Bacteria Growing Experiments in Petri Plates

Introduction

Bacteria are microorganisms that grow everywhere. We can collect and grow them in
specially prepared petri dishes. Blood agar or tryptic soy agar with 5% sheep's blood is an
excellent medium for supplying bacteria with nutrients and an environment in which we can
see them grow.
Sterile powdered agar with nutrients can be mixed with water, heated and then poured
into empty petri plates or ready-to-use dishes can be purchased. The undigestible agar is a
gelatin-like substance with a semi solid surface on which the bacteria can grow while they
consume the added nutrients (like sheep's blood). In fact, this is why gelatin itself does not
make a good growing medium. Some bacteria can digest gelatin, which is a protein derived
from animal tissue. This destroys the growing surface in the petri plate making it unsuitable
as a bacteria growth medium.
CAUTION. Most bacteria collected in the environment will not be harmful. However, once
they multiply into millions of colonies in a petri dish they become more of a hazard. Be sure
to protect open cuts with rubber gloves and never ingest or breathe in growing bacteria.
Keep growing petri dishes taped closed until your experiment is done. Then you should
safely destroy the fuzzy bacteria colonies using bleach.
Below are general outlines of three types of experiments involving bacteria growth. They
are offered to assist in designing your own experiment or project.

Experiment 1: Direct Contact

Discussion.
 In this type of experiment, bacteria is transferred directly to the prepared petri plate via
direct contact. You can test the effectiveness of different soaps by treating different petri
dishes with "dirty" hands before washing and "clean" hands after washing. Or, you can
press a variety of common objects like coins, combs, etc. on different plates and compare
the bacteria growth that results.

What you need.

 Prepared petri plates containing agar medium and nutrients.
The Science Company has these available at this link.
 Bacteria on hands, paws, etc.
 Wax pencil for labeling dishes.
 Masking tape.
 Bleach.

What to do.

1. Prepared petri dishes should be refrigerated until used and always stored upside
down (i.e media in upper dish, cover on bottom). This keeps condensation which
forms in the lid from dropping onto and disrupting the bacteria growing surface.
2. When ready to use, let dishes come to room temperature before taking samples
(about one hour).
3. Without tearing the agar surface, inoculate the dish by gently pressing fingers, finger
nails, coin, etc onto agar surface. (Direct contact of lips or tongue is NOT a good
idea.)
4. Replace cover on dish, tape closed, and label each dish so you know the source of
the bacteria. Store upside down.
5. Let grow in undisturbed warm location. Bacteria can grow at any temperature from
about ambient room temperature (hopefully around 70°F) all the way up to about
100°F. Do not place in sunlight or on a heating register.
6. You should see growth within a couple of days. The dishes will start to smell which
means the bacteria are growing.
7. Make observations and keep records of what you see growing in each dish. Can you
make any conclusions about what objects had the most bacteria?
8. Before disposing of dishes in the trash the bacteria should be destroyed. Pour a
small amount of household bleach over the colonies while holding dish over sink.
Caution - do not allow bleach to touch your skin, eyes or clothes. It will burn!

Experiment 2: Collected bacteria samples

Discussion.
Use a sterilized inoculating loop or sterile swabs to collect bacteria from different
locations and then streak each petri dish with your sample. This involves a bit more
technique than Experiment 1 but offers a wider choice of bacteria sampling locations.
Swabs can be run over doorknobs, bathroom fixtures, animal mouths, etc.

What you need.

 Prepared petri dishes containing agar medium and nutrients.
The Science Company has these available at this link.
 Bacteria collected from doorknobs, bathroom fixtures, etc.
 Wax pencil for labeling dishes.
 Masking tape.
 Sterile swabs or inoculating loop.
 Alcohol burner (source of flame to sterilize inoculating loop).
 Bleach.

What to do.

