美国FDA细菌学分析手册第八版(BAM)沙门氏菌(2011版)

BAM: Salmonella

NOTICE:

If you are looking for BAM Chapter 5: Salmonella (December 2007 Edition) that is incorporated by reference in 21 CFR Parts 16 and 118: Federal Register Final Rule1 (July 9, 2009, 74 FR 33030): Prevention of Salmonella Enteritidis in Shell Eggs During Production, Storage, and Transportation, please use these versions of the BAM Salmonella Chapter2 (PDF, 189 Kb) and Appendix 1: Rapid Methods for Detecting Foodborne Pathogens3 (PDF, 195 Kb).

The most recent Edition of BAM Chapter 5: Salmonella is available below this notice.

November 2011 Version

Bacteriological Analytical Manual

Chapter 5

Salmonella

Authors: Wallace H. Andrews and Thomas Hammack

Revision History:

?November 2011 - Addition to Section C: Preparation of foods for isolation of Salmonella: Leafy green vegetables and herbs.

?February 2011 – Removed link to Appendix 1: Rapid Methods for Detecting Foodborne Pathogens (now archived).

?December 2007 – Mamey pulp method added, and Section D revised.

?June 2006 – Eggs method revised for shell eggs and liquid whole eggs.

?April 2003 – Frog legs method, Lactic casein, Rennet casein, Sodium caseinate and Rabbit carcass methods revised, top ears and other dog chew

toys added. Removed section A.25, Mechanical shaker.

?October 25, 2001 – Extension of the applicability of the orange juice method in section C.19 to apple juice and apple cider.

?1999-DEC, 2000-MAR, and 2000-AUG Final revision on 2000-NOV-14 (see the Introduction for a summary of changes).

To obtain a copy of a prior version not currently posted, please contact Thomas Hammack

Chapter Contents

?Introduction

?Equipment and Materials

?Media and Reagents

?Preparation of foods for isolation of Salmonella

?Isolation of Salmonella

?Identification of Salmonella

?References

Introduction

Several changes are being introduced in this edition of BAM (8th Edition). The first change involves the expanded use of Rappaport-Vassiliadis (RV) medium4 for foods with both high and low levels of competitive microflora. In the previous edition, RV medium was recommended only for the analysis of shrimp. Based on the completion of AOAC precollaborative (5, 6) and collaborative (7, 8) studies, RV medium is now being recommended for the analysis of high microbial and low microbial load foods. RV medium replaces selenite cystine (SC) broth for the analysis of all foods, except guar gum. In addition, RV medium replaces lauryl tryptose broth for use with dry active yeast. Tetrathionate (TT)5 broth continues to be used as the second selective enrichment broth. However, TT broth is to be incubated at 43°C for the analysis of high microbial load foods and at 35°C for the analysis of low microbial load foods, including guar gum.

The second change involves the option of refrigerating incubated preenrichments and selective enrichments of low-moisture foods for up to 72 h. With this option, sample analyses can be initiated as late as Wednesday or Thursday without weekend work being involved.

The third change involves reducing the period of incubation of the lysine iron agar (LIA)6 slants. In the former edition (BAM-7), triple sugar iron agar (TSI)7 and LIA slants were incubated at 35°C for 24 ± 2 h and 48 ± 2 h, respectively. Unpublished data have demonstrated that the 48 h reading of LIA slants is without diagnostic value. Of 193 LIA slants examined, all gave definitive results within 24 ± 2 h of incubation.

No significant changes altered the final test result when the slants were incubated an additional 24 h. Thus, both the TSI and LIA slants are now incubated for 24 ± 2 h.

The fourth change involves the procedure for surface disinfection of shell eggs. In the previous edition (BAM-7), egg shells were surface-disinfected by soaking in 0.1% mercuric chloride solution for 1 h followed by soaking in 70% ethanol for 30 min. Mercuric chloride is classified as a hazardous waste, and is expensive to dispose of according to Environmental Protection Agency guidelines. In this edition (BAM-8) egg shells are now surface-disinfected by soaking for at least 10 sec in a 3:1 solution consisting of 3 parts of 70% alcohol (ethyl or isopropyl) to 1 part of iodine/potassium iodide solution.

The fifth change involves the sample preparation of eggs. Egg contents (yolk and albumen) are thoroughly mixed before analysis. After mixing the egg contents, 25 g (ml) are added to 225 ml trypticase (tryptic) soy broth supplemented with ferrous sulfate.

A method for the analysis of guar gum has been included. When guar gum is preenriched at a 1:9 sample/broth ratio, a highly viscous, nonpipettable mixture results. Addition of the enzyme cellulase to the preenrichment medium, however, results in a readily pipettable mixture.

A method for orange juice (pasteurized and unpasteurized) has been included due to recent orange juice-related outbreaks.

The directions for picking colonies from the selective plating agars have been made more explicit to reflect the intent of the method. In the absence of typical or suspect colonies on the selective plating agars, it is recommended that atypical colonies be picked to TSI and LIA slants. This recommendation is based on the fact that up to 4% of all Salmonella cultures isolated by FDA analysts from certain foods, especially seafoods, during the past several years have been atypical.

Finally, since the publication of BAM-7, a 6-way comparison was conducted of the relative effectiveness of the three selective plating agars recommended in the BAM (bismuth sulfite8, Hektoen enteric9, and xylose lysine desoxycholate agars10) and three relatively new agars (EF-18, xylose lysine Tergitol 4, and Rambach agars). Our results (9) indicated no advantage in replacing any of the BAM-recommended agars with one or more of the newer agars. Thus, the combination of selective plating agars recommended in BAM-7 remains unchanged.

A.Equipment and Materials

1.Blender and sterile blender jars (see Chapter 111)

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

https://www.wendangku.net/doc/5819283849.html,mp (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 (Nasco cat # B01299WA), or equivalent.

26.Swabs, non-bactericidal, cotton-tipped.

B.Media12 and Reagents13

For preparation of media and reagents, refer to Methods 967.25-967.28 in

Official Methods of Analysis (1).

https://www.wendangku.net/doc/5819283849.html,ctose broth (M7414)

2.Nonfat dry milk (reconstituted) (M11115)

3.Selenite cystine (SC) broth (M13416)

4.Tetrathionate (TT) broth (M14517)

5.Rappaport-Vassiliadis (RV) medium (M13218). NOTE: RV medium

must be made from its individual ingredients. Commercial

formulations are not acceptable.

6.Xylose lysine desoxycholate (XLD) agar (M17919)

7.Hektoen enteric (HE) agar (M6120)

8.Bismuth sulfite (BS) agar (M1921)

9.Triple sugar iron agar (TSI) (M14922)

10.Tryptone (tryptophane) broth (M16423)

11.Trypticase (tryptic) soy broth (M15424)

12.Trypticase soy broth with ferrous sulfate (M18625)

13.Trypticase soy-tryptose broth (M16026)

14.MR-VP broth (M10427)

15.Simmons citrate agar (M13828)

16.Urea broth (M17129)

17.Urea broth (rapid) (M17230)

18.Malonate broth (M9231)

19.Lysine iron agar (LIA) (Edwards and Fife) (M8932)

20.Lysine decarboxylase broth (M87)33

21.Motility test medium (semisolid) (M10334)

22.Potassium cyanide (KCN) broth (M12635)

23.Phenol red carbohydrate broth (M12136)

24.Purple carbohydrate broth (M13037)

25.MacConkey agar (M9138)

26.Nutrient broth (M11439)

27.Brain heart infusion (BHI) broth (M2440)

28.Papain solution, 5% (M56a41)

29.Cellulase solution, 1% (M18742)

30.Tryptose blood agar base (M16643)

31.Universal preenrichment broth (M18844)

32.Universal preenrichment broth (without ferric ammonium citrate)

(M188a45)

33.Buffered peptone water (M19246)

34.Dey-Engley broth (M19347)

35.Potassium sulfite powder, anhydrous

36.Chlorine solution, 200 ppm, containing 0.1% sodium dodecyl sulfate

(R12a48)

37.Ethanol, 70% (R2349)

38.Kovacs' reagent (R3850)

39.Voges-Proskauer (VP) test reagents (R8951)

40.Creatine phosphate crystals

41.Potassium hydroxide solution, 40% (R6552)

42.1 N Sodium hydroxide solution (R7353)

43.1 N Hydrochloric acid (R3654)

44.Brilliant green dye solution, 1% (R855)

45.Bromcresol purple dye solution, 0.2% (R956)

46.Methyl red indicator (R4457)

47.Sterile distilled water

48.Tergitol Anionic 7 (R7858)

49.Triton X-100 (R8659)

50.Physiological saline solution, 0.85% (sterile) (R6360)

51.Formalinized physiological saline solution (R2761)

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

The following methods are based on the analysis of a 25 g analytical unit at a 1:9 sample/broth ratio. Depending on the extent of compositing, add enough broth to maintain this 1:9 ratio unless otherwise indicated. For samples not analyzed on an exact weight basis, e.g., frog legs, refer to the specific method for instructions.

1.Dried egg yolk, dried egg whites, dried whole eggs, liquid milk

(skim milk, 2% fat milk, whole, and buttermilk), and prepared

powdered mixes (cake, cookie, doughnut, biscuit, and bread),

infant formula, and oral or tube feedings containing egg.

Preferably, do not thaw frozen samples before analysis. If frozen

sample must be tempered to obtain analytical portion, thaw suitable

portion as rapidly as possible to minimize increase in number of

competing organisms or to reduce potential of injuring Salmonella

organisms. Thaw below 45°C for 15 min with continuous agitation in

thermostatically controlled water bath or thaw within 18 h at 2-5°C.

Aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar

(500 ml) or other appropriate container. For nonpowdered samples,

add 225 ml sterile lactose broth65. If product is powdered, add about 15

ml sterile lactose broth and stir with sterile glass rod, spoon, or tongue

depressor to smooth suspension. Add 3 additional portions of lactose

broth, 10, 10, and 190 ml, for total of 225 ml. Stir thoroughly until

sample is suspended without lumps. Cap jar securely and let stand 60 ±

5 min at room temperature. Mix well by swirling and determine pH

with test paper. Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1 N

NaOH or 1 N HCl. Cap jar securely and mix well before determining final pH. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35°C.

