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LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/2.03 Appendix 2


PREPARATION OF PRE-REDUCED MEDIA FOR GROWING ANAEROBES
Example

Reproduced from Holdeman, L.V., E.P. Cato, and W.E.C. Moore. 1977.
Anaerobe Laboratory Manual, 4th ed.. Virginia Polytechnic Institute and State University, Blacksburg.

When preparing pre-reduced media, one wants to:

1) drive off oxygen and partially reduce the ingredients by boiling,

2) then further reduce with cysteine, and

3) keep the medium oxygen-free thereafter by flushing with oxygen-free gas and storing in tightly-stoppered tubes (or bottles) containing oxygen-free gas.

The media are boiled until the resazurin Eh indicator changes from blue to pink (or for 10 minutes for dilution blanks). After boiling, media are cooled. During cooling oxygen-free CO2 ìs bubbled through the medium to introduce CO2 into the medium and to keep out air.

Reducing agent is then added to lower the Eh of the medium still further. The reducing agent (cysteine hydrochloride) is added after the partial reduction by boiling so that there will be minimum oxidation of the reducing agent. Oxidized reducing agent can be toxic for some fastidious anaerobes.

The pH of the autoclaved medium should be about 7.0 ( 0.2 unit acceptable for most purposes). Media are only lightly buffered so that acid production from carbohydrates can be detected by changes in pH. Since the carbohydrates are autoclaved in the media, it is desirable to have the pH near neutrality during autoclaving to prevent possible denaturation of the carbohydrates during the autoclaving process.

After reducing agent is added to the medium, the pH is adjusted with 8 N NaOH and CO2. The pH often changes duríng autoclaving. To attain final pH of about 7, we adjust pH with NaOH to 0.1 to 0.2 pH units above the desired autoclaving pH, then lower the pH to the value given in column 4 of the table by bubbling C02 through the medium. Once the desired pre- autoclaving pH is attained, switch to oxygen-free nitrogen to prevent further lowering of the pH of the medium.

The temperature at which the medium is tubed may affect the final pH. If all media are tubed at the same temperature, the final pH will be more reproducible. If media are tubed too warm, a vacuum will develop at room temperature and air will be drawn into the tubes when they are opened. If media are tubed cold, pressure will develop at room temperature and tend to loosen stoppers during storage. Therefore, it is most practical to tube at or near room temperature.

Resazurin in medium may turn pink when the NaOH is added because the NaOH solution contains oxygen. If this happens, the resazurin should turn back to colourless upon standing because of the action of the reducing agent in the medium. Media that do not contain reducing sugars (e.g., dilution blanks, PY, etc.) almost always turn pink when the NaOH is added. If desired, this may be prevented by heating the NaOH carefully (it's caustic) before use.

Any tubes that are pink after autoclaving should be discarded.

STEPS IN MEDIA PREPARATION

1. Weigh out dry ingredients. These may be weighed immediately before use or pre- weighed and stored for a few days in stoppered tubes. [Standard ingredients may be measured out in calibrated dippers. These should be re-calibrated for new lots of ingredients.]

2. Place dry ingredients in flask. Add water, salts solution, and resazurin. Use a flask that is only slightly larger than the volume of ingredients in it so that there will not be much air space between the top of the fluid and the top of the flask (e.g., 500 ml in 750 ml flask or 750 ml in 1 litre flask).

3. Fit a removable chimney on the flask to prevent media from boiling over. A satisfactory chimney can be made from a 16 oz polypropylene bottle, pyrex glass joint (standard taper 24/40, inner part), and stopper to fit flask. Punch hole (ca 1/2 inch diameter) in bottom of plastic bottle. Tape mouth of bottle to one end of glass joint. Insert other end of glass joint into hole drilled in rubber stopper.

4. Add a few glass beads or boiling stones to the flask or use a magnetic stirring hot plate to obtain an even boil.

5. Boil medium in flask with chimney until the resazurin turns from blue to pink or colourless (usually takes 5 to 10 min).

6 . Remove flask from heat and immediately replace chimney with a 2-hole stopper and a cannula (in one hole of the stopper) which delivers a stream of oxygen-free CO2 into the medium. The flow of CO2 should cause gentle bubbling (sufficient to exclude air).

7. Cool the medium to room temperature in an ice bath (all the while bubbling with CO2). When cool remove the flask from the ice bath.

8. Add cysteine.

9. Using 8N NaOH (or 5N HCl), adjust pH to 7.0-7.1 (for most media). Continue bubbling C02 through the medium until pH is lowered 0.1 to 0.2 pH unit (to 6.9 for most media). Media should be pH 7.0 (+ 0.2 unit) after autoclaving. [See table. Adjust pH to about 0.2 unit above that given in column 4, then attain the value given in column 4 by bubbling CO2 through the medium. The values given may have to be adjusted for use in your laboratory. The desired result is to have medium around pH 7 after autoclaving.]

10. Switch to oxygen-free nitrogen after pH has reached the desired value and continue to bubble nitrogen in order to exclude air.

11. Dispense media into tubes that are being flushed with oxygen-free nitrogen. Use of medium pump and 2 cannulas soldered together (1 from medium pump and 1 from oxygen-free nitrogen source) facilitates dispensing medium. Purge pump and tubing of all air bubbles before dispensing into tubes. [ Let new tubing stand with boiled water and cysteine solution in it overnight before use or place tubing assembly in anaerobe jar for ca 18 h before use. Amber gum rubber tubing and glass tubing have the least tendency to oxidize the medium.)

12. Stopper with rubber stoppers (e.g., Fisher 14-130) as cannulas are withdrawn from the tube (or bottle).

13. Place rack of stoppered tubes in press for autoclaving.

14. Autoclave at 121C (15 lbs); use fast exhaust.

15. Cool. Remove from rack, label and store at room temperature protected from sunlight which inactivates resazurin. Media are satisfactory for use until they become oxidized (resazurin in the medium turns pink).

To prepare tubes of pre-reduced agar medium, add agar to the tubes to give the final concentration of agar desired. Add pre-reduced broth medium to the agar in the tubes, and autoclave. After removing from the autoclave, mix by inverting the tubes in the press several times. The agar may be added to the tubes with a calibrated measuring device, which should be checked each time a new bottle of agar is opened to be certain that the material has the same weight: volume ratio as the previous lot. Gelatin, or any other solidifying agent, is also added to each tube before the broth is dispensed.

Use at least 2% agar for roll tubes for streaking (more is alright). For agar medium that is to be used for 'pour tubes', do not use more than 2% agar because the medium will solidify before the tubes are inoculated and spun if cooled to 47 to 50 C before inoculation; higher temperatures may be lethal for some of the organisms present.

People who dispense pre-reduced media should always wear the chain mail glove to avoid serious cuts. If you do not do this or require it of people who make media, be sure that your insurance is paid up and adequate! Breakage is more of a problem in media prep than anywhere else, because if tubes are going to break they will either do it when the stoppers are being inserted for the first time or during autoclaving. We have had no serious breaks from normal manipulations in the rest of the laboratory since we started using the heavy-wall tube.

Note: Certain media may require other gas phases!


Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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Copyright CABRI, 1998

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