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.

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Gram-negative cells losetheCrystal violet-Grams 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. Dextrose is the energy source used by the microorganisms(http://www.sigmaaldrich.com)
The next agar medium that is to be produced is Actinomycete Isolation Medium (AIM) that is used for the isolation of antibiotic-producing bacteria bacterium. And the third agar media to be produced is the Luria Bertani (LB) Lennox formulation. nutritionally rich medium, was developed for the growth and maintenance of pure cultures of recombinant strains of E. coli(http://www.talron.com). Obtained from the lab manual here are the instructions on producing the three above mentioned media.

Yeast Peptone Dextrose (YPD) – Amp agar
(For the isolation of antibiotic-producing fungi)
Per 1 Liter
Dextrose 20 grams
Peptone 10 grams
Yeast extract 5 grams
Agar 15 grams
Adjust to pH 5 and autoclave.

When cool (50C) add: 0.2 gram ampicillin (bacteriocide)
Actinomycete Isolation Medium (AIM)
(For the isolation of antibiotic-producing bacteria)
Per 1 Liter
Glycerol 5.0 grams
NaProprionate 4.0 grams
NaCaseinate 2.0 gram
K2HPO4 1.0 gram
l-Asparagine 0.1 gram
MgSO47H2O 0.1 gram
Fe2(SO4)36H2O 0.01 gram
Agar 15 grams
Autoclave. When cool (50C) add:
100 uL 10,000X Benzimidizole stock (final conc. 0.01 ppm) (fungicide)
1 mL of 2% filter sterilized cycloheximide.

Luria Bertani (LB ) Agar – Lennox formulation
Per 1 Liter
Tryptone 10 grams
Yeast Extract 5 grams
NaCl 5 grams
Agar 15 grams
Autoclave. Pour plates after cooling (50C).


After the medias are produced and the soil sample is obtained the student then must create a soil slurry sample so that a serial dilution can take place. To obtain a soil slurry the student must place the soil sample into 5ml of sterile PBS into a small container to vortex with a standard automated vortex. The mixture should settle for at least five minuets and while the soil is settling the student can set up the agar plates to plate out the dilution (see figure one for an example of how to perform a serial dilution). The student is to have seven LB agar plates labeled from 10-2 thru 10-8, The student also should have three AIM and three YPD agar plates each being labeled 10-2 thru 10-4. The serial dilution starts with the original slurry mixture and takes 0.1ml of that solution to a 0.9 ml PBS solution in a small container. This first new solution is 10-1 of the original sample and 0.1 ml of this solution is to be transferred onto one AIM,YPD, and LB agar plate and the plates are labeled 10-2 due to the fact that what is plated onto these three agar plates are 10-2 diluted from the original sample. Going back to the 0.1ml slurry and 0.9 ml PBS solution labeled 10-1 and take 0.1 ml of that solution to another container with 0.9ml PBS.

From this second container 0.1ml is to be transferred again to the three agar media and since the solution is again diluted down the agar plates are numbered 10-3. This procedure is to be done so that a final dilution of 10-7 is obtained for the small containers and all of these new dilutions are to be plated onto the LB agar media labeled up to 10-8 . With the AIM and YPD media the dilution is only done once more to achieve a finial dilution of 10-4 . Also as a side note the finial volume of the PBS and slurry mixture in each small container determines the dilution. With these finial dilutions ends week number one and all of the agar plates are to be incubated upright at room temperature for one week.
With the second week the students are to record growth from the soil dilutions prepared a week earlier and to observe the various bacterium that have grown. Enumeration of the microorganisms can now occur (See table one for results obtained) and if fewer than 30 colonies are present it is not a statistically viable amount to be considered significant for this study so one could label the table TFTC for Too few to count, also if more than 300 colonies are present then the number is to large and is TNTC for Too numerous to count. On the plates with a countable amount of colonies the student can estimate the number of organisms cfu/mL in the original soil sample. After a table containing the enumeration of the various agar plates is obtained then the student can contrast the microbial growth observed on the various media. The student then is to select appropriate colonies and restreak growth for culture isolation on the same selective media. And concluding week to is the Gram stain of the selected microorganisms that are to be replated. The following procedure to perform gram stains was obtained from the lab manual.
1. Prepare a heat-fixed smear. Leave the slide clamped in the large clothespin.

