[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fOwcMLh9FgpOLJSkYtARzWY-EVV_wXWreg5NlTzUXELg":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":47},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":36,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":39,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"body":43,"faq":44,"tags":45,"related":46},"bioburden-testing-purpose-procedure-and-accepted-level","Bioburden Testing: Purpose, Procedure, and Accepted Level",null,"Ashma Shrestha","2022-11-16","2025-12-29",false,"general-microbiology","Bioburden describes the number of viable microorganisms present in a product or on a sterile barrier system. The bioburden may be introduced by various sources like raw materials, environment, cleaning processes, and manufacturing and assembling components.\n\n**Bioburden testing is a recommended method for determining the total quantity and types of viable microorganisms in unsterilized material or products before performing sterilization. It is a crucial method for selecting the correct process of sterilizing, finalizing, and distributing materials and products.**\n\n**The vital step in bioburden testing is the collection of samples or recovery methods of microorganisms from products and medical devices. Bioburden testing is highly used in pharmaceutical industries, manufacturers of medical devices, and cosmetics industries.**\n\n![Bioburden Testing - Bioburden testing](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FBioburden-testing.jpg)\n\u003Cfigcaption>Figure: Bioburden testing\u003C\u002Ffigcaption>\n\n## Purpose of Bioburden Testing\n\nThe bioburden testing methods isolate and enumerate viable microorganisms before sterilizing any materials and products. These testing methods have many different purposes in different laboratories, which are as follows:\n\n1. It helps to figure out the correct way of sterilizing the raw materials and water used to produce various products.\n2. It also helps determine the number of viable microorganisms in the medical devices after production and before use.\n3. Bioburden testing helps maintain laboratory standards as per the rules or protocols followed.\n\n## Procedure of Bioburden Testing\n\nBioburden testing is the method of determining total viable count (TVC). Bioburden testing can be carried out as TAMC (total aerobic microbial count) or a combination of TAMC and TYMC (total yeast and mold count). For TAMC microbial pour plate and [spread plate method](\u002Fspread-plate-technique\u002F) are used.\n\n![ - Steps of Bioburden Testing](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FBio-burden-testing-steps.png)\n\u003Cfigcaption>Figure: Steps of Bioburden Testing\u003C\u002Ffigcaption>\n\nThe procedure of bioburden testing for manufacturing (raw materials and packaging practices) in or on a medical device has the following four distinct stages:**Microorganism recovery, Enumeration of microorganisms, Bioburden characterization, Validating the method.**\n\n### Microorganism Recovery\n\nThere are many ways of recovering microbial samples based on the material types to be tested.\n\n1. For water samples, filtration followed by plating is preferred.\n2. Sometimes materials are shaken ultrasonically, after which the substance is filtered and placed in an agar medium.\n3. Sometimes stomaching, rinsing, and flushing are done, followed by filtration and plating on an agar medium.\n4. If nothing of the above methods works, direct swabbing or contact plate technique is usable.\n5. The agar plate is then incubated in two ways.\n\nTwo media methods: Fungal-specific media like [SDA (Sarbouraud dextrose agar)](\u002Fsabouraud-dextrose-agar-sda-principle-composition-uses-colony-morphology\u002F) is incubated at 25 ℃ for 2-7 days. Another TAMC-specific media like [TSA (trypticase soy agar)](\u002Ftryptic-soy-agar-tsa-composition-preparation-uses\u002F) is incubated at 37 ℃ for 18-48 hours.\nOne media dual temperature method: All-purpose media like SCD (soybean casein digest) medium is used and incubated firstly at 30-35 ℃ for 2-3 days and then at 20-25 ℃ for 5-7 days. It is a preferable method in areas with low bioburden.\n\n### Enumeration of the Microorganisms\n\nThe cultured microorganisms are then enumerated by using either [MPN](\u002Fprobable-number-mpn-test-principle-procedure-results\u002F) (most probable number), membrane filtration, or aerobic plate count method. The microbial load is expressed in CFU (colony forming unit), which helps to develop a standard level.\n\n### Bioburden Characterization\n\nThe identification of microorganisms is made using various methods. For the identification of bacteria, observation of colony morphology, Gram staining, and other differential staining for studying cell morphology and performing different biochemical tests is usually done. Observing colony morphology and different types of fungal staining methods is used to identify yeast and molds.