Biochemical Identification of Gram Negative Bacteria (LEC/LAB) Flashcards
1
Q
Gram stain reaction and morphology of Enterobacteriaceae
A
Gram Negative (-) Bacilli and Cocobacilli
2
Q
Aerotolerance of Enterobacteriaceae
A
Facultative Anaerobes
3
Q
Enterobacteriaceae do not produce ________.
A
Cytochrome C oxidase
4
Q
Which member of Enterobacteriaceae produces cytochrome c oxidase?
A
Plesiomonas spp.
5
Q
TRUE OR FALSE.
Enterobacteriaceae family produces cytochrome C oxidase.
A
False
6
Q
Which carbohydrate/s can be fermented by the members of Enterobacteriaceae?
A
Glucose
7
Q
Are Enterobacteriaceae motile?
A
Yes (at room temperature)
8
Q
At which temperature are Enterobacteriaceae motile?
A
Room Temperature (20-25 C)
9
Q
Which member/s of the Enterobacteriaceae family are not motile at room temperature?
A
MNEMONIC: SKY
* Shigella spp.
* Klebsiella spp.
* Yersinia spp.
10
Q
Specimen of Choice for Enterobacteriaceae
A
Fecal/Stool Specimens
Collected within first 4 days of illness & before administration of ATBs
11
Q
Specimen Collection
Fecal/stool specimens are collected within ____ of illness and before administration of antibiotics.
A
first 4 days
12
Q
Specimen Collection
Specimens should be processed within ____ of collection.
A
1-2 hours
13
Q
Specimen Collection
What should be done if the specimen cannot be processed within the ideal time frame (1-2 hours)?
A
Placed in a transport medium at the temperature of 4 C
14
Q
Specimen Collection
Specimens in a transport medium must be kept at a temperature of ____.
A
4 C
15
Q
Specimen Collection
Transport medium for Enterobacteriaceae
A
- Cary-Blair
- Amies
- Stuart
16
Q
Which step in biochemical identification of Enterobacteriaceae is not helpful in identifying enteric pathogens?
A
Direct Smear Examination/Direct Microscopic Examination/Gram Staining
17
Q
Which specimen sample is not helpful in direct smear preparation for identifying enteric pathogens?
A
Stool
18
Q
Direct smear preparation of stool samples may reveal the presence of ______.
A
inflammatory cells
19
Q
Enterobacteriaceae grow at an optimal temperature of ______.
A
35 - 37 C
20
Q
Which organism/s is/are being described?
Grow at low temperatures (1-5 C)
A
- Serratia spp.
- Yersinia spp.
21
Q
Serratia spp. and Yersinia spp. grow at a temperature of ______.
A
1 - 5 C
22
Q
Which organism/s is/are being described?
Grow at high temperatures (45-50 C)
A
Escherichia coli
TIP: Read it as Escherichia colHIGH
23
Q
Escherichia coli grows at a temperature of _____.
A
45 - 50 C
24
Q
Colonies of Enterobacteriaceae are visible after ____ of incubation.
A
18 - 24 hours
25
Common culture media used or Enterobacteriaceae
* BAP
* CAP
* MacConkey Agar
| **EMB, HEA**
26
Colonial appearance of Enterobacteriaceae on BAP and CAP
Large, gray, and smooth
27
# **Which organism/s is/are being described?**
Most likely to have mucoid colonies
* *Klebsiella* spp.
* *Enterobacter* spp.
28
*Klebsiella* spp. and *Enterobacter* spp. are most likely to have ____ colonies.
mucoid
29
A factor that contributes to the mucoid appearance of *Klebsiella* spp. and *Enterobacter* spp. is the _____.
Polysaccharide Capsule
30
# **Which organism/s is/are being described?**
Most likely to produce a "swarm" on blood and chocolate agars
**ACRONYM: MVP**
* *Proteus **m**irabilis*
* *Proteus **v**ulgaris*
* *Proteus **p**enneri*
31
*Proteus mirabilis*, *Proteus vulgaris*, and *Proteus penneri* are most likely to produce a ____ on blood and chocolate agars.
swarm
32
# **Which organism/s is/are being described?**
Pinpoint colonies at 5% SBA after 24 hours of incubation
*Yersinia pestis*
33
*Y. pestis* colonial appearance on 5% SBA after 24 hours of incubation
Pinpoint
34
Which culture medium is utilized to produce pinpoint *Y. pestis* colonies?
