MMBB255 Week 6
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Biochemical and Metabolic Properties of Microbes
I. Objective
The goal of this experiment is to characterize an organism based on its ability to produce proteins that allow
it to carry out a set of unique reactions, its motility, and oxygen requirements. You will also follow up on the
growth curve and unknown two. The Photographic Atlas for the Microbiology Laboratory will be useful.
II. Background
Microorganisms can be classified, or distinguished from one another, by the ability to (1) grow on different
substrates and/or production of different end products, (2) produce specific enzymes, (3) use oxygen, or (4) be
motile. For example, certain microbes can use different carbohydrates as sources of energy and/or carbon.
Because such variability exists in carbohydrate utilization between different microbes, this can aid in the group,
genus, or species identification. We will examine the following methods for classification:
A. Carbohydrate utilization: acid and/or gas production
B. Enzyme production
C. Aerobic or anaerobic mode of growth
D. Motility
A. Carbohydrate utilization
Carbohydrates can be fermented or oxidized via aerobic or anaerobic respiration. When a carbohydrate
is fermented, acid, and sometimes gas, is produced. The most common end product of carbohydrate
fermentation is lactic acid (lactate). Other acids include formic and acetic acid. Microbes including
Streptococcus and Lactobacillus can produce lactic acid, formic acid, and/or ethanol. Enteric organisms (see
below) can produce lactic acid, formic acid, succinic acid, as well as ethanol and gasses CO2 and H2.
Acid production can be detected by addition of a pH indicator, such as phenol red or brom cresol purple
to the medium. Phenol red (PR) turns yellow in acidic conditions (slightly under pH 7.0) while at neutral or
basic pH it is red. Gas production can be monitored by addition of a small tube, called a Durham tube,
which has been inverted in the carbohydrate-containing growth medium to trap gas bubbles. If gas is
produced, it typically is CO2 or H2.
Carbohydrates that are used in microbial classification include the following classes:
a) Monosaccharides are simple sugars with 1 to 6 carbons. For example, tetroses are 4 carbon sugars,
such as erythrose. Pentoses are 5 carbon sugars and include ribose, xylose, arabinose, and ribulose.
Hexoses are 6 carbon sugars and include glucose (also called dextrose), galactose, mannose, and
fructose (also called levulose).
b) Polysaccharides are polymers of monosaccharides. For example, a disaccharide (di = two) contains
two monosaccharide units. Some examples include sucrose (also called saccharose) composed of the
hexoses glucose and fructose, maltose composed of two units of the hexose glucose, and lactose
composed of the hexoses glucose and galactose.
c) Alcohol sugars are polyhydric alcohols that are reduction products of a monosaccharide. Some
examples include adonitol, dulcitol, mannitol, and sorbitol.
Carbohydrate utilization is important in distinguishing
Enterobacteriaceae (enteric bacteria) are
among the members of the family Enterobacteriaceae
(the enteric bacteria). The Enterobacteriaceae is a family
facultatively anaerobic, oxidase negative,
of organisms that are facultatively anaerobic, oxidase
Gram negative rods that ferment glucose.
negative, Gram negative rods that ferment glucose. This
family includes the genera Escherichia, Salmonella,
Proteus, Enterobacter, Serratia, Yersinia, Edwardsiella, Providencia, Hafnia, Citrobacter, Shigella, and
Klebsiella, to name a few. Of the above genera the only lactose fermenters are Escherichia coli, Klebsiella,
Citrobacter, and Enterobacter.
B. Enzyme production
Listed below are some brief descriptions of enzymes you might find in a microbe. The ability of an
organism to produce or express a particular enzyme can be assayed (tested) in the laboratory using simple
biochemical reagents and substrates. Several of these tests require only small amounts of cellular material
and may take only a few minutes to interpret. Subsets of microbes can then be classified into groups based
on their ability to produce a positive or negative reaction. The results of biochemical tests are used with
other data, including the Gram reaction, to identify an organism by name. These tests play critical roles in
the swift identification of bacteria causing infection.
