Quality control methods for

Pretreatment of the material being examined 
Pretreat the material as described in the "Test for specific microorganisms", page 
64, but in place of lactose broth use buffered sodium chloride-peptone solution 
pH 7.0, or another suitable medium shown not to have antimicrobial activity 
under the conditions of the test. 
Membrane filtration 
Use membrane filters with a nominal pore size of not greater than 0.45 µm, the 
effectiveness of which in retaining bacteria has been established. For example, 
cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions, 
and cellulose acetate filters for strongly alcoholic solutions. The technique 
described uses filter discs of about 50mm in diameter. For filters of a different 
diameter, adjust the volumes of the dilutions and washings accordingly. Sterilize 
the filtration apparatus and the membrane by appropriate means. They are 

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Quality control methods for medicinal plant materials 
designed to permit the solution being examined to be introduced and filtered 
under aseptic conditions, and the membrane to be transferred to the culture 
medium. 
Transfer 10ml or a solution containing 1g of the material to each of two 
membrane filters and filter immediately. If necessary, dilute the pretreated 
material to obtain an expected colony count of 10-100. Wash each membrane, 
filtering three or more successive quantities of approximately 100ml of a suitable 
liquid such as buffered sodium chloride-peptone solution, pH 7.0. For fatty 
materials, a suitable surfactant may be added, such as polysorbate 20R or 
polysorbate 80R. Transfer one of the membrane filters, intended primarily for 
the enumeration of bacteria, to the surface of a plate with casein-soybean digest 
agar and the other, intended primarily for the enumeration of fungi, to the 
surface of a plate with Sabouraud glucose agar with antibiotics. Incubate the 
plates for 5 days, unless a more reliable count can be obtained otherwise, at 30-
35°C for the detection of bacteria and at 20-25°C for the detection of fungi. Count 
the number of colonies formed. Calculate the number of microorganisms per g 
or per ml of the material tested, if necessary counting bacteria and fungi 
separately. 
Plate count 
For bacteria. Use Petri dishes 9-10 cm in diameter. To one dish add a mixture of 
1 ml of the pretreated material and about 15 ml of liquefied casein-soybean 
digest agar at a temperature not exceeding 45°C. Alternatively, spread the 
pretreated material on the surface of the solidified medium in a Petri dish. If 
necessary, dilute the pretreated material as described above to obtain an 
expected colony count of not more than 300. Prepare at least two dishes using 
the same dilution and incubate them at 30-35°C for 5 days, unless a more reliable 
count is obtained in a shorter period of time. Count the number of colonies 
formed and calculate the results using the plate with the largest number of 
colonies, up to a maximum of 300. 
For fungi. Use Petri dishes 9-10 cm in diameter. To one dish add a mixture of 1 
ml of the pretreated material and about 15 ml of liquefied Sabouraud glucose 
agar with antibiotics at a temperature not exceeding 45°C. Alternatively, spread 
the pretreated material on the surface of the solidified medium in a Petri dish. If 
necessary, dilute the pretreated material as described above to obtain an 
expected colony count of not more than 100. Prepare at least two dishes using 
the same dilution and incubate them at 20-25°C for 5 days, unless a more reliable 
count is obtained in a shorter period of time. Count the number of colonies 
formed and calculate the results using the dish with not more than 100 colonies. 
Serial dilution 
Prepare a series of 12 tubes each containing 9-10ml of soybean-casein digest me-
dium. To each of the first three tubes add 1 ml of the 1:10 dilution of dissolved, 
homogenized material prepared as described on pages 64-65. To the next three 
tubes add 1 ml of a 1:100 dilution of the material and to the next three tubes add 
1 ml of a 1:1000 dilution of the material. To the last three tubes add 1 ml of the 
diluent. Incubate the tubes at 30-35°C for at least 5 days. No microbial growth 

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Quality control methods for medicinal plant materials 
should appear in the last three tubes. If the reading of the results is difficult or 
uncertain owing to the nature of the material being examined, prepare a 
subculture in a liquid or a solid medium, and evaluate the results after a further 
period of incubation. Determine the most probable number of microorganisms 
per g or ml of the material using Table 8. 
If, for the first column, the number of tubes showing microbial growth is two or 
less, the most probable number of microorganisms per g or per ml is less than 
100.