The Microbiology of Music


The Microbiology of Music – Describing a Microbe Found in a Flute Headjoint

Chaya Pike

Biol 342, Spring 2017



It has long been the practice of well-meaning parents to encourage their offspring to pick up a musical instrument, in the hopes that it will make them smarter, more disciplined, or more attractive to selective schools. Whether or not any of these hopes are realized, few parents likely recognize the potential dangers lurking within the wind instruments their children play. Several authors have documented respiratory infections, some of them serious, contracted from saxophones (Lodha & Sharma, 1988; Metzger et al., 2010), trombones (Metersky et al., 2010), and other woodwind and brass instruments (Glass, Conrad, Kohler, & Bullard, 2011; Marshall & Levy, 2011; Rackley & Meltzer, 2011). While not all microorganisms are inherently pathogenic to humans, the warm, moist (and rarely cleaned) interior surfaces of wind instruments provide a prime environment for a wide variety of pathogenic and non-pathogenic microbes to grow.

The objective of this paper is to isolate, characterize, and identify a slow-growing bacterium collected from the interior surface of a metal flute headjoint. Methods used to identify the bacterium include a battery of physiological testing and genomic sequencing.

Intriguingly, this microbe was one of only two from the sample capable of growing under room-temperature conditions on TSA. This is consistent with previous studies that have found reduced microbial loads on non-reed wind instruments compared to reed instruments (Marshall & Levy, 2011).


Sample collection and isolation

Two damp, sterile swabs were used to collect microbial samples from the inside surface of a metal flute headjoint, just below the embouchure hole. The flute had been played but not cleaned approximately twenty-four hours previously. The swabs were then used to inoculate a trypticase soy agar (TSA) plate which was then incubated at room temperature for five days.

Once visible colonies had formed, a single colony from the TSA plate was used to inoculate a fresh TSA plate using the streak plate method. This was repeated once more to obtain a pure culture.

Morphological and physiological characterization

The pure culture was examined for details about colony size, color, shape, distribution, and other relevant colony morphological characteristics. A Gram stain was performed on the isolate using the procedure outlined in Lab Handout 4, and the stained isolate was examined under the microscope for the aforementioned morphological characteristics, as well as to determine if the isolate was Gram positive or Gram negative.

Catalase and oxidase tests were performed to determine the enzymes used by the isolate in the electron transport chain, and a suite of 21 physiological tests (including tests for fermentation of glucose, mannitol, sorbitol, and others) were performed using an API 20E test strip. Protocols for each of these tests, described in Lab Handout 6, was followed exactly.

DNA extraction and analysis

A trypticase soy broth was inoculated with the isolate, but did not yield enough cells to perform a proper genomic DNA extraction, so additional colonies from an agar plate were added to the broth culture immediately prior to DNA extraction. Excepting this detail, genomic DNA extraction protocol described in Lab Handout 5 was followed. Using a PowerSoil DNA kit, DNA was isolated from the liquid culture and sequenced using the Illumina MiSeq DNA sequencer.

Genomic data produced during DNA sequencing was analyzed using Illumina BaseSpace. The genome was assembled using the SPAdes Genomic Assembler tool, taxonomic assignments were made using the Kraken Metagenomics tool, and functional genes were identified using the Prokka Gene Annotation tool. Additional analyses were performed using BLAST nucleotide alignment, and Dr. Eric Collins conducted analysis using Bandage software to assemble the isolate genome.


Morphology and physiological tests

The isolate colonies appeared to be perfectly circular, and quite small (<2mm diameter). They were a consistent matte coral-pink color, and projected slightly from the surface of the agar plate. The colonies were incredibly slow-growing, and it took three days for the initial environmental sample to demonstrate visible microbial growth. Growth rates did not appear to be affected by temperature, and the isolate was similarly slow-growing in 37oC incubator and 4oC  refrigerator alike. When viewed under the microscope, the cell morphology was difficult to discern – the cells could either be irregularly shaped and arranged in chains, or elongate with constrictions along each cell (Figure 1).

