Microbiological assay and methods

Microbial assay

• Microorganisms have found widespread uses in the performance of bioassays for:
  • Determining the concentration of certain compounds (e.g., amino-acids, vitamins and some antibiotics) in complex chemical mixtures or in body fluids.
  • Diagnosing certain diseases.
  • Testing chemicals for potential mutagenicity or carcinogenicity.
  • Monitoring purposes involving the use of immobilized enzymes.
  • Sterility testing of antibiotics.
• Microbiological assays are used during production to determine the potency and quality control.
• These are used to determine the pharmacokinetics of drugs in animal and human.
• In antimicrobial chemotherapy to monitor, in managing, controlling the chemotherapeutic agents.

Microbiological assay of antibiotics

• The potency of an antibiotic is estimated by comparing the inhibition of growth of sensitive micro-organisms produced by known concentrations of the antibiotic to be examined and a reference substance.
• The reference substances used in the assays are substances whose activity has been precisely determined with reference to the corresponding international standard or international reference preparation.
• 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 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=0.05.
• 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 percent and not more than 105 percent of the estimated potency.

Methods of microbiological assay of antibiotics:

• Following methods are used for microbiological assays:

1. Disc diffusion method (cylindrical cup plate method):

• Use petri dishes or rectangular trays filled to a depth of 3-4 mm with a culture medium that has previously been inoculated with a suitable inoculum of a susceptible test organism prepared as described below.
• The nutrient agar may be composed of two separate layers of which only the upper one may be inoculated.
• The concentration of the inoculum should be so selected that the sharpest zones of inhibition and suitable dose-response at different concentrations of the standard are obtained.
• When using the inoculum, an inoculated medium containing 1ml of inoculum per 100ml of the culture medium is usually suitable.
• When the inoculum consists of a suspension of vegetative organisms, the temperature of the molten agar medium must not exceed 48-50^oC at the time of inoculation.
• The dishes or trays should be especially selected with flat bottoms.
• During the filling they should be placed on a flat, horizontal surface so as to ensure that the layer of the medium will be of uniform thickness.
• With some test organisms, the procedure maybe improved if the inoculated plates are allowed to dry for 30 minutes at room temperature before use, or refrigerated at 4^oC for several hours.
• For the application of the test solution, small sterile cylinders of uniform size, approximately 10mm high and having an internal diameter of approximately 5mm, made of suitable material such as glass, porcelain, or stainless steel, are placed on the surface of the inoculated medium.
• Instead of cylinders, holes 8-10mm in diameter may be bored in medium with a previously sterilized borer.
• Other methods of application of the test solution may also be used.
• The arrangement on the plate should be such that overlapping of zones is avoided.
• Solutions of the reference material of known concentration and corresponding dilutions of the test substance, presumed to be of approximately the same concentration, are prepared in a sterile buffer of a suitable pH value.
• To assess the validity of the assay at least 3 different doses of the reference material should be used together with an equal number of doses of the test substance which has the same presumed activity as the solutions of the reference material.
• The dose levels used should be in geometric progression, for example, by preparing a series of dilutions in the ratio 2:1.
• Once the relationship between the logarithm of concentration of the antibiotic and the diameter of the zone of inhibition has been shown to be approximately rectilinear for the system used, routine assays may be carried out using only 2 concentrations of the reference material and 2 dilutions of the test substance.
• Where a monograph gives directions for the initial preparation of a solution of the substance, this solution is then diluted as necessary with the appropriate sterile buffer.
• The solutions of the reference material and the test substance are preferably arranged in the form of a Latin square when rectangular trays are employed.
• When petri dishes are used, the solutions are arranged on each dish so that the solutions of the reference material and those of the test substance alternate around the dish and are placed in such a manner that the highest concentrations of the reference material and of the test substance are not adjacent.
• The solutions are placed in the cylinders or holes by means of a pipette that delivers a uniform volume of liquid.
• When the holes are used, the delivered volume should be sufficient to fill them almost completely.
• The plates are incubated at a suitable temperature, the selected temperature being controlled at +/- 0.5^oC, for approximately 16 hours, and the diameters or areas of the zones of inhibition produced by the varied concentrations of the standard and of the test substance are measured accurately, preferably to the nearest 0.1 mm of the actual zone size, by using a suitable measuring device.
• From the results, the potency of the tested substance is calculated.