1. Prepared petri dishes should be refrigerated until used and always stored upside
down (i.e media in upper dish, cover on bottom). This keeps condensation which
forms in the lid from dropping onto and disrupting the bacteria growing surface.
2. When ready to use, let dishes come to room temperature before taking samples
(about one hour).
3. Collect bacteria from each location using one swab (or resterilized innoculating loop)
for each new spot.
4. Inoculate each dish by streaking a pattern gently across the entire agar surface
without tearing into it. Another common technique is to divide each plate into four
quadrants by marking the lid with a cross. Streak your sample in straight lines
starting in quadrant 1. Generally, after a few days, quadrant one will show the most
growth. Depending on bacteria abundance on the swab, quadrant 4 may show no
grow or only a few colonies. It is sometimes easier to distinguish different bacteria
types in this low growth, less cluttered area.
5. Replace cover on dish, tape closed, and label each dish so you know the source of
the bacteria. Store upside down.
6. Let grow in undisturbed warm location, ideally in an environment around 100° F (37°
C) - not in sunlight or on a heating register.
7. You should see growth within a couple of days. The dishes will start to smell which
means the bacteria are growing.
8. Make observations and keep records of what you see growing in each dish. Can you
make any conclusions about what locations had the most bacteria?
9. Before disposing of dishes in the trash the bacteria should be destroyed. Pour a
small amount of household bleach over the colonies while holding dish over sink.
Caution - do not allow bleach to touch your skin, eyes or clothes. It will burn!

Experiment 3: Testing the effectiveness of bacteria killing agents

Discussion.
In this type of project, a petri dish is inoculated with bacteria then a paper disk (filter
paper) treated with an antiseptic agent is placed in the dish. After several days, a halo
develops around the paper disk indicating a zone of no growth. Comparisons can be made
between different antibacterial agents.

What you need.

 Prepared petri dishes containing agar medium and nutrients.
The Science Company has these available at this link.
 Bacteria collected from doorknobs, bathroom fixtures, etc.
 Wax pencil for labeling dishes.
 Masking tape.
 Sterile swabs or inoculating loop.
 Alcohol burner (source of flame to sterilize inoculating loop).
 Antibacterial agent (soaps, disinfectants, etc.).
 Sterile water.
 Test tubes, 12 x 75mm.
 Filter paper or paper towel.
 Small containers in which to soak paper disks.
 Hole punch.
 Tweezers.
 Ruler.
 Bleach.

What to do.

1. Prepared petri dishes should be refrigerated until used and always stored upside
down (i.e media in upper dish, cover on bottom). This keeps condensation which
forms in the lid from dropping onto and disrupting the bacteria growing surface.
2. Prepare sterilized water by boiling water and letting cool to room temperature.
3. When ready to use, let petri dishes come to room temperature before taking samples
(about one hour).
4. Prepare antiseptic disks by using a hole punch to create paper disks out of a piece
of filter paper or paper towel. Soak one disk in each antibacterial agent to be tested.
Set aside until step 6.
5. Collect bacteria from each location using one swab for each new spot.
6. Fill a small test tube partly full of sterilized water. Dip bacteria laden swab into water.
This will transfer some of the bacteria you collected into the water. Now, inoculate a
petri dish by pouring the water into the dish so the entire surface is covered. Pour
out excess water. Repeat for each bacteria sample using fresh water and clean test
tube each time.
7. Place a pretreated antiseptic disk in each inoculated petri dish.
 8. Replace cover on dish, tape closed, store upside down. Be sure to label each petri
dish with a name or number.
9. Let grow in undisturbed warm location, ideally in an environment around 100° F (37°
C) - not in sunlight or on a heating register.
10. You should see growth within a couple of days. You should also see a "halo" around
each disk indicating a no growth zone. Measure and compare the size of the kill
zone to determine effectiveness of each antibacterial agent.
11. Before disposing of dishes in the trash the bacteria should be destroyed. Pour a
small amount of household bleach over the colonies while holding dish over sink.
Caution - do not allow bleach to touch your skin, eyes or clothes. It will burn!

Notes on maintaining stock cultures, and preparing actively growing cultures for use by students.

Stock cultures

Instead of re-purchasing whenever needed, it may be convenient to maintain a stock of a pure

culture. Most of those recommended are relatively easy to maintain on the appropriate growth
medium, but maintenance of stock cultures needs to be well-organised with attention to detail.

