PEPPER

INTERNATIONAL PEPPER COMMUNITY MANUAL OF METHODS OF ANALYSIS

1. PREPARATION OF SAMPLE


2. DETERMINATION OF MOISTURE (DISTILLATION METHOD)


3. LIGHT BERRIES IN BLACK AND WHITE PEPPER


4. DETERMINATION OF BULK DENSITY


5. DETERMINATION OF EXTRANEOUS MATTER IN BLACK AND WHITE PEPPER


6. DETERMINATION OF WHOLE INSECTS, (DEAD OR LIVE) IN BLACK AND WHITE PEPPER


7. DETERMINATION OF EXCRETA (MAMMALIAN OR OTHERS) IN BLACK AND WHITE PEPPER


8. DETERMINATION OF INSECT DEFILED BERRIES IN BLACK AND WHITE PEPPER


9. DETERMINATION OF MOULD IN BLACK AND WHITE PEPPER


10. DETERMINATION OF GREY AND BLACK BERRIES IN WHITE PEPPER


11. SALMONELLA SAMPLING PLAN


12. SALMONELLA

Source: International Pepper community

PREPARATION OF SAMPLE

Method No.1.0
Purpose: To prepare homogeneous sample for the determination of moisture in black and white pepper.

1. Apparatus:
   1. Sample splitter/dividing apparatus capable of reducing samples into equal portions.
   
   
   2. Mill capable of grinding sample to particle size U.S. standard No.20/850 micron sieve.
   
   
   3. U.S. Standard No.20/850 micron sieve


2. Procedure:
   1. Mix the samples from a given lot, then split the mixed sample until 100g-200g remains using the sample splitter/deviding apparatus or by coning and quartering.
   
   
   2. Grind the sample to pass through the US Standard No.20/850 micron sieve. At least 99% of sample should pass through the sieve to avoid variation in the composition of the sample. Grind the sample at a rate that does not excessively heat the sample and cause evaporation of essential oil.
   
   
   3. Store the sample in tightly sealed container until the analysis is performed. (Store in refrigerator if the analysis is not performed immediately).
   
   
   4. Mix the sample prior to removing aliquot for analysis. (Allow the sample to return to ambient temperature prior to opening for analysis if stored in refrigerator.

DETERMINATION OF MOISTURE

(DISTILLATION METHOD)
Method No. 2.0
Purpose: To determine the moisture content in black and white pepper by co-distillation with toluene.

1. Apparatus:
   1. Distillation unit with ground glass joints constructed and assembled as shown in Fig.1 with
      1. 500 ml round bottom flask with a T.S. 24/29 joint
      
      
      2. West condenser with drip tip, 400 mm in length with a T.S. 19/26 joint.
      
      
      3. Dean and Stark Water estimation trap, TS 24/29 joint, 10 mL capacity graduated in 0.1 mL intervals.
   
   
   2. Heat source capable of refluxing toluene in the above apparatus. An electric heating mantle with a variable power control or heating mantle supported by a variable speed stirring plate and egg shaped teflon covered stir bar can also be used. If not using string plate add boiling chips.
   
   
   3. Nylon bristle brush, ½ inch in diameter or a wire loop, long enough to extend through the condenser (approx. 450 mm).
   
   
   4. Analytical balance of sensitivity 0.01g


2. Reagents:
   1. Toluene (Laboratory Reagent Grade)


3. Preparation of Sample:
   1. Prepare sample as given in Method No.1.0


4. Procedure:
   1. Weigh aliquot of sample sufficient to yield 2-4 mL of water (about 40g)
   
   
   2. Transfer sample quantitatively to distillation flask and add sufficient toluene to cover the sample completely and to middle of distillation flask. Add a stir bar or boiling chips.
   
   
   3. Assemble the apparatus, and fill the trap with toluene by pouring through the condenser until it just fills the trap and begins to flow into the flask. Insert a loose non-absorbing cotton plug into the top of the condenser to prevent condensation of atmospheric moisture into the condenser.
   
   
   4. Bring to boil and reflux at about 2 drops per second until most of the water has been collected in the trap, then increase the reflux rate to about 4 drops per second.
   
