沙门氏菌RVS肉汤培养基

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RVS肉汤产品基本信息

培养基名称: RVS肉汤,沙门氏菌RVS肉汤培养基,胰蛋白胨氯化镁孔雀绿肉汤培养基
英文名称:
RVS Broth,Rappaport-Vassiliadis Soy Peptone Broth
 
培养基类型: 选择性培养基
产品目录号:
M721-03
 
产品规格: 250g      查看其它规格
保存条件:
2-30度,避光,密封,阴凉干燥处保存。
 
产品性状: 黄色粉末或浅绿色粉末。
液体为孔雀蓝色,透明。
注意事项:
避免摄入、呼入、皮肤接触。配制时在通风橱中进行,戴口罩、手套、护目镜。
 
相关产品: RV肉汤培养基



产品描述:


氯化镁孔雀绿肉汤(Rappaport-Vassiliadis Enrichment Broth)简称RVS肉汤,是沙门氏菌选择性增菌肉汤培养基,是液体加富型培养基。多个对比研究表明,RV增菌肉汤培养基在分离增菌沙门氏菌方面要比其它选择性增菌培养基效果好,如TT肉汤(tetrathionate-brilliant green broth)、亚硒酸肉汤培养基。

RVS肉汤培养基

RVS肉汤培养基的工作原理:


Rappaport-Vassiliadis沙门氏菌增菌肉汤培养基的选择性基于沙门氏菌以下4个特点:与其它肠杆菌科细菌相比,沙门氏菌能够承受相对较高的渗透压;能够在pH较低环境下增菌;对孔雀绿更有耐受力;营养需求相对较低。


RV增菌肉汤是对Rappaport配方的改良。本配方的特点是减少了孔雀绿的使用量。配方中的高浓度的氯化镁抑制变形杆菌和大肠杆菌的生长,孔雀绿主要作用是抑制大肠菌群。低pH值可以抑制除沙门氏菌外的其它细菌。本配方中原来的胰酪蛋白的被大豆蛋白胨替代,研究表明,大豆蛋白胨更适合沙门氏菌的营养需求。





RVS肉汤培养基的用途:


1.RVS肉汤培养基被推荐用于从食品和环境样品(如饮用水)中分离沙门氏菌的选择性增菌培养基。

2. 氯化镁孔雀绿肉汤也可以直接用于从人的粪便样品中分离沙门氏菌,不需要预增菌,但是接种量必须小。

3. RV增菌肉汤培养基被USP推荐用于微生物限度测试。

4. 需要注意的是RVS肉汤不适用于分离伤寒沙门氏菌。当分离样品中的伤寒沙门氏菌时,要采用四硫磺酸盐肉汤(Tetrathionate Broth)进行选择性增菌。





RVS肉汤培养基配方与配制方法




培养基成分: 含量:g/L
大豆蛋白胨 4.5
氯化钠 8.0
磷酸二氢钾 0.6
磷酸氢二钾 0.4
无水氯化镁(六水氯化镁) 13.58(29.0)
孔雀绿 0.036
pH
5.2± 0.2

配制方法:

1. 称取本品27.1g,加1000ml去离子水,温和加热搅拌至完全溶解。分装10ml到螺口试管中。

2. 115°C,高温高压灭菌15min。

3. 待培养基冷却后加入新生霉素。(可选择)




实验方法


当用RVS肉汤增菌时,接种量必须足够小,不能干扰其选择性。一般推荐的接种量为培养基体积的1/100~1/2000。

鉴于该培养基成分的温度敏感性,美国农业部USDA规定在检测食品中的沙门氏菌时,RVS肉汤培养基不能使用脱水培养基,Biofeng提供非脱水型预混培养基,即能解决这个问题也使用方便。




实验方法



在使用前,确保液体培养基温度恢复到室温。

1. 转移25g固体样品或25ml液体样品到无菌容器中,加入225ml无菌的RVS肉汤培养基。用匀浆器均质。也可以每隔一分钟震荡几下,如此持续30min,确保样品均匀分散到培养基中。35ºC,培养24 小时。

