Methods and Procedures in Diagnostic Microbiology Flashcards
1
Q
Bacteria capable of growth on this medium produce the enzyme acylamidase, which deaminates acetamide to release ammonia.
A
Acetamide Utilization Test
2
Q
Positive color of Acetamide Utilization Test
A
Deamination of the acetamide resulting in a BLUE color.
3
Q
POS organism in Acetamide Utilization Test
A
P.aeruginosa
4
Q
NEG reaction in Acetamide Utilization Test
A
No color change/Green
5
Q
NEG organism in Acetamide Utilization Test
A
E.coli
6
Q
Purpose:
Differentiate microorganisms based on the ability to use acetamide the sole source of carbon.
A
Acetamide Utilization Test
7
Q
Principle:
Organism capable of using sodium acetate grow in the medium, resulting in an alkaline pH, turning the bromothymol blue ind. from green to blue.
A
Acetate Utilization Test
8
Q
POS reaction in Acetate Utilization Test
A
Medium becomes alkalinized (BLUE) as a result of the growth and use of acetate.
9
Q
POS organism in Acetate Utilization Test
A
E.coli
10
Q
NEG reaction in Acetate Utilization Test
A
No color change/Green
11
Q
NEG organism in Acetate Utilization Test
A
S.sonnei
12
Q
Purpose:
Differentiate microorganisms based on the ability to use acetate as the sole source of carbon. Generally used to differentiate Shigella from E. coli.
A
Acetate Utilization Test
13
Q
Principle
➢ Bacitracin inhibits the synthesis of bacterial cell walls.
➢ A disk (TaxoA) impregnated with a small amount of bacitracin (0.04 units) is placed on an agar plate.
➢ After incubation the inoculated plates are examined for zone of inhibition.
A
Bacitracin Susceptibility Test
14
Q
POS reaction in Bacitracin Susceptibility Test
A
Any zone of inhibition >10 mm
15
Q
POS organism in Bacitracin Susceptibility Test
A
S. pyogenes
M. luteus
16
Q
NEG reaction in Bacitracin Susceptibility Test
A
No zone of inhibition (resistant)
17
Q
NEG organism in Bacitracin Susceptibility Test
A
S. agalactiae
S. aureus
18
Q
This test is used to provide presumptive identification and differentiation of:
➢ Beta-hemolytic group A streptococci (S. pyogenes are susceptible) from other beta hemolytic streptococci.
➢ Also used to distinguished staphylococci (resistant) from micrococci (susceptible)
A
Bacitracin Susceptibility Test
19
Q
This test is used for the presumptive identification of enterococci and organism in the Streptococcus bovis group. The test differentiates enterococci and group D streptococci from non-group D viridans streptococci.
A
Bile Esculin Test
20
Q
➢ Gram-positive other than some streptococci and enterococci are inhibited by the bile salts in this medium.
➢ Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin.
➢ Esculetin reacts with Fe3+ and forms a dark brown to black precipitate.
A
Bile Esculin Test
21
Q
POS reaction in Bile Esculin Test
A
Growth and blackening of the agar slant
22
Q
POS organism in Bile Esculin Test
A
E.faecalis
23
Q
NEG reaction in Bile Esculin Test
A
Growth and no blackening of medium, no growth
24
Q
NEG organism in Bile Esculin Test
A
E. coli (growth, no color change)
S. pyogenes (no growth, no color change)
25
This test differentiates Streptococcus pneumoniae (positive-soluble) from alpha-hemolytic streptococci (negative-insoluble)
Bile Solubility Test
26
➢ Bile or a solution of a bile salt (ex. sodium desoxycholate) rapidly lyses pneumococcal colonies.
➢ Lysis depends on the presence of an intracellular autolytic enzyme, amidase.
Bile Solubility Test
27
POS reaction in Bile Solubility Test
Colony DISINTEGRATES; an imprint of the LYSED colony may remain in the zone
28
POS organism in Bile Solubility Test
S.pneumoniae
29
NEG reaction in Bile Solubility Test
Intact colonies
30
NEG organism in Bile Solubility Test
E.faecalis
31
This is a rapid test to detect the enzyme butyrate esterase, to aid identification of Moraxella (Branhamella) catarrhalis.
Butyrate disk
32
➢ Organisms capable of producing butyrate esterase hydrolyze bromochlorindolyl butyrate.
➢ Hydrolysis of the substrate in the presence of butyrate esterase releases indoxyl.