2. Cover the smear with Crystal violet solution. Allow it to stain for 1 minute.

Gently rinse the excess reagent with tap water.

3. Cover the smear with Grams iodine solution. Allow it to stain 1 minute.

Gently rinse the excess iodine off with tap water.

4. Decolorize by tilting the slide over the sink and dripping a few drops of Decolorizer
(acetone-alcohol) over the slide. Immediately rinse the Decolorizer off with tap water.

5. Counterstain by covering the smear with Safranin. Allow to counterstain 30 seconds to 1 minute. Gently rinse the excess stain off with tap water.

6. Blot the slide dry using Bibulous paper.

7. Place one drop of immersion oil on area of smear ( NO cover slip! )
8. Examine stained bacterial smear using the microscope.


Week three starts with Performing the gram-stain if not performed during week two. After that has been accomplished the student is to obtain the media used to re-plate there microorganism onto and test the microorganism ability to possibly produce antibiotics against E. coli a gram negative bacterium and B. mycoides a gram positive bacterium. I chose to replate three different microorganisms onto two different plates, The organisms labled one and two were replated onto the YPD media and the organism labled 3 was replated onto the LB media during week two so sufficient growth had occurred.


The student is to obtain two more plates and on one plate the student is to spread a bacterial lawn of E.coli and B. mycoides. This is done by obtaining a broth of E. Coli and
Starting with week number four the student Is to observe for any zones of inhibition, and this is done by observing the two agar plates and looking for any rings around any of the plugs (see figure four and five again). To measure any zones take the diameter of the plug and subtract that from the difference of the total diameter of the zone of inhibition. The next task that should be performed during week number four is choosing the microorganisms that had good zones of inhibition and constructing a multiple susceptibility plate. The following directions for creating a Susceptibility plate were obtained from the lab manual.

To create a Susceptibility Plate
1. Take a loop of your antibiotic producing organism and suspend in a sterile 1.5mL
microcentrifuge tube with 500uL of sterile PBS.

2. Vortex closed tubes until cells are evenly distributed in PBS.

3. Spread plate this on the appropriate agar medium.

4. Place appropriate antibiotic discs on agar medium with sterile forceps.

5. Incubate upright at room temperature.


A key note that must be taken is that the susceptibility plates must be on the same media that the antibiotic producing microorganism was located on. The four antibiotics used were Tetracycline TE30, Chlorophenicol C30, Chlorophenicol K30, and Ampicillin AM10. With the production of the Susceptibility plates ends week four.

The only task left for week five is to record the Results if any for the Susceptibility plates done during week four. If there are no zones of inhibition around an antibiotic disk this infers that the isolate organism is producing that said antibiotic and if there is a zone of inhibition around one of the disk this is saying that the microorganism is susceptible to the antibiotic present in the disk.(see figures 6 and 7)
Results and Disscusion:
Figure 1: Serial Dilution
I did not obtain any plate dilution factors due to the large or small amount of colonies present on my agar plated media. This could be accounted for by stating that there was a very large amount of microbes present in the soil and the dilutions did not dilute enough of the microorganism out or human error could also account for Not having any statistically significant plates to use.
The bacterium that I replated was obtained from the YPD 10-3 media and it gave rise to bacterium 1 & 3, The other bacterium that I obtained came from LB 10-5 . The two bacterium from the YPD plate varied in color the bacterium that I refer to as one had a reddish color had a circular form a raised elevation and a entire margin, The bacterium that I refer to as 3 Had a grey appearance a circular form a raised elevation and the margin appeared to be entire. The bacterium that I obtained from the LB media had a white appearance had a circular form a faintly raised elevation and the margin was entire.

Here are the results of 1 & 3 being replated
Figure 2: The bacteria I assigned as 1 is on the left, and the bacteria I assigned as 3 appears on the right and this is on YPD media
References:
-Microbiology Laboratory Manual
-http://www.talron.co.il/index.php?module=pagemaster&page user op=view page&PAGE id=8&MMN position=9:2
-http://www.sigmaaldrich.com/img/assests/6840/95762_YPD_Agar_17119_83055_ 1777 .pdf