EXERCISE 4

TECHNIQUES FOR ISOLATING PURE CULTURES

Objectives:

Students should be able to

1. define pure culture and colony-forming unit

2. compare the advantages and disadvantages of the pour plate, spread plate, and streak
plate procedures

3. isolate an organism in pure culture using a pour plate, spread plate, and streak plate
procedures

4. determine which technique is appropriate for various situations

Until 1883, bacteria were grown in the laboratory only in liquid broth or on the surface of
fruits and vegetables. One of the most critical discoveries in microbiology was the
development of a solid nutritional medium for isolating pure cultures by Robert Koch.
Microbes could be spread across the solid surface and separated from each other. When these
individual organisms reproduced, they formed colonies. When each colony-forming unit
(CFU) represents the progeny of a single bacterium, it is a pure culture. The first widely used
solidifying agent was gelatin, which unfortunately melts at temperatures above 30°C. (In the
summer, it may be impossible to work with gelatin in an unairconditioned room.) Because of
this limitation, agar, a more stable algal product, replaced gelatin.

In microbiology, pure culture is a laboratory culture containing a single species of organism.

Or we can say that a pure culture is a cell population with only one species. Only in rare cases,
microbiological populations occur alone in nature. In almost all cases, microorganisms of one
species are mixed with one or more other species; it is commonly called "a mixed culture." A
pure culture is usually derived from a mixed culture (containing many species) by separating
the individual cells. When individual cells multiply, each will form an individually distinct
colony. Then they may be used to establish new cultures with the assurance that only one type
of organism will be present. To characterize, identify, differentiate, propagate, study, or
perform antimicrobial susceptibility testing on a microorganism, we must first isolate the
targeted microorganism from the other species to which it does not belong. That is essential
because it is impossible to study a given type of microorganism's characteristics when
contaminants are present. Only with pure cultures, the properties of individual types of
microorganisms can be examined and understood.

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Below, you will see figures of both the mixed culture and pure plate:

This plate is contaminated This plate is clean

Bacterial
Contamination

Culture

Fungal
Contamination

Microbiologists have developed special techniques and pieces of equipment to isolate and
grow pure cultures of microorganisms free from contaminating forms. Bacteria can be
separated by one of the following methods: streak plate, spread plate, or pour plate. In general
pure cultures are best obtained by using solid media. We must learn all three methods to
isolate colonies. Remember to use the aseptic technique to prevent contamination from the
environment.

1. POUR PLATE TECHNIQUE

Purpose:

We will use this technique as a part of the plate count method of enumerating bacteria.

Principle:

A pour plate is a method of melted agar inoculation followed by petri dish incubation. With
the pour plate method, the bacteria are mixed with melted agar until evenly distributed and
separated throughout the liquid. The melted agar is then poured into a sterile empty plate and
allowed to solidify. After incubation, discrete bacterial colonies can then be found growing
both on the agar and in the agar.

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PROCEDURE

1. Label the bottoms of sterile empty Petri plates with the date, your name, dilution factors,
and the name of microorganism used.

2. Place nutrient agar deeps into the boiling water bath for melting.

3. Suspend the microorganisms in broth culture by vortex mixing. Make serial dilutions of the
broth culture. Transfer 1mL of broth culture to 9 mL saline for 10-fold dilutions and 0.1 mL
of it to 9.9 mL saline for 100-fold dilutions. Be sure to flame the culture tube and dilution
tubes' mouth before and after pipetting (obey the aseptic conditions).

3. Vortex the diluted culture and transfer 0.1 mL of it to the melted deep, aseptically. Take
and gently shake the freshly inoculated deep, remove the plug, flame the mouth of nutrient
agar vessel (melted and held at 45 °C), and carefully pour agar contents into the petri dish.
Flame the mouth of the tube. Do not keep the agar out of the water bath for an extended
period.