MMBB255 Week 6
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1. Catalase: H2O2 (hydrogen
A Few Examples
peroxide) is produced as an
Catalase Positive
Catalase Negative
oxidation product during growth
Micrococcus and Staphylococcus
Streptococcus
on carbohydrates in the presence
Bacillus
Clostridium
of oxygen and is toxic if allowed
Listeria
monocytogenes
and
some
to accumulate. Many organisms
Erysipelothrix
Corynebacterium sp.
make the enzyme catalase to
remove hydrogen peroxide. The catalase test
The catalase test is useful to differentiate
measures the ability of an organism to produce
the enzyme catalase that degrades H2O2 to H2O
between the Staphylococci/Micrococci and the
and O2. Active catalase is a homotetramer (four
Streptococci (see the table above).
identical subunits) with two heme groups
containing trivalent iron (ferric, Fe3+). To see if an organism makes catalase, H2O2 is added to cells. If
catalase is present, dioxygen gas is liberated and bubbles will be produced. Note that two molecules of
H2O2 are needed because this is an oxidation-reduction reaction in which one molecule of H2O2 serves as
the substrate and the other serves as a donor. Water is the reduced product and O2 is the oxidized product.
2. Oxidase: The enzyme oxidase is part of the electron transfer system used by some organisms that use
molecular oxygen as a
A Few Examples
terminal electron acceptor.
Oxidase Positive
Oxidase Negative
Oxidase interacts with the
Neisseria, Moraxella
Acinetobacter
membrane bound
Aeromonas, Alcaligenes,
Enterobacteriaceae
cytochromes and delivers
Flavobacterium, Pseudomonas, Vibrio
electrons from the
cytochromes to O2. As a result, H2O2 or
The oxidase test is a useful test for distinguishing
H2O is generated. Strict anaerobes do not
between the Gram negative rods Pseudomonas
use O2 and hence do not possess the
oxidase enzyme. Most Gram positive
and the Enterobacteriaceae.
bacteria are oxidase negative as well as the
members of the family Enterobacteriaceae.
3. Gelatinase (an extracellular
A Few Examples
enzyme): This test determines
Gelatinase
Positive
Gelatinase Negative
whether an organism produces
a proteolytic-like enzyme that
Staphylococcus epidermidis + slow
Staphylococcus aureus
can liquefy or digest gelatin, a
Micrococcus + slow
protein produced by the
Corynebacterium sp. (most)
Listeria monocytogenes
hydrolysis of collagen. Proteins
are too large to be taken up by
microbes, so gelatinases (and other proteases) are secreted into the environment where they break
proteins into peptides and amino acids, which can be taken up by the cell.
4. Amylase (an extracellular enzyme): This test measures whether an organism produces amylase, an
enzyme that breaks down starch, a glucose polymer. Amylase hydrolyses starch into dextrins (smaller
polysaccharides) and maltose (disaccharides), which now can be imported into the cell.
5. Lipase, lecithinase, proteases (extracellular enzymes): Lipases are enzymes that break down
triglycerides (lipids) into their constituent glycerol and fatty acids. Lecithinase breaks down lecithin
which is a modified phospholipid; one of the fatty acids has a special modification. Cells can use these
components as energy or to produce other cellular components. Proteases are enzymes that break down
(hydrolyze) proteins and peptides.
6. Urease: The enzyme urease is an
A Few Examples
amidase produced by certain eukaryotes
and prokaryotes. It plays an important
Urease Positive
Urease Negative
role in the decomposition of organic
Klebsiella
Escherichia
compounds. Because not all microbes
produce this enzyme, a simple test for
Proteus
Providencia
this enzyme can be used to distinguish
Bordetella bronchiseptica
Bordetella pertussis
between various microbes. For example,
urease activity is very characteristic of all
Yersinia pseudotuberculosis,
Yersinia pestis
Proteus spp. This test aids in rapid
Y. enterocolitica
detection of lactose-nonfermenting
enteric organisms such as Proteus vulgaris, an opportunistic pathogen.
