteria that can break phosphodiester bonds – Streak organisms with heavy inoculum on DNase plate – Incubate for 18 to 24 hours. – Add 1N HCl to surface of the plate. DNA precipitates but if DNase then clear halo

Question 28

Indole Production Description:

Answer 28

* Indole is a degradation product of tryptophan (amino acid) * Organisms that possess tryptophanase can deaminate tryptophan resulting in indole. * Bacteria are inoculated into tryptophan or peptone broth and incubated for 48 hours before indole testing can be done

Question 29

Ehrlich’s indole test – more sensitive method

Answer 29

– Indole extracted by xylene – Add xylene and shake tube well – Add Ehrlich’s reagent (para- dimethylaminobenzaldehyde, PDAB) Positive: red

Question 30

Kovac’s – less sensitive method

Answer 30

Add five drops of Kovac’s reagent (PDAB) to culture. Positive: red

Question 31

LIA Slant:

Answer 31

Contains lysine, glucose, ferric ammonium citrate, and sodium thiosulfate * pH indicator bromocresol purple – normally light purple

Question 32

LIA slant: Deamination

Answer 32

Plum / Reddish color with yellow butt because of glucose fermentation

Question 33

Motility:

Answer 33

Observing growth in a semi-solid medium * Agar of 0.4% or less Stab single line into media * Examine movement away from stab line

Question 34

LIA slant: Decarboxylation

Answer 34

* Butt dark purple (needs anaerobic) * Butt with black precipitate H2S + decarboxylation

Question 35

Motility-Indole-Ornithine (MIO) Agar Description:

Answer 35

– Semi-solid – Used to detect motility, indole, and ornithine decarboxylase production – Useful in differentiation Klebsiella spp. from Enterobacter and Serratia spp.

Question 36

Nitrate and Nitrite Reduction:

Answer 36

Reduction of Nitrate (NO3) to Nitrite (NO2) – Add N,N-dimethyl-α-naphthylamine (NNDN) + sulfanilic acid – Examine for red color = nitrite present Nitrate - nitrite + sulfanilic acid + NNDN - diazo red dye Test verification: add zinc if red color is only now produced, then nitrate was not reduced.

Question 37

Oxidase Description:

Answer 37

* Presence of cytochrome oxidase system * Helpful in differentiating between the Enterobacteriaceae that are mostly oxidase negative and pseudomonads, also Neisseria spp. * Modified oxidase is used to distinguish Staphylococcus from Micrococcus.

Question 38

Kovac’s Oxidase Test:

Answer 38

Kovac’s oxidase test uses 0.5% or 1% aqueous solution of tetramethyl-p-phenylenediamine dihydrochloride 1) Add drop of reagent to filter paper. 2) Use wooden applicator stick to rub a bacterial colony onto the moistened filter paper. 3) Development of a lavender color within 10 to 15 seconds constitutes a positive test

Question 39

Sulfide-Indole-Motility (SIM) Agar:

Answer 39

* Semi-solid * Helpful in differentiating gram-negative bacteria in the Enterobacteriaceae family * Performed via a straight stab of inoculum in center of medium

Question 40

SIM agar results:

Answer 40

– Cloudiness spreading from inoculum = motility – Production of H2S = black precipitate – Pink to red color after addition of Kovac’s reagent is positive for indole.

Question 41

Urease Purpose and Procedure Highlights:

Answer 41

* Used to determine whether a microorganism can hydrolyze urea, releasing a sufficient amount of ammonia to produce a color change by a pH indicator * Urease hydrolyzes urea to form ammonia, water, and CO2 * Surface of agar slant is inoculated (not stabbed)

Question 42

Principles of Identification:

Answer 42

* pH-based reactions * Enzyme-based reactions * Utilization of carbon sources * Visual detection of bacterial growth * Molecular assays based on characterization of molecules

Question 43

API 20E:

Answer 43

System for the identification of gram-negative fermentative bacteria (the family Enterobacteriaceae)

Question 44

API 20E system highlights:

Answer 44

– Consists of 20 cupules attached to plastic strip each with a specific lyophilized, pH-based substrate – A liquid bacterial suspension is used to rehydrate the cupules

Question 45

API 20E characteristics:

Answer 45

* Some cupules require mineral oil overlay. * Principles of the tests are the same for similar tests performed in test tubes. * Strip is incubated for 18 to 24 hours. * Reagents are added as appropriate * Oxidase test also done, separately * Results are recorded and a 7-digit code profile number is determined. * The code profile number is checked against a database provided by manufacturer for identification; additional testing may be needed. * Accuracy for common isolates is 87.7% * Additional multitest systems are available to identify a variety of organisms

Question 46

Rapid Identification Systems Commercial Kits:

Answer 46

Use chromogenic or fluorogenic substrates * Colorless until cleaved by microbial enzyme – Based on enzymes * Due to amplification of effect, substrates change color quickly. -- Some results may be reached after 2 to 6 hours of incubation

Question 47

Most automated systems use:

Answer 47

– Turbidity – Colorimetry – Fluorescent technology

Question 48

Automated Identification Systems Examples:

Answer 48

* MicroScan System * TREK Diagnostic System * Vitek 2 System * BD Phoenix Automated Microbiology System * Biolog OmniLog ID System

Question 49

Triple sugar iron and Kligler iron agars are useful in determining the ability of bacteria to utilize ___

Answer 49

certain carbohydrates and to produce H2S

Question 50

The methyl red and Voges-Proskauer tests are used to determine

Answer 50

end products of glucose fermentation

Question 51

Decarboxylase, dihydrolases, and deaminases are enzymes used by bacteria to

Answer 51

metabolize aminos acids and can be used in identification

Question 52

Numerous tests, such as citrate, DNase, indole, nitrate reduction, oxidase, and urease are

Answer 52

important in the identification of gram-negative bacteria

Question 53

Manual multitest systems have improved the identification of bacteria by

Answer 53

simplifying inoculation of many different biochemical tests and producing numeric codes that can be compared with numbers in a database

Question 54

Rapid tests often use

Answer 54

chromogenic or fluorogenic substrates to assay for preformed bacterial enzymes

Question 55

Automated microbial identification systems offer

Answer 55

accurate, rapid, identifications with less hands-on time by laboratory scientists