➢ Indoxyl in the presence of oxygen spontaneously form indigo, a blue to blue- violet color.
Butyrate disk
33
POS reaction in Butyrate disk
Development of a blue color during the 5-minute incubation period
34
POS organism in Butyrate disk
M.catarrhalis
35
NEG reaction in Butyrate disk
No color change/Indigo
36
NEG organism in Butyrate disk
N.gonorrhoeae
37
This test is used to differentiate group B streptococci (Streptococcus agalactiae-positive) from other streptococci. L. monocytogenes also produces a positive CAMP reaction.
Christie, Atkins, and Munch-Peterson (CAMP) test
38
➢ Certain organisms produce a diffusible extracellular hemolytic protein (CAMP factor) that acts synergistically with the beta-lysin of Staphylococcus aureus to cause enhanced lysis or red blood cells.
➢ The group B streptococci are streaked perpendicular to a streak of S. aureus on sheep blood agar.
Christie, Atkins, and Munch-Peterson (CAMP) test
39
POS reaction in CAMP test
Enhanced hemolysis is indicated by an ARROWHEAD-SHAPED zone of beta- hemolysis at the juncture of the two organisms.
40
NEG reaction in CAMP test
No enhancement of hemolysis
41
POS organism in CAMP test
S. agalactiae
42
NEG organism in CAMP test
S. pyogenes
43
This test differentiates catalase-positive micrococcal and staphylococcal species from catalase-negative streptococcal species.
Catalase Test
44
➢ Aerobic and facultative aerobic organisms produce two toxins during normal metabolism, hydrogen peroxide (H2O2) and superoxide radical (O2-).
➢ These organisms have the enzyme catalase.
➢ Catalase converts H2O2 to water and oxygen.
Catalase test
45
POS reaction in Catalase test
Copious bubbles are produced
46
NEG reaction in Catalase test
No or few bubbles are produced
47
POS organism in Catalase test
S. aureus
48
NEG organism in Catalase test
S.pyogenes
49
This test is primarily used to isolate and purify Pseudomonas aeruginosa from contaminated specimens.
Cetrimide agar
50
➢ Used to determine the ability of an organism to grow in the presence of cetrimide (cetyltrimethylammonium bromide), a toxic substance that inhibits the growth of many bacteria by causing the release of nitrogen and phosphorus, which slows or kills the organism.
➢ Pseudomonas aeruginosa is malupit and lodi
(haha ) and is resistant to cetrimide.
Cetrimide agar
51
POS reaction in Cetrimide agar
Growth, variation in color of colonies.
52
POS organism in Cetrimide agar
P.aeruginosa
53
NEG reaction in Cetrimide agar
No growth
54
NEG organism in Cetrimide agar
Escherichia coli
55
Used to identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source.
Citrate Utilization
56
➢ Bacteria that can grow in this medium produce the enzyme citrate-permease
➢ Citrate-permease, capable of converting citrate to pyruvate.
➢ Pyruvate is used in the production of energy.
➢ Bacteria capable of growth use citrate to convert ammonium phosphate to ammonia and ammonium hydroxide, creating an alkaline environment.
Citrate Utilization
57
POS reaction in Citrate Utilization
Growth on the medium, with or without a change in the color of the indicator. GREEN to BLUE color.
58
POS organism in Citrate Utilization
E.aerogenes
59
NEG reaction in Citrate Utilization
Absence of growth
60
NEG organism in Citrate Utilization
Escherichia coli
61
This test is used to differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci (negative).
Coagulase Test
62
S. aureus form of coagulase that bound to the bacterial cell wall and reacts directly with fibrinogen.
• Detected by the Slide test.
Bound coagulase or clumping factor
63
POS reaction in Slide Coagulase test
Macroscopic clumping in 10 seconds in plasma, no clumping in saline or water drops
64
NEG reaction in Slide Coagulase test
No clumping in either drop.
65
Preferred coagulase plasma in Slide test
rabbit plasma with EDTA
66
An extracellular protein enzyme that causes formation of a clot when colonies are incubated.
• Detected by the tube test.
Free coagulase
67
➢ Bacterial colony is emulsified in 0.5 mL of rabbit plasma (with EDTA) to give a milky suspension.
➢ Incubate at 35-37 C in ambient air for 4 hours.
