264 BACTERIOLOGICAL EXAMINATION OF MATERIAL 
Examination by Staining.— A Gram-stained preparation of sputum, 
buccal or pharyngeal material usually contains a variety of micro- 
organisms comprising cocci, spiral forms, and even fungi and yeasts. 
Many of the organisms may be normal inhabitants of the buccal 
cavity, and of the pathogenic organisms, pneumococci, streptococci 
and occasionally diphtheria bacilli are found. Usually clinical signs 
or an abnormal appearance of the sputum, mouth or throat lead to 
a microscopic examination of the material from this region and, as a 
rule, the nature of the symptomatology is a reliable guide to the stain 
to be used. Among the organisms which stain by Gram's method, 
pneumococci, streptococci, staphylococci. Micrococcus tetragenus and 
occasionally Diplococcus crassus are the more common spherical 
organisms. Micrococcus catarrhalis. the meningococcus and para- 
meningococci are the only Gram-negative cocci, so far as is known. 
Of the Gram-staining bacilli, the diphtheria and pseudodiphtheria 
bacilli together with B. subtilis and rarely B. anthracis may be found. 
The bacillus of Friedlander, typhoid, influenza, pertussis, plague and 
glanders bacilli are Gram-negative. B. fusiformis and Vincent's 
spirillum are Gram-negative as well. They color somewhat indistinctly 
with LofRer's methylene blue, but very distinctly and characteristically 
with Wright's or Giemsa's stain. Mouth spirals and Treponema 
pallidum are best stained with the latter method. Tubercle, leprosy 
and nasal secretion bacilli (Karlinski) stain with the acid-fast stain. 
Higher bacteria and moulds are occasionally identified in material 
from the buccal cavity. Actinomyces, Oidium albicans, aspergillus, 
mucor, streptothrix and yeasts have been detected. The virus of 
poliomyelitis has also been demonstrated in material from the naso- 
pharynx which has been freed from bacteria by passage through a 
Berkefeld filter and injected into a monkey. Recently the virus of 
lethargic encephalitis has also been recovered from the spinal fluid in 
the same manner so it is stated. 
For the routine examination of sputum, three stains are ordinarily 
employed— Ziehl-Neelsen for tubercle bacilli, Loffler's alkaline methyl- 
ene blue or Albert's stain for diphtheria, pseudodiphtheria, and fusiform 
bacilli (and Vincent's spirillum), and the Gram stain, using dilute carbol 
fuchsin as a counterstain for pneumococci, streptococci, influenza and 
pertussis bacilli principally. Smith's stain for sputum (see page 207) 
is advantageous for pneumonic sputum. 
The organisms mentioned previously (page 106), but not detailed in 
the routine examination of sputum, are of comparatively rare occur- 
rence. They must be studied by purely cultural methods. 
Cultural Methods.— Antiseptic gargles should not be used for two 
or more hours before collecting sputum or material from the mouth 
or pharynx for cultural examination. Sputum or exudate, obtained 
in a suitable manner, is first washed through six or seven portions of 
sterile salt solution, if its cohesiveness permits, to remove or diminish 
surface contamination. For a majority of bacteria, freshly prepared 
