FACULTAD DE VETERINARIA

HIGIENE Y SEGURIDAD
ALIMENTARIA
 
Avda. de las Ciencias, s/n
E-10003-Cáceres (Spain)
E Mail: higiene@unex.es
http://higiene.unex.es

LABORATORY CLASSES
FOOD HYGIENE AND SAFETY II

Academic year 2021/2022

INDEX
SAFETY RULES IN THE MICROBIOLOGY LABORATORY................................................................. 2 
1.  MYCOTOXIN DETERMINATION BY CHROMATOGRAPHIC TECHNIQUES IN RIPENED FOODS ......... 3 
2.  ENZYME IMMUNOASSAY FOR DETECTING AFLATOXINS IN RIPENED FOODS ............................ 5 
3.  MOULD AND YEAST COUNT ON INTERMEDIATE MOISTURE FOODS ......................................... 7 
4.  DETECTION OF LISTERIA MONOCYTOGENES IN READY-TO EAT FOODS BY CONVENTIONAL
AND MOLECULAR TECHNIQUES ................................................................................................ 8 

5.  ENUMERATION OF COLIFORMS AND ESCHERICHIA COLI IN BIVALVE MOLLUSCS ................... 12 
6.  QUANTIFICATION OF SULFITE-REDUCING CLOSTRIDIA IN COOKED MEAT PRODUCTS ............. 15 
7.  EGG QUALITY EVALUATION ............................................................................................ 16 
8.  TOTAL VOLATILE BASIC NITROGEN (TVB-N) DETERMINATION IN FISH AND FISH PRODUCTS.. 18 
9.  DETERMINATION OF HYDROXYMETHYLFURFURAL (HMF) IN HONEY.................................... 20 

Student’s name: Ana Libertad Fdez León

 2

SAFETY RULES IN THE MICROBIOLOGY LABORATORY

The general safety rules must be observed. They are available at:
https://www.unex.es/conoce-la-uex/centros/veterinaria/informacion-academica/normativas/NormasseguridadLaboratorios.pdf
The load of microorganisms present in the lab is higher than that commonly present in other
environments, thus leading to more severe infections with different signs from those of the usual
disease.
Microorganisms can infect the body by different routes:
Ingestion. Due to surfaces (fingers, equipment) contaminated by spillage or splashing that are
not detected or not properly disinfected.
Inhalation. Aerosols are formed during common handling.
Injection. A sharp object (wire, shards of glass) punctures the skin. Microorganisms can also
enter the body through minuscule cuts or abrasions that can go unnoticed.
Eye absorption. By splashing contaminated liquids in the eye, or by touching the eyes after
contact with contaminated surfaces.
To prevent infections, the following measures must be observed:
Do not chew gum, drink, or eat in the lab.
Do not pipette by mouth. Use adjustable pipettes, rubber bulb or pipette pump.
Do not put things in your mouth (pen, felt-tip pen, etc.).
Each time you use glassware, be sure to check it for chips and cracks. Notify your lab instructor
of any damaged glassware.
Make sure you always follow the proper procedures for disposing lab waste.
All material in contact with microorganisms, especially the inoculation loop and pipette tips, must
be properly disposed of or sterilized immediately after use.
Report all injuries, accidents and broken glass right away, even if the incident seems small or
unimportant.
If there is a spill of a microbial culture, cover the spill with absorbent material (paper, cloth)
soaked in suitable disinfectant (80% ethanol) and leave for 30 min. Transfer all contaminated
material to an appropriate waste container and remove for a complete decontamination in an
autoclave.
Keep laboratory coats properly fastened.
Gloves must be used and, when necessary, wear safety glasses or goggles.
Do not wear the lab coat or gloves outside the lab. Set aside the lab coat in a separate plastic
bag and wash it at your earliest convenience.
Cover wounds, cuts, and abrasions in exposed parts of the body with waterproof bandage.
Do not leave notebooks, pens, mobile phones or any other object in the working area.
Keep your area neat and organized. Leave your work area clean and gas turned off before
leaving the laboratory.
Wash your hands immediately after handling contaminated materials and prior to leaving the
laboratory.

