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

Current USP<2021> and <2022> Testinghttp://www.gibraltarlabsinc.com/services/microbiology.html

Proprietary and Confidential © Gibraltar Laboratories, Inc.

Rupinder Puar

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.

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.

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.

<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.

Membrane Filtration

Plate count method

Multiple Tube Method

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.

MPN Chart

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.

Mossel-Enterobacteria Enrichment Broth

VRBG Agar (E.coli)

MPN chart for Enterobacteria

<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.

Sa on VJ agar

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

Sa on MSA

Staphylococcus aureus has yellow colonies with yellow zones

Sa on BP agar

Staphylococcus aureus has black shiny colonies with 2-5mm clear zones

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.

Coagulase test

Coagulase test

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.

Salmonella on BGA

an uninoculated plate

Salmonella on BGA . The colonies are small, opaque, pink or white. Can also be transparent and colorless.

Salmonella on XLD

Uninoculated plate

Salmonella on XLD is red, with or without black centers

HEKTOEN ENTERIC AGAR Salmonella on HE agar is Blue-green, with or without black centers

Salmonella on Hektoen Enteric Agar

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.

TSI slant

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.

MacConkey agar

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.

LEMB agar Uninoculated LEMB agar

E coli on LEMB

Escherichia coli on LEMB

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.

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.

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.

Retest• For confirming a doubtful result on

any of the tests using 10g sample, a retest using 25g sample can be conducted.

3737

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GIBRALTAR LABORATORIES

122 Fairfield Road 16 Montesano Road

(Shipping/Receiving)Fairfield, New Jersey 07004(973) 227-6882

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