This document summarizes a student laboratory experiment to identify two unknown bacterial strains, one gram-positive and one gram-negative, through a series of biochemical tests over two weeks. The student's gram-positive strain was identified as Enterococcus faecalis and the gram-negative strain as Salmonella typhimurium based on results from tests including gram staining, catalase, oxidase, hemolytic activity, salt tolerance, bile-esculin hydrolysis, DNase production, and sugar fermentation patterns. A variety of selective and differential media were used to isolate and characterize the bacteria through observation of colony morphology and biochemical reactions.

1. Report for “Unknown” Identification Project
Jessica Olivares
Francisco Alarcon-Chaidez
12/02/2015
Negative and Positive Identification
Of Unknown Bacterial Strains Report
Introduction/Purpose
The objective of this lab was to classify the unknown bacterial strains provided utilizing a number
of biochemical procedures. In this lab each student was assigned two different strands of bacteria,
one strand being a Gram negative bacteria and the other a Gram positive bacteria. Classifying and
knowing the bacteria we had was done through a series of biochemical test. This lab project took
2 weeks of lab experimentation. Depending on each test the unknown strain transitioned to another
test whether it was a positive result or a negative result we would go from there to another test and
soon to finally identifying both of our bacteria’s. By following the procedures and methods of
inoculation the results I obtained showed that my unknown strains were positive bacteria
enterococcus faecalis, and negative bacteria Salmonella typhimurium.
Materials
Types of media used included broth tube of mixed culture unknown bacteria strain #92. Used
selective and differential media, for our unknown bacteria, such as slants, broth, and semisolid
agar plates to identify negative and positive bacteria. To describe the morphology such as the
growth, color, texture, and shape of our unknown strain we inoculated our bacteria into a
differential media grown on a TSA plate. We used selective media MacConkey Agar and
Mannitol Salt Agar for isolating and inhibit growth of the gram negative bacteria and gram
positive bacteria. MacConkey agar is used for the isolation of gram-negative bacteria and
variates lactose fermenting from lactose non-fermenting gram-negative bacteria we also used the
gram staining technique to view the selected bacteria under the microscope. Catalase test was
used to identify catalase negative bacteria the results were facultative in the production of
bubbles and bacteria that test negative do not require oxygen as a terminal electron acceptor
which anaerobes only ferment. Likewise following this we used the oxidase test as well.
Following this we also used Gram positive test to identify unknown such as the identification of
Hemolysis, Bile Esculin, DNase and the salt broth test. Gram-negative bacteria have a very thin
peptidoglycan layer that is in between an inner cell membrane and a bacterial outer membrane
fallowing the gram positive test we also conducted many of the gram negative test used to
identify the correct gram negative sample.
Methods
The lab project took 2 weeks of observation and identification of our unknown strains which began
from Tuesday 11/10/2015-Thursday 11/19/2015 and consisted of four days of working with the
bacteria sample. On the first day each student picked up an unknown mixed broth tube culture that

2. was selected to work for two weeks, my unknown bacteria was number 92. This sample was then
inoculated by streaking for the isolation on a TSA agar plate. We then put the inoculated plate
media into the incubator for the next lab period.
On the second lab day we took our TSA plate and inoculated the TSA plate into two mini Nutrient
Agar plates to store for next the following week, the same day we collected our results from our
TSA agar plate and we streaked our growth of bacteria from our TSA plates to be used for the
gram staining technique in order to isolate both of the organisms. We then had to inoculate the two
species to identify if our bacteria’s were Gram negative or Gram positive because from there we
would follow charts that were specific for Gram negative or Gram positive bacteria. Gram staining
is a method of determining bacterial species into two groups, it distinguishes bacteria by the
chemical and physical properties of their cell walls which detects the peptidoglycan, Gram positive
results in a purple/blue color while a Gram negative results in a pink/red color. Gram staining takes
roughly around 5-8 minutes if done correctly. After gram staining we also conducted the catalase
test which used a small sample of our unknown bacteria this test for a catalase enzyme I added a
drop of hydrogen peroxide and positive bubbling occurs to differentiate between staphylococcus
which is catalase positive and streptococcus which is catalase negative, an oxidase test was also
conducted the same day. After initial gram staining was done TSA plate was used to also streak
MAC Agar plates and MSA plates to test for positive and negative bacteria. We then disposed the
TSA plate and incubated the mini NA plates for the next lab period.
On day three we used are MAC and MSA bacterial growth results to gather the test needed to
identify both the species. We used the identification report sheet to figure out what test we needed
to conduct for the negative and positive bacteria. We again inoculated the media appropriately
using the mini NA plates that were made from the last lab period. After everything had been
properly followed and each test was conducted accordingly we then incubated all the test we
needed and disposed the 2 NA plates because we no longer needed for the experiment.
On the fourth and last day of this lab project we gathered all of our results from the incubator and
recorded them into our data sheets for proper identification. We then examined all of our
biochemical test and filled out our report sheet. After, we cleaned up our entire work station and
disposed all our media and we finished by using our identification chart to match our results with
the identity of our microbe. Finally we checked with our professor to see if our results were correct.
Results and Conclusions
From my results I obtained my unknown sample #92 Enterococcus faecalis as my positive
bacteria and Salmonella typhimurium as my negative bacteria. Below I have provide a data sheet
that illustrates my observations which also shows the conducted test and results of both of my
unknown species.