5% SBA
35
Pinpoint colonies of *Y. pestis* on 5% SBA is produced after ____ of incubation.
24 hours
36
# **Which organism/s is/are being described?**
Rough, cauliflower appearance of colonies at 5% SBA after 48 hours of incubation
*Yersinia pestis*
37
*Y. pestis* colonial appearance on 5% SBA after 48 hours of incubation
Rough, cauliflower
38
Which culture medium is utilized to produce rough, cauliflower appearance of *Y. pestis* colonies?
5% SBA
39
Rough, cauliflower appearing colonies of *Y. pestis* on 5% SBA is produced after ____ of incubation.
48 hours
40
Characteristic of *Yersinia pestis* grown in broth cultures
Stalactite Pattern
41
# **Which organism/s is/are being described?**
Exhibits a **"stalactite pattern"** when grown in broth cultures
*Yersinia pestis*
42
# **Which organism/s is/are being described?**
It produces **"bull's-eye"** colonies
*Yersinia enterocolitica*
43
*Yersinia entercolitica* produces ____ colonies
bull's-eye
44
Mucoid colonies form a ____ when a loop tries to pick it up.
string
45
Rapid Lactose Fermenters
**MNEMONIC: EKE**
* ***E**scherichia coli*
* ***K**lebsiella* spp.
* ***E**nterobacter* spp.
46
Rapid Lactose Fermenters ferment lactose within _____.
18 - 24 hours
47
________ ferment lactose within 18-24 hours.
Rapid Lactose Fermenters
48
Late Lactose Fermenters
* *Hafnia* spp.
* *Serratia* spp.
* *Citrobacter* spp.
* *Salmonella arizonae*
* *Shigella sonnei*
* *Yersinia enterocolitica*
49
Late Lactose Fermenters ferment lactose within _____.
48 - 72 hours
50
________ ferment lactose within 48-72 hours.
Late Lactose Fermenters
51
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.
52
Which medium is best used for characterizing gram (-) bacilli?
MacConkey Agar
53
What kind of medium (according to function) is MAC agar?
Selective and Differential
54
How is MAC agar selective?
Selective for gram negative (-) enteric bacilli
55
How is MAC agar differential?
Differentiates lactose from non-lactose fermenters
56
Lactose fermenters will color the MAC agar _____.
Dark Pink
57
Non-Lactose fermenters will color the MAC agar _____.
Colorless
58
The red pigment of *Serratia marcescens* on MAC agar is due to the _______.
Production of Prodigiosin
59
Which species produces a greenish-metallic sheen on EMB agar?
*Escherichia coli*
60
# **Which Biochemical Test?**
To determine glucose and lactose, or sucrose utilization and hydrogen sulfide production.
* Triple Sugar Iron Agar (TSI)
* Kligler Iron Agar (KIA)
61
Purpose of TIA and KIA Test
To determine glucose and lactose, or sucrose utilization and hydrogen sulfide production.
62
# **Which Biochemical Test?**
To determine the end-products of glucose fermentation.
* Methyl Red
* Voges-Proskauer
63
Purpose of Methyl Red and VP Test
To determine the end-products of glucose fermentation
64
# **Which Biochemical Test?**
To determine whether an indole was formed from tryptophan by tryptophanase.
