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1st week.

Microbial culture
Introduction:

This lab introduces an introduction to plating and culturing E. coli (MG1655) on LB agar

plates such that single cells can be isolated from one another. Each cell then reproduces to

form a visible colony composed of genetically identical clones. Streaking cells to obtain

individual colonies is usually the first step in genetic manipulations of microorganisms.

Using cells derived from a single colony minimizes the chance of using a cell mass

contaminated with a foreign microorganism.

HANDLING AND DISPOSAL OF E. coli

E. coli is a normal part of the bacterial fauna of the human gut. It is not considered

pathogenic and is rarely associated with any illness in healthy individuals. MG1655 and

others commonly used lab strains, are ineffective in colonizing the human gut. Adherence to

simple guidelines for handling and disposal makes work with E. coli a non-threatening

experience.

1. To avoid contamination, always re-flame inoculating loop or cell spreader on final time

before placing it on the lab bench/countertop.

2. Keep nose and mouth away from tip end when pipetting suspension culture to avoid

inhaling any aerosol that might be created.

3. Do not over-incubate plates. E. coli is generally the only organism that will appear on

plates incubated from 15-20 hours at 37C. However, with longer incubation,

contaminating bacteria and slower-growing fungi can arise. If plates cannot be observed

following initial incubation, refrigerate them to retard growth of contaminants.

4. Collect treatment bacterial cultures and tubes, pipettes, and micropipette tips that have
come into contact with the cultures. Disinfect these materials as soon as possible after use.

Contaminants, often odorous and sometimes potentially pathogenic, are readily cultured

over a period of several days at room temperature.

- Disinfect by using a 10% bleach solution. Immerse contaminated pipettes, tips, and

tubes (opened) directly into sink or tub containing bleach solution. Bacterial plates

should be disposed of properly by placing them in a sealed container. Allow materials

to stand in bleach for 15 minutes or more. Then drain excess bleach solution, seal

materials in plastic bag, and dispose in accordance with local regulations.

5. Wipe down lab bench/countertop with 70% ethanol at the end of the lab.

6. Always WASH YOUR HANDS before leaving the lab.

PREPARING LB MEDIUM and LB AGAR PLATES

1. Weigh out:

A. 10 g tryptone (an assortment of peptides from digested casein cheese protein)

B. 5 g of yeast extract

C. 10 g of NaCl

D. 15 of agar (for LB agar plates, add 15 g/L agar before autoclaving)

(We use a prepared mixture of these instead.)

2. Dissolve all ingredients in 800 ml deionized water in a bottle/beaker/flask that has been

rinsed with distilled water, preferably using a magnetic stir bar.

3. Adjust the pH of the medium to 7.0 using 1N NaOH and bring volume up to 1 liter.

4. Autoclave solution of 15 minutes at 121 C.

5. After the sterilization run in the autoclave, let solution to cool down so that the bottle(s)

can be held in bare hands (55-60C). If solution cools too long and the agar begins to

solidify, re-melt by briefly heating it in a microwave for a few minutes.


6. While agar is cooling, wipe down the lab bench with 70% ethanol to sterilize working

area.

7. Continue using sterilized lab bench. Mark culture plate bottoms with the date and

description of the media (e.g., LB or LB/amp).

8. When the agar flask is cool enough to hold to 55 C, add antibiotic if needed (50 ug/L of

Amp or Kan) and lift lid of culture plate only enough to pour solution. Quickly pour in

agar to just cover plate bottom (~25-30 mL). Tilt the plate to spread the agar, and

immediately replace lid.

9. To remove any bubbles in the surface of the poured agar, let agar solidify undisturbed for

5-10 minutes.

10. Incubate plates lid side down for several hours at 37C (overnight recommended). This

dries the agar, limiting condensation when plates are stored under refrigeration. It also

allows the ready detection of any contaminated plates.

11. Store at 4C; plates should last for 3 months.

PLATE-STREAKING

1. Use a permanent marker to label the bottom of each plate with your name and the date

(of inoculation). Each plate will have been previously marked to indicate whether it is

plain LB agar, along with the date of preparation.

2. Take two LB plates. Mark plate

3. Hold the inoculating loop like a pencil, and sterilize the loop in the alcohol lamp flame

until it glows red hot. Then continue to pass the lower shaft through the flame.

4. Cool for 5 seconds. To avoid contamination, do not place inoculating loop on lab bench.

5. Use one of the following techniques to scrap up E. coli.

6. If working from a culture plate:


a. Remove the lid from the E. coli culture plate with your free hand. Do not place lid on

the lab bench! Hold the lid face down just above the culture plate to help prevent

contaminants from falling on the plate or lid.

b. Stab inoculating loop into a clear area of the agar several times to cool.

c. Use the loop tip to scrap up a visible cell mass from a colony.

