Biochemical Identification of Gram Negative Bacteria (LEC/LAB) Flashcards
Gram stain reaction and morphology of Enterobacteriaceae
Gram Negative (-) Bacilli and Cocobacilli
Aerotolerance of Enterobacteriaceae
Facultative Anaerobes
Enterobacteriaceae do not produce ________.
Cytochrome C oxidase
Which member of Enterobacteriaceae produces cytochrome c oxidase?
Plesiomonas spp.
TRUE OR FALSE.
Enterobacteriaceae family produces cytochrome C oxidase.
False
Which carbohydrate/s can be fermented by the members of Enterobacteriaceae?
Glucose
Are Enterobacteriaceae motile?
Yes (at room temperature)
At which temperature are Enterobacteriaceae motile?
Room Temperature (20-25 C)
Which member/s of the Enterobacteriaceae family are not motile at room temperature?
MNEMONIC: SKY
* Shigella spp.
* Klebsiella spp.
* Yersinia spp.
Specimen of Choice for Enterobacteriaceae
Fecal/Stool Specimens
Collected within first 4 days of illness & before administration of ATBs
Specimen Collection
Fecal/stool specimens are collected within ____ of illness and before administration of antibiotics.
first 4 days
Specimen Collection
Specimens should be processed within ____ of collection.
1-2 hours
Specimen Collection
What should be done if the specimen cannot be processed within the ideal time frame (1-2 hours)?
Placed in a transport medium at the temperature of 4 C
Specimen Collection
Specimens in a transport medium must be kept at a temperature of ____.
4 C
Specimen Collection
Transport medium for Enterobacteriaceae
- Cary-Blair
- Amies
- Stuart
Which step in biochemical identification of Enterobacteriaceae is not helpful in identifying enteric pathogens?
Direct Smear Examination/Direct Microscopic Examination/Gram Staining
Which specimen sample is not helpful in direct smear preparation for identifying enteric pathogens?
Stool
Direct smear preparation of stool samples may reveal the presence of ______.
inflammatory cells
Enterobacteriaceae grow at an optimal temperature of ______.
35 - 37 C
Which organism/s is/are being described?
Grow at low temperatures (1-5 C)
- Serratia spp.
- Yersinia spp.
Serratia spp. and Yersinia spp. grow at a temperature of ______.
1 - 5 C
Which organism/s is/are being described?
Grow at high temperatures (45-50 C)
Escherichia coli
TIP: Read it as Escherichia colHIGH
Escherichia coli grows at a temperature of _____.
45 - 50 C
Colonies of Enterobacteriaceae are visible after ____ of incubation.
18 - 24 hours
Common culture media used or Enterobacteriaceae
- BAP
- CAP
- MacConkey Agar
EMB, HEA
Colonial appearance of Enterobacteriaceae on BAP and CAP
Large, gray, and smooth
Which organism/s is/are being described?
Most likely to have mucoid colonies
- Klebsiella spp.
- Enterobacter spp.
Klebsiella spp. and Enterobacter spp. are most likely to have ____ colonies.
mucoid
A factor that contributes to the mucoid appearance of Klebsiella spp. and Enterobacter spp. is the _____.
Polysaccharide Capsule
Which organism/s is/are being described?
Most likely to produce a “swarm” on blood and chocolate agars
ACRONYM: MVP
* Proteus mirabilis
* Proteus vulgaris
* Proteus penneri
Proteus mirabilis, Proteus vulgaris, and Proteus penneri are most likely to produce a ____ on blood and chocolate agars.
swarm
Which organism/s is/are being described?
Pinpoint colonies at 5% SBA after 24 hours of incubation
Yersinia pestis
Y. pestis colonial appearance on 5% SBA after 24 hours of incubation
Pinpoint
Which culture medium is utilized to produce pinpoint Y. pestis colonies?
5% SBA
Pinpoint colonies of Y. pestis on 5% SBA is produced after ____ of incubation.
24 hours
Which organism/s is/are being described?
Rough, cauliflower appearance of colonies at 5% SBA after 48 hours of incubation
Yersinia pestis
Y. pestis colonial appearance on 5% SBA after 48 hours of incubation
Rough, cauliflower
Which culture medium is utilized to produce rough, cauliflower appearance of Y. pestis colonies?
5% SBA
Rough, cauliflower appearing colonies of Y. pestis on 5% SBA is produced after ____ of incubation.
48 hours
Characteristic of Yersinia pestis grown in broth cultures
Stalactite Pattern
Which organism/s is/are being described?
Exhibits a “stalactite pattern” when grown in broth cultures
Yersinia pestis
Which organism/s is/are being described?
