The document discusses methods for assessing new antibiotics through microbiological assays. It describes how the minimum inhibitory concentration (MIC) can be determined using either liquid or solid dilution methods. The liquid dilution method involves setting up a series of test tubes with doubling dilutions of the antibiotic being tested and incubating with a test microorganism. The solid dilution method incorporates the antibiotic dilutions into an agar plate which is then inoculated. After incubation, the MIC is the lowest concentration that inhibits microbial growth. These assays help establish the efficacy and potency of new antibiotics.
2. Introduction:
• Principle
• The microbiological or microbial assay is a type of biological assay in which the relative
potency of activity of a compound is determined by measuring the amount required for
producing the predicted effect on a suitable test organism under standard conditions.
• Vitamins are essentially required for the growth and multiplication of microorganisms
(which are highly sensitive even to small amounts of growth factors).
• These test microorganisms can synthesis the factor being assayed which forms the basis of
microbiological assay of vitamins and amino acids.
• The test microorganisms used for the standardization of water-soluble vitamins are given in
table 1.1.
4. Microbiological Assay of Cynocobalamin (Vitamin B12):
• Microbiological assay of Vitamin B12 is can be performed by
using one of the following methods,
• Tritrimetric Method.
• Turbidimetric Method.
5. Tritrimetric Method:
• Preparation of Standard Cynocobalmine stock solution:
• The standard stock solution is prepared by adding a sufficient amount of 25% alcohol
to an accurately weighed quantity of U.S.P. cyanocobalamin RS.
• The solution obtained having a known concentration of 1.0µg/ml is stored in a
refrigerator for not more than two months.
• Preparation of Basal Medium Stock Solution:
• Composition of basal medium stock solution is as follows
6. • Cysteine and tryptophan are dissolved in hydrochloric acid and then the next 8 solutions
are added.
• To the resultant solution, 100ml water is added and mixed.
• To this mixture, dextrose, sodium acetate, and ascorbic acid are added.
• The solution is filtered and polysorbate 80 solution is added.
• The pH is adjusted between 5.5 and 6.0 using 1 N sodium hydroxide, and volume is
adjusted to 250ml with purified water.
7. Ingredient Quantity
L-Cystine 0.1gm
L-Tryptophan 0.05gm
1N Hydrochloric acid 5 ml
Adenine-guanine-uracil solution 5ml
Xanthine solution 5 ml
Vitamin solution I 10 ml
Vitamin solution II 10 ml
Salt solution A 5 ml
Salt solution B 5 ml
Asparagine solution 5 ml
Acid-hydrolysed casein solution 25 ml
Dextrose (anhydrous) 10 gm
Sodium Acetate (anhydrous) 5 gm
Ascorbic Acid 1 gm
Polysorbate 80 Solution 5 ml
Table 1.2: Basal Medium Ingredients
8. Test Solution of the material to be assayed
• Take an accurate amount of material to be assayed and dissolve it in
water.
• Add dilute HCL or solution of sodium hydroxide to adjust pH (6.0)
and add water to make up the final volume
9. Preparation of inoculum:
• The inoculum is prepared by transferring cells from the stock culture of the organism to
two sterile tubes, each containing 10ml culture medium.
• These culture tubes are then incubated for 16-24 hours at 30-40 ± 0.5°C temperature.
• The incubated tubes are aseptically centrifuged, and the supernatant is decanted.
• The cells from the culture are suspended and mixed with a 5ml sterile suspension
medium.
• The volume is adjusted using sterile suspension medium so that 1 in 20 dilutions in
saline TS produces 70% transmittance on a spectrophotometer at 530nm wavelength,
and read against saline TS set at 100% transmittance.
• 1 in 400 dilution of the adjusted suspension is prepared using basal medium stock
solution to use for the test inoculum.
10. Procedure of Titrimetric method:
• Clean ten test tubes and add 0.0 ml, 0.5 ml, 1 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3 ml, 4 ml,
4.5 ml and 5 ml respectively of standard cynocobalamine solution (1.0µg/ml).
• To each test tube add 5 ml of basic medium stock solution.
• Adjust final volume (10ml) using water.
• Take another four test tubes and add 1 ml, 2 ml, 3 ml, 4 ml respectively of test
solution to be assayed.
• To each test tube add 5 ml of basal stock medium solution and make up final volume (10ml) with
water.
• Autoclave all the test tubes @ 121℃ for 5 minutes.
• Cool all test tubes at room temperature and then inoculate one drop of inoculum (
Lactobacillus leichmanni ATCC 7830).
• Incubate all tubes at temperature between 30-37 ℃.
• Titrate contents of each test tube electrometrically or using Bromothymol blue as an
indicator to obtain average titration values.
• Plot the graph of average titration values vs std cynocobalamine solution and
determine concentration of activity per ml of test solution.
11. Turbidimetric Method:
• Everything is the same as the Titrimetric method; additionally it contains two
test tubes to which neither standard nor a test cyanocobalamin solution is
added.
• Incubate all test tubes at 30-37 ℃ for 16 - 24 hours.