3. Table 1: The following table has a list of my data recording that were observed during the lab
inoculation of the two species.
For identification of culture
(morphology-growth, color,
texture, shape.Etc.)
Unknown ID Report
Grown on TSA Completely filled with bacterial growth colonies cannot yet be
distinguished which is gram positive or gram negative species.
Grown on MAC (inhibits
negative bacteria)
Pink to red growth with or without bile precipitate, some colonies
were barely pink.
Grown on MSA (inhibits
positive bacteria)
Yellow growth and halo showed up as positive bacteria.
Viewed under microscope
(gram stain)
Gram-positive bacteria species inoculated to identify if our
bacteria’s were Gram negative or Gram positive bacteria.
Chemical and physical properties of their cell walls detected the
peptidoglycan, Gram positive results in a purple/blue color while a
Gram negative results in a pink/red color.
Gram-positive bacteria appeared a bit more red than pink
Gram-negative bacteria appeared a really deep purple color

4. Table 2: By
conducting the
preliminary test I first
streaked the media on
a glass slide. And did
tconducted the gram stains for the species. The catalase test created no bubbles which ment
ngative sample bacteria.
Table 2A: The Hemolysis test is where a
bacteria is inoculated onto blood agar which
uses bacteria sample to test how much
hemolysins the bacteria
produces which is an exotoxin that destroys
red blood cells (RBCs); and in the case of
my bacteria it tested out gamma.
Figure 1: Shows the DNase sample of my inoculated bacteria. The DNase agar contains nutrients
for the bacteria or DNA and uses methyl green as an indicator.
:
Preliminary Test
Gram Stain + -
Catalase + -
Oxidase + -
Gram Positive Test
Hemolysis α β γ
6.5% Salt Broth + -
Bile Esculin + -
DNase + -

5. Figure 2: Shows the hemolysis sample collected of my inoculated bacteria. The blood agar
showed the growth of the bacteria which appears as a pink growth colonies. It showed the
exotoxins that destroys the red blood cells.
Table 2B: Gram negative bacteria was inoculated and put into different vials that was isolated
using differential media. The gram negative test identified the correct microbe.
Gram Negative
Tests
TSI SLANT ALK ACID
BUTT ALK ACID
GAS + -
𝑯 𝟐 𝑺 + -
SIM 𝑯 𝟐 𝑺 + -
INDOLE + -
MOTOLITY + -
PR-GLUCOSE pH + -
GAS + -
PR-SUCROSE pH + -
GAS + -
PR-LACTOSE pH + -
GAS + -
DECARBOXYLASE LYSINE + -
ORINTHINE + -
MR + -
VP + -
CITRATE + -
UREASE + -
PHENYLALANINE DEAMINASE + -

6. Table 3: The proper identification was done by only using the TSI Slants, SIM medium. & The
Decarboxylase.
Table 4: The following table was identified by conducting the appropriate gram stain results
fallowed by the catalase test, oxidase test and the Hemolysis test.