In Vitro Antimicrobial screening

Procedure for In Vitro Antimicrobial screening

Introduction

Any chemical substance or biological agent that destroys or suppresses the growth of microorganism is called antimicrobial agent. Antimicrobial screening of a crude extract or pure compound isolated from natural sources is essential to ascertain its activity against various types of pathogenic organisms.

 Antimicrobial activity of any plant can be detected by observing the growth response of several of various microorganisms to the plant extract, which is placed in contact with them. In general Antimicrobial screening is undertaken in two phages: a primary qualitative assay to detect the presence or absence of activity and a secondary assay which quantities the relative potency, expressed as Minimum Inhibitory Concentration (MIC) value, of a pure compound, an important method in the further development of a new Antimicrobial compound.

The primary assay can be done in three ways as

a.    Diffusion method

b.    Dilution method

c.     Bioautographic method

Among these methods, the disc Diffusion method is widely acceptable for the preliminary evaluation of Antimicrobial activity. Disc Diffusion is essentially a qualitative or semi- qualitative test indicating the sensitivity or resistance of microorganisms to the test materials. However no distinction between bacteriostatic or bacteriosidal activity can be made by this method.

Principle of diffusion method

Diffusion is based on the ability of a drug to a diffuse from a confined source through the nutrient agar medium and creates concentration gradient. If agar is seeded with a sensitive organism, a zone of inhibition will result where the concentration exceeds the minimum concentration (MIC) for that particular organism.

In this method, measured amount of the test samples are dissolved in definite volumes of solvent to give solutions of known concentration (µg/ml). Then sterile filter paper discs (5 mm diameters) are impregnated with known amounts of the test substances and dried. The dried discs are places on plates (Petri dishes, 120mm diameters) containing a suitable medium (nutrient agar) seeded with the test organisms. These plates are kept at low temperature (4ºC) for 24 hours to allow maximum diffusion. A number of events take place simultaneously which include-

·         The dried discs absorb water from the agar medium and the material under test is dissolved.

·         The test material diffuses from the discs to the surrounding medium according to the physical law that controls the diffusion of molecules through agar gel.

·         There is a gradual change of taste material concentration on the agar surrounding each disc.

The plates are then kept in an incubator (37ºC) for 12-18 hours to allow the growth of microorganism. If the test material has antimicrobial activity, it will inhibit the growth of the microorganism, giving a clear, distinct zone called “Zone of Inhibition”. The antimicrobial activity of the test agent is determined in term of millimeter by measuring the diameter of the zone of inhibition. The greater the zone of inhibition the greater the activity of the test material against the test organism.

The principles factors which determine the size of the zone of inhibition are –

• Intensive antimicrobial susceptibility of the test sample.
• Growth rate of the test organism.
• Diffusion rate of the test sample which is related to its water solubility.
• Concentration of the test organisms incubated in the medium.
• Concentration of the test sample per disc.
• Thickness of the test medium in the Petri discs.

Test Materials

In our present study, the antibacterial activity of methanolic extracts was investigated in compares with standard kanamycin (30 µg/disc) antibiotic agent a number of pathogenic Gram-positive and Gram-negative bacteria.

Test Organism

Both Gram-positive and Gram-negative stains of bacteria were used as the test organism to observe the anti-bacterial activity of the compounds. The bacterial steins used for this investigation are listed in the Table. These organisms were collected from the Microbiology research laboratory, Department of Pharmacy, Southeast University of Dhaka. The pure of which was previously collected from the Microbiology Department of Dhaka University.

Table: List of pathogenic bacterial strains

Gram-positive	Gram-negative
Staphylococus aereus Bacillus megaterium Bacillus subtilis Sarcina lutea	1. Salmonella paratyphi 2. Salmonella typhi 3. Escherichia coli 4. Shigella dysenteriae 5. Vibrio minicus 6. Vibrio parahemolyticus 7. Shigella boydii 8. Psedomonas aeruginosa

Apparatus and Reagents

• Filter paper discs (5mm in diameter)
• Petri dishes
• Refrigerator
• Test tubes
• Sterile forceps
• Sterile cotton
• Incubating loop
• Bunsen burner
• Micropipette(10-100µl)
• Laminar air flow unit (Biocraft’s Scientific Industries, India)
• Autoclave(|YX-280B 18L)
• Incubator(OSK-9636, Japan)
• Nutrient agar media(DIFCD)
• Ethanol
• Vials
• Standard disc(Kanamycin 30 µg/disc)
• 
  

Sterilization Procedure

Petri dishes and other glass wares were sterilized by autoclaving at a temperature 126ºC and a pressure of 20 lbs/sq inches for 20 minutes. Blank discs first kept in a covered Petridis and then subjected to dry heat sterilization at 180ºC for 1 hour. Latter they were kept in laminar hood under UV light for 30 minutes. UV light was switched on before one hour working in laminar hood to avoid any accidental contamination.

