1. Ryla Best
Bio2420.105
Marc Carpenter
Table of Contents
Pg.2---Lab 4: Enterobacteriaceae Biochemical Test
Pg.5---Lab 5: Bergey’s Manual
Pg.6---Lab 6: Biochemical activities of bacteria 2
Pg.10---Appendix
Pg. 1

2. Lab 4: Enterobacteriaceae Biochemical Test
I. Objective/Purpose
The purpose of this lab was to be able to perform different biochemical tests on different
enterobacteriaceae and determine their results.
II. Materials
Test Tube
Test Tube rack
Inoculating loop and needle
Sterile distilled water
Bunsen burner
Tubing
Marker
Petri dish
Hektoen Enteric Medium (HEM) plate
Eosin Methylene Blue (EMB) plate
MacConkey plate
SIM semisolid deeps (Sulfur Indole Motility)
MR-VP broths (Indole, Methyl Red, Voges-Proskauer)
SCA slants (Simmons Citrate Agar)
TSI slants (Triple Sugar Iron)
Urea slants
Kovacs’ reagent
Barritt’s reagent
40% KOH
Bacillus megaterium (Bm)
Kocuria rhizophila (Kr)
Escherichia coli (Ec)
Proteus vulgaris (Pv)
Klebsiella pneumoniae (Kp)
III. Methods
To perform the biochemical tests on the Hektoen, EMB, and MacConkey plates,
the directions from the Enterobacteriaceae Biochemical Test Week 1 handout were
followed and the loop/streak technique was used. The SIM deep instructions were used
on page 157 (steps 1-3) of the Microbiology lab book by John P. Harley. The needle stab
technique was used on page 90 Fig.14.5 of the lab book. The MR-VP broths and the SCA
slants procedure was from page 165-166 Fig. 25.1-25.5 of the lab book. The TSI slants
steps were used from page 140-141 and the urea slants procedure from page 196-197 of
the book. The (Kp) was inoculated under the hood.
IV. Results
Pg. 2

3. TSI
K
r
K
p
P
v
E
c
Slant A A A A
Butt A A A A
Gas + + + +
H2S + - + -
SIM
Bacteria Motility/Indole
Pv +/+
Bm -/-
Ec -/+
Kr -/-
Urea
Bacteria Growth/Urease
Ec -
Pv + strong
Kp + weak
Kr + very weak
EMB
Bacteria Growth
Bm +
Pv -
Pg. 3
SCA
Bacteria Citrate
Kp +
Ec -
Pv +
Kr -
MR-VP
Bacteria Methyl Red/Voges-Proskauer
Bm -/-
Kr -/-
Ec +/-
Pv +/-
Hektoen
Bacteria Growth
Bm -
Pv -
Kr green
Ec coliform

4. Kr -
Ec +
V. Discussion
All the bacteria listed above are in the Enterobacteriaceae family which are all
GG and some are facultative rods. The TSI (Triple Sugar Iron) agar slant test was used to
portray the difference of Enterobacteriaceae from other gram negative bacteria by how
they catabolize glucose, lactose, or sucrose and release sulfides from ammonium sulfate
or sodium thiosulfate. All the bacteria tested for the TSI test (Kr, Kp, Pv, Ec) showed
them all being acidic from the butt and gas end by turning from a red to yellow color as
well as showing gas production by splitting of the agar from the needle stab. This is due
to the glucose, lactose and/or sucrose fermentation of high sugar concentrations. The Kr
and Pv showed H2S formation by observing the blackening of the agar unlike Kp and Ec
with no change of that matter. If the slant remained red with a yellow butt then it had
limited glucose levels and acid formation to be able to increase the pH. If both the slant
and butt remained red, then none of the sugars were fermented and neither gas nor H2S
were produced.
The SCA (Simmons Citrate Agar) test determines if the bacteria uses citrate as a
sole carbon source by having citrate permease that facilitates transport of citrate into the
bacterium that is converted to pyruvic acid and CO2. The Kp and Pv showed positive
citrate by turning the agar from green to blue while the Ec and Kr remained blue. The pH
change is due to the CO2 combined with sodium and water to form sodium carbonate
which is an alkaline product that raises the pH level. Negative citrate like Ec and Kr also
showed no signs of growth.
The SIM (Sulfur Indole Motility) test used to determine the bacteria’s ability to
reduce sulfur-containing amino acids or inorganic sulfur-containing compounds to
produce a gaseous hydrogen sulfide and to determine indole production as well as
motility. Only Pv out of the other bacteria (Bm, Ec, Kr), showed signs of motility by
producing a black precipitate which indicated H2S production and growth was not
restricted to the stab line. No motility is indicated when none of these changes are
observed. The Indole test is used to determine the bacteria’s ability to hydrolyze
tryptophan and produce metabolic products like indole, pyruvic acid and ammonia. Pv
and Ec produced a red layer indicating the presence of indole due to adding the Kovacs’
reagent while the Bm and Kr remained colorless, therefore, tryptophan was not
hydrolyzed.
The MR-VP broth test consisted of using methyl red has a pH indicator to detect
the change of acidity to products such as lactic, acetic, and formic acids. It also uses the
Voges-Proskauer test to identify bacteria that ferment glucose to produce a 2,3-butanediol
compound in the medium. Ec and Pv produced a red layer indicating that they were
Pg. 4
MacConkey
Bacteria Growth
Bm -
Pv -
Kr -
Ec +