Continue as in D, 1-11 , below.

2.Eggs

a.Shell eggs. Remove any adherent material from the shell

surface. Disinfect eggs with 3:1 solution consisting of 3 parts of

70% alcohol (ethyl or isopropyl) to 1 part iodine/potassium

iodide solution. Prepare 70% alcohol solution either by diluting

700 ml 100% alcohol with sterile distilled water for a final

volume of 1,000 ml or by diluting 700 ml 95% alcohol with

sterile distilled water for a final volume of 950 ml. Prepare

iodine/potassium iodide solution by dissolving 100 g potassium

iodide in 200-300 ml sterile distilled water. Add 50 g iodine

and heat gently with constant mixing until the iodine is

dissolved. Dilute the iodine/potassium iodide solution to 1,000

ml with sterile distilled water. Store iodine/potassium iodide

solution in amber glass-stoppered bottle in the dark. Prepare the

disinfection solution by adding 250 ml iodine/potassium iodide

solution to 750 ml 70% alcohol solution and mix well.

Submerge eggs in disinfection solution for at least 10 seconds.

Remove eggs and allow to air dry. Eggs with chipped, cracked,

or broken shells are not included in the sample. Each sample

shall consist of twenty (20) eggs cracked aseptically into a

Whirl-Pak bag, for a total of fifty (50) samples per poultry

house. Eggs are cracked aseptically by gloved hands, with a

change of gloves between samples. Mix samples thoroughly by

gloved hands, with a change of gloves between samples. Mix

samples thoroughly by hand until yolks are completely mixed

with the albumen. Samples are held at room temperature

(20-24°C) for 96 ± 2 h. After 96 ± 2 h, remove 25 ml portion

from each sample of pooled eggs, and preenrich 25 ml test

portion in 225 ml sterile trypticase soy broth (TSB)

supplemented with ferrous sulfate66 (35 mg ferrous sulfate

added to 1000 ml TSB) and mix well by swirling. Let stand 60

± 5 min at room temperature. Mix well by swirling and

determine pH with test paper. Adjust pH, if necessary, to 6.8 ±

0.2. Incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

b.Liquid whole eggs (homogenized). Combine fifteen (15) 25

ml test portions into a 375 ml composite contained in a 6-liter

Erlenmeyer flask. Composites are held at room temperature

(20-24°C) for 96 ± 2 h. After 96 ± 2 h, add 3,375 ml sterile

TSB supplemented with ferrous sulfate67, as described above,

and mix well by swirling. Let stand 60 ± 5 min at room

temperature. Mix well by swirling and determine pH with test

paper. Adjust pH, if necessary, to 6.8 ± 0.2. Incubate 24 ± 2 h

at 35°C. Continue as in D, 1-11, below.

c.Hard-boiled eggs (chicken, duck, and others). If the egg

shells are still intact, disinfect the shells as described above and

aseptically separate the shells from the eggs. Pulverize the eggs

(egg yolk solids and egg white solids) aseptically and weigh 25

g into a sterile 500 ml Erlenmeyer flask or other appropriate

container. Add 225 ml TSB68 (without ferrous sulfate) and mix

well by swirling. Continue as described above.

3.Nonfat dry milk

a.Instant. Aseptically weigh 25 g sample into sterile beaker (250

ml) or other appropriate container. Using sterile glass or paper

funnel (made with tape to withstand autoclaving), pour 25 g

analytical unit gently and slowly over surface of 225 ml

brilliant green water contained in sterile 500 ml Erlenmeyer

flask or other appropriate container. Alternatively, 25 g

analytical units may be composited and poured over the surface

of proportionately larger volumes of brilliant green water.

Prepare brilliant green water by adding 2 ml 1% brilliant green

dye solution69 per 1000 ml sterile distilled water. Let container

stand undisturbed for 60 ± 5 min. Incubate loosely capped

container, without mixing or pH adjustment, for 24 ± 2 h at

35°C. Continue as in D, 1-11, below.

b.Non-Instant. Examine as described for instant nonfat dry milk,

except that the 25 g analytical units may not be composited.

4.Dry whole milk. Examine as described for instant nonfat dry milk,

except that the 25 g analytical units may not be composited.

5.Casein

https://www.wendangku.net/doc/5819283849.html,ctic casein. Aseptically weigh 25 g sample into sterile

beaker (250 ml) or other appropriate container. Using sterile

glass or paper funnel (made with tape to withstand autoclaving),

pour 25 g analytical unit gently and slowly over the surface of

225 ml Universal Preenrichment broth contained in sterile 500

ml Erlenmeyer flask or other appropriate container. Analytical

units (25 g) may be composited. Let container stand

undisturbed 60 ± 5 min. Incubate loosely capped container,

without mixing or pH adjustment, for 24 ± 2 h at 35°C.

Continue as in D, 1-11, below.

b.Rennet casein. Aseptically weigh 25 g sample into sterile

beaker (250 ml) or other appropriate container. Using sterile

glass or paper funnel (made with tape to withstand autoclaving),

pour 25 g analytical unit gently and slowly over the surface of

225 ml lactose broth contained in sterile 500 ml Erlenmeyer

flask or other appropriate container. Analytical units (25 g) may

be composited. Let container stand undisturbed 60 ± 5 min.

Incubate loosely capped container, without mixing or pH

adjustment, for 24 ± 2 h at 35°C. Continue as in D, 1-11,

below.

c.Sodium caseinate. Aseptically weigh 25 g sample into sterile,

wide-mouth, screw-cap jar (500 ml) or other appropriate

container. Add 225 ml sterile lactose broth and mix well.

Analytical units may be composited. Let stand 60 min at room

temperature with jar securely capped. Mix well by swirling and

determine pH with test paper. Adjust pH, if necessary, to 6.8 ±

0.2. Loosen jar about 1/4 turn and incubate 24 ± 2 h at 35°C.

Continue as in D, 1-11, below.

6.Soy flour. Examine as described for rennet casein, except 25 g

analytical units (25 g) may not be composited.

7.Egg-containing products (noodles, egg rolls, macaroni, spaghetti),

cheese, dough, prepared salads (ham, egg, chicken, tuna, turkey), fresh, frozen, or dried fruits and vegetables, nut meats,

crustaceans (shrimp, crab, crayfish, langostinos, lobster), and fish.

Preferably, do not thaw frozen samples before analysis. If frozen

sample must be tempered to obtain analytical portion, thaw below

45°C for <15 min with continuous agitation in thermostatically

controlled water bath or thaw within 18 h at 2-5°C.

Aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile lactose broth70 and blend 2 min. Aseptically transfer

homogenized mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container and let stand 60 ± 5 min at room

temperature with jar securely capped. Mix well by swirling and

determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2.

Mix well and loosen jar cap about 1/4 turn. Incubate 24 ± 2 h at 35°C.

Continue as in D, 1-11, below.

8.Dried yeast (active and inactive yeast). Aseptically weigh 25 g

sample into sterile, wide-mouth, screw-cap jar (500 ml) or other

appropriate container. Add 225 ml sterile trypticase soy broth71. Mix well to form smooth suspension. Let stand 60 ± 5 min at room

temperature with jar securely capped. Mix well by swirling and

determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2,

mixing well before determining final pH. Loosen jar cap 1/4 turn and incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

9.Frosting and topping mixes. Aseptically weigh 25 g sample into

sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate

container. Add 225 ml nutrient broth72 and mix well. Cap jar securely and let stand 60 ± 5 min at room temperature. Mix well by swirling

and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2.

Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

10.Spices

a.Black pepper, white pepper, celery seed or flakes, chili

powder, cumin, paprika, parsley flakes, rosemary, sesame

seed, thyme, and vegetable flakes.

Aseptically weigh 25 g sample into sterile, wide-mouth,

screw-cap jar (500 ml) or other appropriate container. Add 225

ml sterile trypticase soy broth (TSB)73 and mix well. Cap jar

securely and let stand 60 ± 5 min at room temperature. Mix

well by swirling and determine pH with test paper. Adjust pH,

if necessary, to 6.8 ± 0.2. Loosen jar cap about l/4 turn and

incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

b.Onion flakes, onion powder, garlic flakes.

Aseptically weigh 25 g sample into sterile, wide-mouth,

screw-cap jar (500 ml) or other appropriate container. Preenrich

sample in TSB74 with added K2SO3 (5 g K2SO3 per 1000 ml

TSB, resulting in final 0.5% K2SO3 concentration). Add K2SO3

to broth before autoclaving 225 ml volumes in 500 ml

Erlenmeyer flasks at 121°C for 15 min. After autoclaving,

aseptically determine and, if necessary, adjust final volume to

225 ml. Add 225 ml sterile TSB with added K2SO3 to sample

and mix well. Continue as in C-10a.

c.Allspice, cinnamon, cloves, and oregano.

At this time there are no known methods for neutralizing the

toxicity of these 4 spices. Dilute them beyond their toxic levels

to examine them. Examine allspice, cinnamon, and oregano at

1:100 sample/broth ratio, and cloves at 1:1000 sample/broth

ratio. Examine leafy condiments at sample/broth ratio greater

than 1:10 because of physical difficulties encountered by

absorption of broth by dehydrated product. Examine these

spices as described in C-10a, above, maintaining recommended

sample/broth ratios.

11.Candy and candy coating (including chocolate). Aseptically weigh

25 g sample into sterile blending container. Add 225 ml sterile,

reconstituted nonfat dry milk75 and blend 2 min. Aseptically transfer homogenized mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container and let stand 60 ± 5 min at room

temperature with jar securely capped. Mix well by swirling and

determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2.

Add 0.45 ml 1% aqueous brilliant green dye solution and mix well.

