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OXOID SALMONELLA LATEX TEST

Code: FT0203

Introduction
The Oxoid Salmonella Latex Test is an agglutination test for the presumptive identification of Salmonella spp. isolated by the Oxoid Rapid Salmonella Test (FT0201).
Additional investigations have shown it can be used to screen presumptive Salmonella colonies isolated on selective agar plates, from both food and clinical samples. The test allows the user to presumptively identify and confirm the presence of Salmonella spp. At the same time, other species such Citrobacter spp. and Proteus spp. can be more rapidly eliminated.

Principle
A wide range of Salmonella flagella antigens have been used to produce polyvalent antisera in rabbits. Salmonella antibodies present in the antisera include :¹
a, b, c, d, eh, Fg, gp, gms, gq, gst, i, k, lv, lw, mt, r, y, z, enz₁₅, enx, 1.2, 1.5, 1.6, 1.7, z₄, z₆, z₁₀, z₂₃, z₂₄, z₂₉, z₃₈ and gpu.
The purified antibodies are used to sensitise the latex particles.
A loopful of material is taken from the top of a positive indicator tube in the Salmonella Rapid Test and mixed with a drop of the latex test reagent. If agglutination of the latex test reagent occurs, then Salmonella is present in the material. Similarly, presumptive Salmonella colonies can be taken from an agar plate, mixed with the latex test reagent and agglutination will occur if Salmonella is present.

Components of the Kit
Test Latex (FT0204)   latex particles sensitised with polyvalent Salmonella antibodies (rabbit IgG). Sufficient for30 tests.
Control Latex (FT0205)   latex particles sensitised with normal rabbit serum globulins. Sufficient for 30 tests.
Positive Control Suspension (FT0206)   a suspension of non-viable Salmonella preserved with 1% formalin (0.5 ml).
NB: The suspension has been checked by a standard procedure to ensure that no viable organisms are present.
Test Cards (DR500G) Ten test cards are provided. Each card   has 6 test circles. If only two test circles are to be used, then the card may be cut and the unused portion saved for later use.
Instruction Leaflet

Material Required but not Supplied
Timer
Microbiological Loop
Disinfectant - sodium hypochlorite solution (> 1.3% w/w) or a suitable proprietary disinfectant used at the manufacturers recommended concentration.

Sample Collection and Preparation
The Salmonella Latex Test Kit can be used in conjunction with the Oxoid Salmonella Rapid Test (FT0201). However additional investigations have shown it can also be used as a rapid screen for presumptive positive colonies.
When using the Oxoid Salmonella Rapid Test (FT0201) take a sample of material from the upper indicator media in the tubes of a presumptive positive result, using a microbiological loop. If one tube is indicator positive, perform the latex test on that one. However, if both tubes give a positive indicator reaction, then test tube A first. Only use tube B for testing if tube A is latex negative.
When using the latex test with suspect colonies grown on selective culture media, it is recommended that any suspect colonies are first streaked on to a fresh non-selective plate in order to obtain a pure culture. Sufficient material can then be taken to perform the test (ie a single isolated colony for both the test latex and control latex.).

Procedure
1. Bring the latex reagents to room temperature. Mix the latex suspensions by vigorous shaking. Expel any excess latex from the dropper pipettes to ensure complete mixing.
2. Dispense one free-falling drop of the test latex onto one of the test circles on the reaction card.
3. Dispense one free-falling drop of the control latex onto an adjacent test circle on the test card.
4. (a) From Oxoid Salmonella Rapid Test
Mix a loopful of material from the upper indicator media of the presumptive positive Salmonella Rapid Test tube, and mix with the test latex drop. Continue mixing with the loop for 10-15 seconds, while spreading the drop to cover most of the reaction area. Mix a loopful of material from the upper indicator media of the presumptive positive Salmonella Rapid Test tube, and mix with the test latex drop. Continue mixing with the loop for 10-15 seconds, while spreading the drop to cover most of the reaction area.
Flame and cool the loop (or use a new sterile plastic loop) and mix a second loopful of material from the same tube with the control latex drop. Continue mixing with the loop for 10-15 seconds, while spreading the drop to cover most of the reaction area.
(b) Colonies Taken From a Selective Agar Plate
Take a suspect colony from the selective agar plate and streak on to a fresh non-selective plate (eg blood agar) and incubate to obtain a pure culture. Mix one colony in a test circle with a drop of the test latex. Using a fresh loop, mix a second colony in a second test circle with a drop of the control latex. Continue mixing for 10 –15 seconds.
5. Gently rock the cards in a circular motion for up to 2 minutes and observe for agglutination. (Do not rock the card for longer than 2 minutes and do not use a magnifying glass to aid reading)
6. Return the latex kit to the refrigerator after use.

Reading and Interpretation
Positive A result is positive if agglutination of the test latex occurs within 2 minutes, and no agglutination of the control latex occurs within 2 minutes.
Negative A result is negative if no agglutination of either the test latex or the control latex occurs within 2 minutes. Reactions occurring after 2 minutes should be ignored.
NB: In some cases, agglutination of both the test latex and control latex may be observed. Such results should be regarded as un-interpretable, and in these cases the culture should be restreaked and checked using biochemical and serological procedures.

Further Identification
A positive agglutination reaction should be regarded as presumptively positive for Salmonella spp. Identification should be confirmed using further standard cultural, biochemical and serological techniques.

Positive Control Suspension
The positive control suspension should be used periodically to check the correct working of the latex reagents. Use a loopful of the non-viable positive control suspension as the test sample. The test latex must give a positive reaction within 30 seconds. The control latex must not show any signs of agglutination during the 2 minutes of rocking.

