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Identification of Unknown Species from Soil Sample

Paper Type: Free Essay Subject: Biology
Wordcount: 3266 words Published: 8th Feb 2020

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In the present examination, a preliminary was done to discover another unknown species microscopic organisms from soil tests gathered from Newyork state in 2019. Isolation of different bacterial colonies from soil sample was carried out. All the isolated bacterial colonies were then screened for their antimicrobial activity against the ESKAPE pathogens.Polymerase chain reaction,Gel elctrophorosis has been doen with that sample. The DNA sequence showing that the species would be Pseudomonas species.other biochemical test such as fermentation test,oxidase test ,catalase test ,gram staining test has been down.the results of biochemical co relates with  DNA sequence which is Psudomonas species.


The purpose of the experiment is to identify the unknown species from soil sample which took in Newyork using various various DNA test and Biochemical tests.July 2015, FDI published a Strategic Analysis Paper entitled Under Our Feet: Soil Microorganisms as Primary Drivers of Essential Ecological Processes. Since the publication of that article there has been a moderate trend toward the study of soils holistically rather than the detailed study of soil

components in isolation.

Just 5% of what is created by green plants is devoured by creatures, however the 95% is devoured by microorganisms. One gram of fruitful soil can contain up to one billion microbes.( Kandeler, E., Stemmer, M., & Gerzabek, M. H. (n.d.). Role of Microorganisms in Carbon Cycling in Soils. Soil Biology Microorganisms in Soils: Roles in Genesis and Functions, 139-157. doi:10.1007/3-540-26609-7_7) There are various kinds of bacteria, and the greater part of them have not been found yet! The greater part of these bacteria are aerobic, implying that they require oxygen from the dirt air. In any case, other bacteria need to live without oxygen, and different sorts can live both with, and without oxygen. The development of these bacteria is constrained by the nourishment that is accessible in the dirt. Microorganisms comprise a significant wellspring of biodiversity in soils and are a vital piece of terrestrial ecosystems (Zhang, Z., Pan, L., & Li, H. (2010). Isolation, identification and characterization of soil microbes which degrade phenolic allelochemicals. Journal of Applied Microbiology,108(5), 1839-1849. doi:10.1111/j.1365-2672.2009.04589.x). They add to major natural capacities, for example, nutrient and gas cycling, biogeochemical processes and the decomposition and transformation of natural matter.Considerable research is being done so as to discover new antimicrobial producing bacteria isolated from soil (Rondon et al., 2000; Crowe and Olsson, 2001; Courtis et al., 2003).


 To study about unknown species in soil, soil has been taken from Newyork state.Local and familiar soils are rich and dynamic ,and remain largely uncharted.we took a sample which encapsulates a good representation of ecosystem of choice and that is rich in biodiversity. After collection of soil sample, the first step is to suspend the bacteria in liquid so that they can be transferred to another medium,leaving behind plant material ,soil animals and minerals from soil.because cosindeeation the kind of vegetation the soil is very important.1 g of soil sample placed into conical tube and added 9 ml of water.


 To know about microbial counts for liquid and soil samples is a common practice.for that serial  dilution has been performed.its also used to find biomass of soil sample ,calculate an antibiotic minimal inhibitory concentration and population density in a liquid culture.Mostly bacteria are numerous,ranging from the tens to the millions in as little as 1 g of sample .so instead is counting cells one by one ,we tried to calculate colony forming units,which give us an approximation of the number is vialbke cells per milliliter or gram of a sample.Fir serial dilution,we added 9ml of water to 1 GM of soil sample.again dilutions made in increments of 10,added 900 micro liters of diligent water into each dilution tube.(900 microliters of diluent  + 100 microliters of specimen transferred =1000 microliter.then 100 microlieters of sample taken from master conical tube and transfered to 900 microliteres of water in Epps drift tube which was 10 ^1 dilution.like that till 10^4 has been done.after serial dilution ,placed 100 microlitera of each dilution to luria broth ,Brain heart infusion agar,tryptic plate Agar.pour plate technique has been used.9 plates were prepared and placed in the incubator for 96 hours at 37 °C



    To find unique characters of an organism ,isolation of single species of bacteria from a mixed culture containing tens or hundreds of species was performed.Pick and patch technique used to make master plates which contains unique bacteria from mixed culture.This technique involves picking bacteria from a mixed culture and patching them onto a fresh plate to form master plate.after that,  picked isolates from master plate and patched to fresh plate.ESKAPE pathogen has been placed in the Center of the plate near.petri plates were placed for 64 hours at 25 °C

TABLE 1.Colony morphology for each plate after  incubation for antibiotic screening

Plate names

LB medium

BHI medium

TSA Medium



Dark yellow circle

Big circle with spotes


Small cicle,pinkish

Flate whole circle

Small in  corner


Slight yellow ,small patch

Big loop

Rod shape


Gel white,half circle

Medium circle near gluster

Small patch


White ,Big cicle

Big separate circle

Small patch


Small pinkish with white dot

Corner circle

Small circle


Half cicle with gel

Waterish ,circle without corner

Could not analyse due to contamination


Big white circle

Irregular shape

Could not analyse due to contamination


Small dopes colourless

Small circle with bubbles

Could not analyse due to contamination


Corner half circle

White small patch

Could not analyse due to contamination


Small pink irregular


Waterish patch

Could not analyse due to contamination


Small round shape

Colourless patch

Could not analyse due to contamination


 ESKAPE pathogens are one of the biggest threats of infectious disease.They are Enterococcus Faecium,Staphylococcus aureus,Klebsiella pneumoniae,Actnetobacter baumannii,Pseudomonas aeruginosa and several species of Enterobacter.For inhibition of this pathogens , seperate lawns of ESKAPE pathogens and made them to grow isolates on lawns .

