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Microbiological Assay of antibiotics

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1 Microbiological Assay of antibiotics
Pharmaceutical Microbiology-I PHR 203 Shahana Sharmin

2 Introduction Microbiological assay is a technique in which the potency or concentration of a compound is assessed by determining its effect on micro-organisms. The principles are discussed by Roberts and Boyce.' Microbiological assay is a legal QC requirement for the assay of a number of antibiotics, in both the British Pharmacopoeia (BP) and United States Pharmacopoeia (USP). Bioassay compares a reference standard and an unknown sample, the two preparations being measured simultaneously.

3 Introduction A lot of preliminary work is necessary for microbiological assays, and if only a small number of samples are expected irregularly, the method is inefficient. Two assay methods are normally used, agar diffusion and tube assays. They have several common features: (a) The compound being assayed must influence the growth of the test organism. (b) A varying response in growth must be produced by addition of varying quantities of the test material. (c) The growth medium must contain an excess of all the compounds required by the test organism for growth. The exception to this is the compound being assayed which should be totally absent from the basic medium. (d) The assumption is made that the compound being assayed is the only growth promoting or inhibiting compound present.

4 Reference standard and units of activity
The potency (activity) of an antibiotic product is expressed as the ratio of the dose that inhibits the growth of a suitable susceptible microorganism to the dose of an International Biological Standard, an International Biological Reference Preparation, or an International Chemical Reference Substance of that antibiotic that produces similar inhibition. Potency of antibiotic can be expressed in “unit” or “μg” of activity per mg of dried material, as stated in Pharmacopeia. “μg” of activity is based on single active ingredient. “unit” is used when there are more than one active ingredient in the antibiotic.


6 Dose response curve The dose-response curve for both test and standard is sigmoidal The distance between the two curves (x) measures the effectiveness of the test material relative to the standard. This is the log potency ratio. The two lines coincide if standard and test materials are identical.

7 Large plate assay using Latin square design
Latin Square Designs are very efficient designs. It allow for two blocking factors. In other words, these designs are used to simultaneously control (or eliminate) two sources of irritation variability. Whenever, you have more than one blocking factor a Latin square design will allow you to remove the variation for these two sources from the error variation. We can remove the variation from our measured response in both directions if we consider both rows and columns as factors in our design.

8 Large plate assay using Latin square design
The Latin Square Design gets its name from the fact that we can write it as a square with Latin letters to correspond to the treatments. The treatment factor levels are the Latin letters in the Latin square design. The number of rows and columns has to correspond to the number of treatment levels. So, if we have four treatments then we would need to have four rows and four columns in order to create a Latin square. This gives us a design where we have each of the treatments and in each row and in each column.

9 Statistical interpretation of microbiological assay methods
The assay must be designed in a way that will permit examination of the validity of the mathematical model on which the potency equation is based. If a parallel-line model is chosen, the 2 log dose-response (or transformed response) lines of the preparation to be examined and the reference preparation must be parallel ; they must be linear over the range of doses used in the calculation. These conditions must be verified by validity tests for a given probability, usually P = Other mathematical models, such as the slope ratio model, may be used provided that proof of validity is demonstrated. Unless otherwise stated in the monograph, the confidence limits (P = 0.95) of the assay for potency are not less than 95 per cent and not more than 105 per cent of the estimated potency.

10 Agar diffusion assay These are usually carried out on plates. The assay system is identical for antibiotics and vitamins. A liquid agar (50 "C) preparation of medium minus the compound to be assayed is inoculated uniformly with a sensitive organism. The agar is poured into a petri dish and is allowed to solidify. The assay material and suitable reference standards are placed in individual reservoirs. These diffuse out into the agar, and the organism grows to form zones after a suitable period of incubation. The zone is one of inhibition (no growth) when antibiotics or preservatives are used. The zone is one of growth or exhibition when vitamins or amino acids are used. The width of the zone, which depends on the concentration of active compound used, forms the quantitative basis of the test.


12 Factors affecting diffusion assay
Agar nutrient content. Cylinder charge volume. Thickness (volume) of the agar layer. Incubation temperature.

13 Turbidimetric Method Reaction tube made from glass or plastic with same height. Spectrophotometric tube has to be sterile. All residues are removed and sterilized before and after use. 1 ml of test solution and standard solution of each doses placed in 3 tubes (triplo) randomly Make 2 control tubes Add 9 ml of inocula into each tubes Place tubes in waterbath or incubator at ( )OC for 2 hrs After incubation, add 0.5 ml of dilute formaldehyde Transmittance or Absorbance at 530 nm

Industrial microbiology is an important area of applied microbiology. It refers to the use of microorganisms in commercial enterprise and, Cheap raw materials are converted to valuable products through the metabolism of microbes. Microbes for this purpose could be exploited in different ways. For instance this includes (i) synthesis of fermentation products as acids, alcohols or other organic compounds (ii) transformation of one compound into another desired type, (iii) the production of enzymes, antibiotics, or insecticides, or (iv) the use of microbes themselves as food.

