Page 18 - Quality control of pharmaceuticals (07-PA 704) practical

A.1) Examination of sample by Membrane Filtration Method:

1 Aseptically connect the rubber tube of sterile manifold to receiver tank and receiver tank
rubber pipe to vacuum pump.

2 Using sterile smooth tip forceps, place a 47 mm diameter 0.45m sterile membrane
filter on the center of the filter support screen. Without disturbing the filter, place the
funnel on top of the filter holder base.

3 Separately transfer 10 ml of pretreated sample from step no. 3.1.3 to each two 90 ml of
sterile 0.1% peptone water. Mix well and transfer the whole quantity of dilution to each
of two membrane filters and filter immediately.

4 Wash both the membrane filter with each 3 x 100 ml of sterile 0.1% peptone water into
the filtration funnel and filter under partial vacuum

5 After completion of filtration process, shut off the vacuum with the help of vacuum
control key.

6 Transfer one of the membrane filters, intended for the enumeration of bacteria, to the
surface of the plate containing SCDA and other, intended for the enumeration of fungi, to
the surface of the plate of SDA.

7 For positive control carry out the same procedure in duplicate except for sample use 100
ml fluid A inoculated with 100 bacterial cells and another 100 ml fluid A is inoculated
with C.albicans intended for identification of total aerobic and fungal count respectively.

8 For negative control carry out the same procedure except for sample use 100 ml sterile
0.1% peptone water.

9 Incubate the plates for 5 days, unless a more reliable count is obtained in shorter time, at
30 to 35°C in the test of bacteria and 20 to 25°C in the test for fungi.

10 Count the number of colonies that are formed. Calculate the number of cfu per gram or
per ml of the sample being examined.

A.2) Examination of sample by Plate Count Method:

1 Use this method for fatty products and product insoluble in water.
2 Use 90 mm sterile petri plate. Take a four-petri plate and label two plates for bacteria and

remaining two for fungi count. Transfer 1 ml quantity of each pretreated dilution sample
solution to each of four petri plates.
3 Add 15 ml of sterile liquefied SCDA at not more than 45°C, in to two plates labeled for
bacterial count.
4 Then add 15 ml of sterile liquefied SDA at not more than 45°C, into two plates labeled
for fungal count.
5 Allow to solidify the plates at room temperature, invert and incubate at 30 to 35°C for 5
days and 20 to 25°C for 5 days respectively.
6 Count the number of colonies that are formed. Calculate the number of cfu per gram or
per ml of the sample being examined.

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