Figure 1: Gram-stained isolate under 1000x magnification

Results of the Gram-stain indicate that the isolate is Gram-negative. Tests for catalase and oxidase were also negative. Results from the API 20E test strip are shown in Table 1. The only positive physiological tests for this isolate were for arginine dihydrolase, gelatinase, and glucose metabolism. Arginine dihydrolase is present in cells that use arginine as a source of carbon and energy, gelatinase is used to break down gelatin into useful sub-compounds, and glucose fermentation enables cells to use glucose as a carbon source and produces acidic byproducts.

Table 1: Results of API 20 E test strip.

Subtest Reaction/Enzyme Result
ONPG beta-galactosidase negative
ADH arginine dihydrolase positive
LDC lysine decarboxylase negative
ODC ornithine decarboxylase negative
CIT citrate utilization negative
H2S H2S production negative
URE urease negative
TDA tryptophane deaminase negative
IND indole production negative
VP acetoin production negative
GEL gelatinase positive
GLU glucose fermentation/oxidation positive
MAN mannitol fermentation/oxidation negative
INO inositol fermentation/oxidation negative
SOR sorbitol fermentation/oxidation negative
RHA rhamnose fermentation/oxidation negative
SAC saccharose fermentation/oxidation negative
MEL melibiose fermentation/oxidation negative
AMY amygdalin fermentation/oxidation negative
ARA arabinose fermentation/oxidation negative


DNA analysis

Genome assembly software yielded 347 contigs of greater than 1000 bp in length from the isolate, with a maximum length of 45702 bp. Functional gene annotation software found 55 tRNAs within the isolate sample, and a total of 2197 coding genes. Metagenomic analysis classified the isolate as Micrococcus luteus, with 98.75% of reads classified to the species level (79.85% of analyzed reads).

As M. luteus is morphologically and physiologically unlike the isolate in question, contamination was suspected. Further bioinformatic analysis conducted by Dr. Eric Collins suggests the isolate is another species within the genus Micrococcus, and likely possesses the mercury(II) reductase gene. This gene enables the microbe to use elemental mercury, NADP+, and H+ as substrates for the generation of NADPH, with Hg2+ as a byproduct.


The morphological, physiological, and genetic results of this study are inconsistent and inconclusive. Morphologically, the isolate cells bear some resemblance to fungal hyphae, but considering that nothing grew on the Sabouraud agar plate, which selects for fungi, this result is highly unlikely. Genetic sequencing suggests the isolate may be the species Micrococcus luteus, but morphological observations and physiological tests do not align with this species identification. M. luteus forms yellow or greenish-yellow colonies on plated cultures, and the cells are cocci arranged in tetrads, while the colonies of this isolate are pinkish-coral colored with irregularly shaped, elongate cells (Kocur, Pacova, & Martinec, 1972). Furthermore, M. luteus is a Gram-positive, oxidase-positive, gelatinase-positive species, while this isolate is Gram-negative, oxidase-negative, and gelatinase-positive (Kocur et al., 1972).

Additional bioinformatic analysis done by Alexis Walker suggest another possible species within the genus Micrococcus M. roseus is similar in color to the isolate, and is also oxidase-negative (Mohana, Thippeswamy, & Abhishek, 2013). However, both M. roseus and M. luteus are catalase positive and negative for arginine dihydrolase, which is inconsistent with the physiological test results of this isolate. A summary of morphological and biochemical characteristics for M. luteus, M. roseus, and this isolate appears in Table 2.


Characteristic M. luteus M. roseus Isolate
pigmentation yellow red pink/coral
cell morphology tetra head coccus tetra head coccus pleiomorphic/ undefined
Gram stain positive positive negative
catalase test positive positive negative
oxidase test positive negative negative
glucose fermentation negative positive positive
gelatin hydrolysis positive negative positive
arginine dihydrolase negative negative positive

Table 2. Morphological and biochemical characteristics of M. luteus, M. roseus, and experimental isolate (Government of Canada, 2011; Kocur et al., 1972; Mohana et al., 2013).