It produces “bull’s-eye” colonies
Yersinia enterocolitica
Yersinia entercolitica produces ____ colonies
bull’s-eye
Mucoid colonies form a ____ when a loop tries to pick it up.
string
Rapid Lactose Fermenters
MNEMONIC: EKE
* Escherichia coli
* Klebsiella spp.
* Enterobacter spp.
Rapid Lactose Fermenters ferment lactose within _____.
18 - 24 hours
________ ferment lactose within 18-24 hours.
Rapid Lactose Fermenters
Late Lactose Fermenters
- Hafnia spp.
- Serratia spp.
- Citrobacter spp.
- Salmonella arizonae
- Shigella sonnei
- Yersinia enterocolitica
Late Lactose Fermenters ferment lactose within _____.
48 - 72 hours
________ ferment lactose within 48-72 hours.
Late Lactose Fermenters
Non-Lactose Fermenters
- All Salmonella EXCEPT S. arizonae
- All Shigella EXCEPT S. sonnei
- All Yersinia EXCEPT Y. enterocolitica
- Proteus spp.
- Providencia spp.
- Morganella spp.
- Edwardsiella spp.
Which medium is best used for characterizing gram (-) bacilli?
MacConkey Agar
What kind of medium (according to function) is MAC agar?
Selective and Differential
How is MAC agar selective?
Selective for gram negative (-) enteric bacilli
How is MAC agar differential?
Differentiates lactose from non-lactose fermenters
Lactose fermenters will color the MAC agar _____.
Dark Pink
Non-Lactose fermenters will color the MAC agar _____.
Colorless
The red pigment of Serratia marcescens on MAC agar is due to the _______.
Production of Prodigiosin
Which species produces a greenish-metallic sheen on EMB agar?
Escherichia coli
Which Biochemical Test?
To determine glucose and lactose, or sucrose utilization and hydrogen sulfide production.
- Triple Sugar Iron Agar (TSI)
- Kligler Iron Agar (KIA)
Purpose of TIA and KIA Test
To determine glucose and lactose, or sucrose utilization and hydrogen sulfide production.
Which Biochemical Test?
To determine the end-products of glucose fermentation.
- Methyl Red
- Voges-Proskauer
Purpose of Methyl Red and VP Test
To determine the end-products of glucose fermentation
Which Biochemical Test?
To determine whether an indole was formed from tryptophan by tryptophanase.
Indole Test
Purpose of Indole Test
To determine whether indole was formed from tryptophan by tryptophanase
Which Biochemical Test?
To determine the hydrolysis of Urea
Urease Test
Purpose of Urease Test
To determine hydrolysis of Urea
Which Biochemical Test?
To determine whether citrate can be used as a sole carbon source
Simmons’ Citrate
Purpose of Simmons’ Citrate
To determine whether citrate can be used as a sole carbon source
Which Biochemical Test?
To determine lysine decarboxylase activity
Lysine Iron Agar (LIA)
Purpose of LIA
To determine lysine decarboxylase activity
Carbohydrate Utilization Tests
- Oxidation-Fermentation Tests
- Triple Sugar Iron Agar Test
- Ortho-Nitrophenyl-B-D-Galactopyranoside Test (ONPG)
Carbohydrate Utilization
Two enzymes necessary for taking up lactose and cleaving it into monosaccharides
- β-galactoside permease (lactose permease)
- β-galactosidase
A transport enzyme which facilitates the entry of lactose in the plasma membrane of bacteria.
β-galactoside permease (lactose permease)
What does β-galactoside permease do?
It acts as a transport enzyme, which facilitates the entry of lactose in the plasma membrane of the bacteria.
β-galactoside permease is also known as _____.
Lactose Permease
It hydrolyzes lactose into glucose and galactose
β-galactosidase
What does β-galactosidase do?
It hydrolyzes lactose into glucose and galactose
RLFs contain the enzyme _________.
β-galactoside permease and β-galactosidase
NLFs contain the enzyme _________.
None
LLFs contain the enzyme ____.
β-galactosidase
Which medium is used for Oxidation-Fermentation Test?
OF Basal Medium
E.g., Hugh-Leifson OFBM
What is the pH indicator for Oxidation-Fermentation Test?
Bromthymol blue
Color of uninoculated OFBM
Green
OFBM color of acid environment
Yellow
OFBM color of alkaline environment
Blue
How is Oxidation-Fermentation Test performed?
- Two tubes of Hugh-Leifson OFBM are inoculated.
- One tube is overlaid with sterile mineral oil to promote an anaerobic environment (closed).
