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
