Preparing Spread Plates Protocols

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Created: Monday, 09 October 2006
Author • Kathryn Wise

Information One method of distributing bacteria evenly over the surface of an agar
plate medium is commonly referred to as the spread plate method.
Classically a small volume of a bacterial suspension is spread evenly over
the agar surface using a sterile bent glass rod as the spreading device.
The goal in evenly distributing the bacterial suspension is typically to
permit the growth of colonies that can subsequently be enumerated (see
Serial Dilution Protocols) and/or sampled following incubation. Each plate
is spread with a single inoculum of the bacterial suspension.

An alternative approach to spreading a single inoculum volume with a

smooth device is to apply a smaller volume and tip the plate, allowing
gravity to distribute the inoculum in a band or track (track method) or to
allow the inoculum to dry in place (drop method). With this alternative
approach, several sample dilutions can be distributed on a single agar
plate.

History

Since the development of the agar plate in Robert Koch's laboratory,

several methods have been used to achieve an even distribution of
bacterial growth on or in the agar. The most common methods used to
achieve this type of distribution are: spread, pour, thin-layer, layered,
and membrane filter (2).

Principles

Using the spread method a small volume of a bacterial suspension is

distributed evenly over the surface of an agar plate using a smooth
sterilized spreader (2). In the case of track plates, gravity is used to
spread the inoculum down the agar in a column forming a track (1).

PROTOCOL: Spread Plates

Agar plates:

Select and prepare an agar medium based upon the type of bacteria to
be enumerated or selected.

After autoclaving, cool the agar to between 45°C and 50°C prior to
pouring the plates to minimize the amount of condensation that forms.
The thickness of the agar should be roughly 0.3 cm, which can be

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 achieved by pouring 15 to 20 ml of media per 100 x 15 mm plate.

Freshly prepared plates do not work as well as dry plates as it takes

longer for the inoculum to absorb into the agar. Plates may be dried by
keeping them at room temperature for roughly 24 hours. Plates will dry
faster in lower humidity so placing them in a laminar flow hood will speed
the drying process. Once dried, plates may be used or refrigerated in
closed bags or containers until required. Refrigerated plates should be
warmed to room temperature prior to use.
Inoculations:

When enumerating colony-forming units (CFUs), plates with between 20

to 300 (or 25 to 250) CFUs can be used to calculate the number of
CFUs/ml of the original sample. Typically a dilution series is prepared,
often a ten-fold dilution series, using a suitable dilutent such as
phosphate-buffered saline. Serial Dilution Protocols

A convenient inoculum volume, in terms of spreading, absorption, and

calculations, is 0.1 ml (100 microliters).

Since some bacteria rapidly attach to the agar surface, the inoculum
should be spread soon after it is applied.

Working from the most dilute suspension to the most concentrated is

advised. Proceeding from most dilute to most concentrated makes it
unnecessary to change pipette tips between the dilutions.

Spreading:

A reusable glass or metal spreader should be flame sterilized by dipping

in alcohol (such as 70% isopropyl or ethanol), shaking off the excess
alcohol, and igniting the residue (Fig. 2, Atlas page). The spreader is
then allowed to cool.

The spreader is placed in contact with the inoculum on the surface of the
plate and positioned to allow the inoculum to run evenly along the length
of the spreader. Even pressure is applied to the spreader and the plate is
spun, on a turntable or by hand, as illustrated in Fig. 3-7 on the Atlas
page.

Alternatively the spreader may be rotated over the agar surface (Fig. 10,
Atlas page).

Avoid spreading the inoculum all the way to the edge of the agar.

The goal is to evenly distribute the inoculum and to allow it to be

absorbed into the agar. The plate, or spreader, should be rotated long
enough to avoid pooling along the spreader once the rotation is stopped.

Avoid disturbing plates for 10 to 20 minutes after spreading. Drying time

varies with the room temperature and humidity.

Incubation:

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 After the spread plates have been permitted to absorb the inocula for 10
to 20 minutes they may be inverted and incubated as desired.

Observe the plates before the colonies have had time to fully develop.
Closely positioned colonies may be difficult to resolve as separate
colonies later. Continue the incubation as necessary.

