Laboratory Maintenance of Moraxella UNIT 6B.

1
catarrhalis
Rachel Balder1 and Eric R. Lafontaine1
1
University of Georgia, Department of Infectious Diseases, Athens, Georgia

ABSTRACT
Moraxella catarrhalis is a Gram-negative bacterium that has recently emerged as the third
leading cause of bacterial ear infections in children. This organism is also responsible
for a variety of upper respiratory tract illnesses in adults, including ∼10% of all cases of
respiratory exacerbations in patients with chronic obstructive pulmonary disease (COPD).
There is interest in studying M. catarrhalis for vaccine development, and this unit
provides guidelines for the laboratory maintenance of the organism. The three Basic
Protocols presented in this unit describe how to culture and prepare M. catarrhalis
cells for use in experiments pertaining to various biological aspects of this important
respiratory pathogen. Curr. Protoc. Microbiol. 11:6B.1.1-6B.1.11.  C 2008 by John Wiley

& Sons, Inc.

Keywords: Moraxella catarrhalis r growth r laboratory r biological assays r
phase variation r viability

INTRODUCTION
This unit describes methods, reagents, and equipment commonly used to grow the Gram-
negative bacterium, Moraxella catarrhalis, in the laboratory. Basic Protocol 1 describes
the preparation of a working stock, which is used for culturing M. catarrhalis on solid
(Basic Protocol 2) or liquid (Basic Protocol 3) medium. Conditions for inoculation,
incubation, passage, and storage are discussed. Antibiotic concentrations used to grow
genetically engineered mutant strains, as well as optical densities of M. catarrhalis
liquid suspensions and their corresponding bacterial concentrations, are presented in
Basic Protocol 1 and 2, respectively. Together, these simple guidelines will facilitate the
use of low-passage M. catarrhalis cells in biological assays, which will improve the
reproducibility of experimental outcomes. With a few exceptions (e.g., media), these
protocols are appropriate for the laboratory maintenance of other bacterial species as
well.

CAUTION: Moraxella catarrhalis is a Biosafety Level 2 (BSL-2) pathogen. Follow all

appropriate guidelines and regulations for the use and handling of pathogenic microor-
ganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.

NOTE: All solutions and equipment that come in contact with M. catarrhalis must be
sterile and aseptic technique must be used to avoid contamination. Refer to APPENDIX 4
for more details.

PREPARATION OF A WORKING M. CATARRHALIS STOCK BASIC

PROTOCOL 1
Passaging of M. catarrhalis should be limited, especially when studying characteris-
tics of the organism that are associated with pathogenesis (e.g., adherence to host cells,
serum resistance, biofilm formation). The expression of several M. catarrhalis molecules
involved in these phenotypic traits has been shown to spontaneously undergo phase vari-
ation. Multiple successive passages will thus increase the risk of studying a bacterial Nonenteric
Gamma
Proteobacteria

Current Protocols in Microbiology 6B.1.1-6B.1.11, November 2008 6B.1.1

Published online November 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc06b01s11 Supplement 11
Copyright C 2008 John Wiley & Sons, Inc.
 population that is expressing reduced levels of, or completely lacking, one or more bio-
logically relevant antigens. The use of a working stock, which consists of M. catarrhalis
cells “revived” from a frozen state using solid medium, will minimize passaging of the
organism. These revived bacteria are stored at 4◦ C and used to start new cultures. By
following these simple guidelines, M. catarrhalis cells are passaged minimally prior to
an experiment.

Materials
Moraxella catarrhalis frozen stock vial (see Support Protocol)
Todd Hewitt agar plates (see recipe)
Wire inoculating loop (see APPENDIX 4A)
37◦ C, 7.5% CO2 incubator
Additional reagents and equipment for aseptic technique and streaking bacteria
(APPENDIX 4A)
1. Thaw a frozen stock vial of M. catarrhalis at room temperature for 10 min.
The entire vial is thawed to reduce the possibility of contamination associated with
scraping or chipping off frozen pieces of the sample.

2. Vortex vial for 30 sec.

3. Transfer 0.1 ml of the M. catarrhalis suspension to an area of a Todd Hewitt agar
plate that is 2 to 3 cm to the side of the center of the plate.
4. Use a wire inoculating loop and aseptic technique to streak bacteria for isolated
colonies (refer to APPENDIX 4A).
Moraxella catarrhalis looses viability upon repeated freezing and thawing. Therefore, do
not put stock vial back in −80◦ C storage for later use. Instead, discard unused bacteria
according to the appropriate institutional biosafety protocols.
Genetically engineered M. catarrhalis mutant strains typically contain the resistance
markers listed in Table 6B.1.1. Thus, where indicated, use Todd Hewitt agar plates
supplemented with the appropriate antibiotic concentration.

