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Horse blood (HL) agar
International Journal of Food Microbiology, 9 (1989) 97-98
97
Elsevier FOOD 00M04
Horse blood (HL) agar
Description and history HL agar utilizes a very thin layer of horse blood to detect the very weak fl-haemolytic activity of Listeria spp. It is a totally non-selective agar and thus is useful for restreaking and purification of 'listeria' colonies picked from selective media such as LPM (q.v.), which are invariably contaminated by other bacteria. The fl-haemolysis produced by Listeria spp. on the agar differs from that of other fl-haemolytic organisms and it thus provides a differential medium for the presumptive identification of the pathogenic Listeria spp.
Composition of Columbia blood agar base (grams) Pancreatic digest of casein Peptone (peptic digest of animal tissue) Yeast extract Beef extract Corn starch Sodium chloride Agar Distilled or deionised water
12.0 5.0 3.0 3.0 1.0 5.0 15.0 1 000.0
Note: Other forrnulae for Columbia base are equally satisfactory.
Preparation Autoclave the Columbia blood agar base for 15 rain at 121°C and cool to 46 ° C. Dispense 10 ml of the Columbia agar base to each 9 cm Petri dish. After the base layer has just solidified but whilst it is still warm, overlay with 5 ml of Columbia agar containing 5% defibrinated horse blood. Tilt gently to spread out the thin blood layer evenly.
Physical properties Appearance pH
Bright red. 7.3 ± 0.2. Discard any contaminated or old discoloured plates.
Shelf life
2-3 weeks in sealed plastic bags at 4 ± 2 ° C.
0168-1605/89/$03.50 © 1989 Elsevier Science Publishers B.V.
98
Inoculation method for samples Pick single or multiple (4-5) presumptive listeria colonies from selective agar using a pure platinum needle and streak for isolated colonies on a single H L plate.
Incubation method At 35 ° C overnight (16-20 h) in air with 5% CO z (optional).
Reading results and interpretation The fl-haemolytic Listeria ivanovii, Listeria monocytogenes and Listeria seeligeri form 2 - 3 m m diameter glass clear colonies with a narrow fl-haemolytic zone visible underneath when the H L plate is viewed by holding it at 90 ° close to a fluorescent table lamp (McClain and Lee, 1988). Listeria colonies also show a blue tint when the H L plate is tilted at a 45 ° angle towards the lamp. This appearance is specific for the fl-haemolytic listeriae. The above colony characteristics cannot be seen if the H L plate is examined with colony counter illumination.
Quality assessment Streak a known culture of Listeria monocytogenes (e.g. A T C C 1 3 9 3 2 / N C T C 10527) on H E agar with each use and discard all the H E plates that fail to give the above colony characteristics after overnight incubation. H L has no selective value.
Reference McClain, D. and Lee, W.H. (1988) Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71,660-664.
97
Elsevier FOOD 00M04
Horse blood (HL) agar
Description and history HL agar utilizes a very thin layer of horse blood to detect the very weak fl-haemolytic activity of Listeria spp. It is a totally non-selective agar and thus is useful for restreaking and purification of 'listeria' colonies picked from selective media such as LPM (q.v.), which are invariably contaminated by other bacteria. The fl-haemolysis produced by Listeria spp. on the agar differs from that of other fl-haemolytic organisms and it thus provides a differential medium for the presumptive identification of the pathogenic Listeria spp.
Composition of Columbia blood agar base (grams) Pancreatic digest of casein Peptone (peptic digest of animal tissue) Yeast extract Beef extract Corn starch Sodium chloride Agar Distilled or deionised water
12.0 5.0 3.0 3.0 1.0 5.0 15.0 1 000.0
Note: Other forrnulae for Columbia base are equally satisfactory.
Preparation Autoclave the Columbia blood agar base for 15 rain at 121°C and cool to 46 ° C. Dispense 10 ml of the Columbia agar base to each 9 cm Petri dish. After the base layer has just solidified but whilst it is still warm, overlay with 5 ml of Columbia agar containing 5% defibrinated horse blood. Tilt gently to spread out the thin blood layer evenly.
Physical properties Appearance pH
Bright red. 7.3 ± 0.2. Discard any contaminated or old discoloured plates.
Shelf life
2-3 weeks in sealed plastic bags at 4 ± 2 ° C.
0168-1605/89/$03.50 © 1989 Elsevier Science Publishers B.V.
98
Inoculation method for samples Pick single or multiple (4-5) presumptive listeria colonies from selective agar using a pure platinum needle and streak for isolated colonies on a single H L plate.
Incubation method At 35 ° C overnight (16-20 h) in air with 5% CO z (optional).
Reading results and interpretation The fl-haemolytic Listeria ivanovii, Listeria monocytogenes and Listeria seeligeri form 2 - 3 m m diameter glass clear colonies with a narrow fl-haemolytic zone visible underneath when the H L plate is viewed by holding it at 90 ° close to a fluorescent table lamp (McClain and Lee, 1988). Listeria colonies also show a blue tint when the H L plate is tilted at a 45 ° angle towards the lamp. This appearance is specific for the fl-haemolytic listeriae. The above colony characteristics cannot be seen if the H L plate is examined with colony counter illumination.
Quality assessment Streak a known culture of Listeria monocytogenes (e.g. A T C C 1 3 9 3 2 / N C T C 10527) on H E agar with each use and discard all the H E plates that fail to give the above colony characteristics after overnight incubation. H L has no selective value.
Reference McClain, D. and Lee, W.H. (1988) Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71,660-664.