Home Cosmetic Science Talk Formulating Standardized microbial challenge cocktail? (Candida, A. Brasiliensis, E. coli, P. Aeruginosa, Staph)

  • Standardized microbial challenge cocktail? (Candida, A. Brasiliensis, E. coli, P. Aeruginosa, Staph)

    Posted by Zink on February 24, 2014 at 9:04 pm

    You can buy microbial growth testing kits, but where do you get standardized microbial challenge “cocktails”? To do a typical screening test. Using those included in USP 51 testing would make most sense? 

    “USP <51>” refers to General Chapter 51 of the United States Pharmacopeia (USP). Chapter 51 describes in detail the USP method for preservative efficacy testing, sometimes called “preservative challenge testing.”

    • Candida albicans (a yeast…yeasts are a form of fungus)
    • Aspergillus brasiliensis (a filamentous mold…also a fungus)
    • Escherichia coli (a bacterium…better known as “E. coli”)
    • Pseudomonas aeruginosa (a bacterium….very problematic industrially)
    • Staphylococcus aureus (a bacterium…better known as “Staph”)
    Having a cocktail such as this would be very useful for initial screening of your formulas, before you send the best candidates of to e.g. USP51 testing which can be expensive.
    Zink replied 10 years, 4 months ago 6 Members · 15 Replies
  • 15 Replies
  • Microformulation

    February 25, 2014 at 9:15 am

    No offense, but I would leave the micro testing to the pros. It is an involved test that is best done by a specialist. Also, using an outside lab gives you third party validation.

    With a well thought out and designed preservative system you should be able to feel comfortable sending your samples for USP 51 testing without attempting the test in-house.

  • The_Microbiologist

    February 25, 2014 at 10:19 am

    HI Zink,

    You can buy germ cocktails similar to what you have outlined from a company called MicroBioLogics.  The brand name is called EZ-PEC. 

    However, I agree with Mark that such testing is best left to the pros and here is why:

    • The EZ-PEC organisms are lyophilized (freeze-dried).  Freeze dried germs have a tendency to disintegrate into small, ultra-lightweight, inhalable particles and go everywhere, so in my opinion lyophilized microbes should only be worked with in a Class II biological safety cabinet.
    • Some of the USP germs are pathogenic, meaning they can cause disease.
    • The lyophilized pellet re-hydration step is something that should be understood by the person doing it to make sure it’s done right.
    • Optimally a person would verify the initial inoculum counts for each organism rather than depend on the manufacturers’ certification.  That requires serial dilution and plating.
    • At the end of the test you have to know how many germs are left in the formula.  That also requires dilution and plating.
    • You have to verify that any germs left in the formula can grow on the agar through a step called a neutralization/recovery validation that is rather technical.
    • The whole process generates dozens of used agar plates, many of which will be loaded with bacterial and fungal colonies.  Each colony will have about 10 million germs, which means the risks posed by the organisms such as Staph aureus are greater in the case of accidental contact.
    • All those agar plates, dilution tubes, etc, are considered “medical waste” in most states and must be disposed of properly, usually with some paperwork to go with it (even in Texas where my lab is located!).

    Hopefully that information is helpful!

  • Zink

    February 25, 2014 at 10:40 pm

    Thanks for the feedback, I agree that USP 51 should be done by pro’s, but sometimes you want to do crude testing before you send your best samples off for expensive $300-$500/sample professional testing which is why you can buy those microbial testing kits with double sided stick for fungal / bact growth.


    Great answer! I have done some microbiology in my training as a molecular biologist, and can get access to Class II biosafety cabinets, but would prefer something easier and safer, it doesn’t need to 100% mimic USP 51, but should consistently challenge for bact and fungi at the very least. I mean, what do people use the home testing kits for anyways?
  • Microformulation

    February 26, 2014 at 9:43 am

    Those stick tests are for the presence of bacteria/yeast/mold as a “snapshot” in a fixed point in time. Very analogous to APC Testing but less accurate. USP 51 is a totally different test. As such the comparison is flawed. I have used those test “stick” in plant sanitation monitoring but again I prefer to send tests out when need be. The concept of “Third Party Validation” is critical in a QA Program.

    Nobody is doing home PET. The sticks roughly allow them to do an analogous test to APC. That is ok for a home tester. It doesn’t pass the level of scrutiny for a Contract Manufacturer.

  • tonyh

    February 26, 2014 at 11:31 am

    Common Usage Test:

    Common Usage Test
    “This test is an alternative to the Challenge Test that is
    recommended to home crafters who are not widely marketing their product.
    It is much less quantitative and rigorous than the challenge test but
    still gives useful information to fit a home crafters budget. To do
    this, first send a product in for testing.  If these counts come out
    zero to low it assures that you are starting with good GMP (good
    manufacturing practices) and your product is without contamination. Now,
    take a second sample from that batch and use and abuse it for several
    days. Make sure you stick  your dirty fingers into it and leave it open –
    enough abuse that you know you are getting bacteria and fungus in the
    product.  Don’t hold back. Send this sample in for testing after a week
    or two. This second test will tell you how your product holds up to that
    insult and gives an idea how effective your preservative is.  The
    counts for the second testing should be as low as the first testing. 
    The cost for this would be that for two APC and fungal/yeast tests

    Shelf Life of a preservative is a difficult thing to assess and can
    really only be done in real time. You might try having your products
    tested ever 3-6 months to estimate a shelf life or determine how long
    their preservative works.”