5. Label the agar side of the Petri dish with the date and the name of the strain. Also,
indicate that this culture corresponds to bacteria “revived” from a frozen stock vial.
6. Incubate for 16 to 20 hr at 37◦ C in the presence of 7.5% CO2 .
If a CO2 incubator is not available, place the agar plate inside a desiccator containing
a lit candle. Seal the jar and allow the candle to go out. This will produce enough CO2
to support the overnight growth of M. catarrhalis. Place the sealed desiccator in an
incubator or a warm room set at 37◦ C.
Table 6B.1.1 Antibiotic Stock Solutions for Use with M. catarrhalis Cultures

Stock Working
Antibiotic Solvent Storage
concentration concentration

Kanamycin Water 10 mg/ml 20 μg/ml Up to 6 months at 4◦ C

Spectinomycin Water 10 mg/ml 20 μg/ml Up to 6 months at 4◦ C
Chloramphenicol Ethanol 10 mg/ml 0.5 μg/ml Up to 6 months at –20◦ C
Zeocina — 100 mg/ml 5 μg/ml Up to 1 year at –20◦ C
Streptomycin Water 10 mg/ml 50 μg/ml Up to 6 months at 4◦ C
a Stock commercially available from Invitrogen (or equivalent). Formulation is proprietary.
Laboratory
Maintenance of
M. catarrhalis

6B.1.2
Supplement 11 Current Protocols in Microbiology
Figure 6B.1.1 Moraxella catarrhalis strain O35E grown on a Todd Hewitt agar plate for 20 hr at
37◦ C.

7. Ascertain that the “revived” bacteria are not contaminated with organisms other than
M. catarrhalis by inspecting the appearance of isolated colonies on the agar plate.
Following a 16- to 20-hr incubation, M. catarrhalis colonies are 1 to 2 mm in diameter,
round, opaque, and convex, with a yellow-tan color (Fig. 6B.1.1). Another useful charac-
teristic of these colonies is that they remain intact while being pushed across the surface
of the agar plate.
If contamination is observed, find an area that is not contaminated and use a wire
inoculating loop and aseptic technique to scrape several M. catarrhalis colonies and
streak them onto a new Todd Hewitt agar plate.

8. Store this agar plate with “revived” cells, which corresponds to the working
M. catarrhalis stock, at 4◦ C for up to 5 days.
This working stock should always be used to start cultures (liquid and solid media) for
biological assays (refer to Basic Protocols 2 and 3). This practice reduces the number of
passages of the strain in the laboratory and will increase the reproducibility of results.
Moraxella catarrhalis does not survive well when stored at 4◦ C for extended periods of
time. After 5 days, the “revived” bacteria should therefore be discarded according to
appropriate institutional biosafety protocols. A fresh working stock should be prepared
as needed.

GROWTH OF M. CATARRHALIS ON SOLID MEDIUM FOR BIOLOGICAL BASIC

ASSAYS PROTOCOL 2
Moraxella catarrhalis grown on solid medium has been used in various biological assays
such as measuring the ability of antibodies to bind to the bacterial surface by flow
cytometry and testing adherence to host cells. Bacteria are scraped off agar plates and
suspended to the desired concentration in liquid, and this suspension is used in biological
assays. This provides an expedient way of preparing cells and yields highly reproducible Nonenteric
Gamma
results. Furthermore, visual inspection of the agar plate prior to setting up a liquid Proteobacteria

6B.1.3
Current Protocols in Microbiology Supplement 11
 suspension can minimize the risk of contamination. Although the authors’ laboratory
routinely grows M. catarrhalis on Todd Hewitt agar plates, other media are commonly
used, including Brain Heart Infusion, Mueller Hinton, chocolate agar, and blood agar
plates. With the exception of transposon mutagenesis, where the authors have found Todd
Hewitt broth to increase efficiency (see Basic Protocol 3), there is no specific advantage
or disadvantage to use one medium over another when growing M. catarrhalis.

Materials
Moraxella catarrhalis working stock (see Basic Protocol 1)
Todd Hewitt agar plates (see recipe)
PBSG (see recipe)
Wire inoculating loop (see APPENDIX 4A)
37◦ C, 7.5% CO2 incubator
12 × 75–mm snap-cap polypropylene tubes, sterile
Disposable inoculating loops, 10 μl (BD or equivalent)
Klett 802 calibrated test tubes (Scienceware/Bel-Art Products) or
spectrophotometer and appropriate cuvettes
Klett colorimeter with Klett color filter KS-54
Additional reagents and equipment for aseptic technique and streaking bacteria
(APPENDIX 4A)
1. Using a wire inoculating loop and aseptic technique (APPENDIX 4A), transfer a loopful
of bacteria from the working M. catarrhalis stock (i.e., the plate prepared in Basic
Protocol 1) to an area near one side of a Todd Hewitt agar plate. Streak to obtain
isolated colonies as illustrated in Figure A.4A.3.
It is not recommended to use only a single colony to propagate M. catarrhalis. This
bacterium expresses several molecules subject to phase variation, and the use of a single
colony for propagation increases the risk of studying a population of cells expressing
reduced levels of one or more of these antigens. The use of a loopful of bacteria will best
facilitate an unbiased approach to the study of M. catarrhalis.

2. Incubate 16 to 20 hr at 37◦ C in the presence of 7.5% CO2 .

3. Using a sterile disposable loop and aseptic technique (also see APPENDIX 4A), scrape a
loopful of freshly grown bacteria from the agar plate and transfer to a 12 × 75–mm
snap-cap tube containing 1 ml of PBSG. Repeat 3 to 5 times.
PBSG (PBS supplemented with 0.15% gelatin) is used to increase the viability of
M. catarrhalis.
Discard the agar plate with unused freshly grown bacteria according to appropriate
institutional biosafety protocols. Do not use this agar plate to start a new culture for
repeating biological assays. Instead, use the working M. catarrhalis stock to propagate
the organism for additional experiments.