7. Streaking

a. Streak 1: Glide inoculating loop tip back and forth across the agar surface to make a

streak across the top of the plate. Avoid gouging agar. Replace lid of plate between

streaks.

b. Streak 2: Re-flame inoculating loop and cool by stabbing it into the agar away from

the first (primary) streak. Draw loop tip through the end of the primary streak and,

without lifting loop, make a zigzag streak across one quarter of the agar surface.

Replace plate lid.

c. Streak 3: Re-flame loop and cool in the agar as described above. Draw loop tip

through the end of the secondary streak, and make another zigzag streak in the

adjacent quarter of the plate without touching the previous streak.

d. Streak 4: Re-flame loop and cool in the agar as described above. Draw tip through

the end of the tertiary streak, and make a final zigzag streak in remaining quarter of

plate.

8. Re-flame the loop, and allow it to cool, before placing it on lab bench. Make it a habit to

always flame the loop one last time

9. Place plates upside down in a 37C incubator and incubate for 15-20 hours. (Plates are

inverted to prevent condensation that might collect on the lids from falling back on the

agar and causing colonies to run together.)


10. Take time for a responsible cleanup.

11. Optimal growth of well-formed, individual colonies is achieved in 15-20 hours. At this

point, colonies should range in diameter from 0.5 mm to 3.0 mm.

PLATE-SPREADING
1. Dip the spreader into the ethanol beaker and briefly pass it through a alcohol lamp flame

to ignite the alcohol. Allow alcohol to burn off away from the flame; the rod of the

spreader will become too hot if left in the flame.

NOTE: Be careful not to ignite the ethanol in the beaker! DO NOT PANIC if the

ethanol is accidentally ignited. Cover the beaker with a Petri dish lid or other cover

to cut off oxygen and rapidly extinguish the fire.

2. Lift the lid of one plate just enough to allow spreading; do not place lid on lab bench.

3. Cool spreader by gently rubbing it on the surface of the agar away from the cell

suspension or by touching it to condensation on the plate lid.

4. Touch the spreader to the cell suspension, and gently drag it back and forth several times

across the surface of the agar. Rotate plate one-quarter turn, and repeat spreading motion.

Try to spread the suspension evenly across agar surface. Be careful not to gouge the agar.
5. Replace plate lid. Return cell spreader to ethanol without flaming

6. Place plates upside down in a 37C incubator and incubate for 15-20 hours. (Plates are

inverted to prevent condensation that might collect on the lids from falling back on the

agar and causing colonies to run together.)

7. Take time for a responsible cleanup.

8. Optimal growth of well-formed, individual colonies is achieved in 15-20 hours. At this

point, colonies should range in diameter from 0.5 mm to 3.0 mm.

LIQUID CELL CULTURE

Introduction:

E. coli has simple nutritional requirements and grows slowly on minimal medium

containing an energy source such as glucose, salts (such as NaCl and MgCl), the vitamin

biotin, and the vitamin thiamine (B1). E. coli synthesizes all necessary vitamins and amino

acids from these precursors and grows rapidly in a complete media, such as LB. Yeast extract

and hydrolyzed milk protein (casein) provide a ready supply of vitamins and amino acids.

A liquid bacterial culture goes through a series of growth phases. For approximately 30 60

minutes following inoculation, there is a lag phase during which there is limited cell growth.

The bacteria begin dividing rapidly during log phase, when the cell number doubles every 20

25 minutes. As nutrients in the media are depleted, the cells nearly stop dividing and the

culture enters stationary phase. During death phase, waste products accumulate and cells

begin to die.

Optimum growth in liquid culture is achieved with continuous agitation, which aerates the

cells, facilitates the exchange of nutrients, and flushes away waste products of metabolism. It

can be safely assumed that a culture in complete medium has reached stationary phase
following overnight incubation with continuous shaking. However, it is not absolutely

essential to grow a culture with shaking. Suspensions can be incubated, without shaking, in a

rack within a 37 C incubator. These cultures will need to incubate for 1 day or more to

obtain an adequate number of cells.

A stationary phase culture will look very cloudy and turbid. Discard any overnight culture

where vigorous growth is not evident. Expect less growth in cultures incubated for 1 2 days

without continuous shaking. To gauge growth, shake the tube to suspend cells that have

settled at the bottom of the tube.

PREPARING LB BROTH STOCK FOR EACH GROUP

1. The following recipe is for 50 mL of media, we may adjust volume depending on class

size

a. 0.5 g tryptone

b. 0.25 g of yeast extract

c. 0.5 g of NaCl (m.w. = 58.44)


(We might use a prepared mixture of these instead)

2. Add all ingredients to a clean 250 mL bottle that has been rinsed with deionized or

distilled water.

