Lab Report

 Enterococcus sp. Streptococcus thermophiles, Micrococcus luteus, and Melissococcus plutonius isolated from contact lens storage.




There are about 125 million contact lens wearers worldwide. (Kay JE, 2007) Aside from the people with one-day use only lenses, most contact lens wearers are required to have contact storage to keep their lenses moisturized and protected from potential contaminants. With the help of cleaning solution, the lens itself can be easily washed and disinfected, however the cases are often easily disregarded and left untreated. According to the American Academy of Ophthalmology (AAO), and the American Optometric Association (AOA), it is recommended to replace the storage case at least every three months as microbes from fingers and surrounding area will eventually build up along the bottom and sides of the cases. Also, since many people keep their cases in the bathroom, there are further chances of having contamination that could eventually be transferred to our eyes.

This lab project focused on isolating microbes from a contact lens case that has been used for about 2 months and deciphering whether the found microbes can potentially cause eye infection. To investigate what types of bacteria may have been building up, inoculated samples went through isolation to pure culture. The pure culture was then sequenced to obtain a partial genome via shotgun metagenomics. Identification of my isolated helped to decipher whether it has potential to cause microbial corneal infection, which is a serious complication of contact lens wear.

The environment which housed my contact lens case in this study was a bathroom shared by 4 people. Myself and 2 more people wear contact lenses and keep the storage case extremely near the bathroom sink or inside the cabinet. The bathroom, although a convenient place to keep a contact lens case, is a very hospitable environment for microbes. Microbes can be introduced by potential splashes of droplets while washing hands near the sin, drying hair or shaving.

From this lab project, Enterococcus was found to be the majority portion growing on my contact lens case. Enterococcus in general can cause from minor infection that can be treated with ease to serious infections such as Endocarditis that requires numerous antibiotic treatment as Enterococcus possess intrinsic nature to be resistant to antibiotic drugs and thus is currently the second leading cause of healthcare associated bacteremia. (Agudelo, N, et al. 2014)



Sample collection and isolation

A sterile swab was used to collect samples from the sides and bottom surfaces of a contact lens case. Since the surface of the case was dry, the swab was first dipped into sterile water then was used to streak the sample surfaces and then the tryptic soy agar (TSA). After the inoculation, the plates were left in room temperature for growth.

Once visible colonies formed on the TSA plate, a single colony was inoculated using the aseptic technique to a new TSA plate. The new TSA plate was then incubated at 37 °C quadrant streak method several times until the microbes on the plate were isolated and pure.

 Identification and characterization

When the isolated colonies were cultured, physical morphology was first recorded by observing uniformity of physical characteristics such as color, size and shape. Then Gram staining technique was used to stain the isolated culture and was placed under a microscope to see if the culture was either Gram positive, negative or Gram variable. Morphology of the culture was noted again, as the 100X magnification provided clearer images of the culture’s physical properties.

Further detailed physiological tests were performed such as fluid thiogylcollate test to determine oxygen class, oxidase test to determine if the isolated sample has cytochrome c oxidase, catalase test to see if the strain has the enzyme catalase and lastly API 20E test which consists of 21 miniaturized physiological tests.

For detailed genomic identification, a tryptic soy broth was inoculated with my isolate using aseptic technique. The DNA was then extracted using a Powersoil DNA kit. Once a pure solution of DNA in buffer was made, it went through DNA Core lab for genome sequencing using the Illumina MiSeq DNA sequencer. The results were analyzed on BaseSpace through SPAdes Genome Assembler (to assemble the genome), Kraken metagenomics (for identification of isolate down to genus and species level) and Prokka Genome Annotation (to annotate genes and determine the function).

Antibiotic susceptibility test

In preparation for the antibiotic susceptibility test, my isolate was transferred to a broth culture then incubated. With fresh isolate the culture was spread evenly throughout the two TSA plates. Using the spring-loaded antibiotic disk dispenser, 8 different antibiotics: piperacillin, cefazolin, oxacillin, cefotaxime, tobramycin, amikacin, clindamycin and trimethoprim were placed on the plates. Once the cultures were grown with antibiotic disks, diameter of the zones of inhibition were measured to determine the susceptibility.