4. Mix the contents of the Petri plate by carefully moving the dish on the benchtop.

5. Allow the agar to solidify and incubate for 24 hours at 37 oC in an inverted position to
prevent condensation.

6. Repeat the procedure for other dilutions.

Advantages of pour plate technique;

1. Pour plates are useful for quantifying microorganisms that grow in a solid medium. The
approximate number of viable bacterial cells in a broth culture can be calculated, and the data
are expressed as the number of colony-forming units per milliliter of the substance that is
examined.

2. Because the "pour plate" embeds colonies in agar, it can supply a sufficiently oxygen-
deficient environment that it can allow the growth and quantification of microaerophiles.

3. To determine the hemolytic activity of embedded colonies of bacteria, such as

streptococci, an agar medium containing blood is used.

4. The organisms will grow as individual colonies within the agar. Up to 1.0 mL can be
plated using this technique as long as the sample contains less than 300 organisms.

But; There are also disadvantages of this technique,

1. The colony morphologies of several bacteria are similar, so that may cause errors.

2. Certain species of some bacteria cannot grow embedded in agar due to anoxygenic
conditions.

3. It may be challenging to pick colonies for further study.

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SPREAD PLATE TECHNIQUE

Purpose:

Enumerating bacteria using spread plate technique

Principle:

The spread plate method involves diluting the bacterial sample in tubes containing sterile
water, saline solution, or broth. Small samples of the diluted bacteria are then pipetted and
delivered onto the agar media's surface in plates. A sterile, bent-glass rod is then used to
spread the bacteria evenly over the entire agar surface. Obtain a Bunsen burner, a container of
70% alcohol, and a bent glass spreader (also known as a rod).

PROCEDURE

1. Label the bottoms of Petri plates containing medium with the sample, date, your name.

2. Place the spreader into the container of alcohol and light the Bunsen burner.

3. Make the 10-fold or 100-fold dilutions from broth culture using sterile physiological saline
tubes (0.85% NaCl in dH2O). Suspend the microorganisms in diluted culture by vortex
mixing. Aseptically draw sample (0.1 mL) into the pipette using a pipette aid.

4. Crack the lid of the Petri plate using your non-dominant hand.

5. Insert the pipette under the Petri plate lid so that the pipette's tip touches the agar surface.
Holding the pipette relatively horizontal will make the flow of liquid easy to control.

6. Pipette sample (usually 0.1 mL) onto the agar surface.

7. Place the pipette into a discard container and remove the pipette aid.

8. Immediately pass the spreader through the Bunsen burner flame to ignite the alcohol that is
adhering to it. Never hold the glass spreader after dipping into alcohol in the flame.

9. Allow the alcohol to burn off, wait for a while spreader has air-cooled, or you can cool it by
touching it to the bottom of the Petri lid.

10. Holding the Petri plate's lid with your non-dominant hand halfway open, use the spreader
to spread the sample evenly over the entire agar surface.

11. Rotate the plate 90 degrees, and continue spreading the sample while moving the spreader
back and forth across the plate.

12. Place the spreader back into the alcohol.

13. Cover the plate and wait several minutes before turning it upside down for incubation.
That will allow the broth to soak into the agar media in the Petri plate so the bacteria will not
drip onto the plate lid.

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14. Incubate the plate agar side up, lid down as directed (at 37 oC for 24 hours).

Advantages of spread plate technique;

1. Spreading a plate is an additional method of quantifying microorganisms on a solid

medium.

2. Instead of embedding microorganisms into agar, as is done with the pour plate method,
liquid cultures are spread on the agar surface using a spreader (a glass rod looks like a hockey
stick).

3. An advantage of spreading a plate over the pour plate method is that cultures are never
exposed to 45 °C+ melted agar temperatures.