I have read and fully understand the safety rules in the laboratory:

(Just chek the box for acceptance)

Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-3

1. MYCOTOXIN DETERMINATION BY CHROMATOGRAPHIC TECHNIQUES IN RIPENED

FOODS
Moulds usually outgrow other microorganisms on ripened foods. Moulds pose a hazard to public
health due to their ability to produce mycotoxins in foods of low or intermediate water activity. To
screen for mycotoxins in foods, rapid methods based on chromatographic techniques can be used.
Maximum levels have been set for certain mycotoxins in foodstuffs of animal origin by some countries:
Food for human consumption (Spanish Real Decreto 475/1988):
ꞏ Aflatoxin B1: 5 µg/kg.
ꞏ Sum of aflatoxin B1, B2,G1 and G2: 10 µg/kg.
Meat products (Italian Ministero della Sanità, 1999)
ꞏ Ochratoxin A: 1 µg/kg.
Mycotoxins extraction
Weigh 100 g of food or fungal culture in a Stomacher bag. Add 800 ml of 0.1% formic acid in
acetonitrile:water (9:1) and 500 ml of hexane. Shake in a Stomacher for 4-5 min. Stir for 1 h in the
darkness. Filter twice through filter paper (Whatman no. 1) with sodium sulphate anhydrous. Take the
mixture to a separating funnel and collect the acetonitrile phase. Evaporate to dryness in a rotary
evaporator at 40 ºC. Resuspend in 0.5 ml HPLC-grade acetonitrile and filter through 0.22 µm
nitrocellulose filter into a glass test tube.
Determination of mycotoxins by Thin Layer Chromatography (TLC)
- Activate the TLC plate at 110°C for 2 h.
- Draw the application line with pencil 1 cm above and parallel to the bottom edge of TLC plate.
- Divide the application line with points every 1 cm.
- Apply 5 µL from the extract to one point at the application line by using a capillary tube.
- Apply 5 µL from each standard (citrinin, cyclopiazonic acid, griseofulvin, ochratoxin A, verrucosidin,
aflatoxin B1 and aflatoxin G1) to each point at the application line with a capillary tube.
- Put the plate into a developing chamber containing toluene-ethyl acetate and 90% formic acid (5:4:1).
- When the mobile phase reaches ¾ of the plate length, take out the plate and let it dry on the bench.
- Visualize mycotoxins under ultraviolet light (UV366 and UV254).
- Identify mycotoxins according to retention factor (Rf) and colour compared to the standards.

Mycotoxins (increasing Rf) Colour under UV light (254 and 366 nm)
Aflatoxin G1 Brilliant turquoise blue-green UV366
Aflatoxin B1 Brilliant blue
Griseofulvin Brilliant blue
Citrinin Brilliant yellow-green (comet)
Ochratoxin A Brilliant blue
Verrucosidine Not detected
Sterigmatocystin Maroon
Cyclopiazonic acid Non detected

Results
Sample identification: Eg

Mycotoxin/s identified: Griseofulvin

Interpretation and decision about the product:
According to retention factor and colour we come to the conclusion that there is
griseofulvin in the sample ''Eg''. This product could be commercialized since, nowadays,
there is no legislation in order to standarize this mycotoxin.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-4

DETERMINATION OF MYCOTOXINS BY HPLC-MS (QUANTITATIVE)

Twenty L from the extract are injected into an HPLC equipped with a reverse-phase column
C18, 25 cm length, 4.6 mm diameter and 5 m particle size. Water (solvent A) and 0.05 %
trifluoroacetic acid in acetonitrile (solvent B) vacuum filtered are used as mobile phase. The
separation is performed at 0.8 mL/min flow, with the following gradient elution:
Gradient used for HPLC
Time (min) Flow (mL/min) Solvent A (%) Solvent B (%)
0,5 0.8 90 10
20 0.8 30 70
30 0.8 30 70
32 0.8 10 90
37 0.8 10 90
39 0.8 1 99
44 0.8 1 99
46 0.8 90 10
51 0.8 90 10
Compounds are detected using a mass detector with a chemical ionization source at
atmospheric pressure (APCI). Positive ions without fragmentation are detected in a mass
range of 50-2000. The molecular ions from mycotoxins can be fragmented. Mycotoxin
identification is carried out by comparison of the retention time, molecular weight, and mass
spectra with those obtained from standards for different mycotoxins. Quantification is
performed with standard curves made from known quantities of mycotoxin standards.
Mycotoxin Molecular ion [M+H]+ Standard curve
Aflatoxin B1 313 y = 5620812x
Aflatoxin G1 329 y = 30350793x
Ochratoxin A 404 y = 103443x - 309176
Patulin 137 y = 8955,7x
Cyclopiazonic acid 337 y = 19497x
Citrinin 251 y = 17572937x
Verrucosidin 417 y = 10930x
Griseofulvin 353 y = 73425x
Sterigmatocystin 325 y = 548553x
Being “y” the peak area and “x” the mycotoxin quantity in ng.

