MICROBIOLOGY LABORATORY 6 Colonies of Erysipelothrix

USTMED ’07 Sec C – AsM; Photos provided by JV.N & MeaM. rhusiopathiae. Colonies are
smooth, small (less than
Amino acid hydrolysis reactions of Nocardia asteroids. All 1.0mm in diameter), and
amino acid reactions are negative on this identification plate: transparent. A greenish color
casein, tyrosine, xanthine and hypoxanthine. and/or α-hemolysis appears as
the colonies age.

Erysipelothrix rhusiopathiae on
tripe sugar iron (TSI) agar.
Erysipelothrix rhusiopathiae
demonstrates H2S production along
the stab line in a TSI agar slant within
48 hours of incubation at 35oC. This is
a helpful feature that separates this
organism from other gram-positive
bacilli.
Colonies of Nocardia asteroides on 5% sheep blood agar.
Colonies initially appear as powdery, and as they mature they
can develop a number of colors, ranging from a chalky-white to
yellow-orange, buff, red, purple, brown and black. Most
colonies have an earthy odor. Amino acid hydrolysis
reactions of
Streptomyces spp. All
reactions are positive on
this identification plate.
Unlike any Nocardia spp.,
Streptomyces spp.
hydrolyze casein,
tyrosine, xanthine, and hypoxanthine. Colonial morphology
resembles the chalky, dry white colonies of Nocardia spp.
Although Streptomyces spp. are urea-positive,
they are nitrate-negative, distinguishing the
genera from Nocardia spp.
Colonies of Nocardia asteroids growing on BAY late (x15).
Colonies are large, dry, bumpy, and heaped after 9 days
Molar tooth appearance of Actinomyces israelii
incubation at 27oC. This morphology has been referred to as
after incubation for 1 week.
glabrous orange form.

Gram stain of Streptomyces spp. (x1250). Streptomyces spp.

are characterized
microscopically by branching,
beaded, gram-positive
filamentous bacilli,
indistinguishable from
Identification of the genus Nocardia with biochemical Nocardia spp. Unlike Nocardia
reactions. Nocardia spp. are aerobic actinomycetes that reduce spp. the partial acid-fast stain
nitrate to nitrite (tube on left), hydrolyze urea (tube in center), is negative.
and grow on the surface of a broth medium, such as the
thioglycolate broth on the right.

Gomori methenamine silver

stain of Actinomyces spp.
(x1250). The slide was prepared
from a tissue biopsy specimen.
In the center is a characteristic
“sulfur granule,” which consists
of a granular microcolony
surrounded by purulent xudate.
The Actinomyces spp. are gram-postive bacilli that vary in size
from short, diphtheroid forms to long, branching filaments.
Unlike Nocardia spp. they are not partially acid-fast.

Gram stain Nocardia spp. (x1250). Nocardia species are Skin punch biopsy with
branching, beaded, filamentous gram-positive bacilli, positive acid fast stain of
approximately 1um in diameter. They can also appear as Mycobacterium leprae.
coccoid or coccobacillary forms. Care should be taken when
examining the slides because of the faint staining properties of
these microorganisms and other actinomycetes.