5. mixed acid fermenters with a pH of 4 while Bm and Kr did not, which indicated that they
were butanediol fermenters that formed butanediol, acetoin, and fewer organic acids with
a pH of 6, remaining the color yellow. All the bacteria (Bm, Kr, Ec, Pv) remained
colorless when the Barritt’s reagent and 40%KOH was added to the broth to detect the
presence of acetoin, which is a precursor in the synthesis of 2,3-butanediol. The presence
of action would be detected if the broth developed a red color. From the pervious test,
Bm and Kr can be concluded that they form butanediol by other means than using
acetoin.
The Urease Test was used to identify bacteria that are able to produce the enzyme
urease that attacks nitrogen and carbon bonds to form ammonia, CO2, and water. All the
bacteria (Pv, Kp, Kr) showed signs of containing urea due to the color change from
orange to pink except Ec which remained orange. Some bacteria reacted faster than
others because of the shade and volume of pink in the tube. (Strongest to weakest: Pv,
Kp, Kr)
MacConkey and Hektoen plates are selective media which select for gram
negative bacteria through fermentation. Gram positive bacteria won’t be able to grow on
them. The HEM agar contained bile, lactose, and salicin, Coliform enteric bacteria appear
as a salmon to orange tone. Salmonella and Shigella appear as bluish-green and H2S
production turned colonies black within 24-28 hours of incubation if acid is produced.
Bm and Pv showed no signs of growth indicating that they are gram positive bacteria. Kr
appeared green which indicates that it’s a differentiated from Shigella of green, moist,
and raised colonies. Ec showed coliforms that had a salmon color. There was no black
center to be seen which concluded that H2S was not produced by any of the bacteria.
MacConkey agar contain bile salts and crystal violet which inhibit the growth of
gram positive organisms and contains lactose. The red color is used as a pH indicator for
when it changes color, lactose is fermented. Fermentation of lactose produces lactic acid
and decreases pH. The colonies appear as opaque and dark pink to a brick red color.
None of the bacteria (Bm, Kr, Pv) shown signs of growth except Ec which turned bright
red indicating that it’s a lactose fermenter. The other bacteria either are gram positive or
not lactose fermenters.
EMB plates are used to detect and isolate gram negative enteric bacteria. Eosin
and Methylene Blue gives a contrast between lactose fermenting colonies and non-lactose
ones. Sucrose was also added to detect bacteria that ferment more than lactose. Pv and Kr
showed no signs of growth so they are non-lactose fermenters. Ec continues to be seen as
a lactose fermenter. Bm surprisingly showed signs of growth even though its gram
positive and from the above tests shown that it wasn’t a lactose fermenter so this growth
might be due to contamination.
VI. References
Harley, J. Laboratory Exercises in Microbiology. Fifth Edition (2010)
Lab 5: Bergey’s Manual
I. Objective/Purpose
Pg. 5

6. The purpose of this lab was to learn how to use the Bergey’s Manual.
II. Materials
Pencil
Bergey’s Manual
V. Discussion
The Bergey’s Manual is used to help identify bacteria by listing their properties
from temperature range, cell wall, mobility, size to its environment and other side
comments. The Manual can be useful to help identify unknown bacteria by having the
characteristics given or discovering them which can lead to unlocking what their genus is.
Lab 6: Biochemical activities of bacteria 2
I. Objective/Purpose
The purpose of this lab was to be able to perform different biochemical tests on different
enterobacteriaceae and determine their results.
II. Materials
Test Tube
Test Tube rack
Inoculating loop and needle
Sterile distilled water
Bunsen burner
Tubing
Marker
Tape
Petri dish
Phenol Red w/ Durham Tube & Dextrose
Phenol Red w/ Durham Tube & Sucrose
Phenol Red w/ Durham Tube & Lactose
Casein Plate
Starch Plate
Litmus Milk Broth
Gelatin Deep
Nitrate Broth w/ Durham Tube
TSA (tropic soy agar) Catalase
Unknown Bacteria #9
Enterotube
Pg. 6