Results
Sample: Ap2 Injected volumen: 20 μL

Identified mycotoxin 1: Aflatoxin G1 Mycotoxin 2: Citrinin

Peak 1 area: 98.847.217 Mycotoxin 1 concentration (g/kg): 0,8 μg/kg

Peak 2 area: 429.221 Mycotoxin 2 concentration (g/kg): 0,0061 μg/kg

Interpretation and decision about the product:
Maximum levels of Aflatoxin G1 in foodstuffs of animal origin in Spain is 10 μg/kg (Spanish
Real Decreto 475/1988). The Aflatoxin G1 concentration we have found is 0,8 μg/kg.
Regarding the Citrinin, there is no legislation in order to standarize this mycotoxin, so this
product could be commercialized.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-5

2. ENZYME IMMUNOASSAY FOR DETECTING AFLATOXINS IN RIPENED FOODS

Aflatoxins are carcinogenic and highly toxic metabolic products from certain species of
Aspergillus moulds. Aflatoxin B1 can be found in different foods, almost always together
with aflatoxins B2, B1, and G2.
Conventional detection methods for mycotoxins are time-consuming and usually require
laborious pretreatment. Immunoenzyme assays can be used for rapid and precise
determination of aflatoxins.
For aflatoxin detection, a competitive ELISA test is carried out in microtiter plates. The
wells are coated with immobilized antibodies raised against aflatoxins. Test samples are
added together with horseradish peroxidase-conjugated aflatoxin. The mycotoxin present
in the sample and the peroxydase-conjugated aflatoxin compete for binding to the
antibody. The conjugated enzyme unbound to the immobilized antibodies is removed by
washing.
The reaction is evidenced by adding a chromogenic substrate, which is converted by the
peroxidase to a blue-coloured substance that can be quantified spectrophotometrically at
550 nm. The absorbance a 550 nm is inversely proportional to the aflatoxin concentration
in the sample.
Procedure
- Weigh 10 g of the sample in a stomacher bag with filter, add 50 mL of 70% methanol
(v/v) and homogenize in the Stomacher.
- In a microtiter plate coated with aflatoxin-specific antibody, add in duplicate:
100 μL of distilled water in a well as negative control
100 μL of a solution of aflatoxin B1 (in methanol) in another well (positive control)
100 μL of each sample in separate remaining wells.
- Add 100 μL/well of peroxidase-conjugated aflatoxin B1, mix and incubate for 30 min at
room temperature to allow the conjugate to bind.
- Decant the content from wells and wash three times with 250 μL of water to remove
unbound conjugate and antigens. After the last wash, invert the plate and gently tap the
plate dry on paper towels.
- Add 200 μL/well of substrate, mix, and incubate in the dark for 10-30 min.
- The presence of aflatoxins in the samples is visually checked by a change of colour.
Additionally, absorbance at 550 nm can be recorded to quantify aflatoxins, using air to
set zero absorbance. The intensity of the colour is directly proportional to the amount of
bound conjugate and inversely proportional to the concentration of aflatoxins in the
sample.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-6

Outline of competitive ELISA for detecting aflatoxins

Anti-aflatoxin antibody-coated wells

Ag E Ag E
Ag Ag E
Addition of sample and conjugate (aflatoxin bound to
Ag Ag E peroxidase)
Ag Ag E

Wash to remove unbound aflatoxin

Addition of the substrate

+ 
Results
Sample identification: SAUSAGE C

Data obtained:
-: there is no blue
+: blue
M: there is no blue
M: there is no blue

Interpretation and decision about the product:

Theres no presence of Aflatoxins in the sample ''sausage C''. Processing is correct in
regard to Aflatoxins. At first, there is no risk in the production chain.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-7

3. MOULD AND YEAST COUNT ON INTERMEDIATE MOISTURE FOODS

Fungal contamination of food is important since some fungi can spoil foods and
produce mycotoxins. Microbiological quality of some foods, such as intermediate moisture
foods, can be evaluated by checking mould and yeast contamination. Limit values for mould
and yeast contamination for some foods are:

Honey 102 cfu/g

Edible fats 102 cfu/g

Procedure:
- Weigh 1 g of honey and homogenise with 9 mL of 0.1 % sterile peptone water (dilution
1/10).
- Take a volume of 1 mL from dilution 1/10 to prepare dilution 1/100 by diluting in 9 mL
of 0.1 % sterile peptone water.
- Pipette 1 mL of each sample dilution (1/10 and 1/100) into a sterile plate.
- Add about 20 mL sterile Rose-Bengal Chloramphenicol medium cooled at 45-47ºC.
- Incubate plates IN UPRIGHT POSITION at 25ºC for 4-5 days.
- Count mould and yeast colonies (plate which contains 0-50 colonies should be used).

