1
Q
urea is a product of
A
decarboxylation of certain amino acids
2
Q
urease
A
urea can be hydrolyzed to ammonia and CO2 by bacteria containing the enzyme urease. urea hydrolysis provides N in a usable form
3
Q
rapid urease+ bacteria
A
proteus, morganella morganii, some providencia stuartii strains
- h. pylori
4
Q
urea agar
A
Urea Agar was formulated to differentiate rapid
urease-positive bacteria from slower urease-positive
and urease-negative bacteria. It contains urea, peptone,
potassium phosphate, glucose, and phenol red. Peptone
and glucose provide essential nutrients for a broad range
of bacteria. Potassium phosphate is a mild buffer used to
resist alkalinization of the medium from peptone metabolism.
5
Q
phenol red colors
A
- yellow or orange below pH
8. 4 and red or pink above - <6.8: yellow
- 6.8 to 7.4: red
- > 7.4: pink to magenta
6
Q
urea broth
A
Urea broth differs from urea agar in two important
ways. First, its only nutrient source is a trace (0.0001%)
of yeast extract. Second, it contains buffers strong enough
to inhibit alkalinization of the medium by all but the
rapid urease-positive organisms mentioned above.
7
Q
urea broth test results
A
- pink – rapid urea hydrolysis, strong urease production
- orange/yellow – no urea hydrolysis; organism doesn’t produce urease or can’t live in broth
8
Q
urea agar test results
A
- rapid urea hydrolysis and strong urease production: all pink within 24h, gelatinase present within 6 days
- slow, weak (w+): partially pink or orange/yellow within 24h, all/partially pink w/in 6 days
- orange or yellow in 6 days: no urea hydrolysis, urea absent
9
Q
nitrate reduction test application
A
many g- bacteria, includig most enterobacteriaceae, contain nitrate reductase.
- differentates them from g- rods that either dont reduce nitrate or reduce it beyond nitrite to N2 or other ocmpounds
10
Q
preparation of phenol red broth
A
- pH adjusted to 7.3 so appears red
- inverted durham tube added to each tube as an indicator of gas production
11
Q
carb fermentation
A
- organic molecule acts as an e- donor (becoming oxidized in the process) and 1 or more of its organic products act as the final e- acceptor
- this term is used rather broadly to include hydrolysis of disaccharides prior to the fermentation
reaction
12
Q
PR broth application
A
used to differentiate members of Entero -
bacteriaceae and to distinguish them from other Gramnegative
rods. also used to distinuish btwn g+ fermenters, such as streptococcus and lactobacillus species
13
Q
PR broth: acid production, __, gas production
A
- acid: lowers pH and turns medium yellow
- deamination of peptone AAs produces NH3, which turns broth pink
- gas: bubble/pocket in tube where broth has been displaced
14
Q
PR broth restuls
A
- A/G: yellow broth w/ bubble – fermentation w/ acid, gas end products
- A/–: yellow w/o bubble – fermentation w/ acid but no gas end products
- –/–: red w/o bubble – no fermentation
- K: pink w/o bubble – degradation of peptone; alkaline end products
15
Q
denitrification
A
nitrate to N2
16
Q
nitrate reductase
A
single step reduction of nitrate to nitrite
17
Q
anaerobic respiration involves
A
the reduction of an inorganic molecule other than oxygen. nitrate reduction is an example
18
Q
nitrate broth
A
undefined medium of beef extract, peptone, K nitrate. inverted durham tube is placed in each broth to trap a portion of any gas produced. no color indicators
19
Q
before a broth can be tested for nitrate reductase activity,
A
it must be examined
for evidence of denitrification. This is simply a visual
inspection for the presence of gas in the Durham tube
(Figure 5-25). If the Durham tube contains gas and the
organism is known not to be a fermenter (as evidenced by
a fermentation test), the test is complete. De nitrifica tion
has taken place. Gas produced in a nitrate reduction test
by an organism capable of fermenting is not deter mina -
tive because the source of the gas is unknow
20
Q
If there is no visual evidence of denitrification,
A
sulfanilic acid (nitrate reagent A) and alpha-naphthylamine
(nitrate reagent B) are added to the medium to test for
nitrate reduction to nitrite. If present, nitrite will form
nitrous acid (HNO2) in the aqueous medium. Nitrous acid
reacts with the added reagents to produce a red, watersoluble
compound
21
Q
nitrate reductase: Zn
A
to catalyze the reduction of
any nitrate (which still may be present as KNO3) to
nitrite. If nitrate is present at the time zinc is added, it
will be converted immediately to nitrite, and the above-
described reaction between nitrous acid and reagents
will follow and turn the medium red. In this instance,
the red color indicates that nitrate was not reduced by
the organism (Figure 5-27). No color change after the
addition of zinc indicates that the organism reduced the
nitrate to NH3, NO, N2O, or some other nongaseous
nitrogenous compound.