2. 转移1ml上清液到合适的加富型肉汤中,如TT肉汤(Tetrathionate Broth)和亚硒酸盐胱氨酸肉汤(Selenite Cystine Broth)。35ºC培养24小时。

3. 用接种环转移上清液到合适的选择性培养基上,如HE琼脂(Hektoen Enteric Agar)、XLD琼脂(XLD Agar)和亚硫酸铋琼脂(Bismuth Sulfite Agar)。在35ºC,培养24小时。

食品中沙门氏菌的分离与鉴别 流程图



沙门氏的分离和鉴定方法


引用《细菌学鉴定手册》2011版

A.Equipment and materials

1.Blender and sterile blender jars (see Chapter 1)
2.Sterile, 16 oz (500 ml) wide-mouth, screw-cap jars, sterile 500 ml Erlenmeyer flasks, sterile 250 ml beakers, sterile glass or paper funnels of appropriate size, and, optionally, containers of appropriate capacity to accommodate composited samples
3.Sterile, bent glass or plastic spreader rods
4.Balance, with weights; 2000 g capacity, sensitivity of 0.1 g
5.Balance, with weights; 120 g capacity, sensitivity of 5 mg
6.Incubator, 35 ± 2 °C
7.Refrigerated incubator or laboratory refrigerator, 4 ± 2°C
8.Water bath, 49 ± 1°C
9.Water bath, circulating, thermostatically-controlled, 43 ± 0.2°C
10.Water bath, circulating, thermostatically-controlled,42 ± 0.2°C
11.Sterile spoons or other appropriate instruments for transferring food samples
12.Sterile culture dishes, 15 x 100 mm, glass or plastic
13.Sterile pipets, 1 ml, with 0.01 ml graduations; 5 and 10 ml, with 0.1 ml graduations
14.Inoculating needle and inoculating loop (about 3 mm id or 10 5l), nichrome, platinum-iridium, chromel wire, or sterile plastic
15.Sterile test or culture tubes, 16 x 150 mm and 20 x 150 mm; serological tubes, 10 x 75 mm or 13 x 100 mm
16.Test or culture tube racks
17.Vortex mixer
18.Sterile shears, large scissors, scalpel, and forceps
19.Lamp (for observing serological reactions)
20.Fisher or Bunsen burner
21.pH test paper (pH range 6-8) with maximum graduations of 0.4 pH units per color change
22.pH meter
23.Plastic bags, 28 x 37 cm, sterile, with resealable tape. (Items 23-24 are needed in the analysis of frog legs and rabbit carcasses.)
24.Plastic beakers, 4 liter, autoclavable, for holding plastic bag during shaking and incubation.
25.Sponges, non-bactericidal, or equivalent.
26.Swabs, non-bactericidal, cotton-tipped.

