Sterility Assurance Testing

Procedural Information

Make FTM tubes

Using 3mL vials and 15mL test tubes.

1. Mix the FTM broth with distilled water as per the package instructions
2. Add polysorbate 80 at a concentration of 40mL/L
3. Add 13.5mL to test tubes
4. Autoclave test tubes

Add the CSP

1. Choose the number of vials to test.
2. Under laminar flow add 1.5mL from each vial to a tube
3. Seal.

Incubate

You will likely be incubating 4-10 tubes.

Tubes should be incubated at 30-35°C (86-95°F) for 14 days¹.

Observe

Cloudy tubes mean contam?

Sterility Assurance Testing Notes Below

We can test the sterility of our finished product if we have some special supplies and tools. For this tek we’re working with two different growth mediums as recommended by USP 71 and the book “Compounding Sterile Preparations.” These are Tryptic Soy Agar (TSA) and Fluid Thioglycollate Medium (FTM)².

Notes Below

What tube size should we be using?

Tube contains:

• 1.5-5mL of CSP (50% of vial)
• 13.5-45mL of FTM (with polysorbate 80)

the volume of the product is not more than 10% of the volume of the medium

For testing 3mL vials, we need 15mL tubes.

USP 71

How many vials to test?

From USP 71

How to neutralize the BA?

If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium¹

BA is neutralized by Polysorbate 80 at a concentration of 40mL/L³

Testing Oily Liquids?

Use media to which have been added a suitable emulsifying agent at a concentration shown to be appropriate in the Method Suitability Test, for example polysorbate 80 at a concentration of 10 g per L.¹

Is TSA even needed aside from fingertip tests? Can just stick with FTM solely?

How much P80 to add?

• 40mL per L for neutralizing BA
• 10g per L for emulsifying

Which is greater? P80 is 1.06g/mL

10g is 9.43mL.

Verifying Your Test is Suitable

After transferring the contents of the container or containers to be tested… to the culture medium, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the medium.

In both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 5 days.

If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification.

If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity, and repeat the Method Suitability Test.

This method suitability is performed (a) when the test for sterility has to be carried out on a new product; and (b) whenever there is a change in the experimental conditions of the test. The method suitability may be performed simultaneously with the Test for Sterility of the Product to be Examined.¹

Since we don’t have access to strains of bacteria aside from what we can just pickup in the kitchen sink, we need to adapt this test for home use.

Supplies

• Laminar flow hood
• Incubator
• Autoclave
• Media bottles
• Tryptic Soy Agar plates
• Fluid Thioglycollate Medium tubes
• Polysorbate 80 as an emulsifier

You can potentially substitute your laminar flow hood here for a still air box, but your results won’t be as consistent or trustworthy.

How to make TSA plates

You can make TSA with neutralizers  if you’d like to perform fingertip testing⁴, a way of verifying how solid your personal aseptic technique is. Do not use neutralizers otherwise.

1. Sterilize empty agar plates
2. Mix the TSA powder with distilled water according to the TSA package.
3. Autoclave in a media bottle
4. When TSA cools to a handling temperature, but before it solidifies, pour the liquid TSA into agar plates under a laminar flow hood and immediately cover.
5. You can optionally add a small piece of tape to keep them shut
6. Let cool fully before use

If you’re using this right away, make sure to set a couple aside to ensure that they were sterilized correctly and aren’t growing bacteria you didn’t mean to introduce.

Using TSA plates

TSA plates should be incubated at 20-35°C (68-95°F) for 14 days⁵.

Using FTM tubes

Heat in a water bath prior to use.¹

FTM tubes should be incubated at 30-35°C (86-95°F) for 14 days¹.

How much product to test

The minimum volume of each CSP [compounded sterile preparation] to be tested is dependent on the volume of the final product. If the product is <1 mL, the entire volume must be tested. If 1-40 mL, then half the total volume is tested. If 40-100 mL, then 20 mL is tested. If the volume is >100 mL, then 10% of the volume is tested (but at least 20 mL).⁶

If the individual containers (for end-user use) have between 1-40mL in them (like what we’re doing here on HRT Mom), then sterility testing is done on 50% of the volume of the containers⁷

“the product is not more than 10% of the volume of the medium”⁷

References

Some information on this page comes from HRT Cat.

View the library page for access to some PDFs.