MMBB255 Week 6
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The urease test measures the ability of a microorganism to split, or hydrolyze, a molecule of urea into
two molecules of ammonia using the enzyme urease. A heavy inoculum of a young culture is added to
urea broth (this media is designed specifically to differentiate Proteus from other enterics). The critical
ingredients are urea and the pH indicator, phenol red. The initial pH of the medium is 6.8 and is slightly
buffered (there is less buffer if you use a urease assay – like urea agar - designed for organisms other than
Proteus). Phenol red is yellow at pH 6.8 (slightly acidic) and red at pH 8.4. Microbes that can make
urease, will hydrolyze urea as shown in the box, resulting in a shift in the pH of the medium toward
alkaline pH. This
H2N
changes the phenol red
urease
indicator to pink (red
C
O
CO2 + H2O
+
2 NH3
+
2 HOH
or cerise). Urea is a
H2N
diamide of carbonic
acid that is split into
Carbon
Urea
Water
Water
Ammonia
carbon dioxide and
Dioxide
ammonia by the urease
enzyme as shown in the box.
C. Aerobic or anaerobic mode of growth (Oxygen Requirements)
A very important requirement of cellular growth and metabolism is the requirement for oxygen.
Microbes can be grouped into five different categories based on their requirement for molecular oxygen.
Strict or obligate aerobes must have O2 for growth because O2 is used as the terminal electron acceptor for
oxidative phosphorylation. In contrast, strict or obligate anaerobes cannot grow in the presence of O2 and
may be killed by trace amounts of O2. Microaerophilic organisms need a small amount of O2 for growth
but too much O2 will kill them. A subset of anaerobes can tolerate O2 but they do not use it. They are called
aerotolerant anaerobes. Facultative anaerobes grow best when O2 is available, but they can also grow,
though not as well, if O2 is not present. We will test several organisms for their growth pattern in medium to
determine if they prefer aerobic or anaerobic conditions. Note that the handling of very strict anaerobes is
beyond the scope of this class.
D. Motility: swimming, swarming, twitching, and gliding
Microbes move in a variety of
A Few Examples
ways that are roughly analogous to
Temp.
Motile
Nonmotile
our walking, running, swimming,
Vibrio
spp.
Acintobacillus
spp.
or crawling movements. Many
Enterobacter
spp.
Klebsiella
spp.
organisms can swim in liquid (such
37°C
Aerogenic Escherichia coli
Anaerogenic Escherichia coli
as lakes or oceans, blood system).
Most swimmers move by rotation
Other Bacillus spp.
Bacillus anthracis
of one or more external appendages
Pseudomonas spp.
Pseudomonas mallei
called flagella. This is a complex
Corynebacterium spp.
Listeria spp.
process that is very well studied.
(C. aquaticum is positive)
22°C
The helical shape of the flagellum
Yersinia pseudotuberculosis
Yersinia pestis
is reminiscent of a motorboat
Y. enterocolitica
propeller; movement results from
rotating one or more flagella. Flagella can rotate very fast when the cell is in liquid medium, allowing for
rapid swimming. When cells with flagella are in more viscous (thicker) medium, such as broth containing
some agar, the rate of swimming is reduced. Some examples of swimmers include Escherichia coli,
Salmonella Typhimurium, and Bordetella pertussis.
Some swimmers can also swarm. Swarming occurs when a swimmer cell enters a viscous environment
or a solid surface. The swimmer then increases in length and makes a large number of flagella. Some
examples include Proteus mirabilis and Serrratia marcescens.
Twitching motility is a slow movement of cells over a surface via type IV pili. The cell throws out a
pilus which attaches to a surface and is subsequently depolymerized so that the cell moves toward the
attachment point. Pathogens, such a Neisseria and Pseudomonas, use twitching motility, as does the soil
bacterium Myxococcus xanthus.