3. Add 50 mL of deionized or distilled water to the bottle.

4. Stir to dissolve dry ingredients.

5. LOOSELY screw on the cap.

6. Autoclave for 15 20 minutes at 121 C.

PREPARING OVERNIGHT CULTURE

LB broth can be considered sterile as long as the solution remains clear. Cloudiness is a sign

of contamination by microbes. Always swirl solution to check for bacterial or fungal cells

that may have settled at the bottom of the flask or bottle.

1. Label a sterile 50 mL test-tube or flask with your name and the date.

2. Use a 10 mL pipette to sterilely transfer 5 mL of LB broth into the tube.

a. Attach pipette pump to pipette.

b. Remove cap of LB stock bottle using little finger of hand holding pipette bulb.

Flame mouth of LB bottle.

c. Withdraw 5 mL of LB. Re-flame mouth of bottle, and replace cap.

d. Remove cap of sterile test tube or flask. Briefly flame mouth of tube, and replace

cap.

3. Locate a well-defined colony 1 4 mm in diameter on a freshly streaked plate.

4. Sterilize inoculating loop in the alcohol lamp until it glows red hot. Then, continue to

pass lower half of its handle through the flame.

5. Cool loop tip by stabbing it several times into agar near the edge of the plate.

6. Use loop to scrape up a visible cell mass from selected colony.


7. Sterilely transfer colony into culture tube:

a. Remove cap of the culture tube using little finger of hand holding loop.

b. Briefly flame mouth of culture tube.

c. Immerse loop tip in broth, and agitate to dislodge cell mass.

d. Briefly re-flame mouth of culture tube, and replace cap.

8. Re-flame loop before placing it on lab bench.

9. Incubate for 12 24 hours at 37 C, preferable with continuous agitation.

10. Take time for a responsible cleanup.

a. Segregate for proper disposal bacterial cultures and tubes, pipettes, and

micropipette tips that have come into contact with the cultures.

b. Disinfect overnight culture and pipettes and tips with 10% bleach solution, or

disinfectant.

c. Wipe down lab bench with soapy water, 10% bleach solution, or disinfectant.

d. Wash hands (as always) before leaving lab.

Bacterial Cell Growth Analysis

Introduction:

This protocol is for preparing a mid-log culture of E. coli. Cells in mid-log growth can

generally be rendered more competent to uptake plasmid DNA that can cells at stationary

phase. Mid-log cells are used in the classic transformation protocol that we will try later in

the year. The protocol begins with an overnight suspension culture of E. coli. Incubation with

agitation has brought the culture to stationary phase and ensures a large number of healthy

cells capable of further reproduction. The object is to subculture a small volume of the

overnight culture in a large volume of fresh nutrient broth. This re-sets the culture to zero

growth, where after a short lag phase, the cells enter the log-growth phase. As a general rule,
1 volume of overnight culture (the inoculums) is added to 100 volumes of fresh LB broth in

an Erlenmeyer flask. To provide good aeration for bacterial growth, the flask volume should

be at least four times the total culture volume.

A shaking incubator is necessary for growing E. coli for competent cells. Proper aeration and

nutrient exchange are essential to achieve vigorous growth; only cells collected during the

middle part of log (mid-log) phase will produce competent cells with a high transformation

frequency.

Timing of the culture to reach mid-log phase is likely to be affected by any change in the

protocol. For example, a culture inoculated with an overnight culture that was grown without

shaking will take longer to reach mid-log phase. Different strains of E. coli display different

growth properties. Different nutrient broths also will affect the growth of the culture.

PROCEDURE

1. Sterilely transfer 1 volume of overnight culture into 100 volumes of LB broth at room

temperature.

2. If using a 1 mL overnight culture:

a. Remove cap from overnight culture tube, and flame mouth. Do not place cap on

lab bench.

b. Remove cap from flask, and flame mouth. Do not place cap on lab bench.

c. Pour 500l overnight culture into a flask. Re-flame mouth of flask, and replace

cap.

3. Incubate at 37 C with continuous shaking.

4. It can be safely assumed that an MG1655 culture has reached OD600 0.3 0.5 after 2

hours of incubation with continuous shaking. However, less ideal conditions often result

in slower growth.
5. Take time for a responsible cleanup.

a. Segregate for proper disposal bacterial cultures and tubes, pipettes, and

micropipette tips that have come into contact with the cultures.

b. Disinfect overnight culture and pipettes and tips with 10% bleach solution, or

disinfectant.

c. Wipe down lab bench with soapy water, 10% bleach solution, or disinfectant.

d. Wash hands (as always) before leaving lab.

Measurement of cell mass using spectrophotometer

1. Blank spectrophotometer with 0.8-1 mL of dew-water or culture medium (i.e. LB) in a

cuvette.

2. Pipette 1 mL sample of your culture into cuvette and take reading

3. Sample can then be diluted appropriately using the following formula.

o Desired OD/Sample OD = % of sample in final volume

4. Retest OD after mixing sample thoroughly (swirling or briefly vortexing) to attain new

OD.