4.

Spread Plate Procedure: Formation of Discrete Bacterial Colonies for Plate

Counts, Enrichment, Selection, or Screening
This technique typically is used to separate microorganisms contained within a small sample
volume, which is spread over the surface of an agar plate, resulting in the formation of discrete
colonies distributed evenly across the agar surface when the appropriate concentration of cells is
plated. In addition to using this technique for viable plate counts, in which the total number of
colony forming units on a single plate is enumerated and used to calculate the concentration of
cells in the tube from which the sample was plated, spread-plating is routinely used in
enrichment, selection, and screening experiments. The desired result for these three experiments
is usually the same as for plate counts, in which a distribution of discrete colonies forms across
the surface of the agar. However, the goal is not to ensure all viable cells form colonies. Instead,
only those cells within a population that have a particular genotype should grow. The spread
plate procedure may be employed over the pour plate technique for an enumeration experiment if
the end goal is to isolate colonies for further analysis because colonies grow accessibly on the
agar surface whereas they become embedded in the agar with the pour plate procedure.
There are two strategies described here for the spread plate procedure. The first (Method A)
involves use of a turntable and glass or metal rod shaped like a hockey stick. The second
(Method B), referred to as the "Copacabana Method", involves shaking pre-sterilized glass
beads. Both facilitate even spreading of cells across the agar surface.
(erin r. sanders, 2011)

Spread Plate Technique- Principle, Procedure and Uses

Spread plate technique is the method of isolation and enumeration of

microorganisms in a mixed culture and distributing it evenly. The technique
makes it easier to quantify bacteria in a solution.

Principle of Spread Plate Technique

The spread plate technique involves using a sterilized spreader with a smooth
surface made of metal or glass to apply a small amount of bacteria suspended
in a solution over a plate. The plate needs to be dry and at room temperature
so that the agar can absorb the bacteria more readily. A successful spread
plate will have a countable number of isolated bacterial colonies evenly
distributed on the plate.

Procedure of Spread Plate Technique

1. Make a dilution series from a sample.
 2. Pipette out 0.1 ml from the appropriate desired dilution series onto the
center of the surface of an agar plate.
3. Dip the L-shaped glass spreader into alcohol.
4. Flame the glass spreader (hockey stick) over a Bunsen burner.
5. Spread the sample evenly over the surface of agar using the sterile glass
spreader, carefully rotating the Petridish underneath at the same time.
6. Incubate the plate at 37°C for 24 hours.
7. Calculate the CFU value of the sample. Once you count the colonies,
multiply by the appropriate dilution factor to determine the number of
CFU/mL in the original sample.

Uses of Spread Plate Technique

1. It is used for viable plate counts, in which the total number of colony
forming units on a single plate is enumerated.
2. It is used to calculate the concentration of cells in the tube from which the
sample was plated.
3. Spread plating is routinely used in enrichment, selection, and screening
experiments.

Limitations of Spread Plate Technique

1. Strick aerobes are favored while microaerophilic tends to glow slower.
2. Crowding of the colonies makes the enumeration difficult.

september 10, 2016 by Sagar Aryal

3. Spread Plate Method:
In this method (Fig. 16.15), the mixed culture or microorganisms is not
diluted in the melted agar medium (unlike the pour plate method); it
is rather diluted in a series of tubes containing sterile liquid, usually,
water or physiological saline.

A drop of so diluted liquid from each tube is placed on the center of an

agar plate and spread evenly over the surface by means of a sterilized
bent-glass-rod. The medium is now incubated.

When the colonies develop on the agar medium plates, it is found that
there are some plates in which well-isolated colonies grow. This
happens as a result of separation of individual microorganisms by
spreading over the drop of diluted liquid on the medium of the plate.

The isolated colonies are picked up and transferred onto fresh medium
to ensure purity. In contrast to pour plate method, only surface
colonies develop in this method and the microorganisms are not
required to withstand the temperature of the melted agar medium.

Article Shared by Manisha Garg

Conclusion:
The spread plate method is a technique to plate a liquid sample containing bacteria so that
the bacteria are easy to count and isolate. A successful spread plate will have a countable
number of isolated bacterial colonies evenly distributed on the plate.
In order to be able to adequately study and characterize a
certainm i c r o o r g a n i s m , m i c r o b i o l o g i s t s n e e d t o s e p a r a t e a n d i s o
l a t e microorganism from a mixed culture of many other microorganismsw i t h w h i c h i t
u s u a l l y s h a r e s i t s e n v i r o n m e n t o r h a b i t a t t o a p u r e culture which is a
population of cells arising from a single cell.Microbiologists routinely rely on
following ways and techniques tomicroorganism cultures.

SPREAD PLATE TECHNIQUE

A mixture of cells or of a cell culture is spread out on the agar surface of a petri dish, with the help of a
"flame-sterilized glass rod-made cell spreader and incubated over a defined time period -usually
between 12 and 48 hours. (the surface-dispersed cells growing to distinctive and visible cell clusters
called colonies.

Advantages

•It is useful for isolating aerobic microorganisms.

•cultures are never exposed to 45 degree Celsius melted agar temperatures.

Disadvantages

•It allows the growth of more microbes and presence of more colony forming units.

•It doesn’t allow the growth of obligate anaerobic microorganisms.

