3. Laboratory Methods in Diagnosis 
a. Direct Methods: 
(1) Preparation of specimen for inocula- 
tion. Certain types of materials, such 
as throat washings, sputum, and feces, 
must be freed of contaminating bac- 
teria and molds before they are used for 
inoculation. Ultra-centrifugation can 
separate the contaminants at one speed 
and concentrate the virus at another. 
Filtration, chemical treatment, and 
especially the addition of antibiotics 
are also useful in removing contami- 
nants. An effort should be made how- 
ever, to collect materials as free from 
contaminants as possible. 
(2) Animal inoculation methods. Various 
species of animals are inoculated by 
different methods (intracerebral, in- 
traperitoneal, intranasal, intravenous, 
etc.), depending on the type of human 
disease being studied and the type of 
material available. Symptoms and 
pathological responses are then ob- 
served. These methods are used in the 
isolation of the agents of rabies, psit- 
tacosis, lymphogranuloma venereum, 
encephalitis, herpes, rickettsial, and 
other diseases. 
(3) Chick embryo inoculation methods. 
Embryonic tissue is more susceptible 
to invasion by most viruses than adult 
tissue. Chick embryos of varying ages 
are inoculated by different routes (yolk 
sac, allantoic sac, amniotic sac, etc.) 
and are observed for death, for specific 
pathological lesions, or as a source of 
antigenic material for specific serolog- 
ical identification against known pos- 
itive immune sera (used in influenza, 
smallpox, mumps, etc.). 
(4) Tissue culture methods. Tissue culture 
represents the use of specific tissues 
in culture where extracellular factors 
of the media can be controlled. This 
method has become the laboratory 
procedure of choice in the study of 
many viruses (those of poliomyelitis, 
influenza, chickenpox, etc.) for isola- 
tion or for antibody determination and 
measurement. 
b. Indirect Methods: 
( 1 ) Examination of tissues for pathological 
change. Certain viruses cause specific 
cellular responses characterized by in- 
clusion bodies, such as Negri bodies 
in rabies, which are diagnostic of active 
infection. Some of the larger viruses — 
lymphogranuloma venereum, for ex- 
ample can actually be seen in tissue 
smears as elementary bodies. Fluores- 
cent antibody techniques have recently 
been applied to the direct examination 
of infected tissues or exudates for the 
rapid identification of specific viral 
antigen. This technique has been most 
widely used in the diagnosis of rabies. 
(2) Complement fixation tests. Specific 
antigens are available for testing 
paired acute and convalescent sera in 
many viral and rickettsial diseases. In 
rickettsial diseases, the complement 
fixation test is more specific than the 
Weil-Felix proteus agglutination pro- 
cedure. 
(3) Red cell agglutination tests. Certain 
viruses cause red blood cells to ag- 
glutinate. The presence of specific anti- 
bodies against that virus will inhibit 
the agglutination. This type of test is 
frequently used in influenza, mumps, 
Newcastle virus disease, certain en- 
cephalitis infections, and rubella. 
(4) Neutralization tests. In the neutraliza- 
tion test, the fact that a given virus has 
been inactivated or “neutralized” by 
specific antibody present in a serum 
specimen is demonstrated by the fail- 
ure of a susceptible animal or tissue 
culture to become infected when in- 
oculated with the serum-virus mixture. 
This procedure can be used with paired 
acute and convalescent sera in any 
virus or rickettsial disease for which 
there is a susceptible animal or an 
appropriate tissue culture system in 
which the etiologic agent will multiply. 
4. Interpretation of Laboratory Results 
a. Virus Isolation: 
Failure to isolate a specific virus from a 
specimen does not rule out that agent, or an 
unsuspected one, as the possible cause of 
the illness in question. When a virus is iso- 
lated, it signifies current infection (which 
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