   
   5. Continue refluxing until two consecutive readings 15 min. apart show no change. Turn off the heat and allow to cool to ambient temperature. Dislodge any water held up in the condenser with a brush or wire loop. Rinse the condenser carefully with about 5 mL toluene.
   
   
   6. Read volume of water in the trap.


5. Calculation:
   1. Moisture, % = Vol. of water (mL) x 100 Correction factor Weight of sample (g)
   
   
   2. Correction factor = ML distilled ML added


6. Reporting:
   
   Report the moisture content to an accuracy of 0.0% (v/wt.)


7. Notes:
   1. The apparatus, including the condenser should be cleaned with potassium dichromate sulfuric acid cleaning solution and rinse with water followed by a rinse with 0.05N KOH solution. Rinse with alcohol and drain for 10 minutes.
   
   
   2. A correction blank for toluene must be conducted periodically by adding mL of distilled water to 150 mL of toluene in the distillation flask and run the method as described above.


8. Reference:
   1. ASTA Method No.2.0, Official Analytical Methods of the American Spices Trade Association, Fourth Edition, 1997.
   
   
   2. Official Methods of Analysis of AOAC International, 16th Edition, Vo.II, 1996.

Fig.1: Moisture Distillation Apparatus

LIGHT BERRIES IN BLACK AND WHITE PEPPER

Method No. 3.0
Purpose: To determine the percentage of light berries in black and white pepper.

1. Apparatus:
   1. Balance of sensitivity 0.01g
   
   
   2. Beaker 600 mL. Griffin, Low form approximately 85mm in diameter and 120 mm in height.
   
   
   3. Blotting paper or other similar absorbant material.


2. Reagents:
   1. Alcohol-water solution with a specific gravity of 0.80 to 0.82 at 250C. The alcohol may be ethanol, denatured ethanol or isopropanol.


3. Procedure:
   1. From the composite sample remove two 50g aliquots from opposite quarters after coning and quartering.
   
   
   2. Weigh the sample in the 600 mL Griffin, Low form beaker and add 300 mL of alcohol-water solution.
   
   
   3. Stir pepper corns with a spoon and allow to settle for 2 minutes. Spoon off the berries that float.
   
   
   4. Repeat the stirring, settling and removal of floating berries until two successive additional stirrings raise no more berries to the surface. (Caution: Some berries may remain suspended some distance below the surface of the liquid. These are not considered as floaters).
   
   
   5. Blot the berries, which were removed to free from excessive liquid and spread them out to dry on a piece of blotting paper or other similar absorbent material.
   
   
   6. Air dry for one hour and weigh the air dried light berries to the nearest 0.01 g. Calculate percentage of light berries.


4. Calculation:
   
   % of light berries = Wt. of light berries (g) X 100 Weight of sample (50g)
   
   The range of two determinations shall be averaged and reported as % light berries. If the difference is greater than 0.8%, determine % light berries in a third aliquot. Average all the three values and report % light berries.


5. Reporting :
   
   Report the light berries content to an accuracy of 0.0% (by wt.)


6. Reference :
   
   ASTA method 14.0, Official Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997.

DETERMINATION OF BULK DENSITY

Method No. 4.0
Purpose: To determine the bulk density of whole black and white pepper.

1. Apparatus:
   1. Apparatus for measuring bulk density consisting of :
      1. Cylinder, of capacity 1L or a cylinder of greater capacity, but equipped with apparatus allowing levelling of the product to the 1L level.
      
      
      2. Hopper, of capacity greater than 1L and equipped with a gate;
      
      
      3. Device, for fixing the hopper above the cylinder at a certain distance, to allow free fall of the product into the cylinder from a constant height. Fig-2 shows the example of such an apparatus.
   
   
   2. Balance – top pan balance of 2 kg. Capacity with a sensitivity of 0.1g.


2. Procedure :
   1. Weigh the empty cylinder and tare the balance.
   
   
   2. Place the cylinder on a horizontal plane and set the hopper on it with the fixing device.
   
   
   3. Pour the pepper into the hopper until it is filled. Open the gate and allow the pepper berries to flow freely into the cylinder until the level slightly exceeds the 1L level.
   
