Tests for Gram-Negative (Enterobact Flashcards
1
Q
Principle of Gram Sure
A
L-alanine-7-amido-4-methylcoumarin + aminopeptidase (from Gram neg) → blue fluorescence
2
Q
Indicator of Gram Sure
A
Longwave UV light
3
Q
Incubation for Gram Sure
A
22-25°C for 5-10 minutes
4
Q
Positive QC for Gram Sure
A
Blue fluorescence (E. coli)
5
Q
Negative QC for Gram Sure
A
No fluorescence (S. aureus)
6
Q
Differentiates Enterobacterales from other GN bacilli
A
Oxidase test (Kovac’s method)
7
Q
Principle of Oxidase Test (Kovac’s Method)
A
Tetramethyl-p-phenylenediamine dihydrochloride + cytochrome oxidase → indophenol
8
Q
Incubation for Oxidase Test (Kovac’s Method)
A
22-25°C, within 10 seconds
9
Q
Positive QC for Oxidase Test
A
Dark purple/Deep blue (P. aeruginosa)
10
Q
Negative QC for Oxidase Test
A
No color change (E. coli)
11
Q
Aid in ID of Gram-negative along with Gram stain
A
Gram Sure
12
Q
Differentiates Enterobacterales from other GN bacilli
A
Oxidase test (Kovac’s method)
13
Q
Positive for all Enterobacteriales/Enterobacteriaceae
A
Nitrate Reduction
14
Q
Principle of nitrate reduction
A
Nitrate + bacterial nitrate reductase → Nitrite
15
Q
Medium for nitrate and nitrite reduction
A
Peptone broth
16
Q
Indicator/Reagent for Solution A in nitrate/nitrite reduction
A
Sulfanilic acid
17
Q
Indicator/Reagent for Solution B in nitrate/nitrite reduction
A
α-naphthylamine
18
Q
Durham tube purpose in nitrate/nitrite
A
N2 gas (detection is positive)
19
Q
Incubation of nitrate and nitrite reduction
A
35-37°C, 48 hours
20
Q
Positive QC for nitrate reduction
A
RED after addition of indicator (E. coli)
21
Q
Negative QC for nitrate reduction
A
Colorless or change of color after zinc dust (Acinetobacter baumanii)
22
Q
Positive QC for nitrite reduction
A
Colorless, gas production (P. mirabilis)
23
Q
Negative QC for nitrite reduction
A
Red, no gas (Acinetobacter baumanii)
24
Q
Tests bacteria for oxidative or fermentative ability
A
Oxidation and Fermentation (CDC Method)
25
Principle for oxidation and fermentation (CDC Method)
6 Carbohydrates: Glucose, Xylose, Mannitol, Maltose, Lactose, Sucrose → Acids
26
Medium for oxidation and fermentation (CDC Method)
Agar deep + 1 carbohydrate, Anaerobic: Sterile melted petrolatum or paraffin oil
27
Indicator/Reagent for oxidation and fermentation (CDC Method)
Phenol red
28
Incubation of OF (CDC Method)
35-37°C, Up to 7 days
29
QC positive for OF method (CDC Method)
Yellow (acids); Oxidizer: P. aeruginosa; Fermenter: E. coli
30
For decarboxylase-producing Enterobacteria
Moeller's decarboxylase test
31
Principle of Moeller's test
Lysine → Cadaverine; Ornithine → Putrescine; Arginine → Citrulline
32
Medium for Moeller's decarboxylase test
Moeller’s decarboxylase medium, add 4mm Mineral oil
33
Indicator of Moeller's decarboxylase test
Bromocresol purple and Cresol red
34
Incubation period for Moeller's decarboxylase test
35-37°C, Examine at 24, 48, 72, and 96 hours
35
Positive QC for Moeller’s Decarboxylase test Lysine (purple)
K. pneumoniae
36
Positive QC for Moeller’s Decarboxylase test Ornithine (purple)
K. aerogenes
37
Positive QC for Moeller’s Decarboxylase test Arginine (purple)
P. aeruginosa
38
Negative QC for Moeller’s Decarboxylase test Arginine
E. coli
39
Negative QC for Moeller’s Decarboxylase test Ornithine
P. vulgaris
40
Negative QC for Moeller’s Decarboxylase test Lysine
C. freundii
41
LIA is a test for
Enterobacterales/Enterobacteriaceae
42
Principle of LIA aerobic and anaerobic
Aerobic Slant: Lysine; Anaerobic Butt: Glucose
43
Purple slant LIA (K)
Lysine decarboxylase
44
Red slant (R) LIA
Lysine deamination
45
Glucose fermentation (A) color
Yellow butt (A)
46
Black precipitate in LIA indicates
H2S production
47
Lysine Iron Agar reactions: Positive Lysine decarboxylation (LDC+)
K/K (ALK/ALK)
48
Lysine Iron Agar reactions: Negative Lysine decarboxylation (LDC-)
K/A (ALK/ACD)
49
Lysine Iron Agar reactions: Positive Lysine deamination (LDA+)
R/A or R/Y
50
Indicator for LIA
Bromocresol Purple; Ferric ammonium citrate (H2S)
51
Incubation for LIA
35-37°C, 18-24 hours
52
Positive QC for LIA Lysine decarboxylation (K/K)
Klebsiella, E. coli, Edwardsiella, Salmonella, Serratia