100 microliter of ESKAPE pathogens added to plates.same procedure performed for all plates.plates were incubated.


 To isolate a pure cultures of antibiotic producers ,incubated plates were measured.Number of isolates in plate ,number of products and measurement of smallest inhibition distance has been calculated.Fir that ,Three 3 products has been chosen and made streak plate in appropriate media from master plate.


 Polymerase Chain Reaction ,Gel Electrophoresis and BLAST has been used to identify producers. PCR is a common techno used to amplify specific regions of DNA for several applications, Including sequencing and genetic analysis. PCR has been done using Econotaq plus master mix which is ready to use master mix contains agarose gel loading buffer.It has a combination of master mix with template DNA,primers and water.PCR contains 3 steps which are denaturion ,annealing and extension.Two colonies has been used for PCR .For three procedures of PCR has desperate temperate settings. Initial denaturation for 2 minutes at 94 °C

 Preheating thermonuclear for 94 °C,Denaturation for 15-30 sec at 94 °C,Annealing for 15-39 sec at 50-60 °C ,Extension for 1 minute at 72 degree Celsius.Final Extension for 5-10 minutes at °C ,Preheating thermonuclear for 94 degree Celsius.

Denaturation for 15-30 sec at 94 ° C,Annealing for 15-39 sec at 50-60 °C , Extension for

1 minute at 72 °C. Final Extension for 5-10 minutes at 72 °C . Preheating thermonuclear for 94

Denaturation for 15-30 sec at 94 °C.Annealing for 15-39 sec at 50-60 degree Celsius.Extension for 1 minute at 72 °C. Final Extension for 5-10 minutes at 72 °C .After that this 5 micro liter of the traction is loaded onto an agarose gel for analysis.Then gel electrophoresis done In epicentre.This tunes has been incubate for 6 minutes at 65 degree Celsius.then tube transferred to heat block for 2 minutes at 98 °C.DNA  has been stored at -20 °Cfor 1 week .DNA sequence used for BLAST to get a maximum identity of relevant species


Image 1.Results  of PCR and Gel Eletrophorosis. from 2 isolates,one osolate shows more active in gel elctrophoroesis(4b)


                  Gram staining was performed followed by MacConkey test to confirm the results.Besides, IMViC test was conducted to identify bacteria that are able to synthesize indole, produce butanediol, detect production of acids formed during metabolism using mixed acid fermentation pathway using pyruvate as a substrate, and utilize citrate as a sole carbon source. SIM agar deep, MR-VP broth, Simmons citrate agar, McConkey agar plates, phenol red glucose broth with Durham tubes, starch agar plates and spirit blue agar plates were the media used to perform the experiment. Concerning the gram staining, the bacteria were spread on two separate slides, after adding the reagents and rinsing the specimen, they were dried observed under 100X immersion oil microscope objective.  For IMViC test, the test tubes were labeled, inoculated and incubated for 24hrs at 37˚C. After incubation, the following reagents were added to appropriate test tubes: Kovacs reagent for Indole test, methyl red for methyl red test, and Barritt’s reagent for VP test. concerning MacConkey agar test, the plates were labeled, and the strains were streaked on agar plates and incubated for 48hrs at 37˚C. the fermentation and β-galactosidase activity were performed on the strains with phenol red glucose broth with Durham tubes, which were placed in a test tube-rack and incubated at 37˚C for 48hrs after being labeled and inoculated. To investigate the ability of the unknown strains to ferment carbohydrates, they were streaked on the starch plates and incubated for 48hrs at 37C, then several drops of Gram’s iodine were added onto the plates. Finally, to study the lipid hydrolysis, the spirit blue plates spot-inoculated with the unknown strains and incubated in an inverted position for more than 24hrs.


     The overall objective is to find whether any new species available  or known species in the soil sample which took at specific place.According to DNA test such as polymerase Chain Reaction ,Gel electrophoresis result of blast shows that the species is pesudomonous species with 99.73 percentage identity.

     Besides Gram stain shows gram negative bacteria with pink rod shape as a result.other than catalase test shows positive with bubbles production and oxidase test reacted positive. macconkey agar shows positive growth and Mannitol agar plays shows negative result.

Table 2.Distance of inhibition from Isolates(in milli meter)

Plate name

Bacillus subtilis

Escherichia coli

Staphylococcus aureus

LB medium.Isolate 1

1 mm

2 mm

1 mm

LB medium.Isolate 2

2 mm

3 mm

1 mm

LB medium.Isolate 3

4 mm

2 mm

2 mm

LB medium.Isolate 4

2 mm

No activity

3 mm


          Although the expected biochemical result shows that the unknown species would be pseudomonas species .antibiotic suspectablity test proves that species has a antibiotic resistance over some antibiotics .Tetracycline 30 (TC30) killed bacteria in that region.its shows that the species would be pseudomonas [7]

But penicillin doesn’t kill any pathogen in antibiotic test.Environmental  factors may affected the result and contamination might be the result for resistance.

           Eosin methylene blue and macconkey agar test were taken second time to get a result according with BLASt result. Because in first time it’s showed against biochemical property of pseudomonas species.Then the second time it shows positive.Lactose fermentation test doesn’t co relate with the above result.Until evidence regarding appropriate species of pseudomonas,it will remain unidentified.


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