Laboratory report of identification of unknown #43 bacteria

Introduction

Bacterial identification is one of the major aspects that are being observed in identification, diagnosis, prevention, and treatment of diseases that are caused by bacteria. In order to identify these bacteria, the bacterial classification has to be followed to the latter to reveal various characteristics of each and how it reacts to some chemicals and the environmental changes. Bacterial classification depends on six main characteristics which include morphology and arrangements, staining characteristics, cultural characteristics, antigenic structures, and base composition of bacterial DNA (Berger and Andrew 50). These six characteristics are often used in identification of any unknown bacteria of interest that might be suspected to cause a certain disease.

The morphology and arrangements of bacteria is classified as:  Cocci, these are oval or round bacteria that may appear in single, pairs, cluster, or chains. Bacilli, these are stick- like with rounded, swollen or square ends, Coccobacilli are short rods, and spiral base bacteria. In terms of staining, bacteria stain either gram positive or gram negative depending on the composition of the cell wall (Perin and Luis 20). This technique uses different dyes and acts as the first approach in bacterial identification since it helps in identifying the bacterial morphology. Different bacteria respond to certain dyes in different manners with distinct color change. In this way, it quite easy to predict which kind of bacteria is present before moving to the next step of identification.

Cultural characteristic are the response that is observed when different bacterial are subjected to certain media. The media contain different food substances that if utilized by the bacteria, forms observable color change that are easy to distinguish. Likewise, biochemical tests assist in identification of substances that are within the living organism. In bacteria, it is the qualitative and quantitative test for substance or chemical such as enzymes that are available in the bacteria (Mourani et al. 7). The tests include catalase tests, oxidase tests, Indole tests, blood agar tests, and Triple sugar iron tests.

All these tests are capable of giving accurate results when the procedure and methodologies are keenly observed. However, there are some bacteria that are closely related such that the bacteriological tests cannot differentiate them. In this way, the last step of bacteriological identification is the observation of the bacterial genome that is the identification by the polymerase chain reaction that accurately identifies the genome of every bacterium. In this experiment, the unknown bacteria #43 was to be identified using the bacterial identification methods.

 

Aim

To identify the unknown # 43 bacteria

Methods

  • Gram staining
  • Biochemical reactions (oxidase, catalase)
  • Culture method (MSA, CNA, blood agar, bile Esculin, and Starch Agar)

Media

MSA Agar, CAN, Starch Agar, TSA, and bile Esculin Agar

Materials

Microscopes, specimen, test tubes, sterile loop, Bunsen burner, and gram stain reagents

Procedure

  • The materials for the procedure were cleaned and made ready. This was to avoid any contamination and also to prevent any delay in the processes that are quick. All the needed media were made as per the instruction to avoid any faulty result that could emerge as a result of poor media preparation.
  • To achieve the correct results, all laboratory machines were calibrated according to the instructions. This included the laboratory and incubators temperatures.
  • The first procedure was the inoculation of the unknown bacteria to the TSA plate. The plate has casein and soya nutrients which facilitates the growth of all types of bacteria including the fastidious bacteria. The main aim of this procedure was to speed up the the growth of the bacteria and to observe their morphology. This was done under aseptic conditions. The results were recorded.
  • After 2 days, gram staining was done to indicate whether the unknown specimen was a gram negative or a gram positive bacterium. The procedure of gram staining was followed as per the laboratory manual. The time limit between additions of each reagent was observed to avoid faulty results. The results were recorded.
  • After gram staining, culturing method was done. The sample was inoculated on the CNA agar. This is a selective medium that inhibits the growth of gram negative bacteria but selects gram positive. The colistin and naladixic are responsible for its function.
  • The sample was then inoculated on the Mannitol Salt agar and the results were observed and recorded. The wire loop was sterilized thoroughly to minimize the rate of contamination.
  • The sample was then inoculated on the blood agar medium to test for the beta and alpha hemolysis that is common with bacteria. The observation was made and recorded.
  • Well prepared starch media was used to investigate the growth of the 6 and lastly, Bile Esculin test was done and the color changes that were observed were recorded.