    FDA - Microbiological Methods for Cosmetics:


    Challenge Testing or Preservative Efficacy Testing
    “This is the most rigorous test to determine whether your
    preservative is working. It involves introducing known bacteria (E.
    Coli, Pseudomonas, and Staph) and Fungi (Aspergillus, Candida) into your
    product. Plate counts are done at various times over a months period
    afterward. If the preservative is working, counts will decrease within 2
    weeks and not increase again after that.

    The FDA does not require any microbiology testing but it is a
    responsible thing to do to protect your formula and your customer. The FDA
    does say that a cosmetic should not be adulterated which is interpreted
    as meaning it should not contain harmful bacteria or fungus.”

    Here is what the FDA guidelines say about bacteria in cosmetic products:
    “Cosmetic products are not expected to be aseptic; however, they must be
    completely free of high-virulence microbial pathogens, and the total
    number of aerobic microorganisms per gram must be low. Since there are
    no widely acceptable standards for numbers, temporary guidelines are
    used instead. For eye-area products, counts should not be greater than
    500 colony forming units (CFU)/g; for non-eye-area products, counts
    should not be greater than 1000 CFU/g. The presence of pathogens would
    be particularly important in evaluating as unacceptable a cosmetic with a
    marginally acceptable count, e.g., 400 CFU/g for an eye-area product.
    Pathogens or opportunistic pathogens whose incidence would be of
    particular concern, especially in eye-area cosmetic products, include S.
    aureus, Streptococcus pyogenes, P. aeruginosa and other species, and
    Klebsiella pneumoniae. Some microbes normally regarded as nonpathogenic
    may be opportunistically pathogenic, e.g., in wounds.” The EU recommendations are similar.

  • Zink

    February 26, 2014 at 1:34 pm

    @Microformulation I think you’re missing my point, my point is to do a rough screen BEFORE sending samples for USP 51 testing, if you’re doing a lot of samples e.g. testing different preservatives, sending all of them to USP 51 testing could get cost prohibitive at > $400/sample.

    @Tonyh useful link to the common usage test, cheap to at $31 for anaerobic bacteria + fungi, but to really test your product you’d still need some microorganisms to contaminate it with in the first place..

  • The_Microbiologist

    February 26, 2014 at 5:55 pm

    Hi Zink,

    For a very rough preservative efficacy screen, here is what I recommend (no joke):

    Make some product.  Then collect some saliva and measure it (saliva, not mucous).  I would do this at least a few hours after brushing, using mouthwash, or eating.  Then add a measured amount of the saliva to your product, say 1 ml per 10 ml product. Then mix it thoroughly, then do an APC using a dipslide.  Then let the product sit at room temp for 1 week.  Then do another APC.  If counts go up, the preservative is not working.  If counts go down substantially, it’s working at least a bit. 

    Do note that oral pathogens may be present at fairly high concentrations (making them more risky) on the dipslide after incubation and should be disposed of in accordance with local laws.

    That’s an inexpensive rough screen for preservative efficacy, though it lacks confirmation of preservative neutralization (which means the germs that appear to be killed may just be inhibited from growing on the dipslide). 

    No ivory tower here, ha ha ha :)


  • The_Microbiologist

    February 26, 2014 at 5:59 pm

    One more note…

    the “common usage test” is not as good as what I proposed because there is no standardization whatsoever of the microbial inoculum.  Most mouths are sort of self-standardizing to a reasonably high concentration of bacteria (usually with nice diversity as well) and none are sterile that I know of.

  • Zink

    February 26, 2014 at 9:14 pm

    That’s great advice, what about taking a large sample of saliva at one point and storing it in the fridge? Lypholizing it? Hehe, joking. I assume it’s best just to take new samples after your rec.

    In any case it seems like the best way to do a rough semi-standardized screen, I might play around with some “””natural””” preservatives to see if any are up to snuff. Then do USP 51 on any promising candidates.

  • Bobzchemist

    February 27, 2014 at 8:52 am

    Could you let us know your results on the “natural” preservatives?

  • Zink

    February 27, 2014 at 7:24 pm

    For now this is just an idea, wondering whether such a product would be commercially viable and how large the “label” benefit would be.

    So it would have to work in emulsion, unlike Leucidal Liquid. But I guess there are other oil compatible ECOcert ones on the market?

    Leucidal LiquidLeucidal Liquid

  • MakingSkincare

    February 28, 2014 at 6:15 am

    A starting Ecocert preservative blend for an emulsion to try:- sodium benzoate 0.35%, potassium sorbate 0.35%, benzyl alcohol 0.2% together with a chelator.  The pH must be brought down to 5 to 5.5. 

  • Zink

    February 28, 2014 at 11:39 pm

    How about using borax and beeswax? I’ve heard that combo can work too. Less “chemical” sounding perhaps.. I know..

    Otherwise there’s EcoMulse:

    Glyceryl Stearate                       55-65%
    Cetearyl Alcohol                        20-30%
    Sodium Stearoyl Lactylate      10-20%

    And NeoDefend: Gluconolactone + Sodium Benzoate (GSB)
  • MakingSkincare

    March 1, 2014 at 7:20 am

    Borax doesn’t have a great name in the “natural” sector and a beeswax/borax w/o emulsion tends to be unstable.

    Yes there are plenty of emulsifiers with Ecocert status to choose from - my personal favourite is glyceryl stearate (the non SE version) with cetearyl glucoside.  
  • Zink

    March 3, 2014 at 1:00 am

    Looking at the cursed whole foods list, I see that Glyceryl Stearate is not on it (but glyceryl isostearate is..) nor is cetearyl glucoside, so I guess it’s both ECO and wholefoods friendly. So the Herbiary sells it as “Sugarmulse. Will get some..

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