Some organisms, particularly soil organisms, move by gliding. Gliding is a translocation over a solid
surface (soil, agar). Gliders do not have flagella or cilia but appear to move by secretion of a material. Some
examples of gliders are Myxococcus xanthus and Beggiotoa spp.
Motility can be used to distinguish between certain genera and to aid in species identification. Note that
motility can vary as a function of temperature. Therefore it is important to measure motility at the
temperature indicated for each organism.
MMBB255 Week 6
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Tuesday’s Procedures:
A. Carbohydrate fermentation and enzymes in carbohydrate catabolism – Work in Pairs.
1. You will be assigned E.coli plus one other organism from the list
Enterobacter aerogenes
to the right. These will be provided as broth cultures. You will
Citrobacter freundii
need to record the results for the other organisms you do not use.
Enterococcus faecalis
2. Obtain and label the following media: (Note that the extra two
Escherichia coli
tubes are for you and your partner to inoculate your second
Klebsiella pneumoniae
unknown)
Proteus mirabilis
4 tubes of PR-lactose with Durham tubes
Pseudomonas aeruginosa
4 tubes of PR-glucose with Durham tubes
Salmonella Typhimurium-see note
4 tubes of PR-sucrose with Durham tubes
3. Inoculate each tube aseptically with your loop. Label each tube
with the name of the organism, the sugar, the date, your name and lab section. The instructor will keep
uninoculated carbohydrate media as negative controls and incubate them along with the inoculated tubes.
4. Incubate all tubes at 37°C for 24 hrs. and examine.
Note: The nomenclature for the Salmonella species has been changed. There are now only three species of
Salmonella, enterica (used to be Salmonella choloraesuis), bongori, and subterranean. Most human
pathogens are serovars of S. enterica subspecies enterica (there are 6 subspecies total). For example
Salmonella Typhimurium is short for S. enterica subspecies enterica serovar Typhimurium; similarly
there is Salmonella Typhi, Paratyphi, Enteritidis short names for the subsp. enterica. Please note how
they are formatted. If you were to hand write them you would underline all italics.
B. Enzymes: Work in Pairs.
i. Extracellular enzyme gelatinase:
1. The bacterial cultures. For these tests, we will use the following microorganisms:
Staphylococcus aureus
Bacillus cereus
Staphylococcus epidermidis
Pseudomonas aeruginosa
2. Inoculate. Using an inoculating needle or loop aseptically stab each organism into a labeled nutrient
gelatin tube. Incubate at 37°C for 48 hrs.
ii. Extracellular enzyme amylase:
1. The bacterial cultures. For these tests, you will use the following organisms:
(you may also test your second unknown if you want, you will need six
sectors)
Staphylococcus aureus
Bacillus cereus
Staphylococcus epidermidis
Pseudomonas aeruginosa
2. Inoculate. Using your inoculating loop aseptically make a line in the
quadrant of a starch agar plate with each of the test organisms (see the
diagram). Label the back and incubate at 37°C for 48 hrs.
iii. Extracellular enzymes lipase, lecithinase, and protease:
1. The bacterial cultures. For these tests, you will use the following organisms:
(you may also test your second unknown if you want, you will need six
sectors)
Staphylococcus aureus
Bacillus cereus
Staphylococcus epidermidis
Pseudomonas aeruginosa
2. Inoculate. Obtain one plates of egg yolk nutrient agar (EY-NA) from the
instructor. The addition of egg yolk at 5-10% (v/v) makes the medium look
milky. Aseptically inoculate a single line for each organism on the plate (see the
diagram). Incubate at 37°C for 24-48 hrs.
MMBB255 Week 6
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iv. The urease test
1. Work with the following bacterial cultures.
Escherichia coli
(plate stock)
Enterobacter aerogenes
(plate stock)
Klebsiella pneumoniae
(plate stock)
Proteus mirabilis
(plate stock)
Your unknown
(plate stock)
Your partners unknown
(plate stock)
2. Obtain and label: 6 tubes of phenol red urea medium (urea broth).
3. Inoculate. Using your inoculating loop, aseptically transfer a heavy inoculum (loopful) of each
organism into a separate tube of urea broth. The lab instructor will keep aside one tube uninoculated
and incubate it at 37°C as a control.
