Bile Salt Irgasan Brilliant Green Agar (BSIBG)

Bile Salt Irgasan Brilliant Green Agar (BSIBG) is a highly selective agar medium that uses bile salts, brilliant green, and Irgasan for the isolation of Aeromonas spp. from a variety of environmental, clinical, and food samples. Aeromonas spp. has been regarded as controversial pathogen and a possible source of food-borne infection with some strains noted as being enteropathogenic. This species of bacteria has also been found to grow and produce virulence factors not only at optimal growth temperature, but at refrigeration temperatures as well. 

Biuret Assay

The Biuret Assay, also known as the Piotrowski Test, is a biochemical assay that allows one to accurately quantify protein concentration within the range of 5-150 mg/mL. The protein sample, irrespective of its composition, is measured through absorbance spectroscopy at 540 nm in conjunction with a known protein concentration sample. 

Cetrimide Agar

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Introduction

Cetrimide Agar, also known as Pseudomonas Cetrimide Agar or Pseudosel Agar, is a selective and differential medium used for the isolation and identification of Pseudomonas aeruginosa from clinical and non-clinical specimens. 

Cetyltrimethylammonium bromide (Cetrimide)

Cetyltrimethylammonium bromide (Cetrimide)

Cetyltrimethylammonium bromide (Cetrimide) is a mixture of different quaternary ammonium salts that serve as the selective agent of this media. The mixture, acting as a detergent, causes a release of nitrogen and phosphorus from the bacteria cell, denaturing the cell membrane for bacteria except for P. aeruginosa. Other species of Pseudomonas are still susceptible to this compound. Cetrimide also serves as way to detect an organisms ability to produce fluorescent pigments. 

Pancreatic digest of gelatin provides nitrogen, carbon, and other vitamins viable for the growth of P. aeruginosa. Glycerol serves as a supplemental carbon source and addition of magnesium chloride and potassium sulphate stimulate the production of pyocyanin and pyoverdin. Agar is the solidifying agent. 

Selectivity

Pseudomonas aeruginosa  via CDC [10]

Pseudomonas aeruginosa via CDC [10]

Pseudomonas aeruginosa is an opportunistic aerobic, gram-negative bacterium found widely throughout nature and is one of the most commercially and biotechnological valuable microorganisms.  

P. aeruginosa produces soluble iron pigments: pyocyanin, pyoverdin, pyomelanin, and pyorubrin. No other species of gram-negative, non-fermenting bacteria produces pyocyanin which is a key element in the selectivity of the agar. When pyoverdin combines with pyocyanin, a bright green phenazine pigment is produced. Cetrimide, potassium sulfate, and magnesium chloride enhance and stimulate the production of both pyocanin and pyoverdin.    

Pyocyanin

Pyocyanin

On the selective agar, the presence of growth indicates a positive reaction.  Examination of individual colonies under ultraviolet light (254 nm) allows for detection of fluorescent pigments. 

 

Reconstitution

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PREPARATION OF CENTRIMIDE AGAR

  1. Add both dry and wet components to distilled water and boil to dissolve completely.

  2. Sterilize media by autoclaving at 121ºC for 15 minutes.

  3. Cool sterilized media to 45-50ºC.

  4. Aseptically dispense into sterile petri dishes and/or other appropriate containers.

Final pH: 7.2 ± 0.2 at 25°C

STORAGE AND SHELF LIFE 

Media is both light and temperature sensitive. Store plates away from direct light at 2-8ºC, or if properly sealed and stored upright, 15-25ºC. Plates may be used for one week when stored in a clean sterile area. Media should not be used if any signs of deterioration, color change, contamination, and/or expiration date has passed.  

STERILIZE ALL BIOHAZARD WASTE BEFORE DISPOSAL.

Quality Control

P. aeruginosa  on Cetrimide Agar via ThermoFischer [12]

P. aeruginosa on Cetrimide Agar via ThermoFischer [12]

After 18-24 hours of incubation (or ≥ 72 hours for negative controls) in a 30-35°C, plates should show isolated colonies in streaked areas, and confluent growth/lawns in areas of heavy inoculation. Colonies may be identified as P. aeruginosa when exhibiting a blue/green pigment fluorescing under short wavelength ultraviolet light. (254 nm) 

Note: Certain strains of P. aeruginosa may not produce pyocyanin. Other species of Pseudomonas do not produce pyocyanin, but will fluoresce under UV light.  Some non-fermenting aerobic spore formers may exhibit a water-soluble tan to brown pigmentation on the medium. Serratia spp. may exhibit pink pigmentation. 

Suitable microorganisms for Quality Control:

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Limitations

  1. Slight yellowing of the medium may occur due to enteric strains and/or environmental variables, however coloration can be distinguished using UV light.

