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Protocol 1.4: Isolation of Auxotrophs–Replica Plating, Toothpicking, or Screening on 2 EM Plates

Protocol 1.4: Isolation of Auxotrophs–Replica Plating, Toothpicking, or Screening on 2 EM Plates

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9 Mutagenized E. coli cells prepared in Protocol 1.3 that are tetracyclineresistant colonies o n Ltetl 5 agar plates

9 Ltetl 5 agar plates

9 minimaltetl 0 agar plates

9 2 EMtetl 0 agar plates

9 sterile toothpicks (To sterilize toothpicks, autoclave for 20 min at a fast

and dry setting.)

9 sterile velvets" Square pieces of cloth about 89in. larger than the petri

plates with a thick nap, such as velvet or velveteen. (As an alternative

to velvets, sterile circular pieces of filter paper with a diameter about

2 mm less than the inside diameter of a petri dish bottom may be used.)

9 platform to put velvets on for replica plating

9 sterile test tubes and rack

9 lX VBC broth, a minimal broth


Replica Plating

1. Select Ltetl 5 plates grown in Protocol 1.3 that have between 50 and 125

well-spaced colonies.

2. Carefully replica plate the colonies onto a minimal plate and another

Lte t p l a t e .

a. Place a sterile velvet on the replica plating platform.

b. Press a n Lte t agar plate with colonies onto the platform. Apply gentle,

uniform pressure to the bottom of the petri plate to ensure that all

colonies on the agar surface touch the velvet. Remove the agar plate.

c. Carefully press a minimaltetl 0 agar plate to the velvet. Again, using

gentle pressure, make sure all areas of the agar surface make contact

with the velvet. Mark the top of the plate with a permanent marker.

Remove the agar plate.

d. Carefully press a new Lt~u5 agar plate to the velvet. Mark the top of

the plate with a permanent marker. Remove the agar plate.

3. Incubate the plates at 40~ overnight.

4. The next day, compare the growth of bacteria on both plates. An auxotroph will not grow on the minimal plate, but will grow on the Lte t


5. When an auxotroph is found, locate the corresponding colony on the

Lt~t plate. With a sterile toothpick or an inoculation loop, transfer some

of the bacterial colony to a n e w Ere t plate. Streak for single-colony

isolation. Incubate the plates at 40~ overnight.



6. Retest that the colony isolated is an auxotroph by streaking single

colonies on a minimalt~t~ 0 agar plate.

Note: When replica plating, use agar plates that are thoroughly dry.

If plates are too wet, the colonies can smear together. Do not apply too much

pressure when transferring the colonies or colonies may run together.


1. Choose Ltetl 5 plates grown in Protocol 1.3 that have too many colonies

to use for replica plating, but still have separate, distinct colonies.

2. Using a sterile toothpick, touch a colony.

3. With the same toothpick, make a 88 streak on the surface of a minimaltetl 0 agar plate.

4. Make a second streak with the toothpick on the corresponding place

on a n Ltetl 5 agar plate.

5. With a new sterile toothpick, touch a new colony.

6. Repeat the process.

7. Incubate the plates at 40~ overnight.

8. Identify streaks that fail to grow on minimal agar plates. Find the

corresponding streak on the Ltetl 5 agar plate.

9. Streak some of that colony for single colonies on a n e w Ltetl 5 agar plate.

Once it has grown, retest by streaking on a minimalt~tlO agar plate.


1. Be sure to make the streaks in the corresponding places on the pair of

plates. At least 40 to 50 different colonies can be streaked on an agar

plate. A template can be used if desired. Examples are given of a

template in Appendix 1. Do not try to pick colonies so close together

that it is extremely difficult to pick only one colony with the toothpick.

2. Used toothpicks can be collected, resterilized, and reused.

3. Once an auxotrophic strain has been isolated, be sure to make a permanent stock of that strain (See Appendix 2). Label the strain clearly.

Screening for Auxotrophs on 2 EM Agar (Minimal Medium Supplemented with a Small Amount of Nutrient Broth) Plates. Crowded plates

containing from 500 to 1000 bacterial colonies are used. The basis of this

screen is that in the limiting amount of nutrients on 2 EM plates containing

a large number of colonies, the growth of auxotrophs will be slowed

or stopped as the crowded plate becomes depleted of the nutrients the

auxotrophs require for growth. Colonies that are tiny compared to the



majority of the colonies may be auxotrophs; such colonies will be picked

and retested.

1. Remove bacteria from a n Lte t plate containing more than 100 colonies

from the mutagenesis protocol by flooding the plate with 5 ml of l x

VBC minimal broth.

2. With a sterile 5-ml pipet, scrape the bacteria off the agar surface.

Remove the bacteria with a pipet and place bacteria in a centrifuge


3. Centrifuge the bacteria for 5 min at 5000 rpm. Decant the buffer from

the bacterial cell pellet.

