Method tor Isolating Legionnaires" Disease Bacterium I'roiii Soil and Water Samples 



growth on each medium, and record as follows: 



sparse growth <10'' CFU/ml 



' moderate growth lO"* to 10* CFU/ml 



heavy growth >10*' CFU/ml 



Use the calculated microbial density or CFU for each water sample as a guide to determine 

 amount of sample to inject into guinea pigs in the procedure described in a later section of this 

 chapter. 



Examine plates at 3 days and daily thereafter for the development of additional colonies 

 resembling the LDB. Colonies of the LDB are usually visible within 4-7 days; however, incubate 

 plates with no growth or well-dispersed colonies of nomial tlora for 14 days before discarding as 

 negative. Confirm isolates suspected of being LDB with tests described elsewhere in this manual. 



Primary Culture of Soil Samples. Culture soil samples on F-G and CYE agar media. To 

 prepare a soil sample for culture, first add 10 gm of soil to 100 ml of sterile distilled water 

 containing 0.5% Tween 60. Then shake the suspension vigorously for 30 min on a mechanical 

 shaker. Let the heavy particles settle for 5-10 min, and then plate the aqueous layer as described 

 for liquid samples using serial 10-fold dilutions through 10"". Examine all plates for LDB as 

 described above for water samples. 



PREPARATION OF SAMPLE AND INOCULATION OF GUINEA PIGS 



Although the guinea pig procedure is a laborious and tedious means of isolating the LDB, 

 researchers have not yet found a satisfactory alternative for working with soil and water samples. 

 Only once in our laboratories has the LDB been isolated directly from a water sample without 

 inoculating guinea pigs. Results of direct FA tests (performed with polyvalent conjugates against 

 all known serogroups) show which samples are most likely to contain the LDB when tested 

 further. Environmental samples are frequently much more contaminated with other microflora 

 than are clinical specimens. If a guinea pig is inoculated with a large number of microbial tlora, 

 the animal is likely to die of causes other than an LDB infection. Therefore, it is important to be 

 aware of the nonnal tlora concentration in the samples when choosing the inoculum for the 

 guinea pigs. We currently do this by culture. Other potentially suitable methods for estimating 

 microbial density of non-LDB which we have not yet evaluated are direct microscopic observa- 

 tion of Gram-stained slides and growth of non-LDB on total plate count media. Before injecting 

 guinea pigs with samples, establish each animal's average baseline temperature from measure- 

 ments taken on each of the 5 days before inoculation. 



Water Samples (See Figure 1). If the CFU/ml described above from cultures of a water 

 sample is less than 10", concentrate the sample. Centrifuge 100 ml at high speed (ca. 2900 X g) 

 for 30 min; discard the supernatant. Resuspend the sediment in 10 ml of sterile water. Mix well, 

 and inoculate two guinea pigs intraperitoneally (IP) with 3 ml each of the sample. When the 

 CFU/ml for a sample is moderate (10'* to 10*), inject 3 ml of the untreated sample into each of 

 two guinea pigs. If the CFU/ml for a sample is high (> 10* ), dilute it to 10* CFU/ml and inject 3 

 ml of the diluted sample. 



Soil Samples (See Figure 2). Suspensions of soil samples to be used as inoculum are prepared 

 as follows: Add 10 gm of soil sample to 100 ml of sterile distilled water containing 0.5% Tween 

 60. Place the mixture on a shaker at moderate speed for 30 min. Allow 5-10 min for the heaviest 

 particles to settle, decant and save the top portion, and discard the heavy sediment. Centrifuge 

 the top portion (approximately 90 ml) of the suspension at 400 X g for 5 min. Carefully remove 

 the supernatant and discard the sediment. Centrifuge the supernatant at 2900 X g for 30 min. 

 Discard this tlnal supernatant, and resuspend the sediment in 10 ml of sterile distilled water. 



87 