Mould and yeast growth is characterised by a woolly and cottony aspect of the colonies.

Results
Sample identification: HONEY C
Exact sample weight (CRM): 1g Diluent volume (Vd): 9 mL

Vol. pipetted into plate 1 (VsD): 1 mL Dil. plate 1 (D): 1/10 Colony count in plate 1 (ND): 14

Vol. pipetted into plate 2 (VsD+1): 1 mL Dil. plate 2 (D+1): 1/100 Colony count in plate 2 (ND+1): 0
Resulting colony count: Choose
Count an option 140 cfu/g or ml
Resulting colony count (scientific notation): Count
Choose an option 1,4 x 10 2
cfu/g or ml
Microbiological criterium: n = 1 c= 0 m= 1 x 10 2
ufc/g o ml M= 1 x 10 2
cfu/g or ml
Interpretation and decision about the product:
The sample ''Honey C'' does not satisfy the mycrobiological criteria (less than 100
ufc/g), we have found 140 ufc/g.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-8

4. DETECTION OF LISTERIA MONOCYTOGENES IN READY-TO EAT FOODS BY

CONVENTIONAL AND MOLECULAR TECHNIQUES

Listeria monocytogenes is a highly infective foodborne bacteria, with infective doses lower
than 103 cfu/g or ml. The most common source for this microorganism in food is cross
contamination at processing plants. In addition, L. monocytogenes grows in a wide range of
temperatures, including refrigeration. All this makes unsafely handled and refrigerated foods
the most likely source for listeriosis.
The standardized methods for the detection and enumeration of L. monocytogenes use
classical microbiological procedures based on culture in selective media, such as ISO
11290-1 and 11290-2 methods. These microbiological procedures require considerable time
for incubations (often several days), as well as confirmation tests for identification of the
investigated microorganism.
A possible way to save time in the analysis of pathogenic microorganisms is the use of
techniques based on the detection of specific nucleic acid sequences. These techniques
have brilliantly solved sensitivity problems and can even quantify the number of cells
present.
Detection of L. monocytogenes by conventional methods
According to ISO 11290-1 y 11290-2, four consecutive steps are needed to investigate this
microorganism.
1. Primary enrichment in Half Fraser Broth, with a reduced concentration of the
selective agents. Mix 25 g of sample with 225 mL Half Fraser Broth into a sterile
Stomacher bag and incubate at 30ºC for 24 h.
2. Secondary enrichment in Fraser Broth, with full concentration of selective
agents. Take 100 μL of homogenised sample (from 1st enrichment) and pipette into a
10 mL sterile Fraser broth tube. Mix and incubate sample at 37ºC for 48 h.
3. Plating out on two selective solid media. From the secondary enrichment broth,
streak a small drop with a sterile inoculation loop on the surface of ALOA or Oxford
agar plates. Alternatively, spread 100 μL over the agar surface using a sterile glass
rod. In both cases, incubate plates at 37ºC for 24-48 h.
4. Isolation and identification (on either ALOA or Oxford agar)
- ALOA agar
The incorporation of the chromogenic substrate X-glucoside for the detection of beta-
glucosidase demonstrates the presence of Listeria spp. A specific phospholipase C enzyme
produced by L. monocytogenes will be also detected. Listeria spp. grow on this medium
producing blue/green colonies, with pathogenic species producing similar coloured
colonies surrounded by a characteristic opaque halo.
- OXFORD agar
This agar contains esculin and ferric ammonium citrate. Since all Listeria spp. hydrolyse
esculin, the addition of ferric ions to the medium will detect the reaction. Blackening of the
colony and surrounding medium in cultures containing esculin-hydrolysing bacteria results
from the formation of 6,7-dihydroxycoumarin that reacts with the ferric ions.
Several confirmation tests may be carried out: catalase (+), Gram staining (+), motility test
(+), haemolysis (+), carbohydrate fermentation test (rhamnose + and xylose -). L.
monocytogenes can be also confirmed by PCR techniques.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-9
Overview of Listeria monocytogenes detection by conventional methods
Primary enrichment: 25 g sample + 225 mL Half Fraser Broth (30ºC, 24h)
Secondary enrichment:100 μL homogenised sample + 10 mL Fraser Broth (37ºC, 48h)
Selective solid media (37ºC, 24 h): ALOA agar (blue-green colonies + opaque halo)
Oxford agar (brown-black colonies + opaque halo)
Confirmation (optional): Catalase +
Gram +
Motility test +
Haemolysis +
Rhamnose +
Xylose -

