-2 Flashcards
gram stain
The Gram stain is a differential stain in which a decolori -
zation step occurs between the appli cation of two basic
stains.
primary stain and counterstain
crystal violent; safranin
mordant
a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material; Iodine is added as a mordant
to enhance crystal violet staining by forming a crystal
violet–iodine complex.
Upon successful completion
of a Gram stain,
Gram-positive cells appear purple and
Gram-negative cells appear reddish-pink
decolorization
is the
most critical step in the procedure. Gram- negative cells
are decolorized by the solution (of variable composition—
generally alcohol or acetone) whereas Gram-positive
cells are not. Gram-negative cells can thus be colorized
by the counterstain safranin.
Electron microscopy and other evidence indicate that
the ability to resist decolorization or not is based on
the
different wall constructions of Gram-positive and Gramnegative
cells. Gram-negative cell walls have a higher
lipid content (because of the outer membrane) and a
thinner peptidoglycan layer than Gram-positive cell walls. The alcohol/acetone in the decolorizer
extracts the lipid, making the Gram-negative wall more
porous and in capable of retaining the crystal violet–
iodine complex, thereby decolorizing it. The thicker
peptidoglycan and greater degree of cross-linking (because
of teichoic acids) trap the crystal violet–iodine complex
more effectively, making the Gram-positive wall less
susceptible to decolorization.
over-decolorize
It is
possible to over-decolorize by leaving the alcohol on
too long and get reddish Gram-positive cells.
under-decolorize
produce purple Gramnegative
cells.
A second source of poor Gram stains is
inconsistency
in preparation of the emulsion. Remember, a good emulsion
dries to a faint haze on the slide.
age of the culture
Age of the culture also affects Gram stain consistency.
Older Gram-positive cultures may lose their ability to resist
decolorization and give an artifactual Gram-negative
result. The genus Bacillus is notorious for this. Staphylococcus
can also be a culprit. Cultures 24 hours old or
less are best for this procedure.
Interpretation of Gram stains can be complicated by
nonbacterial elements. For instance,
stain crystals from
an old or improperly made stain solution can disrupt the
field or stain precipitate may be mistakenly
identified as bacteria.
mycolic acids
The presence of mycolic acids in the cell walls of acidfast
organisms is the cytological basis for the acid-fast
differential stain. Mycolic acid is a waxy substance that
gives acid-fast cells a higher affinity for the primary stain
and resistance to decolorization by an acid alcohol
solution.
ZN v K
A variety of acid-fast staining procedures are
employed, two of which are the Ziehl-Neelsen (ZN)
method and the Kinyoun (K) method. These differ primarily
in that the ZN method uses heat as part of the
staining process, whereas the K method is a “cold” stain.
- K uses a more lipid-soluble and concentrated carbolfuchsin. These properties allow the stain to penetrate
the acid-fast walls without the use of heat but make
this method slightly less sensitive than the ZN method.
acid-fast stain
The acid-fast stain is a differential stain used to detect
cells capable of retaining a primary stain when treated
with an acid alcohol.
AFB
Acid-fast stains are useful in identifying acid-fast
bacilli (AFB) and in rapid, preliminary diagnosis of tuberculosis
(with greater than 90% predictive value from
sputum samples). It also can be performed on patient
samples to track the progress of antibiotic therapy and determine the degree of contagiousness. A prescribed
number of microscopic fields is examined and the number
of AFB is determined and reported using a standard
scoring system.
why dont typical stains work with acid fast cells
The waxy wall of acid-fast cells repels typical aqueous
stains. (As a result, most acid-fast positive organisms are
only weakly Gram-positive.)
carbolfuchsin
phenolic compound that’s lipid-soluble and penetrates
the waxy cell wall
ZN method
carbolfuchsin used as primary stain. Staining by carbolfuchsin is
further enhanced by steam-heating the preparation to
melt the wax and allow the stain to move into the cell.
Acid alcohol is used to decolorize nonacid-fast cells;
acid-fast cells resist this decolorization. A counterstain,
such as methylene blue, then is applied.
upon successful completion of ZN method
Acid-fast cells are
reddish-purple; non acid-fast cells are blue
kinyoun method
uses a slightly
more lipid-soluble and concentrated carbolfuchsin as the
primary stain.
Decolorization with acid alcohol is followed by a contrasting
counterstain, such as brilliant green
or methylene blue.
endospore
An endospore is a dormant form of the bacterium that
allows it to survive poor environmental conditions.
Spores are resistant to heat and chemicals because of a
tough outer covering made of the protein keratin. The
keratin also resists staining, so extreme measures must
be taken to stain the spore.
cause of sporulation
Sporulation is done in response
to nutrient depletion, and so is characteristic of older cultures.
upon completion of schaeffer fulton spore stain
spores are green, and vegetative and spore mother
cells are red.
spore characteristics
Spores may be located in the middle of the cell
(central), at the end of the cell (terminal), or between
the end and middle of the cell (subterminal). Spores also
may be differentiated based on shape—either spherical
or elliptical (oval)—and size relative to the cell (i.e.,
whether they cause the cell to look swollen or not).
spore stain
The spore stain is a differential stain used to detect the
presence and location of spores in bacterial cells.
Schaeffer-Fulton
method
a primary stain of malachite
green is forced into the spore by steaming the bacterial
emulsion. Alternatively, malachite green can be left on
the slide for 15 minutes or more to stain the spores.
Malachite green is water-soluble and has a low affinity
for cellular material, so vegetative cells and spore mother
cells can be decolorized with water and counterstained
with safranin
Only a
few genera produce spores. Among them are
Bacillus and Clostridium. Most members of Bacillus are
soil, freshwater, or marine sap rophytes, but a few are
pathogens, such as B. anthracis, the causal agent of
anthrax. Most members of Clostridium are soil or
aquatic saprophytes or inhabitants of human intestines,
but four pathogens are fairly well known:
C. tetani: tetanus
C. botulinum: botulism
C. perfringens: gas gangrene
C. difficile: pseudomembranous colitis
the acid fast stain is impt in identifying
bacteria in the genus Mycobacterium,
some of which are pathogens (e.g., M. leprae and
M. tuberculosis, causative agents of leprosy and tuber -
culosis, respectively). also members of the actinomycete genus nocardia are partially acid-fast. Oocysts
of coccidian parasites, such as Crypto sporidium and
Isospora, are also acid-fast.