B.Media and reagents

For preparation of media and reagents, refer to Methods 967.25-967.28 in Official Methods of Analysis (1).
1.Lactose broth (M74)
2.Nonfat dry milk (reconstituted) (M111)
3.Selenite cystine (SC) broth (M134)
4.Tetrathionate (TT) broth (M145)
5.Rappaport-Vassiliadis (RV) medium (M132). NOTE: RV medium must be made from its individual ingredients. Commercial formulations are not acceptable.
6.Xylose lysine desoxycholate (XLD) agar (M179)
7.Hektoen enteric (HE) agar (M61)
8.Bismuth sulfite (BS) agar (M19)
9.Triple sugar iron agar (TSI) (M149)
10.Tryptone (tryptophane) broth (M164)
11.Trypticase (tryptic) soy broth (M154)
12.Trypticase soy broth with ferrous sulfate (M186)
13.Trypticase soy-tryptose broth (M160)
14.MR-VP broth (M104)
15.Simmons citrate agar (M138)
16.Urea broth (M171)
17.Urea broth (rapid) (M172)
18.Malonate broth (M92)
19.Lysine iron agar (LIA) (Edwards and Fife) (M89)
20.Lysine decarboxylase broth (M87)
21.Motility test medium (semisolid) (M103)
22.Potassium cyanide (KCN) broth (M126)
23.Phenol red carbohydrate broth (M121)
24.Purple carbohydrate broth (M130)
25.MacConkey agar (M91)
26.Nutrient broth (M114)
27.Brain heart infusion (BHI) broth (M24)
28.Papain solution, 5% (M56a)
29.Cellulase solution, 1% (M187)
30.Tryptose blood agar base (M166)
31.Universal preenrichment broth (M188)
32.Universal preenrichment broth (without ferric ammonium citrate) (M188a)
33.Buffered peptone water (M192)
34.Dey-Engley broth (M193)
35.Potassium sulfite powder, anhydrous
36.Chlorine solution, 200 ppm, containing 0.1% sodium dodecyl sulfate (R12a)
37.Ethanol, 70% (R23)
38.Kovacs' reagent (R38)
39.Voges-Proskauer (VP) test reagents (R89)
40.Creatine phosphate crystals
41.Potassium hydroxide solution, 40% (R65)
42.1 N Sodium hydroxide solution (R73)
43.1 N Hydrochloric acid (R36)
44.Brilliant green dye solution, 1% (R8)
45.Bromcresol purple dye solution, 0.2% (R9)
46.Methyl red indicator (R44)
47.Sterile distilled water
48.Tergitol Anionic 7 (R78)
49.Triton X-100 (R86)
50.Physiological saline solution, 0.85% (sterile) (R63)
51.Formalinized physiological saline solution (R27)
52.Salmonella polyvalent somatic (O) antiserum
53.Salmonella polyvalent flagellar (H) antiserum
54.Salmonella somatic group (O) antisera: A, B, C1, C2, C3, D1, D2, E1, E2, E3, E4, F, G, H, I, Vi, and other groups, as appropriate
55.Salmonella Spicer-Edwards flagellar (H) antisera

C.Preparation of foods for isolation of Salmonella


D.Isolation of Salmonella


1.Tighten lid and gently shake incubated sample.
Guar gum and foods suspected to be contaminated with S. Typhi. Transfer 1 ml mixture to 10 ml selenite cystine (SC) broth and another 1 ml mixture to 10 ml TT broth 98. Vortex.All other foods. Transfer 0.1 ml mixture to 10 ml Rappaport-Vassiliadis (RV) medium and another 1 ml mixture to 10 ml tetrathionate (TT) broth. Vortex.

2.Incubate selective enrichment media as follows:
Foods with a high microbial load. Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating, thermostatically-controlled, water bath). Incubate TT broth 24 ± 2 h at 43 ± 0.2°C (circulating, thermostatically-controlled, water bath).

Foods with a low microbial load (except guar gum and foods suspected to be contaminated with S. Typhi). Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulating, thermostatically controlled, water bath). Incubate TT broth 24 ± 2 h at 35 ± 2.0°C.
Guar gum and foods suspected to be contaminated with S. Typhi. Incubate SC and TT broths 24 ± 2 h at 35°C.


3.Mix (vortex, if tube) and streak 3 mm loopful (10 μl) incubated TT broth on bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Prepare BS plates the day before streaking and store in dark at room temperature until streaked.

4.Repeat with 3 mm loopful (10 μl) of RV medium (for samples of high and low microbial load foods) and of SC broth (for guar gum).

5.Refer to 994.04 in Official Methods of Analysis (1) for option of refrigerating incubated sample preenrichments and incubated sample selective enrichments (SC and TT broths only) of low moisture foods. This option allows sample analyses to be initiated as late as Thursday while still avoiding weekend work.

6.Incubate plates 24 ± 2 h at 35°C.

