Quality control methods for

Table 5 
Determination of Enterobacteriaceae and certain other Gram-negative bacteria 
Result for each 
quantity or volume 
Probable number of 
bacteria per g of material 
1.0 g or 
1.0 ml
0.1g or
0.1 ml 
0.01 g or 
0.01 ml 
+ 
+ 
+ 
More than 10
2
+ 
+ 
− 
Less than 10
2
but more than 10 
+ 
− 
− 
Less than 10 but more than 1 
− 
− 
− 
Less than 1 
Prepare a subculture on a plate with MacConkey agar and incubate at 43-45°C 
for 18-24 hours. Growth of red, generally non-mucoid colonies of Gram-negative 
rods, sometimes surrounded by a reddish zone of precipitation, indicates the 
possible presence of E. coli. This may be confirmed by the formation of indole at 
43.5-44.5°C or by other biochemical reactions. The material passes the test if no 
such colonies are detected or if the confirmatory biochemical reactions are 
negative. 
Salmonella spp. 
Incubate the solution, suspension or emulsion of the pretreated material 
prepared as described above at 35-37°C for 5-24 hours, as appropriate for 
enrichment. 
Primary test 
Transfer 10 ml of the enrichment culture to 100 ml of tetrathionate bile brilliant 
green broth and incubate at 42-43°C for 18-24 hours. Prepare subcultures on at 
least two of the following three agar media: deoxycholate citrate agar; xylose, 
lysine, deoxycholate agar; and brilliant green agar. Incubate at 35-37°C for 24-48 
hours. Carry out the secondary test if any colonies are produced that conform to 
the description given in Table 6. 
Table 6 
Description of Salmonella colonies appearing on different culture media 
Medium 
Description of colony 
Deoxycholate citrate agar 
Well developed, colourless 
Xylose, lysine, 
deoxycholate agar 
Well developed, red, with or without 
black centres deoxycholate agar 
Brilliant green agar 
Small, transparent and colourless, or 
opaque, pink or white (frequently 
surrounded by a pink to red zone) 

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Quality control methods for medicinal plant materials 
Secondary test 
Prepare a subculture of any colonies showing the characteristics described in 
Table 6 on the surface of triple sugar iron agar using the deep inoculation 
technique. This can be achieved by first inoculating the inclined surface of the 
culture medium followed by a stab culture with the same inoculating needle and 
incubating at 35-37°C for 18-24 hours. The test is positive for the presence of 
Salmonella spp. if a change of colour from red to yellow is observed in the deep 
culture (but not in the surface culture), usually with the formation of gas with or 
without production of hydrogen sulfide in the agar. Confirmation is obtained by 
appropriate biochemical and serological tests. 
The material being examined passes the test if cultures of the type described do 
not appear in the primary test, or if the confirmatory biochemical and serological 
tests in the secondary test are negative.