APT AGAR
For the cultivation and enumeration of heterofermentative Lactobacilli.
Code: CB1/8852
Typical formula g/l
| Tryptone | 12.50 |
| Yeast extract | 7.60 |
| Glucose | 10.00 |
| Sodium citrate | 5.00 |
| Sodium chloride | 5.00 |
| Dipotassium phosphate | 6.00 |
| Manganous chloride | 0.14 |
| Magnesium sulphate | 0.80 |
| Ferrous sulphate | 0.04 |
| Sorbitan mono. | 0.20 |
| Agar | 15.00 |
| Thiamine HCl |
0.10 mg |
pH 6.7 +/- 0.2
Directions
Suspend 46.2 g of APT Broth or 61.29g of APT Agar in 1000 ml of cold distilled water; heat to boiling, distribute and sterilise by autoclaving at 121°C for 15 minutes. Do not overheat.
Description
Evans and Niven studied APT Agar and APT Broth for the cultivation of heterofermentative lactobacilli that produce greening of cured meat products. These formulae are also used for the conservation and preparation of the Lactobacillus viridescens ATCC 12706 strain inoculums, which is the test organism in the microbiological assay of thiamine according to the method described by Deibel et al. A.P.H.A. recommends the use of APT Agar for the detection of lactobacilli in foodstuffs.
Method
The technique suggested is the standard plate count; the details change according to the material to be tested.
|
Materials to be tested |
Diluent |
Incubation |
|
Sauerkraut |
Distilled water |
320C for 3 days |
|
Fruit juices |
Distilled water |
320C for 6 days |
|
Salted/canned meat |
Phosphate buffer |
210C for 6 days |
For the detection of H2O2 producing strains, APT Agar may be prepared with MnO2 suspend 20g of MnO2 in 200 ml of APT Broth, distribute 10ml in tubes and sterilise by autoclaving at 121°C for 15 minutes. To 100ml of APT Agar add 10ml of MnO2 suspension. Prepare the plates with a 15ml base layer of APT Agar without MnO2 leave the medium to solidify then add 15 ml of a surface layer of APT Agar with MnO2. Inoculate the sample and the sample dilutions onto the surface of the medium. H2O2 producing lactobacilli grow with colonies surrounded by a transparent halo.
As these media are non-selective and permit the growth of contaminants, the presumptive diagnosis of the presence of lactobacilli should be confirmed by microscopic and biochemical examinations.
APHA moreover, recommends an artificial pollution test to confirm the diagnosis of bacterial greening of canned meats. Transfer a few colonies from the APT Agar plates to APT Broth tubes and incubate at 32°C for 24 hrs. Prepare a moist sterile chamber (Petri dish with filter paper imbued with sterile water) and put a slice of the test material in this chamber under aseptic conditions. Inoculate the surface with a loopful of Broth culture in APT Broth; incubate at 32°C for 24 hours and observe whether the meat has greened. If it occurs and if an uninoculated control specimen is found to be unchanged, the diagnosis is confirmed. The presence of greening due to exceeding nitrites is to be distinguished from the bacterial greening by carrying out identification tests and assays of nitrites with the standard reagents.
User quality assurance (APT Agar + MnO2 : 30°C-5 days)
Productivity control
L.viridescens ATCC 12706: growth, colonies with transparent halo
L.brevis ATCC 14869: growth, colonies with transparent halo
L.sakie ATCC 215521: growth, colonies without transparent halo
L.mesenteroides DSM 20241: growth, colonies without transparent halo
P.damnosus ATCC 29358: growth, colonies without transparent halo
Storage
Dehydrated media: 15-30°C
User prepared plates and tubes: up to 7 days at 2-8°C
References
• APHA (1966) - Recommended Methods for the Microbiological Examination of Foods. 2nd. edition.
• D’Aubert S. (1963) Ann. Microbiol., 8, 189
• Deibel, RH., Evans, J.B. & Niven, C.P. Jr. (1957) - J. Bact., 74, 818-821.
• Niven, C.F. Jr. & Evans, J.B. (1957) - J. Bact., 73, 758-759.
ciation, Leatherhead, U.K.