Coagulase Tube test
68
POS reaction in Coagulase Tube test
Clot of any size
69
POS organism in Coagulase Tube test
S.aureus
70
NEG reaction in Coagulase Tube test
No clot
71
NEG organism in Coagulase Tube test
S.epidermidis
72
This test is used to differentiate decarboxylase- producing Enterobacteriaceae from other gram- negative rods.
Decarboxylase Tests (Moeller’s method)
73
➢ This test measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form amine.
➢ Decarboxylation of amino acid results in an alkaline pH and a color change from orange to purple. (Take note that this method uses bromocresol purple)
Decarboxylase Tests (Moeller’s method)
74
POS reaction in Decarboxylase Tests (Moeller’s method)
Alkaline (PURPLE) color change compared with the control tube
75
NEG reaction in Decarboxylase Tests (Moeller’s method)
No color change or acid (YELLOW) color in test and control tube.
Growth in control tube
76
POS organism in Decarboxylase Tests (Moeller’s method)
Lysine: K. pneumoniae
Ornithine: E. aerogenes
Arginine: E. cloacae
77
NEG organism in Decarboxylase Tests (Moeller’s method)
Lysine: E. cloacae
Ornithine: K. pneumoniae
Arginine: K. pneumoniae
78
This test is used to differentiate organisms based on the production of deoxyribonuclease (DNase):
➢ Serratia (positive) vs. Enterobacter (negative)
➢ Staphylococcus aureus (positive) from others
➢ Moraxella catarrhalis (positive) from Neisseria (negative)
DNA hydrolysis
79
➢ Determine the ability of an organism to hydrolyze DNA.
➢ The medium is pale green because of the DNA-methyl green complex.
➢ If the organism growing on the medium hydrolyses DNA, the green color fades and the colony is surrounded by a colorless zone.
DNA hydrolysis
80
POS reaction in DNA hydrolysis
When DNA is hydrolyzed, methyl green is released and combines with highly polymerized DNA at a pH of 7.5. TURNING THE MEDIUM COLORLESS.
81
POS organism in DNA hydrolysis
S.aureus
82
NEG reaction in DNA hydrolysis
If no degradation of DNA occurs, the medium remains GREEN
83
NEG organism in DNA hydrolysis
E.coli
84
This test is used for the presumptive identification and differentiation of Enterobacteriaceae.
Esculin hydrolysis
85
➢ This test is used to determine whether an organism is able to hydrolyze the glycoside esculin.
➢ Esculin is hydrolyzed to esculetin, which reacts with Fe3+ and forms a dark brown to black precipitate.
Esculin hydrolysis
86
POS reaction in Esculin hydrolysis
Blackened medium + loss of fluorescence under the wood’s lamp
87
POS organism in Esculin hydrolysis
E.faecalis
88
NEG reaction in Esculin hydrolysis
No blackening + No loss of fluorescence. Or Slight blackening + No loss of fluorescence
89
NEG organism in Esculin hydrolysis
E.coli
90
It is used to differentiate organism based on their ability to ferment carbohydrates.
Fermentation media
91
It is is used to differentiate enteric bacteria from coryneform.
Andrade formula
92
➢ A fermentation medium consists of basal medium containing a single carbohydrate (glucose, lactose, or sucrose) for fermentation.
➢ Color indicator detects the formation of acids.
➢ A Durham tube is placed to capture gas produced by metabolism.
Fermentation Media: Peptone w/ Andrade
93
POS reaction in Fermentation Media: Peptone w/ Andrade
Indicator change to PINK with or without gas
94
NEG reaction in Fermentation Media: Peptone w/ Andrade
Growth, but no color change. Medium remains clear to straw colored.
95
POS organism in Fermentation Media: Peptone w/ Andrade
Dextrose:
➢ E. coli (positive with gas)
➢ S. flexneri (positive, no gas)
96
NEG organism in Fermentation Media: Peptone w/ Andrade
Dextrose:
Moraxella osloensis
97
It is used to differentiate enterococci from streptococci
Bromocresol purple formula
98
POS reaction in Fermentation Media: BHI w/ Bromocresol
Indicator change to YELLOW
99
NEG reaction in Fermentation Media: BHI w/ Bromocresol
Growth, but no color change. Remain purple
100
POS organism in Fermentation Media: BHI w/ Bromocresol
Dextrose:
➢ E. coli (positive with gas)
101
NEG organism in Fermentation Media: BHI w/ Bromocresol
Dextrose:
Moraxella osloensis
102
This technique is used to visualize the presence and arrangement of flagella for the presumptive identification of motile species.