Date stamp new cultures on arrival from the supplier.

Cultures on streak plates are not suitable as stock cultures. Cultures of bacteria and fungi are
normally kept in Universal/ McCartney bottles or (for a short time) in test tubes in which the agar has
been allowed to set at a slope. Dispense the agar in the normal way (5 ml is sufficient in a
Universal bottle) and, after autoclaving, prop the bottle (or tube) at an angle, for example, around the
edges of a pile of bench mats, until the agar has set. Make sure that the sloping agar does not quite
reach the neck of the bottle (or tube). Caps should remain loose until the agar slope has solidified. If
a lot of slopes have to be prepared, it might be worthwhile making a d-i-y construction to hold the
containers at the correct angle.

Slope cultures in bottles with screw-caps (rather than cotton wool plugs or plastic lids) are
preferred because the cap reduces evaporation and dehydration and the caps cannot be
accidentally knocked off. Slope cultures are also preferred to broth (liquid medium) cultures because
the first sign of contamination is more readily noticed on an agar surface.

Prepare two stock cultures; a ‘permanent’ stock which is opened once only (to prepare the next
two stock cultures) and a ‘working’ stock for taking sub-cultures for class. If a particular culture is
used a lot in a short period, it is much more convenient to prepare several working sub-cultures at
once.

Incubate at an appropriate temperature until there is good growth. For growing strict aerobes it may
be necessary to slightly loosen the cap for incubation (but close securely before storage) if there is
insufficient air in the headspace. If you are not sure whether the strain is a strict aerobe or not, leave
the cap loose until the culture is established.

As soon as there is adequate growth, and the caps are screwed tight, the cultures may be stored in
a refrigerator (one in which human foodstuffs are never kept), but they will remain viable in either a
cupboard or a drawer at room temperature. A dark place at a temperature of 10-15 °C is ideal.

Be prepared to transfer most cultures four times a year to maintain viability. Label each culture
clearly including the date of transfer. Do not destroy old cultures until the new subcultures have
become established. Some bacteria need more frequent sub-culturing to maintain viability (for
example Lactobacillus sp).

Checking purity of cultures

It is sensible to check purity on suspicion of contamination of the working stock, and of the
permanent stock when preparing new stock cultures. Evidence of purity is given by the uniformity of
colony form on a dilution streak plate, cell form on stained microscope slides, and consistency with
the appearance of the original culture.

If a culture becomes contaminated, go back to the working stock or permanent stock cultures, or buy
in fresh supplies.

Preparing cultures to use in investigations

Microbial cultures cannot be taken from a shelf and instantly be ready for use. It is necessary to
begin to prepare cultures well in advance otherwise the outcome might not be as expected. The key
is to transfer cultures several times in advance to ensure that they are growing well and are
presented as young, fully-active cultures on the day of the practical class.

To transfer cultures, scrape a small amount of bacteria or yeast cells off the agar slope of a culture
using a sterile loop. Wipe the loop over a fresh agar slope to seed a new culture directly or,
preferably, shake the loop in a small volume of sterile nutrient broth. After allowing the transferred
cells time to multiply until the broth becomes cloudy (1-2 days, or longer with slower-growing
organisms or large volumes of broth), the microbes are ready to be used for investigations or to
prepare new agar-slope cultures.

Progress of growth can be followed by observation with the naked eye, looking for growth on an agar
surface or turbidity in a broth culture. It is usual to grow moulds on the surface of an agar medium,
allowing an incubation period of several days to a week.

Use a sterile loop, syringe or Pasteur pipette to transfer bacteria or yeast cells from the broth.

The main points to observe are:

 use of an adequate amount of inoculum

  an appropriate culture medium

 an appropriate incubation temperature

 adequate aeration for a strictly-aerobic organism in a single large volume (more than 20
cm3) of liquid culture.

Suggestions and tips

1 It will save time preparing large numbers of cultures of bacteria and yeast for a class if the
inoculum is taken by Pasteur pipette from a well-growing, turbid broth culture. A line of growth on a
slope culture inoculated by a wire loop is easy for students to observe – but you can achieve almost
the same effect with a pipette. With a broth (rather than a slope) the risk of spills is greater, so
slopes are preferred with younger or inexperienced students.

