Running head: The Discovery of Unknown 33 & 34

The Discovery of Unknown 33 & 34

Suffolk Community College

Introduction

The Discovery of Unknown 33 & 34

The objective of this experiment is to identify the organisms of two unknown bacterial
cultures. Students must identify the species of the unknown bacteria by utilizing the techniques
and information learned in previous laboratory exercises. These techniques include streaking for
isolation, Gram staining, and specific biochemical tests. Students are given a map known as a
dichotomous key, a guide in determining the identity of their unknown sample.
Identifying microbes using a series of biochemical tests, like those performed by
students, are used in a clinical setting for several important reasons other than taxonomy. It is
used to determine susceptibility to antimicrobial drugs, gain information for future treatments,
identify pathogens in terms of their potential danger, aid epidemiologists in tracing sources of
infections, and to accumulate data of interest to those studying infectious diseases (Tortora, G. J.,
Funke, B. R., & Case, C. L., 2016).
Results
The first steps to identifying the two unknown microorganisms in tube 33 & 34 is to
perform a Gram Stain, prepare a MacConkey Agar plate, and subculture each unknown on to a
Blood Heart Infusion Agar slant.
Table 1.1: Gram Staining result for unknown 33
PURPOSE
REAGENT

OBSERVATION

RESULT

Identify and classify bacteria as

Crystal violet, Iodine,

Pink rod rod-shaped

Gram Negative Bacilli

gram negative or gram positive.

alcohol, & Safranin

bacterium

Table 1.2: Gram Staining result for unknown 34

PURPOSE
REAGENT
Identify and classify bacteria as
Crystal violet, Iodine,

OBSERVATION
Purple cocci that groups in

gram negative or gram positive

pairs and short chains

alcohol, & Safranin

RESULT
Gram Positive cocci

Streaking for isolation is a method used to obtain a pure culture of a bacteria on a

medium. The MacConkey (MAC) Agar plate is a selective and differential culture medium used
here to isolate gram-negative bacteria and identify a lactose or non-lactose fermenter. The Brain

The Discovery of Unknown 33 & 34

Heart Infusion Agar (BHIA) slant is a general-purpose medium used for the isolation,
cultivation, and maintenance of a variety of microorganisms (Beishir, L., Cappuccino, J.,
Sherman, N., Johnson, T. R., & Case, C. L. 2015). BHIA slant also allow students to observe
bacterium growth pattern for more clues. The unknown microbes are inoculated using aseptic
technique and placed in each media. The now inoculated MAC Agar plate and BHIA slant of
unknown 33 & 34 are then incubated for 48 hours at 37 C for optimal growth.
Table 2.1: Streaking for Isolation result for unknown 33
CULTURE MEDIA PURPOSE
REAGENT

OBSERVATION

RESULT

MAC AGAR

Demonstrate the

Crystal violet dye,

Bright pink halo, bile

Ferment lactose-

PLATE

ability of a gram-

lactose, neutral red

precipitant (haze) around the

produced an acidic

negative bacterium

indicator, and bile

colonies, and pink colony

environment that

to metabolize

salts

growth. Shiny mucoid colonies

appears pink. Gram

that are slightly raised

BHIA SLANT

Lactose
Used for

Brain heart infusion,

A spreading growth pattern.

Neg.
Facultative

maintenance and

peptone, and glucose

Appeared as a cloudy film

anaerobe.

preservation of pure

components.

over the whole surface of

cultures

slant.

Table 2.2: Streaking for Isolation result for unknown 34

CULTURE MEDIA
PURPOSE
REAGENT
MAC AGAR
Demonstrate the
Crystal violet dye,
PLATE

BHIA SLANT

OBSERVATION
No Growth presence

RESULT
The organism is

ability of a gram-

lactose, neutral red

inhibited by the

negative bacterium

indicator, and bile

bile salts and

to metabolize

salts

crystal violets -

Lactose
Used for

Brain heart infusion,

A zig-zagged pattern. Less

Gram Pos.
Facultative

maintenance and

peptone, and glucose

growth observed on the

anaerobic.

preservation of pure

components.

surface of slant.

