Microbiology Practical: Disinfectants, Antibiotics, and Fermentation

This microbiology practical assignment explores the effectiveness of disinfectants, the production of antibiotics, and fermentation processes. It begins by examining the properties of Virkon and DISINFX D-125, comparing their efficacy against various microorganisms using the Kelsey Sykes test and tube dilution methods. The experiment investigates the optimal concentrations of disinfectants A, B, and C. Additionally, the assignment assesses penicillin production through zone of inhibition measurements and analyzes citric acid production via agitated and static methods, including absorbance readings and standard curves. The results are analyzed using tube dilution and gradient plate methods, discussing the accuracy and advantages of each. The discussion section compares the results with published studies and highlights the importance of agitation in citric acid production. The assignment offers insights into the practical application of microbiology techniques and the evaluation of antimicrobial agents.

Microbiology
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Introduction:
Peroxygenic acid is the component of Vikron disinfectant. Virkon disinfectant can be used
agaisnt multiple microorganisms like viruses, some fungi, and bacteria. Virkon acts by
carrying out oxidation of proteins and constituents of cell protoplasm. It leads to the enzyme
inhibition and damage to the cell wall integrity. DISINFX D-125 is another multipurpose
disinfectant which can kill approximately 135 microorganisms. Virkon has the ability to kill
20 microorganisms. Both of these disinfectants can kill microorganisms with contact time of
10 minutes. Constituents of Virkon are potassium peroxymonosulfate and sodium chloride-
based disinfectant. Constituent of DISNFX D-125 is a quaternary ammonium disinfectant
(Moslehifard et al., 2015). Virkon was experimentally compared with the sodium
hypochloride in dental casts contaminated with Staphylococcus aureus, Pseudomonas
aeruginosa, Bacillus subtilis and Candida albicans. It was evident that 1 % Virkon was
required for killing S. aureus, P aeruginosa, and Candida albicans. 3 % Virkon soluion was
required for killing B. subtilis. 1 % Virkon solution was more effective as compared to the
0.525 % sodium hypochlorite solution in killing S. aureus, P. aeruginosa, and C. albicans.
However, there was no difference between 1 % Virkon and 0.525 % sodium hypochlorite in
killing B. subtilis. However based on low toxicity and good environmental compatibility,
Virkon is considered as better as compared to the sodium hypochlorite (Moslehifard et al.,
2015). Penicillin G (Benzyl penicillin) can be obtained from the Penicillium chrysogenum by
the fermentation process by growing on the supplemented Corn Steep Liquor. Citric acid can
be obtained by the fermentation of Aspergillus niger and yield of citric acid can be increased
by using carbohydrates rich mollases.
Results:
Kelsey Sykes test for Disinfectants A, B and C:
Initially, three different disinfectants A (50 %), B (100 %) and C (50 %) were used against P.
aeruginosa. All these disinfectants proved effective in reducing microbial growth. A (50 %
and C (50 %) showed no microorganisms in all the three series of testing. B (100 %) showed
no microorganisms series 1 and 3 tests, while in series 2, one tube exhibited positive growth
for microorganisms.
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Table 1 : Dsinfectant A, B and C in Kelsey Sykes Test.
1 2 3 4 5 6 7 8
Test
no.
Date Disinfectant
concentration
CFU/ml
test
organism
Organism growth in recovery
medium after specified time
Result
Series 1 Series 2 Series 3
1 A (50% ) 3.3x108 - - - - Pass
2 B (100%) 3.3x108 - - + - - - - - Pass
3 C ( 50% ) 3.3x108 - - - - - - Pass
Kelsey Sykes test for range of disinfectants A, B and C:
For the evaluation of optimum concentration of disinfectants, A, B and C to eradicate all the
microorganisms, these disinfectants were diluted. Disinfectant A (0.5 and 0.6 %) showed
microorganisms survival in all the tubes. Disinfectant B (1.0 %) didn’t showed
microorganisms survival in series 1, while it showed microorganisms survival in series 2 and
3. Disinfectant B (1.2 %) exhibited no growth in all the tubes of series 1 and 3 tubes with no
growth in series 2 and all tubes with growth in series 3. Disinfectant C (1.5 %) didn’t showed
any microbial survival in series 1 and 2, however it showed microbial survival in 2 tubes in
series 3. Disinfectant C (1.8 %) is most effective among all dilutions because it demonstrated
no microbial survival in all the three series.
Table 2: Dilutions of disinfectant A, B and C in Kelsey Sykes Test.
1 2 3 4 5 6 7 8
Test
no.
Date Disinfectant
conc.