   
   4. Level the pepper using the cut off blade attached to the cylinder.
   
   
   5. Remove the hopper and its support and weigh the cylinder filled with the pepper.
   
   
   6. Carry out the above experiment three times.


3. Calculation:
   
   The bulk density of pepper, expressed in grams per litre, is given by the mass of pepper contained in the cylinder. The arithmetic mean of three determinations are taken as the result.


4. Reporting:
   
   Report the bulk density as g/L to an accuracy of nearest whole number.


5. Notes:
   1. The difference between the results of two determinations carried out in rapid succession by the same analyst using the same apparatus shall not exceed 5 g per litre.
   
   


6. Reference:
   1. International Standard ISO 959-1 and ISO 959-2, 1989.

   Fig.2: Apparatus for determination of bulk density

DETERMINATION OF EXTRANEOUS MATTER

IN BLACK AND WHITE PEPPER
Method No. 5.0
Purpose: To determine the amount of extraneous matter in black and white pepper.

1. Apparatus:
   1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear inch or 2 mm square opening).
   
   
   2. Balance of sensitivity 0.01 g.
   
   
   3. Tweezers


2. Procedure:
   1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time, with a good light and against a white back ground into a standard pepper sieve. Pick out any bird, rodent or animal excreta. Do not remove other extraneous/foreign material at this time.
   
   
   2. Shake the sieve moderately back and forth 10 times. Examine the siftings collected on white back ground for live and dead insects and for excreta and separate them.
   
   
   3. Accumulate the siftings. Remove small berries of pepper that pass through the pepper sieve. Weigh the siftings to the nearest 0.1 g. and calculate the percentage of the extraneous/foreign matter by sifting.
   
   
   4. From the composite sample remove two 100g aliquots (A & C) from opposite quarters after coning and quartering.
   
   
   5. Hand pick for any sticks, stones, stems, foreign seeds, other extraneous matter.
   
   
   6. Weigh the pickings to the nearest 0.1 g and calculate the percentage of extraneous/foreign matter by picking.


3. Calculation:
   
   % of extraneous/ foreign matter by sifting (Es) = Wt of siftings (g) X 100 Wt of sub-sample (g)
   
   % of extraneous/foreign matter by hand picking (Ep) = A (in g) + C (in g)2
   
   % of extraneous/foreign matter in the sample = Es + Ep


4. Reporting:
   
   Report the average % of extraneous matter into the sample to an accuracy of 0.0% by wt.


5. Reference:
   1. ASTA Method No.14.0; Official Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997.
   
   
   2. FDA Technical Bulletin No.5, Macro analytical Procedures Manual, 1984, Chapter V.

DETERMINATION OF WHOLE INSECTS, (DEAD OR LIVE)

IN BLACK AND WHITE PEPPER
Method No. 6.0
Purpose: To determine the number of whole insects (dead or live) in black and white pepper.

1. Apparatus:
   1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear inch or 2 mm square opening).
   
   
   2. Tweezers


2. Procedure :
   1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time, with a good light and against a white back ground into a standard pepper sieve. Pick out any bird, rodent or animal excreta. Do not remove other extraneous/foreign matter at this time.
   
   
   2. Shake the sieve moderately back and forth. Examine the siftings collected or white back ground for live and dead insects.
   
   
   3. Count the number of whole insects live or dead in each sub-sample and record.


3. Reporting :
   
   Report the total number of whole insects live/dead in all the sub-samples.


4. Reference :
   1. ASTA Method No.14.0; Official Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997.
   
   
   2. FDA Technical Bulletin No.5, Macro analytical Procedures Manual, 1984, Chapter V.

DETERMINATION OF EXCRETA (MAMMALIAN OR OTHERS)

IN BLACK AND WHITE PEPPER
Method No. 7.0
Purpose: To determine the amount of mammalian or other excreta present in black and white pepper.

1. Apparatus :
   1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear inch or 2 mm square opening).
   
   
   2. Balance of sensitivity 0.01 mg.
   
   
   3. Tweezers


2. Procedure :
   1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time, with a good light and against a white back ground into a standard pepper sieve. Pick out any bird, rodent or animal excreta. Weigh to the nearest 0.1 mg and record. Do not remove other extraneous/foreign matter at this time.
   