4. Compared to the pour plate technique, the spread plate may be easier to pick colonies for
further study.

Quantifying Bacteria by Spread and Pour Plate Techniques

The number of bacteria in a solution can be readily quantified using both the spread plate and
pour plate techniques. The sample is appropriately diluted in the spread plate technique, and a
small aliquot is transferred to an agar plate. The bacteria are then distributed evenly over the
surface by spreading with a spreader. In the case of the pour plate technique, the sample is
appropriately diluted and a small aliquot transferred to melted agar, then is poured a sterile

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 Petri plate, as mentioned previously. After colonies are grown, they are counted, and the
number of bacteria in the original sample is calculated.

The first step of the method is to prepare serial dilutions of bacteria in physiological saline
(0.85% NaCl in distilled water).

SERIAL DILUTION

The purpose:

Bacteria commonly grow up to densities around 109 CFU/mL, although the maximum
densities vary tremendously depending on the bacteria's species and the media they are
growing in. Therefore, to get readily countable numbers of bacteria, we have to make a wide
range of dilutions and assay them to have one or two dilutions with countable numbers. We
do this by making serial 10-fold dilutions of the bacteria that cover the whole probable range
of concentrations. We then transfer 0.1 mL of each dilution to an agar plate, which in effect
makes another 10-fold dilution since the final units are CFU/mL, and we are only streaking
0.1 mL.

Procedure:

1. You must change your pipette tip after every dilution and discard the tip into the benches'
waste cans. It would be best to loosen all the lids (do not remove). We will use a starting
solution of a 1% bacterial solution for practice, which should be added to the 9 mL 0.85 %
saline solution, as shown below.

2. Label the tubes for the different dilutions 10 -1,10-2, 10-3, 10-4,10-5, 10-6, then using a 1mL
pipette.

3. Add 1ml of concentrated bacterial solution to 9 mL of physiological saline (0.85% NaCl in

distilled water) = 10-1

Add 1mL of 10-1 solution to 9mL of physiological saline = 10-2

Add 1mL of 10-2 solution to 9mL of physiological saline = 10-3
Add 1mL of 10-3 solution to 9mL of physiological saline = 10-4
Add 1mL of 10-4 solution to 9mL of physiological saline = 10-5
Add 1mL of 10-5 solution to 9mL of physiological saline = 10-6
1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

conc. 10-1 10-2 10-3 10-4 10-5 10-6

E.coli stock

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 1 ml into 9 mL = 1:10 dilution

Or you can make 100-fold dilution by adding 0.1mL bacterial solution to 9.9mL of saline
solution, for example.

0.1 ml into 9.9 mL = 1:100 dilution

Add 0.1 mL of stock solution to 9.9 mL of physiological saline = 10 -2

Add 0.1 mL of 10-2 solution to 9.9 mL of physiological saline = 10--4
Add 0.1 mL of 10-3 solution to 9.9 mL of physiological saline = 10-5

0.1 mL 0.1 mL

conc. 10-2 10-3 10-4 10-5

E.coli stock

1 mL 0.1 mL

Dilutions of microorganisms are not usually visibly different unless pigmented.

For quantifying the bacteria per mL of culture, the endpoint of the analysis is the number of
colony-forming units per mL (CFU/mL) since we are counting the number of colonies rather
than the actual number of bacteria. We are assuming that each viable bacteria in the
suspension will form an individual colony. That would be a valid assumption only if all the
techniques were correctly applied. CFU/mL is a more useful determination than counting all
the bacteria under a microscope. In many bacterial populations, a significant number will be
dead cells and thus of no interest.

In the dilutions of bacteria from 10 -1 to 10-6, some of the colonies on the plates will be too
dense and should be recorded as TNTC (too numerous to count), in other words, UC-
uncountable- that is over 300 colonies/plate.

Firstly, turn the agar plate upside down and count the colonies by looking through the agar.
To be confident of not double-counting a colony, touch the plate's underside at each colony
counted, with a marker pen, thus ensuring that you count each colony only once.