Indole Test
65
Purpose of Indole Test
To determine whether indole was formed from tryptophan by tryptophanase
66
# **Which Biochemical Test?**
To determine the hydrolysis of Urea
Urease Test
67
Purpose of Urease Test
To determine hydrolysis of Urea
68
# **Which Biochemical Test?**
To determine whether citrate can be used as a sole carbon source
Simmons' Citrate
69
Purpose of Simmons' Citrate
To determine whether citrate can be used as a sole carbon source
70
# **Which Biochemical Test?**
To determine lysine decarboxylase activity
Lysine Iron Agar (LIA)
71
Purpose of LIA
To determine lysine decarboxylase activity
72
Carbohydrate Utilization Tests
* Oxidation-Fermentation Tests
* Triple Sugar Iron Agar Test
* Ortho-Nitrophenyl-B-D-Galactopyranoside Test (ONPG)
73
# **Carbohydrate Utilization**
Two enzymes necessary for taking up lactose and cleaving it into monosaccharides
* β-galactoside permease (lactose permease)
* β-galactosidase
74
A transport enzyme which facilitates the entry of lactose in the plasma membrane of bacteria.
β-galactoside permease (lactose permease)
75
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.
76
β-galactoside permease is also known as _____.
Lactose Permease
77
It hydrolyzes lactose into glucose and galactose
β-galactosidase
78
What does β-galactosidase do?
It hydrolyzes lactose into glucose and galactose
79
RLFs contain the enzyme _________.
β-galactoside permease and β-galactosidase
80
NLFs contain the enzyme _________.
None
81
LLFs contain the enzyme ____.
β-galactosidase
82
Which medium is used for Oxidation-Fermentation Test?
OF Basal Medium
| **E.g., Hugh-Leifson OFBM**
83
What is the pH indicator for Oxidation-Fermentation Test?
Bromthymol blue
84
Color of uninoculated OFBM
Green
85
OFBM color of acid environment
Yellow
86
OFBM color of alkaline environment
Blue
87
How is Oxidation-Fermentation Test performed?
1. Two tubes of Hugh-Leifson OFBM are inoculated.
2. One tube is overlaid with sterile mineral oil to promote an anaerobic environment (closed).
3. The other tube is without mineral oil to promote an aerobic environment (open).
88
What is added to the OFBM medium to promote an anaerobic environment?
Sterile mineral oil
89
Sterile mineral oil is used for ______.
promoting an anaerobic environment
90
# **Reactions in OF Test**
When acid is produced on both tubes, it means that _______.
The organism is both an oxidizer and fermenter
91
# **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
92
# **Reactions in OF Test**
When acid is produced only in the open tube, it means that _______.
The organism is an oxidizer
93
OFBM contains ____.
1% Carbohydrate
94
Composition of Triple Sugar Iron Agar (TIA)
* 10 parts Lactose
* 10 parts Sucrose
* 1 part Glucose
* Peptone
95
What is the pH indicator for Triple Sugar Iron Agar (TSI)?
Phenol red
96
What is the H2S indicator for Triple Sugar Iron Agar (TSI)?
Ferrous sulfate and Sodium thiosulfate
97
If the pH indicator of TSI turns yellow, it means that ______.
it is acidic
98
When the pH of TSI drops to 6.8, it means that ______.
it is acidic
99
What is the color of TSI when carbohydrate is utilized?
Yellow
100
What is the color of TSI when carbohydrate is not utilized?
Red
101
What is the color of TSI when there is a production of H2S?
Black
102
The butt portion of the TSI determines if the organism is able to ferment _____.
Glucose
103
Which portion of the TSI determines if the organism is a glucose fermenter?
Butt
104
Which portion of the TSI is anaerobic?
Butt
105
The slant portion of the TSI determines if the organism is able to ferment _____.
Sucrose/Lactose
106
Which portion of the TSI determines if the organism is a sucrose/lactose fermenter?
Slant
107
Which portion of the TSI is aerobic?
Slant
108
# **Reactions on TSI Agar**
**Slant:** Acid (yellow)
**Butt:** Acid (yellow)
A/A
Lactose/Sucrose and Glucose Fermenter
109
# **Reactions on TSI Agar**
**Slant:** Alkaline (red)
**Butt:** Acid (yellow)
K/A
Glucose Fermenter
110
# **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
111
# **Interpret the TSI Result**
**Slant:** Alkaline (red)
**Butt:** Acid (yellow)
**H2S Production:** +
K/A, H2S
112
For H2S production to occur, it requires an _____ environment.
acidic
113
What is the indicator for gas production in TSI?