7.Examine plates for presence of colonies that may be Salmonella.
TYPICAL Salmonella COLONY MORPHOLOGY
Pick 2 or more colonies of Salmonella from each selective agar after 24 ± 2 h incubation. Typical Salmonella colonies are as follows:

a.Hektoen enteric (HE) agar. Blue-green to blue colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.

b.Xylose lysine desoxycholate (XLD) agar. Pink colonies with or without black centers. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.

c.Bismuth sulfite (BS) agar. Brown, gray, or black colonies; sometimes they have a metallic sheen. Surrounding medium is usually brown at first, but may turn black in time with increased incubation, producing the so-called halo effect.
If typical colonies are present on the BS agar after 24 ± 2 h incubation, then pick 2 or more colonies. Irrespective of whether or not BS agar plates are picked at 24 ± 2 h, reincubate BS agar plates an additional 24 ± 2 h. After 48 ± 2 h incubation, pick 2 or more typical colonies, if present, from the BS agar plates, only if colonies picked from the BS agar plates incubated for 24 ± 2 h give atypical reactions in triple sugar iron agar (TSI) and lysine iron agar (LIA) that result in culture being discarded as not being Salmonella . See sections D.9 and D.10, below, for details in interpreting TSI and LIA reactions.
ATYPICAL Salmonella COLONY MORPHOLOGY
In the absence of typical or suspicious Salmonella colonies, search for atypical Salmonella colonies as follows:

d.HE and XLD agars. Atypically a few Salmonella cultures produce yellow colonies with or without black centers on HE and XLD agars. In the absence of typical Salmonella colonies on HE or XLD agars after 24 ± 2 h incubation, then pick 2 or more atypical Salmonellacolonies.

e.BS agar. Atypically some strains produce green colonies with little or no darkening of the surrounding medium. If typical or suspicious colonies are notpresent on BS agar after 24 ± 2 h, then do not pick any colonies but reincubate an additional 24 ± 2 h. If typical or suspicious colonies are not present after 48 ± 2 h incubation, then pick 2 or more atypical colonies.

SUGGESTED CONTROL CULTURES

In addition to the positive control cultures (typical Salmonella), 3 additional Salmonella cultures are recommended to assist in the selection of atypical Salmonella colony morphology on selective agars. These cultures are a lactose-positive, H2S-positive S. diarizonae (ATCC 12325) and a lactose-negative, H2S-negative S. abortus equi (ATCC 9842); OR a lactose-positive, H2S-negative S. diarizonae (ATCC 29934). These cultures may be obtained from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.

8.Lightly touch the very center of the colony to be picked with sterile inoculating needle and inoculate TSI slant by streaking slant and stabbing butt. Without flaming, inoculate LIA slant by stabbing butt twice and then streaking slant. Since lysine decarboxylation reaction is strictly anaerobic, the LIA slants must have deep butt (4 cm). Store picked selective agar plates at 5-8°C.

9.Incubate TSI and LIA slants at 35°C for 24 ± 2 h. Cap tubes loosely to maintain aerobic conditions while incubating slants to prevent excessive H2S production. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in butt of tube. Consider only distinct yellow in butt of tube as acidic (negative) reaction. Do not eliminate cultures that produce discoloration in butt of tube solely on this basis. Most Salmonella cultures produce H2S in LIA. Some non- Salmonella cultures produce a brick-red reaction in LIA slants.

10.All cultures that give an alkaline butt in LIA, regardless of TSI reaction, should be retained as potential Salmonella isolates and submitted for biochemical and serological tests. Cultures that give an acid butt in LIA and an alkaline slant and acid butt in TSI should also be considered potential Salmonella isolates and should be submitted for biochemical and serological tests. Cultures that give an acid butt in LIA and an acid slant and acid butt in TSI may be discarded as not being Salmonella . Test retained, presumed-positive TSI cultures as directed in D-11, below, to determine if they are Salmonella species, including S. arizonae. If TSI cultures fail to give typical reactions for Salmonella (alkaline slant and acid butt) pick additional suspicious colonies from selective medium plate not giving presumed-positive culture and inoculate TSI and LIA slants as described in D-8, above.