Results
Sample: CHEESE C

Colony morphology: Oxford agar: black colonies

ALOA agar: green colonies without halo
Primary interpretation:
Oxford agar: there is Listeria spp.
ALOA agar there is not Listeria monocytogenes

Confirmation tests (if required):

Interpretation and decision about the product

Listeria monocytogenes was not found in sample ''cheese C'', so this product could be
commercialized (from the point of view of Listeria, since we have not tested other
microorganisms).

L. monocytogenes
ALOA Oxford
Listeria spp.

L. innocua
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-10

DETECTION OF L. MONOCYTOGENES BY POLYMERASE CHAIN REACTION (PCR)

1. DNA extraction from cultures

- Take 100 μL of homogenised sample (from 2nd enrichment) and pipette into a 1.5 mL
sterile Eppendorf tube.
- Heat the sample at 98 ºC for 10 min (heater block).
- Cool down the sample at -80 ºC for 5 min and keep on ice until use (10 μL will be used
for PCR).
2. PCR
The PCR mixture will be made into sterile Eppendorf tubes adding all the following
components for the reaction.

PCR components and volume per reaction

10x Mg free PCR buffer: 20
dNTPs (1 mM): 10
C(+) (5’-TTACGAATTAAAAAGGAGCG-3’) (10 µM): 10 µL
D(-) (5’-TTAAATCAGCAGGGGTCTTT-3’) (10 µM): 10 µL
Taq DNA Polymerase (0.2 U/μL): 10 µL
DNA Template extracted from culture medium: 10 µL
Water: 30 µL
Total volume: 100 µL

Vortex the PCR mixture and place the PCR tubes on the thermal cycler and run the
following thermal cycling program:

94 ºC 2 minutes 1 cycle
94 ºC 30 seconds
50 ºC 1 minute 35 cycles
72 ºC 1 minute
72 ºC 5 minutes 1 cycle

3. Detection of PCR products

Detection of PCR products will be done by 1% (w/v) agarose gel electrophoresis.
Preparation of agarose gel
- Mix 0.3 g of agarose with 30 mL of 1xTAE buffer on a flask.
- Melt the agarose in a microwave oven until it dissolves.
- Cool down the solution at 55-60ºC and add 3 µL of ethidium bromide.
- Pour the gel into the gel-casting tray.
- Insert the comb immediately and wait until gel is solidified.
- Remove the comb and pour enough 1xTAE buffer into the electrophoresis tank
(the liquid level should be above the top of the gel and not overflow).
Preparation of samples to be loaded into the gel
Mix 10 μL of each amplification product with 3 μL of a loading buffer by passing the mixed
solution up and down several times through the pipette tip into a sterile Eppendorf tube or a
piece of Parafilm M. Carefully load all the 13 µL of the PCR product/loading dye mixture into
a well of the gel (make sure you keep track of what sample is being loaded into each well).
Electrophoresis
- Plug the electrodes from the gel box into the power supply.
- Run at 100 V for about 20-45 min using 1xTAE as electrophoresis running buffer.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-11

Visualisation of PCR products

Visualize DNA fragments bound to ethidium bromide under UV light by placing the gel on a
transilluminator. Positive samples to L. monocytogenes are detected by the presence of a
161 bp amplification product. Estimate the size of the PCR product by comparison against
the molecular weight ladder.

Results
Sample: CHEESE C

Data obtained
Positive control did not appear properly.

