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The Examination and Identification of the F1 Organism Segregated from 10 Other

Organisms with a Dichotomous Key

Abstract: After a scientist has gone missing, a rack of unknown organisms in lysogenic broth

only identifiable by codes were left behind. Due to the critical nature of their work, students

were tasked with identifying the organisms left behind utilizing Bergeys Manual of

Determinative Bacteriology (Holt et al., 1994) to create a dichotomous key. This report focuses

on the organism coded F1 and utilized Gram staining, agar deep, and Voges-Proskauer tests

to determine the organism was Enterobacter dissolvens. This conclusion was supported by

secondary testing and verification with KOH and MALDI TOF experiments.

Introduction: In October of 2017, a microbiologist was working for a lab at Simon Fraser

University until their strange disappearance after working late one night. Seemingly prepared

for this, the individual left behind instructions that were critical to their work in case a medical

emergency happened. Local law enforcement discovered these notes shortly after the

disappearance as well as a rack of lysogenic broth (LB) solutions with codes written on them in

the workspace of the missing. Interestingly, the work and notes of the microbiologist were

found to be extremely encrypted and inaccessible and their lab notebook was found with pages

torn out. In the interests of the missing, students were assigned to identify the coded

organisms left behind so that work may continue when the microbiologist returns or if

someone else is tasked to take on their assignments. One page left behind in the lab notebook

was a list of the organisms that were found in the lab, but with no indication as to which

organism is which (Table 1). A dichotomous key was designed using Bergeys Manual of

Systemic Bacteriology (Holt et al., 1994) to investigate the properties of the organism coded as
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F1. Using the key, bacteria could be organized and systematically determined via experiments

involving biochemical tests, oxygen requirements, and endospore and Gram staining.

Phenotype and colony morphology of all 11 organisms was also examined prior to testing with

plates found in the lab and later compared with resulting streak plates of LB for similarities.

Materials and Methods: The broth culture of F1 was initially used to create a wet mount of

the organism in question and was examined using Kohler illumination and oil immersion

microscopy at a total magnification of 1000x with a compound light microscope (Albright et al.,

2017). After observation, the organism was then plated using streak plate methods on tryptic

soy agar (TSA) and incubated at 37C and 25C for 24 hours (Albright et al., 2017). Following the

creation of culture on a plate, the dichotomous key (Fig. 1) was utilized to a gram stain was

conducted using the outline provided in the lab manual and was confirmed using a 3% KOH test

(Albright et al., 2017). An agar deep test was then administered to examine oxygen

requirement properties of the organism followed by a Voges-Proskauer Test with MR-VP media

(Albright et al., 2017). The results of these tests gave an indication of what organism was

residing in the broth labelled F1 and were confirmed by a MALDI TOF analysis utilizing a mass

spectrometer (Fig. 2). All protocols were followed directly as dictated in the Fall 2017, BISC 303

Lab manual (Albright et al., 2017).

Results: The wet mount observed determined the F1 organism was motile and the TSA plates

showed growth on the 37C plate was plentiful while the 25C plate only exhibited moderate

amounts of growth (Fig. 2). Gram staining revealed that unknown F1 consisted of pink rods

approximately 1 m long in length which occasionally clustered together (Fig. 3). This indicates

that the unknown is a gram-negative organism. To clarify the observation, a 3% KOH test was
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completed which resulted in the sample becoming slightly white in colour, viscous, and with a

string-like consistency when the inoculating loop was lifted from the slide containing the

sample, giving more evidence that the organism is gram-negative due to the positive KOH test

result.

An agar deep tube was inoculated for 48 hours with the unknown and cloudiness in the media

was present throughout the entirety of the length of the stab (Fig 4). The depth of the growth

present in the experiment suggests that the organism can grow in aerobic and anaerobic

conditions, indicating that it is a facultative anaerobe. Of note is the observation that the agar

media at the very end of the test tube was raised from its original position.

After incubation for 24 hours, the addition of Barritts A (alpha-naphthol) and B (40% KOH)

reagents to an inoculated MR-VP broth resulted in a colour change from yellow to red

concluding a positive test result within 1 minute of reagent addition. The test result indicates

the intermediate compound Acetoin was present and provided a turning point for

identification.

MALDI TOF identification results were positive for Enterobacter dissolvens in one of the two

sections used for testing and based on a scoring basis implying results were highly probable

(Table 2). Colonies appeared circular, flat, with an entire margin, smooth, glistening, slimy, and

semi-transparent with a yellow tint similar to the agar media. These results were comparable to

plate morphology observations taken from available TSA plated Enterobacter dissolvens prior to

experimentation.
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Discussion: The tests followed the structure of the dichotomous key (Fig. 1) and revealed that

the unknown F1 was a gram-negative organism consisting of a rod structure which was

confirmed by the positive result of a KOH test. Further investigation with an agar deep revealed

that F1 is a facultative anaerobe, and TSA plates indicated optimal growth was at 37C. A

Voges-Proskauer test yielded a positive result, which indicated Enterobacter dissolvens as the

organism of interest, consistent with information provided by Bergeys Manual of

Determinative Bacteriology (Fig. 1). The result was confirmed with a MALDI TOF test that

provided highly probable results reiterating the conclusion that the organism was Enterobacter

dissolvens (Table 2). One possible issue is the 2 sections used on the plate for MALDI TOF

analysis gave only gave 1 clear result, while the other section provided unreliable identification.