\n\nBesides these methods, molecular methods like PCR and blotting techniques are also performed to characterize microorganisms.\n\n### Validate the Method\n\nUsing calculated correction factor(s) during the recovery of bioburden helps calculate the bioburden estimate from the raw sterilization count. The correction factor helps to validate the recovery method.\n\nA correction factor is a numerical value provided by recovery efficiency (RE). The RE helps in assessing the efficiency of the extraction method. According to ISO 11737-1, the RE is measured in two ways; **inoculated recovery and repetitive extraction (exhaustive recovery)**.\n\n**In the inoculated method,** the materials have low bioburden or are pre-sterilized, which reduces the effect of other competitive microorganisms in order to obtain an accurate count of the inoculated organism. The RE is the ratio of the colony count of the target microorganism recovered to the positive control. It is the preferred method. Usually, a spore-forming microorganism like *Bacillus*is applied to the material and allowed to dry for this method.\n\n**Recovery Efficiency (RE)= CFU recovered in one rinse\u002FInoculation Population**\n\n**In the repetitive method,** product samples are rinsed (three to five times). The viable bioburden is measured after every extraction. The RE is calculated by comparing the total number of colonies recovered (from all the rinses) to the first rinse. This method is unsuitable for powders, gels, or any other dissolving material.\n\n**Recovery Efficiency (RE)= Total rinse CFU\u002FFirst Rinse CFU**\n\n**Correction factor= 1\u002FRE**\n\n### Things to consider\n\nWhile performing a bioburden, the following factors need to be considered:\n\n1. The time of sampling should be at the end of processing and packaging.\n2. The sample should be from homogenous bulk.\n3. Aseptic handling and technique are required.\n4. Clean and sterile media and containers should be used.\n5. A correct method of sampling, selection, and identification is necessary.\n6. Validation of the processes after every test is a must.\n7. A proper method of circulating alert and action levels should be established.\n8. The expiry time of the sample can influence the validity of the test.\n\n### Acceptable Level for Bioburden Testing\n\nAccording to EMA (European medicine association), not more than 10 CFU per 100 ml is an acceptable level for bioburden testing in the samples obtained through filtration. However, these levels vary based on region, company, and industry.\n\n### Common Microorganism Found\n\nThe microorganism commonly found during bioburden testing is bacterial species. Sometimes yeast and mold may also be recovered.\n\n1. *Staphylococcus* species\n2. *Bacillus* species\n3. Coliform (in water sample): *Escherichia coli* is the most common coliform obtained as bioburden from water samples.\n4. Yeast\n5. Mold\n\n## Application of Bioburden Testing\n\nBioburden testing is the estimation of microorganisms in samples like containers, medical devices, water, in-process samples, final bulk products before sterilization, product contact surfaces, or materials that require frequent assessment of the bioload. It is also common practice to perform bioburden testing to validate sterilization methods. Some of the fields that commonly apply bioburden testing are:\n\n1. **Microbiology laboratories:** These laboratories constantly deals with microorganism but also require sterile zone for working. So, bioburden testing is widely used to determine the total aerobic microbial count (TAMC).\n2. **Medical products and device manufacturing companies:** These companies must produce materials and devices that contain very few or almost non-bioload because these come in constant contact with immunocompromised persons. So, bioburden testing of these products pre-sterilization is preferred and recommended. It is essential to validate the sterilization method.\n3. **Pharmaceutical industries:** Final bulk of oral drugs before sterilization, water used for producing drugs, containers, and surfaces are constantly used as samples for bioburden testing in pharmaceutical industries.\n4. **Cosmetic Industries:** The raw materials for cosmetic and bulk products before sterilization are subjected to bioburden testing. It is also performed on the equipment used to produce those cosmetic products.\n\n## Difference Between Microbial Limit Test and Bioburden Testing\n\nBioburden testing is a quantitative method that is performed in non-sterile, pre-sterile, unfiltered and filtered samples. Microbial limit test (MLT) is a qualitative and quantitative method performed for non-sterile materials.\n\nUsually, MLT is the method of determining the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) separately. In bioburden testing, the total viable count (TVC) is calculated as a whole.