11.Apply biochemical and serological identification tests to:
a.
Three presumptive TSI cultures recovered from set of plates streaked from RV medium (or SC broth for guar gum), if present, and 3 presumptive TSI agar cultures recovered from plates streaked from TT broth, if present.
b.
If 3 presumptive-positive TSI cultures are not isolated from one set of agar plates, test other presumptive-positive TSI agar cultures, if isolated, by bioche mical and serological tests. Examine a minimum of 6 TSI cultures for each 25 g analytical unit or each 375 g composite.
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E.Identification of Salmonella


1.Mixed cultures. Streak TSI agar cultures that appear to be mixed on MacConkey agar, HE agar, or XLD agar. Incubate plates 24 ± 2 h at 35°C. Examine plates for presence of colonies suspected to be Salmonella.
a.
MacConkey agar. Typical colonies appear transparent and colorless, sometimes with dark center. Colonies of Salmonella will clear areas of precipitated bile caused by other organisms sometimes present.
b.
Hektoen enteric (HE) agar. See D-7a, above.
c.
Xylose lysine desoxycholate (XLD) agar. See D-7b, above. Transfer at least 2 colonies suspected to be Salmonella to TSI and LIA slants as described in D-7, above, and continue as in D-9, above.


2.Pure cultures
a.
Urease test (conventional). With sterile needle, inoculate growth from each presumed-positive TSI slant culture into tubes of urea broth. Since occasional, uninoculated tubes of urea broth turn purple-red (positive test) on standing, include uninoculated tube of this broth as control. Incubate 24 ± 2 h at 35°C.
b.
Optional urease test (rapid). Transfer two 3-mm loopfuls of growth from each presumed-positive TSI slant culture into tubes of rapid urea broth. Incubate 2 h in 37 ± 0.5°C water bath. Discard all cultures giving positive test. Retain for further study all cultures that give negative test (no change in color of medium).

3.
Serological polyvalent flagellar (H) test
a.
Perform the polyvalent flagellar (H) test at this point, or later, as described in E-5, below. Inoculate growth from each urease-negative TSI agar slant into either 1) BHI broth and incubate 4-6 h at 35°C until visible growth occurs (to test on same day); or 2) trypticase soy-tryptose broth and incubate 24 ± 2 h at 35°C (to test on following day). Add 2.5 ml formalinized physiological saline solution to 5 ml of either broth culture.
b.
Select 2 formalinized broth cultures and test with Salmonella polyvalent flagellar (H) antisera. Place 0.5 ml of appropriately diluted Salmonella polyvalent flagellar (H) antiserum in 10 x 75 mm or 13 x 100 mm serological test tube. Add 0.5 ml antigen to be tested. Prepare saline control by mixing 0.5 ml formalinized physiological saline solution with 0.5 ml formalinized antigen. Incubate mixtures in 48-50°C water bath. Observe at 15 min intervals and read final results in 1 h.
Positive--agglutination in test mixture and no agglutination in control.
Negative--no agglutination in test mixture and no agglutination in control.
Nonspecific--agglutination in both test mixture and control. Test the cultures giving such results with Spicer-Edwards antisera.

4.
Spicer-Edwards serological test. Use this test as an alternative to the polyvalent flagellar (H) test. It may also be used with cultures giving nonspecific agglutination in polyvalent flagellar (H) test. Perform Spicer-Edwards flagellar (H) antisera test as described in E, 3b, above. Perform additional biochemical tests (E, 5a-c, below) on cultures giving positive flagellar test results. If both formalinized broth cultures are negative, perform serological tests on 4 additional broth cultures (E, 3a, above). If possible, obtain 2 positive cultures for additional biochemical testing E, 5a-c, below). If all urease-negative TSI cultures from sample give negative serological flagellar (H) test results, perform additional biochemical tests E, 5a-c, below).


5.
Testing of urease-negative cultures
a.
Lysine decarboxylase broth. If LIA test was satisfactory, it need not be repeated. Use lysine decarboxylase broth for final determination of lysine decarboxylase if culture gives doubtful LIA reaction. Inoculate broth with small amount of growth from TSI slant suspicious for Salmonella . Replace cap tightly and incubate 48 ± 2 h at 35°C but examine at 24 h intervals. Salmonella species cause alkaline reaction indicated by purple color throughout medium. Negative test is indicated by yellow color throughout medium. If medium appears discolored (neither purple nor yellow) add a few drops of 0.2% bromcresol purple dye and re-read tube reactions.
b.
Phenol red dulcitol broth or purple broth base with 0.5% dulcitol. Inoculate broth with small amount of growth from TSI culture. Replace cap loosely and incubate 48 ± 2 h at 35°C, but examine after 24 h. Most Salmonella species give positive test, indicated by gas formation in inner fermentation vial and acid pH(yellow) of medium. Production of acid should be interpreted as a positive reaction. Negative test is indicated by no gas formation in inner fermentation vial and red (with phenol red as indicator) or purple (with bromcresol purple as indicator) color throughout medium.
c.
Tryptone (or tryptophane) broth. Inoculate broth with small growth from TSI agar culture. Incubate 24 ± 2 h at 35°C and proceed as follows:

