Media used
nutrient agar, LB broth
Extracts
spices were collected from the market. Sample was grinded, by mortar and pestle, and sieved to obtain fine powder. Names of the Indian spices used in the study are mentioned in Table 1, along with their few details. Lowering the particle size increases surface contact between samples and extraction solvents. So, smaller the particle size, better it will soak into the solvent.8
Table 1
Names of all the spices chosen for the study with their scientific name, name of the family, English name, common name and the plant part which is used in the research
SCIENTIFIC NAME
|
FAMILY NAME
|
ENGLISH NAME
|
COMMON NAME
|
PLANT PART USED
|
Piper nigrum
|
Piperaceae
|
Black pepper
|
Kali mirch
|
Seeds
|
Trachyspermum ammi
|
Umbellifers
|
Carom
|
Ajwain
|
Seeds
|
Cinnamon verum
|
Laurels
|
Cinnamon
|
Dalchini
|
Bark
|
Syzygium aromaticum
|
Myrtle
|
Clove
|
Laung
|
Bud
|
Cuminum cyminum
|
Umbellifers
|
Cumin
|
Jeera
|
Seeds
|
Extracts of these five spices were prepared by soaking a small amount of sample in the selected solvent overnight and later filtered with the help of Whatman paper.
Since fresh samples are fragile and tend to deteriorate faster, dried samples of spices are taken.8
Preparation of extracts:
Aqueous extract- 2 g of finely powdered sample added to 20 ml sterilized distilled water (10% concentration) and left overnight.
Ethanol extract- 2 g of finely powdered sample added to 10 ml ethanol (20% concentration) and left overnight.
Figure 1 shows all the spice extracts prepared. First, these mixtures were filtered with the help of funnel and Whatman paper and then using micron-filters, to remove all the residue. After measuring the volumes of the extracts obtained, they are stored in respective glass bottles.8
Bacterial Strains used:
Two very common food borne pathogens were used to test the antimicrobial effect of spices, a) Staphylococcus aureus and b) Esterichia coli. The effect of spices was also tested on the environmental microbes.
E. coli is an enteric gram-negative, multitalented Bacillus. E. coli assuredly breeds in humans and in animal gut lumen in mass culture.
Staphylococcus aureus is a Gram-positive, round-shaped bacterium that is a member of the Firmicutes, and it is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin.9
ANTIMICROBIAL ACTIVITY OF SPICES
Agar dilution method
Preparation of plates:
For aqueous extracts
7 plates for each spice sample are prepared, by agar dilution method.
A mixture of diluted extract (of concentrations 0.5,1, 2, 4, 8 mg/ml) and nutrient agar was poured in 5 plates while one was negative, with antibiotic kanamycin, and one was positive, with no extract and only nutrient medium.
For ethanolic extracts
with the same method, plates of different spices are prepared under different dilutions or concentrations.
Concentrations for cumin and cinnamon extracts: 0.5, 1, 2, 4, 8 mg/ml
Concentrations for black pepper and clove: 0.1, 0.2, 0.4, 0.8, 1.6 mg/ml
For better results the procedure was repeated for black pepper and clove, this time at concentrations 1, 2, 4, 6, 8 mg/ml.
Disk diffusion method
Prepare 12 (6 plates with three divisions and 6 plates with two divisions) agar plates and inoculate 6 with E. coli and 6 with S. aureus, with the help of spreader, as shown in Fig. 2. Disks with concentration 100%, 75% and 50% of each ethanol extract are placed in respective plates.10
Inoculation:
Inoculum was prepared by adding 500 microlitres of strain to 50 ml LB broth. Inoculated broth was incubated at 37OC for 24hr.
Each petri plate was divided into half, with a marker and inoculated with 200 microlitres of Staphylococcus aureus on one half and 200 microlitres E. coli on the other half with help of micropipette.
These plates were then left in the incubator at 37 degree Celsius for 24 hrs.