Bioinformatic analysis done by Dr. Eric Collins suggests that the genus Micrococcus may be a viable identity for this isolate, even if M. luteus and M. roseus are not. Species within the genus Micrococcus are ubiquitous, and can be found on human skin and any surfaces human skin has been in contact with, which potentially includes the surfaces of a musical instrument (Government of Canada, 2011). However, all known species of Micrococcus are Gram-positive, which this isolate is not (Government of Canada, 2011; Mohana et al., 2013).

Additionally, Dr. Collins’ analysis identified the mercury(II) reductase (merA) gene as a likely component of the isolate genome. The merA gene is part of the mer operon, which reduces Hg(II) to Hg(0) and operates optimally at a slightly basic pH and temperatures between 37℃ and 45℃ (Giovanella, Cabral, Bento, Gianello, & Camargo, 2016). This isolate was sampled from the inside of a flute headjoint composed of silver and nickel, both of which are known to have some inhibitory effect on bacterial growth (Argueta-Figueroa, Morales-Luckie, Scougall-Vilchis, & Olea-Mejía, 2014/8; Clement & Jarrett, 1994). It is possible that the isolate used a similar resistance mechanism to the merA gene to live in a silver and nickel enriched environment, but that mechanism was not found and is purely conjectural.

This isolate is extremely slow-growing, and ultimately did not survive in plated culture for more than seven weeks. As such, it was not possible to streak a sufficient number of plates to guarantee that the culture was pure, though it visually appeared free from contaminants. It is likely that M. luteus was a contaminant in the culture or was introduced as a contaminant during the DNA extraction process. Additionally, because the cultures used for physiological tests were two weeks old or older, the results of those tests may be invalid.

In conclusion, the results of morphological observations, physiological tests, and genomic sequencing are inconsistent, and these inconsistencies could be due to incorrect lab techniques, contamination, or other factors. In future, when characterizing this microbe, it would be useful to test different growth media and incubation temperatures, as TSA and 37℃ were clearly not favorable growing conditions for this organism. Additionally, it would be fruitful to extract and sequence several samples of DNA from multiple colonies, to increase the probability of identifying non-contaminant sequences.


Argueta-Figueroa, L., Morales-Luckie, R. A., Scougall-Vilchis, R. J., & Olea-Mejía, O. F. (2014/8). Synthesis, characterization and antibacterial activity of copper, nickel and bimetallic Cu—Ni nanoparticles for potential use in dental materials. Progress in Natural Science: Materials International, 24(4), 321—328.

Clement, J. L., & Jarrett, P. S. (1994). Antibacterial silver. Metal-Based Drugs, 1(5-6), 467—482.

Giovanella, P., Cabral, L., Bento, F. M., Gianello, C., & Camargo, F. A. O. (2016). Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A. New Biotechnology, 33(1), 216—223.

Glass, R. T., Conrad, R. S., Kohler, G. A., & Bullard, J. W. (2011). Evaluation of the microbial flora found in woodwind and brass instruments and their potential to transmit diseases. General Dentistry, 59(2), 100—7; quiz 108—9.

Government of Canada, P. H. A. of C. (2011, April 19). Micrococcus Pathogen Safety Data Sheet. Retrieved April 12, 2017, from

Kocur, M., Pacova, Z., & Martinec, T. (1972). Taxonomic Status of Micrococcus luteus (Schroeter 1872) Cohn 1872, and Designation of the Neotype Strain. International Journal of Systematic Bacteriology, 22(4), 218—223.

Lodha, S., & Sharma, O. P. (1988). Hypersensitivity pneumonitis in a saxophone player. Chest, 93(6), 1322.

