Techniques for Identifying Unknown Bacteria

Section I: Introduction

  Microorganism surrounds the environment which makes them part of our health and well-being, but sometime, they can become harmful and the ability to correctly identify the harmful species is extremely useful in applied microbiology. New technologies and discoveries are happening everyday, but basic microbiology lab techniques and concepts are needed to perform an accurate bacteria species identification and understanding how these techniques work is also important. A few of these concepts include the aseptic method, microscopic examination, selective and differential media and the importance of having a positive and negative control.

The aseptic technique is the most basic and crucial method of culture transfer in order to have the most accurate test results as possible. This technique consist of a transfer method from various types of media by using a microincinerator sterilizer device that produces high heat which reduces the risk of contamination while performing a culture transfer. An inoculating loop or needle is the instrument used to perform the transfer and has to be inserted in the microincinerator before and after each transfer of culture. The aseptic method is crucial in order to isolate pure cultures, as the inoculating loop is sterilized between each step of the culture transfer which eliminates contaminants and leads to the growth of pure culture (1). Contaminants that are present in growth culture can lead to misinterpretation of results and ultimately sabotage the identification of the unknown bacteria, for this reason, using the aseptic method is the most crucial method of culture transfer.

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Microscopic examination is another crucial technique that needs to be mastered as it allows to observe cultures with greater magnification and resolution. This technology permits to distinguish the morphology of the unknown culture and ultimately helps eliminating bacteria that do not fit the characteristics observed via the microscope (2). The microscope has evolved from when it was first introduced by Van Leewenhoek whom was the first scientist to accurately describe bacteria morphology (2). Nowdays, there are several kinds of microscopes such as electron microscope or light microscope, the latter has several subcategories where the bright field microscope is the most popular in a typical microbiology laboratory setting. Bright field microscope allows to see specimen with light coming from underneath and makes it darker than its surroundings (2). Using the differential Gram staining method prior to microscopic observation is often done to give additional information such as classification of the bacteria as Gram positive or Gram negative (1). Knowing the classification also sets the stage for which subsequent tests will be performed in order to determine the identity of the unknown bacteria.

To perform a series of tests, the unknown culture needs to grow on an appropriate media in order to receive the right nutrients and ensure its growth and survival, this is where the use of differential and selective media are employed.  A selective medium contains all the basic nutrients needed, but also has a few components added that inhibits the growth of undesired bacteria (l). A differential medium allows to distinguish between species that are capable of performing certain biochemical processes, which further assists in the identification process of the unknown culture (1) It is important to note that some media can be classified as both selective and differential.

While performing the tests, keeping track of the positive and negative controls is crucial because it is used as a baseline to compare the unknown test results. A positive control for a test means the bacteria performs the reaction tested and a negative control means the bacteria is not capable of performing the reaction tested. Having these controls as a guideline is essential as it eliminates confusion in test results obtained from the unknow culture, and consequently, narrows down possible errors while identifying the unknown culture.

With all the possible species the bacteria domain can have, a sytematic bacterial classification can be challenging to come up with. A useful tool for bacterial classification is the The bacterial species concept which implies that bacteria with a common ancestor share similar physical traits as well as sharing similar genes (3). The biological concept takes it a step further where it implies that biological species should be able to reproduce by interbreeding and cannot breed with a non-self breed due reproduction isolation mechanisms (3). The biological concept is accepted for Eukaryotyic species classification, because genes are transferred from parents to offsrings, but in the case of bacteria this process is not observed, instead bacteria transfer genes via Horizontal Gene Transfer (1). The bacterial species concept is more suited to categorize bacteria species as the concept bases itself on DNA-DNA hybridization, 16S rRNA sequence data and horizontal gene transfer.

 DNA-DNA hybridization is among pioneered technique to measure how close the genomes of two bacteria are (4). This technique calls for precise manipulation such as denaturation and blending the two genome to see how much of it anneals together, the threshold of 70% DNA-DNA hybridization between is used to indicate that the two genomes are closely related (4). Althought the DNA-DNA hybridization is a good indicator of bacterial relatedness, it is not the most precise, because bacteria species can show a wide range of genome variety (3). To employ a more recent technology, scientits can use the 16S rRNA sequence comparison in addition to DNA-DNA hybridization method. Ribosomal RNA is highly conserved so it only makes sense that it evolves at a slow pace, which limits the genome variety issue when trying to find out bacterial relatedness (3). The 16S rRNA sequence is also easier to access and updated as it is a computerized database, but it is not infallible to the genome variability, in part due to bacterial horizontal gene transfer. Horizontal gene transfer as mentioned above, is the way bacteria exchange genome information and unlike Eukaryote that use lateral gene transfer, bacteria do not transfer genes from parents to offsprings (1). Horizontal gene transfer can be done via transformation, conjugation or transduciton, but regardless of the mechanism, the end result is the same. When bacterial transfer genes from one bacterium to another, it ultimately increases genomic variabiltiy. Despite all the effort mentioned above to compare bacterial genome whether it is through DNA-DNA hybridization or 16S rRNA sequencing, classification of bacteria remains a big challenge. In order to overcome these challenges, novel method called genomic-phylogenetic species concept (GPSC) is used. This concept uses both genomic and phylogenic analysis are combined to give a better outcome (5). GPSC is efficient and the most promessing because not only it analyzes DNA, RNA and protein, but it also looks at the phylogeny of the bacteria, which gives insight on the common ancestor and can relate species together even if they are not in the same location (staley)

