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Comparison of traditional and modern approaches to the identification of bacteria

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Introduction

Comparison of traditional and modern approaches to the identification of bacteria The traditional techniques of biochemical testing in the form of API strips was compared to modern techniques of Polymerase Chain Reaction (PCR) for the accurate identification of bacteria. The PCR technique would make use of 16S rRNA databases from Internet search sites. It was found that both techniques were, in most cases, able to identify each bacterium to at least the genus level. INTRODUCTION Identifying a particular strain of bacteria is very useful since it opens up a wealth of knowledge about it. For instance, if one knows that the bacteria is a member of the genus Micrococcus of the Actinobacteria class, then one would be able to find that it is; Gram-Positive, non-motile, has a G + C content of 70%, is aerobic, it is found mainly on mammalian skin etc. If the bacterium was pathogenic, such as Streptococcus pneumoniae, then one could find that it has the potential to cause meningitis, so one could take precautions against this. Identification of a bacterium can also establish the bacteria's position on a phylogenic tree, and thus decipher the immediate relatives. The traditional methods for identification of bacteria generally required the isolation and growth of bacteria. Bacterial growth is usually characterised by the appearance of colonies on media. Then isolation of the bacteria required can be achieved using selective media. ...read more.

Middle

This enables a large quantity of DNA to be sequenced (more than 1x106) in less than a few hours since each cycle produces an exponential increase for sequenced DNA. The sequences can then be compared to the thousands of already sequenced 16s rRNA genes from the databases available. The result will be displayed with a percentage match next to the name of a bacteria strain, for instance Staphylococcus auerus 91.48%. This would mean that the DNA inputted into the system from the strain analysed had a 91.48% match with the DNA in the database. This suggests one could be quite confident that this was Staphylococcus auerus and almost positive that it was from the Staphylococcus genus. METHODS Preparation of unknown bacteria. A known strain of bacteria was isolated and grown into colonies. Each colony is grown from a single bacterium and is thus a clone of this bacterium. This was prepared by the demonstrators such that they knew what each of the strains was, but I was not informed until the end. Preparation of API strip. API 20E (BioMerieux) strip was inoculated with a test strain by using a pipette to remove a single colony from a pre-made agar containing one species of unknown bacteria. The colony was then mixed into a universal tube containing 5 ml of sterile saline by rubbing the pipette on the wall of the tube. ...read more.

Conclusion

As Dr J. Lodge instructed the class what each strain was, it was then known that this was incorrect, but it demonstrates how 16S rRNA is highly conserved across the microbial world. Therefore, caution must be taken not to accept the highest match given on face value, but to compare to that of the second and third matches. Overall, this method is a useful taxonomic tool. PCR of the 16S rRNA can be performed relatively quickly, with only small amounts of DNA As little as, 10-9g is needed. This technique is now automated in many laboratories and the DNA can be amplified up in less than one hour.This means that many 16S rRNA molecules can be compared in anyone day and thus it is a very quick and reliable method of classifying organisms. Strain Name Score A Enterobacter clocae 16S rRNA gene 0.803 A Enterobacter clocae 256-36 0.797 A Seratia marcescens 16S rRNA gene 0.792 B Pseudomonas aeroginosa 16S rRNA gene 0.831 B Pseudomonas sp. 16S rRNA gene 0.831 F Citrobacter freundi 16S rRNA gene 0.876 F Citrobacter freundi CDC 621-64 0.823 F Citrobacter freundi DSM 300 39 0.823 Figure 3. The scores above refer to how good the match is, with 1.000 a perfect base pair to base pair match and 0.000 being no match at all. Therefore, the highest score for each strain should be the closest match that the database can come up with. In each case the database identified the correct genus, i.e., A: Enterobacter; B: Pseudomonas; F: Citrobacter. ...read more.

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