The Gram stain revealed that the bacterium was Gram variable and under the microscope and was generally cocci shaped and clustered together. Taxonomic results generated from Kraken Metagenomics revealed that my isolate was Enterococcus and these attributes fitted well with the physical description of Enterococcus from numerous literatures.

Physiological tests included fluid thioglycolate test, oxidase test, catalase test and API 20E strip test. The results for fluid thioglycolate test indicated that my bacterium is a facultative anaerobe as it grew throughout the tube but heavily on oxidized area. For the oxidase and catalase tests, my bacterium showed positive results meaning it can use oxygen as a terminal electron acceptor in respiration and will produce catalase, protecting it from the toxic by-products of oxygen metabolism.

From the API 20E test, my bacterium showed 5 positive results. CIT was positive, indicating the utilization of citrate as the only carbon source. The URE test was positive which tested for the enzyme urease. The TDA test was positive which indicates that the enzyme tryptophan deaminase was detected. The VP test was positive meaning that acetoin or 2,3-butanediol were formed from fermentation. Lastly, a positive GEL test indicated that my bacterium is capable of producing gelatinase enzyme which liquefies gelatin.

From BaseSpace Sequence Hub, the taxonomic results generated from Kraken Metagenomics application yielded 95% reads up to genus level. (Figure 1) From running nucleotide BLAST, different genuses that incorporated the remaining 5% were also found which were: Streptococcus thermophiles, Micrococcus luteus, and Melissococcus plutonius. Along with numbers of different genus, there were also numerous species of Enterococcus present as well.

Figure 1. Classification Statistics generated from Kraken Metagenomics

Upon analysis of my bacterium’s antibiotic susceptiblity, 8 different antibiotics were usded and later analyzed for its inhibition zone diameter (Figure 2)

Antibiotics Measured Inhibition Zone Diameter Susceptibility
Piperacillian 48mm Susceptible
Cefazolin 50mm Susceptible
Oxacillin 18mm Susceptible
Cefatoxime 52m Susceptible
Tobramycin 14mm Intermediate
Amikacin 24mm Susceptible
Trimethoprim 14mm Intermediate
Clindamycin 20mm Intermediate

Figure 2. Potential susceptibility of 8 different antibiotics tested on Enterococcus




There were numerous differences between the taxonomic identification and associated literature and results from Gram staining and physiological tests for this microbe. The genus Enterococcus is a Gram-positive, and oxidase and catalase negative cocci. (Castillo-Rojas et al, 2013) This disagrees with majority of the results I acquired from physiological testing as my isolate showed both catalase and oxidase positive.

There are few explanation for this phenomenon. First, my isolated sample, after the bioinformatics analysis using Kraken Metagenomics turned out to be impure. Although the microscopic morphology (all cocci-shaped) looked identical from a TSA plate, there existed numerous species of Enterococcus. In addition to my analyses using Kraken, further taxonomic identification provided via BLAST analysis, revealed there were also species from different genera such as Micrococcus, Streptococcus, and Melissococcus. Thus, it is probable that the numerous contaminants are likely what skewed the physiological test. All the identified species but Micrococcus have both oxidase and catalase negative and Gram-positive and Micrococcus was the only species with both catalase and oxidase positive result. Considering my physiological result, it is probable that Micrococcus leutus heavily influenced the data.

For antibiotic susceptibility, Enterococci are intrinsically resistant to many commonly used antimicrobial agents and show decreased susceptibility to penicillin and ampicillin. Also, they are intrinsically resistant to clindamycin (Miller et al. 2014) which was used during the lab. However, the result disagreed with the literature as my isolate showed susceptibility to almost every antibiotic that was tested. Clindamycin was analyzed as intermediate by observing the zone of inhibition which somewhat fits with the literature.

The reason why antibiotic susceptibility test disagreed with the literature could be because my microbe had numerous Enterococcus species.  It is common for strains of species not showing resistance when another strains do because different strains could have adapted or mutated differently to their fitting situations.