Loosen jar caps 1/4 turn and incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

12.Coconut. Aseptically weigh 25 g sample into sterile, wide-mouth,

screw-cap jar (500 ml) or other appropriate container. Add 225 ml

sterile lactose broth76, shake well, and let stand 60 ± 5 min at room

temperature with jar securely capped. Mix well by swirling and

determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2.

Add up to 2.25 ml steamed (15 min) Tergitol Anionic 777 and mix

well. Alternatively, use steamed (15 min) Triton X-10078. Limit use of these surfactants to minimum quantity needed to initiate foaming. For Triton X-100 this quantity may be as little as 2 or 3 drops. Loosen jar cap about l/4 turn and incubate 24 ± 2 h at 35°C. Continue as in D,

1-11, below.

13. Food dyes and food coloring substances. For dyes with pH 6.0 or

above (10% aqueous suspension), use method described for dried

whole eggs (C-l, above). For laked dyes or dyes with pH below 6.0, aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate container. Add 225 ml tetrathionate

broth79 without brilliant green dye. Mix well and let stand 60 ± 5 min at room temperature with jar securely capped. Using pH meter, adjust pH to 6.8 ± 0.2. Add 2.25 ml 0.1% brilliant green dye solution80 and mix thoroughly by swirling. Loosen jar cap about 1/4 turn and incubate

24 ± 2 h at 35°C. Continue as in D, 3-11, below.

14.Gelatin. Aseptically weigh 25 g sample into sterile, wide-mouth,

screw-cap jar (500 ml) or other appropriate container. Add 225 ml

sterile lactose broth81 and 5 ml 5% aqueous papain solution82 and mix well. Cap jar securely and incubate at 35°C for 60 ± 5 min. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Loosen jar cap about 1/4 turn and incubate 24 ± 2 h at

35°C. Continue as in D, 1-11, below.

15.Meats, meat substitutes, meat by-products, animal substances,

glandular products, and meals (fish, meat, bone). Aseptically weigh

25 g sample into sterile blending container. Add 225 ml sterile lactose

broth83 and blend 2 min. Aseptically transfer homogenized mixture to sterile wide-mouth, screw-cap jar (500 ml) or other appropriate

container and let stand 60 ± 5 min at room temperature with jar

securely capped. If mixture is powder or is ground or comminuted,

blending may be omitted. For samples that do not require blending,

add lactose broth and mix thoroughly; let stand for 60 ± 5 min at room temperature with jar securely capped.

Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add up to 2.25 ml steamed (15 min) Tergitol Anionic 7 and mix well. Alternatively, use steamed (15 min) Triton X-100. Limit use of these surfactants to minimum quantity needed to initiate foaming. Actual quantity will depend on composition of test material. Surfactants will not be needed in analysis of powdered

glandular products. Loosen jar caps 1/4 turn and incubate sample

mixtures 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

16.Frog legs. (This method is used for all domestic and imported frog

legs.) Place 15 pairs of frog legs into sterile plastic bag and cover with sterile lactose broth at a 1:9 sample-to-broth (g/ml) ratio (see A, 23-24, above). If single legs are estimated to average 25 g or more, examine only one leg of each of 15 pairs. Place bag in large plastic beaker or other suitable container. Mix well and let stand 60 ± 5 min at room

temperature. Mix well by swirling and determine pH with test paper.

Adjust pH, if necessary, to 6.8 ± 0.2. Place plastic bag containing the frog legs and lactose broth into plastic beaker or other suitable

container. Incubate 24 ± 2 h at 35°C. Continue examination as in D, 1-11, below.

17.Rabbit carcasses. (This method is used for all domestic and imported

rabbit carcasses.) Place rabbit carcass into sterile plastic bag . Place

bag in beaker or other suitable container. Add sterile lactose broth at a 1:9 sample-to-broth (g/ml) ratio to cover carcass (see A, 23-24, above).

Mix well by swirling and let stand 60 ± 5 min at room temperature.

Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Incubate 24 ± 2 h at 35° C . Continue

examination as in D, 1-11, below.

18.Guar gum. Aseptically weigh 25 g sample into sterile beaker (250 ml)

or other appropriate container. Prepare a 1.0% cellulase solution (add 1

g cellulase to 99 ml sterile distilled water). Dispense into 150 ml

bottles. (Cellulase solution may be stored at 2-5°C for up to 2 weeks).

Add 225 ml sterile lactose broth84 and 2.25 ml sterile 1% cellulase

solution to sterile, wide-mouth, screw-cap jar (500 ml) or other

appropriate container. While vigorously stirring the cellulase/lactose broth with magnetic stirrer, pour 25 g analytical unit quickly through sterile glass funnel into the cellulase/lactose broth. Cap jar securely

and let stand 60 ± 5 min at room temperature. Incubate loosely capped container without pH adjustment, for 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

19.Orange juice (pasteurized and unpasteurized), apple cider

(pasteurized and unpasteurized), and apple juice (pasteurized)

Aseptically add 25 ml sample to 225 ml Universal preenrichment

broth85 in a sterile, wide mouth, screw-capped jar (500 ml) or other

appropriate container. Swirl the flask contents thoroughly. Cap jar

securely and let stand 60 ± 5 min at room temperature. Do not adjust pH. Incubate loosely capped container for 24 ± 2 h at 35°C. Continue as in D, 1-11, below (treat as a low microbial load food).

20.Pig ears and other types of dog chew pieces. Place 1 piece (or 2-3

pieces if smaller sizes) from each sample unit into sterile plastic bag.

Place bag into large beaker or other suitable container. Add sterile

lactose broth at a 1:9 sample-to-broth (g/ml) ratio to cover pieces (see A, 23-24, above). Mix well by swirling and let stand 60 ± 5 min at

room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Add either steamed (15 min) Tergitol Anionic 7 or steamed (15 min) Triton X-100 up to a 1% concentration. For example, if 225 ml lactose broth is added, the

maximum volume of added surfactant is 2.25 ml. Limit use of these surfactants to minimum quantity to initiate foaming. Incubate 24 ± 2 h at 35° C. Continue examination as in D, 1-11, below.

21.Cantaloupes. Preferably, do not thaw frozen samples before analysis.

If frozen sample must be tempered to obtain analytical portion, thaw below 45°C for <15 min with continuous agitation in thermostatically controlled water bath or thaw within 18 h at 2-5°C.

For comminuted or cut fruit, aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile Universal preenrichment

broth86 (UP) and blend 2 min. Aseptically transfer homogenized

mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other

appropriate container and let stand 60 ± 5 min at room temperature with jar securely capped. Do not adjust pH. Mix well and loosen jar cap about 1/4 turn. Incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

For whole cantaloupes, do not rinse even if there is visible dirt.

Examine the cantaloupes "as is".

Place the cantaloupe into a sterile plastic bag. Add enough UP87 broth to allow the cantaloupe to float. The volume of UP88 broth may be 1.5 times the weight of the cantaloupes. For example, cantaloupes

weighing 1500 g will probably need a volume of approximately 2250 ml UP89 broth to float. Add more broth, if necessary. Place the plastic bag, with cantaloupes and UP90 broth, into a 5 liter beaker, or other appropriate container, for support during incubation. Allow the

open-end flap of the plastic bag to "fold over" so as to form a secure, but not air-tight, closure during incubation.

Let stand for 60 ± 5 min at room temperature. Do not adjust pH.

Incubate slightly opened bag, containing cantaloupe, for 24 ± 2 h at

35°C. Continue as in D, 1-11, below.

22.Mangoes. Preferably, do not thaw frozen samples before analysis. If

frozen sample must be tempered to obtain analytical portion, thaw

below 45°C for <15 min with continuous agitation in thermostatically controlled water bath or thaw within 18 h at 2-5°C.

For comminuted or cut fruit, aseptically weigh 25 g sample into sterile blending container. Add 225 ml sterile buffered peptone water

(BPW)91 and blend 2 min. Aseptically transfer homogenized mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate

container and let stand 60 ± 5 min at room temperature with jar

securely capped. Mix well by swirling and determine pH with test

paper. Adjust pH, if necessary, to 6.8 ± 0.2. Mix well and loosen jar cap about 1/4 turn. Incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

For whole mangoes, do not rinse even if there is visible dirt. Examine the mangoes "as is".

Place the mango into a sterile plastic bag. Add enough BPW92 to allow the mango to float. The volume of BPW93 may be 1.0 times the weight of the mangoes. For example, mangoes weighing 500 g will probably need a volume of approximately 500 ml BPW94 broth to float. Add

more broth, if necessary. Place the plastic bag, with mangoes and

BPW95 broth, into a 5 liter beaker, or other appropriate container, for support during incubation.

Let stand for 60 ± 5 min at room temperature. Adjust pH to 6.8 ± 0.2, if necessary. Incubate slightly opened bag for 24 ± 2 h at 35°C.

Continue as in D, 1-11, below.

23.Tomatoes. For comminuted or cut fruit, aseptically weigh 25 g sample

into sterile blending container. Add 225 ml sterile buffered peptone

water and blend 2 min. Aseptically transfer homogenized mixture to sterile, wide-mouth, screw-cap jar (500 ml) or other appropriate

container and let stand 60 ± 5 min at room temperature with jar

securely capped. Mix well by swirling and determine pH with test

paper. Adjust pH, if necessary, to 6.8 ± 0.2. Mix well and loosen jar cap about 1/4 turn. Incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

For whole tomatoes, do not rinse even if there is visible dirt. Examine the tomatoes "as is".

Place the tomato into a sterile plastic bag or other suitable container (sterile foil covered beaker can be used). Add enough UP96 broth to

allow the tomato to float. The volume of UP97 broth may be 1.0 times the weight of the tomato. For example, tomatoes weighing 300 g will probably need a volume of approximately 300 ml UP98 broth to float.

Add more, if necessary. Place the plastic bag (if used), with tomato and UP99 broth, into a sterile beaker (beaker size is dependent on the size of the tomato), or other appropriate container, for support during

incubation. Allow the open-end flap of the plastic bag to "fold over" so as to form a secure, but not air-tight, closure during incubation.

Let stand for 60 ± 5 min at room temperature. Do not adjust pH.