Acid Fast Staining

Materials for Acid Fast Staining


Kinyoun’s Carbol
Fuchsin Acid
Alcohol
Methylene Blue
Kinyoun’s acid fast
stain (x1500).
Mycobacteria are
readily stained with
carbol fuchsin,
which binds the
mycolic acid in their
lipid-rich cell walls.
This stain cannot be
removed
(decolorized) with acid alcohol and, therefore, the
microorganisms are referred to as acid-fast. There are two
common acid-fast stains, Ziehl-Neelsen (ZN), and Kinyoun’s.
The difference is that the ZN stain requires heat during the
staining process because the phenol concentration used is less
than in the Kinyoun’s method. The decolorizer, acid alcohol,
and the counterstain, methylene blue, are the same for both
methods. When stained, acid-fast bacilli stain red and the
background is blue, as shown here.
M. Tuberculosis stained
with fluorochrome stain.
M. tuberculosis stained
with Kinyoun acid-fast
stain.
Mycobacterium
tuberculosis
Stain used:
- Kinyoun’s Acid
fast stain
Clinical specimen used:
- Sputum
Results:
Positive for acid fast
bacilli. acid fast stain of direct smear to show acid fast bacilli
staining deep red (arrow A) and non-acid fast bacilli cells
staining blue with the counter stain methylene blue (arrow B)
oun acid-fast stain. Nocardia species
st when stained with a modified Kinyoun stain
as the decolorizing agent. This
o distinguish this mircroorganism from other
. species of
Mycobacterium
tuberculosis
Clinical specimen used:
Sputum
Stain used: kinyoun’s acid
fast staining
Acid Fast Bacilli appears as
red bacilli (arrow A). The pus cells and the non-acid fast bacilli
stained with counter stain which is the methylene blue (arrow
B).
Photochromogen M.
kansasii colonies when
exposed to light have
strong yellow color.
Colonies of M.
tuberculosis growing on
Middlebrook agar.
Cording characteristics of
M. tuberculosis.
Colonies of M. tuberculosis
growing on Middlebrook agar.
Scotochromogen M. gordonae
with yellow colonies.
M. Tuberculosis colonices on
Lowenstein-Jensen agar after 8
weeks of incubation.
Mycobacterium tuberculosis on
Lowenstein Jensen medium (LJ)
Mycobacterium tuberculosis colonies
on Lowenstein-Jensen media. The
organism grows in culture as slowly
developing, rough colonies with a
characteristic buff color. Under
optimal conditions, colonies are
recognizable in 3 weeks, but some
strains may require 4 to 6 weeks. LJ
media, an egg-based media containing
malachite green to inhibit growth of
contaminating bacteria, is commonly
used in clinical laboratories. Other
mycobacterial isolation media include
Middlebrook 7H11, Middlebrook 7H10
(used predominantly for susceptibility
testing), mycobactosel, and
Middlebrook 7H9 broth. For optimal
recovery, a combination of liquid and solid media is
recommended.
M. tuberculosis on LJ agar slant
LJ agar has been used as the standard for isolating
Mycobacteria. It contains coagulated whole eggs, glycerol,
potato flour, and salts. Malachite green is added to inhibit the
growth of contaminating bacteria. The
disadvantage of this medium is that it
becomes hydrolyzed when contaminants
do grow on it, and the culture must be
discarded. Illustrated here are rough,
half-colored colonies that appeared
w/in 3 weeks typical of M. tuberculosis.
Colonies of
Bacillus spp. on
5% sheep blood
agar. Some
Bacillus are nonhemolytic with mucoid
and spreading colonies resembling the
Enterobacteriaceae and Pseudomonas
spp. Colonies of Bacillus anthracis, the major human pathogen,
are usually nonhemolytic. This figure shows and environmental
Bacillus sp. that rarely causes human infections.
Gelatin hydrolysis test. Many Bacillus
spp. secrete proteolytic enzymes that
can hydrolyze gelatin. In the method
shown in this figure, exposed,
undeveloped x-ray film is used as the
substrate for detection of gelatinase
activity. Hydrolysis destroys the film’s
gelatin coating, leaving only the clear
photographic film where the strip was
 immersed in the organism suspension.

Sputum smear stained with
Gram’s stain shows
neutrophils, amorphous
debris, and filamentous,
beaded, branched gram-
positive bacilli (oil
immersion)
Colonies of Bacteroides
fragilis on Bacteroides bile
esculin (BBE) agar. This
medium contains gentamicin to
inhibit facultative gram-
negative bacilli. Bile salts
inhibit most anaerobes other
than those of the B. fragilis
group, which grow as large,
gray to black colonies due to
their hydrolysis of esculin in the medium.