   
   2. Shake the pepper sieve moderately back and forth 10 times. Examine the siftings collected on white back ground for excreta. If excreta is present combine the same along with the pickings.


3. Calculation :
   
   Excreta (mammalian or others) present in the sample = Wt. of combined excreta (mammalian/others) in mg across the sub-samples analyzed


4. Reporting :
   
   Report the total amount of excreta (mammalian/others) in mg.


5. Reference :
   1. ASTA Method No.14.0; Official Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997.
   
   
   2. FDA Technical Bulletin No.5, Macro analytical Procedures Manual, 1984, Chapter V.

DETERMINATION OF INSECT DEFILED BERRIES

IN BLACK AND WHITE PEPPER
Method No. 8.0
Purpose: To determine the percentage of insect infested/defiled berries in black and white pepper.

1. Apparatus:
   1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear inch or 2 mm square opening).
   
   
   2. Balance of sensitivity 0.01 g
   
   
   3. Tweezers


2. Procedure :
   1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time, with a good light and against a white back ground into a standard pepper sieve. Pick out any bird, rodent or animal excreta. Do not remove other extraneous/foreign matter at this time.
   
   
   2. Shake the sieve moderately back and forth 10 times to remove the siftings.
   
   
   3. Examine the entire sample for insect defiled/infested berries. Collect all the defiled/infested berries and record the weight.


3. Calculation :
   
   Percentage of insect defiled/infested = Wt. of infested berries Weight of sample x 100


4. Reporting:
   
   Report the average percentage of insect defiled/infested berries to an accuracy of 0.00% by wt.


5. Note:
   1. Pepper may be infested by insects in the growing area during production and harvest. Insects may also attack pepper during storage. Examples of insect infestation and damage range from webbing and frass, insect tunnels or evidence of surface feeding on the pepper berries.


6. Reference:
   1. ASTA Method No.14.0, Official Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997.
   
   
   2. FDA Technical Bulletin No.5, Macro analytical Procedures Manual, 1984, Chapter V.

DETERMINATION OF MOULD IN BLACK AND WHITE PEPPER

Method No. 9.0
Purpose: To determine the percentage of mouldy berries in black and white pepper.

1. Apparatus:
   1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear inch or 2 mm square opening).
   
   
   2. Balance of sensitivity 0.01 g.
   
   
   3. Tweezers


2. Procedure :
   1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time, with a good light and against a white back ground into a standard pepper sieve. Pick out any bird, rodent or animal excreta. Do not remove other extraneous/foreign matter at this time.
   
   
   2. Shake the sieve moderately back and forth 10 tmes to remove the siftings.
   
   
   3. Mix the sub-sample on sieve and weigh 50 g of aliquot into a pan. Hand pick mouldy berries and record the weight.


3. Calculation :
   
   Percentage of mouldy pepper = (Wt. of mouldy berries / 50 g) x 100


4. Reporting :
   
   Report the average percentage of mouldy pepper in all the sub-samples to an accuracy of 0.00% by wt.


5. Note:
   1. Pepper berries bearing mould on more than ¼ of its surface is considered as mouldy.


6. Reference:
   1. ASTA Method No.14.0; Official Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997.
   
   
   2. FDA Technical Bulletin No.5, Macro analytical Procedures Manual, 1984, Chapter V.

DETERMINATION OF GREY AND BLACK BERRIES IN WHITE PEPPER

Method No. 10.0
Purpose: To determine the percentage of grey and black berries in white pepper.

1. Apparatus:
   1. Balance of sensitivity 0.01 g.
   
   
   2. Tweezers


2. Procedure:
   1. From the composite sample remove two 50 g. aliquots from opposite quarters after coning and quartering.
   
   
   2. Pick-out grey and black berries.
   
   
   3. Weigh to the nearest 0.01 g.
   
   
   4. Calculate the percentage of grey and black berries.


3. Calculation:
   
   Percentage of grey & black berries = (Wt. of grey and black berries (g) / Wt. of Sample (50 g.)) X 100


4. Reporting:
   
   Report the average of two determinations to an accuracy of 0.0% (by wt.)