Incubate slightly opened bag for 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

24.Environmental testing. Sample environmental surfaces with sterile

swabs or sponges. Place the swab/sponge in a sterile Whirl-pak bag, or equivalent, that contains enough Dey-Engley (DE) broth100 to cover the swab/sponge.

Transport swabs/sponges in an insulated transport container with

frozen gel packs to keep the samples cold, but not frozen. If samples cannot be processed immediately, refrigerate at 4 ± 2°C. Start sample analysis within 48 ± 2 h of collection.

Add swab/sponge to 225 ml lactose broth in a sterile, wide mouth,

screw-capped jar (500 ml) or other appropriate container. Swirl the

flask contents thoroughly. Cap jar securely and let stand 60 ± 5 min at room temperature. Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ± 0.2. Incubate 24 ± 2 h at 35°C.

Continue examination as in D, 1-11, below.

25.Alfalfa seeds and mung beans. Aseptically weigh 25g alfalfa seeds or

mung beans into a sterile 500 mL Erlenmeyer flask. Aseptically add 225 mL lactose broth to the test portion and swirl the Erlenmeyer flask.

Cover the mouth of the Erlenmeyer flask with sterile aluminum foil

and allow contents to stand at room temperature for 60 ± 5 min. Adjust the pH of the culture to 6.8 ± 0.2, if necessary. Incubate for 24 ± 2h at

35 ± 2°C. Continue as in D, 1-11, below (treat as high microbial load

food).

26.Mamey pulp. If frozen, sample must be tempered to obtain analytical

portion. Thaw below 45°C for <15 min with continuous agitation in

thermostatically controlled water bath or thaw within 18 h at 2-5°C.

For mamey pulp, suspected to be contaminated with S. Typhi,

aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar

(500 ml) or other appropriate container. Add 225 ml sterile Universal

Preenrichment broth without ferric ammonium citrate101, mix by

swirling, and let stand 60 ± 5 min at room temperature with jar

securely capped. Do not adjust pH. Mix well and loosen jar cap about

1/4 turn. Incubate 24 ± 2 h at 35°C. Continue as in D, 1-11, below.

Treat as a low microbial load food.

For mamey pulp, NOT suspected to be contaminated with S. Typhi,

aseptically weigh 25 g sample into sterile, wide-mouth, screw-cap jar

(500 ml) or other appropriate container. Add 225 ml sterile Universal

Preenrichment broth, mix by swirling, and let stand 60 ± 5 min at room

temperature with jar securely capped. Do not adjust pH. Mix well and

loosen jar cap about 1/4 turn. Incubate 24 ± 2 h at 35°C. Continue as in

D, 1-11, below.

27.Leafy green vegetables and herbs (baby spinach, Romaine lettuce,

cilantro, curly parsley, Italian parsley, culantro, cabbage, and

basil). Aseptically weigh 25 g into a sterile wide mouth Erlenmeyer

flask or other appropriate container. Add 225 mL lactose broth and

manually mix contents by vigorously swirling the flask 25 times

clockwise and 25 times counterclockwise. Allow the flask to stand at

room temperature for 60 ± 5.0 minutes, measure the pH and adjust it to

6.8 ± 0.2 with 1N NaOH or 1N HCl, if necessary. Incubate at 35o ±

2.0o C for 24 ± 2.0 hours and continue as in D, 1-11, below.

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 102and

another 1 ml mixture to 10 ml TT broth 103. Vortex.

All other foods. Transfer 0.1 ml mixture to 10 ml

Rappaport-Vassiliadis (RV) medium104 and another 1 ml mixture to 10

ml tetrathionate (TT) broth105. 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) agar106, xylose lysine desoxycholate

(XLD) agar107, and Hektoen enteric (HE) agar108. 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 Salmonella colonies.

e.BS agar. Atypically some strains produce green colonies with

little or no darkening of the surrounding medium. If typical or

suspicious colonies are not present 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 Collection109, 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.

E. Identification of Salmonella

1.Mixed cultures. Streak TSI agar cultures that appear to be mixed on

MacConkey agar110, HE agar111, or XLD agar112. 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 broth113. 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 broth114. 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 broth115 and

incubate 4-6 h at 35°C until visible growth occurs (to test on

same day); or 2) trypticase soy-tryptose broth116 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.

沙门氏菌的检验

沙门氏菌得检验 食品学院14食品质量与安全１班 刘文敏柳基炜卫杰恒温紫君 2 2 2 ２ 摘要:本实验采用GB／T4７89。4-２０10得检测方法测定鸡场中得沙门氏菌、通过本实验学习沙门氏菌得检测方法与技术,了解沙门氏菌得一些生化特性;本实验先用显色培养基找出可疑菌落,再做生化试验找出可疑得典型性得沙门氏菌,再通过血清学试验最终确定就是否为沙门氏菌属。 关键词:沙门氏菌接种生化试验血清学鉴定 前言 沙门氏菌病就是公共卫生学中具有重要意义得人畜共患病种之一,其病原沙门氏菌属于肠道细菌科。沙门氏菌就是一个统称,泛指 20００多种有紧密连系得细菌,包括引起食物中毒,导致胃肠炎、伤寒与副伤寒得细菌。虽然只有少数人因沙门氏菌而患病,但就是,在世界范围内得细菌性食物中毒事件中,由沙门氏菌引起得占大多数。因此,采用科学、合理得方法检验食品中沙门氏菌,已经成为了人们最关心得问题之一［１]。国标法(GB4789。４-２0１０)就是目前中国规定得食品中沙门氏菌得标准检测方法,也就是基层实验室普遍采用得检测方法,它根据沙门氏菌得生长特点与生化特性,采取前增菌、增菌、分离、生化试验与血清学鉴定5个步骤进行[２]。 1材料与方法 1、1实验材料 1、１.1仪器设备 均质器、三角烧瓶、平皿、玻璃棒、接种棒 恒温培养箱:36℃±１℃,4２℃±1℃ 吸管:1 mＬ(具 0.０1 mL刻度)、10ｍL(具0、1mL刻度或微量移液器及吸头 电子天平ＰL６02－S,梅特勒—托利多仪器(上海)有限公司; 手提式不锈钢压力蒸汽灭菌锅SYＱ—DSＸ-2８0B,上海申安医疗器械厂 1。1。2试剂药品

鸡肠、靛基质试剂、沙门氏菌Ｏ与H诊断血清、AＰI20E生化试剂盒或VIＴＥKGNI 生化鉴定卡 1.1。３培养基 蛋白胨水(BPW)、四硫磺酸钠煌绿(TTB)、亚硒酸盐胱氨酸(SC)增菌液、亚硫酸铋(BS)琼脂、HE琼脂、三糖铁琼脂、蛋白胨水、尿素琼脂、氰化钾、氰化钾对照、赖氨酸脱羧酶、赖氨酸脱羧酶对照、甘露醇、山梨醇、β—D半乳糖苷(ONPＧ)培养基 1、2 实验方法 １.２。1培养基得制备 1、2。１.1培养基得配制步骤 蛋白胨水(ＢＰＷ):称取蛋白胨１０ｇ、氯化钠5g、磷酸氢二钠9g、磷酸二氢钠1.5g、蒸馏水10０0ｍｌ,将各成分加入蒸馏水中,搅混均匀,静置约 10 miｎ,煮沸溶解,调节pH,高压灭菌 12１℃,1５ｍin。分装10瓶,每瓶９0ml 四硫磺酸钠煌绿(TTB):高压灭菌 121 ℃,15 min灭菌冷却后至30℃,每100ml 基础培养液加碘液2ml,煌绿液１ｍｌ 1。2.1、2配制培养基得注意事项 (1)按照说明书上得用量进行换算,称取准确分量得合成培养基粉末; (2)加热煮沸溶解培养基时,留意锅内水位得变化,水位下降可再添加适量得水,以免水分蒸发过多,导致后面分装不够量; (３)往试管中放小导管时,注意处理气泡、 1。2。2 沙门氏菌群检测 1、2.2.1沙门氏菌检测程序

沙门氏菌检验

沙门氏菌的检验 1．目的 规范沙门氏菌检测方法，使产品检验有据可依。 2．消毒灭菌要求 微生物检测用的玻璃仪器、金属用具及培养基、被污染和接种的培养物等，必须经灭菌后方能使用。 注：本实验采用湿热灭菌法，吸管、培养皿、培养基等盖好塞子并包好瓶口在高压灭菌锅中按要求的温度和压力灭菌，一般是121℃（）下灭菌20min。 3．原理 沙门氏菌的检验分四个连续阶段： 4．操作步骤 准备工作 配制实验所需的缓冲蛋白胨水、亚硒酸盐胱氨酸培养基（无需灭菌）、HE培养基、三糖铁培养基等，并将准备好的均质杯、吸管、培养皿、大试管等一起灭菌。 前增菌 在无菌环境下，称取25g待检样品放入盛有225ml灭菌好的缓冲蛋白胨水中，然后放到36±1℃的恒温培养箱内进行前增菌4-6h; 增菌 在无菌环境下，用灭菌好的吸管吸取10ml前增菌液接种与100ml亚硒酸盐胱氨酸培养基中进行二次增菌，恒温培养箱36±1℃，培养18-24h; 分离培养 将增菌培养液摇匀，以无菌操作，用直径3mm的接种环挑取一环，划线于表面无凝结水的BS和SS琼脂平板各一个，于36±1℃培养18-24h。观察各个平板上有无典型或可疑沙门氏菌属的菌落。如无典型或可疑菌落，应再继续培养24±2h。然后观察培养的平板（黄色的菌落是大肠杆菌；蓝绿色或蓝色，产硫化氢，菌落中心黑色或几乎全黑色为可疑沙门氏菌）。 沙门氏菌属各亚属在其他选择性琼脂平板的菌落特征 生化实验 用灭菌好的接种针在培养平板上挑取可疑的沙门氏菌单菌落，接种到三糖铁培养基上，恒温培养箱36±1℃，培养18-24h; 典型沙门氏菌培养物斜面显红色（碱性），底端显黄色（酸性），有气体产生，形成硫化氢（琼脂变黑）。 三糖铁培养基变化表 肠杆菌科各属在三糖铁琼脂内的反应结果

