Investigating the correlation of antimicrobial effect of Ocimum tenuiflorum (Tulsi) on the growth of Saccharomyces cerevisiae (yeast)

Being an avid nature lover, going back or tracing back to nature has always been something that fascinates me and I am passionate about. This time of pandemic has especially made it clear that depending on the nature for our resources and sustainable development is inevitable. “Tulsi”- a common Indian herb is a popular plant in any common Hindu Indian household family. I was under the impression that it is just another plant in my garden in the balcony with an additional religious value until I saw my grandmother recommending me to eat a tincture made out of the leaf of this plant and honey to cure my cough and cold. As an age-old tradition, this plant is acclaimed to have potential medicinal benefits. However, what bothered me was that are they equally or more effective than the common allopathic drugs that we consume. How would one know the correct dosage amount of the herbs that someone would need to consume? Does it become more effective if consumed in larger quantities? To understand all these, I did some further research published in some acclaimed journals on medicinal biology and clinical biology and all of them indicated that this plant has a pronounced antimicrobial effect on both gram positive and gram- negative bacteria. Multiple evidences collected from various experimental data shows that the plant contains essential oils and can even boost the immunity system of a human body which can be accounted for its antimicrobial in nature. Now, the objective was to realize that how this antimicrobial nature and action would depend on the percentage concentration of the extract used. Thus, I decided to carry out my Chemistry Extended Essay on this research question.

“Tulsi” or “Indian Tulsi” is one of the most common Indian medicinal herb. Tulsi is considered as a “adaptogen” because of it’s ability to allow the body to withstand stress and also aids in the process of “homeostasis”. It belongs to the biological genus of – “Lamiaceae”. The scientific name of the plant is – “Ocimum tenuiflorum”. This plant has been found to show both antimicrobial and antibacterial activities. Both in vitro and in vivo studies support this. This extract has been reported to have inhibitory effects towards the growth of bacteria, viruses and fungus. Tulsi extract can also prevents the body from the toxic action of “pollutant heavy metals” which includes but is not limited to Chromium, Iron, Lead. Moreover, it has also shown anti- oxidant and can thus consumes or destroys radicals which eventually protects the cell from radioactive damages. It also promotes the secretion of the enzyme “Cytochrome P450” which allows the deactivation of toxic chemicals in the liver. The medicinal values of Tulsi extract is mainly attributed to the presence of “essential oils – Eugenol” within the plant body. However, apart from this, there are other components which can also behave as potential phytochemicals with antibacterial and antimicrobial property. This list includes but not limited to oleanolic acid, “β- caryophyllene”, “Carvacrol” and many more. This compounds imparts anti-oxidant, anti-inflammatory and even pain reducing abilities in the plant extract.

Saccharomyces cerevisiae is a eukaryotic single celled micro-organism. Alcoholic fermentation is a process in which sugar uses an enzyme secreted by yeast to produce ethanol and carbon dioxide. Based on the manufacturing process, yeast are categorized into different categories – baker’s yeast, active dry yeast, instant yeast and many more. This categorization depends on the steps followed and the percentage of water in the medium or culture where they are grown. Eventually, this alters their dry mass as well. Yeast is “70% water, 50% protein, 10 % fat and ash and 40 % carbohydrate”. Though yeast is available in various forms, the microorganism when taken in solid state displays the maximum synthesis of protein which is a fundamental requirement to monitor its growth using spectrophotometry.

The growth of yeast and microbes can be monitored in various ways – measuring the turbidity, measuring absorbance or optical density at 420 nm, plate count, measuring dry weight and many more. In this investigation, the absorbance of the yeast solution was recorded at 420 nm. A glass cuvette was used and a UV-Visible spectrophotometer was used. As per literature, absorbance of 1.000 ± 0.001 abs at 420 nm, the cell density recorded is 1.00 × 10⁸ cells in 1.00 cm³ of the solution.

Literature review:

The literature review refers to a research paper titled – “Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study” by Pooja Agarwal in the Indian Journal of Dental Research. Tulsi extract of 15 different concentration was applied against the Streptococcus mutans. To compare the antimicrobial effect, the zone of inhibition was recorded. The result reported that the Tulsi extract displays antimicrobial action and the maximum antimicrobial was at 40.00 % concentration with a zone of inhibition of 22 mm.

There is no correlation between the antimicrobial effect of the neutral, alcoholic extracts of Tulsi leaves and the percent strength of the extract used. Any correlation detected is an outcome of a random error.

Alternate hypotheses:

There is a correlation between the antimicrobial effect of the neutral, alcoholic extracts of Tulsi leaves and the percent strength of the extract used. Any correlation detected is an outcome of a random error.

