sediment cultured. Streak the blood agar
plates so as to obtain well isolated colonies
and inoculate a tube of enriched infusion
broth. If heavily contaminated, inoculate
Pike's medium and culture after overnight
incubation.
After decontaminating the skin with 4% iodine
followed by wiping with 70% alcohol, draw about
10 ml. of blood, using a sterile syringe, and
add it to a flask containing 2 ml. of 3% sterile
sodium citrate of 1.0 mg. of heparin. After
mixing, transfer the specimen aseptically to
100 ml. of blood culture broth, a Loeffler slant
or a blood agar plate. If a plate is used, limit
the inoculum to a small area of the medium and
transport to the laboratory where streaking for
isolation can be done properly with a wire loop.
Do not rub the swab over the entire plate. Do
not mail dry swabs or Petri dishes. Addition
of antagonists to media for blood cultures,
from patients undergoing chemotherapy, is not
usually necessary nor is the addition of
penicillinase needed in cultures from patients
receiving penicillin.
Refrigerate the specimen as much of the time
as possible before it is cultured. If this
is impossible, mix it with 1/3 volume of
glycerol. Culture suitable dilutions in
blood agar pour plates in duplicate, mixing
the sample and melted medium in the plate so
as to get isolated colonies. Incubate one
plate aerobically and one under anaerobic
conditions.
b. Specimens for streptococcus typing
Very few laboratories are in a position to type
streptococci. If isolates are to be sent to CDC
for this purpose, they should be submitted as
pure cultures on blood agar slants only after
consultation with the Streptococcus Unit of the
Laboratory Branch.
Streptococcus typing by fluorescent antibody
(FA) technic
Smears made directly from the patient are un-
satisfactory for examination by FA technic.
For this examination, the swab should be placed
in Todd-Hewitt broth for enrichment purposes
2 to 3 hours at 37° C. before the FA smears
are made.
6. Hemophilus Infections of the Pharynx or Conjunctiva
Specimens in which hemophilic bacteria may be present
should be examined in a local laboratory, if possible,
because these organisms do not withstand shipment well.
Isolation of the organisms may be successful from
blood, sputum, throat and conjunctival swabs, and
from purulent exudates from joints, spinal fluid,
and the genitourinary tract. Since these bacteria are
fastidious, attention must be paid to their growth re-
quirements. A laboratory that is not prepared to meet
these requirements should not attempt cultivation since
the results will be unreliable.
Specimens for the isolation of the agent
Blood, sputum, throat swabs, and purulent exudates
are collected in the usual manner with due attention
to aseptic technic where practicable. A small,
sterile, dry cotton swab may be gently rotated over
the conjunctiva or pharnygeal mucosa, obtaining some
of the exudate, if present. The swab should be
immediately placed in a tube of semisolid agar for
transport to the laboratory where it is incubated
for about 4 hours after which plates of transparent
agar and blood are streaked for isolation of the
organism. Blood should be inoculated into a flask
of broth with a large surface area. Sputum and
throat specimens are cultured on one of the trans-
parent agars as well as blood agar. The swab should
not be left in the tube of semisolid agar during
incubation.
Leptospirosis should be considered in all cases of
febrile illness of unknown origin and it may suggest
aseptic meningitis, or nonparalytic poliomyelitis.
Rats, dogs, cattle, swine, and many wild animals are
common animal reservoirs. Infection is transmitted to
man by urine containing the agent and occurs when
leptospires enter through the mucous membranes of the
mouth, nose, throat, eyes, lungs, or abraded skin.
Specimens for the isolation of the agent
Blood, urine, and cerebrospinal fluid may be
collected for culture and/or animal inoculation
with a view to recovery of the organism. Blood
taken during the febrile stage of illness is the
most reliable for culture, but spinal fluid
collected within the first 10 days of illness
may also be cultured. Inoculate the freshly-drawn
specimen directly into several tubes of a suitable
semisolid medium such as that of Fletcher. Multiple
tubes of media should be used and small inocula, 1
to 3 drops of blood to 5 ml. of medium, planted.
As Fletcher's medium remains stable for 3 to 4
months, it may be supplied to the physician by
the laboratory. It can be inoculated at the bed-
side, and shipped by mail. Voided urine may be
cultured directly into semisolid medium if first
diluted. Five 10-fold dilutions of the urine in
buffered saline or a suitable broth are first pre-
pared. Since quantitative dilutions are not necessary,
a single 2.0 ml. syringe with a 20-gauge needle may
be used to prepare these dilutions in the syringe.
Draw up 0.1 ml. of urine and 0.9 ml. of diluent for
the first dilution; expel all but 0.1 ml. of this
dilution, planting one drop onto 5 ml. of medium.