4. Vortex this concentrated bacterial suspension at high speed until homogeneous.

Moraxella catarrhalis cells autoagglutinate; therefore, it is important to dissociate ag-
gregates before performing assays. An alternative to vortexing is to repeatedly pipet the
suspension up and down through a 1-ml pipet tip until it has reached homogeneity.
5. Use a sterile Klett tube containing 5 ml PBSG to zero the Klett colorimeter. To this
tube, aseptically add 25 μl of the concentrated bacterial suspension (from step 3).
A spectrophotometer can be used as a substitute for a Klett colorimeter. Refer to
Table 6B.1.2 for optical densities and corresponding bacterial concentrations.
Laboratory
Maintenance of 6. Gently mix by vortexing (medium speed) and measure the density of the suspension.
M. catarrhalis Repeat until the desired bacterial cell concentration is obtained (see Table 6B.1.2).
6B.1.4
Supplement 11 Current Protocols in Microbiology
 Table 6B.1.2 Optical Densities of M. catarrhalis Liquid Suspensions
and Corresponding Bacterial Concentrations

Klett reading Bacterial concentration Absorbance

(KS-54 green filter) (cfu/ml) (600 nm)

50 2.0 × 107 0.09

150 6.0 × 10 7
0.3
230 1.0 × 10 8
0.45
300 1.5 × 10 8
0.600

GROWTH OF M. CATARRHALIS IN LIQUID MEDIUM FOR BIOLOGICAL BASIC

ASSAYS PROTOCOL 3
Broth-grown M. catarrhalis has been used in various experiments including preparation
of electrocompetent cells for transposon mutagenesis, complement-mediated killing tests,
and growth curves. Plate-grown bacteria are scraped off agar plates, suspended to a low
optical density in broth medium, and then cultured until a target bacterial concentration
and/or phase of growth is reached. Several broths have been described for growing
M. catarrhalis such as Brain Heart Infusion, Mueller Hinton, and chemically defined
medium (CDM). The authors favor the use of Todd Hewitt broth because it was found to
substantially increase the efficiency of transposon mutagenesis experiments.

Materials
M. catarrhalis working stock (see Basic Protocol 1)
Todd Hewitt agar plates (see recipe)
PBSG (see recipe)
Todd Hewitt broth (see recipe)
Wire inoculating loop (see APPENDIX 4A)
37◦ C incubator (no CO2 added) with shaker
37◦ C, 7.5% CO2 incubator
12 × 75–mm snap-cap polypropylene tubes, sterile
300- to 500-ml Nephelo culture flask, with 14 × 130–mm sidearm (Bellco
Biotechnology or equivalent)
Klett colorimeter with Klett color filter KS-54
Additional reagents and equipment for aseptic technique and streaking plates
(APPENDIX 4A)
1. Using a wire inoculating loop and aseptic technique (APPENDIX 4A), transfer a loopful
of bacteria from the working M. catarrhalis stock (i.e., the plate prepared in Basic
Protocol 1) to an area near one side of a Todd Hewitt agar plate. Streak to obtain
isolated colonies as illustrated in Figure A.4A.3.
As an alternative, M. catarrhalis can be grown in Todd Hewitt broth for 16 to 20 hr. This
overnight culture can then be diluted into fresh medium as described in step 5. However,
we recommend using plate-grown bacteria, as visual inspection of the agar plate prior to
setting up the broth culture will help reduce the risk of contamination.

2. Incubate for 16 to 20 hr at 37◦ C in the presence of 7.5% CO2 .

3. Using a sterile disposable loop and aseptic technique, scrape a loopful of freshly
grown bacteria from the agar plate. Suspend bacteria in a small snap-cap tube
containing 1 ml of PBSG. Repeat 3 to 5 times. Vortex this concentrated bacterial Nonenteric
suspension at high speed until homogeneous. Gamma
Proteobacteria

6B.1.5
Current Protocols in Microbiology Supplement 11
 Discard the agar plate with unused freshly grown bacteria according to appropriate
institutional biosafety protocols.
4. Add desired volume of Todd Hewitt broth (15 to 30 ml) to a sterile Nephelo culture
flask (300 to 500 ml size) with a 14 × 130–mm sidearm.
The Nephelo flask is a specialized flask with a built-in sidearm that makes it easy to assess
the culture density during growth without exposing the culture to potential contaminants
outside the flask.

5. Carefully tilt the flask to fill the sidearm with broth. Place the sidearm in the Klett
colorimeter to zero. Tilt the flask back to return the broth to the bottom of the culture
flask.
When appropriate, supplement the Todd Hewitt broth with the indicated antibiotic con-
centration (Table 6B.1.1).

6. To this flask, aseptically add 25 μl of the concentrated bacterial suspension (step 3).
Mix by gently swirling the flask.
7. Fill the sidearm with broth as described in the annotation to step 4, then measure the
optical density. Repeat until the density reaches 35 to 50 Klett units.
8. Place the sidearm flask in a shaking incubator set at 37◦ C (CO2 not used) and a speed
of 200 rpm. Incubate until the desired bacterial cell concentration (Table 6B.1.2) or
growth phase is attained.
A spectrophotometer can be used in place of a Klett colorimeter. Refer to Table 6B.1.2
for optical densities and corresponding bacterial concentrations.
Under the conditions stated above, an optical density of 125 to 150 Klett units should
be reached within 3 to 3.5 hr of incubation, and will correspond to the mid–log phase
of growth; M. catarrhalis cells will enter stationary phase after ∼6 hr (300 to 350
Klett units). These values will need to be empirically determined when modifying culture
conditions (e.g., using a different culture medium or growth temperature) or studying
genetically engineered mutant strains that may have slower growth rates.