沙门氏菌的检验

沙门氏菌的检验 食品学院14食品质量与安全1班 刘文敏柳基炜卫杰恒温紫君 2 2 2 2 摘要：本实验采用GB/T4789.4-2010的检测方法测定鸡场中的沙门氏菌。通过本实验学习沙门氏菌的检测方法和技术，了解沙门氏菌的一些生化特性；本实验先用显色培养基找出可疑菌落，再做生化试验找出可疑的典型性的沙门氏菌，再通过血清学试验最终确定是否为沙门氏菌属。 关键词：沙门氏菌接种生化试验血清学鉴定 前言 沙门氏菌病是公共卫生学中具有重要意义的人畜共患病种之一，其病原沙门氏菌属于肠道细菌科。沙门氏菌是一个统称，泛指 2000 多种有紧密连系的细菌，包括引起食物中毒，导致胃肠炎、伤寒和副伤寒的细菌。虽然只有少数人因沙门氏菌而患病，但是，在世界范围内的细菌性食物中毒事件中，由沙门氏菌引起的占大多数。因此，采用科学、合理的方法检验食品中沙门氏菌，已经成为了人们最关心的问题之一[1]。国标法(GB4789.4-2010)是目前中国规定的食品中沙门氏菌的标准检测方法,也是基层实验室普遍采用的检测方法,它根据沙门氏菌的生长特点和生化特性,采取前增菌、增菌、分离、生化试验和血清学鉴定5个步骤进行[2]。 1材料与方法 1.1实验材料 1.1.1仪器设备 均质器、三角烧瓶、平皿、玻璃棒、接种棒 恒温培养箱：36℃±1℃，42℃±1℃ 吸管：1 mL（具 0.01 mL刻度）、10mL（具0.1mL刻度或微量移液器及吸头

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沙门氏菌的检验

沙门氏菌的检验 2.2 恒温培养箱：36 ℃±1 ℃，42 ℃±1 ℃。 2.3 均质器。 2.4 振荡器。 2.5天平：感量0.1 g。 2.6 无菌锥形瓶:容量500 mL，250 mL。 2.7 无菌吸管：1 mL（具0.01 mL 刻度）、10 mL（具0.1 mL 刻度）或微量移液器及吸头。 2.8 无菌培养皿：直径90 mm。 2.9 无菌试管：3 mm×50 mm、10 mm×75 mm。 2.10 无菌毛细管。 2.11 pH 计或pH 比色管或精密pH 试纸。 3 培养基和试剂 3.1 缓冲蛋白胨水（BPW）：见附录A 中A.1。 3.2 四硫磺酸钠煌绿（TTB）增菌液：见附录A 中A.2。 3.3 亚硒酸盐胱氨酸（SC）增菌液：见附录A 中A.3。 3.4 亚硫酸铋（BS）琼脂：见附录A 中A.4。 3.5 HE 琼脂：见附录A 中A.5。 3.6 木糖赖氨酸脱氧胆盐（XLD）琼脂：见附录A 中A.6。 3.8 三糖铁（TSI）琼脂：见附录A 中A.7。 3.9 蛋白胨水、靛基质试剂：见附录A 中A.8。 3.10 尿素琼脂（pH 7.2）：见附录A 中A.9。 3.11 氰化钾（KCN）培养基：见附录A 中A.10。 3.12 赖氨酸脱羧酶试验培养基：见附录A 中A.11。 3.13 糖发酵管：见附录A 中A.12。 3.14 邻硝基酚β-D 半乳糖苷（ONPG）培养基：见附录A 中A.13。 3.15 半固体琼脂：见附录A 中A.14。 3.16 丙二酸钠培养基：见附录A 中A.15。 1 前增菌 称取25 g（mL）样品放入盛有225 mL BPW 的无菌均质杯中，以8 000 r/min～10 000 r/min 均质 1 min～ 2 min，或置于盛有225 mL BPW 的无菌均质袋中，用拍击式均质器拍打1 min～2 min。 2 增菌 轻轻摇动培养过的样品混合物，移取1 mL，转种于10 mL TTB 内，于42 ℃±1 ℃培养18 h～24h。同时，另取1 mL，转种于10 mL SC 内，于36 ℃±1 ℃培养18 h～24 h。 3 分离 分别用接种环取增菌液1 环，划线接种于一个BS 琼脂平板和一个XLD 琼脂平板（或HE 琼脂平板或沙门氏菌属显色培养基平板）。于36 ℃±1 ℃分别培养18 h～24 h （XLD 琼脂平板、

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1目的P URPOSE 规范888物料、产品的沙门氏菌检验操作，确保检验结果的可靠性。 2范围SCOPE 适用于888物料、产品的沙门氏菌检验工作。 3责任RESPONSIBILITY 微生物检验员严格执行本规程，品质部负责人监督执行。 4程序PROCEDURE 定义和原理 沙门氏菌广泛存在于动物的肠道和内脏，以及被粪便污染的水和土壤中，是细菌性食物中毒的主要病因。本方法利用沙门氏菌呈辛酸酯酶阳性，而氧化酶和脂肪酶均呈阴性的特性，对物料、产品进行沙门氏菌检验。 材料和设备 紫外灯：波长366nm，功率不小于6W。 放大镜：3至4倍。 毛细滴管。 LX-B35L压力蒸汽灭菌锅。 无菌的培养皿。 无菌的接种环。 培养基和试剂 SCDLP液体培养基：按《化妆品卫生规范》（2007版）或使用商品培养基干粉配制。 四硫磺酸钠煌绿（TTB）增菌液：按—2010附录配制或使用商品试剂。 SS琼脂培养基（含1%蔗糖）：牛肉浸膏(或牛肉粉)，蛋白胨，乳糖，蔗糖，胆盐，柠檬酸钠，硫代硫酸钠，柠檬酸铁，煌绿，中性红，琼脂，蒸馏水1000mL。 HE琼脂培养基：按—2010附录或使用商品培养基干粉配制。 玉米油维多利亚蓝琼脂培养基：灭菌后的基础培养基冷却至50℃左右，边摇边加入玉米油乳化液。倾注平皿，制成平板。基础培养基及玉米油乳化液配方如下： a）基础培养基：蛋白胨5g，酵母浸粉3g，氯化钠5g，琼脂13g，水900mL，。将 各成分加入水中，加热溶解。调节，116℃15min高压灭菌。 b）玉米油乳化液：玉米油100mL，%维多利亚蓝水溶液100mL，%琼脂溶液80mL， 吐温801mL。玉米油加维多利亚蓝水溶液混合，边振动边在沸水中加热溶化。然后， 放入分液漏斗中，弃去蓝色水部分，再将着色的脂肪用水洗1-2次。将着色脂肪20mL 加入810mL琼脂溶液中，再加1mL吐温80，116℃15min高压灭菌。冷却后用超声 波乳化。

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食品中沙门氏菌检验操作规范 1 检验依据 本方法参照GB4789.4-2010《食品安全国家标准食品微生物学检验沙门氏菌检验》。 2 设备和材料 除微生物实验室常规灭菌及培养设备外，其他设备和材料如下： 2.1 冰箱：2 ℃～5 ℃。 2.2 恒温培养箱：36 ℃±1 ℃，42 ℃±1 ℃。 2.3 均质器。 2.4 振荡器。 2.5 电子天平：感量0.1 g。 2.6 无菌锥形瓶:容量500 mL，250 mL。 2.7 无菌吸管：1 mL（具0.01 mL 刻度）、10 mL（具0.1 mL 刻度）或微量移液器及吸头。 2.8 无菌培养皿：直径90 mm。 2.9 无菌试管：3 mm×50 mm、10 mm×75 mm。 2.10 无菌毛细管。 2.11 pH 计或pH 比色管或精密pH 试纸。 2.12 全自动微生物生化鉴定系统。 3 培养基和试剂（按说明书配置或灭菌） 3.1 缓冲蛋白胨水（BPW）； 3.2 四硫磺酸钠煌绿（TTB）增菌液； 3.3 亚硒酸盐胱氨酸（SC）增菌液； 3.4 亚硫酸铋（BS）琼脂； 3.5 HE 琼脂； 3.6 木糖赖氨酸脱氧胆盐（XLD）琼脂； 3.7 沙门氏菌属显色培养基； 3.8 三糖铁（TSI）琼脂； 3.9 蛋白胨水、靛基质试剂； 3.10 尿素琼脂（pH 7.2）； 3.11 氰化钾（KCN）培养基； 3.12 赖氨酸脱羧酶试验培养基； 3.13 糖发酵管； 3.14 邻硝基酚β-D 半乳糖苷（ONPG）培养基；

3.15 半固体琼脂； 3.16 丙二酸钠培养基； 3.17 沙门氏菌O 和H 诊断血清； 3.18 生化鉴定试剂盒。 4 检验程序 沙门氏菌检验程序见图1。 图1 沙门氏菌检验程序 5 操作步骤 5.1 前增菌 称取25 g（mL）样品放入盛有225 mL BPW 的无菌均质杯中，以8 000 r/min～10 000 r/min 均质1 min～2 min，或置于盛有225 mL BPW 的无菌均质袋中，用拍击式均质器拍打1 min～2 min。若样品为液态，不需要均质，振荡混匀。如需测定pH 值，用1 mol/mL 无菌NaOH 或HCl 调pH 至6.8±0.2。无菌操作将样品转至500 mL 锥形瓶中，如使用均质袋，可直接进行培养，于36 ℃±1 ℃培养8 h～18h。

沙门氏菌检验(现用)