Percent strength of the neutral, alcoholic extract of Tulsi: The extract of Tulsi leaves was prepared by washing the leaves under running stream of water, turned into a paste using a mortar and pestle and filtered out to get an extract free from lumps. Phosphate buffer was added to the extract to make it neutral. A pH meter was used to check the pH level. The pH was maintained at a level of 7.00. The total volume was kept constant at 10.00 cm³ by adding requisite volume of isopropanol. The percentage strength used are – 10.00 %, 20.00%, 30.00%, 40.00% and 50.00%. For, a 10.00% extract, 1.00 ± 0.05 cm³ of Tulsi extract was mixed with phosphate buffer and isopropanol till the final volume is 10.00 ± 0.05 cm³. As reported in the literature, the lowest percentage strengths of an antimicrobial solutions is 30.00%7. Thus, the range of percentage strength of the antimicrobial solution has been varied from 10.00% to 50.00%. The independent variable is a continuous set of variable as each values differ from the other one by 10.00 units and that allows a scope of more accurate and precise comparison.

Percentage inhibition of growth of yeast Yeast is a microorganism. It has certain organic molecules as a part of it’s cellular composition which displays maximum absorbance in the UV region at 420 nm⁸. Thus, higher the absorbance more the amount of yeast grown. A UV-Visible spectrophotometer was used to measure the absorbance. The percentage inhibition of growth of yeast was measured according to the formula given below: Percentage inhibition =

Temperature: The antimicrobial effect of Tulsi is mainly due to the phytochemical eucalyptol along with other essential oils which acts as an enzyme to inhibit the reproduction of yeast and thereby retards the cell density and growth of it9. This is an enzymatic process and is dependent on temperature. Variation of temperature, specifically keeping the temperature at a low value or at a high value may cause the enzyme to either react very slow or get denatured respectively. Thus, a constant temperature of 40.0 ± 0.5°C was maintained using a hot water bath as the optimum temperature of Tulsi extract for pharmacological effect has been reported to be at 40.0 °C10. A laboratory thermometer was used to control and monitor the temperature.

pH: Like temperature, pH plays a major role in controlling the antimicrobial action of an enzyme. As reported in medical researches, the antimicrobial effect of natural extracts especially essential oils and tarpenoids have been reported to be at the neutral point; pH of 7.00^11. Thus, phosphate buffer was used to control the pH and maintain a constant pH of 7.00 ± 0.01 in all cases. A pH meter was used to monitor the pH.

Mass of yeast: Yeast is acting as a substrate in this investigation. More the mass of yeast used, lesser the action of definite volume of extract or concentration of antimicrobial solution. Moreover, the absorbance of the yeast solution has been used as a quantitative tool to monitor the growth of yeast. More the concentration of yeast used, higher the absorbance recorded as per Beer-Lambert law. Thus, in all cases the same mass of yeast was added and the value was kept constant at 1.00 ± 0.01g. A digital mass balance was used for this.

Part-A: Sterilization of apparatus

A day prior to the experiment, 7 conical flasks were autoclaved which later was kept in a laminar air flow in order to maintain sterilization to ensure no contamination.

Part-B: Preparation of Tulsi extract

• 10-12 Tulsi leaves from the plant was plucked and washed gently under running water to ensure any sand or dust particles were cleaned.
• Using a pestle and mortar, the leaves were crushed in order to make it into anextract and the mixture was filtered into a beaker making sure the solutionobtained is lump free.
• A 100 cm³ of glass beaker was taken.
• A 10.00 cm³ graduated pipette was taken to transfer 1.00 ± 0.05 cm³ of Tulsi
  extract into a beaker.
• A pH meter was taken and dipped inside the beaker.
• A dropper was used to add phosphate buffer unless the pH of the solution was
  recorded as 7.00.
• A dropper was used to add isopropanol until the volume of the solution was till
  10.00 cm^3.
• Steps 1-7 were repeated using 2.00 ± 0.05 cm^3, 3.00 ± 0.05 cm^3, 4.00 ± 0.05
  cm³ and 5.00 ± 0.05 cm³ of Tulsi extracts to prepare 20.00 %, 30.00%,
  40.00% and 50.00%.