SUPPORT PREPARATION OF M. CATARRHALIS FROZEN STOCKS

PROTOCOL
Materials
M. catarrhalis growing on agar plate (e.g., clinical isolate, purchased strain, or
working stock needing to be restocked)
M. catarrhalis freezing medium (see recipe)
Disposable inoculating loops, 10 μl (BD or equivalent)
2-ml cryovials (Wheaton or equivalent)
−80◦ C freezer or liquid nitrogen tank
1. Using a sterile disposable inoculating loop, scrape a loopful of M. catarrhalis cells
from the surface of an agar plate.
It is not recommended to use only a single colony to make frozen stocks because
M. catarrhalis expresses several phase-variable molecules. The use of a single colony
increases the risk of archiving a bacterial population that lacks expression of one or more
of these antigens. The use of a loopful of bacteria to make frozen stocks will best facilitate
an unbiased approach to the study of M. catarrhalis.

2. Transfer M. catarrhalis cells to a cryovial containing 0.3 ml of freezing medium.

3. Vortex the suspension for 1 min and store in a −80◦ C freezer or a liquid nitrogen
Laboratory tank. Make several frozen stock vials for each M. catarrhalis strain.
Maintenance of
M. catarrhalis Making several frozen stocks is necessary because vials should not be refrozen after
thawing, due to a decrease in the viability of the organism (refer to Basic Protocol 1).
6B.1.6
Supplement 11 Current Protocols in Microbiology
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX. Filter-sterilization is a preference of the
authors for its efficiency; however, autoclaving can also be used to sterilize the liquid media in
this unit.

M. catarrhalis freezing medium

To 100 ml of distilled water, add 9 g of BD BBL Brain Heart Infusion and stir at
room temperature to dissolve. Add 100 ml of 100% glycerol (Fisher BioReagents)
and stir at room temperature to mix. Sterilize by filtration using a low protein-
binding 0.22-μm filter (e.g., Millipore Stericup ExpressPlus 0.22-μm filter units).
Store indefinitely at room temperature.

Todd Hewitt agar plates

To 1000 ml of distilled water, add 30 g of Bacto Todd Hewitt broth (BD Biosciences)
and stir at room temperature to dissolve. Add 15 g of Bacto Agar (BD Biosciences)
and stir with heat to dissolve. Autoclave for 20 min and cool to 50◦ C. Add antibiotics
when necessary (see Table 6B.1.1) and gently stir to evenly disperse all ingredients.
Pour into sterile 100 × 15–mm petri dishes and let dry at room temperature for 1
to 2 days. Store agar plates at 4◦ C for up to 2 months.
Also see APPENDIX 4A for more general information regarding the preparation of agar plates

Todd Hewitt broth

To 1000 ml of distilled water, add 30 g of Bacto Todd Hewitt broth and stir at room
temperature to dissolve. Sterilize by filtration using a low protein-binding 0.22-μm
filter (e.g., Millipore Stericup ExpressPlus 0.22-μm filter units). Store indefinitely
at room temperature.

Phosphate-buffered saline (PBS) supplemented with 0.15% (w/v) gelatin (PBSG)

To 1000 ml of PBS (APPENDIX 2A), add 1.5 g of gelatin (bovine, Sigma), and stir
with heat to dissolve. Sterilize by filtration using a low protein-binding 0.22-μm
filter (e.g., Millipore Stericup ExpressPlus 0.22-μm filter units). Let cool to room
temperature before use. Store indefinitely at room temperature.