2. Turbidometric method:

• Inoculate a suitable medium with a suspension of the chosen micro-organism having a sensitivity to the antibiotic to be examined such that a sufficiently large inhibition of microbial growth occurs in the conditions of the test.
• Use a known quantity of the suspension chosen so as to obtain a readily measurable opacity after an incubation period of about 4h.
• Use the inoculated medium immediately after its preparation.
• Using the solvent and the buffer solution indicated prepare solutions of the reference substance and of the antibiotic to be examined having known concentrations presumed to be an equal activity.
• In order that the validity of the assay may be assesses, use not fewer than 3 doses of the reference substance and 3 doses of the antibiotic to be examined which has the same presumed activity as the doses of the reference substance.
• It is preferable to use a series of doses in geometric progression. In order to obtain the required linearity, it may be necessary to select from a large number 3 consecutive doses for the reference substance and the antibiotic to be examined.
• Distribute an equal volume of each of the solutions into identical test-tubes and add to each tube an equal volume of inoculated medium (for example, 1 ml of the solution and 9ml of the medium).
• For the assay of tyrothricin, add 0.1ml of the solution to 9.9ml of inoculated medium.
• Prepare at the same time 2 control tubes without antibiotic, both containing the inoculated medium and to one of which is added immediately 0.5ml of formaldehyde R.
• These tubes are used to set the optical apparatus used to measure the growth.
• Place all the tubes, randomly distributed or in a Latin square or randomized block arrangement, in a water-bath or suitable apparatus fitted with a means of bringing all the tubes rapidly to the appropriate incubation temperature.
•  Maintain them at that temperature for 3h to 4h, taking precautions to ensure uniformity of temperature and identical incubation time.
• After incubation, stop the growth of the micro-organisms by adding 0.5ml of formaldehyde R to each tube or by heat treatment and measure the opacity of 3 significant figures using suitable optical apparatus.
• Alternatively use a method which allows the opacity of each tube to be measured after exactly the same period of incubation.
• Calculate the potency using appropriate statistical methods.
• Linearity of the dose-response relationship, transformed or untransformed, is often obtained only over a very limited range.
• It is this range which must be used in calculating the activity and it must include at least 3 consecutive doses in order to permit linearity to be verified.
• In routine assays when the linearity of the system has been demonstrated over an adequate number of experiments using a three-point assay, a two-point assay may be sufficient, subject to agreement by the competent authority.
• However, in all cases of dispute, a three-point assay must be applied.
• Use in each assay the number of replications per dose sufficient to ensure the required precision.
• The assay may be repeated and the results combined statistically to obtain the required precision and to ascertain whether the potency of the antibiotic to be examined is not less than the minimum required.

Besides these methods other commonly used microbiological assays:

 3. Urease Assay:

• This assay is used for the assay of those antibiotics which inhibits the protein synthesis.
• Aminoglycosides such as streptomycin, amikacin, kanamycin, gentamicin, netilmicin, and macrolides such as erythromycin, azithromycin, clarithromycin.
• Certain micro-organisms like Proteus mirabilis produce enzyme urease.
• Production of urease indicated by increase in pH of the media.
• Antibiotics that inhibit protein synthesis, inhibit urea production so there is no further increase in pH.
• In this way, urease assay is done. This has very limited application.

 4. Luciferase Assay:

• Luciferase is the enzyme that acts on luciferin protein produced by different micro-organisms presence of ATP and gives bioluminescence.
• This assay can be used to detect the very low amount of ATP present in bacterial culture.
• Drugs like Aminoglycosides, inhibits ATP or reduce the level of ATP and luminescence doesn’t occur.
• If antibiotics inhibit the growth of micro-organisms, there is no ATP and no luminescence occurs.

5. Radioenzymatic Assay:

• To detect the resistance of bacteria against different antibiotic especially aminoglycosides and chloramphenicol.
• If bacteria are resistant to these agents, they produce specific enzymes like aminoglycoside acetyl transferase (AAC), aminoglycoside adenyl transferase (AAD) and aminoglycoside phosphotransferase (APH) and thus becomes resistant to antibiotics.
• In this case for aminoglycoside acetyl transferase (AAC) is radiolabelling agent ¹⁴C carbon isotope (with the help of cofactor [ 1- ¹⁴C] acetyl co-enzyme A) and for AAD and APH ³H (by [2- ³H] ATP) are used to detect the production of these aminoglycoside inactivating enzymes by micro-organisms.
• If micro-organisms produced AAC, AAD, or APH, these enzymes become radiolabelled with their specific isotopes ¹⁴C/ ³H and give specific color change.
• If color change is observed resistance to aminoglycoside is inferred.

Microbiological assay and methods