Formation of bubbles or spliiting (space) of the medium in butt
114
_____ is indicated by the formation of bubbles or splitting (space) of the medium in butt.
Gas production
115
H2S producing organisms in TSI agar
**MNEMONIC: SPACE**
* ***S**higella*
* ***P**roteus*
* ***A**rizona*
* ***C**itrobacter*
* ***E**dwardsiella*
116
# **Which Biochemical Test?**
To determine the ability of an organism to produce β-Galactosidase
Ortho-Nitrophenyl-β-D-Galactopyranoside Test (ONPG)
117
What is the purpose of Ortho-Nitrophenyl-β-D-Galactopyranoside Test (ONPG)?
To determine the ability of an organism to produce β-Galactosidase
118
____ hydrolyzes ONPG.
β-Galactosidase
119
β-Galactosidase hydrolyzes ONPG into _______.
galactose and o-nitrophenol
120
The colorless compound in ONPG test
ONPG
121
The yellow-colored compound in ONPG test
galactose and o-nitrophenol
122
If the organism is a non-lactose fermenter, the ONPG is _____.
colorless
123
If the organism is a lactose fermenter, the ONPG is _____.
yellow
124
Positive results for ONPG test can generally be seen within ______.
6 hours
125
Steps in performing ONPG test
1. Inoculate the organism in the tube containing 0.85% sterile saline.
2. Place ONPG disk
3. Incubate for 4 hours at 37 C
4. Observe color change
126
The ONPG tube contains ______.
0.85% sterile saline
127
Incubation period for ONPG test
4 hours at 37 C
128
Tests Under Glucose Metabolism and its Metabolic Products
* Methyl Red
* Voges-Proskauer
129
Which pathway is utilized for Methyl Red Test?
Mixed Acid Fermentation Pathway
130
If glucose is metabolized by the Mixed Acid Fermentation Pathway, ____ are produced, which results in a low pH.
stable acid end-products
131
If glucose is metabolized by the Mixed Acid Fermentation Pathway, stable acid end-products are produced, which results in a ____.
low pH (4.4)
132
Reagent for MR Test
Methyl Red
133
Positive result for MR Test
Red color
134
Negative result for MR Test
Yellow color
135
Incubation period for MRVP Test
48 hours at 35-37 C
136
Enumerate the steps for MR Test
1. Inoculate organism in MRVP Broth
2. Incubate for 48 hours at 35-37 C
3. Divide the tubes into two (1 vor VP)
4. Add MR reagent
5. Observe color change
137
# **Which Biochemical Test?**
To determine the ability of an organism to convert acid products into acetoin and 2,3-butanediol.
Voges-Proskauer Test
138
Purpose of VP Test
To detect the organism's ability to convert acid products to acetoin and 2,3-butanediol.
139
VP test determines the ability of an organism to convert acid products to ________.
acetoin and 2,3-butanediol
140
Which pathway is used for VP Test?
Butylene Glycol Pathway
141
It is added as a catalyst or color intensifier for VP Test.
α-naphthol
142
α-naphthol function in VP Test
Catalyst and Color intensifier
143
Reagent for VP Test
40% KOH or NaOH
144
Positive result for VP Test
Red color
145
Negative result for VP Test
Yellow color
146
Enumerate the steps for VP Test
1. Inoculate organism in MRVP Broth
2. Incubate for 48 hours at 35-37 C
3. Divide the tubes into two (1 vor VP)
4. Add α-naphthol
5. Add 40% KOH or NaOH
6. Observe color change
147
Amino Acid Utilization Tests
* Lysine Iron Agar (LIA)
* Phenylalanine Deaminase Test (PAD)
148
# **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)
149
Purpose of Lysine Iron Agar (LIA)
To differentiate gram (-) bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S)
150
pH indicator for LIA
Bromcresol purple
151
The butt portion of the LIA determines if the organism is able to _____.
Decarboxylate lsyine
152
Which portion of the LIA determines if the organism can decarboxylate lysine?