Marshall, B., & Levy, S. (2011). Microbial contamination of musical wind instruments. International Journal of Environmental Health Research, 21(4), 275—285.

Metersky, M. L., Bean, S. B., Meyer, J. D., Mutambudzi, M., Brown-Elliott, B. A., Wechsler, M. E., & Wallace, R. J., Jr. (2010). Trombone player’s lung: a probable new cause of hypersensitivity pneumonitis. Chest, 138(3), 754—756.

Metzger, F., Haccuria, A., Reboux, G., Nolard, N., Dalphin, J.-C., & De Vuyst, P. (2010). Hypersensitivity pneumonitis due to molds in a saxophone player. Chest, 138(3), 724—726.

Mohana, D. C., Thippeswamy, S., & Abhishek, R. U. (2013). Antioxidant, antibacterial, and ultraviolet-protective properties of carotenoids isolated from Micrococcus spp. Radiation Protection and Environment, 36(4), 168.

Rackley, C. R., & Meltzer, E. B. (2011). Throw caution to the wind instruments. Chest, 139(3), 729; author reply 729—30.


Gram Skein Cheater’s Gloves

Hello all! This art project comes to you from Chaya, the person who sits in the front row and knits during lectures. As you may have guessed, I knit something for my art project, and you’ll find pictures and my artist’s statement on my Ravelry project page. The artist’s statement will be towards the bottom of the page, and you can click the pictures to make them bigger.


Updating the Hygiene Hypothesis

From  The New York Times  on June 3rd, 2016 – Educate Your Immune System


This article summarizes recent research done on the development of autoimmune diseases (Type I diabetes, celiac disease, severe allergies, etc.) in children who grew up in different microbial environments – represented by households in Finland, Estonia, and the Karelia region of Russia. Studies found that, when factors such as diet and breastfeeding were controlled for, toddlers  who grew up in Finland were four times as likely as those in Karelia to develop precursors for Type I diabetes, and the two groups had very few similarities between their microbiomes. Karelia is a significantly poorer area than most of Finland, and many households drink untreated well water, so researchers hypothesized that early exposure to microbes from the environment “taught” the toddlers’ immune systems how to respond appropriately to common environmental pathogens, and so they developed fewer autoimmune issues.


We’ve discussed acquired immunity in class, but this area of research takes it a bit farther, and suggests that our microbiomes and  when we are exposed to certain microbes may play a larger role in our immune development than previously thought. Other studies mentioned in this article found that children who were exposed to certain pathogens at a young age were much less likely to develop autoimmune diseases than those that first encountered the same pathogens as teenagers or adults. This updates the hygiene hypothesis (which I think we discussed briefly?), which essentially says that people exposed to fewer kinds of microbes during their development tend to be sicker than those that were exposed to a wider variety of microbes.

Critical Analysis:

I appreciated  the angle this article took, describing autoimmune diseases and decreased exposure to diverse microbial communities as an issue of the 21st century. The author did an excellent job of defining terms and ideas that may be foreign to the lay reader, and I think this article is accessible to a wide range of audiences. However, the article implicitly assumed that the relationship between early microbial exposure and autoimmune disease was proven, and I don’t think any of the studies examined in the piece proved a causal relationship. Popular science writing needs to be careful not to assume causation when it has not been proven!


How might we (ethically) prove a link between childhood microbial exposure and autoimmune disease?

Air Pollution + Biofilms = Disease?

From  The Atlantic on April 12th, 2017 – Air Pollution Might Make Dangerous Bacteria Harder to Kill


This article discusses a recent study that examined the effects of black carbon (a major component of air pollution) on the growth and antibiotic resistance of common opportunistic pathogens within the human microbiome –  Staphylococcus aureus  and  Streptococcus pneumoniae. The researchers found that the addition of black carbon to plated cultures of the two species changed the morphology of their respective biofilms and increased their antibiotic resistance, as well as increasing their pathogenicity when applied to the nasal mucosa of mice.