Incubation in closed humidified containers will help avoid problems with

plates drying out when working with slow-growing colonies.

Counting and Selection:

After appropriate incubation, plates are inspected. When plating a

dilution series, the growth on the plates should reflect the predictable
drop in CFUs/plate as illustrated in this picture (Fig. 20, Atlas page) of a
10-fold dilution series prepared from an overnight broth culture
of Escherichia coli.

FIG. 20. Picture of spread plates showing bacterial growth (E. coli, 40
hours, room temperature) on five plates prepared from a ten-fold
dilution series. Care was taken to avoid spreading to the edges of the
plates as it is more difficult to count colonies along the edge of the agar.

Duplicate or triplicate plates with 30 to 300 CFUs/plate are used to

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 calculate CFUs/ml.

Plates with well isolated colonies may be inspected and, if desired,

colonies "picked" to establish new cultures.

PROTOCOL: Track Plate Method

Agar plates:

Select and prepare an agar medium based upon the type of bacteria to
be enumerated or selected.

After autoclaving, cool the agar to between 45°C and 50°C prior to
pouring the plates to minimize the amount of condensation forming. The
thickness of the agar should be roughly 0.3 cm, which can be achieved
by pouring 15 to 20 ml of media per plate. Square plates, 100 x 15 mm,
with 13 mm grids or 100 mm round petri dishes may be used.

Freshly prepared plates do not work as well as dry plates as it takes

longer for the inocula to absorb into the agar. Plates may be dried by
keeping them at room temperature for roughly 24 hours. Plates will dry
faster at lower humidity so placing them in a laminar flow hood will speed
the drying process. Once dried, plates may be used or refrigerated in
closed bags or containers until required. Refrigerated plates should be
warmed to room temperature prior to use.

Inoculations:

When enumerating colony-forming units, tracks with 20 to 200 CFUs can

be used to calculate the number of CFUs/ml of the original sample.
Typically a dilution series is prepared, often a ten-fold dilution series,
using a suitable dilutent such as phosphate-buffered saline. Serial
Dilution Protocols

A convenient inoculate volume, in terms of spreading, absorption, and

calculations, is 0.01 ml (10 microliters). Some bacteria rapidly attach to
the agar surface so the plates should be tipped soon after delivery.
Working from the most dilute suspension to the most concentrated is
advised.

Six tracks will comfortably fit on a square agar plate and three or four
tracks will fit on a round plate. Use a marker to establish the places to
initially dispense inocula onto a round plate.

Spreading:

Inocula are placed in the appropriate squares or on the appropriate

designated spots. To speed up the inoculation process and minimize the
number of pipette tip changes, add the inocula to the plate in ascending
order of bacterial concentration (most dilute to most concentrated).

Once the dilutions have been applied, the plate is tipped at roughly a 60°
angle and then the angle is adjusted (45° to 90°) to control the flow of

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 the inocula forming the tracks as illustrated in Fig. 33 on the Atlas page.

When the "fastest" track comes to within 0.5 cm of the edge of the agar,
the plate may be tipped backwards slightly (Fig. 37, Atlas page) to avoid
pooling at the base of the tracks.

The plates should be left undisturbed for 5 to 10 minutes.

Incubation:

After the tracks have been permitted to absorb into the agar for 5 to 10
minutes, the plates should be inverted and incubated as desired.

Observe the plates before the colonies have had time to fully develop.
Closely positioned colonies may be difficult to resolve as separate
colonies later. Continue the incubation as necessary.

Incubation in closed humidified containers will help avoid problems with

plates drying out when working with slow-growing colonies.

Counting and Selection:

After appropriate incubation, plates are inspected. When plating a

dilution series, the growth on the plates should reflect the predictable
drop in CFUs/plate as illustrated in this picture (Fig. 38, Atlas page).

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 FIG. 38. Picture of four replica plates showing E. coli (room
temperature, 40 hours) growing in four tracks.

Duplicate or triplicate tracks with 20 to 200 CFUs/plate are used to

calculate CFUs/ml.

Plates with well isolated colonies may be inspected and colonies "picked"
to establish new cultures.

