Interpretation of physiological and biochemical properties of an unknown microorganism for

identification via cross-referencing to the biochemical profiles of five bacteria

Jasper Fisher

Lab Partners: N/A

Lab Section 4

November 15, 2020

Abstract

The purpose of this experiment was to identify the unknown microorganism #100 using multiple

differential tests to indicate its physiological and biochemical properties. The tests performed on

the unknown include: the Gram Stain, differential and selective growth on Endo Agar, Eosin

Methylene Blue (EMB) Agar, and Phenylethyl Alcohol (PEA) Agar, the SIM Agar tube (which

incorporates tests for Indole, sulfur reduction, and motility), Kligler’s Iron Agar test (which

incorporates tests for lactose and glucose fermentation, gas production, and sulfur reduction), the

Methyl Red test for fermentation type, the Vogues-Proskauer test for acetoin production, the

Citrate test for alternative carbon source utilization, and the Gelatin Hydrolysis for the presence

of gelatinases in the unknown. Biochemical profiles for five known species were provided for

identification following the completion of the unknown’s profile; these species include

Escherichia coli, Enterobacter aerogenes, Citrobacter freundii, Proteus murabilis, and Serratia

marcescens. Additional completion of the RapID SS/u system for the unknown was completed to

include additional test results for cross-referencing and determining the identity of the unknown.

The unknown #100 was discovered to be a motile, Gram-negative, facultative anaerobic bacilli

that is a mixed-acid fermenter capable of fermenting glucose and citrate, as well as having the

capability to perform sulfur reduction. With this analysis and cross-referencing these

physiological and biochemical characteristics of the unknown to the biochemical profiles of the

known bacteria, it was concluded that the identity of the unknown was Citrobacter freundii.
 Introduction

Identification of organisms based on their biochemical and physiological properties is

essential in multiple applications, notably in clinical laboratories and specimen collection for

health care. Identification tests in medical settings have varying degrees of specificity and

sensitivity to pathogens, with tests high in both of these criteria being able to identify a pathogen

with a high degree of reliability (Madigan et al., 2018). In addition to visual and microscopic

observations of cultured colonies, an example of a direct methodology of identification used in

this report is the Gram-stain test, which directly examines the cell-wall composition of bacteria

through a staining procedure (Madigan et al., 2018; Leboffe & Pierce, 2011). Another method of

assessing the Gram characteristic of a microorganism is by culturing bacteria on various

selective and differential media and observing their growth patterns; for example, the selective

media may permit the growth of Gram-negative bacteria while inhibiting the growth of Gram-

positive bacteria (Leboffe & Pierce, 2011). By combining the results of a wide variety of

biochemical and physiological tests, both indirect and direct methods, unknown organisms can

be determined with a relatively high degree of accuracy by cross-referencing the results of the

unknown to the documented results of other known species.

A common technique for identifying unknown organisms when physiological

observations prove insufficient on their own for reliable identification is to assess various

indirect biochemical tests, such as cellular respiration behaviours. Microorganisms have

mechanisms of cellular respiration that fall into one of 5 categories based on their oxygenic

requirements: aerotolerant anaerobe, facultative anaerobe, strict anaerobe, strict aerobe, or

microaerophile. In organisms capable of performing aerobic respiration, such as microaerophiles

and strict aerobes, oxygen acts as the final electron acceptor in the respiration process (Madigan
et al., 2018; Silverthorn, 2019). Conversely, anaerobic respiration, including aerotolerant and

strict anaerobes, is supported by final electron acceptors that are not oxygen, such as nitrite,

ferric iron, sulfate, and carbon dioxide (Madigan et al., 2018; Silverthorn, 2018). An important

final electron acceptor in anaerobic respiration for this study is inorganic sulfur compounds, such

as SO4-2, which is much less energetically favourable as an electron acceptor in comparison to

oxygen; bacteria capable of sulfur-reduction reduce the compounds to H2S through either

assimilative or dissimilative reduction (Madigan et al., 2018). This reduction and the following

electron transport are what drive the proton motive force for ATP synthesis (Madigan et al.,

2018; Silverthorn et al., 2019).