4. Incubate at 37°C. Check your reactions after about 4-6 hours if possible. Fast positive organisms, such
as Proteus, may turn pink in a few hours. Check for a positive test at 24 hr.
Date and time that tubes were inoculated: _____________ time________________
C. Oxygen requirements – Work in Pairs.
1. The bacterial cultures.
Escherichia coli
Pseudomonas aeruginosa
Clostridium sporogenes
Streptococcus agalactiae
2. Three methods will be used to monitor the oxygen needs of the four bacterial strains:
i. Method 1. Agar deeps.
Obtain four tubes of TSA (trypticase soy agar) deeps, tempered at 55°C. Remove one tube from the
55°C bath and aseptically add two drops of an organism into the tube using a sterile pasteur pipette.
Immediately and vigorously roll the tube between the palms of your hands to evenly distribute the cells
throughout the agar. Then place the tube into a bucket of ice to solidify the medium. Repeat with the
other three cultures. Incubate at 37 °C for 48 hrs.
ii. Method 2. Thioglycolate broth.
Obtain four screw-capped tubes of thioglycollate medium. The medium should be slightly pink or blue
in the upper one-fourth of the tube due to the presence of resazurin or methylene blue respectively,
both redox indicators (they turn color when there is oxygen). Sodium thioglycolate, the active
ingredient, binds to O2. Aseptically inoculate each of the tubes, taking care to stab to the bottom of the
tube with your inoculating loop or needle - try not to introduce air. Don’t shake or tighten the cap
(you want air to get in the tube but not the medium). Incubate at 37°C for 18-24 hrs (check Wed).
iii. Method 3. Gas-Pak Jar.
Obtain two TSA plates from the lab instructor. Aseptically streak a single line of each of the four
organisms on both of the plates and your unknowns. [Be sure to label each quadrant correctly].
Immediately place one plate in the Gas-Pak Jar. It will be activated and placed at 37°C for 48 hrs.
Incubate the other plate at 37°C in the air for 48 hours.
The Gas-Pak works as follows: To activate the system, water will be added to a reagent in the GasPak to generate CO2 and H2. The jar contains a small amount of palladium that then catalyzes the
conversion of O2 and H2 to H2O. This removes sufficient O2 to make an environment favorable for
anaerobes. The jar also includes a redox indicator called methylene blue to check for anaerobiasis. It
is colorless when reduced and blue when oxidized.
D. Motility – Work in Pairs.
Motility will be tested by examining the distance cells migrate in semisolid medium. Remember motility
may also be checked via a hanging drop or wet-mount method. Standard agar medium that we have been
using is considered “solid” medium because it contains 1.5% w/v (1.5 g per 100 ml) agar. The semisolid
medium has only 0.5% w/v agar (swarming medium has even less, 0.3 % w/v agar) and contains all the
nutrients required for organisms to grow. The semisolid medium may also contain an indicator of cells with
active respiration called 2,3,5-tetraphenyltetrazolium chloride (TTC). This allows easier observation of where
the organisms are growing since it turns red when reduced by an active electron transport chain (it is
colorless when oxidized).
MMBB255 Week 6
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1. The bacterial cultures. Obtain these strains from your instructor.
Escherichia coli
Klebsiella pneumoniae
Your unknown
Your partners unknown
2. Inoculate by stabbing. Label each sectors, then aseptically stab each
organism with an inoculating needle into the center of a quadrant of
semisolid medium, see the diagram to the right.
3. Incubate. Incubate the plate upright (do not invert) at 30°C and examine
for motility (movement away from the stab inside the agar – not the surface)
after 24 and then 48 hrs.
E. Growth curve - Finish
1. Record your statistically valid plate counts (between 25-250), calculate the total viable cell
concentration, fill out the table below, and enter the calculated cfu/ml into the computer.
time point
dilution
x dilution
÷ by volume
calculated
# of colonies
elapsed min.
used
factor
plated
cfu/ml
2. You will get graphing paper and all sections’ growth curve data next lab period. You should make a
growth curve of each growth condition and figure out the doubling time from the graph. Get help if you
do not know how to do this.