COMMENTARY
Background Information the interpretation of experimental outcomes.
This issue is discussed further under Critical
Maintenance of M. catarrhalis Parameters and Troubleshooting.
The protocols described in this unit pro-
Common applications utilizing M.
vide the necessary information to culture
catarrhalis grown on solid medium include
M. catarrhalis in the laboratory. Specifically,
isolation of chromosomal DNA (Balder
they describe how to prepare frozen stocks for
et al., 2007), whole-cell protein preparations
long-term storage of the organism (Support
(Bullard et al., 2005), and removal of loosely
Protocol) and how to revive bacteria from this
associated outer membrane proteins by
frozen state (Basic Protocol 1), as well as how
mechanical shearing (Luke et al., 2004). In
to culture M. catarrhalis using solid (Basic
addition, several functional assays can be per-
Protocol 2) and liquid (Basic Protocol 3) me-
formed using M. catarrhalis cells cultured on
dia. Basic Protocol 1 describes how to revive
agar plates. This list includes, but is not limited
bacteria stored at −80◦ C and how to pre-
to, assessing attachment of the organism to
pare a working stock for the propagation of
eukaryotic cells (Holm et al., 2003), analyzing
M. catarrhalis. The use of this working stock
the binding of antibodies to bacteria by flow
to inoculate cultures limits the number of pas-
cytometry (Lipski et al., 2007), measuring Nonenteric
sages that occur prior to using the organism
cell-associated enzymatic activity (Timpe Gamma
in experiments, which in turn reduces the Proteobacteria
et al., 2003), and determining the organism’s
likeliness of phase variability, complicating
6B.1.7
Current Protocols in Microbiology Supplement 11
 ability to use different iron sources (Bonnah
et al., 1999). Basic Protocol 2 describes how
to grow and prepare M. catarrhalis cells to
perform these experiments. There are also
several applications in which broth-grown
bacteria can be used, such as isolation of outer
membrane vesicles (Murphy and Loeb, 1989),
bactericidal assays (Attia et al., 2005), growth
curves (Furano and Campagnari, 2003; Holm
et al., 2004), biofilm assays (Pearson et al.,
2006), iron-uptake studies (Aebi et al., 1996),
and electroporation (Holm et al., 2003;
Pearson and Hansen, 2007). The information
required to propagate M. catarrhalis for
setting up these assays is presented in Basic
Protocol 3.
Significance of M. catarrhalis
Moraxella catarrhalis is a Gram-negative,
unencapsulated diplococcus that causes oti-
tis media (middle ear infections) in children
and respiratory tract diseases in adults. Orig-
inally described in 1896, this bacterium was
long considered a commensal of the upper
respiratory tract and referred to as Neisseria
catarrhalis. This classification was in part due
to difficulties differentiating M. catarrhalis
from other nonpathogenic Neisseria species
such as Neisseria cinerea, and it was not
until the later part of the 20th century that
the organism emerged as an important hu-
man pathogen. Based on DNA hybridization
studies, M. catarrhalis was temporarily clas-
sified in the 1970s to a new genus and called
Branhamella catarrhalis. However, the organ-
ism was reassigned to the Moraxella genus in
1984, and DNA sequence analyses have thus
far validated this classification (Catlin, 1990;
Murphy, 1996; Christensen, 1999; Karalus and
Campagnari, 2000; Verduin et al., 2002).
Several criteria are used to identify M.
catarrhalis in clinical specimens from the
respiratory tract, including Gram stain,
colony morphology, oxidase production,
and lack of glucose, maltose, sucrose,
lactose, or fructose fermentation, as well
as a lack of pigmentation (i.e., gray-white
colonies) on blood agar. Additional tests
are necessary to distinguish M. catarrhalis
from commensal Neisseria species, and
those include the reduction of nitrate and
nitrite, DNase production, and tributyrin
hydrolysis (Doern and Morse, 1980; Catlin,
1990; Singh et al., 1997; Christensen, 1999;
Verduin et al., 2002). The latter is mediated
Laboratory by the M. catarrhalis esterase/phospholipase
Maintenance of B McaP (Timpe et al., 2003; Lipski et al.,
M. catarrhalis
6B.1.8
Supplement 11
2007), and is the basis of several tests
that are commercially available for rapidly
identifying the organism, such as Gibson
ID- M. CAT (Gibson http://www.gibsonlabs.
com/) and BactiCard Neisseria (Remel).
Moraxella catarrhalis contributes to ∼15%
of all cases of bacterial ear infections (Cripps
et al., 2005, Giebink et al., 2005) and ∼10%
of respiratory tract exacerbations in patients
with chronic obstructive pulmonary disease
(COPD; Murphy et al., 2005). These ail-
ments impose a significant financial burden
on the U.S. healthcare system, amounting
to ∼$5 and ∼$18 billion a year for treat-
ment of ear infections and COPD exacer-
bations, respectively (Murphy, 1996; Karalus
and Campagnari, 2000; Klein, 2000; Sethi and
Murphy, 2001; Murphy et al., 2005). Otitis me-
dia is primarily a disease of early childhood.
Patients commonly experience persistent pain
and upper respiratory symptoms. Other indi-
cators may include fever, nausea, and vom-
iting. The tympanic membrane of patients is