- The other tube is without mineral oil to promote an aerobic environment (open).
What is added to the OFBM medium to promote an anaerobic environment?
Sterile mineral oil
Sterile mineral oil is used for ______.
promoting an anaerobic environment
Reactions in OF Test
When acid is produced on both tubes, it means that _______.
The organism is both an oxidizer and fermenter
Reactions in OF Test
When acid is produced only in the closed tube, it means that _______.
The organism is a fermenter and possible an obligate anaerobe
Reactions in OF Test
When acid is produced only in the open tube, it means that _______.
The organism is an oxidizer
OFBM contains ____.
1% Carbohydrate
Composition of Triple Sugar Iron Agar (TIA)
- 10 parts Lactose
- 10 parts Sucrose
- 1 part Glucose
- Peptone
What is the pH indicator for Triple Sugar Iron Agar (TSI)?
Phenol red
What is the H2S indicator for Triple Sugar Iron Agar (TSI)?
Ferrous sulfate and Sodium thiosulfate
If the pH indicator of TSI turns yellow, it means that ______.
it is acidic
When the pH of TSI drops to 6.8, it means that ______.
it is acidic
What is the color of TSI when carbohydrate is utilized?
Yellow
What is the color of TSI when carbohydrate is not utilized?
Red
What is the color of TSI when there is a production of H2S?
Black
The butt portion of the TSI determines if the organism is able to ferment _____.
Glucose
Which portion of the TSI determines if the organism is a glucose fermenter?
Butt
Which portion of the TSI is anaerobic?
Butt
The slant portion of the TSI determines if the organism is able to ferment _____.
Sucrose/Lactose
Which portion of the TSI determines if the organism is a sucrose/lactose fermenter?
Slant
Which portion of the TSI is aerobic?
Slant
Reactions on TSI Agar
Slant: Acid (yellow)
Butt: Acid (yellow)
A/A
Lactose/Sucrose and Glucose Fermenter
Reactions on TSI Agar
Slant: Alkaline (red)
Butt: Acid (yellow)
K/A
Glucose Fermenter
Reactions on TSI Agar
Slant: Alkaline (red)
Butt: Alkaline (red)
K/K
No fermentation
It means that the organism is not a member of the Enterobacteriaceae Fam
Interpret the TSI Result
Slant: Alkaline (red)
Butt: Acid (yellow)
H2S Production: +
K/A, H2S
For H2S production to occur, it requires an _____ environment.
acidic
What is the indicator for gas production in TSI?
Formation of bubbles or spliiting (space) of the medium in butt
_____ is indicated by the formation of bubbles or splitting (space) of the medium in butt.
Gas production
H2S producing organisms in TSI agar
MNEMONIC: SPACE
* Shigella
* Proteus
* Arizona
* Citrobacter
* Edwardsiella
Which Biochemical Test?
To determine the ability of an organism to produce β-Galactosidase
Ortho-Nitrophenyl-β-D-Galactopyranoside Test (ONPG)
What is the purpose of Ortho-Nitrophenyl-β-D-Galactopyranoside Test (ONPG)?
To determine the ability of an organism to produce β-Galactosidase
____ hydrolyzes ONPG.
β-Galactosidase
β-Galactosidase hydrolyzes ONPG into _______.
galactose and o-nitrophenol
The colorless compound in ONPG test
ONPG
The yellow-colored compound in ONPG test
galactose and o-nitrophenol
If the organism is a non-lactose fermenter, the ONPG is _____.
colorless
If the organism is a lactose fermenter, the ONPG is _____.
yellow
Positive results for ONPG test can generally be seen within ______.
6 hours
Steps in performing ONPG test
- Inoculate the organism in the tube containing 0.85% sterile saline.
- Place ONPG disk
- Incubate for 4 hours at 37 C
- Observe color change
The ONPG tube contains ______.
0.85% sterile saline
Incubation period for ONPG test
4 hours at 37 C
Tests Under Glucose Metabolism and its Metabolic Products
- Methyl Red
- Voges-Proskauer
Which pathway is utilized for Methyl Red Test?
Mixed Acid Fermentation Pathway
If glucose is metabolized by the Mixed Acid Fermentation Pathway, ____ are produced, which results in a low pH.
stable acid end-products
If glucose is metabolized by the Mixed Acid Fermentation Pathway, stable acid end-products are produced, which results in a ____.
low pH (4.4)
Reagent for MR Test
Methyl Red
Positive result for MR Test
Red color
Negative result for MR Test
Yellow color
Incubation period for MRVP Test
48 hours at 35-37 C
Enumerate the steps for MR Test
- Inoculate organism in MRVP Broth
- Incubate for 48 hours at 35-37 C
- Divide the tubes into two (1 vor VP)
- Add MR reagent
- Observe color change
Which Biochemical Test?