Colonies of Bacillus spp. on Colonies of C. diphtheriae on

5% sheep blood agar are Tinsdale agar. A selective
smooth, round, and medium should be used along with
surrounded by a zone of sheep blood agar whenever
beta hemolysis. Coryebacterium diphtheriae is
suspected, as well as to
differentiate C. diphtheriae from
other corynebacteriua. ON the
selective medium cystine tellurite
agar or potassium tellurite (Tinsdale) agar, the colonies of
Growth of Bacillus spp. on Corynebacterium diphtheriae have a gun-metal, gray-black
egg yolk agar. Reaction on appearance.
egg yolk agar is one of the
characteristics used to Sputum stained with Gram’s stain
identify Bacillus spp. shows many neutrophils,
Bacillus cereus is one of amorphous debris, and
many species that produce coryneform gram-positive bacilli
the enzyme lecithinase, (oil immersion)
demonstrated by a zone of
opacity (whitish color in the agar) extending away from the
bacterial growth, as shown on the left. Lipase activity can also
be detected on egg yolk agar, demonstrated by an oily looking Esculin hydrolysis test. Most
sheen on the surface of the bacterial growth but not extending strains of Listeria monocytogenes
onto the agar, as shown on the right. can hydrolyze esculin in less than
2 hours. This photograph shows
Gram stain of Bacillus spp. positive reactions for bile esculin
Large gram positive bacilli (tube on left) and hippurate
with squared-off ends (right).
measuring 0.8 x 7.5 um.
Occurring singly and in Corynebacterium diphtheriae
chains.

Clostridium botulinum
Colonies of Bacteroides
fragilis on Brucella blood
agar. Bacteroides spp. as
well as other anaerobic
gram-negative bacilli grow
best on this nonselective
mediu. Agar must be
supplemented with hemin
and vitamin K1 to support Reverse CAMP test for
the growth of many species presumptive identification
of anaerobes. These colonies appear as circular, entire and of Clostridium perfringens. C. perfringens is streaked verticall
raised. and Streptococcus agalactiae is streaked horizontally in this
test. The hemolysis of the clostridium is synergistically
Colonies of Listeria enhanced by the hemolysin of the streptococcus in an
monocytogenes on 5% sheep arrowhead-shaped pattern.
blood agar. Colonies of
Listeria monocytogenes are Clostridium tetani on
small (less than 1mm in Brucella blood agar.
diameter), smooth, irregular, Growth on agar is
and translucent. The colonies characterized by
are surrounded by a very swarming, such that
characteristic narrow zone of beta hemolysis on 5% sheep blood growth will cover the
agar and may be confused with group B beta-hemolytic entire plate in a thin film
streptococci. Differentiating characteristics include Listeria’s within a few days.
narrow zone of beta hemolysis, positive catalase production,
and growth on bile and hydrolysis of esculin. Both species
hydrolyze hippurate.

Clostridium perfringens
on Brucella blood agar.
Colonies display the
Colonies of C. double-zone of beta
diphtheriae on 5% hemolysis typical of this
sheep blood agar. pecies. Colonies are large
Corynebacterium with peaked centers and
diphtheriae grow well on irregular edges after 48
5% sheep blood agar as hours incubation.
whitish, opaque colonies
 Clostridium difficile and C.
perfringens on egg yolk agar.
The C. perfringens (upper half of
plate) produces abundant
lecithinase, while the C. difficile
(lower half) grows but shows no
reaction.

Growth of clostridia on egg yolk

agar. The production of the
enzymes lecithinase, opaque
white precipitate extending from
the colony into the medium right
side), and lipase, iridescent
sheen on surface of colony (left
side) is used to differentiated
among Clostridium species and
to help identify some
Fusobacterium species.

Gram stain of
Clostridium spp. (x1250).
Cells are parallel-sided,
long, thin, gram-variable,
and some show swollen
ends indicative of spore
formation.

Gram stain of Clostridium

paraputrificum (x1250). This
microorganism displays
terminal, swollen spores and
the gram-variable staining
typical of Clostridium species.
Spores may not take up the
stain so they may appear as
clear areas.

-fin-

ustmedc3@yahoogroups.com
audrey_cl@yahoo.com

c3, thank you talaga for the support! Pagpasensyahan niyo na

‘to, I know this could have been helpful for the first practicals..
didn’t realize kasi na kasama sya sa scope.. anyway, enjoy na
lang the pics… jayveeh, ate mea, thanks for the digital pics,
wala ako nagagawang notes without them! Leigh, milo, anne,
thanks sa tulong! Bait niyo talaga! eto na siguro yung hardest
notes na ginawa ko, sobrang daming pictures!!!! I hate making
lab notes! Argggghhhh!!!

-auds-