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PHARMACEUTICAL MICROBIOLOGY
(BP303T)
Unit-IV
Part-3
Microbiological Assay of Vitamin & Amino acid
Assessment of a New Antibiotic:
Name: Ms. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY
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.
Sr
No.
Vitamin Test Microorganism Incubati
on Temp
(℃)
Assay pH
1 Vitamin B12 Lactobacillus leichamannii ATCC
7830
37 6.1
Poteriochromonas stipitata ATCC 11532 30 5.5
2 Vitamin B6 Saccharomyces uvarum ATCC 9080 30 4.5
Tetrahymena thermophila ATCC 30008 30 6.1
3 Riboflavin Lactobacillus casei ATCC 7469 37 6.8
Tetrahymena thermophila ATCC
30008
30 6.1
4 Thiamine Lactobacillus viridescens ATCC 12706 30 6.0
Ochromonas danica ATCC 30004 30 5.5
5 Biotin Lactobacillus plantarum ATCC 8014 37 6.8
Ochromonas danica ATCC 30004 30 5.5
6 Niacin Lactobacillus plantarum ATCC 8014 37 6.8
Tetrahymena thermophila ATCC 30008 30 6.1
7 Pentothenic Acid Lactobacillus plantarum ATCC 8014 37 6.7
Tetrahymena thermophila ATCC
30008
30 6.1
Table 1.1 Test Microorganisms and Conditions for the Microbial Assay of Vitamins
 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.
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
• 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.
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
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
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.
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.
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.
 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.
• 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.
• 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.
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
• 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
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
• 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.
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.
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.
THANK YOU

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Microbiological Assay of Vitamin & Amino acid Assessment of a New Antibiotic: PHARMACEUTICAL MICROBIOLOGY (BP303T) Unit-IV Part-3

  • 1. PHARMACEUTICAL MICROBIOLOGY (BP303T) Unit-IV Part-3 Microbiological Assay of Vitamin & Amino acid Assessment of a New Antibiotic: Name: Ms. Pooja Deepak Bhandare Assistant Professor G H RAISONI UNIVERSITY SCHOOL OF PHARMACY
  • 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.
  • 3. Sr No. Vitamin Test Microorganism Incubati on Temp (℃) Assay pH 1 Vitamin B12 Lactobacillus leichamannii ATCC 7830 37 6.1 Poteriochromonas stipitata ATCC 11532 30 5.5 2 Vitamin B6 Saccharomyces uvarum ATCC 9080 30 4.5 Tetrahymena thermophila ATCC 30008 30 6.1 3 Riboflavin Lactobacillus casei ATCC 7469 37 6.8 Tetrahymena thermophila ATCC 30008 30 6.1 4 Thiamine Lactobacillus viridescens ATCC 12706 30 6.0 Ochromonas danica ATCC 30004 30 5.5 5 Biotin Lactobacillus plantarum ATCC 8014 37 6.8 Ochromonas danica ATCC 30004 30 5.5 6 Niacin Lactobacillus plantarum ATCC 8014 37 6.8 Tetrahymena thermophila ATCC 30008 30 6.1 7 Pentothenic Acid Lactobacillus plantarum ATCC 8014 37 6.7 Tetrahymena thermophila ATCC 30008 30 6.1 Table 1.1 Test Microorganisms and Conditions for the Microbial Assay of Vitamins
  • 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.