• By using an “Uninoculated Blank Tube” adjust transmittance at 640 mµ to
100% in the photoelectric colorimeter.
• Thoroughly mix contents of each test tube and record transmittance.
• Plot graph of transmittance against concentration of cyanocobalamin solution.
• Draw a smooth curve and calculate the concentration of test solution of
cynocobalamine.
12. Microbiological assay of Amino acids.
• The microbiological or microbial assay is a type of biological assay in which
the relative potency of activity of a compound is determined by measuring the
amount required for producing the predicted effect on a suitable test organism
under standard conditions.
• Microbiological assays are time-consuming and also not suitable for all amino
acids.
• Amino acids are essential for the growth and replication of some
microorganisms.
• Many strains of such microbes depend on a particular amino acid.
• Thus, if such microorganisms are cultured using a small amount of that particular
amino acid, a limited degree of growth will be observed, which is measured by
turbidimetry or by measuring the increased lactic acid production by either
microtitration or pH change.
13. • Guthrie and Susi introduced a modified version of microbiological
assay utilising diffusion in gels in clinical biochemistry laboratories for
screening blood Samples having increased phenylalanine levels.
• This test was named as the Guthrie test (after its inventors) and is the
most widely used microbiological assay method of an amino acid.
• This bacterial inhibition assay is based on the ability of phenylalanine to
counteract the effects of β-2- theienylalanine (a competitive metabolic
antagonist on the growth of a special strain of Bacillus subtilis which
depends on phenylalanine for its growth.
14. • The test is carried out on an agar layer in which a mixture of the
suspension of B. subtilis spores, minimum amount of growth nutrients,
and a fixed amount of β-2 thienylalanine is added.
• Filter paper discs of 4 mm diameter are soaked in blood and placed on the
agar surface along with the blood soaked discs of phenylalanine
standards.
• These agar plates are incubated at 37°C overnight.
• Bacterial growth is observed only when phenylalanine concentration in
the blood discs is sufficient to overcome the effects of β-2 thienylalanine.
• Growth is observed in zones of growth around each disc.
• The next day diameter of each zone of growth is measured and related to
phenylalanine concentration.
15. Assessment of a New Antibiotic.
Introduction:
• Antimicrobial Resistance (AMR) is a way through which microbes change their ways
through which a particular antibiotic is acting on them making it ineffective.
• The AMR is giving rise to tougher microbial strains, some of them are even resistant
to all the antibiotics available today we call such stains as superbugs.
• If new antibiotics are not introduced, the patients will die due to the previously
treatable infections.
• The antibiotics are assessed in many ways like their efficiency, side effects, health
gains, spectrum etc
16. • Minimum Inhibitory Concentration is a one of criteria that establishes
efficiency of an antibiotic.
• Minimum Inhibitory Concentration is the minimum concentration at
which an antibiotic inhibits the growth of microorganism but not
necessarily kills it.
• MIC the term is applicable to antibiotics, disinfectants, antiseptics and
preservatives.
• MIC of an antibiotic is tested either by one of the following ways,
• Liquid Dilution Method.
• Solid Dilution Method
17. Liquid Dilution Method (Test Tube Method)
• Twelve test tubes are taken and are labelled as per table 1.1.
Tube Number Volume of Double
Strength Medium
(ml)
Volume of Test
Chemical (ml)
Volume of Sterile
Water (ml)
0 (Uninoculated) 5 0.0 5
0 (Control) 5 0.0 5
1 5 0.5 4.5
2 5 1.0 4.0
3 5 1.5 3.5
4 5 2.0 3.0
5 5 2.5 2.5
6 5 3.0 2.0
7 5 3.5 1.5
8 5 4.0 1.0
9 5 4.5 0.5
10 5 5.0 0.0
18. • Double strength medium is added in all test tubes.
• First test tube (Uninoculated) inoculum is not added and is used to check
sterility of the medium used.
• In all other remaining eleven test tubes 3-4 drops of inoculum is added to
reach 105- 106cells/ ml concentration of the test microorganism.
• In all test tubes except “Uninoculated” and “Control” chemical agent
under test are added from 0.5 ml to 5.0 ml.
• The final volume of 10 ml is adjusted using sterile water in all test
tubes.
• Contents of all test tubes are mixed well and incubated @ 37℃ for 2-3
days.
• After incubation all tests tubes are examined for growth of microorganism
and the minimum inhibitory concentration is calculated.
• It is necessary to carry out a preliminary experiment to determine the range
of MIC of the chemical agent under test.
19. Solid Dilution Method:
• In this method the chemical agent under test is mixed in the molten agar and
then is poured in the petri dish.
• After solidification, the inoculum is spread on the surface of the agar
medium.
• All the plates are incubated @ 37℃ for 2-3 days.
• After incubation all petri dishes are examined for growth of microorganism
and the minimum inhibitory concentration is calculated.
20. Advantages
1. Several microorganisms can be studied once by using a multipoint
inoculator.
2. Contaminations can easily be detected as colony features are
distinctive here rather than turbidity difference.
