10.09 Flashcards
growth patterns in broth
- uniform fine turbidity
- Some organisms float on top of
the medium and produce a type of surface membrane
called a pellicle - sediment
- flocculent (clumps)
fastidious
An organism that relies heavily on the
environment to supply ready-made organic compounds
is referred to as fastidious
Fluid Thioglycollate Medium esp well adapted for
cultivation of strict an aerobes and
microaerophiles.
When inoculating
Thioglycollate Broth, it helps to
dip the loop all
the way to the bottom of the tube and gently mix
the broth with the loop as you remove it
anaerobic system purpose
cultivating anaerobic
and microaerophilic bacteria.
MSA
Mannitol Salt Agar (MSA) contains the carbohydrate
mannitol, 7.5% sodium chloride (NaCl), and the pH
indicator phenol red. used for isolation and differentiation
of Staphylococcus aureus.
phenol red
Phenol red is yellow below pH 6.8,
red at pH 7.4 to 8.4, and pink at pH 8.4 and above. Phenol
red indicates whether fermentation has taken place
mannitol
Mannitol provides the substrate for fermentation and
makes the medium differential.
NaCl
Sodium chloride makes
the medium selective because its concentration is high
enough to dehydrate and kill most bacteria. Staphylococci
thrive in the medium, largely because of their
adaptation to salty habitats such as human skin.
most staphylococci
Most staphylococci are able to grow on MSA, but
do not ferment the mannitol, so the growth appears pink
or red and the medium remains unchanged.
s aureus
Staphylococcus
aureus ferments the mannitol, which produces
acids and lowers the pH of the medium (Figure 4-6).
The result is formation of bright yellow colonies usually
surrounded by a yellow halo
msa result: poor/no growth
organism inhibited by nacl. not staphyloccocus
msa result: good growth
organism not inhibited by nacl. is staphyloccocus
msa result: yellow growth or halo
organism produces acid from mannitol fermentation. possible pathogenis s aureus
msa result: red, no halo
Organism does not ferment mannitol. No reaction
- Staphylococcus other than s aureus
mackonkey agar
a selective and differential medium
containing lactose, bile salts, neutral red, and crystal
violet. used to isolate and differentiate
members of the Enterobacteriaceae based on the ability to ferment lactose
bile salts
inhibit growth of
Gram-positive bacteria.
crystal violet
inhibit growth of
Gram-positive bacteria.Formulations without
crystal violet allow growth of Enterococcus and some
species of Staphylococcus, which ferment the lactose and
appear pink on the medium.
neutral red dye
Neutral red dye is a pH indicator
that is colorless above a pH of 6.8 and red at a pH less
than 6.8. Acid accumulating from lactose fermentation
turns the dye red.
lactose fermenters mackonkey agar
Lactose fermenters turn a shade of
red on MacConkey Agar, whereas lactose non fermenters
retain their normal color or the color of the medium
mc agar: poor/no growth
Organism is inhibited by crystal violet and/or bile. G+
mc agar: good growth
Organism is not inhibited by crystal violet or bile. G-
mc agar: pink/red, w/ or w/o bile precipitate
Organism produces acid from lactose fermentation; probable coliform
mc agar: “colorless” (not red/pink) growth
org doesn’t ferment lactose. no rxn
- noncoliform
coliform
gram-negative rod-shaped bacteria. a subgroup of Enterobacteriaceae (Escherichia coli being the
most prominent member) that produce gas from lactose fermentation
eosin methylene blue agar
Eosin Methylene Blue (EMB) Agar is a complex (chemically
undefined), selective, and differential medium. It
contains peptone, lactose, sucrose, and the dyes eosin Y
and methylene blue. used for the isolation of fecal coliforms.
peptone
The peptone provides a complex
mixture of carbon, nitrogen and other nutritional components.
sugars
The sugars are included to encourage growth of
enteric bacteria and to differentiate them based on color
reactions created when combined with the dyes. Lactose
supports coliforms such as Escherichia coli and sucrose
supports pathogens such as Proteus or Salmonella species
emb agar dyes
The purpose of the dyes is twofold, 1) they inhibit
the growth of Gram-positive organisms and 2) they react
with vigorous lactose fermenters
emb agar: poor/no growth
Organism is inhibited by eosin and methylene blue. G+
emb agar: good growth
Organism is not inhibited by eosin and methylene blue. G-
emb agar: pink, mucoid growth
Organism ferments lactose with little acid production. possible coliform
emb agar: dark growth
Organism ferments lactose and/or sucrose with acid production. PROBABLE coliform
emb agar: whats dark growth
purple to black, w/ or w/o metallic sheen
emb agar: colorless
Organism does not ferment lactose or sucrose. no rxn
- noncoliform
emb agar: whats colorless
no pink, purple, or metallic sheen
flavoprotein
One carrier molecule in the ETC called flavoprotein
can bypass the next carrier in the chain and transfer electrons
directly to oxygen (Figure 5-15). This alternative
pathway produces two highly potent cellular toxins—
hydrogen peroxide (H2O2) and superoxide radical (O2-)
superoxide dismutase
catalyzes conversion of superoxide radicals
(the more lethal of the two compounds) to hydrogen
peroxide
catalase
Catalase converts hydrogen peroxide into
water and gaseous oxygen
Bacteria that produce catalase can be detected easily
using typical store-grade hydrogen peroxide. When
hydrogen peroxide is added to a catalase-positive culture, O2 gas bubbles form immediately . If no bubbles appear, the organism is
catalase-negative.
