Tamarindus indica Seed Extract as a Mordant in Gram Staining

Overview Gram Staining: Differentiates gram positive from gram negative bacteria (Histopathologic techniques by Gregorios,M.D)

The staining response of a certain bacteria depends on its cell wall components(http://www.ruf.rice.edu/~bioslabs/bios318/staining.htm)

Overview

Overview Mordant Tanic acid, Iodine, etc., Forms a mordant-dye-protein complex which can not be decolorized in gram positive bacteria but is extracted by alcohol in gram negative bacteria(Histopathologic techniques by Gregorios, M.D)

Overview Mordant In Gram stain: It is added after the crystal violet and before the alcohol in the gram reaction.

Why Gram Stain? According to Henrys, it is one of the most valuable procedures performed by the microbiology laboratory. It provides information that it is used for selecting appropriate antimicrobial theraphy.

Why Grams Iodine? One of the disadvantages of using Grams Iodine is its hazardous components that can cause irritation to the users. Also, according to the Material Safety Data Sheet of the Grams Iodine, prolonged exposure may cause harm to the respiratory and nervous systems as well

Why Grams Iodine?It shows that Iodine is a toxic reagent. An alternative reagent should be replaced in Grams iodine

Tamarindus indica (Tamarind) seed extract as mordant

Tamarindus indica (Tamarind) An abundant plant in the Philippines that is cultivated between the months of April to October Used for curing asthma and fever Its constituents includes tannin, citric, acetic, pectin, butyric and oxalic acid

Tannin Repels many organisms that cause wood decay Used to preserve leather(Nabors, 2005)

Tannic Acid Commercial form of Tannin Weak acid Has mordant properties

Main ProblemIs the Tamarind (Tamarindus indica) Seed Extract effective as a Mordant in Gram Staining?

Sub Problems What are the physical and chemical properties of the seed husk extract in comparison with the Grams Iodine? Will the physical and chemical properties of the tannic acid from the seed husk extract level or exceed those of the Grams Iodine?

Sub Problems Using spectrophotometry, what is the concentration of the tannic acid from the seeds husk extract? Will the concentration of the tannic acid from the seed husk extract level or exceed those of the Grams Iodine concentration?

General ObjectiveTo prove the effectiveness of Tamarind (Tamarindus indica) Seed Extract as Mordant in Gram Staining.

Specific Objectives To be able to use tamarind leaves extract as a mordant in Grams staining To compare the effectiveness of tamarind leaves extract as a mordant in Grams staining to Grams iodine To determine the best solvent concentration to be used in Tamarind extract. To identify the concentration of tannic acid that will be effective to be used as mordant in Grams staining.

SignificanceFor the scientific community It can be a stepping stone in the evolution of the use of the local resources for medical studies. It can also give an idea to medical professions to come up with a new way or reagents of staining.

SignificanceFor the agricultural community The success of this study will be noted as advancement not only in the scientific studies but also for the agricultural growth of our country. Tamarind is one of the abundant crops in the Philippines and through this study, more opportunities will be developed for the use of more Tamarind products.

SignificanceFor the general public The use of the organic raw material will be promoted which can be in replacement of the chemically produced Grams Iodine.

SignificanceFor the medical technology field The medical technologist will be able to find an alternative for Grams iodine which can be a cause of many problems in their health. It can be the start of finding some alternative reagents for the toxic ones used in the laboratory for the benefit of the medical technologist.

Research Design Experimental (True Exerimental)

MethodologyI. Collection II. Extraction ii.1. Aqueous: water ii.2. Basic/ Alkaline: dioxane ii.3. Acidic: acetic acid ii.4. Alcoholic: ethanol

MethodologyIII. Measurement of Effectiveness III.i. Collection of BacteriaE. coli and S. Aureus from ATCCNutrition broth

Centrifuge: harvest bacteria

HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid)

MethodologyIII.ii. Test tube Preparation

MethodologyIV. Calculations v.1. Absorbance v.2. Relative color strength to the nearest 0.1%

Calculations Beer Lamberts law: A = log10(1/T ) (1) A is the absorbance at wavelength of minimum transmittance T is the transmittance (in decimal fraction form, where 100% = 1.0) at wavelength of minimum transmittance.

Calculations Relative color strength to the nearest 0.1% Fs = 100 (A2C1/A1C2)

Fs = 100 (A2/A1) Fs is relative color strength in percent C is the dye concentration subscript 1 refers to the reference dye subscript 2 refers to the dye under test Assuming C1 = C2, the following simplified equation is obtained:

Gantt ChartOBJECTIVES 1. Topic proposal 2. Title proposal 3. Approval of proposals 4.RRL 5. Chapter 1 draft 6. Revision of chapter 1 and RRL 7. Chapter 3 draft 8. Revision of chapter 3 9. Preparation for experimentation 10. Final draft of chapters 1, 2, and 3. JUNE JULY AUG.

SEPT

OCT

Gantt ChartOBJECTIVES 11. Experimentation 12. Analysis and interpretation of data 13. Chapters 4 and 5 draft 14. Revisions of chapter 4 and 5 15. Final manuscript 16. Oral defense NOV OCT DEC JAN FEB