This could be due to an error in technique when placing the samples on the plate. The colony

morphology is consistent with results obtained from TSA plates containing E. dissolvens

observed prior to plating the unknown.

A couple of factors could be further investigated with future studies of this organism. F1 was

capable of growing both in 25C and 37C incubators over a 24 hour period, and could be tested

in different temperature conditions to determine if 37C is truly the most optimal growing

temperature, or if it can grow at higher than 37C or lower than 25C. Further biochemical

testing may also be done to determine why the bottom of the agar deep had lifted and if a gas

was present. MALDI TOF tests could be repeated to ensure that the sample wasnt an outlier

and provide further evidence for the identification of the unknown F1. Some literature (Holt

et al., 1994) claims other tests can be used to give further evidence that identification was

correct, including looking for a positive Simmons Citrate test, positive ornithine test, and
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negative lysine test. It is also important to recognize that in 1986, Enterobacter dissolvens was

moved from the Erwinia genus into Enterobacter due to biochemical testing considering it to be

different enough for the movement (Brenner et al., 1986). More recent literature (Hoffmann et

al., 2005) believes the change must be reversed, as phylogenetic analysis has determined

extreme similarity between Enterobacter dissolvens and Enterobacter cloacae and evidence

constitutes E. dissolvens should become a subspecies of E. cloacae. Further research has found

that E. dissolvens produces high quality yields of 2,3-butanediol, which is a frequently used

chemical with a variety of applications, such as creating solvents for resins (Wang et al., 2012).

It may therefore be useful for the microbiologist to utilize in their work

The purpose of this study set out to identify the unknown F1 through a variety of biochemical

testing, microscopy, oxygen requirement analysis, and staining. Test results and conclusions

such as gram staining and Voges-Proskauer testing were followed up with secondary

experiments to verify results, including a KOH test and MALDI TOF analysis, respectively. The

evidence collected is conclusive enough to declare that F1 is Enterobacter dissolvens and the

missing microbiologist may return to their duties as normal once they return, or their regular

work may proceed with another individual.

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Literature Cited:

Albright, L., Moore, M., Hollmann, P. (2017). BISC 303 Microbiology Laboratory Manual. pp. 13-
15, 19-20, 28-30, 36-39, 61.
Brenner, D. J., McWhorter, A. C., Kai, A., Steigerwalt, A. G., & Farmer, J. J. (1986). Enterobacter
asburiae sp. nov., a new species found in clinical specimens, and reassignment of Erwinia
dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens
comb. nov. and Enterobacter nimipressuralis comb. nov. Journal of Clinical Microbiology, 23(6),
11141120.
Hoffmann, Stindl, Ludwig, Stumpf, Mehlen, Heesemann, . . . Roggenkamp. (2005).
Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E. cloacae subspecies
dissolvens comb. nov. and emended description of Enterobacter asburiae and Enterobacter
kobei. Systematic and Applied Microbiology, 28(3), 196-205.

Holt, J., Krieg, N., Sneath, P., Staley, J., Williams, S. (1994). Bergeys Manual of Determinative
Bacteriology. 9th ed. Lippincott Williams & Wilkins, Maryland.
Wang, Ailong, Xu, Youqiang, Ma, Cuiqing, Gao, Chao, Li, Lixiang, Wang, Yu, . . . Cascales, Eric.
(2012). Efficient 2,3-Butanediol Production from Cassava Powder by a Crop-Biomass-Utilizer,
Enterobacter cloacae subsp. dissolvens SDM (2,3-Butanediol Production). PLoS ONE, 7(7),
E40442.
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Figures and tables:

Figure 1 Dichotomous key Created from Bergeys Manual of Determinative Bacteriology

Descriptions
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Figure 2 Left, an image of 37C incubated unknown F1. Right, an image of 25C incubated
unknown F1. Black marks are redacted (handwriting) information for identity protection.
Growth is much more prevalent on the left image incubated at higher temperature.

Figure 3 Compound microscope image of unknown F1 after Gram staining, revealing pink
rod structures in a cluster. Length of ~1m.
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Figure 4 Unknown F1 inoculated in agar deep media for 24 hours. Growth was visible on the
surface and throughout the length of the agar, as well as gas production at the bottom of the
tube.

Table 1 - List of Organisms Found in the Laboratory that

may be Potential Unknowns
Isolated Organisms in Lysogeny Broth Solution
Escherichia coli Enterobacter dissolvens
Staphylococcus carnosus Pseudomonas fluorescens
Agrobacterium tumefaciens Bacillus coagulans
Lactococcus lactis Micrococcus luteus
Serratia fonticola Enterococcus casseliflavus
Bacillus subtillis
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Table 2 MALDI TOF Results Presented for Unknown F1 Samples