沙门氏菌的检验 1．目的 规沙门氏菌检测方法，使产品检验有据可依。 2．消毒灭菌要求 微生物检测用的玻璃仪器、金属用具及培养基、被污染和接种的培养物等，必须经灭菌后方能使用。 注：本实验采用湿热灭菌法，吸管、培养皿、培养基等盖好塞子并包好瓶口在高压灭菌锅中按要求的温度和压力灭菌，一般是121℃（1.5MPa）下灭菌20min。3．原理 沙门氏菌的检验分四个连续阶段： 4．操作步骤 4.1 准备工作 配制实验所需的缓冲蛋白胨水、亚硒酸盐胱氨酸培养基（无需灭菌）、HE培养基、三糖铁培养基等，并将准备好的均质杯、吸管、培养皿、大试管等一起灭菌。 4.2 前增菌 在无菌环境下，称取25g待检样品放入盛有225ml灭菌好的缓冲蛋白胨水中，

然后放到36±1℃的恒温培养箱进行前增菌4-6h; 4.3 增菌 在无菌环境下，用灭菌好的吸管吸取10ml前增菌液接种与100ml亚硒酸盐胱氨酸培养基中进行二次增菌，恒温培养箱36±1℃，培养18-24h; 4.4 分离培养 将增菌培养液摇匀，以无菌操作，用直径3mm的接种环挑取一环，划线于表面无凝结水的BS和SS琼脂平板各一个，于36±1℃培养18-24h。观察各个平板上有无典型或可疑沙门氏菌属的菌落。如无典型或可疑菌落，应再继续培养24±2h。然后观察培养的平板（黄色的菌落是大肠杆菌；蓝绿色或蓝色，产硫化氢，菌落中心黑色或几乎全黑色为可疑沙门氏菌）。 沙门氏菌属各亚属在其他选择性琼脂平板的菌落特征 4.5 生化实验 用灭菌好的接种针在培养平板上挑取可疑的沙门氏菌单菌落，接种到三糖铁培养基上，恒温培养箱36±1℃，培养18-24h; 典型沙门氏菌培养物斜面显红色（碱性），底端显黄色（酸性），有气体产生，形成硫化氢（琼脂变黑）。 三糖铁培养基变化表

沙门氏菌检测

沙门氏菌检测 1 设备和材料 除微生物实验室常规灭菌及培养设备外，其他设备和材料如下： 1.1 冰箱：2℃～5℃。 1.2 恒温培养箱：36℃±1℃，42℃±1℃。 1.3 均质器。 1.4 振荡器。 1.5 电子天平：感量0.1g。 1.6 无菌锥形瓶:容量500ml，250ml。 1.7 无菌吸管：1ml（具0.01ml刻度）、10ml（具0.1ml刻度）或微量移液器及吸头。 1.8 无菌培养皿：直径90mm。 1.9 无菌试管：3mm×50mm、10mm×75mm。 1.10 无菌毛细管。 1.11 pH计或pH比色管或精密pH试纸。 1.12 全自动微生物生化鉴定系统。 2 培养基和试剂 2.1 缓冲蛋白胨水（BPW）：见附录中1。 2.2 四硫磺酸钠煌绿（TTB）增菌液：见附录中2。 2.3 亚硒酸盐胱氨酸（SC）增菌液：见附录中3。 2.4 亚硫酸铋（BS）琼脂：见附录中4。 2.5 HE琼脂：见附录中5。 2.6 木糖赖氨酸脱氧胆盐（XLD）琼脂：见附录中6。 2.7 沙门氏菌属显色培养基。 2.8 三糖铁（TSI）琼脂：见附录中7。 2.9 蛋白胨水、靛基质试剂：见附录中8。 2.10 尿素琼脂（pH7.2）：见附录中9。 2.11 氰化钾（KCN）培养基：见附录中10。 2.12 赖氨酸脱羧酶试验培养基：见附录中11。

2.13 糖发酵管：见附录中12。 2.14 邻硝基酚β-D半乳糖苷（ONPG）培养基：见附录中13。 2.15 半固体琼脂：见附录中14。 2.16 丙二酸钠培养基：见附录中15。 2.17 沙门氏菌O和H诊断血清。 2.18 生化鉴定试剂盒。 3 操作方法 3.1 试验前准备 3.1.1 将供试品及所有已灭菌的平皿、锥形瓶、匀浆杯、试管、量筒、吸管（1ml、10ml）、稀释剂等移至操作室内。准备好足够用量，避免操作中出入操作间。3.1.2 开启无菌室紫外杀菌灯和洁净工作台的空气过滤装置30min。 3.1.3 操作人员用肥皂洗手，关闭紫外杀菌灯，进入缓冲间，换工作鞋，用消毒液洗手或用乙醇棉球擦手，穿戴好无菌衣、帽、口罩、手套等。 3.1.4 操作前先用乙醇棉球擦手、工作台面，再用乙醇棉球擦拭供试品瓶、盒、袋等的开口处周围，待干后用灭菌剪刀或镊子将供试品启封。 3.2 前增菌 称取25g（ml）样品放入盛有225ml BPW的无菌均质杯中，以8000r/min～10000r/min均质1min～2min，或置于盛有225ml BPW的无菌均质袋中，用拍击式均质器拍打1min～2min。若样品为液态，不需要均质，振荡混匀。如需测定pH 值，用1mol/ml无菌NaOH或HCl调pH至6.8±0.2。无菌操作将样品转至500ml锥形瓶中，如使用均质袋，可直接进行培养，于36℃±1℃培养8 h～18h。如为冷冻产品,应在45℃以下不超过15min,或2℃～5℃不超过18h解冻。 3.3 增菌 轻轻摇动培养过的样品混合物，移取1ml，转种于10ml TTB内，于42℃±1℃培养18 h～24h。同时，另取1ml，转种于10ml SC内，于36℃±1℃培养18h～24h。 3.4 分离 分别用接种环取增菌液1环，划线接种于一个BS琼脂平板和一个XLD琼脂平板（或HE琼脂平板或沙门氏菌属显色培养基平板）。于36℃±1℃分别培养18h～24h（XLD琼脂平板、HE琼脂平板、沙门氏菌属显色培养基平板）或40h～48h

沙门氏菌检验方法

沙门氏菌检验方法 关于GB 4789.04-2010 食品安全国家标准食品微生物学检验沙门氏菌检验解决方案 GB 4789.04-2010 沙门氏菌检验流程图 环凯针对标准的修改，扩充完善了微生物检测试剂，推出了成套的沙门氏菌检验解决方案，为生产企业提供方便。 功能阶段实验 序号 产品编号产品名称规格类别 前增菌1023130缓冲蛋白胨水（B PW）250g瓶（干粉）

CP0680缓冲蛋白胨水（B PW）225 mL×10袋盒（袋装）CP0290缓冲蛋白胨水（B PW）225 mL×6瓶盒（瓶装）CP0560A缓冲蛋白胨水（B PW）9 ml×20支盒（管装） 选择性增菌2 023030四硫磺酸钠煌绿增菌液基础（TTB）250g瓶（干粉）SR0040 四硫磺酸钠煌绿增菌液配套试剂 （碘液、煌绿各一支添加于100ml培养基） 2×5支/盒盒（西林瓶）CP0300四硫磺酸钠煌绿增菌液（TTB）10 ml×20支盒（管装）3 023040亚硒酸盐胱氨酸增菌液（SC）250g瓶（干粉）CP0050亚硒酸盐胱氨酸增菌液（SC）10 ml×20支盒（管装） 选择性分离4023050亚硫酸铋琼脂（BS）（配送指示剂）250g瓶（干粉）5 023070HE琼脂培养基250g瓶（干粉）CP0110HE琼脂培养基90mm×20盒（平板） 6 029999木糖-赖氨酸-去氧胆酸盐琼脂（XLD）250g瓶（干粉）029999P木糖-赖氨酸-去氧胆酸盐琼脂（XLD） 300 mL/袋 ×10袋 盒（袋装） 颗粒培养基CP0180木糖-赖氨酸-去氧胆酸盐琼脂（XLD）90mm×20盒（平板）7 CRM004沙门氏菌显色培养基1000 mL瓶（干粉）CP0560A沙门氏菌显色培养基平板90mm×20盒（平板）8HM42沙门氏菌乳胶凝集试剂盒50 tests套 生理生化 9 022080三糖铁琼脂（TSI）250g瓶（干粉）CP0080三糖铁斜面20支盒（管装）075750三糖铁20支盒（西林瓶） 10 022110蛋白胨水 (靛基质培养基，色氨酸肉汤)100g瓶（干粉）075240蛋白胨水 (靛基质培养基，色氨酸肉汤)20支盒（西林瓶）029230靛基质试剂10 mL×1支盒 11 023090尿素琼脂培养基基础100g瓶（干粉）02910040%无菌尿素溶液 2 mL×10支盒（西林瓶）075150尿素琼脂培养基20支盒（西林瓶）12 075330氰化钾生长实验管20支盒（西林瓶）075340氰化钾对照管20支盒（西林瓶）13 075280赖氨酸脱羧酶培养基20支盒（西林瓶）075290氨基酸脱羧酶对照培养基20支盒（西林瓶）029110无菌石蜡油10 mL×1支瓶14075090山梨醇20支盒（西林瓶）15075040甘露醇20支盒（西林瓶）16075180β-半乳糖苷 ONPG20支盒（西林瓶）17071740沙门氏菌生化鉴定试剂盒10种×10支盒（西林瓶）18075100卫矛醇20支盒（西林瓶）19075140水杨素20支盒（西林瓶）20075190丙二酸盐20支盒（西林瓶）

沙门氏菌检验

沙门氏菌检验 范围1.本法适用于食品中沙门氏菌的检验。 2.设备和材料 处微生物实验室常规灭菌及培养设备外，其他设备和材料如下：2.1冰箱：2℃～5℃。 2.2恒温培养箱：36℃±1℃，42℃±1℃。 2.3均质器。 2.4振荡器。 2.5电子天平：感量0.1g。 2.6无菌锥形瓶：容量500mL，250mL。 2.7无菌吸管：1mL（具0.01mL刻度）、10mL（具0.1mL刻度）或微量移液器及吸头。 2.8无菌培养皿：直径90mm。 2.9无菌试管：3mm×50mm、10mm×75mm。 2.10无菌毛细管。 2.11pH计或pH比色管或精密pH试纸。 2.12全自动微生物生化鉴定系统。 3.培养基和试剂 3.1缓冲蛋白胨水（BPW）。 ）增菌液。TTB四硫磺酸钠煌绿（3.2．

