Evaluation of Virucidal activity and Preservatives

Evaluation of virucidal activity and preservatives

Intended learning objectives

At the end of this lecture, the student will be able to:

• List the methods for determination of virucidal activity of disinfectants

• Explain the methods for the determination of virucidal activity

• Describe the challenge test for evaluation of preservatives

Evaluation of Virucidal Activity

• The testing of disinfectants for virucidal activity is not easy      

• Viruses are obligate intracellular parasites and are unable in artificial culture media to grow

• They require some other system employing living cells                              

Suggested test viruses include

• Rotavirus, adenovirus, poliovirus, herpes simplex viruses, human immunodeficiency virus, pox viruses and papovavirus

• Appropriate facilities for handling such pathogens are essential

• The virus is grown in an appropriate cell line that is then mixed with water containing an organic load and the disinfectant under test

• After appropriate time, residual viral infectivity is determined using a

         • Tissue culture/plaque assay

         • Other system (e.g. animal host, molecular assay for some specific viral component)

         • Tissue culture or egg inoculation

         • Bacteriophage evaluation method

         • Plaque assays

         • Duck hepatitis B virus (DHVB) method

         • Acceptable animal model

• A reduction of infectivity by a factor of 104 has been regarded as evidence of acceptable virucidal activity

• For viruses which cannot be grown in the laboratory (e.g. hepatitis B), naturally infected cells/tissues must be used


• Such procedures are costly

• Time-consuming

• Must be appropriately controlled to exclude factors such as disinfectant killing of the cell system or test animal

Evaluation of Preservatives

• Preservatives are widely employed in the cosmetic and pharmaceutical industries as well as in a variety of other manufacturing industries

• The inhibitory or bactericidal activity of the chemical to be used as the preservative can be evaluated using an appropriate in vitro test system

• Its continued activity when combined with the other ingredients in the final manufactured product must be established

Evaluation of Preservatives - Challenge test

• The final preserved product is deliberately inoculated with a suitable environmental microorganism

• Fungal, e.g.  candida,  or  bacterial,  e.g.  Staphylococcus  aureus, Escherichia coli, Pseudomonas aeruginosa

• For preparations with a high sucrose content, the osmophilic yeast Zygosaccharomyces rouxii is a consideration

• The subsequent survival (inhibition), death or growth of the inoculum is then assessed using viable count techniques

Evaluation of Mycobactericidal activity

• They are hydrophobic in nature, hence difficult to prepare homogeneous suspensions of mycobacteria

• M.tuberculosis are slow growing pathogenic strains hence a non- pathogenic M.terrae is used as an indicator organism

• General bacteriostatic and bactericidal evaluations are carried out

Evaluation of Sporicidal activity

• Sporicidal activity can be determined against spores in liquid suspension or against spores dried on carriers

• General bacteriostatic and bactericidal evaluations are carried out

• Since they are spores, sufficient germination time must be given considering that damaged spores require even more time for germination

Evaluation of Antifungal activity

• Fungi may be potential pathogens, which occurs as contaminants in pharmaceutical products

• Fungicidal activity – general procedure, suitable culture media

• Fungitatic activity – Solid dilution test Liquid dilution test Gradient – plate technique


Sl. No.

Evaluation of



Virucidal activity

  The test virus us exposed to virucidal agent

  Residual viral infectivity is determined using a

suitable method


Solid Disinfectant

  Dusting the powders onto the surface of seeded

agar plates

  Extent of growth is then observed following





  Bacterial or fungal airborne ‘suspensions’ can be created in a

closed chamber

  Exposed to the disinfectant

  Airborne microbial population is then sampled at regular

intervals using an appropriate



  Challenge test

  Final preserved product is deliberately inoculated with a

suitable environmental microorganism

  subsequent survival (inhibition), death or growth of the

inoculum is then assessed using viable count techniques



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