often red, thickened, and bulging. Addition-
ally, a purulent exudate can also be observed in
some instances. Recurrence of ear infections is
a problem and can have serious consequences
including hearing loss and delays in the devel-
opment of communication skills (Klein, 2000;
Cripps et al., 2005; Giebink et al., 2005).
Moraxella catarrhalis is also a significant
cause of respiratory tract infections in
patients with COPD. This obstructive disorder
severely impairs airflow and is experienced
by patients with chronic bronchitis and/or
emphysema. COPD is most commonly
diagnosed in elderly smokers or individu-
als with other chronic lung problems. M.
catarrhalis generally causes tracheobronchitis
or pneumonia in these patients, and symptoms
include coughing, purulent sputum, and, in
the case of pneumonia, infiltrates in the lower
lobes that are observable by X ray. These
infections have been shown to contribute to
the progression of COPD, which is one of
the leading causes of death worldwide (Sethi
and Murphy, 2001; Sethi et al. 2002; Murphy
et al., 2005). M. catarrhalis is highly resistant
to penicillin-based antimicrobials, as >90%
of clinical isolates produce β-lactamases, and
antibiotics suggested for treatment include
second- and third-generation cephalosporins,
erythromycin, amoxicillin-clavulanate,
fluoroquinolones, and trimethroprim-
sulfamethoxazole (Klugman, 1996; Manninen
et al., 1997; Jacobs et al., 2004; Murray et al.,
2005).
Current Protocols in Microbiology
Critical Parameters and
Troubleshooting
Phase variation in M. catarrhalis
Moraxella catarrhalis possesses several
molecules that may spontaneously undergo
phase variation such that their expression is
abolished or substantially diminished (i.e.,
phase-variant molecules). These antigens,
which include Hag (Pearson et al., 2002;
Mollenkvist et al., 2003), UspA1 (Lafontaine
et al., 2001), UspA2 (Attia and Hansen, 2006),
and UspA2H (Wang et al., 2007), are impor-
tant for several biological functions associated
with bacterial pathogenesis, such as adher-
ence to epithelial cells, biofilm formation, and
serum resistance. Additional measures must
therefore be taken when handling the organ-
ism in order to avoid studying pure cultures
of M. catarrhalis phase variants. One of these
is to limit the passage of strains in the labo-
ratory by utilizing a working stock (described
in Basic Protocol 1). This practice allows ver-
ification that revived bacteria are not contami-
nated, ensures that M. catarrhalis is minimally
passaged prior to performing experiments, and
has been determined by the authors to increase
the reproducibility of biological assay results.
Another important precaution to avoid the
study of a phase-variant population is to re-
frain from using a single colony to propagate
M. catarrhalis. Although not in conformity
with common microbiological practices, this
will reduce the risk of inadvertently selecting
a variant on the agar plate that is not expressing
wild-type levels of phase-variable molecules.
Propagating a single colony is often difficult
to circumvent in some experiments, such as
the construction of isogenic mutant strains or
transposon mutagenesis of M. catarrhalis. In
cases where a single colony must be passaged,
it is recommended to verify that phase-variable
proteins are expressed at wild-type levels by
western blotting. This practice will ascertain
that a particular phenotypic trait is associated
with the gene under study, as opposed to be-
ing the consequence of phase variation in a
different gene product.
Viability of M. catarrhalis
The viability of M. catarrhalis becomes
an issue with respect to several common lab-
oratory procedures. For example, there are
many bacteria for which a frozen stock can
be thawed and the necessary amount removed
and streaked onto an agar plate; following this,
the vial is refrozen for later use. This is not
an option when handling M. catarrhalis, as
Current Protocols in Microbiology
the viability of the organism greatly decreases
after refreezing. The use of a working stock
(Basic Protocol 1) allows for the propagation
of cultures from revived bacteria for several
consecutive days without having to repeatedly
revive and restock the organism. However, M.
catarrhalis does not survive as well, or for
as long, as some other bacteria (e.g., E. coli)
when stored at 4◦ C for extended periods. Thus,
after 5 days of storage under these conditions,
the working stock should be disposed of ac-
cording to institutional guidelines and a new
working stock of revived M. catarrhalis cells
should be prepared as needed.
Another variation of common laboratory
practices to improve the viability of M.
catarrhalis is supplementing PBS-based so-
lutions with gelatin (see Reagents and Solu-
tions). Phosphate-buffered saline is used as a
basic buffer to suspend bacteria for a variety
of biological protocols and cell preparations.
M. catarrhalis does not survive well in PBS,
but the addition of 0.15% (w/v) gelatin in-
creases the organism’s viability (Aebi et al.,
1998). Despite the addition of gelatin, it is
still important to work in an efficient manner.
Specifically, do not let more than 30 min elapse
between suspension of M. catarrhalis in PBSG