cultures

The introductory tests of unknown 33 has determined thus far that it is a gram-negative
rod, a facultative anaerobe, and a lactose fermenter. In following the dichotomous key chart, a set

The Discovery of Unknown 33 & 34

of biochemical tests known as IMViC are performed. IMViC is a series of tests designed to
differentiate the species of Enterobacteriaceae family (Beishir, L., Cappuccino, J., Sherman, N.,
Johnson, T. R., & Case, C. L., 2015). The key gives students three possible outcomes of species
in the Enterobacteriaceae family; Escherichia coli, Enterobacter cloacae, and Klebsiella
pneumoniae. The specific medium for each test are inoculated with unknown 33 and after
incubation a distinctive reagent is added to each tube. The results of each IMViC test assist in
drawing a conclusion. Two additional biochemical analysis are performed along with IMViC test
to confirm the identity of unknown 33. Urease and Motility Test.
3.1 IMViC Biochemical test result of unknown 33
TEST PERFORMED
SUBSTRATE
Indole Test for Indole production
Tryptophan

ENZYME
Tryptophanase

Methyl Red- Detects the

Glucose

concentration of acid
Voges-Proskauer- Detects presence

Glucose

a-naphthol

of acetoin.
Citrate- Ability to use citrate

Citrate

Citrase

PRODUCT
Indole

RESULT
Positive-red ring

Lactic or acetic acid

on top noted
Positive

An absence of

Negative

Acetylacetylmethylcarbinols

Absence of sodium

Negative-

carbonate
3.2 Biochemical test result of unknown 33
TEST PERFORMED
SUBSTRATE
Urease Test Test the ability to
Urea
break down urea with urease.
Motility Test Observe medium

ENZYME
Urease

Semi-solid agar

PRODUCT
No presence of ammonia

RESULT
Negative

Growth confined to stab-

Non-motile

line and surrounding

turbidity. (Movement/flagellum)

medium is clear.

The beginning sequences of tests to bacterial unknown 34 concludes that it is a grampositive coccus and a facultative anaerobe. Catalase Test are performed to establish if the culture
is a Staphylococcus sp. or a Streptococcus sp.
4.1 Biochemical Test Result of unknown 34: Catalase Test
TEST PERFORMED
SUBSTRATE ENZYME
Catalase Test- If catalase is present
Hydrogen
Catalase
peroxide

PRODUCT
No presence of the enzyme

RESULT
Negative -no

catalase

reaction

The Discovery of Unknown 33 & 34

The negative Catalase Test determines unknown 34 is a Streptococcus sp. To distinguish

34 between S. pyogenes, S. lactis, and S. faecalis, four-specific test must be implemented (figure
4.2). The Bacitracin disks are placed into a heavy inoculated Blood Agar plate (BAP) to observe
the microbes response. The specific media are incubated and interpreted within 48 hours.
4.2 Biochemical Test Results for unknown 34
TEST PERFORMED
SUBSTRATE
Tolerance Test (Osmotic
6.5% NaCl

PRODUCT
No dextrose fermentation

RESULT
Negative no

Pressure) The ability of a

(acid reaction). Organism does

growth

microbe to tolerate a salt

not tolerant salt.

environment.
BHIA Plate incubated at

Brain heart infusion,

45 C Growth

peptone, and glucose

BAP/Bacitracin Test

components.
Nutritious material

resistant/susceptible

like blood

BAP/ Hemolysis Detects

RBC

hemolytic activity

ENZYME

______

_______________

Negative no
growth

There is no diminished growth

Resistant - no

______

in the area surrounding area of

zone of inhibition

Hemolysins

the bacitracin disk

No lysis. Organism will grow,

noted
Gamma- no

but will not lyse

greenish color or
clearing observed

Conclusion
Unknown 33 is Escherichia coli the ubiquitous, gut-dwelling bacteria. Beginning with
Gram Staining, A series of different reagents are added to the heat fixed culture; crystal violet,

iodine, ethanol (distinguishes gram-positive from gram-negative cells) and lastly safranin
(Beishir, L., Cappuccino, J., Sherman, N., Johnson, T. R., & Case, C. L., 2015). The gramnegative cells appear pink a characteristic of Escherichia coli. The rod shape is also noted and a
telling sign of E. coli. The family Enterobacteriaceae is a medically important gram-negative
bacillus. They have been described to cause human infection, but the species, Escherichia coli is
a major cause of gastroenteritis. In the interim of Gram Staining, my unknown numbered slides

