Laboratory diagnosis of Gram

positive non spore forming

bacilli

By
Janan M. Salih Almlaly
Msc. Medical Microbiology
Medicine college/ Duhok University
2011
Lactobacillus acidophilus
 Lactobacillus
acidophilus
 Lactobacillus acidophilus, informally known as
"Doderlein's bacillus" colonizes the vagina, because
glycogen is produced which provides the bacteria with
a source of sugar that they ferment to lactic acid .

 These bacteria create an acidic environment (A normal

vaginal pH, between 3.8 and 4.2 which inhibits growth
of many bacterial species which can lead to urogenital
infections.
 Lactobacillus
acidophilus
Laboratory diagnosis
1. Direct gram stain only from clinical
specimens. They are large Gram
positive Pleomorphic.

Gram positive bacilli

2. Colonial Morphology (Pleomorphic)
Multiple colonial morphologies, It
varies ranging from pinpoint, alpha
alpha hemolytic
hemolytic colonies resembling
Streptococci to rough, grey color .

Colonies of Lactobacillus spp. On blood agar

 Lactobacillus
acidophilus

Gram stain of Lactobacillus spp. in vaginal secretions,

appears as long, non-spore forming, gram-positive bacilli.
Also may appear as short, pleomorphic coccobacilli.
 Lactobacillus
acidophilus

Lactobacillus species and a vaginal

squaemous epithelial cell. CDC

Lactobacillus acidophilus colonizes the vaginal epithelium during child-bearing

years and establishes the low pH that inhibits the growth of pathogens.
 Gardnerella vaginalis
Clue cells Epithelial cells
Gardnerella vaginalis
Clinical diagnosis
 Minimum requirement of bacterial vaginosis is
the presence of at least 3 of the following criteria:

1- Abnormal vaginal discharge

2- Vaginal pH > 4.5
3- Clue cells
4- Fishy odor of amine, when a drop of 10% KoH is added
to vaginal secretions.
Mobiluncus is a recently characterized genus of curved, motile, gram-negative,
anaerobic, rod-shaped bacteria Its organisms are found in the human vagina,
particularly in association with Gardnerella vaginalis in cases of bacterial
vaginosis.
Gardnerella vaginalis

Clue cells

Clue cells in wet preparation

which are epithelial cells covered in bacteria
 Wet
preparation
for
vaginal
secretion
Gardnerella vaginalis
Laboratory diagnosis

1. Culture is not recommended because it is normally

found in 20-40% of healthy women. Gardnerella
vaginalis grows as small, circular, convex, gray
colonies on chocolate agar .

2. Gram stain is the method of choice for diagnosis of

bacterial vaginosis. Gardnerella is a small, gram-
positive to gram-variable staining rod shape.
 It has a gram-positive cell wall, but because the cell
wall is so thin it can appear either gram-positive or
gram-negative under the microscope. It is associated
microscopically with clue cells, which are
epithelial cells covered in bacteria
Gardnerella vaginalis

3. Typical smears show clue cells covered

with small gram negative rods accompanied
by mixed flora of large numbers of small
gram negative and gram variable rods with
coccobacilli, no lactobacilli.
Actinomyces israelii
Actinomyces israelii
Morphology & identification:
Microscopical diagnosis

The organisms are filamentous, branching & beaded, gram-

positive bacilli, pleomorphic non-spore forming, non acid fast
facultative anaerobic grow best with increase Co2, or
microaerophilic bacilli.

Actinomyces are fastidious bacteria that require enriched

culture media, may be aided in growth by 6% to 10% ambient
CO2, It takes 3 to 10 or more days to grow them in culture.
Actinomyces israelii

"Sulfur granule": colony of Actinomyces israelii

showing slender, peripheral Gram stain positive
filaments surrounding a Gram stain
Sulfur granules
Actinomyces israelii

 The sulfur granules found in tissue are

yellowish in appearance, up to 1 mm in size,
and are composed of macrophages, other
tissue cells, fibrin, and the bacteria.
Infection with Actinomyces
Actinomyces israelii
Cultural characterization

 Specimens are cultured in thioglycolate broth and

on blood agar plates, incubated an aerobically with
increase CO2 at 37 C.

 On enriched medium, colonies usually appear in 24-

28 hours, but may take 2 weeks to grow.

 A. israelii produce colonies that are opaque, white,

peaked & may resemble molar tooth.
 (x10)

These are the characteristic “molar tooth” colonies

of Actinmyces israelli. That develop in 1-2 weeks on
agar plates. These colonies are raised with irregular
margin.
Gram stain of Lactobacillus spp.
Appears as long, gram-positive bacilli, non-spore
forming. Also may appear as short, pleomorphic
coccobacilli.
Gram stain of Gardnerella vaginalis
It is a small, gram-positive to gram-variable staining
short bacilli shape, non-spore forming.
Direct Gram stain of Gardnerella vaginalis showing
clue cells (which are epithelial cells covered in
bacteria). The bacteria Gardnerella vaginalis
gram-positive to gram-variable.
Gram stain of Actinomyces israelii showing
filamentous, branching & beaded, gram-positive
bacilli, pleomorphic non-spore forming.