Thursday’s Procedures:
A. Carbohydrate fermentation and β-galactosidase activity – Work in Pairs.
1. Record Fermentation Data. After 24 hrs, record your results in the table below. Note: if for some
reason you do not observe your cultures within about 24 hrs, make a note of the time and date below. The
“Ferm?” heading in the table below is your conclusions that the organism does or does not ferment that
sugar- see the background.
Date that tubes were inoculated: _____________ time: ________________
Date that observations were taken: _____________ time: ________________
Lactose
Sample
Neg. Control
E. aerogenes
C. freundii
E. faecalis
E. coli
K. pneumoniae
P. mirabilis
P. aeruginosa
S. Typhimurium
Your Unknown
Growth
&
Color
Gas
Glucose
Ferm?
Growth
&
Color
Gas
Sucrose
Ferm?
Growth &
Color
Gas
Ferm?
MMBB255 Week 6
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2. Assay for β-galactosidase activity. The enzyme β-galactosidase breaks lactose into its two
monosaccharide components, glucose and galactose. Production of the enzyme ß-galactosidase is
regulated by the presence of the substrate, lactose, and the absence of the more favorable substrate,
glucose (you may want read about the Lac operon in your text). To demonstrate this, you will determine
the amount of β-galactosidase produced by Escherichia coli grown in glucose vs. lactose.
a. Mix your E. coli lactose and glucose PR tubes to disperse the cells. Label four 1.5 ml eppendorf (eppi)
tubes and fill them according to the table below. You do not need to be aseptic. Follow the flow chart
below and note the footnotes about the various solutions:
Tube 1
Add 1.5 ml PR-glucose
cells to an eppi tube
Tube 2
Add 1.5 ml PR-lactose cells
to an eppi tube
Tube 3 (Blank)
Add 1.5 ml PR-glucose
cells to an eppi tube
Tube 4 (Blank)
Add 1.5 ml PR-lactose cells
to an eppi tube
↓
Centrifuge @ 12,000 RPMs for 4 min
Discard supernatant into originating PR tube.
↓
Add 1 ml of 0.1% SDS1
Mix to get all the cells resuspended and then transfer all the liquid to a spectrophotometer tube.
Add 2 ml Z buffer2
and 1 ml ONPG3
↓
Add 2 ml Z buffer
and 1 ml ONPG
Add 2 ml Z buffer
and 1 ml H2O
Add 2 ml Z buffer
and 1 ml H2O
↓
Incubate at 37°C for 5 min.
↓
Add 1ml of 1M Na2CO34
Read Abs (OD) at 420 nm in the spectrophotometer.
b. Calculate the units of ß-galactosidase activity as follows: β-gal Units = (A420nm * 213)/Time(min.)
Units for glucose-grown cells:____________
Units for lactose-grown cells: _____________
What can you conclude from these results about the regulation of the gene (lacZ) that encodes for
β-galactosidase?
B. More biochemical (enzyme) tests used to identify microbes.
a. The catalase test. This test must be performed on fresh cultures (<24 hr old) to be accurate.
1. Cultures. Use the following organisms from solid (agar) plate medium.
Staphylococcus aureus
Micrococcus luteus
Streptococcus sp. Gp B or Enterococcus faecalis
Your unknown from the air grown GasPak experiment plates.
2. Smearing. Using your sterile loop (or sterile toothpicks), scrape off a small section of a colony
(enough to see with the unaided eye) and smear it onto a glass slide.
3. Add peroxide. Add a drop of the H2O2 solution and watch for the appearance of bubbles. Any bubble
is indicative of gas production and is a positive catalase reaction. Sometimes it takes a minute to see
bubbles.
4. Test your unknown. Repeat with your unknown organism.
1
To lyse the cells and release the enzyme.