Interpretation and decision about the product:

We cannot come up to a conclusion, since the results were not clear.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-12

5. ENUMERATION OF COLIFORMS AND ESCHERICHIA COLI IN BIVALVE MOLLUSCS

E. coli normally lives in the intestines and is shed in the faeces of man and food-producing
animals. The presence of E. coli is an indicator of faecal contamination and potentially
pathogenic bacteria. It is recommended using E. coli rather than faecal coliforms to
indicate faecal contamination in shellfish harvesting areas.
Microbiological criteria related to E. coli in foods (Reg. 2073/2005) includes the following:
Minced meat: m= 50 cfu/g, M = 500 cfu/g, n = 5, c = 2
Meat preparations: m= 500 cfu/g, M = 5 000 cfu/g, n = 5, c = 2
Cheeses made from milk or whey that has undergone heat treatment:
m = 100 cfu/g, M = 1 000 cfu/g, n = 5, c = 2
Butter and cream from milk that has undergone a heat treatment lower than pasteurisation
m = 10 cfu/ml, M = 100 cfu/ml, n = 5, c = 2
Shelled and shucked products of cooked crustaceans and molluscan shellfish
m = 1 cfu/ml, M = 10 cfu/ml, n = 5, c = 2
Live bivalve molluscs: m = 230 MPN/100 g, M = 700 MPN/100 g, n = 5, c = 1
The usual method to enumerate E. coli in live bivalve molluscs is by the liquid-medium
culture technique and calculation of the most probable number (MPN). The method is
suitable for the enumeration of E. coli cells that may have been subjected to stress due to
dehydration, freezing, and exposure to a saline (such as marine) environment or damage by
disinfectants, such as chlorine-containing products. It is particularly useful when the number
of expected cells is rather low.
MPN Procedure (based on ISO 16649):
a) Sample preparation for shellfish. Weigh 5 g (±0.1 g) of shellfish flesh and intervalvular
fluid and mix with 45 ml sterile peptone water and homogenize. From this 1/10 or 10-1 initial
suspension, prepare a 1/100 or 10-2 dilution.
b) Inoculation and incubation. Place 3 series of 3 test tubes each in a tube rack,
containing 10 ml of double strength Brilliant Green Bile Broth (BGBB) in the first series, and
10 ml single strength BGBB in the rest. The simultaneous presence of bile and brilliant
green inhibits almost all Gram-positive bacteria and Gram-negative other than coliforms.
Add 10 ml of the 1/10 suspension to each of three tubes containing double strength BGBB.
Add 1 ml of the 1/10 suspension to each of three tubes containing single strength BGBB.
Add 1 ml of the 1/100 suspension to each of three tubes containing single strength BGBB.
Incubate all inoculated tubes at 37ºC for 24 h. Examine each tube for
any trace of gas in Durham’s tube due to lactose fermentation,
leading to acid and gas formation in presence of bile salts.
c) Calculation of results. Growth of coliform bacteria is shown by
turbidity and gas production in the Durham tubes due to lactose
fermentation. Count the number of tubes containing gas at each
dilution and use the table to obtain MNP. Report the result as MPN of
E. coli per 100 g.
Confirmation of E. coli (based on ISO 16649-3):
From the BGBB tubes with acid production, subculture by streaking
with a loop onto TBX agar and incubate at 44 ºC for 24 h. TBX agar
contains a chromogenic ingredient, X-glucoronide, which detects the
presence of the enzyme glucuronidase. Most E. coli can be
differentiated from other coliforms by glucuronidase activity.
After incubation, examine the TBX agar for the presence of blue or
blue-green colonies, indicating the presence of E. coli.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-13

SUMMARY OF COLIFORMS AND E. coli INVESTIGATION

Weigh 5 g of shellfish and prepare 1/10 and 1/100 dilutions.

Prepare 3 series of three tubes each with: 10 ml from 1/10 dilution in double strength BGBB
1 ml from 1/10 dilution in single strength BGBB
1 ml from 1/100 dilution in single strength BGBB
Incubate at 37 ºC, 24 h.
Confirmation of E. coli: TBX agar, blue/blue green colonies, 44ºC, 24 h
Results are satisfactory, if all the five values observed are ≤ 230 MPN/100 g or
if only one of the five values observed is > 230 but ≤ 700 MPN/100 g
unsatisfactory, if any of the five values observed are > 700 MPN/100 g or
if at least two of the five values observed are > 230 MPN/100 g.

Results

Sample: MUSSEL C Exact amount of sample: 5g

Results: No. of + tubes in dilution 10:10: 2 1:10: 2 1:100: 0

MPN: 2'1 cfu/ml o g MPN: 210 cfu/100 g

Preliminary interpretation:
We cannot come up to a conclusion yet, confirmatory tests are needed.

Results from confirmatory tests

The sample ''Cheese C'' is positive to E. coli, since we can see blue-green colonies in the
TBX agar.

