216 Inzemational Jtnernal of Food Microbiology. 17 (1993) 216-219 g'~ 1993 Elsevier Science Publishers B.V. All rights re . f red 0168-16{)5/93/$06.00

FOOD 05MG

Dominguez Rodriguez LSAMm agar

Description and history

LSAMm listeria seleetivc agar was developed by Blaoco ct al. (1989) and Domingucz et al. (1990) for the detection of/3-haemolysis of Listeria spp. by a blood overlay procedure. This medium is a modification of the selective plating medium proposed by Dominguez et al. (1984) incorporating more effective ~lective agents proposed by other authors. LSAMm has proved to b c a highly selective agar inhibiting the natural microflora of many different samples (raw milk, cheese, meat, faeces, silage), Direct plating of silage samples n,'tturally contaminated with Listeria monocytogenes on Oxford, MOX and LPM agars showed only a 4A-4.8 log reduction of non-listeria bacteria from the silage, while LSAMm and PALCAM agars yielded a 6,8 log reduction of the non-listcria background bacteria (Fernandez- Garayzabal el al., 1992a; Vazquez-Boland et al., 1992). LSAMm promotes the growth of listeria p re~nt in the samples with minimal inhibition and simultaneously provides enough information to allow an easy and reliable identification of the listcria colonies, which facilitates the isolation and counting of lisleria directly on this selective agar (Dominguez et al., 1990). The use of potassium tellurite and brain heart infusion agar in LSAMm greatly enhances the /3-hacmolysis of Listeria monocytogenes due to listcriolysin O (Fernandez- Garayzabal c t a l . , 1992b). LSAMm agar should therefore b c a useful medium for the detection and enumeration of Listeria spp. in highly contaminated food and fccdstuffs.

Composition (grams)

Brain heart infusion agar 52.0 (follow specific BHI medium instructions) Ae~ulin 0.75 Iron ( l i d ammonium citrate * 0.5 Lithium chloride 15.0 Potassium tcllurite * 0.04 Acriflavine HCI 0,005 Polymath B sulphate 0.01 Ceftazidime pcntahydrate * 0.02 Distilled or deionised water 1000.0

Preparation

Suspend the ingredients, except those marked * in 900 ml of distiUcd water in a bottle or flask containing a magnetic stirring bar and autoclave the medium for 12 rain at 121°C. Iron

217

(111) ammonium citrate is sterilized (121°C/12 rain) ~parately in 85 ml of distilled water. Cool the basal medium in a water bath to 46°C, then add the heat sterilized iron (Ill) ammonium citrate and the following filter sterilized solutions: l0 ml of 0.2% .,aglium ecftazidime and 5 ml of 0.8% potassium tel!urite. Dispense L5 ml per 9 cm Pctri dish.

Physical properties

Appearance

pH

Shelf life

Ready to use medium Component parts

Light yellow or tan. Dark colnur indicates overheating and decomposition of either ac~ulin or telluritc. 7.4 + 0.2.

3 weeks at 4 + 2°C. Ccftazidim¢ ~lution can bc stored at -20oc for ~vcral months, potassium tellurite solution at 4 _+ 2°C for one month.

lnm'ulalion meth+alfi~rsnmples

I. From c,~';zhmcnt broths, streak a ka)pful of the cnricl mcnt broth on LSAMm agar to obtain isolated colonies on the plates.

2. For direct plating and counting of _> I(X) cfu/g of Listeria spp. growing on solid samples such as silage und many kinds of fixgls at the retail level, make homogcnatc suspensions of the food or silage samples and then spread plate {}.1 ml of each dilution on LSAMm agar. In liquid samples the dcr.+~clion limit is _> 10 cfu/ml of Listeria spp.

hwnbation meth¢~l

At 37°C for 48 h in air.

Reading of results and inlt, rpn.tatitm

After 48 h incubation Listeria spp. typically form 1.5 mm grey-green colonies with a dark brown centre surrounded by a black halo. Catalase, Iclluritc and ac~ulin positive colonies on LSAMm are in most cases Listeria spp.

Qlealily a,T, sx'ssm~,nt

(i) Pnaluctirity Test strains Listeria monocy:ogenes scrervar I /2a (ATCC 19111/

NCTC 7973). Listeria monocytogenes serovar 4b (ATCC 13932/CEC'1" 935/NCTC 10527). Listeria iranorii scrovar 5 (ATCC 19119/CECT 913/NCTC 11846/SLCC 2379).

218

Supplementary strains

Inoculation method

(ii) Selecticity Test strains

Listeria innocua serovar 6a (ATCC 33090/CECT 910/NCTC 11288/SLCC 3379). l_isteria monocytogenes serovar 4a (ATCC 19114/CECT 934/NCTC 5214). Listeriu seeligeri serovar I /2b (ATCC 35967/CECT 917/CIP 100 100/NCTC I I856/SLCC 3954). Listeria welshimeri (CIP 1857).

Modified Miles-Misra or streaking.

Enterococcas faecalis (CECT 4176/NCIMB 8260/NCTC 8213). Proteus mirabilis (ATCC 29906/CECT 4168/NCTC 11938).

Supplementary strains Bacillus cereus (ATCC 11778/CCM 869/CECT 193/ DSM 345/NClMB 8012/NCTC 10320). Brochothrix thermosphacta (ATCC 11509/CECT 847/ DSM 20171/NCIMB 10018/NCTC 10822). Carnobacterium piscicola (ATCC 35586 /CECT 4020/NCDO 2762/NCIMB 2264).