All of the bacteria assessed in this report are facultative anaerobes, indicating that

respiration can be performed in both anoxic and oxygen-rich environments. As such, all bacteria

examined are capable of performing cellular respiration with oxygen in addition to other

molecules as the final electron acceptor (Madigan et al., 2018). Determining the cellular

respiration pathways used in bacteria is an efficient method to identify and differentiate species,

which can be assessed by finding what specific final electron acceptor is used in cellular

respiration.

In addition to cellular respiration, fermentation capability is a useful tool in identifying

bacteria. Fermentation is a diverse anaerobic process that is performed primarily on sugars in

order to yield energy in the form of ATP, however some organisms are capable of fermenting

other compounds such as amino acids or aromatic compounds (Madigan, 2018). In typical sugar

fermentation, glucose enters the first stage, glycolysis, directly; glucose undergoes a redox

reaction to form two molecules of pyruvate, which then undergoes redox again to result in the

fermentation end-products (Madigan et al., 2018). An example of this is lactic acid fermentation,
in which pyruvate is transformed into lactic acid in addition to the production of 2 ATP

molecules (Madigan et al., 2018; Silverthorn, 2019). Another fermentation process relevant to

this report is Mixed-Acid fermentation, which is typically characterized by the fermentation end-

products of CO2, hydrogen ions, acetic acid, lactic acid, and succinic acid, however specific end-

products can vary between species (Madigan et al., 2018). A variant of Mixed-Acid fermentation

found in this report is Butanediol Mixed-Acid fermentation, which results in the eventual

conversion of pyruvate to acetoin, and finally to 2,3-Butanediol (Madigan et al., 2018). As

bacterial species can be differentiated based on their fermentation pathways, biochemical tests to

identify fermentation mechanisms are essential for bacterial identification; in determining the

identity of the unknown organism #100, five tests that detect fermentation mechanisms are

performed: Aerotolerance (FTM tube), Sulfur reduction (SIM tube), Kliger’s Iron Agar test,

Citrate test, and the Methyl Red-Vogues Proskauer procedure.

By combining the results of multiple biochemical tests, a characteristic profile can be

made for the species in question. Rather than performing multiple identification tests on an

unknown, multi-test systems such as the RapID SS/u are useful in creating a panel of tests that

can be cross-referenced with a database of organisms to quickly determine the unknown’s

identity with a high degree of accuracy (ThermoFisher Scientific, 2020). Catalogues such as this

document the biochemical and physiological profiles of many microorganisms for reference

when identifying an unknown. Taking into account the individual tests performed (see results in

Tables 2 & 3 in appendix) in parallel with the RapID SS/u identification system, it was

determined that the unknown microorganism #100 is Citrobacter freundii.

 Materials and Methods

Procedures followed are as stated in the BIOL 2P98 Lab 4 Lab Manual (Carpenter-

Cleland, 2020). No changes were made to the provided procedure.

Results

Examination of the physical characteristics of the unknown organism indicated that the

species is Gram negative (as indicated by a pink/red hue following the Gram stain procedure).

Individual cells are bacilli in shape and tend to be arranged singularly. When cultured, the

unknown has circular colonies with smooth margins and appears white, opaque, and moist.

Growth in the FTM tube is located throughout the colourless region with no pink at the surface,

indicating the unknown is a facultative anaerobe. Differential and Selective media indicated

growth on the EMB and Endo agars, with no growth occurring on the PEA agar.

Based on the black precipitate in the Sulfur reduction, the red slant/yellow butt of the

glucose fermentation in KIA media, and the blue colour in the citrate tests, the unknown

organism demonstrates that it is capable of using a wide variety of carbon sources for energy

production, both aerobically and anaerobically.

The Indole test in the SIM Agar tube remains colourless at the surface following the

addition of Kovac’s reagent, indicating the species is incapable of producing indole. This tube

also reveals cultures that have grown away from the inoculating stab line, indicating motility.