Flagella stain: Wet mount technique
103
➢ RYU (Remel, Lenexa, Kansas) flagella stain is used.
➢ Cells with flagella are observed at 100x (oil)
Flagella stain: Wet mount technique
104
E. coli flagella
Peritrichous
105
Polar flagella
P. aeruginosa
106
Negative flagella
K. pneumoniae
107
The production of gelatinases capable of hydrolyzing gelatin is used as a presumptive test for the identification of various organisms, including Staphylococcus, Enterobacteriaceae, and some gram-positive bacilli.
Gelatin Hydrolysis
108
➢ Test to determine the ability to produce gelatinase that liquefy gelatin (a component of vertebrate connective tissue)
➢ In this test, instead of agarose, gelatin is used as the solidifying agent.
➢ WHEN AN ORGANISM PRODUCES GELATINASE, the enzyme liquefies the growth medium.
Gelatin Hydrolysis
109
POS reaction in Gelatin Hydrolysis
Partial or total liquefaction at 4C within 14 days.
110
NEG reaction in Gelatin Hydrolysis
Complete solidification of the tube at 4C.
111
POS organism in Gelatin Hydrolysis
B.subtilis
112
NEG organism in Gelatin Hydrolysis
E.coli
113
This test is used to differentiate a pyocyanogenic pseudomonads (as in P. aeruginosa) from another Pseudomonas.
Growth at 42 C
114
➢ Used to determine the ability of an organism to grow at 42C.
Growth at 42 C
115
POS reaction in Growth at 42 C
Good growth at both 35C and 42 C
116
NEG reaction in Growth at 42 C
No growth at 42C, but good growth at 35C.
117
(+) Good growth at both 35C and 42 C
P.aeruginosa
118
(-) No growth at 42C, but good growth at 35C.
Pseudomonas fluorescens
119
This test is used to detect the presence of the enzyme hippuricase.
Hippurate test
120
➢ Hippuricase hydrolyzes hippuric acid in the test into glycine and benzoic acid.
➢ Glycine is deaminated by the oxidizing agent ninhydrin, which is reduced during the process.
➢ The end products of ninhydrin oxidation react 22 to form a purple-colored compound.
Hippurate test
121
POS reaction in Hippurate test
Deep purple color
122
NEG reaction in Hippurate test
Colorless or slightly yellow pink
123
POS organism in Hippurate test
S.agalactiae
124
NEG organism in Hippurate test
S.pyogenes
125
This test is used to identify organism that produce the enzyme tryptophanase.
Indole production
126
Used determine the capability of an organism to hydrolyze tryptophan to indole.
Bacteria with tryptophanase are capable of hydrolyzing tryptophan to pyruvate, ammonia, and indole.
Indole production
127
Reagents used to detect indole
Kovac’s reagent (dimethylamine-
benzaldehyde and HCl)
— Produces red color with indole.
Ehrlich’s reagent (Kovac’s + absolute ethyl alcohol)
— More sensitive for detecting small
amounts of indole.
— Technique of choice for anaerobes.
128
POS reaction in Indole production
Pink- to wine-colored ring after addition of appropriate reagent
129
NEG reaction in Indole production
No color change after addition of the appropriate reagent
130
Kovac’s Indole Method (+)
E. coli
131
Kovac’s Indole Method (-)
K.pneumoniae
132
Ehrlich’s Indole method (+)
H.influenzae
133
Ehrlich’s Indole method (-)
H. parainfluenza
134
Ehrlich’s Indole method (Anaerobic) (+)
Porphyromonas assacharolytica
135
Ehrlich’s Indole method (Anaerobic) (-)
Bacteroides fragilis
136
This test is used for the presumptive identification of catalase-negative gram-positive cocci.
LAP test
137
➢ The LAP disk is a rapid test for the detection of the enzyme leucine aminopeptidase (LAP).
➢ Leucine-beta-naphthylamide-impregnated disks serve as substrate for LAP.
➢ After hydrolysis by LAP the resulting beta- naphthylamine produces RED COLOR upon addition of cinnamaldehyde reagent.
LAP test
138
POS reaction in LAP test
Development of a red color within 1 minute after adding cinnamaldehyde reagent
139
NEG reaction in LAP test
No color change or development of a slight yellow color.
140
POS organism in LAP test
Enterococcus faecalis
141
NEG organism in LAP test
Aerococcus viridans