The bacteria that were identified through bioinformatics seemed reasonable as I could see why each of them were present on the surfaces of the contact storage. Enterococcus, which inhabits in GI tract or contaminated water, could have been transferred from water droplets from washing hands from myself and 3 other people as I do keep my contact storage extremely close to the sink. Streptococcus thermophiles are microbes that reside in dairy product such as yogurt and milk. I do consume lots of dairy products daily. Micrococcus luetus are found often on mammalian skin and mouth. Considering the location of where my contact storage is, I wasn’t surprised that this microbe was also present. The most unusual microbe was the Melissococcus plutonius which is a pathogenic agent of European foulbrood (Okumura et al. 2012) killing honeybee larvae.

Out of these identified microbes, only Enterococcus faecalis can cause endophthalmitis which is an inflammation to an eye that can potentially cause permeant eye sight damage. However, literatures have mentioned that the cases of endopthalmitis were only occurring to patients who recently underwent eye-related surgery with Enterococcus faecalis contaminated surgical equipment. (Rishi et al. 2009) From this study, Enterococcus that was found on my contact storage cannot cause eye inflammation and so do the other microbes that were also found.

In conclusion, the lab project was successful as isolation and identification of microbes from contact storage revealed multiple species of Enterococcus and different genus. However, further research needs to be done to completely isolate and culture microbes as catalase and oxidase test for my culture did not agree with the literature results. Future researches about possible microbes living inside of contact storage would be interesting as this portion is not exposed to the environment and usually is filled up with cleaning solution that most claims to kill 99% of the bacteria.


Literature Cited


Agudelo, N. I., M. M. Huycke, M. S. Gilmore, D. B. Clewell, Y. Ike, and N. Shankar. “Enterococcal Disease, Epidemiology, and Implications for Treatment.”  National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 28 Apr. 2017.

Castillo-Rojas, Gonzalo, Marisa Mazari-Hiríart, Sergio Ponce De León, Rosa I. Amieva-Fernández, Raúl A. Agis-Juárez, Johannes Huebner, and Yolanda López-Vidal. “Comparison of Enterococcus Faecium and Enterococcus Faecalis Strains Isolated from Water and Clinical Samples: Antimicrobial Susceptibility and Genetic Relationships.”PLOS ONE. Public Library of Science, 1 Apr. 2013.

Kay, JE. “Development of Contact Lenses and Their Worldwide Use.” NCBI, Nov. 2007.

Miller William, Munita M Jose, and Arias A Cesar “Mechanisms of Antibiotic Resistance in Enterococci’ NCBI 15 May. 2015.

Okumuraab, Kayo, Rie Araicd, Masatoshi Okurae, Teruo Kirikaeb, Daisuke Takamatsuce, and Makoto Osakie* And. “Kayo Okumura.”  Journal of Bacteriology. N.p., 01 June 2012.

Rishi, E., P. Rishi, K. Nandi, D. Shroff, and KL Therese. “Endophthalmitis Caused by Enterococcus Faecalis: A Case Series.”  NCBI. Retina, 9 Feb. 2009.

A2: Babies, honey, and botulism

Babies, honey, and botulism


Summary: Honey is often sold as a raw agricultural product, meaning it is not pasteurized. This could mean that C. botulinum contaminated honey could be sold in the market. As parents feed contaminated honey to infants, baby’s immature gut provides a hospitable environment for C. botulinum to grow. Since there aren’t many other microbes in infant’s gut, C. botulinum doesn’t get out-competed by other species of bacteria and will grow spores that will produce botulinum toxin. Botulinum toxin blocks nerve impulses and weakens muscle. Due to this symptom, 60% of the babies have trouble breathing and could lead to death.

Connections: We learned that human gut microbes take long time to build and what’s left behind are usually bacteria that benefits mutually between us and the bacteria. Or sometimes we consume steady consumption of probiotics to alter our gut microbe to our benefit. Infants on the other hand, have not developed such a complex gut microbes and when potentially resilient and dangerous bacteria are introduced, they will grow exponentially without any competition making it the dominant portion of the gut microbes.

Critical Analysis: I really liked this article because it tied what we learned about human microbiome to real life and potentially dangerous event. Although infant botulism is rare these days, there are still some cases in developing countries. For instance, Korea about 6 decades ago after the Korean War, was super poor and in ruins. So honey was extremely rare. Parents would try to feed their infants with honey first rather than themselves because they thought it was going to be good for the babies and wanted to feed anything good to their children. This led to numerous incidents of infant botulism and infant’s death. Even nowadays without doctor’s warning or concept of C. botunlium bacteria and its interaction with gut microbes, it is easy to accidentally feed infants with honey.