The Kliger’s Iron Agar test resulted in a red slant portion with a yellow butt, suggesting the

bacteria is able to ferment glucose but not lactose within the 24-hour incubation period. There

are no cracks or lifting of the agar in this tube, indicating that the unknown does not produce gas

in this reaction. Two tests, the Kliger’s Iron Agar test and the H2S test indicate that the organism

is capable of reducing sulfur. The Methyl Red test in the MRVP tube reveals a red top, indicating
the unknown is capable of performing mixed-acid fermentation. The Vogues-Proskaur test in the

MRVP tube is yellow/brown in colour, indicating a negative result for acetoin production. The

Gelatin Hydrolysis test resulted in a solid nutrient gelatin medium, indicating the unknown does

not have gelatinases present.

Table 4. Physiological and biochemical characteristics of unknown organism #100

Test Observation/Result of test Interpretation/Meaning

Gram Stain Colour: Pink/red Circle: GRAM + or -

Cell length (m) 1.0-3.0 No further interpretation of

cell length required
Cell shape & Bacilli/rod, single No further interpretation of
arrangement cellular characteristics
required
Cultural Whole colony shape: circular No further interpretation of
characteristics Edge/margin: entire/smooth cultural characteristics
Optical Characteristic: Opaque required
Colour: White
Growth on selective Endo: Yes Circle: GRAM + or -
medium EMB: Yes
PEA: No
Oxygen requirement Describe growth in FTM: No pink, Facultative anaerobe
growth throughout colourless region

Citrate Test Blue present on medium Bacteria can use citrate as

sole source of carbon
Indole (SIM) No red present, remains yellow Bacteria cannot produce
indole
Voges-Proskauer Yellow/brown colour, no red present Negative
Test
Methyl Red Test Red present at surface Bacteria is a mixed-acid
fermenter
Glucose Yellow butt Bacteria can ferment glucose
fermentation
(KIA after 24 hours)
Lactose Red slant Bacteria cannot ferment
fermentation lactose
(KIA after 24 hours)
Sulfur reduction H2S SIM: Black precipitate Bacteria capable of reducing
(SIM or KIA after sulfur
48 hours) KIA: Black bottom
Gelatin hydrolysis Solid gelatin medium Bacteria does not have
 gelatinases present
Motility (SIM) Radial growth away from stab line Bacteria is motile

Figure 1. Dichotomous key identification flow chart to identify the unknown organism #100

Gelatin Hydrolysis Test

Citrobacter freundii
Enterobacter aerogenes Proteus mirabilis
Escherichia coli Serratia marcescens

Red Test H2S Test

Escherichia coli
Citrobacter freundii Serratia Proteus
marcescens mirabilis
Enterobacter
aerogenes
Indole Test

Citrobacter Escherichia
freundii coli

Performing the RapID SS/u tests revealed positive results in for only the GMS and A2

tests (see Figure 2 in Appendix). With only two positive tests in the RapID SS/u, the organism

had the potential to be all but one of the provided Gram-negative bacilli (Escherichia coli, which

was negative in the A2 test). By combining the results of the RapID SS/u tests, differential
staining, and biochemical tests performed, the identity of unknown organism #100 is likely

Citrobacter freundii.

Discussion

Based on the results of the biochemical, differential, and RapID SS/u tests performed, it

is probable that the identity of unknown organism #100 is Citrobacter freundii. The first step in

reaching this conclusion was to analyse the biochemical, physiological, and differential growth

characteristics of five provided known microorganisms: Escherichia coli, Enterobacter

aerogenes, Citrobacter freundii, Proteus mirabilis, and Serratia marcescens. The biochemical,

differential, and physiological characteristics were summarized into a table that included the

results that each of these organisms would present on each of the tests being performed, as well

as describing their physical and growth characteristics (see Table 2 in appendix; test results

gathered from Garrity et al., 2005, Madigan et al., 2018, and Leboffe & Pierce, 2011). An

additional table was produced to describe the characteristics of all tests to be performed,

including the indicators and media used, the purpose of the test, and the criteria that indicate a

positive or negative result (see Table 3 in appendix, test information gathered from Leboffe &

Pierce, 2011).