Butt
153
The slant portion of the LIA determines if the organism is able to _____.
Deaminate lysine
154
Which portion of the LIA determines if the organism can deaminate lysine?
Slant
155
(+) result for Lysine Deaminase
Burgundy/Red color
156
(-) result for Lysine Deaminase
Purple color
157
(+) result for Lysine Decarboxylase
Purple color
158
(-) result for Lysine Decarboxylase
Yellow color
159
When the butt portion of LIA turns purple, it means that ____ is produced.
Cadaverine
160
It neutralizes the organic acid fromed during glucose fermentation
Cadaverine
161
What does Cadaverine do?
It It neutralizes the organic acid fromed during glucose fermentation
162
When the butt portion of LIA turns yellow, it means that _____.
Glucose is fermented
163
How is LIA performed?
1. 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.
2. Cap the tube tightly and incubate for 18-24 hours at 35-37 C.
164
# **Reactions on LIA**
**Slant:** Alkaline (purple)
**Butt:** Alkaline (purple)
K/K
Lysine decarboxylation and no fermentation of glucose
165
# **Reactions on LIA**
**Slant:** Alkaline (purple)
**Butt:** Acid (yellow)
K/A
Glucose fermentation
166
# **Reactions on LIA Agar**
**Slant:** Red
**Butt:** Acid (yellow)
R/A
Lysine deamination and glucose fermentation
167
Deaminase (+) members of Enterobacteriaceae Family
**ACRONYM: PPM**
* ***P**roteus* spp.
* ***P**rovidencia* spp.
* ***M**organella* spp.
168
H2S (+) members of Enterobacteriaceae in LIA
**ACRONYM: SACE**
* ***S**almonella* spp.
* ***A**rizona* spp.
* ***C**itrobacter* spp.
* ***E**dwardsiella* spp.
169
# **Which Biochemical Test?**
To determine whether an organism possesses the enzyme that deaminates phenylalanine to phenylpyruvic acid.
Phenylalanine Deaminase Test (PAD)
170
Purpose of PAD
To determine whether an organism possesses the enzyme that deaminates phenylalanine to phenylpyruvic acid
171
Which enzyme deaminates phenylalanine?
Phenylalanine deaminase
172
When phenylalanine is deaminated, it produces _____.
Phenylpyruvic acid
173
(+) result for PAD
Green color
174
How is PAD performed?
1. Inoculate a bacterial colony over the surface of the slant.
2. After incubation, add 10% FeCl3
3. Observe color change
175
PAD contains ____.
0.2% concentration of Phenylalanine
176
What reagent is used for PAD?
10% FeCl3
177
Which medium is used for SIM?
SIM agar
178
What type of agar is used for SIM according to consistency?
semi-solid
179
What are the 3 factors that we observe in SIM?
* Sulfide production
* Indole production
* Motility
180
Sulfide production in SIM agar is indicated by the presence of _______.
black precipitate
181
Purpose of Indole in SIM
To determine which organisms possess the enzyme Tryptophanase
182
Which enzyme deaminates Tryptophan?
Trytophanase
183
What does Trytophanase do?
It is an enzyme that deaminates Trytophan
184
(-) result for Indole in SIM
Pink color
185
What are the intermediate degradation products when Tryptophan is deaminated?
Indole, pyruvic acid, and ammonia
186
What is the reagent used for SIM?
0.5 mL Ehrlich's or Kovac's Reagent
187
(+) result for Indole in SIM
Presence of a red ring
188
Motility in SIM agar is indicated by _______.
Cloudiness or Turbidity of solution
189
Which medium is used for Citrate Utilization?
Simmon's Citrate Agar
190
Which is used as a sole carbon source by organisms (+) for Citrate Utilization?
Sodium citrate
191
Which is used as a sole nitrogen source by organisms (+) for Citrate Utilization?
Ammonium salts
192
pH indicator for Citrate Utilization
Bromthymol blue
193
(+) result for Citrate Utilization
Blue or growth
194
(-) result for Citrate Utilization
Green color
195
Incubation period for Citrate Utilization
35-37 for 7 days
196
______ hydrolyzes Urea
Urease
197
What does Urease do?
