microbiological tests in spice industry

MICROBIOLOGY

Microbial infections of concern with respect to spices:

1. Bacterial infections of concern with respect to spices.

v Highest health hazard

• Salmonella typhi

• E.coli (pathogenic)

v Moderate health hazard

•Clostridium botulinum

• Bacillus cereus

• Other salmonella

2. Microbial intoxications

v Bacterial

• Bacillus cereus

• Staphylococcus aureus

• Clostridium botulinum

v Fungal

• Mycotoxins namely aflatoxins

Determination:

• Aerobic mesophilic bacterial count

• Yeast and mold count

• Indicators bacterial count the coliforms.

General procedures for microbial quality assessment:

ENUMERATION:

•Preparation of food homogenate

•Preparation of dilution

•Dispensing of required inoculums into

Petri plates

•Mixing with appropriate agar medium for

preparation of the gel.

•Incubation of plates

•Counting of colonies and interpretation

DETECTION:

•Enrichment with specific growth

Medium

•Plating on selective agar medium

•Detection of particular bacterial colony

on plates

•Biochemical conformation

•Serological conformation

VIBRIO CHOLARAE

MEDIUM:

1) Alkaline peptone water

2) TCBS agar

PROCEDURE:

Prepare 225ml alkaline peptone water, 25g sample is added to it. The solution is incubating 6 hours at 37°C. The prepared solution is loop to TCBS agar in petri plate and incubates 24 hours at 37°C. After incubation observe the growth of yellow colony.

SALMONELLA

DETECTION & IDENTIFICATION

INTRODUCTION:

Salmonella are gram negative nonsporing, facultative anaerobes that belong to the family enterobacteria. They are usually motive and produce both acid and gas from glucose. This rod- shaped bacteria is of important because it can cause food borne salmonellas (food poisoning) over 2,300 servers have been indentified and are distinguished according to their antigenic stricture. A wide variety of foods have been involved in food borne salmonellas, however, egg, poultry , meat and products have been the main vehicles.

SCOPE:

This method is applicable to processed and unprocessed foods and environmental samples. Only the pre-enrichment protocols ground and whole spices and herbs fruits, vegetables, seasoning blends, cheese, cheese bread, flour, flour based blends, onion, garlic & liquid whole eggs have been outlined. Salmonella is pathogen. It is imperative that standard laboratory safely practices be followed. Handle all inoculated media as bio-hazard materials

MEDIUM:

TSB

RV

TTB

XLD

BSA

HEA

TSI

LIA

SALMONELLA CONVENTIONAL

Pre enrichment (225ml/RSB, 25g

sample kept room temperature

for the (60+/5) 18-24 hr incubation)

			RV (0.1 sample – 10 ml media) TTB (1 ml sample)

Enrichment

Selective media

TSI, LIA media

Glucose Lysine Bio chemical identification (IMVIC Test)

XLD:

Suspect colonies are pink or red with or without black center of completely black, same atypical colonies are yellow with or without black center and should be picked for confirmation.

BSA:

Suspect colonies are dark brown, gray or block usually with surrounding black 30 ml and metallic sheet with little or nor darkening of the surrounding medium, same a typical colonies are green with little or nor darkening of the surrounding medium should be picker for confirmation.

HEA:

Suspect colonies are blue green with or without black centers or completely black, same typical colonies are yellow with or without black centers and should be picked for confirmation.

BIOCHEMICAL IDENTIFICATION:

SUBJECT:

IMVIC Test

INTRODUCTION:

An important group of tests to distinguish between the coliform bacteria as well as other organisms in the family enterobacteriaceae is known by the acronym

IMVIC, these tests determine whether an organism has the ability to produce:

a) Indole from tryptophan (I)

b) Sufficient acid to reduce the medium ph to below 4.4, the point of the indicator methyl red (M)

c) Acetoin (acetyl methyl carbind-voges proskaner)

d) The ability to and utilize citrate (C)

These tests may be used for the bio chemical conformation of E. coli as well as acid in the identification if the rapid.

SCOPE:

This method is applicable to all microbiological laboratories.

VIDAS- SALMONELLA (SLM):

VIDAS Salmonella is an automated quantitative test for use on the VIDAS instruments, for the detection of salmonella in food and environmental specimens, using the ELRA technique (Enzyme Linked Fluorescent Assay).

PRINCIPLE:

VIDAS salmonella is an enzyme immune assay for use on the automated VIDAS system for the detection of salmonella antigens using the ELFA. This solid phase Receptacle (SPR) serves as the solid phase as well as the pipetting device the interior of the SPR is coated with anti-salmonella antibodies soaked and pre-dispensed in the sealed reagent strips.

TECRA-SALMONELLA:

225ml mpw + 25g sample

¯ 16-20 hours incubation

Loading to kit (wells)

VIDAS (Vital Immune Diagnostic Assay System)

225ml BPW + 25 g sample (18-24 hours at 36°)

¯

1ml 0.1ml

TTB RV media (6-8 hr incubation)

1 ml

¯

m broth (post enrichment) 42-2°C

incubation (18-24 hrs)

¯

1ml +1ml 1ml -15 boiling water

then cool

T-tube

(95+100°c) ¯

VIDAS ®loading 0.5 ml to vidas

¯

XLD, BSA to confirm

All of the assay steps are performed automatically by the instrument. The reaction media is cycled in and out of the SPR several times.

