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The Identification Of Bacillus Subtilis The Mystery Bacterium Biology

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    Assorted communities of microorganisms consisting of prokaryotes such as bacteriums, every bit good as eukaryotes such as Fungis, Protozoa, algae, and roundworms are found in all kinds of natural environments ( Robertson and Egger, 2008 ) . In peculiar micro-organisms are rather abundant in dirt environments and are an of import portion of the dirt microbic community ( Prescott et al. , 2005 ) . However merely a fraction of these bugs that make up dirt biomass have been cultured. The microbic community which constitutes dirts makes important parts to biogeochemical cycling every bit good as the C, N and S rhythms ( Prescott et al. , 2005 ) . Soil micro-organisms are besides indispensable for decomposition and the recycling of indispensable foods that are locked in supermolecules of organic stuff ( Robertson and Egger, 2008 ) .

    Microbes can be classified based on a broad scope of features that allow them to last and boom in changing environments. A major categorization is based on O demand ; with O dependant bugs being aerophilic while anoxygenic bugs being anaerobiotic. Aerobic bugs are farther classified based on the grade of O demand. This categorization has a major impact on where these bugs are distributed with in the dirt stratum. Other of import environmental conditions and factors that affect bugs include: temperature, pH, and osmotic force per unit area.

    The aim of this experiment is to insulate and place a bacteria from one of two dirt samples provided: coarse woody dust ( CWD ) or forest dirt ( FS ) . This will be achieved by obtaining pure civilizations of the bacteria and executing legion trials to find Gram discoloration, settlement and cell morphology, biochemical activity and optimum environmental conditions. The information gathered from these trials is important in finding the genus of the unknown bacterial isolate.

    Material and Methods:

    The Identification of Soil Bacteria experiment was performed over a four-week period and experimental processs were based on the instructions outlined in the Biology 203 Laboratory Manual ( Robertson and Egger, 2008 ) . In hebdomad 1, two dirt samples, coarse woody dust ( CWD ) and forest dirt ( FS ) were cultured aseptically on nutrient-containing media.

    In hebdomad 2, selected bacteriums were isolated from the assorted populations by sub-culturing to obtain pure civilizations. A compound microscope was used to find cellular agreement of the bacteriums and settlement features described through direct observation. Gram staining was performed on the civilized bacteriums to find whether the bacteria was Gram positive or Gram negative.

    In hebdomad 3, assorted biochemical trials were performed to analyse the bacterial isolates for specific biochemical activities involved in the cycling of C, N and S. The bacteriums were tested for amylum hydrolysis, production of H2S and motility. The bacteriums were besides tested to uncover their N cycling capacity. This would find if the bacteria was able to execute ammonification ( release of NH3 ) , nitrification ( the oxidization of ammonium hydroxide ) , or denitrification ( decrease of nitrate ) . The catalase trial was performed to find whether the bacteria was aerophilic or anaerobiotic.

    In the concluding hebdomad, hebdomad 4, the bacteriums were tested to find how environmental factors such as temperature, pH and osmotic force per unit area affect the growing of these dirt bug. The findings of these optimum conditions for each bacteria were recorded and the bacteria was identified based on all observations and consequences collected over the four-week period which was interpreted utilizing scientific literature ( Robertson and Egger, 2008 ) .


    The bacteriums were cultured on agar home base media and observed to depict settlement morphology. The undermentioned features were found: irregular/filamentous signifier, raised lift, undulate border, dull visual aspect, opaque optical belongings, pick colour, and unsmooth texture ( Table 1 ) . The bacteria turned violet when Gram stained bespeaking a Gram positive bug. The bacteria showed individual and clustered rods when observed under the compound microscope at 1000X magnification. The rods were ~1.0Aµm broad and ~2.0Aµm long ( Table 1 ) .

    Biochemical testing showed that the bacteria was motile and able to hydrolyse amylum. The bacteria tested negative for ammonification as no colour alteration was found in the peptone stock after Nessler ‘s Reagent was added ( Table 1 ) . However, the bacteria was capable of nitrification and denitrification. In the denitrification trial reagents A & A ; B were added to the nitrate stock and a ruddy colour alteration was observed ( Table 1 ) . This is a positive consequence for denitrification as nitrate ( NO3- ) is reduced to nitrite ( NO2- ) by the enzyme nitrate reductase ( Robertson and Egger, 2008 ) . For the nitrification trial, the nitrite stock turned a deep blue colour after the add-on of diphenylamine reagent and sulphuric acid ( Table 1 ) . This is a positive consequence for nitrification as ammonium hydroxide ( NH3/NH4+ ) was oxidized to nitrate ( NO3- ) by Nitrobacter ( Robertson and Egger, 2008 ) . The bacteria was tested for motility and H2S decrease in SIM deeps. Although the bacterium was motile it was nevertheless unable to bring forth a black precipitate which is characteristic of H2S decrease ( Table 1 ) . For the catalase trial legion bubbles formed when H peroxide was added which shows that the bacteria is aerophilic. The bacteria grew most optimally at 220C, pH 5 and at all concentrations including over 5 % which indicated that the bug is mesophilic, acidophilous, and halophilic ( Table 1 ) .