1.
1) Potassium cyanide (KCN) broth. Transfer 3 mm loopful of 24 h tryptophane broth culture to KCN broth. Heat rim of tube so that good seal is formed when tube is stoppered with wax-coated cork. Incubate 48 ± 2 h at 35°C but examine after 24 h. Interpret growth (indicated by turbidity) as positive. Most Salmonella species do not grow in this medium, as indicated by lack of turbidity.

2.
2) Malonate broth. Transfer 3 mm loopful of 24 h tryptone broth culture to malonate broth. Since occasional uninoculated tubes of malonate broth turn blue (positive test) on standing, include uninoculated tube of this broth as control. Incubate 48 ± 2 h at 35°C, but examine after 24 h. Most Salmonella species cultures give negative test (green or unchanged color) in this broth.

3.
3) Indole test. Transfer 5 ml of 24 h tryptophane broth culture to empty test tube. Add 0.2-0.3 ml Kovacs' reagent. Most Salmonella cultures give negative test (lack of deep red color at surface of broth). Record intermediate shades of orange and pink as ±.

4.
4) Serological flagellar (H) tests for Salmonella . If either polyvalent flagellar (H) test (E-3, above) or the Spicer-Edwards flagellar (H) test tube test (E-4, above) has not already been performed, either test may be performed here.

5.
5) Discard as not Salmonella any culture that shows either positive indole test and negative serological flagellar (H) test, or positive KCN test and negative lysine decarboxylase test.

6.
Serological somatic (O) tests for Salmonella. (Pre-test all antisera to Salmonella with known cultures.)
a.Polyvalent somatic (O) test. Using wax pencil, mark off 2 sections about 1 x 2 cm each on inside of glass or plastic petri dish (15 x 100 mm). Commercially available sectioned slides may be used. Emulsify 3 mm loopful of culture from 24-48 h TSI slant or, preferably, tryptose blood agar base (without blood) with 2 ml 0.85% saline. Add 1 drop of culture suspension to upper portion of each rectangular crayon-marked section. Add 1 drop of saline solution to lower part of one section only. Add 1 drop of Salmonella polyvalent somatic (O) antiserum to other section only. With clean sterile transfer loop or needle, mix culture suspension with saline solution for one section and repeat for other section containing antiserum. Tilt mixtures in back-and-forth motion for 1 min and observe against dark background in good illumination. Consider any degree of agglutination a positive reaction. Classify polyvalent somatic (O) test results as follows:
Positive--agglutination in test mixture; no agglutination in saline control.
Negative--no agglutination in test mixture; no agglutination in saline control.
Nonspecific--agglutination in test and in control mixtures. Perform further biochemical and serological tests as described in Edwards and Ewing's Identification of Enterobacteriaceae (2).
b.Somatic (O) group tests. Test as in E-6a, above, using individual group somatic (O) antisera including Vi, if available, in place of Salmonella polyvalent somatic (O) antiserum. For special treatment of cultures giving positive Vi agglutination reaction, refer to sec. 967.28B in Official Methods of Analysis (1). Recordcultures that give positive agglutination with individual somatic (O) antiserum as positive for that group. Record cultures that do not react with individual somatic (O) antiserum as negative for that group.