To determine the ability of an organism to convert acid products into acetoin and 2,3-butanediol.
Voges-Proskauer Test
Purpose of VP Test
To detect the organism’s ability to convert acid products to acetoin and 2,3-butanediol.
VP test determines the ability of an organism to convert acid products to ________.
acetoin and 2,3-butanediol
Which pathway is used for VP Test?
Butylene Glycol Pathway
It is added as a catalyst or color intensifier for VP Test.
α-naphthol
α-naphthol function in VP Test
Catalyst and Color intensifier
Reagent for VP Test
40% KOH or NaOH
Positive result for VP Test
Red color
Negative result for VP Test
Yellow color
Enumerate the steps for VP Test
- Inoculate organism in MRVP Broth
- Incubate for 48 hours at 35-37 C
- Divide the tubes into two (1 vor VP)
- Add α-naphthol
- Add 40% KOH or NaOH
- Observe color change
Amino Acid Utilization Tests
- Lysine Iron Agar (LIA)
- Phenylalanine Deaminase Test (PAD)
Which Biochemical Test?
To differentiate gram (-) bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S).
Lysine Iron Agar (LIA)
Purpose of Lysine Iron Agar (LIA)
To differentiate gram (-) bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S)
pH indicator for LIA
Bromcresol purple
The butt portion of the LIA determines if the organism is able to _____.
Decarboxylate lsyine
Which portion of the LIA determines if the organism can decarboxylate lysine?
Butt
The slant portion of the LIA determines if the organism is able to _____.
Deaminate lysine
Which portion of the LIA determines if the organism can deaminate lysine?
Slant
(+) result for Lysine Deaminase
Burgundy/Red color
(-) result for Lysine Deaminase
Purple color
(+) result for Lysine Decarboxylase
Purple color
(-) result for Lysine Decarboxylase
Yellow color
When the butt portion of LIA turns purple, it means that ____ is produced.
Cadaverine
It neutralizes the organic acid fromed during glucose fermentation
Cadaverine
What does Cadaverine do?
It It neutralizes the organic acid fromed during glucose fermentation
When the butt portion of LIA turns yellow, it means that _____.
Glucose is fermented
How is LIA performed?
- Using an inoculating needle, inoculate LIA by TWICE stabbing through the center of the medium to the bottom of the tube and then streaking the slant.
- Cap the tube tightly and incubate for 18-24 hours at 35-37 C.
Reactions on LIA
Slant: Alkaline (purple)
Butt: Alkaline (purple)
K/K
Lysine decarboxylation and no fermentation of glucose
Reactions on LIA
Slant: Alkaline (purple)
Butt: Acid (yellow)
K/A
Glucose fermentation
Reactions on LIA Agar
Slant: Red
Butt: Acid (yellow)
R/A
Lysine deamination and glucose fermentation
Deaminase (+) members of Enterobacteriaceae Family
ACRONYM: PPM
* Proteus spp.
* Providencia spp.
* Morganella spp.
H2S (+) members of Enterobacteriaceae in LIA
ACRONYM: SACE
* Salmonella spp.
* Arizona spp.
* Citrobacter spp.
* Edwardsiella spp.
Which Biochemical Test?
To determine whether an organism possesses the enzyme that deaminates phenylalanine to phenylpyruvic acid.
Phenylalanine Deaminase Test (PAD)
Purpose of PAD
To determine whether an organism possesses the enzyme that deaminates phenylalanine to phenylpyruvic acid
Which enzyme deaminates phenylalanine?
Phenylalanine deaminase
When phenylalanine is deaminated, it produces _____.
Phenylpyruvic acid
(+) result for PAD
Green color
How is PAD performed?
- Inoculate a bacterial colony over the surface of the slant.
- After incubation, add 10% FeCl3
- Observe color change
PAD contains ____.
0.2% concentration of Phenylalanine
What reagent is used for PAD?
10% FeCl3
Which medium is used for SIM?
SIM agar
What type of agar is used for SIM according to consistency?
semi-solid
What are the 3 factors that we observe in SIM?
- Sulfide production
- Indole production
- Motility
Sulfide production in SIM agar is indicated by the presence of _______.
black precipitate
Purpose of Indole in SIM
To determine which organisms possess the enzyme Tryptophanase
Which enzyme deaminates Tryptophan?
Trytophanase
What does Trytophanase do?
It is an enzyme that deaminates Trytophan
(-) result for Indole in SIM
Pink color
What are the intermediate degradation products when Tryptophan is deaminated?