0.1M sodium phosphate, 5mM MgCl2 at pH7.3; supplies buffer and needed salt for the enzyme to work.
3
4 mg/ml o-nitrophenyl-β-D-galactopyranoside (which is colorless) is hydrolyzed by β-galactosidase to o-nitrophenol
(which is yellow) and galactose. This is considered a lactose analog since the enzyme thinks it is lactose.
4
This increases the pH to >9, which stops the reaction and maximizes the absorbance at 420 nm.
2
MMBB255 Week 6
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Results of the Catalase Test
Positive
Negative
Staphylococcus aureus
Micrococcus luteus
Streptococcus sp. Gp B or Enterococcus faecalis
your unknown
b. The Oxidase DrySlideTM test. This test must be performed on fresh cultures (<24 hr) to be accurate.
1. Test the following organisms for oxidase:
Escherichia coli
Micrococcus luteus
Pseudomonas aeruginosa
Pseudomonas fluorescens
Your unknown from the air grown GasPak experiment plates.
2. Obtain a DrySlideTM Oxidase slide from the lab instructor. This 2x2 inch slide has four plastic film
reaction areas that contain N,N,N’,N’-tetramethyl-para-phenylenediamine dihydrochloride (TMPD),
gelatin and ascorbic acid. This is an oxidation-reduction reaction. TMPD is colorless when reduced
and purple (blue) when oxidized. Ascorbic acid acts as a stabilizer by being a reducing agent.
3. Apply fresh cells. Scrape up a small glob of cells using a toothpick (do not use any implement
containing iron such as your inoculating loop or needle) and rub the cells in one corner of the Oxidase
slide film reaction areas and look for a color change from clear to bluish-purple. This reaction must
happen within 20 seconds; any change after 20 seconds must be considered negative. You can easily
test about 16 different samples on one slide. Pigmented organisms will contribute to the final color
and you should take this into account. Also
mucoid and heavily pigmented cells will
Results of the Oxidase Test
delay the reaction somewhat; therefore
Positive
Negative
read within 30 seconds if you have one (M.
Escherichia coli
luteus is an example).
Pseudomonas aeruginosa
4. Test your unknown. Repeat with your
Micrococcus luteus
unknown organism from the air grown
GasPak experiment plates. When you and
Pseudomonas fluorescens
your partner have completed your tests
Your unknown
remember to pass the slide to another pair
of students in the lab.
C. Extracellular enzyme gelatinase – Almost Finished.
If an organism has the enzyme gelatinase, the gelatin in the nutrient gelatin tubes will be broken down and
once chilled it will be liquid; it will never ‘set up’ or solidify like gelatin normally does.
1. Transfer the inoculated gelatin cultures to 4°C. This will allow the gelatin to harden unless it has
been broken down by the bacteria. After about 30 min, remove the tubes and shake them. If gelatinase
has been produced, the samples will be fluid. These are considered fast positive reactions. For samples
that are not positive after
Results of the Gelatinase Test
48 hrs, return them to the
After 48 hrs
After 5 days
incubator for 5 more days
(fast positive)
(slow positive)
and recheck them during
Staphylococcus aureus
the next lab period; if they
become liquefied they are
Bacillus cereus
considered slow positive
Staphylococcus epidermidis
reactions.
Pseudomonas aeruginosa
2. Record your results in the
table.
MMBB255 Week 6
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D. Extracellular enzyme amylase - Finished.
1. Obtain your inoculated plate and stain with Gram’s iodine. Iodine will react with intact starch
(also called amylose) to produce a blue-black product. However, if the microbe has hydrolyzed the
starch due to the enzyme amylase, a clear zone will remain after you flood the area with iodine.