2 Regular practice of aseptic technique is necessary to ensure that the manipulations involved in
culture transfer and handling sterile solutions become second nature.

3 The choice of loop or pipette for transfers between test tubes and screw cap bottles depends on
whether they contain agar slopes, liquid media or sterile solutions.

4 To inoculate a tube or bottle from a separate colony on a plate, a wire loop is usually satisfactory.
A straight wire is sometimes needed for small colonies (for example in pure cultures
of Streptococcus and Lactobacillus) or on plates used to isolate cultures from natural samples.

Read our standard health & safety guidance

Carry out a full risk assessment before planning any work in microbiology (see note 1 for more
details).

1 Before embarking on any practical microbiological investigation carry out a full risk assessment.
For detailed safety information on the use of microorganisms in schools and colleges, refer to Basic
Practical Microbiology – A Manual (BPM) which is available, free, from the Society for General
Microbiology (email education@sgm.ac.uk) or go to the safety area of the SGM website
(www.microbiologyonline.org.uk/safety.html) or refer to the CLEAPSS Laboratory Handbook, section
15.2 and 15.12.

Bacterial Coworkers
Mathematicians have to work with numbers. Marine biologists all get wet. When you decide to
pursue a career as a microbiologist, eventually you have to work with microorganisms. But,
performing experiments on living things far too small to see can be difficult. It is not as easy as
snorkeling to observe reef fish or putting white mice in cages. You have to plan and control every
aspect of your new pet bacteria's lives. At the same time, you have to diligently protect your
specimens from being contaminated by all the rogue bacteria that are all around you, covering every
surface, and colonizing your own body. In this lesson, we will hit on some of the basic aspects of
growing bacteria in a lab.

Culturing Vocabulary
One skill that every microbiologist has to master is how to properly grow bacteria without letting all
those environmental bacteria contaminate your work. But, let's get a few vocab words out of the way
first. Bacterial culturing is a process of growing bacteria. Oftentimes, if you are trying to identify a
microbe causing a disease or investigating the populations in an environmental sample, you begin
with a mixed culture. A mixed cultureis a bacterial culture made up of more than one species of
bacteria. You could have as few as two or as many as hundreds of species. It is your job to take this
mixed culture and isolate the one species of interest.

Mixed culture of bacteria

Notice above how many of the colonies look different? These are all different species. What you
want is to make that mixed culture into a pure culture. A pure culture is a bacterial culture made up
of only one species. Notice below that all of the growth looks the same.

Pure culture of bacteria

You want to have a pure culture so you know with certainty that any observations you make are a
result of that one species of interest and not some other unknown contaminant species. There are
many different techniques you can use to isolate a species, but most involve growing the bacteria on
or in media. So, what is media?

Growth Media
A medium (plural media) is a liquid or gel designed to support the growth of a bacterial culture. The
liquid or gel has to contain everything that the bacteria will need for growth and cell division. Leaving
out even one, seemingly insignificant, ingredient from your medium can prevent all growth. This can
be a very frustrating problem, especially if you spent hours painstakingly setting up your culture, only
to see absolutely nothing growing. Today, most labs buy commercially prepared dry media that
contains all needed nutrients. All you have to do is add sterile water!
In addition to the nutrients and growth factors needed for life, there are many additional compounds
you can add to your media. For more information, see the lesson titled 'Differential and Selective
Media in Microbiology.'

Preparation of media
and cultures
Culture media

The method for the preparation of basic microbiology media is given below. In situations
where preparation is uneconomic in time, prepared, sterilized media (liquid and solid) are
available from the major school science equipment suppliers. Sterilization is at 121 °C (15
lb in ˉ²) for 15 minutes. pH values are 7.0 unless stated otherwise.

Note: Allow 15 cm³ of agar for each Petri dish and 5-10 cm³ of broth for each McCartney
bottle. All cotton wool plugs should be made of non-absorbent cotton wool. Plastic or metal
caps may also be used.