Part-C: Measuring the growth of yeast

• A 100 cm³ conical flask was taken and placed on a water bath.
• The temperature of the water bath was set at 30.0 ± 0.5°C. A thermometer was used to check the temperature.
• A watch glass was placed on the digital mass balance and the reading of the
  balance was adjusted to 0.00 ± 0.01 g.
• Using a spatula, 1.00 ± 0.01 g of yeast powder was added to the watch glass.
• The exact mass of yeast weighed was transferred from the watch glass to the
  conical flask.
• 10.00 ± 0.05 cm³ of distilled water was added to the yeast in the conical flask
  using a graduated pipette.
• Using a graduated pipette, 5.00 ± 0.05 cm³ of 10.00 % Tulsi extract was added
  to the same conical flask.
• The stop-watch was started.
• 5.00 ± 0.05 cm³ of phosphate buffer and 5.00 ± 0.05 cm³ of isopropanol was
  added to a test tube using a graduated pipette.
• Using a graduated pipette, 1.00 ± 0.05 cm³ of this mixture of buffer and
  alcohol was transferred to a cuvette.
• The cuvette was inserted into the spectrophotometer, the wavelength was
  adjusted to 420 nm and the absorbance of the sample was adjusted to 0.000±
  0.001 abs to calibrate the spectrophotometer.
• As soon as the stop-watch reads 30.00 minutes, a pipette was used to
  transfer 1.00 ± 0.05 cm³ of the yeast solution into a cuvette.
• The cuvette was inserted into the spectrophotometer, the wavelength was
  adjusted to 420 nm and the absorbance was recorded.
• Steps 1-13 were repeated for four more times.
• Steps 1-15 were repeated with other solutions of Tulsi extract. For blank
  solution (0.00 % Tulsi extract), 5.00 ± 0.05 cm³ of distilled water was used
  instead of Tulsi extract.

• A laboratory coat was worn at all times.
• The hands were covered with gloves and face with mask as exposure to yeast
  may cause irritations.
• Hair was always tied up.
• Any eatables were not allowed inside the laboratory.

After usage, the unused Tulsi extract was used to prepare herbal tea in home instead of wasting them. The yeast solutions were disposed into a safety disposal bin and enough alcohol was added before the disposal to stop the growth of yeast.

Tulsi is a plant of immense use. It has potential medicinal use. Yeast is also an
important ingredient for various baked food products and moreover it is a
microorganism. So, wasting them is unethical. Thus, the investigation has been
designed in a way to minimize their uses and use the lowest possible amount.

Sample calculation:

For 0.00% Tulsi extract,

Mean absorbance = \(\frac{sum\ of\ five\ trials}{5}=\frac{1.750+1.752+1.748+1.752+1.752}{5}\) = 1.751 ± 0.001 

For 10.00 % Tulsi extract, 

Percentage inhibition of growth of yeast

\(=\frac{1.751-1.650}{1.51}\) × 100 = 5.757 %

Impact of uncertainty

For 10.00 % Tulsi extract,

Absorbance at 420 nm for 0.00 % Tulsi extract = 1.751 ± 0.001 abs

Absorbance at 420 nm for 10.00 % Tulsi extract = 1.650 ± 0.001 abs

Difference of absorbance = (1.751 ± 0.001) – (1.650 ± 0.001 )

= 0.101 ± (0.001 + 0.001) abs = 0.101 ± 0.002 abs

Percentage error in percentage inhibition of growth

\(=\big(\frac{0.002} {0.101}+{\frac{0.001}{1.650}\big)}\) × 100 = ±2.04

As there is only one dependent variable and only one category of the dependent variable, the only inferential statistical test that can be used here is the Regression Test.

\({\sum} X = 150\)

\({\sum} Y= 170.835\)

\({\sum} XY= 6181.820 \)

\({\sum} X^2= 5500.000\)

\({\sum} Y^2= 7104.625\)

\(r=\frac{(n×\sum XY)-(\sum X- \sum Y )}{\sqrt{[n\sum X^2}-(\sum X)^2][\sqrt{[n\sum Y^2}-(\sum Y)^2]}\)

\(=\frac{(5×6181.820)-(150×170.835)}{\sqrt{[5×5500.000-(150)^2][5×7104.625-(170.835)^2}}=0.9876\)

The value of regression coefficient is 0.9876. This indicates that there is a strong correlation between the independent and the dependent variable. The percent strength of the Tulsi extract used and the percentage inhibition of the growth of yeast are strongly correlated with each other. Thus, the null hypotheses may be discarded and the alternate hypotheses must be accepted.

How does the antimicrobial effect of neutral alcoholic extract of Ocimum tenuiflorum (Tulsi leaves), on the growth of Saccharomyces cerevisiae (yeast), measured in terms of percentage inhibition of growth of yeast depends on the percentage strength of the Tulsi extract used – 10%, 20%, 30%, 40% and 50%, determined spectrophotometrically?