Questions: The article did not mention at which age can an infant form enough stable gut microbes to intake honey.

Painting With Microbes

Patrick the starfish!
Saitama from OnePunch Man

I decided to paint my favorite cartoon characters – Patrick the starfish and Saitama from OnePunch Man. To paint Patrick, I choose to work with TSA plate because the medium can grow almost every microbes that were given with colors that I wanted. So with TSA plate and S. marcescens (pink colored) I painted Patrick’s outline and it turned out okay!! I wanted to draw his eyes but was scared that microbe would overgrow and ruin the painting so i didn’t.

For Saitama painting, I really hoped the color of the outline would be something more darker, but it did not turn out as I hoped. I’m happy with it though

Overuse of antibiotics brings risks for bees, and for us

Title: Overuse of antibiotics brings risks for bees, and for us


Summary:  Researchers have found that overuse of antibiotics on bees and their hives to prevent  bacterial infections that can affect larvae is actually killing the gut bacteria of the bees. With all the beneficial bacteria decimated, it was easier for harmful bacteria to settle in.

Researchers conducted an experiment by having two groups. One was the control group with bees feeding off from normal honey syrup and the second group was the bees feeding off from a honey syrup with antibiotics mixed in it. After three weeks, they have found that only 1/3 of the population of bees (that were eating honey syrup with antibiotics) survived while the control group had 2/3 population of bees still in the hive.

Connection: Bacteria in our guts and bees don’t form overnight. Clusters of bacteria and biofilms take long period to form in environment such as gut as some die out and the ones that survives will be the bacteria with ability to provide beneficial factors to the host. Disrupting such delicately formed clusters with their own system with a  drug can be extremely harmful. Overuse of antibiotics is a serious issue and the beekeepers should consider how much and how often they are going to treat bees with antibiotics.

Questions: This article explained about harmful effects of antibiotics which might deter beekeepers from using them to prevent potential bacterial infection on larvae. What other solutions are out there that could replace antibiotics yet won’t disrupt bees’ gut microbes?

Prokaryotic Microbes with Eukaryote-like Genes Found

Title: Prokaryotic Microbes with Eukaryote-like Genes Found


Summary: Deep sea microbe (Archaea) was found near the hydro-thermal vent. By using the deep metagenomics techniques, researchers identified the new group of archaea which possessed hallmarks of eukaroytic cells.

Connections: Throughout the class we discussed about similarities and differences amongst the three Bacteria, Archea and Eukarya cells. Archea, although it was grouped as prokaryotes, shared similarities  with Eukaryotic cells such as histones associated DNA, lack of peptidoglycan in cell wall, and Methionine as the initial amino acid for protein synthesis. From this, scientists deduced that the archea and eukayara would have had similar ancestors.

Critical Analysis: From this discovery, the researchers have found similarities between archea and eukarya that was not yet discovered. The researchers found that the newly analyzed archea harbored a suite of genes found in eukaroytes  which are typically used to remodel intracellular membranes to form vesicles. This could have enabled ancestral microbes to evolve into more complex eukaroytic cells. This supports the long-standing hypothesis that archaea are the ancestors of eukaryotes, and helped to fill an evolutionary gap between the two groups.

Questions: The article does mention  that the exact  functions of these eukaryote-like genes in newly found archea  remain still unknown. Although they are similar in sequence, the researchers mentioned that the archaeal genes may be involved in different cellular processes.

Assignment 1: Introduction

Hi my name is Inho Yeo and I’m from South Korea 🙂

I’m a junior pursuing a biological science degree. I’ve been always interested in viruses, diseases, and other dangerous microbes as well as human immunology so I’m super pumped to learn about those in this course :DDDD YAY

I love to travel. I generally save up money during the school year by literally trying to not buy anything – living the typical college student life with ramen noodles. Then once the school is over I hit off to different countries with few of the cash I have left while serving in  Korean military two years ago. I love visiting other countries to see their landmarks and beautiful natures 😀

This was taken at ‘Gardens by the Bay’ in Singapore during this winter break!! The weather was AMAZING