UST – Faculty of Pharmacy

Member # Class # 5,6,7,8

Group # 2

Name: Rafael Arreola, Pompeyo Barroga, Carmelina Beltran, Marielle Bocalan Section 2BPH

EXERCISE 6A
MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS
(MICROBIAL ENUMERATION TEST)

OBJECTIVES:
1. To understand how the microbial enumeration test is performed using pour-plate method for a
non-sterile drug preparation.
2. To understand the importance of Method Suitability Tests.
3. To interpret outcomes based on expected test results and rationalize them.

Laboratory Scenario Analysis: Read the following statements. Interpret the following results and give the
rationale.

1. In growth promotion test, TAMC in one plate is 30 cfu.

Interpretation:
The medium still exhibits antimicrobial action

Rationale:
Having only 30 cfu means that there is not much growth

2. In growth promotion test, TYMC in one plate is 150 cfu.

Interpretation:
The growth of the promotion test which is 150 cfu TYMC in the one plate will pass the test

Rationale:
The cfu control in the experiment is 100 cfu while our test product’s cfu is 30. When we equate the %
Difference they will equal to 50% which falls between the mean count of 50-200% meaning that this
TYMC in the one plate passed the test

3. In suitability of counting method, TAMC in one plate is 400 cfu.

Interpretation:
The TAMC 400 cfu in the one plate will fail the counting method which can cause a procedure change

Rationale:
Stated before the mean count for the cfu is from 50-200%. When we apply our % Difference equation
for this we will get a cfu percentage of 300% which is over the mean count. Since it does not fall in the
criteria this means that it has failed the counting method suitability test

Calculations: Explain how you will prepare 100 cfu/mL bacterial suspension. Present in diagram if necessary
and show all calculations involved.
Flask A
(x) (0.5 mL) = (100 cfu/mL)(100 mL)
x= 2.0 x 104 cfu/mL
Flask B
(1.5 x 108 cfu/mL)(x) = (2.0 x 104 cfu/mL)(100 mL)
x= 0.0133 mL
 UST – Faculty of Pharmacy

Member # Class #
Group # 2

Name: Rafael Arreola, Pompeyo Barroga, Carmelina Beltran, Marielle Bocalan Section 2BPH

EXERCISE 6B
MICROBIOLOGICAL EXAMINATION OF NON-STERILE PRODUCTS
(TEST FOR SPECIFIED MICROORGANISMS)

OBJECTIVES:
1. To understand how the test for specified microorganisms is performed using surface-spread method
for a non-sterile drug preparation.
2. To understand the importance of Inhibitory and Indicative Test.
3. To interpret outcomes based on expected test results and rationalize them.

Laboratory Scenario Analysis:

Read the following statements. Fill-in the possible effect of the procedure and give the rationale.
1. The outer surface of the product container was not disinfected.
Effect:
Not disinfecting the outer surface of a drug product’s packaging will mean that it is most likely
covered in microorganisms. It could, therefore, have the possibility of causing illness. When
product testing, this could contaminate and affect the results of total aerobic microbial count
(TAMC) and total combined yeasts and molds count (TYMC).

Rationale:
Microorganisms are known to cause disease. If such products are distributed without being
disinfected, patients might contract another disease.

During product testing, the product is dissolved in a Soybean-Casein Digest Broth and is
transferred to the appropriate agar. Soybean Casein Digest Agar is used for TAMC, while
Sabouraud Dextrose Agar is used for TYMC. If there is growth on these plates, it indicates the
presence of bacteria or fungi, and should be counted for the TAMC and TYMC. However, since
the product container was not disinfected prior to testing, there could be microorganisms from
the outer surface that contaminated the plates, leading to a false result.

2. The analyst unknowingly contaminated the product with Staphylococcus aureus organisms.
Effect:
This will cause the transmission of Staphylococcus aureus to patients who will take the
product. Unfortunately, S. aureus has the possibility of causing illnesses, ranging from skin
infections, food poisoning, gastroenteritis, toxic shock syndrome, and others.
Rationale:
S. aureus is a pathogenic bacteria, and are dangerous to patients with weakened immune
systems, like neonates and postoperative patients. Infection with S. aureus is especially
dangerous if it has developed antimicrobial resistance.

Tests to ensure that the product is not contaminated should be performed by the analyst, who
is not yet aware of the contamination. When the result for contaminants is positive, the
products made in the same batch must be discarded.
Read the following statements. Interpret the following results and give the rationale.
1. In an inhibitory test, a change in color occurred.
Interpretation:
The change in color is an indication of bacterial growth. The growth of test microorganisms has
occurred, indicating that it is not a positive result.

Rationale:
It is not a positive result because the inhibitory test is for the inhibition of microorganisms. An
indication that there is microbial growth is the change in color. The result would be positive if there are
no changes, meaning no microbial growth has occurred.

2. In an indicative test, donut-shaped, dark pink colonies appeared.

Interpretation:
In MacConkey Agar, the appearance of donut-shaped, dark pink colonies indicates the presence of
lactose fermenting bacteria, such as E. coli, Klebsiella, and Enterobacter.

Rationale:
With the result, the course of action would be to reject and dispose of the product. The presence of
these bacteria would contaminate the product, which could lead to detrimental effects on the health of
the person if the product is used.

3. In product testing, no colonies appeared.

Interpretation:
No colonies appearing indicates the absence of E. coli in the product. However, this does not necessarily
mean that the product passes product testing.

Rationale:
The three criteria needed to either accept or reject a batch are the (1) total aerobic microbial count
(TAMC), (2) total combined yeasts and molds count (TYMC), and (3) presence or absence of specified
microorganisms. These depend on the route of administration of the product. If one of these exceeds
the acceptance criteria for TAMC or TYMC, or if it contains the specified microorganism indicated, then
the batch must be rejected.

Therefore, the suitability of the test should also be checked by simply increasing the volume of diluent
or culture medium, by incorporating neutralizing agents into the diluent, and by other methods. This is
because the chosen media may or may not have been appropriate for growing bacteria. The growth of
fungi should be checked as well. There is also no indication of E. coli being the specified microorganism
for the product being tested.