SAFETY

The ASM advocates that students must successfully demonstrate the

ability to explain and practice safe laboratory techniques. For more
information, read the laboratory safety section of the ASM Curriculum
Recommendations: Introductory Course in Microbiology and
the Guidelines for Biosafety in Teaching Laboratories.

Make sure the container of alcohol is covered when not in use and that it
is kept out of the vicinity of the spreader once the alcohol-dipped
spreader is ignited.

COMMENTS AND TIPS

Spread Plates

Make sure plates are sufficiently dry prior to use.

McFarland standards are very useful in estimating cell densities so that

an appropriate dilution series can be prepared and the dilutions to be
plated determined. Prepare dilutions to deliver an estimated 30 to 300
colonies on at least one plate.

Plates should be prepared in duplicate or triplicate.

Do not delay in spreading the inoculum once it has been applied to the
plate since some cells will rapidly attach to the agar, especially if the
plate agar is nice and dry.

Avoid spreading the inoculum to the edge of the agar as it is more

difficult to inspect and count colonies along the agar's edge.

Once the dilution series has been made, inoculate plates within 30
minutes to minimize changes in the number of cells in each dilution due
to cell division or death.

Make sure even pressure is applied to the spreader so that fluid is evenly
distributed along its length as the plate or spreader is rotated.

Once the dilutions are made, work backwards spreading the most dilute
samples first.

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 When making your own spreaders do not make the spreading edge too
long, it should conveniently fit into the alcohol container as well as the
plate. Fire-polish the end of the spreader the student will hold. Bending
the glass to form a triangle rather than an "L" will help assure only
smooth even surfaces touch the agar and minimize pooling.

Distributing the organisms by rotating the spreader rather than the plate
tends to cause more pooling of the inoculum.

Sterile glass or plastic beads are sometimes used to spread the inoculum
over the surface of an agar plate when an even distribution of colonies is
not an important outcome.

Track plates

Use 0.01 ml (10 microliters) or 0.005 ml (5 microliters) as the inoculum

volume.

An ordinary 100 mm round petri dish may comfortably hold three to four
tracks. If for your enumeration you plate triplicates, one plate per
dilution works well.

Saves on plates and media as well as time.

Reduces labeling errors.

Gridded square plates are great but not necessary

Use 20 to 200 visible colonies per track as the countable range rather
than the 30 to 300 or 25 to 250 ranges commonly used with classic
spread plates.

Quickly apply the inocula onto a plate.

Tip the plate keeping one edge in contact with the bench top and sharpen
or reduce the angle as needed to encourage the inocula to run down the
plate forming tracks.

Do not let the tracks run to the edge of the agar.

Once the tracks have been formed, tip the plate back slightly so that the
fluid does not collect or pool at the base of the track.

Let the plates sit 5 to 10 minutes before inverting and incubating them.
Drying time varies with the room temperature and humidity.

ACKNOWLEDGMENTS

Minnesota State University—Moorhead undergraduates Nicole True and

Rachel Aanenson made valuable suggestions and contributions
throughout the development of the images for this Atlas and the
preparation of the protocol.

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 REFERENCES

1. Jett, B. D., K. L. Hatter, M. M. Huycke, and M. S. Gilmore. 1997.

Simplified agar plate method for quantifying viable bacteria.
BioTechniques 23:648–650.
2. Koch, A. C. 1994. Growth measurement, p. 254–257. In P. Gerhardt,
R. G. E. Murray, W. A. Wood, and N. R. Krieg, (ed.), Methods for general
and molecular bacteriology. ASM Press, Washington, D.C.

REVIEWERS

This resource was peer-reviewed at ASM Conference for Undergraduate

Educators 2006.

Participating reviewers:

Jason C. Baker
Missouri Western State University, St Joseph, MI

Robert E. Brader
Lancaster General College of Nursing and Health Sciences, Lancaster, PA

Sylvia Franke
Skidmore College, Saratoga Springs, NY

William R. Huddleston
University of Calgary, Calgary, Canada

Kevin Sorensen
Snow College, Ephraim, UT

Anh Hue T. Tu
Georgia Southwestern State University, Americus, GA

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