Microbiological Media Preparation

Microbiological Media Preparation

 

The chance observation in 1881 of bacteria growing on the surface of a spoiling slice of boiled potato led Robert Koch to devise the technique called single colony isolation. He correctly deduced that each spot, or colony, of bacteria had grown as
a clone from a single contaminating bacterium. He subsequently developed the use of agar, a polysaccharide derived from certain red algae, to solidify nutrient liquid media. This allowed a specimen to be spread across the surface of the medium and grown to produce single colonies, thus isolating the various microorganisms which might be present. The power and elegance of this technique will be explored in later exercises.

Agar is particularly suited as a solidifying agent because it will not melt until the medium is heated to near boiling(95°C-100°C), but will remain melted until cooled to around 42°C. It is not degraded by the vast majority of bacteria, and therefore maintains its structure during bacterial growth, and because it is not a nutrient for bacteria, it also allows strict
control of growth factors.

There is a wide variety of media which are used in microbiology, but the procedures used in their preparation are generally the same. They include weighing out the dehydrated media, dissolving it in dH2O, sterilizing it (usually by autoclaving), pouring the plates, preincubation to check for contamination and to dry out the plates, and storage.

Because medium boils up during decompression from autoclaving, we will autoclave only 600 mL of medium in a 1 liter bottle. You will need to calculate the amount of powder needed per 600 mL of medium since the directions on reagent bottles are for 1 full liter. Teams of 3-4 students will prepare a media assigned to them. [Have media in autoclave within 75 minutes of start of class in order to have plates poured by the end of a three hour lecture/lab.]

Equipment
1000 mL beaker or vessel into which to weigh dry media
Balance
spirit thermometer, -10°C to 110°C
heat source, hot pads
1000 mL bottle with cap
funnel
autoclave

Supplies
dehydrated media
600 mL dH2O
16-20 sterile petri dishes

WEIGH OUT THE DRY MEDIA:

1000 ml beaker with a scale being level

1. Tare out the 1000 mL beaker, use counterweights if necessary, record tare weight.

Step 2 Bellow, setting weight.

2. Sum the desired mass of the dehydrated medium + tare weight, set balance to read the sum.

Adding Reggent

3. Add dry reagent with care to beaker with a clean spatula. After each addition, gently tap the edge of beaker with spatula to judge progress. When close to the desired weight, tilt and roll the reagent bottle, tap its neck to sprinkle in the final amount until equal swings are achieved. Do not remove any excess back to the reagent bottle. Replace the cover immediately on the reagent bottle (it is hygroscopic). Record the actual amount added.

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dehydrated media has been weighed out

4. If there are more than a single substance in your medium, repeat steps 2 & 3 for other dry materials. (Use each new apparent weight as the new tare, repeat the sequence of steps.)
Check again to be sure all reagent are securely capped.

Adding water into a beaker

5. Add dH20 to the beaker with stirring, break up clumps of powder, q.s. to the desired volume (200 mL here).

DISSOLVE THE MEDIA:

Stirring the contents inside the beaker

6. Stirring with a thermometer, heat to boiling: do not allow to boil over, nor to burn on bottom. Have hot pads available. (Microwave in cycles of heating and stirring, do not boil over.)

Bring contents inside the beaker to a boil.

7. The media should clarify near boiling (95-100 C).

Pour into a 1000 mL bottle with a funnel. Cap loosely,

8. Pour into a 1000 mL bottle with a funnel. Cap loosely, label bottle with name of medium and your group’s name, place in autoclave.

AUTOCLAVE: (After all student groups have placed their loosely capped bottles in autoclave)

place in autoclave

9. Place dissolved, loosely capped media in the autoclave.

 

10. Set for slow exhaust, autoclave the medium at 15 lbs. pressure for 15 minutes (for most media). This takes at least 45 minutes.

11. Remove from autoclave (CAUTION: HOT STEAM), allow to cool to 50-60°C (feels hot, but possible to hold).*

POURING PLATES:

remove plates from sterile bag

12. Open a sterile package of petri dishes preserving bag for later storage.

Mark the Plates

13. Mark the sides of the dishes according to code lines to indicate the type of media they contain (or label the petri dishes on bottom in small letters: plate type, date.)

