cUSP 2021-2022 Microbial Enumeration Tests for Nutritional and Dietary Supplements

Gibraltar Laboratories performs cUSP Chapter 2021-2022 Microbial Enumeration Tests for Nutritional and Dietary Supplements which provides the estimation of viable aerobic microorganisms present in nutritional supplements of all kinds.

1. Current USP<2021> and < 2022> Testing http://www.gibraltarlabsinc.com/services/microbiology.html Proprietary and Confidential © Gibraltar Laboratories, Inc. Rupinder Puar

2. USP<2021> Microbial Enumeration Tests for Nutritional and Dietary Supplements Provides tests for the estimation of viable aerobic microorganisms present in nutritional supplements of all kinds. Preparatory testing required prior to conducting routine tests .

3. Preparatory test Use 10g or mL of sample. Organisms used for 2021 prep test include: Escherichia coli, Bacillus subtilis, and Staphylococcus aureus for Total aerobic microbial count. Candida albicans and Aspergillus brasiliensis for Total combined Molds and Yeasts Count. There should be greater than 70% bioburden recovery in comparison to the control medium.

4. Prep test cont. For Enterobacterial count(Bile Tolerant Gram Negative Bacteria) appropriate dilutions of Escherichia coli and Salmonella typhimurium are used. Failure of the organisms to grow in the relevant medium invalidates the test. The test can then be performed by modifications in the procedure by either an increase in the volume of the diluent , by addition of sufficient inactivating agents in the diluent, or by doing both.

5. <2021> Can be performed by either method: Membrane Filtration, Plate Count method, and Multiple Tube (MPN) method Fluid soybean casein digest media used as the diluent. Soybean Casein Digest Agar used for TAMC and Sabouraud Dextrose Agar used for TYMC. Incubation for TAMC is 30-35°C for 48-72 hours and TYMC is at 20-25C for 5-7 days.

6. Membrane Filtration

7. Plate count method

8. Multiple Tube Method

9. MPN Cont. Most probable number of microorganisms per g or mL of sample calculated based on the tubes with growth. By reference to the MPN chart.

10. MPN Chart

11. Enterobacteria Count Sample diluted in Fluid Soybean Casein Digest Medium. (10g or mL to make 1:10 dilution) Pre-incubate for 2-5 hours at 20-25C Inoculate suitable quantities of Mossel-Enterobacteria Enrichment Broth to contain 0.1, 0.01 and 0.001g or mL of product. Incubate at 30-35C for 24-48h. Subculture to Violet Red Bile Agar with Glucose and Lactose (VRBG). Red or reddish colonies of Gram Negative Bacteria indicate presence of Enterobacteria. Determine MPN from chart.

12. Mossel-Enterobacteria Enrichment Broth

13. VRBG Agar (E.coli)

14. MPN chart for Enterobacteria

15. <2022> Testing For Staphylococcus aureus 1:10 dilution(10g product) in FSCD. Incubate at 30-35C for 18-24h. Streak a loopful on the surface of Vogel-Johnson, Mannitol-Salt-Agar and Baird Parker Agar Medium. Incubate plates at 30-35C for 24-48h.

16. Sa on VJ agar Uninoculated Plate Sa on VJ agar: Black surrounded by Yellow zone

17. Sa on MSA Staphylococcus aureus has yellow colonies with yellow zones

18. Sa on BP agar Staphylococcus aureus has black shiny colonies with 2-5mm clear zones

19. Confirmatory test If characteristic colonies present: Perform coagulase test by transferring suspect colonies to separate tubes containing 5mL rabbit plasma, or any other mammalian plasma. Incubate in a water bath at 37C. Examine for coagulation after 3 hours or at suitable intervals up to 24hours. Negative reaction indicates absence of Sa .

20. Coagulase test

21. Coagulase test

22. Test for Salmonella species Incubate primary enrichment at 30-35C for 18-24h. Pipet 1mL into 10mL RVSE broth, incubate at 30-35C for 18-24hrs. Streak onto Brilliant Green agar, Hektoen Enteric agar and Xylose Lysine Desoxycholate agar. Incubate plates at 30-35C for 24-48hrs.

23. Salmonella on BGA an uninoculated plat e Salmonella on BGA . The colonies are small, opaque, pink or white. Can also be transparent and colorless.

24. Salmonella on XLD Uninoculated plate Salmonella on XLD is red, with or without black centers

25. Hektoen Enteric Agar Salmonella on HE agar is Blue-green, with or without black centers Salmonella on Hektoen Enteric Agar

26. Confirmatory Test If suspect colonies found: Transfer to Slant of Triple Sugar Iron Agar Medium First streak the surface of the slant, then stab the wire well beneath the surface. Incubate at 30-35C for 24-48hrs. No Red alkaline slants and yellow acid butts with or without concomitant blackening of butts(from Hydrogen sulfide production) indicates absence of Salmonella.

27. TSI slant

28. Test for Escherichia coli Incubate primary enrichment at 30-35C for 24-48h. Pipet 1mL into 10mL MacConkey Broth, incubate at 42-44C for 24-48hrs. Streak onto MacConkey Agar. Incubate plates at 30-35C for 18-24hrs. Brick red colonies with or without a zone of precipitated bile.

29. MacConkey agar

30. Confirmatory test Transfer suspect colonies to Levine Eosin-Methylene Blue Agar (LEMB). Incubate at 30-35C for 24-48hrs. Look for metallic sheen under reflected light, and blue black appearance under transmitted light. No such colonies indicates absence of Escherichia coli .

31. LEMB agar Uninoculated LEMB agar E coli on LEMB

32. Escherichia coli on LEMB

33. Test for Clostridium species Take two equal portions of 1:10 preparation. Heat one for 10minutes at 80°C and cool rapidly. Transfer 10mL of each portion to separate 100mL Reinforced medium for Clostridia(RMC) Incubate anaerobially at 35-37C for 48 hrs. Subculture to Columbia agar Medium with Gentamicin. Incubate plates anaerobically for 48hrs at 35-37C. Examine plates for growth, No growth indicates absence of Clostridium species.

34. Confirmatory tests If growth occurs: Transfer colony to two Columbia agar Plates. Incubate one aerobically and other anaerobically at 35-37C for 48hrs. The occurrence of only anaerobic growth indicates presence of Clostridium species. Perform catalase test by transferring suspect colonies to glass slides. Apply a drop of Hydrogen Peroxide.

35. Confirmatory test cont. The reaction is negative if no gas bubbles evolve. The product is negative for Clostridium if there is no anaerobic growth and catalase reaction is positive.

36. Retest For confirming a doubtful result on any of the tests using 10g sample, a retest using 25g sample can be conducted.

37. CONTACT US Gibraltar Laboratories is conveniently located GIBRALTAR LABORATORIES 122 Fairfield Road 16 Montesano Road (Shipping/Receiving) Fairfield, New Jersey 07004 [phone] tkiran @gibraltarlabsinc.com www. gibraltarlabsinc .com Facebook.com/ GBLinc Twitter.com/ GBLinc

38. THANK YOU! Proprietary and Confidential © Gibraltar Laboratories, Inc. www. gibraltarlabsinc .com / [phone]