PHARMACEUTICAL MICROBIOLOGY LABORATORY MANUAL

UST - Faculty of Pharmacy

Name: Group # Section:

EXPERIMENT 10
ANTIMICROBIAL EFFECTIVENESS TEST

PRODUCT: 30 mL nasal solution

DATA:
Initial Calculated Calculated Calculated
calculated concentration concentration concentration at
Container with:
concentration at 7 days at 14 days 28 days
(CFU/mL) (CFU/mL) (CFU/mL) (CFU/mL)

Candida albicans 3.9 x 105 6.3 x 103 1.1 x 102 50

Aspergillus niger 6.0 x 105 6.2 x 104 1.3 x 104 7.2 x 102

Escherichia coli 2.0 x 105 9.1 x 103 7.4 x 102 55

Pseudomonas
3.6 x 105 7.2 x 103 1.2 x 102 70
aeruginosa

Staphylococcus
6.0 x 105 1.5 x 105 5.8 x 104 1.4 x 104
aureus

CALCULATIONS:
Container with: Log of initial Log of Log of Log of
calculated calculated calculated calculated
concentration concentration concentration concentration
(CFU/mL) at 7 days at 14 days at 28 days
(CFU/mL) (CFU/mL) (CFU/mL)

Candida albicans 5.5911 3.7993 2.0414 1.6990

Aspergillus niger 5.7782 4.7924 4.1139 2.8573

Escherichia coli 5.3010 3.9590 2.8692 1.7404

Pseudomonas
5.5563 3.8573 2.0792 1.8451
aeruginosa

Staphylococcus
5.7782 5.1761 4.7634 4.1461
aureus
 Container with: Log reduction Log reduction Log reduction
at 7 days at 14 days at 28 days

Candida albicans 1.7918 3.5497 3.8921

Aspergillus niger 0.9858 1.6643 2.9209

Escherichia coli 1.3420 2.4318 3.5606

Pseudomonas
1.6990 3.4771 3.7112
aeruginosa

Staphylococcus aureus 0.6021 1.0148 1.6321

*the highlighted column represents data used for interpretation.

SHOW ALL SOLUTIONS:

*the highlighted column represents data used for interpretation.

INTERPRETATION OF RESULTS:
 ● Candida albicans: It passed the AET because there was no increase of more than 0.5 log10 units from 14 days to
28 days. As stated in the criteria under Category 2 for yeast and molds, the value at 14 days should not be more
than 0.5 log10 units higher than the value at 28 days.
● Aspergillus niger: It passed the AET because there was no increase of more than 0.5 log10 units from 14 days to
28 days. As stated in the criteria under Category 2 for yeast and molds, the value at 14 days should not be more
than 0.5 log10 units higher than the value at 28 days.
● Escherichia coli: The log reduction value of Escherichia coli from the initial calculated count at 14 days met the
criteria, thus it passed the AET. The product sample belongs to category 2 and the criteria for bacteria under
this category says that the log reduction result should not be less than 2.0 log reduction from the initial count at
14 days, and no increase from the 14 days’ count at 28 days. Based on the results, the log reduction was not less
than 2.0 and there was no increase from the 14 days count at 28 days.
● Pseudomonas aeruginosa: The log reduction value of Pseudomonas aeruginosa from the initial calculated count
at 14 days met the criteria, thus it passed the AET. The product sample belongs to category 2 and the criteria for
bacteria under this category says that the log reduction result should not be less than 2.0 log reduction from the
initial count at 14 days, and no increase from the 14 days’ count at 28 days. Based on the results, the log
reduction was not less than 2.0 and there was no increase from the 14 days count at 28 days.
● Staphylococcus aureus: The log reduction result of Staphylococcus aureus did not meet the criteria, thus it did
not pass the AET. The product sample belongs to category 2 and the criteria for bacteria under this category
says that the log reduction result should not be less than 2.0 log reduction from the initial count at 14 days, and
no increase from the 14 days’ count at 28 days. Based on the results, the log reduction value from the initial
calculated count at 14 days was less than 2.0.

CONCLUSION (ASSUME THAT THIS TEST IS PERFORMED DURING STABILITY TESTING):

Non-sterile nasal solution was classified under category 2 of the product. Based on the criteria for tested
microorganisms for category 2, the antimicrobial preservative of 30 mL non-sterile nasal solution eradicated
almost all of the inoculated microorganisms but still it failed or it is not efficient in protecting the product against
Staphylococcus aureus because the log reduction result is below the minimum criteria. Thus, the product is not
efficient and cannot be accepted.

ANSWER THE FOLLOWING QUESTIONS:

1. AET is called differently in European Pharmacopeia and Japanese Pharmacopeia, what are these
names?
In European Pharmacopeia, AET is referred to as Efficacy Antimicrobial Preservation while in Japanese
Pharmacopeia, it is known as Preservation Effectiveness Test.

2. Give the test organisms of this test in European Pharmacopeia.

The test organisms of Antimicrobial Effectiveness Test stated in European Pharmacopeia include Candida
albicans (ATCC No. 10231; NCPF 3179; IP48.72), Aspergillus brasiliensis (ATCC No. 16404; IMI 149007; IP 1431.83),
Escherichia coli (ATCC No. 8739; NCIMB 8545; CIP 53.126) which is used for all oral preparations, Pseudomonas
aeruginosa (ATCC No. 9027; NCIMB 8628; CIP 82.118), Staphylococcus aureus (ATCC No. 6538; NCTC 10788; NCIMB
9518; CIP 4.83), and Zygosaccharomyces rouxii (NCYC 381; IP 2021.92) which is used for oral preparations that
contains a high concentration of sugar.

3. What are the common neutralizing agents and methods used to inactivate the innate
antimicrobial activity of a product?
There are several neutralizing and methods used for the inactivation of antimicrobial activity. Chemical
inhibition, dilution, and filtration and washing are commonly employed methods for neutralizing antimicrobial
properties. On the other hand, a variety of neutralizing agents exists for this matter, namely, Bisulfate (under
Glutaraldehyde and Mercurial which targets non-sporing bacteria), Glycine (under Aldehydes which targets growing
cells), Lecithin (under QAC’s, Parabens and Bis-biguanides which targets bacteria), Mg+2 or Ca+2 (under EDTA),
Polysorbate (under QAC’s Iodine, Parabens), Thioglycolate (under Mercuria which targets Staphylococci and Spores)
and Thiosulfate (under Mercurial, Halogens and Aldehydes which targets Staphylococci).

REFERENCES:
Efficacy of Antimicrobial Preservation. (2011). Retrieved November 17, 2021 from
https://www.drugfuture.com/Pharmacopoeia/EP7/DATA/50103E.PDF
(n.d.). Retrieved from
https://online.uspnf.com/uspnf/document/1_GUID-48912B84-F603-4473-8250-EEE8533D39FF_3_en-US
?source=Quick Search&highlight=1227