3.1. Cultivation

As P. larvae is a spore-forming bacterium, its isolation from biological samples is typically preceded by a heat treatment step to kill all vegetative microorganisms. This step significantly reduces the risk that P. larvae colonies will be masked by competitors. Different genotypes of P. larvae show variation in germination ability, and their response to heat treatment is variable (Forsgren et al., 2008). MYPGP agar (Dingman and Stahly, 1983) is routinely used to cultivate P. larvae for AFB diagnosis. This medium makes incubation under CO2 unnecessary although the presence of 5 % CO2 significantly increases germination (Nordstrom and Fries, 1995). Contaminants of the genera Bacillus and Brevibacillus, as well as other Paenibacillus species, are inhibited by nalidixic acid (Hornitzky and Clark, 1991) and pipemidic acid (Alippi, 1991; 1995). Apart from brood samples, food stores (honey, pollen and royal jelly), adult workers, and wax debris can also be used to detect the presence of P. larvae spores.

         The outline of the cultivation procedure starting from brood samples is as follows:

1. Prepare an aqueous solution containing P. larvae spores by taking twice samples with a sterile swab from a brood comb (each time multiple brood cells should be sampled), and subsequently suspending them in 5 ml of phosphate buffered saline (PBS).

2. Incubate different aliquots of the spore suspension at 80, 85, 90, 95 and 100°C  for 10 min (Forsgren et al., 2008).

         P. larvae occurs in two forms: vegetative cells and spores. Only spores are infectious to honey bees. While P. larvae sporulates and grows efficiently in the haemolymph of bee larvae, most strains grow poorly in artificial media. Different culture media have been developed for P. larvae cultivation. MYPGP agar (Dingman and Stahly, 1983) yielded the highest percentage of spore recovery, while J-agar (Hornitzky and Nicholls, 1993), brain heart infusion agar (BHI) (Gochnauer, 1973), Columbia sheep blood agar (CSA) (Hornitzky and Karlovskis, 1989) proved to be less efficient in this respect (Nordström and Fries, 1995). Other media used for the cultivation of P. larvae are PLA agar (Schuch et al., 2001) and T-HCl-YGP agar (Steinkraus and Morse, 1996). PLA medium shows superior plating efficacy and also the advantage of inhibiting the majority of micro-organisms normally present in the hive and in bee products. T-HCl-YGP agar is the medium of choice for cultivation P. larvae starting from honey (Steinkraus and Morse, 1996). When starting from diseased larvae, nalidixic acid is necessary to avoid growth of P. alvei. When starting from other sources (e.g. honey), pipemidic acid prevents contamination with other spore-forming bacteria.

 

       MYPGP agar (per litre):

  • 10 g Mueller-Hinton broth (Oxoid CM0405)
  • 15 g yeast extract
  • 3 g K2HPO4
  • 1 g Na-pyruvate
  • 20 g agar
  •  Autoclave at 121°C/15 min.
  •  Add 20 ml 10 % glucose (autoclaved separately).

 

       BHI agar:

  • Suspend 47 g brain heart infusion agar (Oxoid CM1136) in 1 litre of distilled water.
  • Autoclave at 121°C for 15 min.
  • Add 1 mg thiamine hydrochloride per litre.

 

       CSA-agar:

  • Dissolve 39 g Columbia blood agar base (Oxoid CM0331) in 1 litre distilled water.
  • Autoclave at 121°C/15 min.
  • Supplement with 50 ml sterile defibrinated blood (at 50°C).

 

       T-HCl-YGP (per litre):

  • 15 g yeast extract
  • 1 g pyruvic acid
  • 200 ml 0.1 M Tris-HCl, pH 7.0
  • 20 g agar
  • Autoclave at 121°C/15 min.
  • Add 40 ml 10 % glucose (autoclaved separately).

 

       J agar (per litre):

  • 5 g tryptone
  • 3 g K2HPO4
  • 15 g yeast extract
  • 20 g agar
  • Adjust pH to 7.3 to 7.5.
  • Autoclave at 121°C/15 min.
  • Add 20 ml 10 % glucose (autoclaved separately).

 

         PLA medium consists of three different media supplemented with egg yolk. Equal quantities (100 ml) of sterile, molten Bacillus cereus selective agar base (Oxoid CM617), trypticase soy agar (Merck 5458) and supplemented nutrient agar (SNA) are combined and mixed. SNA is composed of (per litre):

  • 23 g nutrient agar
  • 6 g yeast extract
  • 3 g meat extract
  • 10 g NaCl
  •  2 g Na2HPO4
  •  Adjust pH to 7.4 ± 0.2.

         All solid media are sterilized at 121 °C for 15 min. After the three molten media are combined 30 ml of 50% egg-yolk suspension is added to form the PLA medium.

         Cool the media to 50°C and add the antibiotics to a final concentration of 20 µg/ml for nalidixic acid and 10 µg/ml for pipemidic acid.

  • Nalidixic acid stock solution (1 mg/ml) is prepared by dissolving 0.1 g in 2 ml of 1 M NaOH and diluting to 100 ml with 0.01 M phosphate buffer (pH 7.2).
  • Pipemidic acid stock solution (2 mg/ml) is prepared by dissolving 0.2 g in 2 ml of 1 M NaOH and then diluting to 100 ml with 0.01 M phosphate buffer (pH 7.2).
  • Both antibiotic solutions are filter sterilized.

         The medium is poured (20 ml) into sterile Petri dishes and plates are dried before use (15 min).

         Plates inoculated with 150 µl heat-shocked spore suspension are incubated at 35°C up to 6 days in either aerobic conditions or under an atmosphere of 5–10 % CO2. Vegetative bacteria are grown overnight at 35°C without heat-shock treatment.

         The outline of the procedure starting from honey samples is as follows:

1. Dilute 20 g of honey in 20 ml PBS.

2. Shake vigorously.

3. Centrifuge the suspension 40 min at 6,000 x g to harvest the spores.

4. Resuspend the pellet in 1 ml of PBS.

5. Heat treat and plate this spore containing aqueous solution as described above.