To determine minimum inhibitory concentration (MIC) and

minimum bactericidal concentration (MBC) of citrus

disinfectant against three microbes I.e, Pseudomonas
aeruginosa, Escherichia coli and Staphylococcus aureus.

Name: Sunaina Gulati

Student Id: 301250792
Course: Microbiology project 1 (BI-207)
Section: 107
Professor name: Mr. Akalate sir
Centennial college
Table of contents:
Introduction Page-
Materials and methods
Results
Discussion
References

List of Tables and Figures:

Table 1- Table with control results of culture, MHB and Citrus disinfectant
Table 2- Table with individual MIC and MBC results.
Table 3- Calculation of % citrus for individual MIC and MBC.
Table 4- Table with class average for all three organisms for MIC and MBC
Table 5- Table for calculation of average % citrus for class MIC and MBC.
 Introduction
With advancements in the technological and scientific era, a lot of new discoveries have been
done and a lot of new microbes have been discovered. Although, not all microbes are helpful to
human beings. Some microbes are human-friendly whereas others can prove to be fatal.
Scientists keep working on ways to eradicate certain harmful microbes. MIC (Minimum
Inhibitory Concentration) test allows to find out the lowest concentration of disinfectant which
can reduce and eliminate the growth of the microbes. When this is done, no growth should be
observed afterward. On the other hand, MBC is a test that decides the lowest concentration of
disinfectant required to kill the harmful microbes over a short period of time. According to the
latest researches, MBC concentrations are found to be two folds to four folds more than MIC
concentrations (Qlabs,2019). The clear difference between the two tests is that MIC will restrict
or slow down the growth of microbes whereas MBC will determine the concentrations required
to kill the microbe. ISA (Iron Sulphate Agar) and MHB (Mueller Hinton Broth) are used
generally for these types of experiments.
In this experiment, these tests were conducted on 3 types of organisms: E.coli, S. aureus and
P.aeruginosa.
Pseudomonas is mostly present everywhere and usually grows in moist environments. The most
common pathogens in humans are P. aeruginosa but infections may occur due to P. putida, P.
acidovorans or P. paucimobilis. P. aeruginosa is mostly present in the axilla and anogenital areas
of human skin but very rarely in stools. The organism is also found in our surroundings in
hospitals, in sinks, antiseptic solutions and urine receptacles. Transmission of the microbe can
easily occur from health care practitioners to patients. It can cause bacteremia, skin infections
and is commonly known to cause respiratory infections as well. (Bush, 2022).

S. aureus is responsible for many clinical diseases. Infections caused by the microbe are common
in both hospital-acquired and community- acquired settings. S. aureus is the causative agent of
many bacterial infections in humans, such as bacteremia, infective endocarditis, osteomyelitis
meningitis and urinary tract infections (Unakal. C, 2022).
Most of the E. coli strains live in the digestive and intestinal tracts of human beings. However,
not all E.coli strains cause disease or infections in humans. Some E.coli strains are a boon to
human health whereas others are harmful. Infections can be caused by eating undercooked meat
products, drinking unpasteurized milk or water and many other reasons. The types of infections
caused by E, coli are gastroenteritis, UTI(Urinary tract infections), Pneumonia and Meningitis in
newborns and blood infection.
It is extremely difficult to determine the efficacy of citrus for microbes, as it varies from one
disinfectant to another. There are numerous ways by which citrus disinfectant acts on microbes,
such as growth restriction and lethal action (Wheat,2001). The properties of disinfectant depends
on their structure and size and especially on the length of the alkyl chain for both positive and
negative strains. There is a wide range of application from manufacturing to scientific purposes,
also used for decontamination of apparatus and medical devices. Disinfectant is usually found in
the category of acidic and alkaline compounds. They are less effective against gram-negative
bacteria in comparison to gram-positive bacteria. They have no activity against bacterial spores
and viruses. The bacterial wall intends stiffness which is again different for gram-positive and
gram-negative bacteria (What is disinfectants and mode of action of disinfection solution? , n.d.).
The objective of the experiment is to test Citrus disinfectant against Pseudomonas aeruginosa,
E.coli and S. aureus. To determine MBC(Minimum bactericidal concentration) and MIC
(Minimum inhibitory concentration) of the disinfectant on these three pathogens.