7. API Strips
Tray
40% KOH
Zinc powder
Gram’s Iodine
Minerial Oil
Escherichia coli (Ec)
Alcaligenes faecalis (Af)
Proteus vulgaris (Pv)
Enterobacter cloacae (Erc)
Bacillus megaterium (Bm)
Lactococcus lactis (Ll)
Staphyloccus epidermidis (Se)
III. Methods
The Enterobacteriaceae Biochemical Test Week2 handout was used to perform
the Litmus Milk Broth and give a layout about the other tests being performed. To
perform the different phenol red with Durham Tube mixtures test, the procedure from
pages 135-136 from the lab book was used. The Casein plate steps were followed from
page 170 of the book and the starch plate was followed from page 148. The needle stab
technique was used on page 90 Fig.14.5 of the lab book for the Gelatin Deeps procedure
on page 174. The Nitrate broth with Durham tube procedure was followed on page 215 of
the lab book and the TSA Catalase test was performed from page 178. The API strip
process was followed on page 221 steps 1-6 and the enterotube procedure on page 228.
IV. Results
See Enterotube and API sheet.
Phenol Red w/ Durhame Tube & Dextrose
Ec Af Pv
Growth + + +
Gas + + +
Color yellow yellow yellow
Phenol Red w/ Durhame Tube & Lactose
Ec Af Pv
Growth + + +
Gas Strong + + +
Color yellow red red
Pg. 7
Phenol Red w/ Durhame Tube & Sucrose
Ec Af Pv
Growt
h
+ + +
Gas + + Strong +
Color orange/yellow yellow yellow

8. Litmus Milk Broth
Ec Pv Bm Ec
Growth + + + +
Gas - - - -
Consistency creamy creamy creamy creamy
Color Dark purple
Growth at
Bottom/light
Dark purple
Growth at
Bottom/light
Dark purple
Growth at
Bottom/light
Dark purple
Growth at
Bottom/cream
Nitrate Broth w/ Durham Tube
Ec Af Pv Se
Growth + + + +
Gas + + + +
Color change w/
Nitrate reagent
red none red red
Color change w/
Zinc
none red none none
Casein Plate
E
c
P
v
Er
c
Growth + + +
Zone of Hydrolysis - - +
V. Discussion
The phenol red as the pH indicator with Durham tube tested for which sugars of
dextrose, sucrose, or lactose could bacteria fermentate from the possible carbohydrates
and if acid is produced. When the tubes are put in the autoclave, the air is expelled from
the tubes which are filled with medium while gas can be produced. The liquid inside the
Durham tubes could entrap the gas to form a bubble. Out of all three sugars, all of the
Pg. 8
Gelatin Deep
E
c
P
v
B
m
Growt
h
+ - -
Liquid - - -
TSA Catalase
E
c
B
m
Er
c
Growt
h
- + +
H2O2 - + +
Starch Plate
E
c
P
v
B
m
Growth + + +
Iodine test - - +

