Spread Plate Method- Definition, Principle,

Procedure, Uses
Spread Plate Method is one of the widely used culture techniques in
microbiology laboratories due to its ease and simplicity. This method is suitable for
aerobic and facultative aerobic microorganisms. It is an easy, simple, and
economical method; however, it requires the sample to be in liquid or suspension.
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What is Spread Plate Method?
The spread plate method is a microbiological laboratory technique for
isolating and counting the viable microorganisms present in a liquid sample
by spreading a certain volume of the sample over an appropriate solidified
culture media.
Following the incubation, in a successful spread plate, there will be the formation
of evenly distributed discrete colonies all over the surface of the culture media. 
This technique is used for isolating and counting the total number of viable
microorganisms (i.e. calculating the colony-forming units per mL (CFU/mL) in the
given sample. It is also for propagating the old culture and mass producing them. It
can be used for all of the culturable bacteria and fungi. 

Spread Plate Method

The sample in the spread plate method must be liquid or in suspension. Before
plating, the samples are serially diluted. If the objective is to count the CFU/mL
then the sample must be diluted to make the microbial load in the sample between
20 – 300 CFU/mL (suitable colony counting range is 20 – 200, some consider it to
be 30 – 300, and in average it is taken as 25 – 250). This can be obtained by pilot
test or by using a different range of diluted samples. If the sample is solid or
semisolid, it must be first emulsified and then serially diluted to reduce microbial
load up to the permitted range.  
0.1 mL of the sample (0.1 to 0.2 mL) is pipetted over the center of the solidified
agar medium and evenly spread over the surface of the medium. The plates are
incubated under the optimum condition following which the numbers of colonies
are counted. If the colonies are uncountable or fused or more than 300 CFU/mL or
less than 20 CFU/mL, it is recommended to repeat the process for getting the
optimum count.  
Objectives of Spread Plate Technique
1. To isolate the microorganisms from the liquid specimen (or suspension)
2. To calculate viable microbial load by counting colony formation unit
(CFU) per mL
3. To isolate the pure culture of microorganisms from a mixed population
4. To isolate microorganisms in discrete colonies in order to study their
colony characters
5. To obtain sufficient growth for conducting antimicrobial sensitivity testing
and biochemical studies
Principle of Spread Plate Method
When a diluted liquid specimen containing one or more microorganisms, same or
different species, is spread over a suitable solid agar media, each of the viable
microorganisms will multiply forming a separate colony. These colonies can be
counted and expressed in terms of the CFU/mL which can be used to calculate the
microbial load in the sample. 
A certain volume of the diluted sample, mostly 0.1 mL (0 to 1 mL can be used), is
dispensed over the surface of a pre-sterilized solid medium. Usually, a bent glass
rod or swab or glass beads are used to spread the sample. Following the incubation
for usually 24 – 48 hours at 37°C, the viable microorganisms in the sample will
grow into discrete visible colonies on the surface of the medium. The visible
colonies can be counted and CFU/mL can be calculated using the following
formula;

Requirements for Spread Plate Method

 1. Liquid Specimen (or suspension of the solid sample)
The sample must be either in liquid form or in suspension form. The solid sample
must be dissolved in a suitable solvent. The solvent must not show any inhibitory
or growth-promoting activity to any of the microorganisms. Also, it must not react
to any component of the culture media. 
The sample must be diluted to an appropriate concentration so that we can get a
well-isolated 20 – 300 CFU/mL per plate after the incubation. 
2. Pre-solidified Suitable Solid Culture Media Plates
Appropriate culture media which supports the growth of all desired or probable
microorganisms in the specimen must be used. The media must be solidified
properly before spreading the diluted sample. The media can be prepared and
stored at 4°C for future use. If freshly prepared, make sure the media is completely
solidified. 
3. Test Tubes
Sterile test tubes are used to serially dilute the sample. 
4. Sterile Distilled Media (or Sterile Broth)
The sample must be serially diluted using sterile distilled water or sterile broth.
They are also used for dissolving the solid or semi-solid sample. 
5. Micropipette
A micropipette of 0.1 mL or 1 mL capacity is required for measuring the sample
during serial dilution and sample inoculation. A sterile graduated pipette can be
used instead of the micropipette.  
6. Spreaders
Spreaders are the tools that are used to spread the inoculum over the surface of the
agar medium. Bent glass rods, glass beads, and cotton swabs are used as a
spreader.
 Bent glass rod: it is a glass rod that is bent at one end forming either “L” or
“J” shaped or forming a closed triangle-like shape at one end. It is often
called a “dolly rod”. It is the most commonly used spreader. Metallic rod is
not preferred because their surface can be delaminated and they take a
longer period to cool down. Glass rods are with smooth surface and can be
easily made in a lab and also they cool down quickly. 
 Glass beads: smooth tiny glass beads, usually of 4 mm diameter, are used to
spread the inoculum in the “Copacabana method”. The sample is dispensed
on the surface of the agar medium. Sterile glass beads are placed over the
medium and the whole plate is shaken so that the beads distribute the
sample evenly all over the agar surface. 
 Sterile cotton swab: a sterile swab may be used, but it is not recommended