3.3亚硒酸盐胱氨酸（SC）增菌液。 3.4亚硫酸铋（BS）琼脂。 3.5HE琼脂。 3.6木糖赖氨酸脱氧胆盐（XLD）琼脂。 3.7沙门氏菌属显色培养基。 3.8三糖铁（TSI）琼脂。 3.9蛋白胨水、靛基质试剂。 3.10尿素琼脂（pH7.2）。 3.11氰化钾（KCN）培养基。 3.12赖氨酸脱羧酶试验培养基。 3.13糖发酵管。 3.14邻硝基苯β-D-半乳糖苷（ONPG）培养基。 3.15半固体琼脂。 3.16丙二酸钠培养基。 3.17沙门氏菌O和H诊断血清。 3.18生化鉴定试剂盒。 4.检验程序 沙门氏菌检验程序见图1。

检样36℃±1℃，18h～ 24h 25g（mL）样品36℃±1～℃，℃±42118h24h ℃，18h～24h 1mL+SC10mL 1mL+TTB10mL 36℃±1℃，40h～48h 36℃±1℃，18h～24h 1沙门氏菌检验程序图操作步骤5. 前增菌5.1称取25g（mL）样品放入盛有225mLBPW的无菌均质杯中，以8000r/min～10000r/min 均质1min～2min，或置于盛有225mLBPW的无菌均质袋中，用拍击式均质器拍打1min～2min。若样品为液态，不需要均质，振荡混匀。如需测定pH值，用1mol/mL无菌NaOH或HCl调pH至6.8±0.2.无菌操作将样品转至500mL锥形瓶中，如使用均质袋，可直接进行培养，于36℃±1℃培养8h～18h。

沙门氏菌检测的基准方法

国际标准 ISO 6579 食品和动物饲料的微生物学 ——沙门氏菌检测的基准方法 内容 1 范围 2 参考标准 3 定义 4 原理 4.1 概要 4.2前增菌——非选择性液体培养基 4.3 增菌——选择性液体培养基 4.4 划平板和鉴定 4.5 确认 5 培养基、试剂和血清 5.1 概要 5.2 培养基和试剂 5.3 血清 6 设备和玻璃器皿 7 采样 8 检验样品的制备 9 程序 9.1 试验样品和原始悬浮液 9.2 非选择性前增菌 9.3 选择性增菌 9.4 划平板和鉴定 9.5 确认 10 结果表示 11 检验报告 12 质量保证 附录A （标准化）程序图表 附录B （标准化）培养基和试剂的成份及准备 附录C （非标准化）实验室间试验结果 参考书目

ISO（国际标准化组织）是国际标准化团体的全世界同盟。通常由ISO技术委员会来执行国际标准的制订工作（ISO成员体）。通常由ISO技术委员会来完成国际标准的准备工作。在委员会中，若对建立的技术委员会有兴趣的话，每个成员体有权作为代表。国际组织、政府或非政府组织，通过ISO联系，也可以参加这项工作。在所有电工标准方面，ISO与国际电工委员会（IEC）紧密合作。 国际标准按照ISO/IEC指南第三章所列的规则草拟。 技术委员会的主要任务是准备国际标准。被技术委员会采用的国际标准草案以投票方式在成员体内运行。至少要75%成员体的投票赞成，才能作为一个国际标准发布。 要引起注意的可能性是:本国际标准的一些要素可能是专利权主体。ISO不应承担鉴定部分或全部的这种专利权。 ISO6579是由食品ISO/TC34技术委员会、微生物SC9分委员会所准备的。 第四版删除或更换了第三版（ISO6579：1993），并已作了技术性修订。 附录A和附录B形成了本国际标准的标准化部分。附录C仅为资料。

沙门氏菌基本知识及检测方法

沙门氏菌属(Salmonella)是肠杆菌科的一个大属，有2000多个血清型，我国发现的约有100个。沙门氏菌广泛存在于猪、牛、羊、家禽、鸟类、鼠类等多种动物的肠道和内脏中。1880年Eberth首先发现伤寒杆菌，1885年Salmon分离到猪霍乱杆菌，由于Salmon发现本属细菌的时间较早，在研究中的贡献较大，遂定名为沙门氏菌属Salmonella 。本属细菌绝大多数成员对人和动物有致病性，能引起人和动物的败血症与胃肠炎，甚至流产，并能引起人类食物中毒，是人类细菌性食物中毒的最主要病原菌之一。 根据沙门氏菌的致病范围，可将其分为三大类群。第一类群： 专门对人致病。 如伤寒沙门氏菌、副伤寒沙门氏菌(甲型、乙型、丙型)。第二类群： 能引起人类食物中毒——食物中毒沙门氏菌群，如鼠伤寒沙门氏菌、猪霍乱沙门氏菌、肠炎沙门氏菌、纽波特沙门氏菌等。第三类群： 专门对动物致病，很少感染人，如马流产沙门氏菌、鸡白痢沙门氏菌。致病性最强的是猪霍乱沙门氏菌(Salmonellacholerae)，其次是鼠伤寒沙门氏菌(Salmonella typhimurium)和肠炎沙门氏菌(Salmonella enteritidis)。 一、xx属的生物学特征： 1.形态染色特性： G-无芽孢杆菌。大小通常为 0.7～ 1.5μm × 2.0～ 5.0μm，菌端钝圆，散在，偶有短丝状，无荚膜，除鸡白痢沙门氏菌和鸡伤寒沙门氏菌外均有周身鞭毛，能运动，绝大多数菌株有菌毛。需氧或兼性厌氧菌，生长温度范围为10～42℃，最适生长温度为37℃，适宜pH为

6.8～ 7.8，对营养要求不高，在普通培养基中生长旺盛，胆盐可促进其生长。 2.培养特性： 需氧或兼性厌氧菌；生长温度范围为10～42℃，最适生长温度为37℃；适宜pH为 6.8～ 7.8；对营养要求不高，在普通培养基中生长旺盛；胆盐可促进其生长。 §普通xx： 圆形、光滑、无色半透明、边缘整齐或不太整齐的中等大小(2～4mm)菌落。鸡白痢、鸡伤寒、猪副伤寒、甲型副伤寒沙门氏菌等只能长成细小菌落。 §xxxx和伊红xxxx(EMB)： 菌落无色半透明 §SSxx和DHLxx： 无色半透明、中心黑色或几乎全部黑色(多数菌株)的菌落 §HExx： 形成蓝绿色或蓝色菌落，产硫化氢菌株菌落中心带黑色。 3.生化特性： 发酵葡萄糖、麦芽糖、甘露醇和山梨醇产气；不发酵乳糖、蔗糖、水杨苷和侧金盏花醇；产生硫化氢；原硝酸盐；不水解尿素；不液化明胶；V-P反应阴性;不产吲哚(靛基质试验阴性)；能利用枸橼酸盐(个别菌株不利用)。 表xx属细菌一般生化特性

沙门氏菌检验

沙门氏菌检验1.范围 本法适用于食品中沙门氏菌的检验。 2.设备和材料 处微生物实验室常规灭菌及培养设备外，其他设备和材料如下：2.1冰箱：2℃～5℃。 2.2恒温培养箱：36℃±1℃，42℃±1℃。 2.3均质器。 2.4振荡器。 2.5电子天平：感量0.1g。 2.6无菌锥形瓶：容量500mL，250mL。 2.7无菌吸管：1mL（具0.01mL刻度）、10mL（具0.1mL刻度）或微量移液器及吸头。 2.8无菌培养皿：直径90mm。 2.9无菌试管：3mm×50mm、10mm×75mm。 2.10无菌毛细管。 2.11pH计或pH比色管或精密pH试纸。 2.12全自动微生物生化鉴定系统。 3.培养基和试剂 3.1缓冲蛋白胨水（BPW）。 3.2四硫磺酸钠煌绿（TTB）增菌液。

3.3亚硒酸盐胱氨酸（SC）增菌液。 3.4亚硫酸铋（BS）琼脂。 3.5HE琼脂。 3.6木糖赖氨酸脱氧胆盐（XLD）琼脂。 3.7沙门氏菌属显色培养基。 3.8三糖铁（TSI）琼脂。 3.9蛋白胨水、靛基质试剂。 3.10尿素琼脂（pH7.2）。 3.11氰化钾（KCN）培养基。 3.12赖氨酸脱羧酶试验培养基。 3.13糖发酵管。 3.14邻硝基苯β-D-半乳糖苷（ONPG）培养基。 3.15半固体琼脂。 3.16丙二酸钠培养基。 3.17沙门氏菌O和H诊断血清。 3.18生化鉴定试剂盒。 4.检验程序 沙门氏菌检验程序见图1。 42℃±1℃，18h～24h

36℃±1℃，40h～48h 36℃±1℃，18h～24h 图1沙门氏菌检验程序 5.操作步骤 5.1前增菌 称取25g（mL）样品放入盛有225mLBPW的无菌均质杯中，以8000r/min～10000r/min均质1min～2min，或置于盛有225mLBPW的无菌均质袋中，用拍击式均质器拍打1min～2min。若样品为液态，不需要均质，振荡混匀。如需测定pH值，用1mol/mL无菌NaOH或HCl调pH至6.8±0.2.无菌操作将样品转至500mL锥形瓶中，如使用均质袋，可直接进行培养，于36℃±1℃培养8h～18h。 如为冷冻产品，应在45℃以下不超过15min，或2℃～5℃不超过18h解冻。 5.2增菌 轻轻摇动培养过的样品混合物，移取1mL，转种于10mLTTB内，于42℃±1℃培养18h～24h。同时，另取1mL转种于10mLSC内，于36℃±1℃培养18h～24h。 5.3分离 分别用接种环取增菌液1环，划线接种于一个BS琼脂平板和一个XLD琼脂平板（或HE琼脂平板或沙门氏菌属显色培养基平板）。与36℃±1℃分别培养18h～24h（XLD平板、HE琼脂平板或沙门氏菌属显色培养基平板）或40h～48h（BS琼脂平板），观察各个平板上生长的菌落，各个平板上的菌落特征见表1。

沙门氏菌检验

沙门氏菌检验 This manuscript was revised by the office on December 10, 2020.