5. Note:
   
   Black/grey berries are berries with or without pericarp, and are dark in color. Those berries with or without pericarp, which are light brown or cream in color should not be considered as black.

SALMONELLA SAMPLING PLAN

Method No. 11.0
PURPOSE: To secure a representative sample of a lot in a manner to avoid contamination and prepare it for Salmonella analysis

1. Instruments for opening containers

Sterile scissors, knives, scalpels, can openers, or other hand tools as required.
2. Sample transfer instruments

Sterile spatulas, spoons, forceps, knives, scissors and swabs as required.
3. Sample containers

Pre-sterilized polyethylene bags or other suitable sterile, non toxic polyethylene bags, that are clean and dry and do not have a viable bacterial count. Sterile glass containers usually are not desirable because of possible breakage and consequential glass contamination of the sampling environment.
4. Microbicide

Medium strength (100 mg/L) hypochlorite solution or other approved disinfectant.
5. Labeling supplies

Pressure-sensitive tapes and labels, tags of adequate size to hold sample information, indelible marking pens.
6. Sample shipping containers

Containers should be made of sturdy corrugated cardboard or other material capable of withstanding abusive shipping conditions.
7. Balance
   Balance width 2000 g capacity having a sensitivity of 0.1 g with a 200 g load.

1. Each sub sample will consist of a minimum of 100 g. Take 100g samples of 30 sub samples at random to ensure that the total sample is representative of the lot. Collect more than one sample unit from large institutional or bulk containers when the number of sample units required exceeds the number of containers in the lot. A sample unit will consist of more than one container when containers are smaller than 100g (e.g., four 25 g containers could constitute a sample unit).

2. Make every effort to use aseptic technique when sampling the pepper to minimize its contamination from hands, nearby materials and air. After sampling, sterilize the sampling tools and equipment to avoid contaminating subsequent samples. Place the sample in sterile plastic bags, label clearly and forward to the microbiological laboratory without delay.

3. When a sample is collected by transferring it to sample containers, a sample control must be submitted which consists of an empty sample container that is exposed to the same conditions under which the sample is collected.

4. Take a 25 g analytical unit at random from each 100g sample unit. When a sample unit consists of more than one container, aseptically mix the contents of each container before taking the 25 g analytical unit. To reduce the analytical workload, the analytical units may be composited. The maximum size of a composite unit is 375 g or 15 analytical units. The minimum number of composite units to be tested for is 2 composite units.

1. Official Microbiological Methods of the American Spice Trade Association (ASTA), First edition, 1976.
2. Compendium of methods for the microbiological examination of foods of the American Public Health Association, Third edition, 1992.
3. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM), Eighth edition, 1998.

SALMONELLA

Method No. 12.0
Purpose: To detect Salmonella in spices and spice products

1. Conical flasks - 500 mL.
2. Test tubes – 25 X 150 mm and 12 X 100mm.
3. Pipettes (sterile) - 1 mL, 5 mL, 10 mL
4. Petri dishes (sterile) (at least 15x90mm)
5. Sterile spoons or spatulas.
6. Incubator, 35 + 1°C
7. Balance, calibrated (with sensitivity of 0.1g)
8. Inoculating loops and needles.
9. Refrigerator, below 8°C
10. Incubator,42±1°C

B. Reagents:

1. Pre-enrichment medium -Trypticase Soya Broth.
2. Enrichment media -
1. Selenite cystine (SC) Broth.
2. Rappaport Vassiliadis (RV)Medium
3. Selective agars -
1. Bismuth Sulfite(BSA) agar
2. Brilliant Green(BGA) agar, Sulphamandelate supplemented.
3. Xylose lysine desoxycholate(XLD) agar.
4. Hektoen enteric (HE) agar.
4. Biochemical media -
1. Triple Sugar Iron (TSI)Agar
2. Lysine Iron (LIA)Agar.
3. IMVIC Media - Peptone water or Tryptone broth, MR-VP medium, Simmons citrate agar.
4. Urea agar.
5. Malonate broth
6. Lysine decarboxylase broth
7. Phenol red dulcitol broth.
8. Phenol red sucrose broth.
5. Salmonella polyvalent sera
1. Salmonella 'O' Antiserum Poly A-I and Vi (Difco)
2. Salmonella 'H'Antiserum Poly A-Z (Difco)
6.  IMVIC reagents.
   1. Kovac's Indole reagent
   2. Methyl Red indicator
   3. Voges-Proskauer test reagents

