Anaerobic cultivation techniques

The appropriate method for the cultivation of anaerobes should be chosen with consideration of the sensitivity of the given organism to oxygen concentration and/or redox value within the media or the surrounding athmosphere.

For the cultivation of bacteria sensitive to even trace amounts of oxygen (e.g. methanogens, sulphate reducing bacteria), the best technique is the use of an anaerobic system, filled with an appropriate gas, and the only contact with the outside world is through a sluice chamber that removes oxygen when materials are introduced into the system. Microbes that are not sensitive to trace amounts of oxygen and are able to survive temporarily high oxygen concentrations (e.g. sampling and sample processing) by forming endospores, can be cultivated in anaerobic jars or in semisolid media containing special reductive agents. However, colonies formed inside “anaerobic” agar deep tubes are difficult to examine and isolate. This problem can be solved by using Marino-plates (better known as Brewer‘s plates), a combination of a Petri-dish and an “anaerobic” agar.

EXERCISE 29: CULTIVATION OF ANAEROBIC BACTERIA IN SODIUM THIOGLYCOLLATE MEDIUM The oldest and most common method for the cultivation of anaerobic bacteria is culturing them deep inreduced semisolid or solid media. The most important property of these nutrient media is the suitably low redox potential.

Different indicators within the culture media serve to test the appropriately low redox potential. The most common indicators are resazurin and methylene blue, which show colour only in an oxidised environment (where resazurin is red and methylene blue is blue). To ensure that oxygen does not get into the culture media either, the inoculated media are sealed during incubation (melted and heat sterilised vaseline, or 1:1 mixture of Vaseline and paraffin is layered on top of the culture media). Semi-solid agar deep tubes are prepared with a small amount of agar, so that these media are appropriate for the cultivation of anaerobes without sealing, since agar itself can reduce convection currents, thus reoxidation; furthermore agar colloid applied in small concentrations is reductive. To lower the redox potential in semisolid media even further, reductive compounds could be used (sodium thioglycollate and cysteine).

Object of study, test organisms:

anaerobic bacteria of soil Materials and equipment:

soil sample

9 mL sterile distilled water in test tubes vortex mixer

sodium thioglycollate containing melted agar medium (see Appendix) pipette, sterile pipette tips

Bunsen burner incubator Practise:

1. Prepare a 10-fold dilution series from the soil sample (see EXERCISE 15) and pipette 1-1 mL from each dilution to sodium thioglycollate containing melted agar medium and label the tubes.

2. Incubate the tubes at 28°C for one week.

3. Check the colonies inside the test tube and count them if possible.

EXERCISE 30: CULTIVATION OF ANAEROBIC BACTERIA USING MARINO PLATES

Marino-plates provide a bigger surface area to examine the colony-forming properties of anaerobic bacteria and also make isolation easier.

Object of study, test organisms:

anaerobic bacteria of soil Materials and equipment:

soil sample

9 mL sterile distilled water in test tubes vortex mixer

melted semisolid “anaerobic” agar (see Appendix as thioglycollate agar) sterile, glass Petri-dishes wrapped into foil

80° C water bath

pipette, sterile pipette tips incubator

Practise:

1. Prepare a 6-member 10-fold dilution series from the soil sample.

2. Put the test tubes containing the dilution series into an 80°C water bath for 10 minutes to select for endospore-forming organisms.

3. Unwrap the glass Petri-dishes under a laminar flow box. Pipette 100 µl from a given dilution into the inner surface of the Petri-dish top. Then pour culture media (cooled down to approximately 45°C) into the Petri-dish and thoroughly mix the diluted sample with the nutrient medium. Put the bottom of the Petri-dish (which has a smaller diameter than the top) into the top before the medium solidifies; thus create a thin culture medium between the two parts of the Petri-dish.

4. Wrap the Marino-plates in foil again and incubate for at least 1-2 days up to one week depending on the results.

5. Examine the plates if bacterial growth can be detected. Observe the individual colonies under a stereo microscope and try to isolate some of the colonies by making stab cultures in agar deep tubes (thrusting an inoculating loop with bacteria deep down into the centre of the agar).

EXERCISE 31: CULTIVATION OF ANAEROBIC BACTERIA IN AN ANAEROBIC JAR

The anaerobic jar is usually transparent and can be sealed; contains a palladium catalyst, a disposable H₂+CO₂ generator and a redox indicator. Cultures are placed into the jar along with an envelope that includes two tablets.

One of the tablets contains NaBH₄that generates hydrogen when it reacts with water; the other tablet contains citric acid and sodium-hydrogen-carbonate that generates CO₂when comes into contact with water. The CO₂ contributes to the growth of fastidious anaerobes. The jar is sealed after water is added to the envelope. In the presence of the palladium catalyst, hydrogen reacts with oxygen to form water. This reaction removes free oxygen from the inner atmosphere of the jar (Fig. 22). The colour change of the indicator refers to the formation of an anaerobic atmosphere.

Fig. 22. The anaerobic jar.(1) clamp (2) cover (3) palladium catalyst (4) H₂+CO₂generator (5) redox indicator (6) culture plates.

Object of study:

Clostridiumspp. from soil Materials and equipment:

soil or sediment sample

9 mL sterile distilled water in test tubes vortex mixer

anaerobic jar redox indicator strip gas generator envelope palladium catalyst scissors

pipette, sterile pipette tips

glass spreader (alcohol for sterilisation)

bismuth sulphite agar medium (see Appendix as Wilson-Blair type agar) Bunsen burner

incubator Practise:

1. Prepare a 6-member 10-fold dilution series from soil sample.

2. Spread each member of the dilution series onto Wilson-Blair type agar medium.

3. Put the inoculated Petri-dishes with their surface down into the anaerobic jar (Fig. 22). Open the redox indicator and place it next to the plates so that the indicator strip can be seen from the outside (the indicator strip will turn blue within seconds).

4. Take care that palladium catalyst has been regenerated (by incineration) or replace with a fresh one.

5. Cut the gas generator envelope with scissors, and add 8-10 mL water with a pipette (according to the manufac-turers’ instructions). Close the jar and screw on the clamp, then put the jar into an incubator at 28°C.

6. After one week of incubation, determine the CFU number for the original sample (see EXERCISE 15).

EXERCISE 32: DEMONSTRATION OF THE ANAEROBIC CHAMBER (GLOVE-BOX)

The anaerobic chamber is a device suitable for the cultivation of strictly anaerobic bacteria (Fig.23). Inside the system, anoxic conditions can be maintained, while microbiological operations (microscopy, isolation, inoculation, etc.) can be performed. At first step, oxygen is removed with vacuum, then a gas mixture (10% H₂, 10% CO₂, 80%

N₂) is introduced into the system with slightly positive pressure. The detection of trace amounts of oxygen is per-formed by methylene blue or resazurin redox indicators, and the elimination of such oxygen is completed by a palladium catalyst. Active carbon inside the anaerobic chamber serves to bind catalyst poisons (e.g. H₂S) and other substances that are toxic for bacterial cells.

Fig. 23. The anaerobic chamber (glove box).Samples are transferred through the interchange (1) into the working chamber (2), where they can be handled from outside through gloves (3).