22
Q
nitrate test results: gas
A
- nonfermenter: Denitrification—production of n2
(NO3 > NO2 > N2) (+) - Fermenter, or status is unknown: Source of gas is unknown; requires addition of reagents
23
Q
nitrate test results: reagents
A
- red: nitrate reduction to nitrite (+)
- no color: Incomplete test; requires the addition of zinc dust
24
Q
nitrate test results: zn
A
- no color: Nitrate reduction to nongaseous nitrogenous compounds) (NO3 > NO2 > nongaseous nitrogenous products) (+)
- red: no nitrate reduction (-)
25
MR-VP broth
Methyl Red and Voges-Proskauer (MR-VP) Broth is a
combination medium used for both Methyl Red (MR)
and Voges-Proskauer (VP) tests. It is a simple solution
containing only peptone, glucose, and a phosphate buffer.
The peptone and glucose provide protein and ferment able
carbohydrate, respectively, and the potassium phosphate
resists pH changes in the medium.
26
MR test
The MR test is designed to detect organisms capable
of performing a mixed acid fermentation, which overcomes
the phosphate buffer in the medium and lowers
the pH (Figure 5-8 and Figure 5-9). The acids produced
by these organisms tend to be stable, whereas acids
produced by other organisms tend to be unstable and
subsequently are converted to more neutral products.
Mixed acid fermentation is verified by the addition
of methyl red indicator dye following incubation.
27
MRVP test application
The Methyl Red and Voges-Proskauer tests are components
of the IMViC battery of tests (Indole, Methyl red,
Voges-Proskauer, and Citrate) used to distinguish between
members of the family Entero bacteriaceae and
differentiate them from other Gram-negative rods.
28
methyl red color
red at pH 4.4 and yellow at pH 6.2. Between these
two pH values, it is various shades of orange. Red color
is the only true indication of a positive result. Orange is
negative or inconclusive. Yellow is negative
29
MR test results
- red: mixed acid fermentation (+)
| - no color change: no mixed acid ferm (-)
30
VP test
The Voges-Proskauer test was designed for organisms
that are able to ferment glucose, but quickly convert
their acid products to acetoin and 2,3-butanediol. Adding VP reagents to the
medium oxidizes the acetoin to diacetyl, which in turn
reacts with guanidine nuclei from peptone to produce a
red color. A positive VP result, therefore, is
red.
31
VP test copper color
No color change (or development of copper color)
after the addition of reagents is negative. The copper
color is a result of interactions between the reagents
and should not be confused with the true red color of a + result
32
VP test results
- red: 2,3-butanediol fermtnation (acetoin produced)
| - no color change: no; no
33
citrate
citrate (citric acid) produced as acetyl
coenzyme A (from the oxidation of pyruvate or the
-oxidation of fatty acids) reacts with oxaloacetate at
the entry to the Krebs cycle. Citrate then is converted
back to oxaloacetate through a complex series of re -
actions, which begins the cycle anew.
34
citrate-permease
In a medium containing citrate as the only available
carbon source, bacteria that possess citrate-permease can
transport the molecules into the cell and enzymatically
convert it to pyruvate. Pyruvate then can be converted
to a variety of products, depending on the pH of the
environment
35
simmons citrate agar
a defined medium that
contains sodium citrate as the sole carbon source and
ammonium phosphate as the sole nitrogen source. Brom thymol blue dye
36
Brom thymol blue dye
green at pH 6.9 and
blue at pH 7.6, is added as an indicator. Bacteria that
survive in the medium and utilize the citrate also convert
the ammonium phosphate to ammonia (NH3) and ammonium
hydroxide (NH4OH), both of which tend to
alkalinize the agar. As the pH goes up, the medium
changes from green to blue (Figure 5-30). Thus, con version
of the medium to blue is a positive citrate test result
37
Occasionally a citrate-positive organism will grow
on a Simmons Citrate slant without producing a change
in color.
In most cases, this is because of incomplete incubation.
In the absence of color change, growth on the slant indicates that citrate is being utilized and is evi dence
of a positive reaction. To avoid confusion between actual
growth and heavy inoculum, which may appear as
growth, citrate slants typically are inoculated lightly
with an inoculating needle rather than a loop.
38
citrate test results
- blue (even a small amount / no color change but growth: citrate utilized
- no color change no growth: citrate not utilized
39
citrate test application
The citrate utilization test is used to determine the ability
of an organism to use citrate as its sole source of carbon.