when we need to count CFU/mL because some microorganisms may be
trapped on the sample that will be absorbed in the swab.  
7. Ethanol (70%) 
70% ethanol is used as a chemical sterilizer to sterilize the spreaders (except cotton
swabs). 
8. Bunsen Burner
It is used to flame the glass spreader in order to sterile them. Besides it can also be
used to make a sterile working zone. 
9. Other Laboratory Facilities 
Procedure of Spread Plate Method
The general procedure of the spread plate method can be summarized as:
1. Arrange all the requirements, put on the PPE, sterilize the work surface,
and allow all the samples and media to come to room temperature if were
refrigerated.  
2. Sample preparation
 Liquid samples must be serially diluted to reduce the microbial load to
the range of 20 – 300 CFU/mL. (If the sample is assumed to be sterile
or the expected microbial load is very low, we can escape the
dilution. The prior pilot test may give an exact value. You can
prepare serial dilution up to 10-10 and use different dilutions.)
 If the sample is solid or semisolid, dissolve it with a suitable solvent
to prepare its suspension. The suspension then should be serially
diluted to reduce the microbial load at the desirable
range. (Generally, 1 gm sample is mixed with 9 ml of solvent to get
the concentration of 10-1 gm/mL.) 
 Arrange the spreader. The glass rod must be sterilized. For this dip the
rod in 70% ethanol solution and flame it over a Bunsen burner. Let
the rod cool. (You can check if the rod is cold enough or not by
touching a corner of solidified media. If you heard a sizzling sound or
if the media melts, then cool the rod further.)
Serial Dilution
Likewise, the beads can be put in a bottle or beaker and then autoclaved to sterilize
them. 
4. Label the plate at its bottom edge with the dilution factor, date, name,
sample ID, and other required information. 
5. The spreading can be done by either of the following two methods, viz.:
Spreading with a bent glass or metal rod
1. Open the lid of the plate and dispense 0.1 mL of the diluted sample in the
center of the Petri plate using a calibrated pipette or micro-pipette. 
2. Using a sterile bent glass rod uniformly spread the sample all over the
plate. 
 If you are using a turntable, spin it slowly and then hold your spreader gently
on the surface of the media touching the sample, and gradually spread the
sample uniformly all over the surface of the media. Moving the rod back
and forth will allow you to spread the sample.
 If you are performing it manually, hold the plate in your left hand (or in your
right if you are left-handed) or you can also put it still on the bench. You
must move the spreader in either a circular path or in a back and forth
motion to spread the sample evenly. At last, move the spreader in a
circular motion around the edge of the plate to make sure that the sample is
spread even at the corner. 
3. Put on the lid, leave the plate in an upright position and allow the sample
to be absorbed for around 5 minutes. Then incubate the plate in an inverted
position.
Spreading with glass beads: “Copacabana Method”
1. Open a portion of the lid of a Petri plate with your thumb and index finger
and dispense about 10 -12 sterile glass beads. 
 2. Dispense 0.1 mL of the diluted sample in the center of the Petri plate using
a calibrated pipette or micro-pipette. 
3. Close the lid and shake the plate in a horizontal motion so that the beads
spread the sample evenly across the surface of the agar medium. Repeat
the shaking 6 – 7 times.
4. Rotate the plate clockwise or counterclockwise by 60° and again shake the
plate in a horizontal motion 6 – 7 times.
5. Once again, rotate the plate in the same direction by 60° and shake the
plate in a horizontal motion 6 – 7 times. (By this time, the sample will be
spread uniformly, but if you feel that the sample is not distributed evenly,
you can repeat the shaking process.)
6. Leave the plate in an upright position and allow the sample to be absorbed
for around 5 minutes.
7. Discard the beads in a container with disinfectant (10% chlorine bleach,
ethanol, etc.)
8. Incubate the plate in an inverted position. 
Result Interpretation of Spread Plate Method
 Following the incubation, the plates are looked for the development of
discrete colonies. Each colony will account for one viable microbial cell or
one colony-forming unit. 
 If the morphology of all the isolated colonies is the same, it indicates that the
specimen contained only one type of microbial genera. However, there
may be different genera or species producing similar types of colonies. So,
perform a further test for their identification before confirming the
presence of only one type of microorganism. 
 If the morphology of isolated colonies is different, then we can conclude that
the sample contained more than one type of genera or species of
microorganism. They can be purified by sub-culturing each colony on a
separate culture media plate using the streak plate method. 
 Count the colonies and calculate CFU/mL by using the formula:

This will give the total number of viable microbial cells present in the given
sample. 
 For optimum count, the number of colonies must be between 20 – 300
CFU/mL. Beyond this limit, the whole procedure must be repeated. If the
number of the colony is less than 20, it is suggested to use the sample of
 lower dilution, whereas, if the total number of colonies exceeds 300, it is
suggested to use the sample of higher dilution on successive repeats. 
 If the colonies are fused or the whole plate is covered with a single colony,
then report as “too numerous to count” (TNTC) and repeat the process of
taking the sample at a higher dilution.
Precautions during Spread Plate Technique
1. Follow proper safety protocols. Treat every unknown or clinical specimen
as hazardous and follow safety accordingly. Make sure that all the tools
and glass wares are sterile. The water or broth used in serial dilution must
be sterile.   
2. Make sure to sterilize the glass rods by dipping them in 70% alcohol and
flaming them before and after their use to spread a specimen. Glass beads
can be sterilized by autoclaving. The spreading beads or the rod must be at
or below 37°C. Don’t use them immediately after flaming; let them cool. 
3. The solvent used to dissolve the solid sample must be sterile and must not
have any growth-supportive or inhibitory effect against any
microorganism. 
4. The sample must be diluted enough so that the viable microbial load falls
between 20 – 300 CFU/mL. Above this range, it will be difficult to count
the colony and the colonies may fuse together. Below this range, the result
is reported as not significant. The process must be repeated under the same
condition if the colony count is below 20 CFU/mL or above 300 CFU/mL. 
5. The sample must be 0.1 mL (0 to 1 mL is the permitted range) for
spreading. If the sample is 1 mL or more, then there will be fused colonies,
and also the sample may not be absorbed by the media leaving it to float
on the surface. 
6. Media should be properly solidified before use. If it is refrigerated, allow it
to come to room temperature. Appropriate media selection is necessary for
proper and complete isolation.
7. Check for any growth or presence of water droplets on the surface of the
media before inoculation. 
8. Accurate measurement of water for serial dilution and accurate
measurement of sample for inoculation is very important. Hence, always
use a micropipette or calibrated pipette. 
9. Labeling each Petri plate with an accurate dilution factor is necessary in
order to make an accurate calculation of the microbial load following the
incubation. Inoculate each plate with the specific specimen whose
concentration corresponds to the labeled dilution factor on the plate. 
10.Incubate the plate in an inverted position in appropriate condition. Check
the plate by 24 hours of incubation, because if delayed over-growth can
 occur and colonies may fuse making the plate difficult to read. If no
growth after 24 hours, incubate it for the next 24 – 48 hours (or more based
on probable microorganism) before reporting no growth and discarding.    
Applications of Spread Plate Technique
1. Used to isolate bacteria and fungi from a given sample
2. Used in antimicrobial sensitivity testing, and enrichment and screening
experiments. 
3. Used to calculate the number of viable microorganisms (i.e. calculate
CFU/mL) in a sample
4. Used in food industries, pharmaceutical industries, soil studies, etc. in
order to isolate and enumerate spoilers or contaminants to check for the
quality of the products. 
5. Used to mass culture the stock culture or fresh specimen
6. Used in clinical laboratories to inoculate the clinical specimens
7. Used to study growth curves, metabolic activities, and biochemical
features of microorganisms, and also the effect of environmental factors on
them.
8. Used in separating pure culture from a mixed culture
9. Used in generating discrete and pure colonies in order to study colony
characters, genetic features, and other biochemical features of the
isolates.   
Advantages of Spread Plate Technique
1. It is a simple, easy, and quick method of culturing microorganisms.
2.  A very low microbial load can be detected.
3. The colony morphology of a microorganism can be studied by this
method. The size of colonies produced when cultured by this method will
be larger than those produced by the sample species using the pour plate
method.  
4. It is a qualitative as well as quantitative isolation method which facilitates
isolation as well as an enumeration (i.e. calculation of CFU/mL) of
microorganisms.
5. It can be used in performing Kirby Bauer antimicrobial sensitivity testing.
6. Any clinical or industrial or environmental samples can be used unless it is
in a liquid state or can be dissolved to prepare a suspension.
7. It is used in preparing and maintaining stock culture. 
8. It is the most appropriate method for culturing the aerobic microorganism. 
9. Even the syntrophic bacteria can be isolated because they both can be
grown in a single plate nearby with distinct colonies of each type. 
 10.There is very least chance of contamination if one used sterile glass beads
as a spreader. This is because all the action will be done by closing the lid
of the culture plate. 
Limitations of Spread Plate Technique
1. It requires extra tools like a spreader.
2. It needs the sample to be in liquid or suspension form and needs to be
serially diluted, hence; it is a little complex process.
3. Solid or semisolid samples must be suspended prior to inoculation. It is
very difficult if the sample is not easily soluble.
4. It doesn’t support sufficient growth of Microaerophiles and anaerobes. 
5. It is unsuitable if the microbial load in the sample is too high. So, the
sample must be serially diluted to reduce the microbial load at 20 – 300
CFU/mL. To get this dilution range, we may even need to do pilot testing.
6. There is a chance of gouging the media during spreading, especially while
using glass rod by one with not enough skill, and if the media is not
properly solidified.
7. Needs to sterilize the beads or glass rods after spreading each plate. If the
sterilization is not enough, then there is a higher chance of cross-
contamination. 
8. It needs specific media pre-solidified before the work. Hence, either we
need prior information about probable microorganisms in the sample, or
we have to have different types of media.