   Solution 1 (a -naphthol solution)
   a - naphthol – 5g
   Alcohol (absolute) – 100 mL

   Solution 2 (40% KOH)
   Potassium hydroxide – 4g
   Creatine - 0.3g
   Distilled water, sufficient quantity to dilute to 100 mL.

7. Sulpha supplement.
8. Potassium sulphite
9. Physiological Saline (NaCl), solution,0.85% (Sterile).
   Note 1:
   All biochemical media prepared are stored in refrigerator for a period of three months. Pre-prepared culture plates are stored in refrigerator for a period of one month.
   Note 2:
   For preparation of media and reagents, refer to FDA BAM – 8^th edition.
10. Reagents for Gram – Staining :
    1. (a) Crystal violet
    2. (b) Gram’s iodine
    3. (c) Decolorising agent eg. Alcohol
    4. (d) Safranin

C. Procedure:

• Pre-enrichment
1. Using aseptic technique homogenize 25 g of food sample with 225 ml of Trypticase Soya broth. If a larger food sample is required, maintain a sample to broth ratio of 1:9.
2. Before incubating allow it to stand for 60 minutes at room temperature.
3. Loosen cap to allow gas to escape and incubate for 24 h at 35+1°C.
• Enrichment:

  1. Transfer aseptically 1 mL of the pre-enrichment broth into test tube containing 10 mL of Selenite Cystine broth and 0.1 mL into 10 mL Rappaport -vassiliadis medium

  2. Incubate SC broth at 35±1°C and RV medium at 42±1°C for 24 hours.

  3. Selective Agars:
     Streak enrichment media onto any two selective agars and incubate at 35+1°C. Check for typical colonies after 24 and 48 h
     Appearance of colonies:

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

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

     3. Bismuth sulphite (BS) agar: Brown, gray, or black colonies some times 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

     4. Brilliant Green agar:
        Salmonella - slightly pink - white opaque colonies surrounded by brilliant red medium.
        Coliforms:
        Yellow-green colony surrounded by yellow green zone.
        Pick well isolated suspected colonies from selective agar plates and proceed with the biochemical tests. Store picked selective agar plates at 5 to 8 °C. (Inoculate the colony into peptone water, incubate at 35+ 1°C for 5 h. Check whether there is growth and then inoculate into the media).

     Do Gram - Stain as below:

     1. In this method of staining, the bacterial films are covered with a solution of the crystal violet stain and allowed to act for 1 minute.
     2. The stain is washed off with water, and a dilute solution of iodine is added and allowed to remain for 1 minute.
     3. Next the slide is treated with alcohol or a mixture of alcohol and acetone until almost all the colour is removed from the film.
     4. Salmonella is Gram - negative, straight sided, rod-shaped bacteria
         
  4. Agar Slants
     1. Pick well-isolated suspected colonies from selective agar plates and inoculate tubes of Triple sugar Iron Agar slant (TSI) and Lysine Iron agar (LIA) slants by first stabbing the butt and then streaking the surface the slants. Suspected colonies that appears to be mixed culture should be sub cultured on BGA plates.
        Urea Slant: Inoculate from selective agar

     2. Incubate at 35 + 1 °C, 24 + 2 h.

     3. Appearance of slants presumptive positive for Salmonella.
        On TSI agar -
        red (alkaline) slant and yellow (acid) butt. Note also whether H₂S (black) or gas (cracking or pockets in agar) is formed.
        On Lysine Iron agar-
        Salmonella - purple (alkaline) slant and butt; may have blackening if H₂S is produced.
        On Urea Slant-
        Formation of red color indicates production of urease (positive test). Salmonella cultures do not produce urease
         