We’ve discussed how bacteria develop resistance to antibiotics in class, and while we don’t know which kinds of antibiotics were tested (other than penicillin), we can conjecture as to the mechanism through which the bacteria developed their resistance. Since I imagine black carbon is not a favored carbon source of bacteria, it may encourage the survival of bacteria with more efflux pumps, to remove the black carbon from the cells.

Critical Analysis:

It’s fascinating that research into the effects of air pollution on the microbes affecting human health was not done until so recently – especially when the effects of air pollution on  disease are  already well-documented. This article didn’t contain any factual errors (as far as I know), and was careful not to generalize the results of mouse studies to humans. The author also did a good job of defining terms that the lay reader may not be familiar with (biofilm, microbiome, etc.), and was careful to represent the results of the study accurately. Now, if only more science writing was this clear!


What mechanisms are used by bacteria to adapt to air pollution that also increase their pathogenicity?

Want to Boost Test Scores? Stop Misinterpreting Science.

From the  Education Week blog “Inside School Research” on May 25th, 2010 – Want to Boost Test Scores? Try Eating Dirt



This blog post (sponsored by a leading periodical in K-12 education) attempts to summarize the findings of this study, which found that the ingestion of a common soil bacterium,  Mycobacterium vaccae,  temporarily improved the anxiety levels and maze navigation abilities of a small group of mice. The blog post suggests that school gardening projects may produce a similar effect in small children.


The study in question examined both mice that had been injected with dead  Mycobacterium, and mice who had ingested live  Mycobacterium.  Each group of mice experienced some effect, which suggests that the compound or compounds responsible for producing the anxiety-reducing and learning-enhancing effects in mice may be a passive component of the bacterial cell wall, rather than an active mechanism that only functions in live microbes.

Critical Analysis:

This blog post has an extremely misleading title, and the text of the post contains major misinterpretations of the scientific study in question. Though the author attempts to say that the study doesn’t mean that children who eat dirt are smarter, it does imply throughout that consuming certain bacteria can change the base level intelligence of a child.  Clearly, this is an absurd claim, and a misinterpretation of the research. Not only is generalizing small animal studies to humans thoroughly unreasonable, it is also entirely possible that the potential reduction in anxiety experienced by the mice is what contributed to their improved maze navigation, and any anxiety-reducing agent would have had a similar effect.

In a world where a 10 year old’s test scores can determine a teacher’s income, we simply cannot justify misusing microbiology to tell  tired educators that the solution to low test scores is to make students play in the school garden.


How can we more effectively communicate the implications of our research to both educators and the public at large?

Microbial Painting

For my microbial painting, I chose to keep things simple in an attempt to reduce unexpected results and produce a visually pleasing piece. However, there was clearly some unexpected cross-contamination! For this piece, I used EMB agar (which selects for gram-negative bacteria and differentiates between lactose fermenters and non-fermenters) as my “canvas” and two strains of bacteria as my “paint.”  Citrobacter freundii,  a strongly acidic fermenter of lactose, turns dark red on this medium, and so I used it to outline and fill my heart. Then, I used  Proteus mirabilis, a less acidic lactose fermenter, as my ” pink paint” for the small dots along the edge of the plate. There was some cross-contamination along the outline of the heart, which I suspect is a result of cells dropping off of the loop when I lifted it over the plate. An unexpected result, but very pretty nonetheless!

Introductory Post

Hello all! My name is Chaya Pike, and I am a junior/senior studying Biology and Elementary Education, with a minor in Marine Science. I love taking the amazing concepts I learn in my science courses and translating them for non-scientists to enjoy – this is why I want to teach, though research interests me as well. Last summer I taught a microscope camp for families at the Sitka Sound Science Center, and it was a blast! I was astonished to discover that children as young as six were able to use compound microscopes effectively. In preparing for the camp, we discovered that we had a collection of almost 2000 prepared slides from Sheldon Jackson College, and above is a picture of me cleaning and sorting just a few of them.