Primary Isolation Using Guinea Pigs and Embryonated Eggs 



Suspend the minced tissue in enougli phosphate buffered sahne (PBS), pH 7.2, to form a 10% 

 suspension. Inoculate aliquots of the suspension onto bacteriologic media which have been suc- 

 cessfully used in growing the LDB and onto other bacteriologic media such as blood agar or 

 Trypticase soy (TS) agar to serve as controls. Place other aliquots of the suspension on glass 

 slides, air dry them, fix them in acetone or 10% neutral Formalin, and evaluate them with the 

 direct FA test. Dispense 1-ml aliquots of the rest of the suspension into labelled 13- X 100-mm 

 screw-capped test tubes; Hash-freeze the samples in a dry ice-alcohol bath, and store them at 

 -70° C as reference materials. 



B. Interpretating FA Results and Direct Culture Attempts 



The four possible combinations of results can be described as follows: 



1. Both the FA results and attempts to culture the organism //; vitro are negative. Such 

 results indicate that the LDB is not present in the specimen, and attempts to isolate other 

 pathogens should be considered. 



2. Both the direct FA results and the in vitro isolation attempts are positive. Such results 

 strongly indicate that the LDB is present in the specimen and that it may have been isolated. 

 However, confirmational testing must be done to verify that the organism in question is the 

 LDB; i.e., the bacterium isolated on agar should have cultural and staining characteristics 

 and direct FA results comparable to those of prototype strains of the LDB. 



3. Direct FA test results for tissue suspensions are negative, but cultural results are positive 

 on media known to support the growth of the LDB. This combination of results must be 

 interpreted cautiously. In some cases the LDB miglit be isolated directly on agar when it 

 cannot be demonstrated with direct FA tests of the tissues. The resolution of such a 

 situation led to the discovery of the second serogroup of LDB (<S'. 10). However, a more 

 likely explanation for this conflicting pattern of direct FA and culture results is that a 

 bacterium other than the LDB has been isolated. In any case, the reason for the discrepant 

 results must be determined and the data reconciled by appropriate staining, FA, and cultural 

 techniques before isolates are reported to be LDB. 



4. Direct FA test results are positive for the LDB, but the organism is not isolated in direct 

 cultures. If this combination of results occurs, attempts should be made to isolate the 

 organism with guinea pigs and embryonated eggs as previously discussed and as described 

 below. 



C. Inoculating Guinea Pigs 



1. Prebleed each guinea pig and store the serum for future reference. Thaw an aliquot of 

 the tissue suspension which was stored at -70° C, and dilute it to approximately 5 ml in 

 PBS. Inoculate (25-gauge needle) each of four adult male guinea pigs (approximately 600 g) 

 iutraperitoneally with a 1-ml aliquot of the tissue suspension. 



2. Observe the guinea pigs daily for signs of illness. Symptoms sometimes include ruffled 

 fur, watery eyes, and prostration. Determine their rectal temperatures at a specified time 

 each day for 10 days. Temperatures >39.5° C are considered to be frank fevers. 



3. Sacrifice febrile guinea pigs on the second day they have frank fevers by exposing them 

 to excessive levels of CO2 vapor. Aseptically remove a portion of each guinea pig's spleen 

 with sterile forceps and scissors. (Use one set of forceps and scissors to open the animal and 

 a second set to remove the spleen.) Grind a piece of the spleen with alundum and dilute in 

 PBS to form a 10% suspension. Dispense aliquots of the guinea pig spleen into labelled test 



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