Microbiological Tests

STERILITY TESTING : of PHARMACEUTICAL PRODUCTS A sterility test may be defined as — ‘a test that critically assesses whether a sterilized pharmaceutical product is free from contaminating microorganisms’.

TEST FOR STERILITY : the wide-spectrum of the pharmaceutical products, both pure and dosage forms, may be accomplished by adopting any one of the following two well-recognized, time tested, and universally accepted methods, namely : (a) Membrane Filtration, and (b) Direct Inoculation

Membrane Filtration A membrane usually found to be quite suitable for sterility testing essentially bears a nominal pore size not more than 0.45 nm

Membrane Filtration (1) The solution of the product under investigation is carefully filtered via a membrane filter that would precisely retain any possible contaminating microorganisms. (2) The resulting membrane is washed in situ to get rid of any possible traces of antibiotic (3) Finally, the segregated microorganisms are transferred to the suitable culture media under perfect aseptic environment.

The direct inoculation procedure introducing test samples directly into the nutrient media. The European Pharmacopoeia (2002) recommends two media for this purpose Fluid mercaptoacetate medium (also known as fluid thioglycollate medium) suitable for the cultivation of anaerobic organisms (incubation temperature 30-35C). Soyabean casein digest medium (also known as tryptone soya broth) which support the growth of both aerobic bacteria (incubation temperature 30-35C) and fungi (incubation temperature 20-25C).

Procedure 1. Using aseptic technique, a sample of pharmaceutical product from the bulk or from each container of the material under test was transferred to a conical flask containing the medium. 2. The conical flask was incubated at 37C. 3. The sample was examined in every 24 h up to 7 days for signs of living organisms.

Methods for antibiotic detection Diffusion methods: -These methods based on the diffusion of antibiotics in agar inoculated with sensitive microorganisms. - The effects of antibiotics are revealed by the production of zones of inhibition.

Three categories of diffusion are employed: 1- One-dimentional (linear) diffusion in test tubes or capillaries. 2-Two- dimentional diffusion e.g., radial diffusion from cups cut in agar layers. 3- Three dimentional diffusion from cylinders or disc located on the surface of the agar layer.

Sensitivity of microorganisms to antibiotics Measurement of the antibiotic sensitivity of an organism in the laboratory is designed to predict whether an infection will respond to treatment with that antibiotic or not.

Principles and Definitions Antibiotic susceptibility testing (in vitro) Bacteriostatic Antibiotics Minimum inhibitory concentration (MIC) Lowest concentration that results in inhibition of visible growth (colonies on a plate or turbidity of liquid culture) Bactericidal Antibiotics Minimum bactericidal concentration (MBC) Lowest concentration that kills 99.9% of the original inoculum

Different concentration of Tetracycline

Minimum Bactericidal Concentration (MBC)

Pyrogen

Chemical Nature of Endotoxin

Biological Properties of Endotoxin

Pyrogen test

C. Bacterial Endotoxins Test (LAL Test) A Limulus amebocyte lysate (LAL) reagent is the basis for an in vitro pyrogen test method that is specific for bacterial endotoxin pyrogen. The LAL reagent was obtained horseshoe crab.

C. Bacterial Endotoxins Test (LAL Test) Equal volumes of test solution and LAL reagent are mixed in glass test tubes. After incubation at 37 C for 1 h, the tubes are observed for clot formation after inverting them. Formation of a solid gel clot that withstands inversion of the tube constitutes a positive test. . .