Indole, pyruvic acid, and ammonia
What is the reagent used for SIM?
0.5 mL Ehrlich’s or Kovac’s Reagent
(+) result for Indole in SIM
Presence of a red ring
Motility in SIM agar is indicated by _______.
Cloudiness or Turbidity of solution
Which medium is used for Citrate Utilization?
Simmon’s Citrate Agar
Which is used as a sole carbon source by organisms (+) for Citrate Utilization?
Sodium citrate
Which is used as a sole nitrogen source by organisms (+) for Citrate Utilization?
Ammonium salts
pH indicator for Citrate Utilization
Bromthymol blue
(+) result for Citrate Utilization
Blue or growth
(-) result for Citrate Utilization
Green color
Incubation period for Citrate Utilization
35-37 for 7 days
______ hydrolyzes Urea
Urease
What does Urease do?
Hydrolyze urea
Urease hydrolyze urea to form ______.
Ammonia, water, and CO2
Which medium is used for Urease Test?
Christensen’s Urea Agar
(+) result for Urease Test
Magenta (pink) color
What is the pH of the (+) result for Urease Test?
8.1
(-) result for Urease Test
No change in color (remains light orange)
What is the original color of the medium used for Urease Test?
Light orange (pH 6.1)
Rapid urease producers
ACRONYM: PPM
* Proteus spp.
* Providencia spp.
* Morganella spp.
Slow urease producers
ACRONYM: CKEYS
* Citrobacter spp.
* Klebsiella spp.
* Enterobacter spp.
* Yersinia spp.
* Serratia spp.
Which Biochemical Test?
To differentiate organisms based on the production of deoxyribonuclease
DNA Hyrolysis Test (DNAse Test Agar)
Purpose of DNA Hydrolysis Test
To differentiate organisms based on the production of deoxyribonuclease
(+) result for DNAse Test
Colorless
(-) result for DNAse Test
Green color
Which organisms are (+) DNAse Test?
- Serratia spp.
- Staphylococcus aureus
- Moraxella catarrhalis
Which Biochemical Test?
To determine the ability of an organism to produce gelatinase
Gelatin Liquefaction
Purpose of Gelatin Liquefaction
To determine the organism’s ability to produce gelatinase
A proteolytic enzyme that breaks down gelatin into amino acids
Gelatinase
What does gelatinase do?
Break down gelatin into amino acids
Gelatinase breaks down gelatin into _______.
amino acids
(+) result for Gelatin Liquefaction
Liquefaction of gelatin
Which is used as a sole carbon source by organisms (+) for Malonate Utilization?
Malonate
Which is used as a nitrogen source by organisms (+) for Malonate Utilization?
Ammonium sulfate
pH indicator for Malonate Utilization
Bromthymol blue
(+) result for Malonate Utilization
Blue color
(-) result for Malonate Utilization
Green color
Which Biochemical Test?
To determine the ability of an organism to reduce nitrate to nitrite and reduce nitrite to nitrogen gas.
Nitrate and Nitrite Reduction
Purpose of Nitrate and Nitrite Reduction
To determine the ability of an organism to reduce nitrate to nitrite and reduce nitrite to nitrogen gas.
What is the reagent used for Nitrate and Nitrite Reduction?
- N,N-dimethyl-α-naphthylamine
- Sulfanilic acid
The reagent used for Nitrate and Nitrite Reduction is added after ______.
24 hours of incubation
(+) result for Nitrate and Nitrite Reduction
Red color
(-) result for Nitrate and Nitrite Reduction
Yellow color
The red color in Nitrate and Nitrite Reduction indicates the presence of _____.
nitrite
Which Biochemical Test?
To determine the presence of cytochrome oxidase
Oxidase Test
Purpose of Oxidase Test
To determine the presence of cytochrome oxidase
What is the reagent used for Oxidase Test?
0.5% or 1% of tetramethyl-p-phenylenediamine dihydrochloride
(+) result for Oxidase Test
Lavender color within 10 to 15 seconds
The (+) result for Oxidase Test appears within ______.
10 to 15 seconds
Measure amount of antimicrobial agent in serum or body fluid
Antimicrobial Concentration Test
Measure of lowest concentration of antimicrobial agent that kills a bacterial isolate.
Minimum Bactericidal Concentration Test
Measure of highest dilution or titer of a patient’s serum
Serum Bactericidal Test
Measure of susceptibility of a bacterial isolate to a combination of two or more antimicrobial agents.
Synergy Test
Measure of rate of killing of bacteria by an antimicrobial agent
Time-kill Assay