Culture Media

The main requirement for the growth of bacteria were as follows-

1. Source of energy such as carbohydrate, protein and nuclic acid.
2. Essential trace elements e.g. Mg, Mn, Fe, and Co
3. Optimum pH of media and
4. Optimum temperature for incubation

A number of culture media are available to demonstrate the antibacterial activity. This are-

ü  Nutrient agar media

ü  Nutrient broth media

ü  Mueller-Hinton agar media

ü  Tryptic soy broth(TSB)

Among these, nutrient agar media is most frequently used and its composition is shown.

Table: Composition of nutrient agar media

Ingredient	Amount(g/l)
Peptic digest of animal tissue	5.0
Sodium chloride	5.0
Beef extract	1.5
Yeast extract	1.5
Agar	15.0
Distilled water	100 ml
pH	7.5 ±1.0 at 25ºC

Preparation of Medium

The instant nutrient agar media was accurately weighted and then reconstituted with distilled water in a conical flask according to specification (2.8% w/v). It was then heated with water bath to dissolve the agar and a transparent solution was obtained.

The prepared media was then transferred in 9ml and 5ml in a number of clean test tubes, respectively to prepare plates and slants. The slants were used for making sub-culture of microorganism, which in turn use for sensitivity tests.

The test tubes were then plugged with cotton and sterilized in an autoclave at temperature of 126ºc and pressure of Ib/sq inch for 15 minutes.

Preparation of Subculture

With the help of a inoculating lop, the test organisms were transferred from the pure culture to the agar slants in a laminar airflow unit. The incubated satins were then incubated at 37º for 18-24 hours to ensure the growth of test organisms. This culture was used for sensitivity test.

Preparation of Test Plates

The test organism was transferred from the subculture to the test tube containing 9ml autoclaved medium with the help of an incubating loop in aseptic area. The test tube was shaken by rotation to get a uniform suspension of the organism. The bacterial suspensions were immediately transferred to the sterile perishes in an aseptic area and were rotated several times, first clockwise and anticlockwise to ensure homogeneous dispersion of the organism into the medium. The depth of media into each Petri dish was approximately 4mm. After plates were cooled to room temperature, it stored in a refrigerator at 4ºC.

Preparation of Discs

Three types of discs were used for antibacterial screening. These were-

a.    Sample discs

b.    Standard discs

c.    Blank/control discs

1. Sample discs: Sterilized filters discs (5 mm in diameter) were taken in a blank Petri dish. Sample solution of the desired concentration was applied on the discs with the help of a micropipette in an aseptic condition. These discs were left for a few minutes in aseptic condition for complete removal of solvent.
2. Standard discs: these were used to compare to the antibacterial activity of test material. In our investigation Kanamycin (30 g/disc) was used as a reference.

3. Blank discs: Only solvent was applied to the disc to determine the antibacterial effects of the solvent used.

Placement of Disc, Diffusion and Incubation

The sample disc and standard antibiotic disc were placed gently on the solidified agar plates freshly seeded with the organism with the help of a sterile forceps to ensure complete contact with medium surface. The arrangement of the disc was such that the discs were no closer than 15 mm to the plate to prevent overlapping the zone of inhibition.

The plates were then inverted and kept in a refrigerator for about 24 hours at 4ºC. This was sufficient time for the material to diffuse to a considerable area of the medium. Finally, the plates were incubated at 37ºC for 12-18 hours.

Determination of the Antimicrobial Activity

After 24 hour incubation, the antimicrobial activities of the test materials were determined by measuring the diameter of the zones of inhibition in millimeter with transparent scale.

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Data accumulated & compiled by:

Dewan Pavel (M. PHARM)

       Mundipharma (Bangladesh) Pvt. Ltd.

       Officer, Quality Assura