After having the base information required to identify the unknown via the tables

mentioned prior, the lab results for unknown organism #100 on each test, as well as its

physiological characteristics when grown on a basic medium, were assessed and catalogued in

Table 1. The test results were provided in the BIOL 2P98 Lab 4 Lab Manual (Carpenter-Cleland,

2020). With the biochemical and physiological data observed, the results of each test were

interpreted by referencing Table 3 to assess whether results were negative or positive and what

these results indicated. Interpretations and observations of the unknown were then cross-
referenced to the results for each of the five knowns in Table 2. Characteristics and test results

that were identical between the unknown and the known species were highlighted by a green cell

and results that were conflicting were marked with a red cell. This provided a clear and concise

visual representation to assess similarities and differences between the species.

Given all biochemical and physiological characteristics of the known and unknown

species had all been documented and cross-referenced, a clear pattern of similarities between the

unknown species and Citrobacter freundii was observed. Of the five known species, Citrobacter

freundii was the only species that did not have any conflicting biochemical test results. With all

tests indicating the same results, a conclusion was reached that the unknown species is likely

Citrobacter freundii.

Using the results in Tables 1 and 2, a dichotomous key identification flowchart was

created to differentiate between the five known species (Figure 1). This key includes the Gelatin

Hydrolysis Test, Methyl Red test, Sulfur Reduction (H2S) test, and the Indole test to

systematically identify the five known species based on their positive or negative results on each

test. Applying the unknown organism #100 to this identification key also leads to the conclusion

that its identity is likely Citrobacter freundii.

Lastly, the unknown was subject to the multi-biochemical test RadID SS/u, with results

provided in the BIOL 2P98 Lab 4 Lab Manual (Carpenter-Cleland, 2020). The results of this test

were much less specific in identifying a specific species as the unknown, as only two positive

results presented – the GMS and A2 tests (see Figure 2 in appendix). As the unknown only

presented two positive results, these criteria also applied to all but one of the provided Gram-

negative bacilli, Escherichia coli (see Figure 3 in appendix). Although a specific identity could

not be distinguished using only 2 positive test results, this test led to the conclusion that the
unknown could not be E. colii, given the conflicting result in the A2 test. As the unknown and E.

coli shared many characteristics on multiple tests, referring to the RapID SS/u system to rule out

E. coli as a possible identity of the unknown was beneficial.

 References

Carpenter-Cleland, C. (2020) BIOL 2P98 2020FW Principles of Microbiology Lab Manual. St.

Catharines, ON: Brock University.

Garrity et. al. (2005) Part B: The Gammaproteobacteria Volume Two The

Proteobacteria of Bergey's Manual of Systematic Bacteriology (2nd edition) published by

Springer in East Lansing, MI. 

Leboffe MJ and Pierce BE (2011) A Photographic Atlas for the Microbiology Laboratory, 4th

edition. Morton Publishing Company, Colorado.

Madigan MT, Bender KS, Buckley DH, Sattley WM, and Stahl DA (2018) Brock Biology of

Microorganisms, 15th edition. Pearson Education, Inc., New York.

Silverthorn, D. U. (2019). Human physiology: An integrated approach, 8th Ed. Pearson

Education, Inc.

ThermoFisher Scientific (n.d.) RapIDTM SS/u System.

https://www.thermofisher.com/order/catalog/product/R8311004?SID=srch-srp-

R8311004#/R8311004?SID=srch-srp-R8311004
 Appendix

Table 2. Physiological and biochemical characteristics of different bacteria. Green cells indicate
shared characteristics with the unknown organism #100. Red cells indicate results that conflict
with the unknown.

Enterobacter
Escherichia

Citrobacter

marcescens
aerogenes
coli DH5

mirabilis

Serratia
freundii

Proteus
inactive

Risk Group 2 2 2 2 2
Cell length (m) 2-6 1.2-3.0 2.0-6.0 1.0-3.0 0.9-2.0
Gram Stain - - - - -
Cell shape & Straight Straight Straight Straight Bacillus/rod
arrangement bacillus/rod bacillus/rod, bacillus/rod: bacillus/rod:
s:singly or single single and swarm
pairs pairs
Colonial Smooth Mucoid and Smooth Uniform film Opaque or
characteristics convex flat, yellow or convex, moist, (swarm) iridescent; white,
(pigment colour) grey moist white colonies translucent or pink, or red
or rough opaque, grey
flat dry dull
wrinkled
Preferred 21-37 30-37 37 (mammal 37 10-36
Temperature intestine)
Range (oC)