Inoculation method Modified Miles-Misra or streaking.

(iii) Characwristic appearance of colonies Grey-green colonies 1.5 mm diameter with a central dark brown area, sur- rounded by a black aesculin Ix)sitivc zone.

Determinatian of haemolytic activity by an orerlay wchnique

This technique is based on the addition of a sheep blood (SRBC) top-layer to the selective plating medium after growth of listcria.

Compositwn of the top layer (grams)

BHI broth 37.0 Agar 7.0 Sodium chloride 8.0 Sheep blood red cells suspension (ml) 50.0 Distilled or deionised water 1000.0

Preparation of the SRBC suspension

25 ml of twice-washed SRBC are mixed with 25 ml saline solution (0.8% NaCI). This suspension is stored at 4°C and used within one week.

Preparation of the top layer

All ingredients, except the SRBC suspension are sterilized (121°(? 15 rain) and stored at 4°C. Just before use, base top layer is boiled, cooled to 45°C and mixed with the SRBC suspension.

219

Reading of results and interpretation

After incubation (37°C/48 h) and recognition of listeria colonies by their morphological characteristics on LSAMm, plates arc cl~llcd (4°C/2h) and subsequently covered gently by an 8 ml SRBC top layer prepared as above. After the add(lion of the top layer, plates arc incubated again (overnight at 30°C) for hacmolysis ,screening.

On LSAMm all colonies morphologically re, tabling lisleria and positive for telluritc, acsculin and catalase (tested by removing a piece of colony prior to the top layer addition), and displaying typical hacmolysis correspond in most cases to Listeria moe~ocytogenes.

Comments

Thc overlay tcchniquc allows the discrimination and specific enumeration by their haemoly- sis of presumptive hacmolylic/pathogcnic listeria colonies, avoiding the possibility of overhx~king the presence of small numbers of pathogenic listcria eolonics on agars with a hcavy non-pathogenic lisleria growth, when only a few co!on(us arc subcultured for further biochcmical identification.

This overlay technique, when u~d with LSAMm has proved to bc an effective procedure for the measurement of Listeria rnonoeytogenes in f(xxis.

Recently published results (Fcrnandcz-Garayzabal et al., 1992b) suggest that the perfor- mance of Ihe SRBC top layer could bc improved by decreasing the concentration of potassium tellurite in LSAMm to 40 rag/I, as the haemolysis of Listeria mmzocytogenes is slightly higher at this concentration and the sclcctivity of LSAMm is not affcctcd.

Selective properties of acriflavinc may vary from lot to lot and manufacturer [o manufac- turer. Bach new batch must be assayed in combination with other selective agents to bc used in the medium to dclcrminu the optimum concentration for use, with regard to the efficiency of selectivity and abscncc of inhibition of Listeria spp, Storage of st(vck ,solutions of acriflavine for periods of more than one month is not rccommcnded.

References

Blanco, M., Fernandcz-Garayzahal, J.F,, Dorningnez, L.. Briones, V,, Vazquez-Boland. J.A., Blanco, J.L., Gareia, J.A. and Suarez, G. (1989) A technique for the direct id~,nlificalion of hacrnolylic-path- ogenic Listeria on ~lcctive platin 8 media, I..¢t[. Appl. Micmbiol. 9, 1~';-128.

Dorninguez Rodriguez, L., Snarez Fernandez, G., Fernandez-Garayzabal, J.F. and Rodrlguez Ferri, E. (1984) New methodology for the isolation of Listeri, rnicrlmrganisms from heavily contaminated environments. Appl. Environ, Microbiol. 47, 1188- 1190.

Dorninguez, L.. Fernandez-Garayzabal, J.F., Blanco, M., Briones, V., Vazqaez-EIoland, J.A., Blanco, J.L. and Suarez, G. (19~O) Overlay teehniqu¢ for direct detection and idcnlifiealion of haemolytl¢ Lisreria on selective plating medium: comparison of five media. Z. Lebensrn. Unters. Forsch. 191, 10-19.

Fernandez-Garayzabal, J.F., Blancx~, M. Vazqucz-Boland, J.A.. Briones. V., Gareia, J.A., Delgado, C., l>amingo. M., Marco, J. and l~)rningucz, L. (1992a) A direct plating rnelhod for monitoring the contamination of Listeria nr~mocytogenres in silage (Unpublished results).

Fernand~z-Garayzabal. J.F.. Delgad~). C., Blanco, M., Vazquez-Yoland, J.A., Briones, V., Suarez, G, and Dorninguez, L. (1992b) Role of polassium tellurite and brain heart infusion in expression of the haernolytic phenotyPe of Listeria spp. on agar plalcs. Appl. Environ. Mierobiol. 58, 434-438.

Vazquez-Boland, J.A., Dorninguez, L., Blanco, M., R~ourt, J., Fernandez-Garayzanal, J,F., Gutierrez: C.B. and Rodrigacz Fcrri, E. (1992) Epidemiolngical invesligalion of a silage-associalcd urine listeric cneephalilis using u new Listeria sclcci.ivc enumeration medium (LSAMrn) and phage typing Am. J. Vet. Rcs. (In press).