Number of
CFU/ml
Organism growth in recovery
medium after specified time
Results
Series 1 Series 2 Series 3
1
2
3
May
1
May
1
May
1
A (0.5 %)
B (1.0%)
C (1.5%)
4.8 × 108
4.8 × 108
4.8 × 108
+++++
- - - - -
- - - - -
+++++
+++++
- - - - -
+++++
+++++
- - ++ -
FAIL
FAIL
PASS
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4
5
6
May
3
May
3
May
3
A (0.6 %)
B (1.2%)
C (1.8%)
3.6 × 108
3.6 × 108
3.6 × 108
+++++
- - - - -
- - - - -
+++++
- - + -+
- - - - -
+++++
+++++
- - - - -
FAIL
PASS
PASS
Tube Dilution MLCs :
Virkon exhibited microbial survival for E. coli at 0.2 % and for S. aureus, and P. aeruginosa
at 0.1 % concentration, however for P. vulgaris it didn’t showed microbial survival at 0.1 %.
Table 3 : Tube Dilution method MLCs for Virkon.
Tube Number
1 2 3 4 5 6 7 8
Virkon conc. % v/v 0% 0.1% 0.2% 0.4% 0.8% 1.2% 1.6% 2%
Growth of test organisms
E. coli + + + - - - - -
S. aureus + + - - - - - -
P. aeruginosa + + - - - - - -
P. vulgaris + - - - - - - -
Gradient plate method:
Virkon produced maximum inhibitory concentration (MIC) for E. coli, S. aureus, P.
aeuriginosa and P. vulgaris of 1.3, 0.88, 1.46 and 1.4 % v/v respectively.
Table 4 : MIC for gradient plate method for Virkon
Organism MIC (%v/v)
E. coli 1.3
S. aureus 0.88
P. aeuriginosa 1.46
P. vulgaris 1.4
Zone of inhibition measurements for penicillin assay :
Static method was found to be more effective in penicillin assay as compared to agitated
method because estimated average zone of inhibition was more in static method as compared
to the agitated method.
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Table 5: Zone of inhibition for penicillin assay.
Agitated in cm Static in cm
0 1.86
1.2 1.2
1.33 1.35
1.51 1.24
1.66 1.30
Graph 1: Zone of inhibition for penicillin assay.
Agitated method Static method
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Zone of inhibition for penicillin assay
Citric acid absorbance readings:
Agitated and static method produced absorbance of 0.768 and 0.307 at 425 nm respectively
for citric acid.
Table 6: Citric acid absorbance at 425 nm.
Absorbance of 425nm
Agitated 0.768
static 0.307
100μg/ml 0.060
200μg/ml 0.077
400μg/ml 0.127
500μg/ml 0.157
Graph 2 : Standard curve citric acid
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50 100 150 200 250 300 350 400 450 500 550
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
f(x) = 0.000251 x + 0.0282
R² = 0.97995022554052
Citric acid
Amount of citric acid produced :
Y = 0.0003x + 0.0282
Amount of citric acid produced by static method :
Y = 0.0003x + 0.0282
0.307 = 0.0003x + 0.0282
0.307 – 0.0282 = 0.0003x
0.0003x = 0.2788
x = 0.2788/0.0003 = 929.3
Amount of citric acid produced by static method = 929.3 μg/ml
Amount of citric acid produced by agitated method :
Y = 0.0003x + 0.0282
0.768 = 0.0003x + 0.0282
0.768 – 0.0282 = 0.0003x
0.7398 = 0.0003x
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x = 0.7398/0.0003 = 2466
Amount of citric acid produced by agitated method = 2466 μg/ml
Graph 3 : Citric acid production
Satic method Agitated method
0
500
1000
1500
2000
2500
3000
Citric acid prodcution
Discussion:
Tube dilution method is based on the serial dilution of disinfectant in a suitable medium
which contains microorganisms. Gradient plate method is based on the application of agar
plate with gradual reduction in the concentration of the disinfectant. In this method, gradient
can be achieved by keeping the antibiotic in the top agar layer, hence its diffusion from top
layer can occur. This diffusion can result in the concentration gradient. From the literature, it
is evident that there is more accuracy in gradient plate method in comparison to the plate
dilution method (Whittet et al., 2013). It is also evident that results obtained through
employing gradient plate method more closely resembles with the spectrophotometric
method. In addition to the gradient plate method has several advantages like its procedure is
simple to perform and it is more economical as compared to the tube dilution method (Block,
2001). Tube dilution method and gradient plate method employed completely varied results
for Virkon. In tube dilution method, Virkon demonstrated more effect against P. vulgaris and
it showed less effect against E. coli. In gradient plate method, Virkon produced more effect
against S. aureus and less effect against P. aeuriginosa. In the literature it has been
established that gradient plate method results should be considered more accurate. In this
practical, Vikron proved more effective against S. aureus and least effective against P.
aeuriginosa. These results should be considered valid results for Vikron. In published studies,
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Vikron was evaluated by suspension test and carrier test instead of gradient plate and tube
dilution method. Hence, it would be difficult to compare results of Vikron from the current
experiment with the previously published results. In published studies, Vikron was tested
against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus
hirae and Mycobacterium smegmatis. Published studies stated that 1 % Virkon is effective
against mere vegetative bacteria, yeasts and viruses (Hernndezf et al., 2000; Van Rooij et al.,
2017).
