Antimicrobial Soil Isolates

Project 1: Antimicrobial Activity of Soil Isolates

John Franklin Farrar

Department of Biology and Microbiology and Biology

Address BOX 22750 BOWEN HALL, RALEIGH, NC, 27607

Abstract:

Isolation and characterization of microorganisms is a practice that aids in

Increasing ones knowledge of a laboratory setting and it helps improve on

Using sterile technique. Isolates of soil microbes can be categorized and

Characterized based on a number of criteria ranging from gram-staining

Which is done for this project to enumeration which is quantitative description

Based on the amount of microbial colonies available. The Antimicrobial

Activity of three different microbes were also tested and results were obtained

For two out of three of the microbes. Two out of three of the tested microbes

Exhibited antimicrobial activity towards the bacteria E. Coli and B. Mycoides

And the results were recorded. Microbes produce various antibiotics and by

Isolating different microbes the antibodies can be tested for.

Introduction:

This lab focuses on the isolation and characterization of an unknown organisms expressing interesting properties in relation to Antimicrobial Activity. At the conclusion of this laboratory experience The researcher would be required to describe the isolates obtained from the soil macroscopic and microscopic appearance, Perform the gram stain of the microorganisms in question and to discriminate the organisms from other microbes that could be contained in the soil and to finally make certain that the organisms have not been exposed to outside sources and be isolated in a pure culture. The researcher must first isolate the microbe and try to grow the organism in a pure culture to commence with testing the organism. The isolation of Microbes in a laboratory and clinical settings are of the up most importance and due to the isolation of different microbes from each other various procedures become open for the researcher to use like six fold dilutions that allow for the quarantined microbe to be reduced down in number to ensure that the test are being run are being tested on just a single type of organism. But before dilutions can occur the microbe must first be liberated from the soil and streaked onto an Agar plate to grow. There are various methods that can be used once the organism has grown onto the agar plate to ensure that the sample collected would only contain one pure organism. Streak plate enumerations also occur once the isolation of a particular microbe has occurred and the data that is received if the amount of organisms on the plate are between 30-300 individual colonies can be used because these are accepted values for Cell plate enumerations. After initial isolation a variety of techniques can be implemented to analyze and characterize the various microorganisms. The Gram Stain is a procedure that is performed in order to determine the difference between two main microbial cell wall types. Gram-positive cells have a cell wall made up mostly of a thick, peptidylglycan layer. These cells retain thecrystal violet color and the acetone alcohol acts as a dehydrating agent causing all peptidoglycan pores toclose and preventing the insoluble Crystal violet-Grams iodine complex from decolorizing. These cells appear PURPLE.

Gram-negative cells losetheCrystal violet-Gram's iodine complex easily because the acetone-alcohol cannot seal the thin peptidylglycan layer to stop the decolorization. These cells lose the complex, but take up the lipid soluble counterstain, Safranin. These cells appear RED/PINK(laboratory Manual). Observing the types of antibiotics that an organism produces is a tool which could help classify and identify a microorganism also. In contrast to the just mentioned point with the aid of antibacterial disk a researcher could identify what antibiotics hinder the growth of the bacteria could help identify the microorganism. Each of these methods may determine one or more aspects that maybe unique to that organism or that could help identify the organism in relationship to other organisms or to help classify the organism into a group of microorganisms that share most of the characteristics displayed by the said organism.

There are four main groups of Antibiotic-producing organisms that this lab will focus on and they are the Actinomyces, the Streptomyces, Bacillus, and Microbial fungi.

Actinomyces is a class of filamentous gram positive that are rod-shaped(lab manual). Actinomyces produce spores in order to spread the bacteria to new environments and flourish. Streptomyces is a genus of the class Actinomyces and are primarily strictly aerobic soil organisms. Bacillus is a genus of gram-positve bacteria prevalent in the soil. Certain Bacillus species produce survival structures called endospores and these endospores differ from those of the Actinomyces class(lab manual). Fungi is also an antibiotic-producing organism and make up for a large amount of the microbial diversity that occurs in the soil environment.

Materials and Methods:

Students were told to bring in soil samples into lab and isolate organisms from the soil. This was done by going to a dry area of land, preferably away from heavy human traffic and to obtain a dry soil sample. The sample was to be placed in a zip lock bag containing CaCO3 with a Ratio being one part CaCO3 (one gram)to nine parts of dry soil( around nine grams). After collected the samples were to be incubated in a dry environment. Prior to the student obtaining soil samples three media in agar plates are to be produced for plating, these media are Yeast Peptone Dextrose (YPD) that is to be used for the isolation of antibiotic-producing fungi. . Yeast Extract and Peptone provide carbon, nitrogen, minerals, vitamins, trace ingredients and other essential growth nutrients. Yeast extract supplies vitamin B-complexes, which stimulate the growth of yeasts and bacteria.