15 Pharmaceutical products of microbial origin
There are a number of medicinal substances which derive from microorganisms, including antibiotics, immunological products and products resulting from recombinant DNA technology. These are dextran, used as a substitute for plasma, certain enzymes and a small group of organic chemicals. Chemicals which continue to be produced by fermentation include citric acid, ethanol, lactic acid, vitamins B2 and B12 and a considerable range of amino acids.

16 Various commercial products of economic value made by microbes are
(i) medicines i.e. pharmaceuticals, including antibiotics, steroids, human protein, vaccines, and vitamins, (ii) organic acids, (iii) amino acids, (vi) enzymes, (v) alcohols, (vi) organic solvents and (vii) synthetic fuels. In addition to these, quite recently potential of microbes could also be realized in (viii) recovery of metals from ores through bioleaching, (ix) recovery of petrol, and (x) single all protein production.

17 Microbiologically controlled environments in the pharmaceutical industry
Nonviable particulate and viable microbiological surveillance are used to evaluate the design and control of a cGMP-manufacturing environment.The nonviable particulate monitoring program plays an important role as it is used on a routine basis to verify the maintenance of air classifications. In general, a comprehensive environmental monitoring program should include scheduled monitoring of - Airborne viable Nonviable particulates Pressure differentials Direction of air flow Temperature and humidity Surface microbial contaminants on personnel and equipment, work tables, floors, and walls.

18 Different methods of microbial monitoring systems for environment in pharmaceutical industry
Sampling airborne microorganisms Passive Monitoring Settling Plates Active Monitoring Slit-to-Agar (STA) Air Sampler (Air through narrow slit, rotational agar plate) Sieve Impactors (Air through a perforated plate/s) Single-Stage (contact plates or Petri dishes) Multi-Stage Cascade (Stacked perforated plates) Sterilizable Atrium (Stainless Steel head collection device)Single-Use Sterile Atrium (Disposable) Centrifugal Propeller Sampler (Agar coated strip) Filtration (Polycarbonate, cellulose acetate, gelatin filters) Impinger (use of liquid medium for particle collection) Real Time Laser-Induced Fluorescence Systems

19 Sampling microorganisms on surfaces
It is important to perform regular sampling of surfaces within the aseptic processing environment, including equipment, walls, floors, and counter tops. Contact Plates: Flat surfaces can be sampled using contact plates. These specialized agar plates are manufactured precisely to ensure a smooth, evenly distributed layer of agar on each plate. Sampling is achieved by gently rolling the domed surface of the agar onto the test area. The plate is then incubated under appropriate conditions to obtain colony counts. Swabs: In an environmental monitoring program, it is important to include the sampling of surfaces that are not flat or are difficult to access, as these areas may be more difficult to clean and disinfect. Swabs are preferred for this purpose. Early commercially available swabs presented a lower microbial recovery (30-50%).

20 Personnel sampling Periodic sampling of clothing (gowns and gloves) is used to measure the effectiveness of aseptic precautions. Gloves can be sampled (prior to removing or replacement) by touching all fingers and thumbs onto the surface of an agar plate. Other garments can be sampled using contact plates or swabs.

21 Different instruments of microbial monitoring systems for environment in pharmaceutical industry
BioCapt SS BioCapt SU MiniCapt remote BioLaz MiniCapt portable Slit-to-Agar (STA) AirTrace Air Sampler

22 Instruments used in microbiology lab
ELISA PCR PCR - Polymerase Chain Reaction A 96 - well microtiter plate  being used for ELISA.

23 ELISA (Enzyme-linked immunosorbent assay)
A test that uses antibodies and color change to identify a substance. ELISA is a popular format of a "wet-lab" type analytic biochemistry assay. It involves at least one antibody with specificity for a particular antigen. ELISA can perform other forms of  ligand binding assays instead of strictly "immuno" assays. COMPONENTS OF ELISA Antibody: IgG fraction of serum. Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues. Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow.

24 Principle The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding (antigen- antibody binding). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding.

25 How does ELISA work? There are variations of the ELISA test but the most basic type consist of an antibody attached to the solid surface. This antibody has affinity for (will latch onto) the substance of interest. For example… Human Chorionic Gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked to an enzyme and sample (blood, urine, etc.) under test are added to the test system. If no HCG is present in the test sample the only HCG with linked enzyme will bind. The more HCG which is present in the test sample the less enzyme linked HCG will bind.

26 Different antigen in sample
Secondary antibody substrate Enzyme Colored product Primary antibody Different antigen in sample

27 PCR (Polymerase Chain reaction)
PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence. PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence. The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. This results in the synthesis of new DNA strands which are complementary to the parent template strands. These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length.




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