Materials and Methods:

MIC procedure:

Concentration of citrus was according to the different bacteria’s. For E. coli the amount of citrus
added was 1.6%, for S. aureus it was 0.8% and for P. aeruginosa the concentration added was
6.4%. The citrus stock solution was provided in a capped bottle. 9 ssMHB and 1 dsMHB tubes
containing 2 ml of MHB solution were labelled ½,¼/1/8......1/1028. The tube dilutions were
done. The last tube’s 2 ml of aliquot was discarded. 2 ml of bacterial culture was added to each
tube using pasteur pipette. Positive controls of the 3 different organisms were checked in
different tubes to check cultural viability of the media. One ds MHB and ss MHB tube was used
as negative controls to check the sterile condition of media. All of this was incubated at 35
degree celsius at 24 hours to observe the results.
MBC procedure:

For obtaining the MBC results, MHA agar (Mueller-Hinton Agar) was used. The MHA plate was
divided into 5 sections using a scale, in relation to MIC results from lower to higher dilution of
MHB test tubes. Later on, streaking was done in the five sections using the five tubes of MHB.
In the end, the plate was incubated at 35 degree celsius for 24 h. The results were recorded after
24 hours.

Results:

Table 1: Control results of MHB, Citrus disinfectant and Culture.

MHB Zero control (only Positive control(2 Negative control (1

MHB) drops of culture) ml of citrus)
Single Strand -ve +ve +ve
Double strand -ve +ve +ve

Table 2: Table with individual MIC and MBC results:

Table 4: MIC class result of Citrus disinfectant against P. aeruginosa. E. coli and S. aureus grown in
MHB incubated at 35 degree celsius at 24 hours.

Microorganis Student MIC MIC MIC Class MBC MBC MBC

m Names Citrus% Average Citrus % Average
Citrus% Citrus%
E.coli (Citrus Fatemeh 1/256 0.00625 0.00625% 1/128 0.0125 0.0125%
stock) 1.6 Yasaman 1/256 0.00625 1/128 0.0125
Babita 1/256 0.00625 1/128 0.0125
Kiran 1/256 0.00625 1/128 0.0125
Sunaina 1/256 0.00625 1/128 0.0125
Gurdas 1/256 0.00625 1/128 0.0125
Staphylococc Jasmin 1/128 0.00625 0.00469% 1/64 0.0125 0.009375
us aureus Jessi 1/256 0.00313 1/128 0.00625 %
(Citrus stock): 1/128 0.00625
0.8)
Alexia 1/256 0.00313 1/128 0.00625
 Preksha 1/256 0.00313 1/128 0.00625
Gunmeet 1/128 0.00625 1//64 0.0125
Yuvraj 1/128 0.00625 1/64 0.0125
Pseudomonas Sakshi 1/64 0.1 0.1% 1/32 0.2 0.2%
Aeruginosa Sourabh 1/64 0.1 1/32 0.2
(Citrus stock: Hirangi 1/64 0.1 1/32 0.2
6.4) Brijal 1/64 0.1 1/32 0.2

Discussion:
The Staphylococcus aureus is more susceptible to the disinfectant in citrus. The amount of citrus
needed to stop bacterial growth is just 0.009375%. Following the Susceptibility of S. aureus is E.
coli, 0.0125% of citrus is required to prevent this bacteria's development, whereas P.Aeruginosa
is more tolerant to citrus concentrations. Higher proportion of Citrus is necessary to prevent the
growth of this bacteria, therefore less of it demonstrates reduced susceptibility in respect to other
microorganisms. 0.2% of citrus is required to prevent the growth of P.aeruginosa.

When personal data is compared with the students who were assigned same organism as mine, it
is clearly visible that % of MIC to control the inhibition of S. aureus is same as percentage of
Babita, Kiran, Jessi and Jasmine. 0.009375% citrus is required to hinder the growth of S. aureus.
My personal MIC results are 4.69*10^3 for S. aureus as the concentration of S. aureus was 0.8
(citrus stock). Whereas the MBC results amount to 9.3*10^3. The concetration for MIC was
1/256 whereas for MBC it was 1/128.
MIC for my organism was 0.009375 which illustrates that this concentration of citrus is requied
to inhibit the growth of the S. aureus bacteria. The percentage of E. coli recorded is 0.0125%
which means this much citrus is required to hinder the growth of E. coli whereas the highest
concentration of citrus required is for P.aeruginosa which amounts to 0.2% of citrus disinfectant.
The effects of citrus disinfectant is more for gram-positive bacteria because of the thick cell-wall
made up of peptidoglycan ,on the other hand gram-negative bacteria has less effects due to citrus
disinfectant when compared to gram-positive bacteria.
References:
Qlabs. (2019, October 14). Minimum Inhibitory (MIC) and Minimum Bactericidal Concentration
(MBC) Evaluations as R&D Tools. Retrieved from https://www.qlaboratories.com/

What is Disinfectants and mode of action of disinfection solution? (n.d.). Retrieved from
https://www.pharmawareness.com/: https://www.pharmawareness.com/ what-isdisinfectantsand-
mode-of-action-of-disinfection-solution/

Bush, L. M., & Vazquez-Pertejo, M. T. (2022, August 4). Pseudomonas and Related Infections.

Merck Manuals Professional Edition.

https://www.merckmanuals.com/en-ca/professional/infectious-diseases/gram-negative-bacilli/

pseudomonas-and-related-infections