Citrate utilization is one part of a test series referred to
as the IMViC (Indole, Methyl Red, Voges-Proskauer and
Citrate tests) that distinguishes between members of the
family Enterobacteriaceae and differentiate them from
other Gram-negative rods.
40
sim medium
The semisolid medium includes
casein and animal tissue as sources of amino acids, an
iron-containing compound, and sulfur in the form of
sodium thiosulfate.
41
SIM medium is used for determination of three bacterial
| activities:
sulfur reduction, indole production from
| tryptophan, and motility.
42
Sulfur reduction to H2S can be accomplished by
bacteria in two different ways, depending on the enzymes
present.
1. The enzyme cysteine desulfurase catalyzes the
putrefaction of the amino acid cysteine to pyruvate
2. The enzyme thiosulfate reductase catalyzes the
reduction of sulfur (in the form of sulfate) at the
end of the anaerobic respiratory electron transport
chain
Both systems produce hydrogen sulfide (H2S) gas.
43
When
| either reaction occurs in SIM medium,
the H2S that is
produced combines with iron, in the form of ferrous
ammonium sulfate, to form ferric sulfide (FeS), a black
precipitate (Figure 5-63). Any blackening of the medium
is an indication of sulfur reduction and a positive test.
No blackening of the medium indicates no sulfur reduction
and a negative reaction
44
indole production
Indole production in the medium is made possible
by the presence of tryptophan (contained in casein and
animal protein). Bacteria possessing the enzyme tryptophanase
can hydrolyze tryptophan to pyruvate, ammonia
(by deamination), and indole
45
kovascs reagent
The hydrolysis of tryptophan in SIM medium can
be detected by the addition of Kovacs’ reagent after a
period of incubation. Kovacs’ reagent contains dimethyl -
aminobenzaldehyde (DMABA) and HCl dissolved in
amyl alcohol. When a few drops of Kovacs’ reagent are added to the tube, DMABA reacts with any indole present
and produces a quinoidal compound that turns the
reagent layer red (Figure 5-66 and Figure 5-67). The formation
of red color in the reagent layer indicates a positive
reaction and the presence of tryptophanase.
46
Determination of motility in SIM medium is made
| possible by
the reduced agar concentration and the
| method of inoculation.
47
motility determination
The medium is inoculated with a
single stab from an inoculating needle. Motile organisms
are able to move about in the semisolid medium and can
be detected by the radiating growth pattern extending
outward in all directions from the central stab line.
Growth that radiates in all directions and appears slightly
fuzzy is an indication of motility (Figure 5-68). This
should not be confused with the (seemingly) spreading
growth produced by lateral movement of the inoculating
needle when stabbing.
48
sim medium application
SIM medium is used to identify bacteria that are capable
of producing indole, using the enzyme tryptophanase. The
Indole Test is one component of the IMViC battery of
tests (Indole, Methyl red, Voges-Proskauer, and Citrate)
used to differentiate the Entero bacteriaceae. SIM medium
also is used to differentiate sulfur-reducing members of Enterobacteriaceae, especially members of the genera
Salmonella, Francisella, and Proteus from the negative
Morganella morganii and Providencia rettgeri. In addition
to the first two functions of SIM, motility is an im-
portant differential characteristic of Enterobacteriaceae
49
indole production results
- red in the alcohol layer of kovacs reagent: tryptophan is broken down into indole and pyruvate (+)
- reagent color unchanged: tryp not broken down (-)
50
motility results
- growth radiating outward from stab line: motility
| - no radiating growth: nonmotile
51
S reduction results
- black in medium: sulfur reduction(H2S production)
| - no black: S not reduced
52
tsia
Triple Sugar Iron Agar (TSIA) is a rich medium designed
to differentiate bacteria on the basis of glucose fermen -
tation, lactose fermentation, sucrose fermentation, and
sulfur reduction. In addition to the three carbohydrates,
it includes animal proteins as sources of carbon and
nitrogen, and both ferrous sulfate and sodium thiosulfate
as sources of oxidized sulfur. Phenol red is the pH indicator,
and the iron in the ferrous sulfate is the hydrogen
sulfide indicator.
53
tsia prearation
The medium is prepared as a shallow agar slant with
a deep butt, thereby providing both aerobic and anaerobic
growth environments. It is inoculated by a stab in the
agar butt followed by a fishtail streak of the slant.