  5. Biochemical tests for confirmation of Salmonella

     1. IMViC tests-inoculate from TSI slants.

        1. Indole test:-
           Inoculate peptone / Tryptone water and incubate for 48 h at 35 + 1 °C. After incubation add 0.5 mL Kovac's Indole reagent, shake well and examine after 1 minute and 5 minutes. A red color in the reagent layer indicates the presence of Indole. A positive reaction indicates break down the amino - acid tryptophan with the formation of Indole which forms a highly alcohol soluble dye complex with Kovac's Indole reagent.
        2. MR and V.P. tests:-
           For both these tests inoculate the above culture into two tubes of MR-VP medium and incubate them for 48 h at 35 + 1 °C.
           
           Methyl red test:
           
           To one of the above incubated tubes add 3-4 drops of 0.04% methyl red solution. A magenta red color, showing the presence of acid, is regarded as a positive reaction, a yellow color, a negative and an orange color shows an equivocal (+) result. Salmonella gives a positive result in this test.
           
           Voges-Proskauer test:
           
           To the second tube of the duplicate culture add 1mL of a -Naphthol solution and 0.5 mL 40% KOH. Shake the tube vigorously, keep it in a sloped position and examine after 1 h and 4 h. The development of an eosin pink color indicates a positive reaction and a light deep orange brown color indicates an equivocal (+) result. (To speed up the reaction add 2-3 crystals of Creatine.)
        3. Citrate Utilization tests:
           Inoculate the culture in a Simmon's citrate agar slant and incubate for 48 h at 35 + 1 °C. Examine the tubes for growth and blue coloration (alkaline reaction).

        Reaction	Salmonella
        indole	negative
        methyl red	positive
        Voges-Proskauer	negative
        Simmons Citrate	positive

        4. Lysine Decarboxylase broth: Inoculate from TSI. Cotton plug on tightly and incubate at 35+ 1°C, 2 days. Examine daily. Growth with no change in color is a positive reaction. Change in color to yellow (acid) is a negative reaction. Salmonella is positive for lysine decarboxylase.

        5. Malonate broth: Inoculate from TSI and incubate at 35+ 1°C, 48 h. A change in color to blue (alkaline) is a positive test for the utilization of malonate. Salmonella give a negative test (green or unchanged colour).

        6. Phenol red dulcitol broth: Inoculate the broth with small amount of growth from TSI slant and incubate for 24-48h at 35 + 1°C. Most Salmonella species give a positive test indicated by gas formation and acidity (yellow) of the medium.

        7. Phenol red Sucrose broth: Inoculate the broth with small amount of growth from TSI slant and incubate for 24-48 h at 35 + 1°C. Salmonella are sucrose-negative.
            

  6. Serological test (Confirmatory test):
     Using Salmonella Polyvalent "O" Antisera & Polyvalent "H" Antisera.

     1. Prepare a dense suspension of the organism by suspending growth from an 18h TSI agar slant in 0.5 mL of 0.85% NaCl solution.

     2. Using a wax pencil, mark off two circles about 1 cm in diameter on a microscopic slide.

     3. Place a drop of Salmonella Polyvalent "O" Antisera in one of the marked circles and a drop of 0.85% NaCl solution in the other circle (This will act as a negative control).

     4. Using a clean dropper, transfer a drop (0.05 mL) bacterial suspension into each of the circle and mix thoroughly by gently rocking for 1- 2 min. (Avoid excessive evaporation).

     5. Positive agglutination will be rapid and complete. A delayed or partial agglutination should be considered negative.

     Repeat slide agglutination test with Polyvalent "H" Antisera in the same way.

  D. Calculation:

  Nil.

  E. Result & Reporting:

  Since no tolerance of Salmonella is established, report results as: "Salmonella absent/present from negative/positive in 25g of sample"

  F. Reference:

  1. Official Microbiological Methods of the American Spice Trade Association (ASTA), First edition, 1976.
  2. Compendium of methods for the microbiological examination of foods of the American Public Health Association, Third edition, 1992.
  3. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM), Eighth edition, 1998.
  4. Practical Food Microbiology, Methods for examination of food for micro-organisms of public health significance, Second edition, 1995.