    Table 1. Summary of the legion trials performed, every bit good as the observations and findings critical in the designation of the unknown dirt bacteria isolate.



    Colony morphology

    Form: irregular/filamentous ; Elevation: raised ; Margin ; undulate ; Appearance: dull ; Optical Property: opaque ; Color: pick ; Texture: rough

    Cell morphology

    Cell Shape: rod ; Cell agreement: some individual, some bunch ; Dimensions: ~1.0Aµm broad and ~2.0Aµm long

    Gram discoloration

    Cells were violet when stained – Gram Positive

    Starch hydrolysis

    Clear zone around bacterial growing when I added. The colour of iodine reaction was yellow/caramel – Positive consequence for Starch Hydrolysis.

    H2S decrease

    No black precipitate produced therefore S is non reduced and no H sulphide production – Negative for H2S decrease.


    Bacterial growing off from the pang line – Positive for motility.


    No colour alteration observed in peptone stock after add-on of Nessler ‘s Reagent – Negative for Ammonification.


    Red colour observed after reagents A & A ; B added – Positive for Denitrification: transition of NO3- to

    NO2- .


    Deep blue colour observed after diphenylamine reagent and sulphuric acid added to nitrite broth – Positive for Nitrification transition of NH3/NH4+ to NO3- .


    Tonss of bubbles formed when H peroxide added – Positive for Catalase, thereforeAerobic.

    Optimum temperature

    Growth greatest when bacteria incubated at 220C – Mesophile

    Optimal pH

    Growth greatest at pH 5 – Acidophil.

    Optimal salt concentration

    Great sum of growing at all salt concentrations – Halophile.


    The unknown bacteria was isolated from FS sample diluted to 10-5 with unfertile deionized H2O and identified harmonizing to genus and to a species of that peculiar genus. The unknown bacteria isolate was identified as Bacillus subtilis.

    Gram staining and microscopic analysis, revealed that the bacteriums were violet Gram positive rods that appeared in individual or cluster signifier. Besides, from direct observation it was seen that the bacteriums besides produced endospores which narrowed our genus possibilities to six: Bacillus, Sporolactobacillus, Clostridium, Desulfotomaculum, Sporosarcina, and Oscillospira ( Sneath et al. 1986 ) . Since the bacteria was bacillar, motile, aerophilic and positive for catalase the genus was determined to be Bacillus since it was the lone one of the six which possessed these characterisitics. The staying informations from the trials and observations was used to find the species of the Bacillus. Positive consequences for starch hydrolysis, denitrification, growing at pH 5, growing at salt concentrations a‰?5 % and mesophilic temperature demands narrowed the individuality of the bacteria down to two species Bacillus subtilis and B. anthracis ( Sneath et al. 1986 ) . B. anthracis was eliminated as possibility because unlike most Bacillus bugs it is non motile, and besides colony morphology best supported B. subtilis ( Sneath et al. 1986 ) .

    The Bacillus genus is capable of organizing endospores, constructions which allow bacteriums to turn and last in unfavourable environmental conditions, as they are immune to inordinate heat, stop deading and dehydration ( Robertson and Egger, 2008 ) . The presence of endospores is important because to gives the bug the ability to make good in diverse sum of home grounds.

    B. subtilis is a bacteria normally found in dirt, H2O beginnings and in association with workss ( Kunst et al. 1997 ) . Bacillus subtilis, can let the debasement of organic polymers in dirt, and hence play a polar function in the biochemical cycling of C and N ( Emmert and Handelsman, 1999 ) . It besides can advance works growing and protect against fungous pathogen onslaught ( Harsh et al. 2004 ) . B. subtilis and its close relations are an of import beginning of industrial enzymes every bit good ( such as amylases and peptidases ) , and much of the commercial involvement in these bacteriums arises from their capacity to release these enzymes at gm per liter concentrations. It has hence been used for the survey of protein secernment and for development as a host for the production of heterologic proteins ( Kunst et al. 1997 ) .

    Other trials which may hold helped to place the bacterial isolate which were non performed include the Voges-Proskauer trial and citrate use trial. The Voges-proskauer trial is a colorimetric process that detects the acetoin precursor of butanediol and is positive with butanediol fermenters. Butanediol agitation is characteristic of Enterobacter and some species of Bacillus ( Prescott et al. , 2005 ) . The citrate use trial determines if citrate is used as the exclusive C beginning ensuing in alkalinization of the medium ( Prescott et al. , 2005 ) . These trials would of hold been helpful because merely a few of the Bacillus species are positive for these trials and this would hold been of import in contracting down the individuality of the bacteria ( Sneath et al. 1986 ) . Some of the trials performed during the experiment provided limited information sing bacterial designation because the consequences were applicable to many bugs. The ammonification and H2S decrease trials could hold been excluded as these consequences were non used in the designation of the bacteria.

    Numerous beginnings of mistake occurred over the four-week period of this experiment when finding the individuality of the unknown bacteria. Contamination may hold occurred due to hapless sterile technique including sterilisation of research lab equipment and work bench. Besides, some of the chemical trials may hold yielded false negative consequences due to deficient bacterial growing in some broth civilizations. Even though there was a possibility of mistakes over the class of the experiment, the aims were met as the bacteria isolated from a wood dirt sample was identified as Bacillus subtilis.

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