The Discovery of Unknown 33 & 34

were accidently stained causing me to be unsure of which unknown was negative or positive.
Unknown 33 was re-tested. The MAC Agar plate test further assisted in clarifying my gramnegative results with bacterial growth. The neutral red pH indicator registered a lactose fermenter
turning the medium pink, unique sign of E. coli. An inoculated tryptophan broth for the Indole
Test showed a red ring on top of the broth indicating a positive reaction when Kovacs reagent

was added. It was also noted that of my choices of the Enterobacteria family, E. coli appeared to
be the only positive Indole Test and negative Citrate Test. Comparably, culture 33 and E. coli
produce indole from tryptophan using the enzyme tryptophanase. Indole is significant in the
intestinal tract as it regulates diverse microbial processes. The Methyl Red test is another useful
test in detecting E. coli because it detects the concentration of acid end products produced during
glucose fermentation. The MR-VP broth contains glucose so possible fermentation can take
place; after incubation, methyl red is added. Unknown 33 turns red immediately indicating E.
coli. A positive Methyl Red Test means enough acid was fermented to overcome the buffering
capacity of the broth, thereby decreasing the pH revealing the production of lactic acid. E. coli is
a facultative anaerobe as well as an enteric bacterium. Oxygen is not readily available in our gut;
therefore, it can ferment glucose to metabolize. The following results concluded to Escherichia
coli as unknown 33.
Unknown 34 is better known as Streptococcus lactis. A bacterium used for manufacturing

dairy products, such as buttermilk and cheeses. Starting with a Gram Stain the culture is
observed to be a gram-positive coccus with no growth prominent on the MAC Agar plate. The
MacConkey Agar consists of bile salts and crystal violets that does not allow gram-positive
microbes to grow (Beishir, L., Cappuccino, J., Sherman, N., Johnson, T. R., & Case, C. L.,
2015). When the Catalase Test is conducted, it can be established that the culture is a

The Discovery of Unknown 33 & 34

streptococcus sp. and has characteristics of S. lactic. Moving forward with the biochemical test,
S. lactis stood out among the streptococcus sp. because the bacterium was bacitracin resistant,
gamma hemolytic, and showed no growth in the presence of salt at a temperature of 45 C. When
S. lactis is added to milk, the bacterium uses enzymes to metabolize the lactose to lactic acid. S.
lactis is not a Group D Streptococci so it cannot metabolize in 6.5 % NaCl agar plate, and
although BHIA is optimal for some bacteria to grow it not as suitable for mesophilic S. lactic
because lactose is not present. An enriched media blood agar has highly nutritious materials for
fastidious S. lactis. The blood agar allows growth and indicates if the streptococcus species is
hemolytic. S. lactis is noted to be a gamma hemolysis because there was no significant change in
its appearance of the medium surrounding the colonies during growth. The evidence of the
various test concludes that unknown 34 is Streptococcus lactis.
References
Bacteriology. Encyclopedia. Issues & Controversies. Infobase Learning, n.d. Web. 29
Nov. 2016. <http://icof.infobaselearning.com/icofencyarticle.aspx?ID=1755>.
Beishir, L., Cappuccino, J., Sherman, N., Johnson, T. R., & Case, C. L. (2015). Microbiology
Laboratory Manual. Boston, MA: Pearson Learning Solution.
Tortora, G. J., Funke, B. R., & Case, C. L. (2016). Microbiology: An introduction. San
Francisco, CA: Benjamin Cummings
Trek, J., Mira, N., & Jarboe, L. (2015). Adaptation and tolerance of bacteria against acetic acid.
Applied Microbiology & Biotechnology, 99(15), 6215-6229. doi:10.1007/s00253-0156762-3

The Discovery of Unknown 33 & 34

The Discovery of Unknown 33 & 34