Group plates along side of table in groups of four.

14. On a sterile field, each student pours at least four plates using sterile technique as demonstrated. (Position stacks of four or five sterile plates near edge of desk, remove and hold cap with little finger, flame lip of bottle, fill bottom plate first, at least half full, repeat, holding lid over plate as you pour.)

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sterilized media is being poured into sterile petri dishes

15. Flame the lip of the bottle whenever the lid is replaced. The last student pours all of the rest of the medium.

Pour media into dishes and empty bottle.

16. Rinse bottle immediately after pouring last plate before the remnant solidifies.

Incubator with plates

17. When plates have solidified, invert, place in 37°C incubator for 48 hours to check for sterility and to dry out excess moisture. Store in labeled plastic bag at 4C°. Pre-warm before using

*The cooling process may be hastened by placing in front of fan, or immersion of the hot bottles of medium into a bucket of
50°C water. (Colder water may cause the bottles to break…)

OTHER IMAGES:

Here is a detailed series showing successive weighing of media when preparing top agar:

Be sure to push the weights all the way to the right before you return the balance to storage.

Taring out a plastic beaker:

Autoclave Use

Autoclave Use

Pasteur showed that once a medium is sterilized, it will remain sterile until contaminated by microorganisms (i.e., they do not appear by spontaneous generation.). Heating is one of the most convenient methods for sterilization. Simple boiling at 100°C, however, is not completely effective for sterilization because many spores can survive this temperature. However, the boiling point of water goes up when exposed to increased pressures. In a pressure cooker where the pressure is 15 pounds/sq inch (1 atmosphere) above standard pressure (760 mm Hg), water boils at 121°C. This temperature kills all life forms in 15 minutes or less. These conditions have become the standard for sterilizing apparatus or small volumes of liquid: i.e., treat over boiling water at 15 pounds pressure for 15 minutes (commonly called “15 pounds for 15 minutes”).

An autoclave is a piece of laboratory apparatus which acts as an automated pressure cooker. The conditions inside are thermostatically controlled so that heat is applied until 121C is achieved, at which time the timer starts, and the temperature is maintained for the selected time.  Here is the cluster of dials and gauges which regulate its use:

ILLUSTRATE THE AUTOCLAVE , LABEL THESE PARTS:
drain valve *
drain pipe
water level door
door latch
slow/fast exhaust* timer*
pressure indicator *
temperature indicator *

* (Enter the appropriate settings or numbers on the dials for the beginning of the timing segment when autoclaving media)

USING THE AUTOCLAVE:

drain_closed_P7140159

Close drain valve.

Pour 4 L (1 gallon) of hot water into the bottom of autoclave.
Fill to the bottom of the lip as shown.

016_place_in_autoclave_P7120137

Load material to be autoclaved, following the appropriate protocol for type of material being sterilized.

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front view of the autoclave

Close door, latch securely by pressing down firmly.

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Select appropriate exhaust: slow for water-containing materials, fast for apparatus.

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Select the time of treatment (15 minutes for small volumes. For containers containing volumes over 1 liter: 30 minutes, over a gallon: 60 minutes.) This begins the cycle.

When the pressure is 1 atmosphere (15 pounds) and the temperature is 121 C, the timer begins to time the process. (Yes, these numbers were not quite acheived when the pictures were taken…)

drain_open_P7140164

After sterilization, when pressure has returned to zero, open up the door with care–the steam will burn your hands or wrists if they are above the door. Remove hot items with the aid of gloves, pour out any melted contaminated agar immediately and rinse glassware in hot water.

 

Open drain valve to empty water in bottom of autoclave (make sure drain is in proper position).

**Use the following procedure for autoclaving the listed class of materials
liquids in bottles: 3/4ths filled, caps on slightly loosened.
clean petri dishes: top side up, in can if desired.
apparatus: wrap in brown paper, fold over edges, staple to hold.
contaminated petri dishes: agar side down, in pan if plastic.

Do not load tall bottles near the front of the autoclave, the door hits them.