• The percentage inhibition of growth of yeast increases from 5.76 % to 49.34 %as the percentage strength of the Tulsi extract used increases from 10.00% to 50.00%.
• In simple words, more the concentration of the Tulsi extract used slower or lesser
  is the growth of the yeast and thus more the antimicrobial effect.
• The use of Tulsi extract has a negative impact on the growth of yeast and thus
  the cell density or the population of yeast decreases which eventually decreases
  the absorbance as well.
• There is a strong correlation between the independent and the dependent
  variable. The percent strength of the Tulsi extract used and the percentage
  inhibition of the growth of yeast are strongly correlated with each other. Thus,
  the null hypotheses may be discarded and the alternate hypotheses must be
  accepted.
• This study was conducted to delineate if Tulsi extract has an antimicrobial effect in terms of the inhibition of the growth of the yeast or not. Moreover, it was also studied that how the antimicrobial effect would vary with the percentage concentration of the Tulsi extract used. The side effects caused by chemotherapeutics has made it essential for medical researches to find suitable biological and phytochemical alternatives. There has been multiple researches on the antimicrobial action of Tulsi extract. In most of the cases, it has been reported that the antimicrobial action of Tulsi is due to the presence of essential oil that it secretes12. Though there are various other mechanisms proposed to establish the antimicrobial action of this plant. One of the most popular review is about the fact that the plant boosts the secretion of T helper cells which enables to host to fight against the infection13. This effect of Tulsi extract is scientifically attributed as the “immunomodulatory effect”. However, in most of the cases, the idea is that Tulsi extracts releases chemicals that can behave as an enzyme inhibitors for the enzymes which promotes the growth of the microbes.

• The independent variable – percentage strength of Tulsi extract solution is in a continuous range. This allows the variable to be in a systematic range and allows more accurate and fair comparison.
• For the Tulsi extract, the same plant and the same part of the plant has been used.
• The experimental design is simple and easy. The procedure can be easily reproduced and the same data can be collected.
•  Apart from the graphical analysis, the statistical analysis has been carried out to establish a relationship between the independent and the dependent variable.
• The value of percentage uncertainty is not significant enough and that indicates high level of accuracy in the data collected.

• The major source of systematic error in this investigation is the nature and source of the Tulsi plant used. The anti-microbial action of the plant is due to the presence of essential oil in the Tulsi plant. The concentration of the oil is definitely not the same in all plants and precisely in all parts of the plant. Thus, variations in the sample used would interfere with the composition of the Tulsi extract used. For an accurate investigation, it is essential to use the Tulsi extract that has the same composition. To do this, it is necessary to use genetically engineered Tulsi plants and leaves.
• There are multiple sources of random error in the investigation. The digital mass balance has an absolute uncertainty associated with it. Moreover, the stop-watch used also has an uncertainty associated with it. These sources of error will reduce the precision of the data collected. Thus, the data has been collected in multiple trials and an arithmetic average has been used.
• The action of Tulsi extracts on yeast is presumably an example of an enzymatic process. This depends on temperature as well as pH. Thus, for an accurate result, it is essential to keep the temperature and pH constant while conducting the investigation. To control the pH, a buffer solution was used and the pH was maintained at a neutral pH of 7.00 in all cases. A water bath can be used to control the temperature. However, that does not ensure that the temperature was constant throughout because even in a water bath the temperature may fluctuate even slightly.
• Isopropanol has been used as a medium for the Tulsi extract. This alcohol is toxic in nature and can itself inhibit the growth of yeast. At a particular concentration or beyond it, alcohol inhibits the process of cell multiplication and displays lethal effect on the cells14. Thus, use of excessive alcohol may result into a methodological limitation as in that case along with the antimicrobial effect of Tulsi extract, the alcohol will also be responsible for the percentage inhibition of growth. To optimize this, usage of minimum amount of alcohol in the medium should be encouraged.

Use of pharmaceutical drugs has various side effects and an expensive process too. Due to the sustainable development goal of better economy, researches to find a more eco-friendly, less economical and biological alternatives of the life-saving allelopathic drugs is a booming research arena now. The medicinal herbs including Tulsi plays a pivotal role in this context. However, the major issue or challenge is about effectiveness. Are the biological alternatives equally effective as the synthetic or generic drugs? I wanted to extend my investigation along the same line. I would like to repeat the same investigation using Tulsi extract and any common synthetic antimicrobial / antifungal drug like Doxycycline and study their effect on the growth of yeast. The growth of yeast can be monitored spectrophotometrically or by measuring the zone of inhibition. Even, the colony count method can also be used. Different mass by volume percentages of the Tulsi extract as well as the alcoholic aqueous extract of the drug can be used. This will allow us to compare and contrast how the action of the plant Tulsi and the drug Doxycycline on the growth of yeast depends on the percentage concentration of the solutions used. It will give an opportunity to delineate that to what extent the biological alternatives can be recommended to be used over the chemical drugs.

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