Chapter 9: Biochemical Testing Flashcards
Stereotyping:
Use of antibodies to detect specific pathogens located on cell surface
Molecular biology is based on:
Based on genotype and nuclei acid sequences
MALDI - TOF MS based on:
Characterization of microbial proteins
Lactose utilization:
Most important carb determination
Can be used to differentiate lactose fermenting vs. Non-lactose fermenters
Lactose is:
Glucose and galactose
Lactose degradation:
Requires b-galacto side permease (transports lactose through cell wall) and b-galactosidase ( breaks lactose to yield subunits). Afterwards, glucose is available for metabolism
Some bacteria lack permease, but still have
B-galactosidase
Two pathways of glucose metabolism are
Embolden - myerhof-parnas pathway and entner-dovdoroft pathway
Fermentation:
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
Oxidation:
Aerobic, glucose to pyruvate to CO2
Generally weak acid producers, acid neutralized by alkaline reaction from peptones
O/F Basal Media (OFBM) Purpose and Description:
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
Triple Sugar Iron Agar (TSI):
- 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
Reactions on TSI or KIA:
must be read at 18 and 24 hours
read as slant/butt
Ortho-Nitrophenyl-β-D-
Galactopyranoside (ONPG) Test Purpose:
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
ONPG test mechanism:
- 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
Steps to perform ONPG Test:
– Make a heavy suspension of bacteria in sterile saline
– Add ONPG disk/tablet.
– Incubate at 35° C.
– Positive results usually seen within 6 hours
Glucose metabolism:
Occurs via the Embden–Meyerhof pathway, produces several intermediate by-products (pyruvic acid which can then degrade into mixed acids as end products)
Enterics take two separate pathways:
mixed acid fermentation and butylene glycol pathway
The methyl red (MR) and Voges–Proskauer (VP) test
detect
end products of glucose fermentation
MRVP Test Procedure Highlights:
- 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
Decarboxylase test:
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
Dihydrolase test:
Arginine - arginine dihydrolase - citrulline - ornithine
- putrescine
Moeller Decarboxylase Base Medium Description:
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
Deaminase Test Description and Procedure:
Amino acids can be metabolized by deaminases that remove an amine (NH2) group
Phenylalanine deaminase (PAD) test:
– Deamination produces phenylpyruvic acid.
– Slant medium
– Inoculate slant and incubate
– Add 10% ferric chloride
- Green indicates positive test (phenylpyruvic acid present).
Citrate Utilization:
- 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.
Deoxyribonuclease (DNase):
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
Indole Production Description:
- 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
Ehrlich’s indole test – more sensitive method
– Indole extracted by xylene
– Add xylene and shake tube well
– Add Ehrlich’s reagent (para-
dimethylaminobenzaldehyde, PDAB)
Positive: red
Kovac’s – less sensitive method
Add five drops of Kovac’s reagent (PDAB) to culture.
Positive: red
LIA Slant:
Contains lysine, glucose, ferric ammonium citrate,
and sodium thiosulfate
- pH indicator bromocresol purple – normally light purple
LIA slant: Deamination
Plum / Reddish color with yellow butt because of glucose fermentation
Motility:
Observing growth in a semi-solid medium
* Agar of 0.4% or less
Stab single line into media
* Examine movement
away from stab line
LIA slant: Decarboxylation
- Butt dark purple (needs anaerobic)
- Butt with black precipitate H2S + decarboxylation
Motility-Indole-Ornithine (MIO) Agar Description:
– Semi-solid
– Used to detect motility, indole, and ornithine decarboxylase production
– Useful in differentiation Klebsiella spp. from
Enterobacter and Serratia spp.
Nitrate and Nitrite Reduction:
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.
Oxidase Description:
- 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.
Kovac’s Oxidase Test:
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
Sulfide-Indole-Motility (SIM) Agar:
- Semi-solid
- Helpful in differentiating gram-negative bacteria in
the Enterobacteriaceae family - Performed via a straight stab of inoculum in center
of medium
SIM agar results:
– Cloudiness spreading from inoculum = motility
– Production of H2S = black precipitate
– Pink to red color after addition of Kovac’s reagent is positive for indole.
Urease Purpose and Procedure Highlights:
- 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)
Principles of Identification:
- pH-based reactions
- Enzyme-based reactions
- Utilization of carbon sources
- Visual detection of bacterial growth
- Molecular assays based on characterization of molecules
API 20E:
System for the identification of gram-negative fermentative bacteria (the family
Enterobacteriaceae)
API 20E system highlights:
– 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
API 20E characteristics:
- 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
Rapid Identification Systems Commercial Kits:
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
Most automated systems use:
– Turbidity
– Colorimetry
– Fluorescent technology
Automated Identification Systems Examples:
- MicroScan System
- TREK Diagnostic System
- Vitek 2 System
- BD Phoenix Automated Microbiology System
- Biolog OmniLog ID System
Triple sugar iron and Kligler iron agars are useful in determining the ability of bacteria to utilize ___
certain carbohydrates and to produce H2S
The methyl red and Voges-Proskauer tests are used to determine
end products of glucose fermentation
Decarboxylase, dihydrolases, and deaminases are enzymes used by bacteria to
metabolize aminos acids and can be used in identification
Numerous tests, such as citrate, DNase, indole, nitrate reduction, oxidase, and urease are
important in the identification of gram-negative bacteria
Manual multitest systems have improved the identification of bacteria by
simplifying inoculation of many different biochemical tests and producing numeric codes that can be compared with numbers in a database
Rapid tests often use
chromogenic or fluorogenic substrates to assay for
preformed bacterial enzymes
Automated microbial identification systems offer
accurate, rapid, identifications with less hands-on time by laboratory scientists