Nutrient agar

Suspend 28 g of nutrient agar powder in 1 litre of distilled water. Bring to the boil to dissolve
completely. Dispense as required and sterilize.
Nutrient broth

Add 13 g of nutrient broth powder to 1 litre of distilled water. Mix well. Dispense as required
and sterilize.

Malt extract agar

Suspend 18g agar powder in 1 litre of distilled water. Bring to the boil to dissolve
completely. Add 15g malt extract per litre. Mix well. Dispense as required and sterilize.

Mannitol yeast extract agar

Suspend 10 g agar in 1 litre of distilled water. Heat to dissolve. Add 0.5 g K 2HPO4 , 0.2g
MgSO4.7H2O, 0.2 g NaCl, 0.2 g CaCl2.6H2O, 10 g mannitol and 0.4 g yeast extract.
Dispense as required and sterilize.

Mannitol yeast extract broth

As above, without agar.

Glucose nutrient broth

Make up nutrient broth as already directed and add 10 g per litre of glucose.

Sugar peptone water

Add 10 g of peptone, 5 g of NaCl, 5 g of sugar and 20 cm³ of Universal indicator to 1 litre of

distilled water; pH should be 7.4. Dispense as required and sterilize.
Tributyrin agar

Supplied ready for use. Heat to melt and dispense aseptically. May be prepared by adding
1% tributyrin to nutrient agar.

Glucose yeast extract broth

Add 10 g of peptone, 5 g of NaCl, 3 g of yeast extract to 1 litre of distilled water. Dispense

as required and sterilize.

Glucose yeast extract lemco broth

Add 10 g of Lemco (meat extract) to glucose yeast extract broth.

Milk agar

Make up nutrient agar as above but using only 900 cm³ of distilled water. Dissolve 20 g of
dried skimmed milk in 100 cm³ of distilled water. Sterilize separately. Transfer the milk to
the agar aseptically after cooling to 45-50 °C. Dispense aseptically.

Starch agar

Suspend 15 g of nutrient agar in 100 cm³ distilled water. Bring to the boil to dissolve
completely. Heat 40 g of soluble starch in 100 cm³ of distilled water to form a suspension.
Allow to cool and then mix with the nutrient agar solution. Dispense and sterilize.
Iodine solution

Dissolve 1 g of iodine crystals and 2 g of potassium iodine in 300 cm³ of distilled water.

Cellulose broth (for Trichoderma reesei)

 800 cm3 distilled water

 0.1 g CaCl2

 0.5 g (NH4)2SO4

 0.5 g yeast extract powder

 0.5 g asparagine

 10 g carboxymethylcellulose

 1.0 g KH2PO4

 pH6.2

Mix ingredients, heat gently, and stir until dissolved.

Growth and Laboratory Maintenance of Vibrio cholerae

Raquel M. Martinez, Christina J. Megli, and Ronald K. Taylor*

Author information ► Copyright and License information ►

The publisher's final edited version of this article is available at Curr Protoc Microbiol

See other articles in PMC that cite the published article.

Abstract
Go to:

INTRODUCTION
The causative agent of the diarrheal disease cholera is the Gram-negative bacterium Vibrio
cholerae. This enteric pathogen naturally inhabits an aquatic environment. Although most
outbreaks occur in areas where water quality and sanitation are poor, V. cholerae can also be
found in the waters all around the world. Cholera is endemic to Asia, Africa and South America.
Additionally, outbreaks have emerged after conflict and natural disasters, such as in Iraq and
Zimbabwe. Of the >200 serogroups, only O1 and O139 are known to cause epidemic disease due
to the presence of the two major virulence factors, toxin co-regulated pilus (TCP) and cholera
toxin (CT). The O1 serogroup is further divided into classical and El Tor biotypes. The classical
biotype was responsible for the first six cholera pandemics, while the El Tor biotype is
responsible for the current pandemic. The distinction between the classical and El Tor biotypes is
based on their biochemical characteristics. For example, the El Tor biotype is naturally resistant
to the antibiotic polymyxin B (see Table 2).