Limitations
The Salmonella Latex Test can be used as an effective screen for distinguishing between Salmonella spp. and other organisms. However, it should be noted that when it is used in conjunction with the Oxoid Salmonella Rapid Test (FT201A), the method is not appropriate for the detection of non-motile strains of Salmonella spp . (incidence < 0.1%) ².
The Salmonella Latex Test can be used as a rapid confirmation screen for presumptive Salmonella spp. from selective agar plates, provided further confirmation and identification work is carried out on those organisms giving a positive latex reaction.
Testing can be done directly from the selective plate but a negative result should also be confirmed, following streaking on to a non-selective plate (eg blood agar) and confirmed again using the latex as directed.
Certain serotypes may only give a weak reaction. In this case, a sweep of colonies from a pure culture should be tested with the latex.
Granular or Stringy Reactions
Occasionally, granular or stringy reactions may be seen due to the particulate nature of the test material. When such reactions occur, they should be interpreted using the following criteria:
The result is considered POSITIVE, when a greater clearing of the blue background occurs with the test reagent when compared to the reaction of the control reagent.
The result is considered NEGATIVE, when there are no significant changes in the clearing of the blue background when using both the test and control reagents.

Precautions
This product is for in vitro diagnostic use only. Do not freeze. Always use reagents with the same lot number. Reagents contain 0.1% sodium azide as preservative. Sodium azide may react with lead or copper plumbing to produce metallic azides which are explosive by contact detonation. To prevent accumulation of azides in plumbing, flush any waste disposal with copious amounts of water immediately.
Dispose of all contaminated materials (test cards, plastic loops etc.,) by immersing in disinfectant. All samples contain potentially hazardous organisms and must be handled using appropriate safety procedures.

Storage Conditions
Store all reagents at 2°C to 8°C.

Stability
All reagents will remain stable until the expiry date shown on the bottle labels. Do not use beyond the expiry date.

Performance Characteristics
( a ) Oxoid Rapid Salmonella Test
Laboratory tests have demonstrated Salmonella with the following antigenic determinants can be recovered from the Oxoid Salmonella Rapid Test and detected by the Oxoid Salmonella Latex Test.
Flagella Antigens
Phase 1:   a, b, c, d, i, k, r, y, z, z₁₀, z₂₉, z₃₈, fg, fgs, fgt, gp, gm, gms, gmst, gmt, gpu, gq, gst, gt, mpt(u), mt, lv, lz₃₈, lz₁₃, z₄ z₂₃, z₄ z₂₄, z₄ z₃₂. 
Phase 2:   1.2, 1.5, 1.6, 1.7, z₆, eh, enx, enz₁₅, lw.
Somatic Antigens
Sub-genus 1: A-Z, 51, 52.
Sub-genus 2: B-G, I, L, M, O, R, S, T, V, W, Z, 52.
(b) Colonies from Selective Agar Plates
Additional investigations have shown the Salmonella Latex Test can be used as a rapid screen for isolates from certain selective media. (It is recommended that laboratories validate this method with their own in-house strains.)
The following ten Salmonella serotypes all gave positive agglutination reactions with the Oxoid Salmonella Latex Test reagent, and negative reactions with the Oxoid Salmonella Latex Control reagent when tested from XLD (Xylose Lysine Desoxycholate) Agar (CM0469), MLCB (Mannitol Lysine Crystal Violet Brilliant Green) Agar (CM0783), SS (Salmonella-Shigella) Agar (CM0099), mSS (modified Salmonella-Shigella) Agar (CM0533), BGA (Brilliant Green Agar) (CM0263), Oxoid Salmonella Chromogenic Medium (CM1007). S. enteritidis, S. poona, S. hadar, S, heidelberg, S. infantis, S. agona,S. typhimurium, S. newport , S. java and S. virchow.
Strains of C. freundii, Shigella spp. , H. alvei, M. morganii, P. vulgaris, P. mirabilis, E. coli, K. pneumoniae, E. amnigenus 2 and Enterobacter spp. all gave negative reactions with the latex. Some strains of H. alvei may give stringy reactions, but this is usually also apparent with the control latex and therefore should not be confused with a true agglutination reaction ³.
In further evaluations using the Oxoid Salmonella Latex Test as a screening test for Salmonella , 562 strains with a typical Salmonella morphology on DCLS (Desoxycholate Lactose Sucrose) Agar (CM0393), Hektoen Enteric Agar (CM0419) and Bismuth Sulphite Agar (CM0201), and 85 known Salmonella serotypes were all sub-cultured on to TSI Agar slants and then tested with the latex, as per the pack insert.
The results are as follows: Table 1

Table 2

These results showed the Oxoid Salmonella Latex Test to be an effective screen for distinguishing between Salmonella spp. and other organisms, demonstrating a sensitivity of 100% and a specificity of 98.7%⁴.

References
1. Data on file at Oxoid.
2. Personal Communication from Dr. A.C. Baird-Parker.
3. Poster presentation: “Validation of a Latex Agglutination Method for Confirmation of Salmonella colonies from Six Selective Media”, I.Fairlamb, T. Organ, Oxoid Ltd., Wade Road , Basingstoke , Hampshire , RG24 8PW .
4. Poster presentation: “Evaluation of the Oxoid Salmonella Latex Test as a Screening Test for Salmonella”, A. M. Paccagnella, Provincial Laboratory, BC Centre for Disease Control, Vancouver , Canada .