ally produce a granular agglutinate, while flagellated or ''H" cells yield a
more ''fluffy" type of clump, and capsulated organisms often produce a
solid button which cannot readily be broken up. As the serum becomes
208 MANUAL OF MICROBIOLOGICAL METHODS
more dilute, the agglutination becomes weaker. The *' titer " of antiserum
is the highest dilution at which clumping can be readily detected. Occa-
sionally, antiserums will be found which fail to agglutinate until the
liigher dilutions are reached. This range of more concentrated serum
where agglutination fails to occur is called the '^prozone."
In instances where it is desirable to compare several antiserums or
several antigens, all determinations should be made at the same time.
Even in the hands of skilled individuals, the agglutination test may yield
variations of one tube among tests done on different days.
Microscopic slide test. The microscopic method offers the advantage
of requiring only small amounts of serum, but at the sacrifice of some
Antiserum dilutions and antigen are prepared as described for the
macroscopic tube test. A loopful of antiserum at a given dilution is
mixed with a loopful of antigen on a cover glass rimmed with petroleum
jelly, and over this is inverted a hollow ground (''hanging-drop") slide.
Controls of antigen plus saline and antigen plus preimmunization serum
are also included. The hanging-drop preparations are allowed to incu-
bate for 15-30 min and examined with low-power objective of the micro-
scope. With some antigens there is a tendency toward spontaneous
clumping; however, this should be detected in one or both of the control
Macroscopic slide test. This procedure is most useful for the serologic
identification of cultures and is widely used for enteric bacteria (Edwards
and Ewing, 1955). When used for this purpose, it is essentially a quaU-
The antigen is prepared by emulsifying growth from a slant culture in a
droplet of saline on a glass slide. The suspension Carbamazepine Tegretol
should be moderately
dense, but precise standardization is not necessary. A droplet of pre-
diluted antiserum is mixed with the antigen, and the slide is rocked back
and forth. With a good antiserum, agglutination should occur rapidly
and the clumps should be clearly discernible to the naked eye.
The quantitative measurement of agglutinin. While the methods
described previously are entirely adequate for many studies of microbial
antigens, it is also possible to measure agglutinin with greater precision.
This is Carbamazepine Tegretol done by adding thoroughly washed bacterial suspension to meas-
ured volumes of antiserum and allowing the agglutinin to combine with
the bacteria. After an equilibrium is reached, the cells are washed free
of extraneous protein, analyzed by a micro-Kjeldahl Carbamazepine Tegretol
procedure, and the
weight of agglutinin nitrogen calculated.
For certain applications, such a method is greatly superior to the less
quantitative technics; however, the proper evaluation of all factors
involved may well require more information than it is possible to impart in
SEROLOGICAL METHODS 209
a chapter of this length. Since the procedure is well presented in Kabat
and Mayer (1948), further description has not been Carbamazepine Tegretol
When a clear solution of a protein or polysaccharide antigen is mixed
with the appropriate antibody or precipitin, the mixture turns cloudy and
then precipitates. This reaction differs in principle from agglutination
chiefly in the size of Carbamazepine Tegretol
the particles involved. In agglutination, the par-
ticles are usually cells and in any event are large enough to be seen under
the microscope, while in the precipitin reaction, the particles are of
Preparation of reagents. It is essential that both antigen and antibody
solution be perfectly clear and free from lipid or insoluble material.
Lipid in serum can usually be avoided by withholding food from the
animal for 24 hr before bleeding. If this precaution has not been taken,
lipid can be removed by storing the serum at 0C for 3-5 days and then
centrifuging at 2,000-5,000 X gravity to bring lipid to the surface. Any
particulate matter other than lipid will be thrown to the bottom of the Carbamazepine Tegretol
centrifuge tube, and the clear serum may be withdrawn with a capillary
pipet. Clarification can also be accomplished by passing the chilled
serum through a precooled Seitz filter containing a clarifying pad (average
pore diameter 5 m) or Carbamazepine Tegretol
a coarse sterilizing pad (average pore diameter 1 ju).
Antigens to be used for qualitative precipitin tests may consist of exu-
date from an infected animal or cell-free fluid from a broth culture. In
other instances, bacteria may be disrupted by mechanical grinding or by
sonic oscillation and the soluble products used as antigen.
For precise work, it mil be necessary to purify the antigen under study
by the physical and chemical procedures customarily employed for pro-
teins, polysaccharides, and other high-molecular-weight compounds.
Concentration of antigen is usually expressed in terms of weight in
milligrams or micrograms per milliliter of solvent when dealing with puri-
fied antigens. In the case of protein or other nitrogen-containing anti-
gens, concentration may be expressed as milligrams or micrograms of
nitrogen based on a micro-Kjeldahl analysis. For crude antigens like
cultural supernates or lysates, concentration is ordinarily expressed in
terms of dilution of the original cultural fluid. This, of course, is less
desirable than expression on a weight basis but is satisfactory for applica-
tions where Carbamazepine Tegretol
strictly quantitative results are unnecessary.
The electrolyte concentration should be maintained within certain
limits. With most systems, a small amount of electrolyte is necessary
for precipitation, and if a great excess is present, precipitation wiU be
210 Carbamazepine Tegretol MANUAL OF MICROBIOLOGICAL METHODS
inhibited. Generally speaking, a satisfactory electrolyte level can be
assured by preparing antigen Carbamazepine Tegretol solutions in 0.9 per cent sodium chloride
solution (''saline"). This is approximately equivalent to 0.15M sodium
chloride. If the electrolyte strength is unknown, the sample should be
dialyzed against saline solution of known strength. The pH should be
maintained near neutrality, at least not lower than 6.5 or higher than 8.0.
The antigen dilution method. Prepare a series of tenfold dilutions of
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