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Bacteriology.
Staining methods:
1. Gram.
Differentiates bacteria basing on the cell wall
structure. Gram-negative bacteria have thin cell wall
with small amount of peptidoglycan (PG), Gram-
positive bacteria have thick cell wall consisting in
90% of PG.
Dyes: crystal violet (penetrates into a PG), contrast
dye: fuchsin (or saphranin), pink/red color.
Gram(+) bacteria are violet, Gram(-) negative are
red or pink.
2. Capsule detection (ink staining)
Mix bacteria with ink, make smear, wait until slide dries. Counterstaining with any
dye, usually fuchsin. Capsule presence is visible as colorles layer between ink
(background) and bacterial cell (red, stained with fuchsin).
Clinically important microorganisms in which one can see capsule using ink staining:
-
Klebsiella pneumoniae
-
Streptococcus pneumoniae
(capsule is not produced by bacteria growing on
artificial media)
-
Bacteroides fragilis
-
Clostridium perfringens
-
Cryptococcus neoformans
(yeast-like fungus)
Klebsiella pneumoniae
Bacteroides fragilis
Cryptococcus neoformans
Streptococcus pneumoniae
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3. Malachite green staining (spore
detection)
Used for initial identification of clostridia
and
Bacillus
spp.
based
on
spore
presence, shape and localization.
Staining procedure: staining with
malachite green (dye heating on the
glass, three times). Cool down the glass
and counterstain with fuchsin. Green
spores are visible (bacterial cells are red).
4. Neisser technique. Identification of
Corynebacterium dyphteriae
.
Technique is used for inclusion bodies detection (so called Ernst-Babes bodies or
volutin grains). Those inclusions (polyphosphates) are characteristic for
C. diphteriae
and don’t exist in other corynebacteria. Staining is performed with crystal violet and
methylene blue mixture (incl. bodies staining), counterstaining with chrysoidin
(yellow).
5. Ziehl-Neelsen stain (acid-fast
stain). Mycobacterium
tuberculosis detection.
Direct staining of sputum smear
when tuberculosis is suspected.
Mycobacteria have specific
composition of cell wall. It contains
mycolic acids (long chain fatty acids)
instead of PG. Mycobacteria are
difficult for Gram staining (“Gram-
ghost”). Ziehl-Neelsen technique
uses carbol fuchsin which
penetrates into thick cell wall of
mycobacteria. Next, acidic alcohol is
added to flush out fuchsin from any
structure except mycobacteria and methylene blue is used as a counterstain.
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Media and differentiating of the bacteria.
Enriched media.
1. Blood agar. Diagnostically, blood agar is important medium used to detect
hemolysins produced by bacteria. Particular importance for initial identification of
streptococci groups A and B, as well as
S. pneumoniae
and viridans group of
streptococci
Bacteria and types of hemolysis.
Types of hemolysis: alpha (partial) – not complete destruction of RBCs and hemoglobin,
green or turbid pale zone around bacterial colonies. Beta (complete) – clearing around the
colonies.
Double hemolysis zone: closer to the colonies – beta
type, around beta hemolysis ring there is zone of
alpha
hemolysis
–
pattern
characteristic
for
Clostridium perfringens
.
Bacteria:
α-hemolysis:
Streptococcus pyogenes
Streptococcus agalactiae
Pseudomonas aeruginosa
β-hemolysis:
Streptococcus pneumoniae
Viridans streptococci (normal flora of throat and upper respiratory tract)
Variable hemolysis (strain-dependent, some strains produce hemolysins, some not):
Staphylococcus aureus
E. coli
Enterococcus
spp.
2. Chocolate agar. Used for fastidious bacteria culturing, especially
Haemophilus
influenzae
,
Neisseria
and
Moraxella
.
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3. Chocolate tellurite agar for
Corynebacterium
culture.
4. Loewenstein-Jensen medium
For the cultivation and
differentiation of Mycobacterium
species. M.tuberculosis appears
as granular ,rough, dry colonies
Differentiating and selective media.
1. Chapman agar (MSA, mannitol salt
agar). For selective culture of
staphylococci and differentiation
between species able to utilize mannitol
(
S. aureus
) and unable to do so
(number of staphylococci from
skin/mucosal flora). MSA contains 7,5%
of NaCl, what is the factor inhibiting
growth of other bacteria. Mannitol-
positive staphylococci grow as a yellow
colonies with yellow zone around
colonies, mannitol-negative bacteria don’t change appearance of the medium.
2. MacConkey agar. Used for selective culture of
Gram-negative rods from
Enterobacteriaceae
family. Inhibiting factor = crystal violet. Medium
differentiates species utilizing and not utilizing
lactose. Lactose positive rods grow as a
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pink/reddish colonies, lactose-negative are colorless. Clinically important
lactose-positive
Enterobacteriaceae
are:
E. coli, Klebsiella pneumoniae
and
some strains of
Enterobacter
spp. and
Citrobacter
spp.
Pseudomonas
aeruginosa
is one from non-enteric bacteria able to grow on MacConkey agar.
P. aeruginosa
appears similarily to lactose-negative fermenting bacteria but it
is non-fermenting rod (aerobic).
3. Hektoen enteric agar (HE). Diagnostic application is similar to MacConkey
agar. Used to assess lactose fermentation and ability to produce hydrogen
sulfide (H2S). Allows for distinguishing among, E. coli, Salmonella and other
gut pathogens including Shigella species.
E. coli
(lactose fermantation,
Salmonella
spp. (lactose negative,
non producing H2S)
H2S producer)
Media for
Salmonella/Shigella
identification:
Wilson-Blair agar (Bizmuth Sulfite Agar, BSA).
Brilliant green and bismuth largely inhibit the
accompanying bacterial flora. Colonies of H
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-S-
positive salmonellae (e.g.
Salmonella typhi
and
S. typhimurium
) exhibit blackening due to the
formation of iron sulfide. Reduction of bismuth
ions to metallic bismuth produces a metallic
lustre around the colonies.
Salmonella-Shigella agar.
Brilliant green, ox bile and
high concentrations of
thiosulfate and citrate largely
inhibit the accompanying
microbial flora. Sulfide
production is detected by
using thiosulfate and iron
ions, the colonies turn black.
The presence of coliform
bacteria is established by
detecting degradation of lactose to acid with the pH indicator.
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