Paper No.

: 14 Pharmaceutical & Biological Analysis

Module : 25 Determination of antimicrobials and disinfectants

Principal Investigator: Dr. Nutan Kaushik, Senior Fellow

The Energy and Resources Institute (TERI), New Delhi

Co-Principal Investigator: Dr. Mohammad Amir, Professor of Pharm. Chemistry,

Jamia Hamdard, New Delhi

Paper Coordinator: Dr. Showkat R. Mir, Assistant professor, Dept. of

Pharmacognosy, Jamia Hamdard, New Delhi.

Content Writer: Dr. B P Panda, Assistant Professor, Dept. of Pharmacognosy,

Jamia Hamdard, New Delhi.

Content Reviewer: Dr. M. Shahar Yar, Associate professor, Dept. of

Pharmaceutical Chemistry, Jamia Hamdard, New Delhi

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Description of Module

Subject Name Analytical Chemistry / Instrumentation

Paper Name Pharmaceutical and Biological Analysis

Module Determination of antimicrobials and disinfectants

Name/Title
Module Id 25

Pre-requisites

Objectives  Definition, properties and extraction of alkaloids

 General methods for the estimation of alkaloids in plants and their
preparations
 Assays for estimation of individual alkaloids

Keywords Alkaloids, Opium, Morphine, Quinine, Reserpine, Codeine, Atropine, Ergotamine,

Estimation of alkaloids, Total alkaloids

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Learning Objectives
• What are antimicrobial substances?
• Death of Microorganism
• Microbiological evaluation of antimicrobials
• Procedure involved in the evaluation of antimicrobials
• Minimum inhibitory concentration of antimicrobials
• Test for new disinfectants
Introduction
Antimicrobial substances include germicide, disinfectant, antiseptics, inhibitors, bacteriostat
/fungistat/virustat, sanitizer and anti-metabolite. Germicides (bactericide/fungicide/virucide)
are chemical substance used to kill bacteria or fungi or viruses example, 8% formaldehyde,
2% Glutaraldehyde , 100% ethylene oxide gas, 10–20% hydrogen peroxide. Disinfectant is a
chemical used to destroy the biological agent that causes disease in man, animal or plants
example, Phenol, o-Phenylphenol, Chloroxylenol, Ethanol, Isopropanol, Iodine,
Chlorine Benzalkonium chloride . Antiseptics are chemical used to prevent sepsis (blood
poisoning), applied to the tissue of humans or animals to inhibit or destroy invading organisms
example Hydrogen peroxide (6%) and Iodine. Inhibitors are the substance that causes the
microorganism to grow at a slower rate than normal. Bacteriostat /Fungistat/ Virustat are
substances that holds the number of bacteria/ fungi /virus at a constant level. Sanitizer is a
chemical that reduces the number of microorganism and destroys any that might cause disease
in man or economically useful animals example Sodium hypochlorite,ChlorineDioxide
Peroxyacetic Acid (PAA) and anti-metabolite is a substance that oppose the action of
metabolite or prevents its assimilation by an microorganism.

Death of a Microorganism
Inability of a microbial cell to produce growth within a given time in a defined medium at a
defined temperature is known as microbial death. The factors that influence the microbial
death are Viability of the microbial cell, Presence of inhibitors in the growth medium,
Inoculum size (Number of microbial cell), Sensitivity of the medium, Incubation temperature,
Oxygen level, pH of the media, Humidity level and Incubation time. During microbial death
Microbial growth Analysis is carried out by microbial respiration i.e. by measuring Oxygen
level/CO2 level in the media, by measuring metabolites concentration, by measuring the

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Optical Density at λ 620nm And by Visual inspection of the microbial growth in culture
media.

Importance of Microbiological evaluations for antimicrobials

1] To estimate the Potency i.e. The difference in the activity of an active and inactive form of
chemical can be determined only by biological assays. The activity of the preparation is
compared to the activity of a standard preparation

2] To obtain the Phenol Coefficient i.e. Determination of the concentration of a disinfectant

required to produce a particular result compared to the activity of pure phenol that gives the
same result.

3] To confirm that the antimicrobials are active at the concentrations recommended for use
also called use –dilution test, the performance of the test disinfectant is compared to that of
hypochlorite.

4] To determine the efficacy and limitation of new antimicrobial substance

Now days before marketing of any new antimicrobial substance, the manufacturer
carry out a series of proving test like Phenol coefficient test , The minimum inhibitory
concentration for range of microorganisms, The effect of organic matter, Acute and chronic
toxicity test.

Evaluation of antimicrobial substance

A. Phenol Coefficient Tests

1. Suspension test that include the Rideal Walker Test and the Chick Martin Test

2. Surface film test that include dilution test and carrier test

B. Nephelometric Method

C. Capacity Tests

D. Minimum inhibitory concentration

Phenol Coefficient Tests

It is the minimum concentration of pure phenol that produces viable sub-cultures at one time
of sampling but nonviable sub-cultures at the next is compared to the minimum concentration
of test disinfectant that produces same result. The test parameters for Phenol Coefficient

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Tests are microbial strain, culture condition, microbial numbers, the temperature and expose
time, the sample size, the time and temperature of incubation are fixed arbitrarily as part of
test specification. Phenol coefficient tests are performed by suspension test and surface film
test.