Interpretation and decision about the product:

n1: 210 ufc/100g ; n2: 430 ufc/100g ; n3: +24.000 ufc/100g ; n4: 48.000 ufc/100g ; n5:
48.000 ufc/100g

The results are unsatisfactory since there is more than one value between 230 MPN/100 g
and 700 MPN/100 g. This product could not be commercialized since it does not comply
the microbiological criteria.
Academic year 2021/2022
Most Probable Number (MPN) index per gram or millilitre using three series of three
tubes each.
Positive tubes
10 ml 1 ml
1:10 1:10
----------------------------------------------------------------------
0 0
0 0
0 1
1 0
1 0
1 1
1 1
1 2
2 0
2 0
2 1
2 1
2 2
2 2
3 0
3 0
3 0
3 1
3 1
3 1
3 2
3 2
3 2
3 3
3 3
3 3
3 3
------------------------------------------------------------------------------
Laboratory work. Food Hygiene and Safety II-14
.
1 ml MPN per
1:100 gram or ml
0 <0.3
1 0.3
0 0.3
0 0.4
1 0.7
0 0.7
1 1.1
0 1.1
0 0.9
1 1.4
0 1.5
1 2.0
0 2.1
1 2.8
0 2.3
1 3.9
2 6.4
0 4.3
1 7.5
2 12
0 9.3
1 15
2 21
0 24
1 48
2 110
3 > 240
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-15

6. QUANTIFICATION OF SULFITE-REDUCING CLOSTRIDIA IN COOKED MEAT

PRODUCTS

Sulfite-reducing anaerobic bacteria belong to Clostridium genus and are endowed with the
ability to reduce sulfite to sulfide. They are highly resistant to extreme environmental
conditions due to their ability to produce heat-resistant spores. Sulfite-reducing clostridia are
commonly used to evaluate the hygienic conditions of water and foods from animal origin.
Their number is low in fresh products obtained under good hygienic practices.
The detection of sulfite-reducing clostridia is based on culture media that contains sodium
sulfite and an iron source, such as Sulfite Polymyxin Sulfadiazine (SPS) agar. The sulfite-
reducing activity leads to sulfide production, which in turn reacts with the iron source to
produce a black ferrous sulfide and the producing colony becomes black.
Clostridium cultures require an oxygen-free environment, which is achieved by
anaerobic cultures, as follows:
- Using anaerobic jars.
- Sealing the test tubes with a paraffin plug.
- Inoculating the tubes by deep pitting
Some reference values for sulfite-reducing clostridia in foods are the following:
Rennet and curdling enzymes for milk 1 cfu/g or ml
Cooked ham and other cooked meat products 1x102 cfu/g

Procedure (based on ISO 15213):

- Weigh 10 grams of food sample and homogenize with 90 mL of 0.1 % sterile
peptone water (dilution 1/10).
- Take a volume of 1 mL from dilution 1/10 to prepare dilution 1/100 by diluting in 9 mL
of 0.1 % sterile peptone water.
- Using a glass pipette, transfer 1 mL of each sample dilution (1/10 and 1/100) into
two separate SPS agar tubes cooled at 45-47 ºC. Insert the pipette tip down to
the tube bottom and slowly drop the inoculum from bottom to top.
- Allow the medium to solidify and plug the tubes with a layer of sterile nutrient agar.
- Incubate the tubes at 37 °C for 24-48 h.
- Count black colonies and multiply by the dilution factor of each tube.

Results:
Sample: MEAT C
Exact sample amount (CRM): 10 g Diluent volume (Vd): 90 mL
Vol. pipetted into tube 1 (VsD): 1 mL Dil. tube 1 (D): 1/10 No. of colonies in tube 1 (ND): +300 ufc/mL
Vol. pipetted into tube 2 (VsD+1): 1 mL Dil. tubo 2 (D+1): 1/100 No. of colonies in tube 2 (ND+1): 3 ufc/mL
Resulting colony count: Choose
Less thanan option 4 cfu/g or ml
Resulting colony count (scientific notation): LessChoose
than an option 4 x 10 2
cfu/g or ml
Microbiological criterium: n = 1 c= 0 m= 1 x 10 2
ufc/g o ml M= 1 x 10 2
ufc/g o ml
Interpretation and decision about the product:
The reference value for sulfite-reducing clostridia in cooked meat products is 1x10^2
cfu/g. We have found 4x10^2 cfu/g, so this product could not be commercialized since it
is not fit for human consumption.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-16