Part of the enrichment broth is dispensed into the reagent strip. The antigen present will bind to the anti-salmonella are during the washing antibodies coating the inferior of the SPR. Unbound components are eliminated during the washing steps. Antibodies conjugated with alkaline phosphate are cycled in and out of the SPR and will bind to any salmonella antigens which are themselves bound to the antibodies on the SPR. A final wash step removes unbound conjugate. During the final detection step, the substrate (4methyl-umbelliferone) the fluorescence of which is measured at 450nm.

At the end of the assay, results are automatically analyzed by the instruments which is calculated a test value for each sample. This value is then compared to internal reference and each result is interpreted (positive, negative).

TUBE MOST PROBABLE NUMBER (MPN) COLIFORM,

FECAL COLIFORM, AND E. COLI DETERMINATION:

INTRODUCTION:

In general, coli form bacteria are represented by 4 genera of the family Enterobacteriaceae, Escherichia, Enterobacter.

They are gram negative, rod-shaped micro organisms that are commonly found in nature and the intestinal tract of animal humans. They have the capacity to ferment lactose with the production of gas and are able to grow in the presence of bile salts. Because of the genera case with which coli forms can be cultivated and differentiated they are used as indicator organisms to assess food safety and sanitation.

The modified to grow and on the ability of fecal coli forms to grow and produce bas at 45-5c in a lactose broth media. Escherichia coli are the coliform of most concern because its presence in food indicted fecal contamination or unsanitary practices during or after production.

SCOPE:

This is the traditional method for enumerating total coli forms, fecal coli forms and E. coli in foods.

DETERMINATION OF E. coli:

MEDIUM:

§ LSB medium

§ EC Broth

§ EMB agar

§ Macon key agar

PROCRDURE:

MPN coli form, fecal coli form, and E. coli determination:

Prepare sample dilutions. Using a sterile 10ml pipette, transfer 10.0 ml of the 10^-1 dilution into three tubes of double strength. Lauryl sulfate broth. This is equivalent to a 10° dilution. Using a sterile 1 or 2.0 ml pipette and working from the highest dilution to the lowest, transfer 1ml of each dilution into each of three tubes of single strength Lauryl sulphate broth. By working from the separate one for each dilution must be employed. Incubate the tubes at 35°C for 48±2 hours. A tube is presumptively positive if there is effervescence when the tube is gently agitated. Confirm each presumptively positive lauryl sulfate tube by transferring a loop full of the broth into a tube of brilliant green bile broth and into a tube of EC broth. Incubate the Brilliat green Bile tubes at 35±1°c for 48±2 hours and incubate the EC broth tubes in a 45.5±-021° water bath for 48±-2 hours. Production of gas in the BGB tubes incubate, confirmed coli form bacteria. Record the number of positive tubes for each dilution.

Tubes evidencing gas production in EC broth are considered positive for fecal coli forms. Record the number of positive tubes for each dilution. To confirm the presence of E. coli in EC positive tubes, streak Levine EMB agar plates with a loop full of broth from each positive tube of EC broth incubate for 24±-2 hours at 35 ±-1°C

After incubation, examine the plates for typical E. coli: colonies: small, wine colored with or without a gram metallic sheath.

STANDARD PLATE COUNT DETERMINATION:

INTRODUCTION:

The standard plate count or total plate count enumerates viable mesophilic aerobes capable of growth on designated media. the method which is highly empirical, is based on the assumption organism. The standard plate count method or aerobic plate count is one of major application of the colony count method. These procedures are based on the assumption that each microbial cell in a sample will form a visible, separate colony when mixed with an agar or other solid medium and permitted to grow. The microorganism found in foods, often represent the number of population with many growth requirements, it can be possible that some organism may not be able to grow under conditions employed consequently, the counts are, at best, an estimate and should not be reported as absolute.

SCOPE:

This method is applicable to all foods standard plate count aerobic mesophilic

MEDIUM-TPC (total plate count agar):

Prepare the buffer solution (make up 34 g phosphate buffer in 1000ml water). Take 100ml to each bottle. This solution is mix well in ‘8’ shaped 25 times. Take clean and sterilized Petri dishes and 1ml mixed solution is poured into the sample. The sample is then mixed clock wise and anticlockwise and up and down manner. It is then stand for setting the solution. Then incubate at 37°C for 48 hour. After two days observe the growth. The PH range is between 7.0 ± 2.

YEAST AND MOLD DETERMINATION:

MEDIUM:

PDA (Potato dextrose agar)

INTRODUCTION:

Both yeast and mold cause varying degrees of deterioration and decomposition of foods. The ability of fungi to spoil many foods is due to part to their relatively versatile environment requirement.

PROCEDURE:

Prepare the buffer solution. Take 100ml to each bottle. This solution is mix well in ‘8’ shaped for 25 times. Take clean and sterilized Petri dishes and 1ml mixed solutions poured into the Petri dishes. Prepared PDA media poured into the sample. The sample is then mixed clockwise and anticlockwise and up and down manner. It is then stand for few minutes on laminar air flow bench for 3-5 days. After five days count the number of yeast and mold.