9. bacteria (Ec, Af, Pv) produced growth and gas by having the color change from red to
yellow while the Durham tube wasn’t filled up with the media which indicated the gas
bubble. Pv showed stronger signs with sucrose while Ec showed stronger signs of lactose
by the darker shade of yellow. However, Af and Pv remained red with a gas bubble when
testing with lactose indicates that they used alcohol fermentation. If the tube was filled
with red media and the media was red then no fermentation occurred and vice versa when
the tube is fully filled with yellow media then just acid production occurred. This can be
used to help identify different microorganisms.
Gelatin Deeps were composed of a soluble mixture of polypeptides which the test
could help identify which bacteria can hydrolyze gelatin by secreting the proteolytic
enzyme gelatinase. Only Ec out of the other bacteria (Pv, Bm) produced growth but none
of them remained a liquid but turned into a gel so they all didn’t hydrolyze gelatinase.
Because Ec is a facultative anaerobe, it’s why it was able to still grow in that
environment. If bacteria did hydrolysis by using gelatinase, then the liquid culture would
flow when tilt. The production of gelatinase can be related to the bacteria having the
ability to break down tissue collagen and spread throughout the host which is a good
indicator to discovering the pathogenicity of the bacteria.
Litmus Milk Broth test contains lactose and the protein casein as well as other
vitamins used to identify some bacteria that ferment lactose, proteolyze casein or degrade
both. The metabolic changes can be observed using the pH indicator purple of litmus. All
of the bacteria (Ec, Pv, Bm, Ll) produced growth and all showed no signs of gas
production. The consistency of all the bacteria were creamy and all remained dark purple
which indicates that all the bacteria had an alkaline reaction due to the decarboxylation of
the casein amino acids and not containing the enzyme beta-galactosidase or used a
different way to fermentate it. If O2 was removed by the bacteria, the litmus would
reduce by accepting electrons and have a white zone at the bottom.
The Nitrate Broth with the Durham tube test shows the growing the bacteria with
the nitrate broth to examine the presence of gas and nitrite ions as well as zinc. Some
bacteria use nitrate as a terminal electron acceptor during anaerobic respiration. The gas
is released from the reduction of nitrate and from the citric acid cycle. The bubbles
indicate the presence of nitrogen gas for only nonfermenters. To determine if nitrates
were reduced past nitrite, zince was added. All the bacteria (Ec, Pv, Se, Af) produced
growth and gas which can be produced from carbohydrate sources. When the nitrate
reagent was added, all the bacteria but Af turned red which showed that they all reduce
nitrate except Af. When the zinc reagent was added, only Af turned red which shows that
nitrates were not reduced to nitrites by the bacteria so it must posses other enzymes that
reduce nitrite to ammonia. If the red color did not appear, then the nitrates were reduced
past the nitrite stage to either ammonia or nitrogen gas.
The TSA Catalase test was used to determine if the bacteria contained
flavoproteins that reduced O2 so it doesn’t kill it and produce hydrogen peroxide which
are toxic, powerful oxidizing agents that destroy cellular structures very rapidly. To
protect against toxic O2 products, obligate aerobes and facultative anaerobes usually
contain the enzyme superoxide dismutase that catalyzes the destruction of superoxide and
either catalase or peroxidase. Bm and Erc produced growth and H2O2 by forming
bubbles of oxygen unlike Ec that showed no growth or H2O2 with no bubbles being
produced so it cant produce enzymes to protect itself against O2 products.
Pg. 9

10. The Casein Plate is a large milk protein incapable of permeating the plasma
membrane of bacteria which is the main reason why milk is white but it first must be
degraded into amino acids. Some bacteria can secrete proteases that catalyze the
hydrolysis (proteolysis) of casein to yield amino acids and then are transported into the
cell and catabolized. All of the bacteria (Ec, Bm, Erc) produced growth but only Erc had
a zone of hydrolysis that is indicated by a clear area surrounding the colony where the
hydrolytic reaction yields soluble amino acids. Bm and Ec had no protease activity and
the colony remained opaque.
The Starch Plate test was used to see if bacteria could hydrolysis starch after
adding Garm’s Iodine. Hydrolases catalyze the splitting of organic molecules into smaller
ones in the presence of water. All of the bacteria (Ec, Pv, Bm) produced growth and only
Bm formed a blue to brown comlplex when iodine was added that showed it hydrolyzed
starch. Ec and Pv remained clear after the iodine was added indicating that the alpha-
amylase was produced by the bacteria and no starch was hydrolyzed.
The Enterotube II System was consisted of a single tube that contained 12
compartments of different solid agar medium with compartments that required some
aerobic conditions to have small openings. This was used to help identify the unknown
bacteria #9 and what properties it consisted of. The ID biocode was 30400 which
indicated that there was gas production from glucose so it can fermentate it, couldn’t
produce H2S but could hydrolyze tryptophan, negative for VP so it didn’t produce
acetoin as well as no production of urease and citrate. With these results, it can be
concluded that the unknown bacteria #9 was Ec.
The API 20E System is a standardized mini biochemical identification tool for
Enterobacteriaceae and other gram-negative bacteria. There are 20 chambers with
cupules consisting of dehydrated substrates and anaerobic conditions are formed with the
addition of sterile mineral oil. The Profile index number was 3442731 which indicated
that beta-galactosidase was hydrolyzed (ONPG) and ornithine, ammonia, carbon dioxide
was produced (ADH), hydrogen sulfide was produced (H2S), gelatin was liquefied by
proteolytic enzymes (GEL), acid formation occurred from Mannitol (MAN), Inositol
(INO), Sorbitol (SOR), Rhamnose (RHA), Sucrose (SAC) and Amygdalin (AMY) which
catalase releases from oxygen gas from hydrogen peroxide. With these results the
unknown bacteria #9 can’t be Ec because it shouldn’t produce H2S. Comparing the 2
tests, it can be concluded that the unknown bacteria was something not used from the
ones listed or there was an error in validating the results.
VI. References
Harley, J. Laboratory Exercises in Microbiology. Fifth Edition (2010)
Pg. 10