Table 2

Antibiotic stock solutions for use with V. cholerae cultures

Reports of non-O1 and non-O139 serogroups have recently emerged in the US. For example,
O75 and O141 have surfaced in the Gulf Coast waters in recent years. The factors that contribute
to virulence are not well understood in these strains. While most non-O1 and non-O139
serogroupss do not carry the tcpand ctx genes, cases of non-O1 and non-O139 serogroups
causing severe cholera-like gastroenteritis have been reported (3, 4, 9).
This unit describes basic techniques to grow and maintain Vibrio cholerae in the laboratory. A
relatively vigorous organism, V. cholerae can be grown in a variety of media formulations over a
range of temperatures. These protocols should provide the reader with the ability to maintain and
grow V. choleraefor use in a variety of assays.
CAUTION: Vibrio cholerae is a Biosafety Level 2 (BSL-2) pathogen. Follow protocols for the
handling of BSL-2 organisms outlined by your institution. For general biosafety information see
UNIT 1A.1 .
Go to:

STRATEGIC PLANNING

Strain Selection
O395, N16961 and C6706 are commonly used laboratory strains of the O1 serogroup. The V.
choleraestrain O395 is of the classical biotype, while the N16961 and C6706 strains are of the El
Tor biotype. Various clinical and environmental strains have been isolated, several of which
have been sequenced (see Table 1). Strain selection is an important aspect of designing your
experiments. For example, El Tor strains are best for the study of biofilms, whereas classical
strains demonstrate a more robust autoagglutination phenotype than El Tor strains. Of note, the
O1 El Tor biotype is responsible for the most recent epidemics of cholera. Thus, many
investigators are using the corresponding strains for their research. Since 2004, there have been
clinical reports of El Tor variant strains that may exhibit properties of both classical and El Tor
biotype strains, suggesting that studies of both biotypes are critical (1, 7, 8).

Table 1

Selected V. cholerae strains for which the genomic sequence is publically available

Growth Conditions
Vibrio cholerae grows well under standard laboratory conditions (LB at 37°C). Vibrio
cholerae is able to grow between 20°C and 45°C. Unlike other bacteria, V. cholerae is unable to
survive at 4°C for extended periods. Store plates at room temperature. V. cholerae is able to
grow in a wide pH range. We suggest using neutral pH (7.0) for maintenance and growth of this
strain. Broth cultures grow best with aeration, therefore a test tube roller or flask shaker are
required for optimal growth and yield. In rich medium, such as LB, the generation time is
approximately 40 minutes during exponential growth. An over night culture reaches densities of
>109 CFU/ml.

Media
V. cholerae is able to grow in a variety of different media. In the following protocols we suggest
growth in Luria Broth (LB). V. cholerae can also be grown in several defined minimal mediums
(see APPENDIX 4A) that are amendable to the study of particular nutrient requirements. Special
media, such as, plasmid broth can be used for isolation of plasmid DNA from broth cultures (see
recipe). TCBS and TTGA agars are commonly used to isolate and identify V. cholerae from
clinical specimens or environmental samples (see UNIT 6A.5.6). UNIT 1.1 and APPENDIX 4A
describe several protocols for preparing commonly used liquid and solid media.

Basic protocol 1: GROWTH OF V. CHOLERAE FROM A FROZEN STOCK

V. cholerae should be preserved in frozen stocks in 30% glycerol. These are typically stored at -
80°C. Growth from a freezer stock is an essential starting point for any experiment. It is
important to streak from a freezer stock onto an agar plate. Placing V. cholerae directly into
liquid media from a freezer stock is not recommended. It is good laboratory practice to initiate
experiments from a single colony, in order to start with a clonal population.

Materials
V. cholerae frozen stock (see Basic Protocol 3)
LB agar plates (see APPENDIX 4A)
Wooden applicator stick, toothpick or inoculating loop, sterile
37°C incubator

1. From a frozen stock, remove a small amount of slightly thawed bacteria.

Do not thaw the frozen stock, as it will decrease the stock viability over time, instead
remove a small chip of ice.