and the use of these cells in experiments for
which the viability of the organism is impor-
tant (e.g., adherence and bactericidal assays).
Autoagglutination
Moraxella catarrhalis cells autoaggluti-
nate, which makes homogeneous suspension
of the organism in liquid crucial to obtain-
ing consistent results (see Basic Protocols 2
and 3). For instance, a freshly-prepared sus-
pension in PBSG can go from turbid to clear
if allowed to stand undisturbed for ∼20 min,
with all bacteria settled at the bottom of the
tube. It is therefore important to vortex sus-
pensions just prior to starting experiments in
order to obtain the intended number of bacte-
rial cells.
Anticipated Results
Basic Protocols 1 and 2 describe cultur-
ing M. catarrhalis on solid medium. Follow-
ing a 16- to 20-hr incubation, colonies will be
1 to 2 mm in diameter, opaque, round, con-
vex, and yellow-tan in color, and will remain
intact when pushed across the surface of the
agar plate (Fig. 6B.1.1). Basic Protocol 3 de-
scribes the growth of M. catarrhalis in liquid
medium. Table 6B.1.2 lists optical densities
and provides the bacterial concentrations cor- Nonenteric
Gamma
responding to these values. Proteobacteria
6B.1.9
Supplement 11
 Time Considerations
Basic Protocol 1 requires the use of Todd
Hewitt agar plates and a frozen M. catarrhalis
stock, which are usually made in advance. The
specific steps described in the protocol should
take ∼15 min.
The first two steps of Basic Protocols 2
and 3 are the same, and consist of streaking
M. catarrhalis on solid medium from the
working stock, which should take ∼5 min, and
incubating this agar plate for 16 to 20 hr at
37◦ C. In Basic Protocol 2, these freshly grown
M. catarrhalis cells are suspended in PBSG,
and this part of the experiment (i.e., steps 3
and 4) should take 10 to 20 min, depending
on the number of suspensions to be prepared.
In Basic Protocol 3, plate-grown bacteria are
first suspended in PBSG and then diluted to a
low optical density in broth. This part of Ba-
sic Protocol 3 (i.e., steps 3 to 5) can also be
accomplished within 10 to 20 min, depend-
ing on the number of cultures to be set up. It
should be noted that Basic Protocols 2 and 3
only describe the preparation of M. catarrhalis
cells to be subsequently used in various types
of assays. Thus, the time necessary to com-
plete an entire experiment will depend on
the specific type of assay that is to be per-
formed with these cells. For example, prepara-
tion of electrocompetent bacteria will typically
take less time than performing a growth-curve
experiment.
Literature Cited
Aebi, C., Stone, B., Beucher, M., Cope, L.D.,
Maciver, I., Thomas, S.E., McCracken, G.H.
Jr., Sparling, P.F., and Hansen, E.J. 1996. Ex-
pression of the CopB outer membrane protein
by Moraxella catarrhalis is regulated by iron
and affects iron acquisition from transferrin and
lactoferrin. Infect. Immun. 64:2024-2030.
Aebi, C., Lafontaine, E.R., Cope, L.D., Latimer,
J.L., Lumbley, S.L., McCracken, G.H., Jr., and
Hansen, E.J. 1998. Phenotypic effect of iso-
genic uspA1 and uspA2 mutations on Moraxella
catarrhalis 035E. Infect. Immun. 66:3113-
3119.
Attia, A.S. and Hansen, E.J. 2006. A conserved
tetranucleotide repeat is necessary for wild-type
expression of the Moraxella catarrhalis UspA2
protein. J. Bacteriol. 188:7840-7852.
Attia, A.S., Lafontaine, E.R., Latimer, J.L., Aebi,
C., Syrogiannopoulos, G.A., and Hansen,
E.J. 2005. The UspA2 protein of Moraxella
catarrhalis is directly involved in the expression
of serum resistance. Infect. Immun. 73:2400-
2410.
Balder, R., Hassel, J., Lipski, S., and Lafontaine,
Laboratory
Maintenance of E.R. 2007. Moraxella catarrhalis strain O35E
M. catarrhalis expresses two filamentous hemagglutinin-like
6B.1.10
Supplement 11
proteins that mediate adherence to human ep-
ithelial cells. Infect. Immun. 75:2765-2775.
Bonnah, R.A., Wong, H., Loosmore, S.M., and
Schryvers, A.B. 1999. Characterization of
Moraxella (Branhamella) catarrhalis lbpB,
lbpA, and lactoferrin receptor orf3 isogenic
mutants. Infect. Immun. 67:1517-1520.
Bullard, B., Lipski, S.L., and Lafontaine, E.R.
2005. Hag directly mediates the adherence of
Moraxella catarrhalis to human middle ear
cells. Infect. Immun. 73:5127-5136.
Catlin, B.W. 1990. Branhamella catarrhalis: An
organism gaining respect as a pathogen. Clin.
Microbiol. Rev. 3:293-320.
Christensen, J.J. 1999. Moraxella (Branhamella)
catarrhalis: Clinical, microbiological and im-
munological features in lower respiratory tract
infections. APMIS 88:1-36.
Cripps, A.W., Otczyk, D.C., and Kyd, J.M. 2005.
Bacterial otitis media: A vaccine preventable
disease? Vaccine 23:2304-2310.
Doern, G.V. and Morse, S.A. 1980. Branhamella
(Neisseria) catarrhalis: Criteria for laboratory
identification. J. Clin. Microbiol. 11:193-195.
Furano, K. and Campagnari, A.A. 2003. Inacti-
vation of the Moraxella catarrhalis 7169 fer-
ric uptake regulator increases susceptibility to
the bactericidal activity of normal human sera.
Infect. Immun. 71:1843-1848.
Giebink, G.S., Kurono, Y., Bakaletz, L.O., Kyd,
J.M., Barenkamp, S.J., Murphy, T.F., Green, B.,
Ogra, P.L., Gu, X.X., Patel, J.A., Heikkinen, T.,
Pelton, S.I., Hotomi, M., and Karma, P. 2005.
Recent advances in otitis media. 6. Vaccine. Ann.