沙门氏菌检验 1.范围 本法适用于食品中沙门氏菌的检验。 2.设备和材料 处微生物实验室常规灭菌及培养设备外，其他设备和材料如下：冰箱：2℃～5℃。 恒温培养箱：36℃±1℃，42℃±1℃。 均质器。 振荡器。 电子天平：感量。 无菌锥形瓶：容量500mL，250mL。 无菌吸管：1mL（具刻度）、10mL（具刻度）或微量移液器及吸头。无菌培养皿：直径90mm。 无菌试管：3mm×50mm、10mm×75mm。 无菌毛细管。 pH计或pH比色管或精密pH试纸。 全自动微生物生化鉴定系统。 3.培养基和试剂 缓冲蛋白胨水（BPW）。 四硫磺酸钠煌绿（TTB）增菌液。 亚硒酸盐胱氨酸（SC）增菌液。 亚硫酸铋（BS）琼脂。 HE琼脂。 木糖赖氨酸脱氧胆盐（XLD）琼脂。 沙门氏菌属显色培养基。 三糖铁（TSI）琼脂。 蛋白胨水、靛基质试剂。 尿素琼脂（）。 氰化钾（KCN）培养基。 赖氨酸脱羧酶试验培养基。 糖发酵管。 邻硝基苯β-D-半乳糖苷（ONPG）培养基。 半固体琼脂。 丙二酸钠培养基。 沙门氏菌O和H诊断血清。 生化鉴定试剂盒。 4.检验程序 沙门氏菌检验程序见图1。

42℃±1℃，18h～24h 36℃±1℃，18h～24h 36℃±1℃，40h～48h 36℃±1℃，18h～24h 图1 沙门氏菌检验程序 5.操作步骤 前增菌 称取25g（mL）样品放入盛有225mL BPW 的无菌均质杯中，以8000r/min～10000r/min均质1min～2min，或置于盛有225mL BPW的无菌均质袋中，用拍击式均质器拍打1min～2min。若样品为液态，不需要均质，振荡混匀。如需测定pH值，用1mol/mL无菌NaOH或HCl调pH至±.无菌操作将样品转至500mL锥形瓶中，如使用均质袋，可直接进行培养，于36℃±1℃培养8h～18h。

沙门氏菌检验

沙门氏菌检验 1.范围 本法适用于食品中沙门氏菌的检验。 2.设备和材料 处微生物实验室常规灭菌及培养设备外，其他设备和材料如下： 2.1 冰箱：2℃～5℃。 2.2 恒温培养箱：36℃±1℃，42℃±1℃。 2.3 均质器。 2.4 振荡器。 2.5 电子天平：感量0.1g。 2.6 无菌锥形瓶：容量500mL，250mL。 2.7 无菌吸管：1mL（具0.01mL刻度）、10mL（具0.1mL刻度）或微量移液器及吸头。 2.8无菌培养皿：直径90mm。 2.9 无菌试管：3mm×50mm、10mm×75mm。 2.10 无菌毛细管。 2.11 pH计或pH比色管或精密pH试纸。 2.12 全自动微生物生化鉴定系统。 3.培养基和试剂 3.1 缓冲蛋白胨水（BPW）。 3.2 四硫磺酸钠煌绿（TTB）增菌液。 3.3 亚硒酸盐胱氨酸（SC）增菌液。 3.4 亚硫酸铋（BS）琼脂。 3.5 HE琼脂。 3.6 木糖赖氨酸脱氧胆盐（XLD）琼脂。 3.7沙门氏菌属显色培养基。 3.8 三糖铁（TSI）琼脂。 3.9 蛋白胨水、靛基质试剂。 3.10 尿素琼脂（pH7.2）。 3.11 氰化钾（KCN）培养基。 3.12 赖氨酸脱羧酶试验培养基。 3.13 糖发酵管。 3.14 邻硝基苯β-D-半乳糖苷（ONPG）培养基。 3.15 半固体琼脂。 3.16 丙二酸钠培养基。 3.17 沙门氏菌O和H诊断血清。 3.18 生化鉴定试剂盒。 4.检验程序 沙门氏菌检验程序见图1。

图1 沙门氏菌检验程序

5.操作步骤 5.1 前增菌 称取25g（mL）样品放入盛有225mL BPW 的无菌均质杯中，以8000r/min～10000r/min 均质1min～2min，或置于盛有225mL BPW的无菌均质袋中，用拍击式均质器拍打1min～2min。若样品为液态，不需要均质，振荡混匀。如需测定pH值，用1mol/mL无菌NaOH或HCl调pH至6.8±0.2.无菌操作将样品转至500mL锥形瓶中，如使用均质袋，可直接进行培养，于36℃±1℃培养8h～18h。 如为冷冻产品，应在45℃以下不超过15min，或2℃～5℃不超过18h解冻。 5.2 增菌 轻轻摇动培养过的样品混合物，移取1mL，转种于10mL TTB内，于42℃±1℃培养18h～24h。同时，另取1mL转种于10mL SC内，于36℃±1℃培养18h～24h。 5.3 分离 分别用接种环取增菌液1环，划线接种于一个BS琼脂平板和一个XLD琼脂平板（或HE 琼脂平板或沙门氏菌属显色培养基平板）。与36℃±1℃分别培养18h～24h（XLD平板、HE 琼脂平板或沙门氏菌属显色培养基平板）或40h～48h（BS琼脂平板），观察各个平板上生长的菌落，各个平板上的菌落特征见表1。 表1 沙门氏菌属在不同选择性琼脂平板上的菌落特征 5.4 生化试验 5.4.1 自选择性琼脂平板上分别挑取2个以上典型或可以菌落，接种三糖铁琼脂，先在斜面划线，再于底层穿刺；接种针不要灭菌，直接接种懒氨酸脱羧酶试验培养基和营养琼脂平板，于36℃±1℃培养18h～24h，必要时可延长至48h。在三糖铁琼脂和懒氨酸脱羧酶试验培养基内，沙门氏菌属的反应结果见表2。 表2 沙门氏菌属在三糖铁琼脂和懒氨酸脱羧酶试验培养基内的反应结果

沙门氏菌的检验

沙门氏菌的检验 食品学院 14食品质量与安全1班 刘文敏柳基炜卫杰恒温紫君 201430520115 201430520116 201430520121 201430520122 摘要：本实验采用GB/T4789.4-2010的检测方法测定鸡场中的沙门氏菌。通过本实验学习沙门氏菌的检测方法和技术，了解沙门氏菌的一些生化特性；本实验先用显色培养基找出可疑菌落，再做生化试验找出可疑的典型性的沙门氏菌，再通过血清学试验最终确定是否为沙门氏菌属。 关键词：沙门氏菌接种生化试验血清学鉴定 前言 沙门氏菌病是公共卫生学中具有重要意义的人畜共患病种之一，其病原沙门氏菌属于肠道细菌科。沙门氏菌是一个统称，泛指 2000 多种有紧密连系的细菌，包括引起食物中毒，导致胃肠炎、伤寒和副伤寒的细菌。虽然只有少数人因沙门氏菌而患病，但是，在世界范围内的细菌性食物中毒事件中，由沙门氏菌引起的占大多数。因此，采用科学、合理的方法检验食品中沙门氏菌，已经成为了人们最关心的问题之一[1]。国标法(GB4789.4-2010)是目前中国规定的食品中沙门氏菌的标准检测方法,也是基层实验室普遍采用的检测方法,它根据沙门氏菌的生长特点和生化特性,采取前增菌、增菌、分离、生化试验和血清学鉴定5个步骤进行[2]。 1材料与方法 1.1实验材料 1.1.1仪器设备 均质器、三角烧瓶、平皿、玻璃棒、接种棒 恒温培养箱：36℃±1℃，42℃±1℃ 吸管：1 mL（具 0.01 mL刻度）、10mL（具0.1mL刻度或微量移液器及吸头

电子天平PL602-S，梅特勒-托利多仪器（上海）有限公司； 手提式不锈钢压力蒸汽灭菌锅SYQ-DSX-280B，上海申安医疗器械厂 1.1.2试剂药品 鸡肠、靛基质试剂、沙门氏菌O和H诊断血清、API20E生化试剂盒或VITEKGNI 生化鉴定卡 1.1.3培养基 蛋白胨水（BPW)、四硫磺酸钠煌绿（TTB）、亚硒酸盐胱氨酸（SC）增菌液、亚硫酸铋（BS）琼脂、HE琼脂、三糖铁琼脂、蛋白胨水、尿素琼脂、氰化钾、氰化钾对照、赖氨酸脱羧酶、赖氨酸脱羧酶对照、甘露醇、山梨醇、β-D半乳糖苷（ONPG）培养基 1.2 实验方法 1.2.1培养基的制备 1.2.1.1培养基的配制步骤 蛋白胨水（BPW）：称取蛋白胨10g、氯化钠5g、磷酸氢二钠9g、磷酸二氢钠1.5g、蒸馏水1000ml，将各成分加入蒸馏水中，搅混均匀，静置约 10 min，煮沸溶解，调节 pH，高压灭菌 121 ℃，15 min。分装10瓶，每瓶90ml 四硫磺酸钠煌绿（TTB）：高压灭菌 121 ℃，15 min灭菌冷却后至30℃，每100ml 基础培养液加碘液2ml，煌绿液1ml 1.2.1.2配制培养基的注意事项 （1)按照说明书上的用量进行换算，称取准确分量的合成培养基粉末； （2）加热煮沸溶解培养基时，留意锅内水位的变化，水位下降可再添加适量的水，以免水分蒸发过多，导致后面分装不够量； （3）往试管中放小导管时，注意处理气泡。 1.2.2 沙门氏菌群检测 1.2.2.1沙门氏菌检测程序