•contamination of the growth can occur.

https://www.academia.edu/19580833/THE_ADVANTAGES_AND_DISADVANTAGES_OF_THE_V
ARIOUS_MICROBIAL_CULTURE_TECHNIQUES

For calculating the colony forming unit bacteria, please see the following text:
colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal cells. In direct microscopic
counts (cell counting using haemocytometer) where all cells, dead and living, are counted,but CFU
measures only viable cells. For convenience the results are given as CFU/mL (colony-forming units
per milliliter) for liquids, and CFU/g (colony-forming units per gram) for solids. CFU can be calculated
using miles and misra method, it is useful to determine the microbiological load and magnitude of
infection in blood and other samples.
Example:
Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of
colonies by the dilution factor The number of colonies per ml reported should reflect the precision of
the method and should not include more than two significant figures.
The CFU/ml can be calculated using the formula:
cfu/ml = (no. of colonies x dilution factor) / volume of culture plate
For example, suppose the plate of the 10^6 dilution yielded a count of 130 colonies. Then, the
number of bacteria in 1 ml of the original sample can be calculated as follows:
Bacteria/ml = (130) x (10^6) = 1.3 × 10^8 or 130,000,000.
CFU/mL Practice Problems - CFU/mL Calculation Examples
Problem 1:
Five ml of Bacterial Culture is added to 45 ml of sterile diluent. From this suspension, two serial,
1/100 dilutions are made, and 0.1 ml is plated onto Plate Count Agar from the last dilution. After
incubation, 137 colonies are counted on the plate. Calculate CFU/mL of the original Sample?
Answer:
First thing we need to know is the Dilution Factor, or how much the original sample is diluted:
here Initially 5mL in 45mL = Final Volume / Sample volume = 50/5 = 10.
Then two serial dilutions of 1/100.
Total Dilution Factor = 10 * 100 *100 = 10^5
CFU/mL = cfu/ml = (no. of colonies x dilution factor) / volume of culture plate
= (137 * 10^5)/0.1
=1.37*10^8
So Total colony forming units = 1.37*10^8 CFU/mL
Converting CFU/mL to Log value
For example,
Total colony forming units = 1.37*10^8 CFU/mL and you want to convert it into Log value,
Just take Log(CFU/mL)
Here, log (1.37*10^8) = 8.13924922.
Useful for expressing log reduction of microbes / biologic log reduction.
For illustration of the figures in this article, please use the following link:
http://technologyinscience.blogspot.co.il/2011/11/cfu-colony-forming-unit-calculation.html#.VpF-
W_krLIW

Spread plate technique is a method employed to plate a liquid sample for the purpose of
isolating or counting the bacteria present in that sample. A perfect spread plate technique
will results visible and isolated colonies of bacteria that are evenly distributed in the plate
and are countable. The technique is most commonly applied for microbial testing of foods or
any other samples or to isolate and identify variety of microbial flora present in the
environmental samples e.g. soil.

A: Serial Dilution
1. Prepare a series of at least 6 test tubes containing 9 ml of sterile distilled water.
2. Using a sterile pipette ,add 1ml of sample in the first tube of the set.Label it as 10-1
3. Mix the contents well by swirling the tube upside down few times.
4. From the first tube, take 1ml of the sample and transfer to second tube. Label it as 10-2.
5. Repeat the procedure with all the remaining tubes labeling them until 10-6.

B. Plating
1. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar
plate.
2. Dip the L-shaped glass spreader (hockey stick) into alcohol.
3. Flame the glass spreader over a bunsen burner.
4. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating
the Petri dish underneath at an angle of 45oat the same time.
5. Incubate the plate at 37°C for 24 hours.
6. Calculate the conlony forming units (CFU) value of the sample. Once you count the colonies, multiply
by the appropriate dilution factor to determine the number of CFU/mL in the original sample.
Calculation of result:

CFU/ml = (no. of colonies x dilution factor) / volume of culture plate

For example, suppose the plate of the 10^6 dilution yielded a count of 130 colonies. Then,
the number of bacteria in 1 ml of the original sample can be calculated as follows:

Bacteria/ml = (130) x (10^6) = 1.3 × 10^8 or 130,000,000.

JULY 28, 2017 BY NISHA RIJAL IN BACTERIOLOGY, BACTERIOLOGY NOTE, MICROBIOLOGY, MICROBIOLOGY FOR
BEGINNERS ·

Spread Plate Method of Isolation

Purpose The Spread Plate technique is used to count the number of bacteria on a Petri Dish.

Principle The spread plate technique is essentially a method to evenly distribute bacteria across the
plate to make the calculation of the number of bacteria colonies easier, especially when estimating

. Each colony is considered "pure," since theoretically, the colony began with an individual cell.

1. Begin by transferring .5ml – 1.0.ml of inoculated nutrient broth to a sterile Petri dish

2. Remove the glass spreader (hockey stick) from the Ethyl Alcohol.

3. Pass the spreader through a Bunsen burner flame to ignite the alcohol. Wait for the alcohol to burn
away. You are not leaving the spreader in the flame. The alcohol sterilizes the spreader. The flame is just
removing the excess alcohol prior to touching the agar.

4. Open the petri dish and use the spreader to evenly distribute the transferred broth over the entire
surface of the plate.

5. Return Hockey Stick to alcohol. 6. Incubate Plate