2. Streak heavily onto agar for isolated colonies (see UNIT 1.3.2).
3. Incubate at 37°C for 16-24 hr.

Basic protocol 2: GROWTH OF V. CHOLERAE IN LIQUID MEDIUM

Broth cultures are commonly used for a variety of experiments such as, growth curves, DNA
extraction, protein analyses, and for the preparation of electrocompetent cells for
transformations. For general laboratory purposes, LB broth is commonly used.

Materials
V. cholerae grown on agar (see Basic Protocol 1)
LB broth (see APPENDIX 4A)
Antibiotics if needed (see Table 1)
Wooden applicator sticks or inoculating loop, sterile
Capped test tubes, sterile
37°C incubator with a mechanism for rotating or shaking cultures

1. Using aseptic technique, add LB to sterile test tube or flask (For example, 5 ml to a 15 X 125 mm
tube).
2. Using a sterile wooden stick or inoculation loop, pick a single colony from the prepared streak
plate.
3. Inoculate the test tube by suspending the colony in the broth.
4. Cover the test tube and vortex for 1-2 sec.
5. Incubate the test tube on a roller for 12-16 hr at 37°C.

See Figure 1 for a standard growth curve of V. cholerae to determine appropriate

incubation time for a particular growth phase.
 Figure 1

Growth curve of V. cholerae O1, classical strain O395, in LB broth

NOTE: If you are preparing a large number of cultures, and all require the same antibiotics, then
it is recommended that you prepare a stock solution of LB with the appropriate antibiotics. This
reduces the number of manipulations.
NOTE: Capped flasks can be used when larger volumes are necessary. The growth of cultures
should be in a flask that can hold the minimum of 5X the volume of broth. Incubate with
shaking.

Basic protocol 3: PREPARATION OF V. CHOLERAE FROZEN STOCKS

V. cholerae should be stored for long-term usage in 30% glycerol at -80°C. We recommend that
strains that are used frequently should be re-frozen after a single passage to have a back-up
stock.

Materials
V. cholerae grown on LB agar containing antibiotics if appropriate (see Basic Protocol 1)
LB broth (see APPENDIX 4A)
50% glycerol (v/v), sterile
4 ml freezer vials, sterile (Wheaton, cat. No. 224882 or Cryovial®)
-
80°C freezer
37°C incubator with a mechanism for rotating or shaking cultures

1. Inoculate a single colony of V. cholerae into 5 ml liquid LB (see Basic Protocol 2).

A larger volume can be used to grow cultures overnight. 5ml is a suggested starting
volume.

2. Incubate at 37°C shaking for 15-16 hr.

3. Remove 1 ml of bacterial culture.
4. Add 1 ml of culture to 1.5 ml 50% glycerol solution.
5. Place suspension into a freezer vial.
6. Mix well by vortexing.
7. Immediately place into -80°C freezer.

Basic protocol 4: PRESERVATION OF V. CHOLERAE IN AGAR STABS

Stabs are useful for strain storage and are also convenient for shipping strains. V. cholerae can be
stored for several years in agar stabs, but for long-term storage a frozen stock should be prepared
(see Basic Protocol 3). See UNIT 1.3.4 for a detailed description on how to prepare and revive
bacteria preserved in stab agar.
Materials
V. cholerae grown on agar containing antibiotics if appropriate
LB agar stab (see recipe)
Inoculating loop, sterile
37°C incubator

1. Using a sterile inoculating loop or flat toothpick, inoculate stab with a single colony (UNIT 1.3.4).
2. Incubate stab overnight at 37°C with the cap of the vial slightly loose.
3. Tightly seal the vial and store in a cool (15° to 22°C), dark place.

Go to:

REAGENTS AND SOLUTIONS

LB broth
To 1L of deionized, distilled water, add 10 g Tryptone, 5 g Yeast Extract, 5 g NaCl and stir using
a magnetic stir bar and plate at room temperature to dissolve. Aliquot appropriately. Autoclave.
NOTE: If the media is to be autoclaved in a culture flask, always use a flask that holds a
minimum of 2X the volume of media. For example, for 1 L of broth, use a 2 L flask.
NOTE: Add antibiotics after the media has cooled.