Oxygen Facultative Facultative Facultative Facultative Facultative

requirement anaerobe anaerobe anaerobe anaerobe anaerobic
(FTM)
Catalase + + + + +
*Glucose A A/G A/G A/G A/G
fermentation (24
hr KIA)
Lactose - A/G V - -
fermentation (24
hr KIA)
H2S (KIA or - - + + -
SIM)
Methyl Red Test + - + + -
Voges-Proskauer - + - V +
Citrate Test - + + V +
 Indole Test + - - - -
Gelatin - - - + +
hydrolysis
Motility (SIM) v + + + +

Table 3. Summary of the biochemical and differential tests used in this report.

Name of Test Purpose/Import Name of Indicator or + Reaction - Reaction

ance Medium Reagent Result Result
Differential For isolating PhenylEthyl Phenylethyl growth No growth
/selective staphylococci Alcohol Agar alcohol
and streptococci interferes with
or selecting out DNA synthesis
Gram - species in Gram -
organisms
Differential For isolating Eosin Dyes eosin Y Gram -: growth Gram +: no
/selective fecal coliforms Methylene and methylene growth
(enteric lactose Blue Agar blue, inhibit Fermenters:
fermenters) growth of Gram metallic green Slow
+ organisms; sheen Fermenter:
peptone, pink
lactose, sucrose
fermentation Non-
Fermenter: no
colour change
Differential For isolating and Endo Agar Colour Lactose Lactose non-
/selective identifying indicators fermenter: fermenter:
presence of sodium sulfite red/pink colourless
coliforms and basic
fuschin
Aerotolerance Cultivate Fluid Resazurin turns Growth only in Growth
anaerobic or Thioglycollate pink when colourless throughout
microaerophilic Medium oxidized bottom tube: aerobic
bacteria (aerobic (reduced):
top, anaerobic anaerobic
bottom) (pink top)

Fermentation Distinguish Kligler’s Iron Glucose + Glucose Glucose and

of glucose & between species Agar lactose fermented: red lactose not
lactose of 24 hr fermentation: slant, yellow fermented: red
Enterobacteriac pH indicator butt slant and butt
eae (and other Phenol red
Gram – bacilli) Lactose (Not from
fermented: Enterobacteria
Ability to yellow slant ceae)
 ferment glucose and butt
and/or lactose
H2S Detect ability to SIM & Ferrous sulfate Black No black
reduce sulfur to Kligler’s Iron reacting with precipitate precipitate
H2S (via Agar H2S (product of
breakdown of cysteine
cysteine) breakdown)

Motility Detect motility SIM Diffuse growth Radial growth Growth limited
of bacteria away from the outward from to the stab line
inoculation stab stab line
Indole Test Identify bacteria SIM Kovacs’ reagent Indole present: Indole not
that can produce (containing red colour at present: no red
indole via DMABA and surface colour
tryptophanase HCl)
hydrolyzing
pyruvate

Methyl Red Detect bacteria MRVP Methyl Red Mixed-Acid Non-Mixed-

that perform indicator dye fermenter: red Acid
mixed-acid fermenter:
fermentation yellow
resulting in
stable acid end
products

Voges Detect if bacteria MRVP Acetoin Produces Does not

Proskauer are capable of produced acetoin: Red produce
producing detected via VP colour acetoin: no
acetoin from reagents colour change
glucose
degradation
(using 2,3-
butanediol
fermentation)

Citrate Assess ability to Simmond’s Bromthymol Citrate- Citrate-

utilization use citrate as the Citrate blue dye permease permeate not
only source of present: blue present: green
carbon (presence (increased pH) pH at or below
of citrate- 6.9)
permease)

Gelatin Assess if Nutrient Gelatin Liquified Solid gelatin

 hydrolysis bacteria can Gelatin consistency gelatin medium medium (no
produce (Gelatinases gelatinases
gelatinases present) present)
(hydrolizes
gelatin)

Figure 2. Unknown #100 RapID SS/u report form.

Figure 3. RapID SS/U Differential Chart with positive tests of the unknown organism circled.