Immunological techniques have been employed in studies concerned with the immunity of insect blood and the characterization of insect pathogens. A simple means for demonstrating the antibacterial action of insect blood is the cylinder-plate technique (Frings et al., 1948). A great variety of serological techniques was used by Briggs (1959) and Stephens (1959) when they investigated humoral immunity in Lepidoptera. Yama-fuji et al. (1958a) immunized silkworms [Bombyx mori (Linnaeus)] against virus disease. Krieg (1955c) found the serological diagnosis of rickettsial infections to be superior to microscopic examination.
Serological investigations of pathogens have predominantly been carried out on viruses (Bergold, 1958a); b u t bacteria crystals (Krywien-czyk and Angus, 1960) and rickettsiae (Krieg, 1955c, 1958c) have been studied to some extent. T h e antigen (virus inclusion bodies, inclusion body protein, virus particles, virus membranes, etc.) is prepared in highly purified form and injected into rabbits or guinea pigs. T h e antibodies have generally been determined in agglutination tests, b u t during the last few years Krywienczyk and associates have worked out other pro
cedures: a modification of Fulton and Dumbell's (1949) complement-fixation technique (Krywienczyk et al., 1958) and an agar-diffusion tech
nique, which is performed on microscopic slides and in which the re
action product appears in the form of a b a n d that is made visible with amidoschwartz (Krywienczyk and Bergold, 1961). A pipette for serological work was described by Krywienczyk and Bergold (1960). General infor
mation on serological techniques may be obtained from Carpenter (1956).
Immunohistochemical staining techniques have not yet been used in insect pathology.
17. TECHNIQUES IN INSECT PATHOLOGY 617 D . Other Biophysical and Biochemical Techniques
In the investigation of diseased insects and their pathogens, many other techniques have been or may be used, such as those for chemical and biochemical analysis, including colorimetric, titrimetric, gasometric, and dilatometric techniques; techniques of enzymology, chromatography, spectroscopy and spectrophotometry, electrophoresis, and radioautogra-phy; tracer techniques; X-ray techniques; and techniques pertaining to sedimentation, diffusion, and viscosity. Since many of them are performed on sections, suspensions of isolated cells or cellular components, or ex
tracts, their use often presupposes the use of other special techniques of microtomy, separation, or extraction. A large n u m b e r of books and greater works provides information on the various subjects (for instance, Glick, 1949, 1954; Eränkö, 1955; Oster and Pollister, 1956; Cowgill and Pardee, 1957; Lederer and Lederer, 1957; Dixon and W e b b , 1958; Fruton and Simmonds, 1958; Overman and Clark, 1960).
W i t h regard to references to biophysical and biochemical techniques in the insect pathology literature, only a few representative publications may be cited. Bergold's (1959b) review on the biochemistry of insect viruses and the review of Heimpel and Angus (1960) on bacterial insecti
cides may be used as sources for references. Procedures for the biochem
ical analysis of rickettsial proteins have been given by Krieg (1958a, 1959b, c), who also extracted R N A from cytoplasmic polyhedra (Krieg, 1959b). Techniques for the preparation and physical examination of virus crystals may be found in Klug et al. (1959), and some for the bio
chemical investigation of polyhedroses in Yamafuji et al. (1958b, 1960).
Several publications inform about preparation and measurement of bacterial enzymes (Chu, 1949; Heimpel, 1955a; Kushner and Heimpel, 1957; Patel and C u t k o m p , 1961). T h e chemical analysis of bacteria and crystals has been treated by Fitz-James (1955), H a n n a y and Fitz-James (1955), Angus (1956c), Fitz-James and Young (1958), and Fitz-James et al.
(1958). Various techniques for pH-determination in insects have been de
scribed and discussed by Heimpel (1955b, 1961) and Heimpel and Angus (1959).
V. CONCLUDING REMARKS
T h i s chapter on techniques may be closed with a few remarks which seem to be a matter of course to many insect pathologists, b u t which the author, after having surveyed a large n u m b e r of publications, believes to have some foundation. N o t only does detailed information on techniques increase the understanding for the investigator's conclusions, b u t it also enables other research workers fully to utilize existing work. O u r present knowledge of techniques is often not extended enough to let us decide
safely which detail is u n i m p o r t a n t , and a skipped fixation time, concen
tration, dosage, etc., may become a serious obstacle for comparing and repeating results. Also, general procedures may differ from country to country. T h u s it is recommended that the description of techniques be kept very detailed rather than too general and incomplete. Fortunately, during the last few years scientific journals have increasingly promoted formats that give a separate section or paragraph to "materials and methods."
Research on techniques is in itself a phase or branch of insect pathol
ogy, and although the n u m b e r of papers dealing exclusively with tech
niques have been increasing recently, hardly more than the surface seems to be scratched. I n general, insect pathologists have tended to, and should, use more and more technical advances from other fields, b u t they have been slow to develop new procedures of their own. T h a t it is important for insect pathologists to recognize and use the techniques and procedures of other sciences and of other branches of entomology, such as insect physiology, as well as develop techniques of their own, has recently been emphasized by Steinhaus (1962). H e advocates that this be done for the study of both infectious and noninfectious diseases and that "Every cognate science and satellite discipline must be utilized to its fullest if insect pathology is to flourish to the extent it can and should."
Unfortunately, many of the commonly used techniques and methods have not been critically evaluated, and little attention has been given to their reliability and limits. For instance, the various techniques of virus infection (leaf feeding, microfeeding, microinjection) have h a d little comparative study. Such considerations as well as others may have motivated Bergold (1958b) when he suggested an international standard
ization of infectivity tests. Similarly, microscopic and electron microscopic studies have h a d little parallel comparison, and there is no published account of an investigator's having studied granulosis-affected blood cells in both dark field and phase contrast for resolving differences in the findings. T h e selective staining techniques for virus inclusion bodies need a critical evaluation, especially since low-magnification electron microscopy apparently provides a m u c h safer means of identification.
Such considerations lead to the field of methodology, which becomes most important in diagnostic work (for instance, Krieg, 1955a; Krieg and Langenbuch, 1956b; Williams, 1957; Wittig, 1959b; and Chapter 16 in this volume).
17. T E C H N I Q U E S I N I N S E C T P A T H O L O G Y 619
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