LB agar
To 1L of LB broth (see above recipe), add 15 g Agar. Heat to dissolve with constant mixing.
Autoclave media.
Let sterile media cool to 50°C. Pour plates into sterile, disposable petri dishes. See APPENDIX
4A.
NOTE: When making agar plates containing antibiotics, autoclave media and let cool to 50°C
prior to adding antibiotics.

Plasmid Broth
To 1 L of deionized, distilled water, add 12 g Tryptone, 24 g Yeast Extract and 5 ml of Glycerol.
Mix thoroughly. Aliquot 90 ml of media into milk dilution (160 ml) bottles. Autoclave.
Before use, add 10 ml of sterile 1M potassium phosphate (KPO4-) buffer. Mix.

1M KPO4- buffer (pH 7.6)

To 300 ml of deionized, distilled water, add 69 g KH2PO4. Adjust pH to 7.6 with KOH. Increase
volume to 500 ml with water. Aliquot buffer into milk dilution (160 ml) bottles. Autoclave. Add
10 ml of buffer to 90 ml of plasmid broth. Mix.

Agar stabs
To 500 ml of deionized, distilled water, add 3 g Agar, 5 g Nutrient Broth and 4 g NaCl. Heat to
dissolve with constant mixing.
Use autoclavable glass vials with screw caps (Wheaton, Cat. No. 224882). Fill the vials ¾ full
with media that has been cooled to 50°C. Loosely screw caps and autoclave for 15 min. Let cool
and tighten caps.
NOTE: If the caps are not properly tightened, then the agar stabs will dry out over time.
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COMMENTARY

Background Information
Vibrio cholerae O1 is the etiological agent of the diarrheal disease cholera. Although
underreported, cholera cases are increasing in number and recent outbreaks have been reported
as having case fatality rates as great as 40%. Cholera plagues many areas in Africa, Asia, South
America and more recently in the Middle East. From the most recent WHO report,
“(cholera) remains a challenge in those countries where access to safe water and adequate
sanitation cannot be guaranteed for all. Almost every developing country is facing either a
cholera outbreak or the threat of an epidemic.”
–WHO 2005

V. cholerae has two major virulence factors, cholera toxin (CT) and the toxin co-regulated pilus
(TCP). Cholera toxin is responsible for causing the secretory diarrhea of the disease cholera by
initiating a signaling cascade in the small intestine. TCP is a type IV pilus that is required for V.
cholerae to colonize the mammalian host. Additionally, motility and biofilm formation
contribute to the fitness of V. cholerae in the environment as well as in the mammalian host.

Critical Parameters and Troubleshooting

If no growth is seen from the initial streaking from the frozen stock or subsequent re-streaks, it is
possible that an insufficient amount of bacteria was selected. Start from the freezer stock using a
larger ice chip or from the agar plate selecting the entire colony to streak upon the plate.
Additionally, if the freezer stock is old or a frequently used stock, it is possible that the stock has
lost viability and the back-up stock should be used. Often times, when no growth is observed, it
is due to the use of an antibiotic that the strain is not resistant to.
Multiple passages of V. cholerae are not recommended, as mutations may accumulate, thus
resulting in the lost of virulence traits.
Autoagglutination
Vibrio cholerae O1 classical biotype can autoagglutinate during growth in broth culture.
Autoagglutination can be induced by growth at 30°C, starting pH 6.5. Occasionally, a strain will
autoagglutinate under normal laboratory conditions (37°C, starting pH 7). Autoagglutination
interferes with reading the optical density of the culture and may cause unwanted results in
different assays. If autoagglutination occurs, then simply vortex the culture, making sure the
bacterial suspension is homogeneous before use.

Anticipated Results
Basic Protocol 1 describes how to grow V. cholerae on solid media. After 16-20 hr of incubation,
colonies will be large, semi-opaque, convex, smooth, round and off-white/tan in color. Basic
Protocol 2 describes how to grow V. cholerae in liquid media. After 12-16 hrs, the cultures
should be turbid. Basic Protocols 3 and 4 describe how to preserve V. cholerae either by freezing
or in agar stabs. Regrowth from the preserved cultures should produce a large number of viable
cells.
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References
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cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique. Int J Med
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