54
tsia wait times
The
incubation period is 18 to 24 hours for carbohydrate
fermentation and up to 48 hours for hydrogen sulfide
reactions. Many reactions in various combinations are
possible
55
When TSIA is inoculated with a glucose-only fermenter,
acid products lower the pH and turn the entire
medium yellow within a few hours. Because glucose
is in short supply (0.1%), it will be exhausted within
about 12 hours. As the glucose diminishes, the organisms
located in the aerobic region (slant) will begin to break
down available amino acids, producing NH3 and raising
the pH. This process, which takes 18 to 24 hours to
complete, is called a reversion and only occurs in the
slant because of the anaerobic conditions in the butt.
Thus, a TSIA with a red slant and yellow butt after a
24-hour incubation period indicates that the organism
ferments glucose but not lactose.
56
tsia: Organisms that are able to ferment glucose and
| lactose and/or sucrose
also turn the medium yellow
throughout. However, because the lactose and sucrose
concentrations are ten times higher than that of glucose,
resulting in greater acid production, both slant and butt
will remain yellow after 24 hours. therefore, a tsia w/ a yellow slant and butt at 24 hours indicates that
the organism ferments glucose and one or both of the
other sugars. Gas produced by fermentation of any of
the carbohydrates will appear as fissures in the medium
or will lift the agar off the bottom of the tube.
57
tsia black
Hydrogen sulfide (H2S) may be produced by the
reduction of thiosulfate in the medium or by the breakdown
of cysteine in the peptone. Ferrous sulfate reacts
with the H2S to form a black precipitate, usually seen in
the butt. Acid conditions must exist for thiosulfate reduction;
therefore, black precipitate in the medium is an
indication of sulfur reduction and fermentation. If the
black precipitate obscures the color of the butt, the color
of the slant determines which carbohydrates have been
fermented
58
tsia: An organism that does not ferment any of the carbohydrates
An organism that does not ferment any of the carbohydrates
but utilizes peptone and amino acids will alkalinize
the medium and turn it red. If the organism can
use the peptone aerobically and anaerobically, both the
slant and butt will appear red.
59
tsia: obligate aerobe
An obligate aerobe will
| turn only the slant red.
60
kia vs tsia
kia no sucrose
| kia is kligler iron agar
61
tsia pplication
TSIA and KIA are primarily used to differentiate members
of Enterobacteriaceae and to distinguish them
from other Gram-negative rods such as Pseudomonas
aeruginosa.
62
tsia yellow/yellow
glucose, lactose, and/or sucrose ferm w/ acid accumulation in slant and butt (A/A)
63
tsia red slant/yellow butt
Glucose fermentation with acid production. Proteins catabolized K/A
aerobically (in the slant) with alkaline products (reversion). (K/A)
64
tsia red/red
No fermentation. Peptone catabolized aerobically and anaerobically K/K
with alkaline products. Not from Enterobacteriaceae.
65
tsia red slant / butt no change
No fermentation. Peptone catabolized aerobically with alkaline
products. Not from Enterobacteriaceae. (K/NC)
66
tsia no change / no change
Organism is growing slowly or not at all. Not from Enterobacteriaceae. (NC/NC)
67
tsia blck precipitate in agar
Sulfur reduction. (An acid condition, from fermentation of glucose
or lactose, exists in the butt even if the yellow color is obscured
by the black precipitate.) (H2S)
68
tsia gas production
Cracks in or lifting of agar
69
koser application
- for the differentiation of Escherichia coli from Enterobacter aerogenes based on citrate utilization
70
koser medium
- Na ammonium phosophat e
- K phosphate
- Mg sulfate
- Na citrate
- final pH 6.7
71
koser principles
Bacteria that are able to use citrate as their carbon source will grow in the medium and cause turbidity. Bacteria unable to utilize citrate will not grow and the broth should remain clear.
72
koser e aero and e coli
- e aero: luxuriant growth; positive rxn, turbidiy
| - e coli: none to poor growth; negative rxn, no turbidity
73
starch application
Starch agar originally was designed for cultivating
Neisseria. It no longer is used for this, but with pH indicators,
it is used to isolate and presumptively identify
Gardner ella vaginalis. It aids in differentiating members
of the genera Corynebacterium, Clostridium, Bacillus,
Bacte roides, Fusobacterium, and Enterococcus, most of
which have () and () species.
74
casease test application
The casein hydrolysis test is used for the cultivation and
differentiation of bacteria that produce the enzyme
casease.
75
oxidase test application
This test is used to identify bacteria containing the respiratory
enzyme cytochrome c oxi dase. Among its many
uses is the presumptive identification of the oxidase-
positive Neisseria. It also can be useful in differentiating
the oxidase-negative Enterobacteriaceae from the oxidasepositive
Pseudomonadaceae.
