Cryptococci and cryptococcosis WORLD OF MICROBIOLOGY AND IMMUNOLOGY142•
To protect against crystal formation and osmotic pres-
sure shock to the bacteria, bacterial suspensions are typically
prepared in a so-called cryoprotectant solution. Glycerol is an
effective cryoprotective agent for many bacteria. For other
bacteria, such as cyanobacteria, methanol and dimethyl sul-
foxide are more suitable.
The microorganisms used in the cryoprotection process
should be in robust health. Bacteria, for example, should be
obtained from the point in their growth cycle where they are
actively growing and divided. In conventional liquid growth
media, this is described as the mid-logarithmic phase of
growth. In older cultures, where nutrients are becoming
depleted and waste products are accumulating, the cells can
deteriorate and change their characteristics.
For bacteria, the cryoprotectant solution is added
directly to an agar cultureof the bacteria of interest and bac-
teria are gently dislodged into the solution. Alternately, bacte-
ria in a liquid culture can be centrifuged and the “pellet” of
bacteria resuspended in the cryoprotectant solution. The
resulting bacterial suspension is then added to several spe-
cially designed cryovials. These are made of plastic that can
withstand the ultralow temperature.
The freezing process is done as quickly as possible to
minimize crystal formation. This is also referred to as “snap
freezing.” Bacterial suspensions in t cryoprotectant are ini-
tially at room temperature. Each suspension is deep-frozen in
a step-wise manner. First, the suspensions are chilled to refrig-
erator temperature. Next, they are stored for a few hours at
–70° C [–94° F]. Finally, racks of cryovials are either put into
the ultralow temperature freezer or plunged into liquid nitro-
gen. The liquid nitrogen almost instantaneously brings the
samples to –196° C [–320.8° F]. Once at this point, the sam-
ples can be stored indefinitely.
Recovery from cryostorage must also be rapid to avoid
crystal formation. Each suspension is warmed rapidly to room
temperature. The bacteria are immediately recovered by cen-
trifugation and the pellet of bacteria is resuspended in fresh
growth medium. The suspension is allowed to adapt to the
new temperature for a few days before being used.
Cryoprotection can be used for other purposes than the
long-term storage of samples. For example, cryoelectron
microscopy involves the rapid freezing of a sample and
examination of portions of the sample in an electro micro-
scopeunder conditions where the ultralow temperature is
maintained. If done correctly, cryoelectron microscopy will
revel features of microorganisms that are not otherwise evi-
dent in conventional electron microscopy. For example, the
watery glycocalyx, which is made of chains of sugar, col-
lapses onto the surface of a bacterium as the sample is dried
out during preparation for conventional electron microscopy.
But glycocalyx structure can be cryopreserved. In another
example, cryoelectron microscopy has also maintained
external structural order on virus particles, allowing
researchers to deduce how these structures function in the
viral infection of tissue.See alsoBacterial ultrastructure; Donnan equilibrium; Quality
control in microbiologyCCryptococci and cryptococcosisRYPTOCOCCI AND CRYPTOCOCCOSIS
Cryptococcusis a yeastthat has a capsule surrounding the
cell. In the yeast classification system, Cryptococcus is a
member of the Phylum Basidimycota, Subphylum Basidi-
mycotina, Order Sporidiales, and Family Sporidiobolaceae.
There are 37 species in the genus Cryptococcus.One of
these, only one species is disease-causing, Cryptococcus neo-
formans. There are three so-called varieties of this species,
based on antigenic differences in the capsule, some differ-
ences in biochemical reactions such as the use of various sug-
ars as nutrients, and in the shape of the spores produced by the
yeast cells. The varieties are Cryptococcus neoformansvar.
gatti, grubii, and neoformans. The latter variety causes the
most cryptococcal infections in humans.
Cryptococcus neoformanshas a worldwide distribution.
It is normally found on plants, fruits and in birds, such as
pigeons and chicken. Transmission via bird waste is a typical
route of human infection.
Cryptococcus neoformanscauses an infection known as
cryptococcosis. Inhalation of the microorganism leads to the
persistent growth in the lungs. For those whose immune sys-
tem is compromised, such as those having Acquired
ImmunodeficiencySyndrome (AIDS), the pulmonary infection
can be life-threatening. In addition, yeast cells may become
distributed elsewhere in the body, leading to inflammationof
nerve lining in the brain (meningitis). A variety of other infec-
tions and symptoms can be present, including infections of the
eye (conjunctivitis), ear (otitis), heart (myocarditis), liver
(hepatitis), and bone (arthritis).
The most common illness caused by the cryptococcal
fungus is cryptococcal meningitis. Those at most risk of devel-
oping cryptococcosis are AIDS patients. Those who have
received an organ, are receiving chemotherapyfor cancer or
have Hodgkin’s disease are also at risk, since frequently their
immune systems are suppressed. As the incidence of AIDS
and the use of immunosupressant drugs have grown over the
past decade, the number of cases of cryptococcosis has risen.
Until then, cases of cryptococcus occurred only rarely. Even
today, those with a well-functioning immune system are sel-
dom at risk for cryptococcosis. For these individuals a slight
skin infection may be the only adverse effect of exposure to
Cryptococcus.
Cryptococcusbegins with the inhalation of Crypto-
coccus neoformans. Likely, the inhaled yeast is weakly encap-
sulated and is relatively small. This allows the cells to pene-
trate into the alveoli of the lungs. There the production of
capsule occurs. The capsule surrounding each yeast cell aids
the cell in avoiding the immune response of the host, particu-
larly the engulfing of the yeast by macrophage cells (which is
called phagocytosis). The capsule is comprised of chains of
sugars, similar to the capsule around bacteria. The capsule of
Cryptococcus neoformansis very negatively charged. Because
cells such as macrophages are also negatively charged, repul-
sive forces will further discourage interaction of macrophages
with the capsular material.
Another important virulence factor of the yeast is an
enzyme called phenol oxidase. The enzyme operates in thewomi_C 5/6/03 2:05 PM Page 142