This document describes sterility testing methods to ensure pharmaceutical preparations are sterile. There are two recommended sterility testing methods: direct inoculation and membrane filtration. In direct inoculation, samples are directly inoculated into fluid thioglycolate medium and tryptic soy broth, which are then incubated. In membrane filtration, samples are filtered through a 0.25 micron membrane and the membrane is placed in the media and incubated. After incubation, turbidity indicates a non-sterile sample while transparent media indicates a sterile sample. These sterility testing methods help validate that pharmaceutical products are free of microbes.

1. Dr. Imran Sajid

2. Experiment: Sterility Testing To Ensure the
Sterility of Pharmaceutical Preparations
Materials Required
• Media (1. FTG: Fluid Thioglycolate Medium (used for the
detection of aerobic and anaerobic bacteria), 2. TSB: Tryptic
Soy Broth (used for the detection of aerobic bacteria, yeast
and mold)
• Standard organisms for growth promotion test: For the
validation of FTG: (Staphyloccocus aureus ATCC 6538,
Pseudomonas aeruginosa ATCC 09027, Clostridium sporogens
ATCC 11437). For the validation of TSB: (Bacillus subtilis ATCC
6633, Candida albicans ATCC 10321, Aspergillus niger ATCC
16404)

3. • Glass Vials (screw capped vials)
• Filtration assembly
• 0.25 micron filters (membrane filters)
• Incubators
• Test samples (Normal saline, Eye drops)
Lab Utilities/Supplies Required

4. FTG (Fluid Thioglycolate Medium)
Components Quantity g/L
L-Cystein 0.5g
Nacl 2.5g
Dextrose 1g
Yeast extract 5g
Pancreatic digest of casein 15g
Sodium thioglycolate/glycolic acid 3.5g
Resazurin sodium salt 10g
Agar 0.75g
Distilled water Volume make up to 1L
pH = 7.1+ - 0.2
Incubation conditions: 32.5°C±2.5°C for 14 days, under aerobic as well as anaerobic
conditions

5. TSB (Tryptic Soy Broth)
Components Quantity g/L
Pancreatic digest of casein 17g
Peptic digest of soybean meal 3g
Dibasic potassium phosphate 2.5g
Nacl 5g
Dextrose 2.5g
Distilled water Volume make up to 1L
pH = 7.3+ - 0.2
Incubation conditions: 22.5°C±2.5°C for 14 days, under aerobic conditions

6. Principle (Introduction/Theoretical Background)
Sterilization of pharmaceutical preparation such as injection,
ampule water (water for injection WFI), normal saline,
ointments, eye drops, some time antibiotics, is necessary to
avoid any type of harmful effects in the users.
For maintenance of sterility these products are either prepared
under sterile conditions or are terminally sterilized .
Sterilization during manufacturing is achieved in sterile
manufacturing areas or reactors which have the controlled air
filtration faculty with HEPA filter and installed UV lamps etc,
while terminal sterilization refers to the manufacturing of
product under normal clean conditions and then passing it
through sterilization method by ultrafiltration of UV
irradiation etc.

7. Once the sterile product is manufactured it is tested for sterility
in the sterile laboratory (usually attached with the
manufacturing area) (Design of sterile laboratory and
manufacturing area is a topic of class assignment).
There are two recommended methods of sterility testing (these
two methods are usually agreed upon by all the
pharmacopeia including USP (US pharmacopeia), BP (British
Pharmacopeia), EU Pharmacopeia, Japnes Pharmacopeia etc)
1. Direct inoculation
2. Membrane filtration method

8. Direct Inoculation
In this method the recommended quantity of the sample is
directly inoculated in the two recommended media (FTG and
TSB), and the media are incubated under recommended
culture conditions for each media.
Membrane filtration method
In this method the recommended quantity of the sample is first
passed through the membrane filter (usually 0.25 micron
filter) on a filtration assembly, the solid or powder samples
such as antibiotics are first dissolved in some sterile diluent
and then are passed through the filtration assembly. Later the
membrane filter is removed from the assembly, and is cut in
to two halves with sterile scalpel, one half is inoculated in the
FTG, while the other half is inoculated in the TSB. The media
are incubated under recommended conditions. After
incubation the appearance of turbidity or growth in the media
is observed

9. Sample specifications
No of articles in a batch Articles to be tested
<100 4 articles
>100 <500 10 articles
>500 <1000 20 articles
> 1000 2% of the articles
Recommendations for liquid samples
Quantity in a vial Quantity to be tested
<1 ml Whole sample
>1 ml <40 ml Half sample
>40 ml <100 ml 20 ml of the samples
> 100 ml 10-20% of the sample

10. Recommendations for solid samples
Quantity in a vial Quantity to be tested
<50 mg Whole sample
>50 mg <300 mg Half sample
>300 mg <5g 150 mg
> 5 g 500 mg

11. Procedure
All the sterility testing activities are performed under aseptic
conditions in a sterile lab
Direct Inoculation Method
1. Prepared the recommended media (FTG and TSB), adjust pH,
dispense in screw capped bottles (15-20 ml broth in each
vial) and autoclave
2. Take the sample vial to be tested, agitate the container, and
aseptically transfer the recommended quantity of the sample
to the recommended media
3. Incubate the media under specified conditions (FTG: 32.5±
2.5°C, for 14 days, under aerobic and anaerobic conditions,
TSB: 22.5±2.5°C for 14 days under aerobic conditions)

12. Membrane Filtration Method
1. Prepare the recommended media (FTG and TSB), adjust pH,
dispense in screw capped bottles (15-20 ml broth in each
vial) and autoclave
2. Take filtration assembly , adjust 0.25 micron membrane filter
in it, wrap the assembly in aluminum foil and autoclave it
3. Take the sample vial to be tested, agitate the container, and
aseptically pass the recommended quantity of the sample
through the filtration assembly
4. Remove the membrane filter from assembly, divide it in two
equal halves with sterile cutter or scissor and transfer one
half in the FTG and other in the TSB
5. Incubate the media under specified conditions (FTG: 32.5±
2.5°C, for 14 days, under aerobic and anaerobic conditions,
TSB: 22.5±2.5°C for 14 days under aerobic conditions)

13. Results Interpretation
1. Turbidity in the media after incubation is the indicative of
microbial growth and contamination and product is
considered as non sterile
2. No turbidity or transparent media after incubation is the
indicative of sterility and product is considered as sterile
3. Proper negative and positive controls are used in both the
methods