Section II: Materials and Methods

 The unknown #118 was given to which a series of chosen tests had to be performed in order to identify the unknown bacteria. The unknown #118 came in tryptic soy broth (TSB) to which a serial dilution followed by an aseptic transfer using inoculating loop onto a Tryptic soy agar plate (TSA) was performed (1). Streaking method onto the TSA plate and incubation for a period of 24-48h at 25ºC was necessary for isolation of colonies (1). Two types of colonies were successfully isolated on the TSA plate, one large and the other small. The large colony was white, round, smooth, shiny, and the small colony was punctiform, also white, round, shiny and smooth.

Microscopic Examination and Gram Staining

Both colonies were subjected to Gram staining followed by microscopic examination under 1000X oil immersion using a bright-filed microscope. Gram staining was performed using crystal violet (1min) primary stain, iodine (1min) mordant, 95% ethanol (12-15sec) decolorizing and dehydrating agent followed by immediate distilled water rinse and  finally, safranin (1min) couterstain (1). Gram positive bacteria retains the primary stain crystal violet even after the decolorizing agent because of its thick peptidoglycan membrane (20-80nm) characteristic and can therefore be observed as dark purple under the microscope (l). On the other hand, Gram negative bacteria has a thin cell wall (2-7nm), the decolorizing agent is able to strip away the primary stain and would appear clear under the microscope, the use of a counterstain such as safranin is necessary to stain it pink for easier microscope observation (1). When the large colony was observed under the microscope, it turned out to be Gram negative with rod shape bacteria and the small colony was Gram positive with  short rod shape bacteria as well. The results were compared to the Gram negative control Escherichia coli and the Gram positive control Staphylococcus epidermidis.

Differential and Selective Media

 Mannitol Salt Agar (MSA) is a differential and selective media that contains high salt concentration (7.5% NaCl) that selects for bacteria capable of sustaining such environement (1). The media also has methyl red as ph indicator that turns yellow under acidic condition which indicates sugar is fermented by bacteria (l). The unkonwn #118 was aseptically transferred from TSB onto MSA plate via streaking method, followed by an incubation of  24-48hrs at 25º (1). Positive control for MSA test is Staphylococcus aureus (BSL-2) showed growth and yellow color on plate due to lowering of pH caused by acid build up from sugar fermentation, and  negative control for MSA test Staphylococcus showed no growth on plate.

 Maconkey (MC) is another differential and selective media that contains lactose, bile salts which inhibits Gram positive bacteria growth (1). The media contains neutral red ph indicator which changes color to pink when lactose fermenter is present due to acid release (1). The unkonwn #118 was aseptically transferred from TSB onto MC plate via streaking method, followed by an incubation of  24-48hrs at 35º (1). Positive control for MC test Escherichia coli showed pink growth due to acid build up caused by lactose fermentation, and negative control Pseudonoms fluorenscens showed colorless growth.

Catalase Test

Catalase test is used to determine if the bacteria is able to break down hydrogen peroxide (H2O2) into oxygen gas (O2) via calase enzyme. A loopful of the unknown #118 was deposited on a clean slide to which a drop of hydrogen peroxide (3%) was added on top (1). If bubbles are present, this would indicate that the catalase enzyme is present and bacteria is capable of breaking down H2O2 as observed in the positive control test Micrococcus, negative control Enterococcus showed no apparent bubbles.

Oxidase Test

 Oxidase test was used to determine if the bacteria has cytochrome c in the elecrtron transport chain (1). This test was perfomed by using an oxiswab imbedded with chromogenic redusing agent (TMPD), the cotton was swabbed inside the TSA plate containing the unknown #118, where a good amount of colony growth was present (1). If cytochrome c was present in the electron transport chian, the TMPD gets oxidized and turned deep purple/blue color just similar to the positive control Pseudonomas fluorenscens. On the other hand, negative control Escherichia coli did not show a change of color due to absence of cytochrome c in the electron transport chain.

Motility test

Motility test was used to give additional morphological information regarding the unknown #118 such as if the bacteria is motile or non-motile. The motility test was performed using an inoculating needle to aseptically transfer form unknown TSB onto a soft agar (0.4%) deep tube (1). The inoculating needle was inserted in the middle  of the soft agar (0.4%) in a precise linear fashion, without breaking the agar, and be removed before hitting the bottom of the deep tube in the same linear fashion. The tube was incubated for 48h at 35ºC (1). The use of soft agar (0.4%) is important in this test, because the bacteria needs to be able to move around in case it is motile, otherwise the bacteria would not be able to move and give a false negative test result. It is also important to not break the agar while performing the test, because this can lead to cloudiness and could be interpreted as a false positive result. The positive control  Proteus mobilis had a cloudy appearance due to the bacteria moving around the agar, and negative control Staphylococcus epidermidis (BSL-2)  was clear with only the stab print clearly visible in the tube.