\n\nBioburden testing follows guidelines from USP (United States Pharmacopeia) 60, 61, and 62. In contrast, MLT follows the guidelines of USP 61 and 62.\n\n## Difference Between Bioburden and Endotoxin Testing\n\n[Endotoxin testing](\u002Fpyrogen-and-bacterial-endotoxin-testing-methods\u002F) determines bacterial endotoxin, a phospholipid found in the outer membrane of gram-negative bacteria. In contrast, bioburden is not limited to determining endotoxin but expands to counting bacteria and fungi.\n\nBioburden testing determines the total number of viable (living) microorganisms. Whereas endotoxin testing detects and quantifies non-viable bacteria as dead gram-negative bacteria release endotoxin.\n\nEndotoxin is performed in sterile and nonpyrogenic devices or equipment, usually, that come in direct or indirect contact with cardiovascular, lymphatic system, or cerebrospinal fluids. At the same time, bioburden testing helps validate the sterilization method and monitor raw materials, packaging, and the environment.\n\nBioburden testing follows USP (United States Pharmacopeia) 60, 61, and 62. But endotoxin testing follows the guidelines of the USP 161.\n\nEndotoxin tests do not require culturing, so a report is generated within a day. At the same time, bioburden testing requires culturing and can take up to 7 to 10 days for reporting.\n\n**References**\n\n- *Bioburden – Eurofins Scientific*. (n.d.). Retrieved November 16, 2022, from [https:\u002F\u002Fwww.eurofins.it\u002Fmedia\u002F91471\u002F40024_eurofins_biolab_ctp_laboratories_test_bioburden.pdf](https:\u002F\u002Fwww.eurofins.it\u002Fmedia\u002F91471\u002F40024_eurofins_biolab_ctp_laboratories_test_bioburden.pdf)\n- Sandle, Tim. (2013). A new standard for bioburden testing: USP chapter in development. GMP review. 12. 10-12. [https:\u002F\u002Fwww.researchgate.net\u002Fpublication\u002F258074441_A_new_standard_for_bioburden_testing_USP_chapter_in_development](https:\u002F\u002Fwww.researchgate.net\u002Fpublication\u002F258074441_A_new_standard_for_bioburden_testing_USP_chapter_in_development)\n- *Pharmaceutical Microbiology Manual*. FOOD AND DRUG ADMINISTRATION OFFICE OF REGULATORY AFFAIRS Office of Regulatory Science. (2020, August 25). Retrieved November 16, 2022, from [https:\u002F\u002Fwww.fda.gov\u002Fmedia\u002F88801\u002Fdownload](https:\u002F\u002Fwww.fda.gov\u002Fmedia\u002F88801\u002Fdownload)\n- Sandle, T. (2016). Bioburden determination. *Pharmaceutical Microbiology*, 81–91. [https:\u002F\u002Fdoi.org\u002F10.1016\u002Fb978-0-08-100022-9.00007-4](https:\u002F\u002Fdoi.org\u002F10.1016\u002Fb978-0-08-100022-9.00007-4)\n- Clontz, L. (2008, October 14). *Microbial limit and Bioburden tests: Validation Approaches and Global Requirements*. Google Books. Retrieved November 16, 2022, from [https:\u002F\u002Fbooks.google.com\u002Fbooks\u002Fabout\u002FMicrobial_Limit_and_Bioburden_Tests.html?id=tNltHmj7-7EC](https:\u002F\u002Fbooks.google.com\u002Fbooks\u002Fabout\u002FMicrobial_Limit_and_Bioburden_Tests.html?id=tNltHmj7-7EC)\n- Yang, H., Li, N., & Chang, S. (2013). A risk-based approach to setting sterile filtration bioburden limits. *PDA Journal of Pharmaceutical Science and Technology*, *67*(6), 601–609. [https:\u002F\u002Fdoi.org\u002F10.5731\u002Fpdajpst.2013.00942](https:\u002F\u002Fdoi.org\u002F10.5731\u002Fpdajpst.2013.00942)\n- Denyer, S. P., Hodges, N., Gorman, S. P., Gilmore, B. F., Hugo, W. B., & Russell, A. D. (2011). Sterilization procedures and sterility assurance. In *Hugo and Russell’s Pharmaceutical Microbiology* (eighth, pp. 370–371). essay, Wiley-Blackwell.",[],[],[],[48,55,61,66,70,74,79,84,88,92],{"slug":49,"name":50,"description":51,"image":52,"body":53,"postCount":54},"acharya-tankeshwar","Acharya Tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",434,{"slug":56,"name":38,"description":57,"image":58,"body":59,"postCount":60},"ashma-shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":62,"name":63,"description":64,"image":37,"body":37,"postCount":65},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":67,"name":68,"description":64,"image":37,"body":37,"postCount":69},"samikshya-acharya","Samikshya Acharya",20,{"slug":71,"name":72,"description":64,"image":37,"body":37,"postCount":73},"alisha-tripathi","Alisha Tripathi",6,{"slug":75,"name":76,"description":77,"image":37,"body":37,"postCount":78},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",9,{"slug":80,"name":81,"description":82,"image":37,"body":37,"postCount":83},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":85,"name":86,"description":64,"image":37,"body":37,"postCount":87},"srijana-khanal","Srijana Khanal",18,{"slug":89,"name":90,"description":82,"image":37,"body":37,"postCount":91},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":93,"name":94,"description":64,"image":37,"body":95,"postCount":96},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]