Biochemical tests

The unknown bacterium was inoculated on a sterile test tube containing hydrogen peroxide. This was to determine whether the bacteria had the catalase enzyme to break hydrogen peroxide to water and oxygen. The results were observed and recorded.

The last test that was done was oxidase test. The bacterium was streaked on nutrient agar and was incubated for 24 hours. The oxidase reagent was observed if there were color changes and the results recorded.

Results

45 was found to be gram positive Cocci

TEST GROWTH APPEARENCE COLOR POSITIVE/NEGATIVE
TSA growth cocci    
MSA No growth   Pink  
CNA growth cocci   Gram positive
BLOOD AGAR     Green  
STARCH MEDIA growth amylase Brown  
BILE ESCULINE TEST growth enterococci Black  
CATALASE TEST

 

OXIDASE TEST

 

      Negative

 

negative

 

Discussion

The first step of the test was the TSA test. This general media supports the growth of many bacteria, hence, it is difficult to conclude which bacteria are being investigated. However, the TSA test facilitated the growth of the known bacteria so that it could be used in the next level. After Gram Staining was done and the result found to be gram positive cocci, it was concluded that the bacteria belongs to micrococcus spp, staphylococcus spp, enterococcus spp, and streptococcus spp since they are the only gram positive cocci bacteria (Shah and Saheer 600). On the next step, Can test confirmed the presence of gram positive bacteria since this medium contains colistin and naladixic that inhibits the growth of gram negative bacteria. In this way, all gram negative bacteria that may lead to faulty results were inhibited.

The catalase test that is used to determine if the bacteria has the catalase enzyme which is capable of converting the hydrogen peroxide to oxygen and water was found to be negative. This suggests that, the bacteria belong to the Streptococcus spp and Enterococcus spp since Staphylococcus and Micrococcus spp tests positive in hydrogen peroxide.  The starch test was negative since there was no consumption of the starch in the media which made the plate black. This still suggested the presence of Streptococcus and Enterococcus species.

From the results, the Bile Esculin test was found to be positive. This selective deferential agar that is usually used to identify and isolate members of the Enterococcus genus especially group D. This is the determinant test of this practical since members of Enterococcus are able to grow in the oxgall and are capable of hydrolyzing Esculin to glucose resulting to the black color. When the test is positive there is the present of Enterococci spp and not Streptococci spp.

Conclusion

Following the results from the experiment, especially the biochemical and the Bile Esculin test, it can be concluded that the unknown #43 was Enterococcus durans. It can be proved following the experiment from the gram staining reaction to the biochemical tests.

Enterococcus durans is one of the Enterococcus Species which was known as Streptococcus durans around 1984. The bacterium is a gram positive, coccus in appearances and oxidase-negative. Colonies on blood agar are smooth, circular, and entire. It requires amino acid and vitamin for its growth in synthetic media. According to Shah and Saheer (500), some strains of Enterococcus affects human directly. For example, durans strain M4 to M5 and their metabolic product such as butyrate induce certain significant ant-inflammatory reactions as well as preservation of some intestinal epithelial integrity (Singh et al. 315). This suggests that, it is important in prophylactic treatment and prevention of inflammatory diseases that affect the bowel of other animals and man.

 

                                                                   Works Cited

Berger, Andrew J., and Andrew J. Qinqyuan Zhu. “Identification Of Oral Bacteria By Raman Microspectroscopy.”Journal Of Modern Optics 50.15-17 (2003): 2375

Mourani, Peter M., et al. “Molecular Identification Of Bacteria In Tracheal Aspirate Fluid From Mechanically Ventilated Preterm Infants.” Plos ONE 6.10 (2011): 1-7.

Perin, Luana Martins, and Luís Augusto Nero. “Antagonistic Lactic Acid Bacteria Isolated From Goat Milk And Identification Of A Novel Nisin Variant Lactococcus Lactis.” BMC Microbiology 14.1 (2014): 1-21

Shah, Haroun N., and Saheer E. Gharbia. “MALDI-TOF MS For Microbial Identification: Years Of Experimental Development To An Established Protocol.” Mass Spectrometry For Microbial Proteomics (2010)

Singh, Rahul Kunwar, Rajesh Sharma, and Shree Parkash Tiwari. Recent Advances In Microbiology. New York: Nova Biomedical, 2013

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