2. Record your results below.
Results of the amylase test
Positive
Negative
(clear zone)
(blue-black)
Staphylococcus aureus
Bacillus cereus
Staphylococcus epidermidis
Pseudomonas aeruginosa
E. Extracellular enzymes lipase, lecithinase, and protease - Finished.
1. Examine the egg yolk nutrient agar plate area around the streaks. If lipase is produced, the
triglycerides will be degraded producing an iridescent sheen around and on the lipase-secreting
colony; it looks like the oil on water sheen. If lecithinase is produced, you will see a white, opaque
zone around the colony. Secretion of proteases will produce a clearing about the streak. An organism
can produce all three enzymes, so look for combinations.
2. Record your results below.
Results of the lipase, lecithinase, and protease tests
Lipase
Lecithinase
Protease
(iridescent)
(white, opaque)
(clearing)
Staphylococcus aureus
Bacillus cereus
Staphylococcus epidermidis
Pseudomonas aeruginosa
F. The urease test - Finished.
1. Examine the color of the urease tubes. At 24 and 48 hrs, record your results in the table below.
Peach to pink colors are considered positive reactions; fast positive organisms, such as Proteus, may
turn pink in a few hours.
2. Record your results below.
Date and time that tubes were inoculated: ___________ time________________
Results of the Urease Test
Color (peach to pink is positive)
4-6 hrs.
24 hrs.
uninoculated control
Escherichia coli
Enterobacter aerogenes
Klebsiella pneumoniae
Proteus mirabilis
Your Unknown
48 hrs.
MMBB255 Week 6
10
G. Aerobic and anaerobic growth – Finished.
1. Results. Record your observations by noting where in the tube it grew and the quantity of growth. From your
observations record your conclusions about the type of oxygen requirements. The definitions for the terms are in
the background reading material.
Agar Deeps
Thioglycolate
Escherichia coli
Pseudomonas aeruginosa
Clostridium sporogenes
Streptococcus agalactiae
Your Unknown
Gas-Pak
Conclusions*
Agar Deeps
XXXXXXXXXXX
Thioglycolate
vs.
Air
Gas-Pak
XXXXXXXXXXX
*be sure to use the correct term: obligate aerobe, obligate anaerobe, microaerophile, aerotolerant, or
facultative anaerobe (see the background).
H. Motility – Finished.
1. Record your results in the diagram. Observe in the agar itself
and not the surface; look from the side of the semi-solid agar
plate. Did the organism move away from the stab line?
2. Which strains are motile?
Organism
E. coli
K. pneumoniae
Your Unknown
I. Unknown Two– Finish.
Non-Motile
Motile
MMBB255 Week 6
11
Study Questions:
1. Be able to determine what is positive and negative for each of the tests. Understand how each test works.
2. Oxidase is very useful to differentiate between which two groups of organisms? What is a positive reaction?
3. Catalase is very useful to differentiate between which two groups of organisms? What is the reaction
catalyzed by this enzyme and what do you see for a positive reaction?
4. What indicates that a sugar is fermented in the PR-sugar test? You may also have what byproduct as seen in
the small inverted test tube? What is that small test tube called?
5. What are the purposes of the various solutions in the β-galactosidase test? Lactose is a disaccharide
comprised of which two monosaccharides? Be able to answer the question given at the end of this
experiment.
6. Enzymes are named after the substrates they work on. Name the substrate for each enzyme. For example
amylase’s substrate is amylose or starch.
7. Where does a microaerophile grow best in a thioglycollate tube? How about an obligate aerobe, obligate
anaerobe, facultative anaerobe, and aerotolerant anaerobe?
8. What kinds of motility are there?
9. How does the Gas-Pak chamber work to give you anaerobic conditions? How do you know if it is working?
10. The first practicum is soon. Here are some typical questions:
Practice #1:
Describe the cellular morphology and arrangement of this gram stain.
What gram reaction is it?
What is the difference between Gram positive eubacteria and Gram negative eubacteria?
Practice #2:
Is this a positive or negative acid-fast stain?
Why do we have to use acid-fast stain for Mycobacteria?
Practice #3:
Determine the O.D. at 420 nm of the yellow sample.
This was from the galactosidase assay we did using the ONPG substrate. What operon activity are we
detecting and is the above sample from the PR-Glucose or PR-Lactose medium?