Kelsey Sykes test can be used to assess the effectiveness of disinfectants to kill the
microorganisms. Based on the presence and absence of microorganism in each tube, results
can be depicted as pass or fail, instead of depicting results as coefficients. If two or more
negative cultures are evident in particular dilution of disinfectant, it should be considered as
pass in the disinfectant test. If first and second challenge with the disinfectant exhibits
negative growth for the microorganism, this particular dilution should be considered as pass.
If third challenge exhibits negative growth for microorganism, this test should not be
considered as pass for that particular dilution of the disinfectant (Kapoor et al., 1998). In the
initial phase of the experiment higher concentrations of the disinfectants like A (50 %), B
(100 %) and C (50 %) were used. All the tested disinfectants at higher concentrations
exhibited negative growth for P. aeruginosa. Hence, these disinfectants at higher
concentrations proved pass for P. aeruginosa. Further step was designed to decide optimum
concentration of disinfectants to kill P. aeruginosa. Hence, these disinfectants were further
diluted and used against P. aeruginosa. Disinfectant A used below 1 % concentration, hence
it exhibited positive results for P. aeruginosa and it proved to be fail. Disinfectants 1 and 2
were used above 1 % concentration, hence these produced negative results for P. aeruginosa
and proved pass. Among dilutions of A, B and C disinfectants, dilution for disinfectant
proved more effective against P. aeruginosa because at higher concentrations none of the
tubes exhibited positive results for P. aeruginosa (Reybrouck, 1992; Singh et al., 2012).
Zone of inhibition of B. subtilis was measured by employing agar diffusion assay to assess
amount of production of penicillin. Absorbance at 425 nm was used as estimate to determine
amount of citric acid production. For estimating penicillin and citric acid production, two
methods like static and agitated methods were used (Smith et al., 1990). Penicillin production
was found to be more in static method as compared to the agitated method. Production of
citric by agitated method was more as compared to the static method. Agitation process is
mainly responsible for the more production of citric acid by agitation method. Agitation can
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increase shear stress on cell wall and aeration in the medium, which would be helpful in
improving secretion of citric acid. Improvement in the amount of dissolved oxygen and
availability of oxygen in the medium can be improved by agitation and aeration. In the
published literature, it has been established that agitation and aeration can improve amount of
citric acid secretion from A. niger grown on various substrates (Kamzolova et al., 2003; Max
et al., 2010).
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References:
Block, S. S. (2001). Disinfection, Sterilization, and Preservation. Lippincott Williams &
Wilkins.
Hernndezf, A., Matas, E., Martró, L., Martı́n, M., and Ausina, V. (2000). Assessment of in-
vitro efficacy of 1% Virkon® against bacteria, fungi, viruses and spores by means of AFNOR
guidelines. Journal of Hospital Infection, 46(3), pp. 203-209.
Moslehifard, E., Lotfipour, F., Robati, Anaraki. M., Shafee, E., Tamjid-Shabestari, S., and
Ghaffari, T. (2015). Efficacy of Disinfection of Dental Stone Casts: Virkon versus Sodium
Hypochlorite. Journal of Dentistry, 2(3), pp. 206-15.
Kamzolova, S.V., Shishkanova, N.V., Morgunov, I.G., and Finogenova, T.V. (2003). Oxygen
requirements for growth and citric acid production of Yarrowia lipolytica. FEMS Yeast
Research, 3, pp. 217-222.
Kapoor, H., Murlidhar, S., Jais, M., Thakur, R., and Aggarwal, P. (1998). Evaluation of
efficacy of Dettol-H in hospital use. Journal of Communicable Diseases, 30(3), pp. 167-70.
Max, B., Salgado, J. M., Rodríguez, N., Cortés, S., et al. (2010). Biotechnological production
of citric acid. Brazilian Journal of Microbiology, 41(4),
http://dx.doi.org/10.1590/S1517-83822010000400005
Reybrouck, G. (1992). The assessment of the bactericidal activity of surface disinfectants. V.
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Singh, M., Sharma, R., Gupta, P. K, Rana, J. K., et al. (2012). Comparative efficacy
evaluation of disinfectants routinely used in hospital practice: India. Indian Journal of
Critical Care Medicine, 16(3), pp. 123–129.
Smith, J.J., Lilly, M.D., and Fox, R.I. (1990). The effect of agitation on the morphology and
penicillin production of Penicillium chrysogenum. Biotechnology Engineering, 35, pp. 1011-
1023.
Van Rooij, P., Pasmans, F., Coen, Y., and Martel, A. (2017). Efficacy of chemical
disinfectants for the containment of the salamander chytrid fungus Batrachochytrium
salamandrivorans. PLoS One, 12(10):e0186269. doi: 10.1371/journal.pone.0186269.
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Pharmaceutical Monographs. Elsevier.
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