53
Positive QC for LIA Lysine deamination and glucose fermentation (R/A or R/Y)
PMP group
54
Quality control K/A LIA
E. coli
55
Quality control R/A LIA
Proteus mirabilis
56
LIA negative for Lysine decarboxylation, positive for glucose fermentation only (K/A)
Citrobacter, Shigella
57
Triple Sugar Iron (TSI) test for Enterobacterales contents (LSG)
Aerobic Slant: Lactose (10%), Sucrose (10%); Anaerobic Butt: Glucose (1%)
58
Medium: TSI Agar reactions
K: Alkaline (Red); A: Acid (Yellow); H2S: Black ppt.; g: Small gas (Bubble); G: Large gas (Cracks)
59
Reaction interpretations for TSI Ⓐ
E. coli
60
Reaction interpretations for TSI K/A, g/G, H2S+
- Salmonella enterica subsp. enterica serovar Typhimurium
61
Reaction interpretations for TSI K/A, H2S+
P. mirabilis
62
Reaction interpretations for TSI K/A
Shigella flexneri
63
Reaction interpretations for TSI K/K
P. aeruginosa
64
Indicators for TSI
Phenol Red (acid production), Ferric ammonium citrate (H2S)
65
Incubation for TSI
35-37°C, 18-24 hours
66
Gelatin hydrolysis test purpose
Positive for Moraxella lacunata, Serratia orodifera, Proteus spp., Pseudomonas
67
Principle of gelatin hydrolysis
Gelatin + gelatinase → liquefaction (refrigerated)
68
QC for gelatin hydrolysis Partial or total liquefaction at 4°C
Bacillus subtilis
69
QC for gelatin hydrolysis Complete solidification at 4°C
E. coli
70
Principle of fermentation media for differentiating enteric bacteria from Coryneforms
Glucose → Pyruvate
71
Medium for fermentation of enteric bacteria from Coryneforms
Peptone medium + 1 drop BHI culture
72
Indicator for fermentation media for enteric bacteria from Coryneforms
Andrade’s indicator
73
Gas detection method in fermentation media for enteric bacteria from Coryneforms
Durham tube
74
Incubation conditions for fermentation media of enteric bacteria from Coryneforms
35-37°C, Up to 7 days
75
QC reactions for fermentation media for enteric bacteria from Coryneforms (positive)
Pink with gas: E. coli; Pink without gas: S. flexneri
76
QC reactions for fermentation media for enteric bacteria from Coryneforms (negative)
Clear or straw, no gas: Coryneforms
77
Fermentation media to differentiate Enterococci from Streptococci medium
Broth + 2 drops of BHI culture
78
Indicator of fermentation media to differentiate Enterococci from Streptococci
Bromocresol purple
79
Incubation period for fermentation media to differentiate Enterococci from Streptococci
35-37°C, 4 days
80
Positive QC for fermentation media to differentiate Enterococci from Streptococci
Yellow with gas: E. coli
81
Negative QC for fermentation media to differentiate Enterococci from Streptococci
Pink, no gas: Moraxella osloensis
82
ONPG test purpose
Distinguishes late lactose fermenter from non-lactose fermenters
83
ONPG test principle
ONPG + β-galactosidase → o-nitrophenol (yellow)
84
Incubation for ONPG test
37°C, 4 hours
85
QC for ONPG test Positive
Yellow: Shigella sonnei
86
Negative QC for ONPG test
Colorless: Salmonella enterica serovar Typhimurium
87
Spot indole test purpose
Rapid ID of indole positive organisms (e.g., E. coli)
88
Spot indole test principle
1% p-dimethylamino-cinnamaldehyde + tryptophanase → blue
89
Incubation for spot indole test
22-25°C, within 30 seconds
90
QC for spot indole test positive
Blue within 20 seconds: E. coli
91
QC for spot indole test negative
Slight pink/No color change: K. pneumoniae
92
Indole positive reaction bacteria in IMViC (PEKPEC)
PMP group E. coli, Klebsiella oxytoca, Plesiomonas, Edwardsiella, Citrobacter koseri
93
Positive for Methyl red in IMViC (CEPSS)
Citrobacter, E. coli, PMP, Salmonella, Shigella
94
IMViC bacteria positive for Voges-Proskauer (Barritt’s method)(KESH)
Klebsiella, Enterobacter, Serratia, Hafnia
95
Acetate Utilization purpose
Differentiate E. coli from Shigella
96
Urease (Christensen’s method) purpose
Presumptive ID of Proteus spp. and other enterobacteria
97
Motility test purpose
Detects motile organisms
98
MUG test purpose
Detection of EHEC/E. coli O157:H7 (no fluorescence)
99
PAD test purpose
Detects PMP/PPM group (Positive)
100
String test purpose
Rapid detection of Vibrio spp.
