Environmental Isolate Lab Report

Abstract

There are several means of identifying microbes that are collected from their natural habitat surrounding because of their diversity in nature.  Some of these means include; test on enzyme activities (hydrolysis), metabolic test patterns and enumeration of a microbial community.  In this experimental study, a microorganism sample was collected from an environment of Asp parking garage which was then inoculated for microbes in the sample to grow.  Later, three distinct and properly isolated colonies of bacteria were sampled and sub-cultured further to obtain pure cultures where one suitable culture was isolated and used for subsequent identification tests. Based on the presumptive findings and identification characteristics such as metabolism, biochemical cycling, and cell morphology, the identity of the environment isolate was determined to be a bacteria of genus Sporosarcina.

Introduction

 The microbial communities are extensively diverse and they are naturally present in almost every surrounding.  In this exercise of environmental Isolate, microorganisms are isolated from their natural habitat and identified through several series of bacterial culture tests.  The swabbed microorganism sample was obtained from Asp parking garage in a doorway of a metal threshold.  The purpose of this experiment exercise was to identify the genus of the swabbed microorganism sample (unknown bacterial culture) through series of  biochemical, cultural characteristics,  and  studied staining procedures such as Catalase, Oxidase, Starch hydrolysis, Casein, Urea, an Gelatin hydrolysis for independent identification of genus from a bacterial culture that is unknown (Kim et al., 2016).

I.A.  Principle

 The method principle of extracting (isolation) of microorganisms from their natural sources entails microorganism dilution in a suitable medium and finally smeared on Petri dishes’ surfaces as suspensions.  Growth of microorganisms on agar (Petri plates) media involves mass formation termed as colonies. Colony refers to discrete entity containing visible and many cells on a given medium preferable solid (Christensen  & Martin, 2017).  An assumption is made that each bacteria cell that is visible will separately develop into a discrete entity of a given colony.  However, multiple cell arrangements are usually formed by an organism and the resultant chains of a colony may contain several bacteria chains and not one bacterium.  Hence, CFUs (Colony Forming Units) is determined on the basis of known dilutions.

Materials and Method

Various experiments were performed throughout the semester to test the morphological and biochemical diversity of our experimental isolates. All of the methods, techniques, and instructions were taken and performed out of “Microbiology Laboratory Theory & Application” Fourth Edition by Michael J. Leboffe and Burton E. Pierce. All information can be found below in Table 1.

Table 1: Various experiments performed and the outcomes compared to known controls.

Results

Table 2:  Identification test results for Sporosarcina  

Test

Results

Gram stain

Positive

Cell shape of the bacteria

Rod

Capsule  and Acid- Fast

No (-)

Formation of Endospore

Yes

Category: Aerotolerance                                   Facultative aerobe

OF-glucose

Fermentation/ weak Oxidation

Glucose: Phenol Red

A/-

Lactose: Phenol Red

-/-

Sucrose: Phenol Red

A/-

Catalase

Positive

Oxidase

Positive

Nitrate reductase

Positive/ no gas

Decarboxylase of Arginine

(-)

Decarboxylase of Lysine

(+)

Decarboxylase of Ornithine

(+)

Methyl Red

–

Voges – Proskauer

+

Citrate Test

Negative

Indole

(-)

Urea Hydrolysis

Negative

Starch Hydrolysis

Positive

Casein

Positive

Gelatin

Positive

 All results performed throughout the semester can be found above in Table 2. These were the primary experiments observed in determining the genus of the EI. When looking through Bergey’s book of Bacteria, the main tests that aided in identifying the genus of my environmental isolate were the Starch, Urea, Casein, and Gelatin Hydrolysis’. Even helping me narrow the search down even more to find my EI shows great similarity with S.halopila. Both have been proven to be positive for all tests besides urea. My EI is supported by the controls it was compared with, which can be found in Table 1, located in the Materials and Methods section.