7.Additional biochemical tests. Classify as Salmonella those cultures which exhibit typical Salmonella reactions for tests 1-11, shown in Table 1. If one TSI culture from 25 g analytical unit is classified as Salmonella, further testing of other TSI cultures from the same 25 g analytical unit is unnecessary. Cultures that contain demonstrable Salmonella antigens as shown by positive Salmonella flagellar (H) test but do not have biochemical characteristics of Salmonella should be purified (E-l, above) and retested, beginning with E-2, above.
Perform the following additional tests on cultures that do not give typical Salmonella reactions for tests 1-11 in Table 1 and that consequently do not classify as Salmonella.

a.Phenol red lactose broth or purple lactose broth.

1) Inoculate broth with small amount of growth from unclassified 24-48 h TSI slant. Incubate 48 ± 2 h at 35°C, but examine after 24 h.
Positive--acid production (yellow) and gas production in inner fermentation vial. Consider production of acid only as positive reaction. Most cultures of Salmonella give negative test result, indicated by no gas formation in inner fermentation vial and red (with phenol red as indicator) or purple (with bromcresol purple as indicator) throughout medium.
2) Discard as not Salmonella, cultures that give positive lactose tests, except cultures that give acid slants in TSI and positive reactions in LIA, or cultures that give positive malonate broth reactions. Perform further tests on these cultures to determine if they are S. arizonae.

b.Phenol red sucrose broth or purple sucrose broth. Follow procedure described in E,7a-1, above. Discard as not Salmonella , cultures that give positive sucrose tests, except those that give acid slants in TSI and positive reactions in LIA.
c.
MR-VP broth. Inoculate medium with small amount of growth from each unclassified TSI slant suspected to contain Salmonella. Incubate 48 ± 2 h at 35°C.

1) Perform Voges-Proskauer (VP) test at room temperature as follows: Transfer 1 ml 48 h culture to test tube and incubate remainder of MR-VP broth an additional 48 h at 35°C. Add 0.6 ml α-naphthol and shake well. Add 0.2 ml 40% KOH solution and shake. To intensify and speed reaction, add a few crystals of creatine. Read results after 4 h; development of pink-to-ruby red color throughout medium is positive test. Most cultures of Salmonella are VP-negative, indicated by absence of development of pink-to-red color throughout broth.

2) Perform methyl red test as follows: To 5 ml of 96 h MR-VP broth, add 5-6 drops of methyl red indicator. Read results immediately. Most Salmonella cultures give positive test, indicated by diffuse red color in medium. A distinct yellow color is negative test. Discard, as not Salmonella, cultures that give positive KCN and VP tests and negative methyl red test.

d.Simmons citrate agar. Inoculate this agar, using needle containing growth from unclassified TSI agar slant. Inoculate by streaking slant and stabbing butt. Incubate 96 ± 2 h at 35°C. Read results as follows:
Positive--presence of growth, usually accompanied by color change from green to blue. Most cultures of Salmonella are citrate-positive.
Negative--no growth or very little growth and no color change.


8.
Classification of cultures. Classify, as Salmonella, cultures that have reaction patterns of Table l. Discard, as not Salmonella, cultures that give results listed in any subdivision of Table 2. Perform additional tests described in Edwards and Ewing's Identification of Enterobacteriaceae (2) to classify any culture that is not clearly identified as Salmonella by classification scheme in Table l or not eliminated as not being Salmonella by test reactions in Table 2. If neither of 2 TSI cultures carried through biochemical tests confirms the isolate as Salmonella, perform biochemical tests, beginning with E-5, on remaining urease-negative TSI cultures from same 25 g analytical unit.