Number of colony forming units on each plate:

10-1 10-2 10-3 10-4 10-5 10-6

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Now calculate the number of CFU/ mL of the original sample.
1 1
# of colonies on plate x x
Dilution of solution Volume of inoculum

Nos. of colonies = 250

Dilution = The reciprocal of 10-4, which is 104 (means that there are 104 fold bacteria in 0.1mL
of original culture)
Volume of innoculum = 0.1mL

250 x 1/10-4 x 1/0.1

2.5 x 106 x 10
2.5 x 107 CFU/mL of original sample.

STREAK PLATE TECHNIQUE

Purpose:

To obtain isolated colonies from a mix culture.

Principle:

The streak plate technique is essentially a method to dilute the number of microorganisms,
decreasing the density. It provides a rapid and straightforward method of diluting the sample
by mechanical means. That allows for individual colonies to be isolated from other colonies.
Each colony is considered "pure," since theoretically, the colony began with an individual
cell. As the loop is streaked across the agar surface, more and more bacteria are rubbed off
until individual separated organisms are deposited on the agar surface. After incubation, the
area at the beginning of the streak pattern will show confluent growth, while the area near the
end of the pattern should show discrete colonies.

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The photo shows an example of a good streak for isolation using the "four corners" method.
The small colonies here are of Staphylococcus epidermidis.

PROCEDURE

1. Before beginning the procedure, label the sterile Petri plates' bottom with your name, date,
and organism.

2. Shake the mixed culture tube gently. Flame the inoculating loop to redness, remove the
tube's plug with the free fingers of the hand holding the inoculating loop, and flame the lip of
the tube.

3. Remove a loopful of the mixed culture after the loop has cooled for at least four seconds.
Take the agar plate in the left hand. Hold the Petri plate so that the bottom rests on the palm,
and the lid can be manipulated with the thumb and third finger. Lift the Petri plate cover and
place the inoculum at the one edge of agar.

4. Begin with inoculating the first, or primary, quadrant of the agar plate, making thick and
parallel lines. Use a light touch. Don't tear or scrape the agar surface. Then, cover the lid.

5. Flame the loop, cool by touching an uninoculated portion of the surface.

6. Now rotate the plate one-quarter of a full turn. Open lid and streak again. Remember: you
are picking up the growth from quadrant one and using this as your inoculum for quadrant
two. Pay attention to touch the streaks of the quadrant only once for the beginning of the
second streak set.

7. Flame loop (before each streak set); rotate plate, and repeat the procedure for quadrants
three and four.

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8. Flame the inoculating loop before putting it down.

9. Hold the agar plate to soak the bacteria and then turn it, label the bottom as directed.

10. Incubate the plate agar side up as directed (at 37 oC for 24 hours).

Note: The proper wrist action and light touch take practice.

The advantage of the streak plate technique is to obtain single colonies on the plate.

This technique's disadvantage is the disability to calculate the CFU/mL due to making dilution
by streaking instead of serial dilution. Because there will be too many colonies at the first two
streak sets that cannot be counted.

Useful Links

http://www-biol.paisley.ac.uk/schools/lab/expts/microbiology.htm

http://www.hendrix.edu/homes/fac/sutherlandM/CellWeb/Techniques/microaseptic.html

http://www.umd.umich.edu/casl/natsci/slc/slconline/ASEP/sld013.htm

http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20102/Bio_2_menu.htm

http://biology.fullerton.edu/courses/biol_302/Web/302labf99/purcult.html

http://biology.fullerton.edu/courses/biol_302/Web/302labf99/manual.html

http://www.austin.cc.tx.us/microbugz/labindex.html

http://www.cplpress.com/contents/C10.htm

http://www.mrc-lmb.cam.ac.uk/genomes/madanm/articles/dnashuff.htm

http://www.ipmofalaska.homestead.com/files/Bt.html

http://www.ext.colostate.edu/pubs/insect/05556.html

http://helios.bto.ed.ac.uk/bto/microbes/bt.htm

http://www.ejbiotechnology.info/content/vol4/issue2/full/5/

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