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Question 1

gram stain

Answer 1

The Gram stain is a differential stain in which a decolori -
zation step occurs between the appli cation of two basic
stains.

Question 2

primary stain and counterstain

Answer 2

crystal violent; safranin

Question 3

mordant

Answer 3

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.

Question 4

Upon successful completion

of a Gram stain,

Answer 4

Gram-positive cells appear purple and

Gram-negative cells appear reddish-pink

Question 5

decolorization

Answer 5

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.

Question 6

Electron microscopy and other evidence indicate that

the ability to resist decolorization or not is based on

Answer 6

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.

Question 7

over-decolorize

Answer 7

It is
possible to over-decolorize by leaving the alcohol on
too long and get reddish Gram-positive cells.

Question 8

under-decolorize

Answer 8

produce purple Gramnegative

cells.

Question 9

A second source of poor Gram stains is

Answer 9

inconsistency
in preparation of the emulsion. Remember, a good emulsion
dries to a faint haze on the slide.

Question 10

age of the culture

Answer 10

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.

Question 11

Interpretation of Gram stains can be complicated by

nonbacterial elements. For instance,

Answer 11

stain crystals from
an old or improperly made stain solution can disrupt the
field or stain precipitate may be mistakenly
identified as bacteria.

Question 12

mycolic acids

Answer 12

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.

Question 13

ZN v K

Answer 13

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.

Question 14

acid-fast stain

Answer 14

The acid-fast stain is a differential stain used to detect
cells capable of retaining a primary stain when treated
with an acid alcohol.

Question 15

AFB

Answer 15

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.

Question 16

why dont typical stains work with acid fast cells

Answer 16

The waxy wall of acid-fast cells repels typical aqueous
stains. (As a result, most acid-fast positive organisms are
only weakly Gram-positive.)

Question 17

carbolfuchsin

Answer 17

phenolic compound that’s lipid-soluble and penetrates

the waxy cell wall

Question 18

ZN method

Answer 18

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.

Question 19

upon successful completion of ZN method

Answer 19

Acid-fast cells are

reddish-purple; non acid-fast cells are blue

Question 20

kinyoun method

Answer 20

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.

Question 21

endospore

Answer 21

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.

Question 22

cause of sporulation

Answer 22

Sporulation is done in response

to nutrient depletion, and so is characteristic of older cultures.

Question 23

upon completion of schaeffer fulton spore stain

Answer 23

spores are green, and vegetative and spore mother

cells are red.

Question 24

spore characteristics

Answer 24

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).

Question 25

spore stain

Answer 25

The spore stain is a differential stain used to detect the

presence and location of spores in bacterial cells.

Question 26

Schaeffer-Fulton

method

Answer 26

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

Question 27

Only a

few genera produce spores. Among them are

Answer 27

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

Question 28

the acid fast stain is impt in identifying

Answer 28

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.