9.
Presumptive generic identification of Salmonella . As alternative to conventional biochemical tube system, use any of 5 commercial biochemical systems (API 20E, Enterotube II, Enterobacteriaceae II, MICRO-ID, or Vitek GNI) for presumptive generic identification of foodborne Salmonella. Choose a commercial system based on a demonstration in analyst's own laboratory of adequate correlation between commercial system and biochemical tube system delineated in this identification section. Commercial biochemical kits should not be used as a substitute for serological tests (l). Assemble supplies and prepare reagents required for the kit. Inoculate each unit according to Method 978.24 (API 20E, Enterotube II, and Enterobacteriaceae II), sec. 989.12 (MICRO-ID), and Method 991.13 (Vitek GNI) in Official Methods of Analysis (1), incubating for time and temperature specified. Add reagents, observe, and record results. For presumptive identification, classify cultures, according to ref. 1, above, as Salmonella or not Salmonella.
For confirmation of cultures presumptively identified as Salmonella, perform the Salmonella serological somatic (O) test (E-6, above) and the Salmonella serological flagellar (H) test (E-3, above) or the Spicer-Edwards flagellar (H) test (E-4, above), and classify cultures according to the following guidelines:
a.Report as Salmonella those cultures classified as presumptive Salmonella with commercial biochemical kits when the culture demonstrates positive Salmonella somatic (O) test and positive Salmonella (H) test.

b.Discard cultures presumptively classified as not Salmonella with commercial biochemical kits when cultures conform to AOAC criteria (1) for classifying cultures as not Salmonella.
c.For cultures that do not conform to a or b, classify according to additional tests specified in E, 2-7, above, or additional tests as specified by Ewing (2), or send to reference typing laboratory for definitive serotyping and identification.


10.
Treatment of cultures giving negative flagellar (H) test. If biochemical reactions of certain flagellar (H)-negative culture strongly suggest that it is Salmonella, the negative flagellar agglutination may be the result of nonmotile organisms or insufficient development of flagellar antigen. Proceed as follows: Inoculate motility test medium in petri dish, using small amount of growth from TSI slant. Inoculate by stabbing medium once about 10 mm from edge of plate to depth of 2-3 mm. Do not stab to bottom of plate or inoculate any other portion. Incubate 24 h at 35°C. If organisms have migrated 40 mm or more, retest as follows: Transfer 3 mm loopful of growth that migrated farthest to trypticase soy-tryptose broth. Repeat either polyvalent flagellar (H) (E-3, above) or Spicer-Edwards (E-4 , above) serological tests. If cultures are not motile after the first 24 h, incubate an additional 24 h at 35°C; if still not motile, incubate up to 5 days at 25°C. Classify culture as nonmotile if above tests are still negative. If flagellar (H)-negative culture is suspected of being a species of Salmonella on the basis of its biochemical reactions, FDA laboratories should submit the culture to
FDA Denver Laboratory Attention Sample Custodian Denver Federal Center, Building 20 6th Avenue & Kipling Streets Denver, CO 80225-0087 (Above address effective October 1, 2004)for further identification and/or serotyping. Laboratories other than FDA should make arrangements with a reference laboratory for the serotyping of Salmonella cultures.


11.
Submission of cultures for serotyping. Submit 1 isolate of each somatic group recovered from each analytical unit, unless otherwise instructed. Submit cultures on BHI agar slants in screw-cap tubes (13 x 100 mm or 16 x 125 mm) with caps secured tightly. Label each tube with sample number, subsample (analytical unit) number, and code, if applicable. Submit a copy of the Collection Report, FD-464, or Import Sample Report, FD-784 for each sample. Place cultures in culture container with official FDA seal. Place accompanying records (E-11, above) inside shipping carton but not within officially sealed culture container. Submit memo or cover letter for each sample number to expedite reporting of results. Prepare cultures for shipment according to requirements for shipment of etiological agents (3). Label secondary shipping container according to ref. 4. Send container by most rapid mail service available. Maintain duplicate cultures of those submitted for serotyping only on those samples under consideration for legal action.
Microbiology Field laboratories should follow the following guidance in sending Salmonella isolates for serotyping:

Isolates from NRL, WEAC, SRL and ARL will be serotyped in ARL:
Arkansas Regional Laboratory 3900 NCTR Road Building 26 Jefferson, AR 72079 Attention: Gwendolyn Anderson Tel # 870-543-4621 Fax# 870-543-4041
Isolates from SAN, PRL-NW, PRL-SW and DEN will be serotyped in DEN
Denver District Laboratory 6th Avenue & Kipling Street DFC Building 20 Denver, CO 80225-0087 Attention: Doris Farmer Tel# 303-236-9604 Fax# 303-236-9675

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