Otol. Rhinol. Laryngol. 194:86-103.
Holm, M.M., Vanlerberg, S.L., Sledjeski, D.D.,
and Lafontaine, E.R. 2003. The Hag protein of
Moraxella catarrhalis strain O35E is associated
with adherence to human lung and middle ear
cells. Infect. Immun. 71:4977-4984.
Holm, M.M., Vanlerberg, S.L., Foley, I.M.,
Sledjeski, D.D., and Lafontaine, E.R. 2004. The
Moraxella catarrhalis porin-like outer mem-
brane protein CD is an adhesin for human lung
cells. Infect. Immun. 72:1906-1913.
Jacobs, M.R., Bajaksouzian, S., Windau, A.,
Good, C.E., Lin, G., Pankuch, G.A., and
Appelbaum, P.C. 2004. Susceptibility of Strep-
tococcus pneumoniae, Haemophilus influenzae,
and Moraxella catarrhalis to 17 oral antimicro-
bial agents based on pharmacodynamic param-
eters: 1998-2001 U.S. Surveillance Study. Clin.
Lab. Med. 24:503-530.
Karalus, R. and Campagnari, A. 2000. Moraxella
catarrhalis: A review of an important hu-
man mucosal pathogen. Microbes Infect. 2:547-
559.
Klein, J.O. 2000. The burden of otitis media.
Vaccine 19:S2-8.
Klugman, K.P. 1996. The clinical relevance of in-
vitro resistance to penicillin, ampicillin, amoxy-
cillin and alternative agents, for the treatment
of community-acquired pneumonia caused by
Current Protocols in Microbiology
 Streptococcus pneumoniae, Haemophilus in-
fluenzae and Moraxella catarrhalis. J. Antimi-
crob. Chemother. 38:133-140.
Lafontaine, E.R., Wagner, N.J., and Hansen, E.J.
2001. Expression of the Moraxella catarrhalis
UspA1 protein undergoes phase variation and is
regulated at the transcriptional level. J. Bacte-
riol. 183:1540-1551.
Lipski, S.L., Akimana, C., Timpe, J.M., Wooten,
R.M., and Lafontaine, E.R. 2007. The Moraxella
catarrhalis autotransporter McaP is a conserved
surface protein that mediates adherence to hu-
man epithelial cells through its N-terminal
passenger domain. Infect. Immun. 75:314-
324.
Luke, N.R., Howlett, A.J., Shao, J., and
Campagnari, A.A. 2004. Expression of type IV
pili by Moraxella catarrhalis is essential for nat-
ural competence and is affected by iron limita-
tion. Infect. Immun. 72:6262-6270.
Manninen, R., Huovinen, P., and Nissinen, A. 1997.
Increasing antimicrobial resistance in Strepto-
coccus pneumoniae, Haemophilus influenzae
and Moraxella catarrhalis in Finland. J. Antimi-
crob. Chemother. 40:387-392.
Mollenkvist, A., Nordstrom, T., Hallden, C.,
Christensen, J.J., Forsgren, A., and Riesbeck, K.
2003. The Moraxella catarrhalis immunoglob-
ulin D-binding protein MID has conserved se-
quences and is regulated by a mechanism cor-
responding to phase variation. J. Bacteriol.
185:2285-2295.
Murphy, T.F. 1996. Branhamella catarrhalis:
Epidemiology, surface antigenic structure, and
immune response. Microbiol. Rev. 60:267-279.
Murphy, T.F. and Loeb, M.R. 1989. Isolation of
the outer membrane of Branhamella catarrhalis.
Microb. Pathog. 6:159-174.
Murphy, T.F., Brauer, A.L., Grant, B.J., and Sethi,
S. 2005. Moraxella catarrhalis in chronic ob-
structive pulmonary disease: Burden of disease
and immune response. Am. J. Respir. Crit. Care
Med. 172:195-199.
Murray, J.J., Emparanza, P., Lesinskas, E.,
Tawadrous, M., and Breen, J.D. 2005. Efficacy
and safety of a novel, single-dose azithromycin
Current Protocols in Microbiology
microsphere formulation versus 10 days of lev-
ofloxacin for the treatment of acute bacterial si-
nusitis in adults. Otolaryngol. Head Neck Surg.
133:194-200.
Pearson, M.M. and Hansen, E.J. 2007. Identifica-
tion of gene products involved in biofilm produc-
tion by Moraxella catarrhalis ETSU-9 in vitro.
Infect. Immun. 75:4316-4325.
Pearson, M.M., Lafontaine, E.R., Wagner, N.J., St.
Geme, J.W. 3rd, and Hansen, E.J. 2002. A hag
mutant of Moraxella catarrhalis strain O35E
is deficient in hemagglutination, autoagglutina-
tion, and immunoglobulin D-binding activities.
Infect. Immun. 70:4523-4533.
Pearson, M.M., Laurence, C.A., Guinn, S.E.,
and Hansen, E.J. 2006. Biofilm formation by
Moraxella catarrhalis in vitro: Roles of the
UspA1 adhesin and the Hag hemagglutinin.
Infect. Immun. 74:1588-1596.
Sethi, S. and Murphy, T.F. 2001. Bacterial infec-
tion in chronic obstructive pulmonary disease in
2000: A state-of-the-art review. Clin. Microbiol.
Rev. 14:336-363.
Sethi, S., Evans, N., Grant, B.J., and Murphy, T.F.
2002. New strains of bacteria and exacerbations
of chronic obstructive pulmonary disease. N.
Engl. J. Med. 347:465-471.
Singh, S., Cisera, K.M., Turnidge, J.D., and
Russell, E.G. 1997. Selection of optimum lab-
oratory tests for the identification of Moraxella
catarrhalis. Pathology 29:206-208.
Timpe, J.M., Holm, M.M., Vanlerberg, S.L., Basrur,
V., and Lafontaine, E.R. 2003. Identification of a
Moraxella catarrhalis outer membrane protein
exhibiting both adhesin and lipolytic activities.
Infect. Immun. 71:4341-4350.
Verduin, C.M., Hol, C., Fleer, A., van Dijk, H., and
van Belkum, A. 2002. Moraxella catarrhalis:
From emerging to established pathogen. Clin.
Microbiol. Rev. 15:125-144.
Wang, W., Pearson, M.M., Attia, A.S., Blick,
R.J., and Hansen, E.J. 2007. A UspA2H-
negative variant of Moraxella catarrhalis strain
O46E has a deletion in a homopolymeric nu-
cleotide repeat common to uspA2H genes.
Infect. Immun. 75:2035-2045.
Nonenteric
Gamma
Proteobacteria
6B.1.11
Supplement 11