  2. P. aeruginosa can lose its fluorescence under ultraviolet light if cultures are left at room temperature. Fluorescence reappears when re-incubated.

  3. Further tests may be necessary for confirmation of P. aeruginosa.


References:

  1. Aryal, Sagar, and Hariprem. “Cetrimide Agar- Composition, Principle, Uses, Preparation and Colony Morphology.” Online Microbiology Notes, 12 June 2018, microbiologyinfo.com/cetrimide-agar-composition-principle-uses-preparation-and-colony-morphology/.

  2. "Cetrimide Agar" Neogen. 03 Apr 2017. http://foodsafety.neogen.com/pdf/acumedia_pi/7222_pi.pdf

  3. "Cetrimide Agar Base." Himedia. Technical Data. Mar 2017. http://himedialabs.com/TD/M024.pdf

  4. “Cetrimide Agar: Composition, Principle, Preparation and Uses -.” Home -, 5 Nov. 2016, microbeonline.com/cetrimide-agar-composition-principle-preparation-uses/.

  5. “CETRIMIDE SELECTIVE AGAR.” Kligler Iron Agar (KIA) - or the Identificaiton of Enteric Bacteria, catalog.hardydiagnostics.com/cp_prod/Content/hugo/CetrimideSelAgar.htm.

  6. “Cetrimide Agar (USP/EP/JP) -Lab M.” Arkom Limited. Endo Agar Base - Lab M, www.labm.com/products/cetrimide-agar-usp-ep-jp.asp.

  7. "Cetrimide Selective Agar" ThermoFischer. 24 Feb 2011. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU452801.pdf

  8. “Cetrimide 388955.” Sigma-Aldrich, The Journal of Pharmacy and Pharmacology, www.sigmaaldrich.com/catalog/product/aldrich/388955?lang=en.

  9. “CM0579, Cetrimide Agar (USP, EP) | Oxoid - Product Detail.” CM0469, X.L.D. Agar | Oxoid - Product Detail, www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0579.

  10. “Healthcare-Associated Infections.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 9 Mar. 2018, www.cdc.gov/hai/organisms/pseudomonas.html.

  11. Jayaseelan, S., Ramaswamy, D. & Dharmaraj, S. World J Microbiol Biotechnol (2014) 30: 1159. https://doi.org/10.1007/s11274-013-1552-5

  12. “Remel™ Cetrimide Selective Agar.” Thermo Fisher Scientific, Thermo Fisher Scientific, www.thermofisher.com/order/catalog/product/R01292.

 

Potato Dextrose Agar (PDA)

Potato Dextrose Agar, often notated as PDA, is a common microbial growth media made from an infusion of potato and dextrose. It is one of the most widely used media for growing fungi and bacteria. PDA can be supplemented with different acids or antibiotics to inhibit bacterial growth that may interfere with yeasts and mold.  It is recommended for isolation and microbial enumeration of yeasts and molds in dairy, cosmetics, and clinical samples. 

Brucella Blood Agar (BBA)

Brucella Blood Agar (commonly notated as BBA or BRU) is a non-selective agar medium used for isolation, quantitation, and partial identification of fastidious anaerobes. This agar was originally developed for the isolation of Brucella spp. from potentially contaminated materials but has since been found to be useful for use in clinical specimens involving strict anaerobes. This agar is typically supplemented with Hemin and Vitamin K1.

Bicinchoninic Acid Assay (BCA)

The Bicinchoninic Acid Assay (BCA), also referred to as the Smiths' Assay, is a biochemical assay designed by Paul Smith in 1985. (Smith, et al., [7])  This assay uses colorimetric detection and quantification to determine the total concentration of protein in a solution by exhibiting a measurable color change. Applications of this assay include studying protein-protein interactions, estimating percent recovery of membrane proteins from cell extracts, high-throughput screening of fusion proteins, measuring column fractions after affinity chromatography and measuring protein covalently bound to agarose supports and protein adsorbed to multiwell plates.

Standard Time-Kill Testing

A Standard Time-Kill, often referred to as STK, Suspension Test, or Time-Kill Kinetics Assay, is an in vitro test which measures the changes in a population of aerobic microorganisms in a pre-determined sampling time while antimicrobial properties are present.  This test method is often used to measure antimicrobial activity in hand sanitizers, antiseptic wound gels, mouth washes, and other products that are expected to have microbiocidal properties [4]. Standard Time-Kill methods are based on the American Society for Testing and Materials method, Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure (E2315-16) [1].

Gram Staining

Gram staining, also called Gram's Method, is named after Hans Christian Gram who developed the method in 1884, which allows for one to distinguish between gram-postive and gram-negative bacteria using a combination of different chemical stains.  In this test, bacteria that retain the crystal violet dye do so because of a thick layer of peptidoglycan. In contrast, negative bacteria do not retain the violet dye and appear as red/pink.