Hydrolyze urea
198
Urease hydrolyze urea to form ______.
Ammonia, water, and CO2
199
Which medium is used for Urease Test?
Christensen's Urea Agar
200
(+) result for Urease Test
Magenta (pink) color
201
What is the pH of the (+) result for Urease Test?
8.1
202
(-) result for Urease Test
No change in color (remains light orange)
203
What is the original color of the medium used for Urease Test?
Light orange (pH 6.1)
204
Rapid urease producers
**ACRONYM: PPM**
* ***P**roteus* spp.
* ***P**rovidencia* spp.
* ***M**organella* spp.
205
Slow urease producers
**ACRONYM: CKEYS**
* ***C**itrobacter* spp.
* ***K**lebsiella* spp.
* ***E**nterobacter* spp.
* ***Y**ersinia* spp.
* ***S**erratia* spp.
206
# **Which Biochemical Test?**
To differentiate organisms based on the production of deoxyribonuclease
DNA Hyrolysis Test (DNAse Test Agar)
207
Purpose of DNA Hydrolysis Test
To differentiate organisms based on the production of deoxyribonuclease
208
(+) result for DNAse Test
Colorless
209
(-) result for DNAse Test
Green color
210
Which organisms are (+) DNAse Test?
* *Serratia* spp.
* *Staphylococcus aureus*
* *Moraxella catarrhalis*
211
# **Which Biochemical Test?**
To determine the ability of an organism to produce gelatinase
Gelatin Liquefaction
212
Purpose of Gelatin Liquefaction
To determine the organism's ability to produce gelatinase
213
A proteolytic enzyme that breaks down gelatin into amino acids
Gelatinase
214
What does gelatinase do?
Break down gelatin into amino acids
215
Gelatinase breaks down gelatin into _______.
amino acids
216
(+) result for Gelatin Liquefaction
Liquefaction of gelatin
217
Which is used as a sole carbon source by organisms (+) for Malonate Utilization?
Malonate
218
Which is used as a nitrogen source by organisms (+) for Malonate Utilization?
Ammonium sulfate
219
pH indicator for Malonate Utilization
Bromthymol blue
220
(+) result for Malonate Utilization
Blue color
221
(-) result for Malonate Utilization
Green color
222
# **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
223
Purpose of Nitrate and Nitrite Reduction
To determine the ability of an organism to reduce nitrate to nitrite and reduce nitrite to nitrogen gas.
224
What is the reagent used for Nitrate and Nitrite Reduction?
* N,N-dimethyl-α-naphthylamine
* Sulfanilic acid
225
The reagent used for Nitrate and Nitrite Reduction is added after ______.
24 hours of incubation
226
(+) result for Nitrate and Nitrite Reduction
Red color
227
(-) result for Nitrate and Nitrite Reduction
Yellow color
228
The red color in Nitrate and Nitrite Reduction indicates the presence of _____.
nitrite
229
# **Which Biochemical Test?**
To determine the presence of cytochrome oxidase
Oxidase Test
230
Purpose of Oxidase Test
To determine the presence of cytochrome oxidase
231
What is the reagent used for Oxidase Test?
0.5% or 1% of tetramethyl-p-phenylenediamine dihydrochloride
232
(+) result for Oxidase Test
Lavender color within 10 to 15 seconds
233
The (+) result for Oxidase Test appears within ______.
10 to 15 seconds
234
Measure amount of antimicrobial agent in serum or body fluid
Antimicrobial Concentration Test
235
Measure of lowest concentration of antimicrobial agent that kills a bacterial isolate.
Minimum Bactericidal Concentration Test
236
Measure of highest dilution or titer of a patient's serum
Serum Bactericidal Test
237
Measure of susceptibility of a bacterial isolate to a combination of two or more antimicrobial agents.
Synergy Test
238
Measure of rate of killing of bacteria by an antimicrobial agent
Time-kill Assay