Flavoproteins, iron-sulfur proteins,
and cytochromes
important ETC molecules unable
to donate protons.
compelx IV
The last
enzyme in the chain, Complex IV, is called cyto chrome c
oxidase because it makes the final electron transfer of
the chain from cytochrome c, residing in the periplasm,
to oxygen inside the cell
chromogenic reducing agent
Chromogenic reducing
agents are chemicals that develop color as they become
oxidized
The oxidase test is designed to identify the presence
of cytochrome c oxidase. It is able to do this because
cytochrome c oxidase has the unique ability to not only
oxidize cytochrome c, but to catalyze the reduction of
cytochrome c by a chromogenic reducing agent called
tetramethyl-p-phenylenediamine.
oxidase test results n intepretations
- dark blue w/in 20s: cytochrome c oxidase present
2. no color change to dark blue w/in 20s: not present
what do u do in oxidase test
In the oxidase test, the reducing reagent is added
directly to bacterial growth on solid media,
or (more conveniently) a bacterial colony is transferred
to paper saturated with the reagent
starch agar
simple plated medium of beef extract,
soluble starch, and agar
When organisms that produce
alpha-amylase and oligo-1,6-glucosidase are cultivated
on starch agar,
they hydrolyze the starch in the areasurrounding their growth.
iodine
Because both starch and its
sugar subunits are virtually invisible in the medium, the
reagent iodine is used to detect the presence or absence
of starch in the vicinity around the bacterial growth.
Iodine reacts with starch and produces a blue or dark
brown color; therefore, any microbial starch hydrolysis
will be revealed as a clear zone surrounding the growth. Iodine will
not color the agar where the growth is, only the area
surrounding the growth.
amylase test results n predictions
clearing around growth = amylase present, n vice versa
casease
Casease is an enzyme that some bacteria produce
to hydrolyze the milk protein casein, the molecule that
gives milk its white color. When broken down into smaller
fragments, the ordinarily white casein loses its opacity
and becomes clear.
casein hydrolysis test results
clearing in agar = casease present, n vice versa
milk agar
The presence of casease can be detected easily with
the test medium Milk Agar. When
plated Milk Agar is inoculated with a casease-positive
organism, secreted casease will diffuse into the medium
around the colonies and create a zone of clearing where
the casein has been hydrolyzed.
Gelatinase Test Results and Interpretations
gelatin liquid = gelatinase present. solid = not present
gelatin
Gelatin is a protein derived from collagen—a component
of vertebrate connective tissue. Gelatinases comprise a
family of extracellular enzymes produced and secreted
by some microorganisms to hydrolyze gelatin. Subsequently,
the cell can take up individual amino acids and
use them for metabolic purposes.
nutrient gelatin
The presence of gelatinases can be detected using
Nutrient Gelatin, which differs
from most other solid media in that the solidifying
agent (gelatin) is also the substrate for enzymatic activity.
Consequently, when a tube of Nutrient Gelatin is stabinoculated
with a gelatinase-positive organism, secreted
gelatinase (or gelatinases) will liquefy the medium.
A slight disadvantage of Nutrient Gelatin is that it
melts at 28°C (82°F). Therefore, inoculated stabs are
typically incubated at 25°C along with an uninoculated
control to verify that any liquefaction is not temperaturerelated.
hemolysin
Several species of Gram-positive cocci produce exotoxins
called hemolysins, which are able to destroy red blood
cells (RBCs) and hemoglobin.
3 types of hemolysis
beta: the complete
destruction of RBCs and hemoglobin, results in a clearing
of the medium around the colonies
alpha: partial, greenish discoloration of the agar around the
colonies
gamma: non-hemolysis; simple growth w/ no change to medium
streak stab technique
. In this
procedure the Blood Agar plate is streaked for isolation
and then stabbed with a loop. The stabs encourage
streptolysin activity because of the reduced oxygen
concentration of the subsurface environment
Blood Agar is used for
isolation and cultivation of many
types of fastidious bacteria. It also is used to differentiate
bacteria based on their hemolytic characteristics, especially
within the genera Streptococcus, Enterococcus, and
Aerococcus.
streptolysins
Hemolysins produced by streptococci are called
streptolysins.
O: oxygen-labile and expresses maxi-
mal activity under anaerobic conditions
S: oxygen-stable but expresses itself optimally under an aerobic
conditions as well.