Discussion

 Based on gram strain, cell and colony morphology, the potential identity of the microorganism was first reduced to genera Staphylococcus, Sporosarcina, Arthrobacter, Sarcina, and Micrococcus.  Identification attributes such as cell morphology, Motility, colony morphology, and ammonification narrowly indicated characteristics of Sporosarcina genus.  These assumptions were further supported by negative indole test, starch hydrolysis, lack of gas production on nitrate reductase test or denitrification, and negative test on Urea hydrolysis.  As much as the results were indicating positive tests on catalase and oxidase of Sporosarcina, there are species of this bacteria which give negative oxidase within the genus.  In addition, the findings showed that aggregation of Sporosarcina cells were of tetrads or diplococci form, but other previously done studies have suggested that some genus species of Sporosarcina occur as single just the way isolated bacterial colony occurred. As the isolated colonies of bacteria, Sporosarcina are either occurring in psychrophilic or psychrotrophic form.  However, there was lack of denitrification for the isolated colonies of bacteria as it is expected for some given genus species of Sporosarcina.  Ammonium ions nitrification into nitrate was an erroneous characteristic since the isolated bacteria is not expected to undergo nitrification process. Typically, the heterotrophic nitrification is a characteristic of nitrifying microbes such as `Nitrosococcus, Nitrobacter and Nitrosomonas (Christensen  & Martin, 2017).

 According to other done studies, the catalase tests for Sporosarcina are always positive. This was one of the facts that strongly helped in identifying the isolated bacterium. Unlike for the Sporosarcina, catalase test will only give negative results for the case of Nitrobacter, Actinomycetes, and Sarcina. Besides this, the mentioned bacteria did not match with identification characteristics from the isolated bacteria after a series of biochemical tests. Since it wasn’t difficult to interpret and standardize catalase test, the positive results found was a clear indication that the isolated bacteria were of Sporosarcina genus. However, when identifying bacteria such as Sporosarcina, the tests’ success, and reliability is put into uttermost consideration.  In addition, some tests technique such as take practice and gram-staining which were used need to be properly conducted. For the purpose of obtaining more precise results, it is significant if several cultures are prepared in test media and duplicate performance of the tests themselves (Gopal et al., 2015).

Conclusion

 To the best possible extent of the employed observations, nutrient, and biochemical tests, the isolated bacteria from the Asp parking garage area was determined to be a genius member of Sporosarcina.  However, all the tests did not comply with expected identity characteristics of the Sporosarcina genus, but the obtained similarity findings between isolated bacteria and Sporosarcina were adequate to establish the genus identity of the microorganism, backed up by looking in Bergey’s Manual of Determinative Bacteriology.  It cannot be certainly determined that the bacteria is of Sporosarcina genus without more RNA or DNA analysis and biochemical testing but the experimental exercise was a success in establishing the most probable identification of the microorganism as Sporosarcina.

Bibliography

Bergey, D. H.Holt, John G., eds. Bergey’s Manual Of Determinative Bacteriology. Philadelphia: Lippincott Williams & Wilkins, 2000. Print.

Cavnas. Canvas.ou.edu. All experimental procedures found here. Spring 2019

Christensen, A., & Martin, G. D. (2017). Identification and bioactive potential of marine microorganisms from selected Florida coastal areas. MicrobiologyOpen, 6(4), e00448.

Gopal, N., Hill, C., Ross, P. R., Beresford, T. P., Fenelon, M. A., & Cotter, P. D. (2015). The prevalence and control of Bacillus and related spore-forming bacteria in the dairy industry. Frontiers in microbiology, 6, 1418.

Kim, H. J., Eom, H. J., Park, C., Jung, J., Shin, B., Kim, W., & Park, W. (2016). Calcium carbonate precipitation by Bacillus and Sporosarcina strains isolated from concrete and analysis of the bacterial community of concrete. J Microbiol Biotechnol, 26, 540-548.

Michael J. Leboffe, Burton E. Pierce. Microbiology Laboratory Theory & Application, 4th Edition. Customized for University of Oklahoma MBIO 3812. 2019. Print