For the second dilution, again draw 0.9 ml. of
diluent into the syringe. Again after expelling
all but 0.1 ml. of the mixture, plant one drop onto
5 ml. of medium. Repeat for the third, fourth, and
fifth dilution. Otherwise, inoculate into such test
animals as weanling hamsters or guinea pigs.
Any pure cultures isolated should be shipped to a reference laboratory in semisolid media for confirmation by serologic tests.
Specimens for serologic tests
Microscopic or macroscopic agglutination tests are
the most common serologic procedures employed for
the diagnosis of leptospirosis. Two specimens of
blood should be taken: one during the first week
or acute phase of illness, the other ten days or
two weeks later to detect a rise in titer. Maximum
titers are reached by the third or fourth week.
While serodiagnostic tests are of value in confirm-
ing past or current leptospiral infection, paradoxical
reactions may occur and determination of the infecting
serotype can be made only by isolation and serologic
identification of the leptospires.
Any organism capable of invading human tissues can
cause infection of the meninges. As used here,
the term meningitis refers to an inflammation
of the meninges, brain, or spinal cord accompanied by increased cell counts in the spinal fluid and other features common to meningitis regardless of etiology. Such infection can be caused by viruses, bacteria, fungi, spirochetes, or protozoa. characteristic of bacterial meningitis is a predominance of polymorphonuclear cells in spinal fluid associated with a decreased dextrose content. Infection with Mycobacterium tuberculosis is an exception to this general rule.
Nine species of bacteria, representative of the groups involved, should be recognized as most often involved in meningitis, namely: 1) Neisseria meningitidis, 2) Hemophilus influenzae, 3) Diplococcus pneumoniae, 4) Streptococcus pyogenes, 5) Staphylococcus aureus, 6) Proteus species, 7) Pseudomonas species, 8) Escherichia coli, 9) Mycobacterium tuberculosis, 10) Listeria monocytogenes and Mimeae. Meningococcus meningitis is the most commonly encountered of the bacterial meningitides; Flavobacterium meningosepticum is the only organism that occurs in epidemics. In serial specimens from an individual, some may be negative and still be followed by others that will yield almost a pure culture. Therefore, carrier surveys in which a single nasopharyngeal culture is made from each individual are of little or no value.
The significance of the meningococcus recovered from the nasopharynx either in epidemic or endemic situations depends on its serologic group and virulence. Accurate serologic typing is therefore of greatest importance.
Specimens to be collected for isolation of the
agent
Spinal fluid, blood, nasopharyngeal swabs and
petechial scrapings and less frequently ventric-
ular, cisternal, or subdural fluid are the speci-
mens usually submitted for laboratory examination.
The time in the course of the infection at which
the specimen is taken, the temperature at which
it is held, and the amount of the inoculum used
for cultures are all important. Spinal fluid
should be taken with the appearance of meningeal
symptoms; blood for culture, as soon as infection
is suspected; petechial scrapings for culture in
doubtful cases and post-mortem. Nasopharyngeal
swabs are cultured for carrier detection. All
specimens for isolation of the meningococcus
should be kept at body temperature in transit
to the laboratory because of the delicacy of
the organism. If nasopharyngeal swabs are not
planted on warm plating media immediately, they
may be transported in a tube containing defibrinated
horse blood or a semisolid medium, but they must
be kept warm at all times. Blood for culture may
be drawn directly into a bottle of warm B-D cul-
ture medium and taken to the laboratory for incuba-
tion. During transit, it should be kept at body
temperature.
b. Specimens for serologic tests
Serial specimens of blood, the first taken during
the first 5 days of illness and the later ones at
4 to 5 day intervals, should be tested.
specimen is to be shipped, the cells should be
removed to prevent hemolysis and to obviate loss
of agglutinin titer in transit.
Epidemic plague has swept over large areas of the world
from time to time, temporarily paralyzing all forms of
human activity. Primarily, plague is a disease of rats
and wild rodents. It is transmitted from animal to animal
by the bites of infected fleas, with man serving only as
an accidental host. The pneumonic type of the disease
can, however, be spread from man to man by droplet
infection without the intervention of an insect vector.
a. Specimens to be collected for isolation of the agent
Bubo fluid, portions of bubo, spleen, bone marrow,
sputum, blood, or ectoparasites may be submitted
for culture in cystine broth or on blood agar slants
or plates. Shipment of original specimens should be
in double containers with screw tops. Identification
of the disease in decayed or mummified carcasses may
be accomplished by agglutination tests, precipitin
tests, or the fluorescent antibody technic.
Diagnosis of plague by FA technic
This procedure appears also to be reliable and
rapid in the examination of bubo exudate, blood
from human cases, or tissue impression smears,
yielding results in one hour. However, if
clinical material is first injected into labora-
tory animals, about two days must be allowed for
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