4. Resuspend the cells in I ml of minimal medium. This stock of mutagenized bacteria can now be tested on 2 EM plates. The 2 EM plates

should be crowded to enhance selection for potential auxotrophs.

A crowded plate should have about 500-1000 bacterial colonies

on it.

5. Make serial dilutions of the mutagenized bacteria in I x VBC minimal


6. Using an ethanol-flamed bent glass rod, spread 0.1 ml of that dilution

estimated to give 500-1000 colonies per plate on a 2 EM plate (minimal medium supplemented with 20 ml of nutrient broth per liter).

Make a series of such plates. Also spread 0.1 ml of bracketing dilutions,

such as 10 times higher and 10 times lower concentrations, on 2 EM


7. Incubate plates overnight at 40~

8. Examine the plates the next day. Growth will be slower on this minimal plate. Examine the plates closely (a dissecting microscope may

be helpful here) for the presence of colonies that are much smaller

than the majority of colonies on the plate. These tiny colonies are

potential auxotrophs. The tiny colonies may also be less opaque than

the average colony because the tiny auxotrophic colonies are not as

thick. The plates may need to be incubated longer before the tiny

colonies are readily visible.

9. On the outside of the petri plate, mark the location of the tiny colonies

with a marking pen.

10. Using sterile toothpicks or an inoculation wire, streak the tiny colonies

onto Lte t plates.

11. Incubate plates overnight at 37~

12. Replica plate or toothpick the colonies onto minimal plates.

Colonies that grow o n Lte t but not on minimal medium are auxotrophs.

These auxotrophs will be tested to characterize their biochemical defects.




Identification of Auxotrophs on Pool Plates


9 auxotrophs isolated in Protocol 1.4 and retested to be auxotrophs,

streaked for single colonies o n Ltetl 5 plates

9 pool plates, one of each of 11 different pools

9 sterile toothpicks or inoculation loop


1. Using sterile toothpicks, transfer a small number of bacteria of each

auxotroph onto each of the 11 pool plates. Many different auxotrophs

can be tested on the same pool plates. Be sure to place each auxotroph

in the same location on each of the pool plates.

2. Also streak a prototroph on each of the pool plates as a positive control.

The prototroph will grow on all of the pool plates.

3. Incubate at 37~ overnight.

4. Observe growth on pool plates the next day. Score the amount of growth

for each auxotroph on each pool plate.

5. Return the plates to 37~ and allow further growth.

6. Score growth again after the second day.

7. By examining the components in the pool plates, identify the type of


Making Pool Plates

The following components should be filter-sterilized because they

may be degraded at the high temperatures used in autoclaving. All other

components may be autoclaved.



Potassium aspartate


Sodium glutamate



Additional comments:



Solutions containing tryptophan should be stored in the dark.

The salts of glutamic acid and aspartic acid rather than the free acids

should be used.

The following components dissolve more readily in acidic solutions.

Use the acid strength indicated to dissolve the component.

Adenine, 0.1 N HC1

Adenosine, 0.1 N HC1

Guanosine, 0.3 N HC1

Phenylalanine, 0.01 N HC1

Stock solutions of each of the individual components can be made

and combined into the pool groups.

Stocks of each amino acid can be made at a concentration of 10 mg/

ml of the L form or 20 mg/ml of the DL form. Adenosine, guanosine,

thymine, and uracil stocks can be made at 5 mg/ml. Stocks of vitamins and

other components can be made at the specific concentrations indicated:

thiamine (1/zg/ml), diaminopimelic acid (DAP, 300/zg/ml), pyridoxine

(50 /zg/ml), nicotinic acid (50 /zg/ml), biotin (1 /zg/ml), pantothenate

(50/zg/ml). Note that vitamins are trace growth factors; that is, vitamins are

required at very low concentrations. Sterilize the components as indicated.

To make the stocks for pool plates, using aseptic or sterile technique,

combine the stocks of individual components: Mix equal volumes of the

stocks of individual components indicated for each pool.

Pool stocks may be stored indefinitely at room temperature or at 4~

Tightly close caps or wrap caps with Parafilm. Wrap pool stocks containing

tryptophan with aluminum foil to keep out light.

Components of Pool Plates a


























Glutamic acid




Aspartic acid

Diaminopimelic acid








Nicotinic acid




~ The components of pools 1 to 5 are listed vertically; the components of pools 6 to 10 are listed

horizontally. How to use the pool plates: If a mutant grows on two pool plates, the auxotrophic requirement

is for the component in common to both pools. For example, a mutant which grows only on pools 3 and

9 is an auxotroph for uracil. If a mutant grows only on one pool plate of plates I to 10, the mutant must

require more than one component from that pool. If a mutant grows only on pool 11, each component

of pool 11 must be tested individually for growth to identify the auxotrophy.

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Protocol 1.4: Isolation of Auxotrophs–Replica Plating, Toothpicking, or Screening on 2 EM Plates

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