Pharmaceutical Manufacturing Handbook: Production and

OINTMENTS AND CREAMS 285
presence of Staphylococcus aureus and Pseudomonas aeruginosa . In some cases,
screening for Escherichia coli, Salmonella species, and total aerobic microbial counts
is recommended by the USP. For instance, clobetasol propionate ointment USP and
mometasone furoate ointment USP are screened for all these organisms. In addition,
preparations meant for rectal, vaginal, and urethral applications are tested for yeasts
and molds [15] .
Test for S. aureus and P. aeruginosa The test sample is mixed with about 100 mL
of fl uid soybean – casein digest (FSBCD) medium and incubated. If microbial growth
is observed, it is inoculated in agar medium by the streaking technique. Vogel –
Johnson agar (VJA) medium is used for S. aureus screening, and cetrimide agar
(CA) medium is used for screening P. aeruginosa . The petridishes are then closed,
inverted, and incubated under appropriate conditions. The appearance of black
colonies surrounded by a yellow zone over VJA medium and greenish colonies in
CA medium indicates the presence of S. aureus and P. aeruginosa , respectively.
Various other agar media are also available for screening these organisms. A coagulase
test is then performed for confi rming the presence of S. aureus and oxidase and
pigment tests for confi rming P. aeruginosa .
Test for Salmonella Species and E. coli The test sample is mixed with about 100 mL
of fl uid lactose (FL) medium and incubated. If microbial growth is observed, the
contents are mixed and 1 mL is transferred to vessels containing 10 mL of fl uid
selinite cystine (FSC) medium and fl uid tetrathionate (FT) medium and incubated
for 12 – 24 h under appropriate conditions. To identify the presence of Salmonella ,
samples from the above two media are streaked over brilliant green agar (BGA)
medium, xylose lysine desoxycholate agar (XLDA) medium, and bismuth sulfi te
agar (BSA) medium and incubated. The appearance of small, transparent or pink -
to - white opaque colonies over BGA medium, red colonies with or without black
centers over XLDA medium, and black or green colonies over BSA medium indicates
the presence of Salmonella . It is further confi rmed in triple sugar iron agar
medium. The presence of E. coli is screened by streaking the samples from FL
medium over MacConkey agar medium. The appearance of brick red colonies indicates
the presence of E. coli . It is further confi rmed using Levine eosin methylene
blue agar medium.
total aerobic microbial counts The plate method or multiple - tube method is
performed to estimate the total count. About 10 g or 10 mL of the test sample is
dissolved or suspended in suffi cient volume of phosphate buffer (pH 7.2), fl uid
soybean casein digest (FSBCD) medium, or fl uid casein digest – soy lecithin –
polysorbate 20 medium to make the fi nal volume 100 mL. In the plate method, about
1 mL of this diluted sample is mixed with molten soybean – casein digest agar
(SBCDA) medium and solidifi ed at room temperature. The plates are inverted and
incubated for two to three days. The number of colonies that are on the surface of
nutrient media are counted. The multiple tube method is performed using sterile
fl uid SBCD medium. The number of colonies formed should not exceed the limits
specifi ed in an individual monograph. For example, clobetasol propionate ointment
USP and hydrocortisone valerate ointment USP contains less than 100 colony -
forming units (CFU) per gram of sample.