101
Principle for indole
Tryptophan + tryptophanase → indole (detected by indicators)
102
Medium for Indole
SIM (20-25°C), Tryptophan broth (35°C)
103
Indicator for indole (positive for Enterobacterales/Enterobacteriaceae)
Kovac’s reagent
104
Composition of Kovac’s reagent
Dimethylamine-benzaldehyde & HCl
105
Composition of Ehrlich’s reagent
Dimethylamine-benzaldehyde & HCl, Absolute ethanol, Xylene
106
Incubation of indole
35-37°C, 48 hours
107
Positive QC for Indole Kovac's reagent
Pink/Wine color ring after addition of Kovac’s reagent (E. coli)
108
Positive QC for Indole Ehrlich’s reagent
H. influenzae and Porphyromonas asaccharolytica
109
Negative QC for Indole Kovac's reagent
K. pneumoniae
110
Negative QC for Indole Ehrlich’s reagent
H. parainfluenzae and Bacteroides fragilis
111
Principle for Methyl Red
Carbohydrate → mixed acids; Acids make pH < 4.4 (positive)
112
Medium for Methyl Red and Voges-Proskauer
MRVP medium or Clark & Lubs medium
113
Incubation for Methyl Red
35-37°C, 48 hours
114
Positive QC for Methyl Red
E. coli
115
Negative QC for Methyl Red
Yellow - K. aerogenes
116
Voges-Proskauer (Barritt’s method) principle
Carbohydrate → 2,3-Butanediol or acetoin (end product)
117
Voges-Proskauer (Barritt’s method) indicator
Solution A: ɑ-naphthol; Solution B: KOH
118
Positive QC for Voges-Proskauer
Red (K. aerogenes)
119
Negative QC for Voges-Proskauer
Yellow (E. coli)
120
Principle for Citrate Utilization Test
Citrate → Ammonium PO4 and Ammonium hydroxide
121
Medium for Citrate Utilization Test
Simmons citrate agar slant
122
Indicator for Citrate Utilization Test
Bromothymol blue
123
Incubation for Citrate Utilization Test
35-37°C, Up to 7 days
124
Positive QC for Citrate Utilization Test
Blue (K. aerogenes)
125
Negative QC for Citrate Utilization Test
Small/No growth; Green (E. coli)
126
Acetate (Malonate, Acetamide) Utilization principle
Ability to use Sodium acetate as sole source of carbon
127
Medium for Acetate Utilization
Acetate slant
128
Indicator for Acetate Utilization
Bromothymol blue
129
Incubation for Acetate Utilization
35-37°C, Up to 7 days
130
Positive QC for Acetate Utilization Test
Growth; Blue (E. coli)
131
Negative QC for Acetate Utilization Test
Small/No growth; Green (S. sonnei)
132
Urease (Christensen’s method) principle
Urea + urease → Ammonium carbonate (alkaline, pH >8.1)
133
Medium for Urease (Christensen’s method)
Urease agar slant + 2 drops BHI culture
134
Indicator for Urease (Christensen’s method)
Phenol Red
135
Incubation for Urease (Christensen’s method)
35-37°C, Up to 7 days
136
Positive QC for Urease (Christensen’s method)
Magenta/Red (P. vulgaris)
137
Other positive organisms for Urease (Christensen’s method)
Klebsiella, PMP group, Nocardia, Rhodococcus, Helicobacter, Ureaplasma, Cryptococcus
138
Negative QC for Urease (Christensen’s method)
Light Orange/Yellow (E. coli)
139
Motility test principle
Motile bacteria produce a diffuse zone of growth
140
Motility test agar
Semisolid Gelatin butt
141
Incubation for Motility test
35-37°C, Up to 7 days
142
Positive QC for Motility test
Spreading (P. vulgaris)
143
Negative QC for Motility test
Colonies remain on site of stab (K. pneumoniae)
144
MUG test principle
4-methylumbelliferyl-β-d-glucuronide + β-d-glucuronidase → 4-methylumbelliferyl
145
Medium for MUG test
MUG disk, add drop of water and incubate at 35°C for 2 hours
146
Indicator for MUG test
366 nm Ultraviolet light
147
Incubation for MUG test
35-37°C, 2 hours, Closed container
148
Positive QC for MUG test
Electric Blue fluorescence (All E. coli except O157:H7)
149
Negative QC for MUG test
No fluorescence (K. pneumoniae, E. coli O157:H7)
150
Principle for PAD test
Phenylalanine + PAD → ammonia and phenylpyruvic acid
151
Medium for PAD test
PAD agar/slant
152
Indicator for PAD test
10% FeCl3
153
Incubation for PAD test
35-37°C, 18-24 hours
154
Positive QC for PAD test
GREEN (P. mirabilis)
155
Negative QC for PAD test
Yellow (E. coli)
156
String test principle
0.5% Sodium deoxycholate lyses DNA of VIBRIO only, forming a string
157
String test medium
In glass slide + bile
158
Incubation period for String test
22-25°C, Within seconds
159
Positive QC for String test
String-like formation between loop and slide (Vibrio spp.)
160
Negative QC for String test
No string formation