The Rideal Walker Test (BS 541:1934)

The microbial strain use for Rideal Walker test is Salmonella typhi, culture is carried
out for 24 hr at temperature 20oC. Subcultures are performed from both the test and phenol at
intervals of 2.5, 5, 7.5 and 10 minutes and the plate is incubated for 48-72 hours at 37°C. The
phenol coefficient is obtained by dividing the lowest concentration of disinfectant showing
growth after 5minutes but not after 7.5 minutes by the lowest concentration of phenol giving
the same result.

Disadvantages of the Rideal-Walker Test

1) No organic matter is included

2) Microorganism Salmonella typhi may not be appropriate. Since it is not very resistant
to disinfectant and not a common pathogen nowadays and can replaced with
Staphylococcus aureus is used

3) Time allowed for disinfection is short.

4) Used to evaluate phenolic type disinfectants only

Chick–Martin Test (BS 808-1938)

This test is more realistic because the reaction takes place in the presence of a controlled
amount of organic matter in the form of a standardized suspension. In this test mixture of S.
typhi or S. aureus + dried yeast (organic load) is used. The test organism and the incubation
conditions are same as the Rideal-Walker test but the reaction temperature is 30oC. The
exposed time is 30 minutes and duplicate samples are taken. The Chick–Martin coefficient is
the mean of the highest concentration preventing growth (in both tube) and the lowest
concentration preventing growth, divided by the same mean for the disinfectant under test.

Difference between The Rideal Walker Test and Chick–Martin Test

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Surface film Test

Use Dilution Test

In this test film of bacteria dried on a suitable surface with organic substance (protein
substance). The organisms used for this test are S. aureus and P. aeruginosa. Carriers use for
this test (stainless steel cylinders) are meticulously cleaned, sterilized by autoclaving in a
solution of aspargine, cooled and inoculated with a test organism by immersing in one of the
culture suspensions. The cylinders are drained on filter paper, dried at 37oC for 40 minutes,
exposed to the use-dilution of the disinfectant for 10 minutes, and cultured to assess the
survival of the bacteria. Use-dilution test is performed to confirm the efficiency of
disinfectant dilution derived from phenol coefficient test.

Carrier test (Robert Koch Method)

In this test A carrier such as a silk or catgut or a penicylinder (a little stick) is contaminated
by submersion in a liquid culture of the test organism. The carrier is then dried and brought in
contact with the disinfectant for a given exposure time and cultured in a nutrient broth. No
growth of the microbes indicates activity of the disinfectant tested whereas growth indicates a
failing.

Limitation

a) The number of bacteria dried on a carrier is hard to standardize

b) The survival of the bacteria on the carrier during drying is not constant.

c) The disinfectants residues were also carried over to the subculture medium.

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Nephelometric Method

This method is proposed by Needham (1947). Objective of this test is The damaged
and untreated organism may not reproduce at same rate. In this method nephelometer is used
for analysis of survival of Salmonella typhi after exposed to disinfectant. Treated Samples
were incubated for 5hr at 370C and un treated samples (3 no.) were incubated for 5hr at 370C.
After incubation the optical density (opacity) of the culture medium is measured by
nephelometer. The result for the untreated organisms the reference curve is plotted and used
for calculating the no. of microorganism in the treated tube. Advantage of this test is short
incubation time (5hr) and limitation of this test is too sensitive.

Capacity Test or Kelsey-Sykes Test (1969)

Principle of Kelsey-Sykes test is the ability to retain activity in the presence of an

increasing load (microbial) is the capacity of the disinfectant. In a capacity test, the
disinfectant is challenged repeatedly by successive additions of bacterial suspension until its
capacity to kill has been exhausted. Microbes use in this test are S. aureus, E. coli, P.
aeruginosa and Proteus vulgaris. Three successive loads of bacteria are added at 0, 10, and
20 minute interval. Test is carried out a temperature 20oC under clean and dirty conditions.
For sub culturing purpose Calibrated pipette are used. Assessments of the test are done based
on kill or not kill. Disadvantage of this test is the procedure is complicated.

Minimum Inhibitory Concentration (MIC)

The MIC of an antibacterial agent for a particular organism is the minimum /lowest
concentration that just prevents the growth of that micro-organism. MIC can be carried out by
liquid dilution method and by solid dilution method. MIC by solid dilution method is
proposed by Cook in year1954, Dilution of antimicrobial are mixed with molten nutrient agar
and poured into Petri disc, after solidification and dry, a drop of inoculums is added.

The advantages of solid dilution method are:

1) Several different micro-organisms can be tested on each plate

2) Precipitation of medium does not interfere with result as like liquid dilution method
3) Contamination of culture plate can be easily detected because of colony features on solid
media are more distinctive
Conclusion

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants
 Specified strains of S. aureus, P. aeruginosa, P. vulgaris and E. coli are usually
recommended and obtained from specified culture bank like ATCC are to be use for test
purpose. Genetic stability of the microorganism must be controlled in order to prevent the
development of resistant strain. The number of microorganism in the inoculums must be
uniform under different test conditions The medium use of the test must be standardized
because its composition influence the growth rates and development of specific metabolic
pathway Strict aseptic condition must be maintained during culture condition and during
medium preparation. In diffusion assay maximum precision result is obtained when medium
and assay solutions have same pH. Therefore buffers must be chosen carefully. The number
of dead and live cell in the inoculums must be controlled, for depletion of assay substance.

Reference:

J. W. Cooper, C Gunn (1972), Cooper and Gunn's Tutorial Pharmacy. Editor, Sidney James
Carter. 6th Edition, Pitman Medical, London, UK

Analytical Chemistry Pharmaceutical & Biological Analysis

/ Instrumentation
Determination of antimicrobials and disinfectants