Aerotolerance test

 Aerotolerance test was performed to determine bacteria oxygen requirment. There are different degree to which bacteria require oxygen, aerotolerance test can differentiate between obligate aerobes, facultative anaerobes and obligate anaerobes (1). This test was performed by using an inoculating needle to aseptically transfer from TSB onto agar deep stab tube followed by an incubation period of 48hrs at 35ºC (1). The inoculating needle was carefully inserted in a linear fashion, and removed before hitting the bottom of the tube via the same line of entry. While inserting the needle, it was important to not crack the agar, because it would lead to false test results (1). The controls displayed three different kinds of results: obligate aerobe Micrococcus luteus only showed growths at the very top of the agar, facultative anaerobe Echerichia coli had growth throughout the tube but mosty on top, and obligate anaerobe Clostridium sporogenes had growth toward the bottom of the tube.

Litmus test

The litmus test was performed to find out if the unknown #118 was part of the order Lactobacillales, which are lactic-acid fermenters or to differentiate the members of the family Enterbacteriaceae (l).  An inoculating loop was used to transfer from TSB onto litmus milk test tube and was incubated for 7-14 days at 35ºC (lab). Litmus milk test tube contained skim milk, oxidation-reduciton indicator, azolitmin pH indicator, and Na2SO3 (1). The skim milk consited of nutrients such as casein and whey proteins (1). A different array of results was observed from the controls, where the pH indicator changed color to red to indicate acidic conditions and blue for basic conditions (1). The redox indicator appeared white or colorless in reduced conditions and purple in oxidized condition (1). Uninoculated control had purple appearance, positive control for lactose fermenters were red/pink because of build up of acid and negative control were purple/blue which indicates alkaline reaction. Positive controls: Escherichia coli entire tube was pink, Lactococcus lactis had a pink acid clot and some white indicating litmus reduction. Negative controls: Bacillus subtilis had light purple, white, dark blue, Pseudomonas fluorenscens had some white at the bottom and purple.

Citrate test

 This test was used to determine if the unknown #118 was able to use citrate as the sole carbon source. There are a few members of the Enterobacteriaceae family that  are capable of fermenting citrate, and they can be differentiates by using the citrate test (1). The test was performed by using an inoculating needle to aseptically transfer from TSB onto Simmons’ citrate slant. The needle stabbed the end of the slant about 5mm deep, then was dragged out of the tube via a zig-zag motion followed by an incubation time of 48hrs at 35º (1). Uninoculated Simmons’ citrate slant are initially green in color and turn blue if citrate fermentation takes place. The positive control Klebsiella mobilis was green after incubation while negative control Escherichia coli remained green.

Methyl red/ Voges Proskauer Tests

 Methyl red (MR) and Voges Proskauer (VP) tests were useful to determine if unknown #118 was capable of fermenting mixed acid or butanediol after it was depleted of all glucose (1). Methyl red test was used to detect acidic end products, where as Voges Proskauer was used to detect neutral end products (1). This test was performed by using an inoculating loop and aseptically transfer from TSB onto MRVP broth tube followed by an incubation time of 48hrs at 35ºC (1). After the incubation time, the mixture was separated in equal amounts into two new test tubes, one designated MR and the other VP. In the MR tube, 10 drops of methyl red were added and set aside, where as 5 drops of KOH and 15 drops of alpha-naphtol were added in the VP tube followed by vigourous mixing (1). Positive control for MR Escherichia coli, a mixed acid fermenter, was a red color and negative contol for MR Klebsiella mobilis was yellow/clear in color. Positive control for VP Klebsiella mobilis, a butanediol fermenter,turned into a red color and negative control Escherichia coli had a yellow/clear color.

References

1. Malwane S, Malwane SD. 2018. A Laboratory Manual Microbiology 12^th ed. Morton Publishing Company, Englewood, CO.

2. Madigan MT, Martinko JM, Stahl DA, Clark DP. 2012. Brock Biology of Microorganisms 13th ed. Pearson Benjamin Cummings, San Francisco, CA.
3. Riley MA, Lizotte-Waniewski M. 2009. Population Genomics and the Bacterial Species Concept. Horizontal Gene Transfer Methods in Molecular Biology 532:367–377.DOI: 10.1007/978-1-60327-853-9_21
4. Goris J, Klappenbach JA, Vandamme P, Coenye T, Konstantinidis KT, Tiedje JM. 2007. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. International Journal of Systematic and Evolutionary Microbiology 57:81–91.DOI: 10.1099/ijs.0.64483-0
5. Staley JT. 2006. The bacterial species dilemma and the genomic-phylogenetic species concept. Philosophical Transactions of the Royal Society B: Biological Sciences 361:1899–1909.DOI: 10.1098/rstb.2006.1914