7. EGG QUALITY EVALUATION

Egg quality is a general term that refers to several standards that define both internal
and external quality. External quality is focused on shell cleanliness, texture and shape,
whereas internal quality refers to egg white (albumen) cleanliness and viscosity, size of the
air cell, yolk shape and yolk strength.
As soon as the egg is laid, its internal quality starts to decrease: the longer the storage time,
the more the internal quality deteriorates. The egg white becomes thinner and the yolk
structure weakens.
Washing or wiping eggs to remove adhering dirt or foreign materials is not permitted under
EU regulations. This is because the egg shell has a natural protective coating, known as the
cuticle or “bloom”, which seals the pores. This cuticle prevents bacteria from getting into the
egg and reduces moisture loss. Washing removes the bloom. The main cause of
microbiological contamination is the washing or wiping of eggs. Wetting the shell allows
microorganisms on the shell to penetrate and multiply inside.
Immersion (brine or float) test: Gently place the egg in the brine (10% NaCl); Fresh eggs
will sink to the bottom and lay flat on their sides. Eggs that are a few weeks old will stand
on one end at the bottom of the beaker. If they float to the surface, they are no longer fresh
enough to eat.
Observation against a strong light source
(candling): To determine egg quality, an
ovoscope is used. The egg is placed at the
opening in the ovoscope and is illuminated.
Under the candling lamp, the yolk appears as
a faint shadow, whereas the embryo would
appear as a dark shadow.
The size of the air cell can be assessed, as
well as blood spots and cracks of the shell
can be observed. The air cell height can be
measured. Eggs with an air cell height of
more than 6 mm are not acceptable. Mobility
of air cell and yolk can also be considered.

UV-A blacklight inspection of eggs: The shells of eggs must be fully developed and
contain no breaks. Eggshell cuticle integrity can be evaluated under UV light. Purple colour
means not damaged cuticle. Patches deprived of cuticle appear whitish or brownish.

Checking quality of the yolk and white: Crack the sample egg open on a plate. The egg
white should be translucent, thick, and clean. Thin, cloudy and watery whites spreading
over a wide distance on the plate reveal unsound eggs. A rounded yolk standing up in the
viscous white is also a sign of fresh eggs. Blood spots may be present on egg yolk, due to
rupture of blood vessels during the formation of the egg in the oviduct. Eggs with blood
spots are fit for consumption.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-17
CLASSIFICATION (Reg. 589/2008, http://data.europa.eu/eli/reg/2008/589/2017-11-25)

Quality Class A or fresh egg (for direct Categoría B (either for food
characteristics human consumption) industry or non-food industry)
Shell and cuticle Normal shape, clean, undamaged
Air space <6 mm, stationary (Extra <4 mm)
Visible on candling as a shadow
only, without clearly discernible
Yolk outline, slightly mobile upon
Eggs which do not meet the
turning the egg, and returning to a
quality characteristics for class A
central position;
White Clear, translucent
Germ Imperceptible development
Foreign matter Not permitted
Foreign smell Not permitted

Tolerance for quality defects

1. Batches of Class A eggs:
(a) at the packing centre, just before dispatch: 5 % of eggs with quality defects;
(b) at the other marketing stages: 7 % of eggs with quality defects.
2. For eggs marketed as ‘extra’ or ‘extra fresh’, no tolerance shall be
allowed for the height of the air space at the time of packing or import.
3. Where the batch checked contains fewer than 180 eggs, the percentages referred to in
paragraph 1 shall be doubled.

Results
Sample: EGG: 3ES1902128C (C.PREF 12/04)
Float test in 10% NaCl solution
This egg floats to the surface.

Candling test:
The yolk appears as a faint shadow.
The air cell height is less than 6 mm, and it is motionless.
Blacklight inspection:
Purple colour is not uniform throughout the eggshell.

White and yolk examination:

We can see a rounded yolk standing up in the viscous white. The white is clear and
translucent.
Interpretation and decision about the product:
After analyzing the freshness of this egg we can come up to the conclusion that it is class
A, it is fit for consumption.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-18

8. TOTAL VOLATILE BASIC NITROGEN (TVB-N) DETERMINATION IN FISH AND FISH

PRODUCTS

Both bacterial activity and chemical reactions due to the autolytic enzymes of fish
tissue result in basic nitrogen compounds, such as ammonia and different volatile amines,
including trimethylamine, dimethylamine, and monomethylamine. This group of nitrogen
compounds, known as Total Volatile Basic Nitrogen (TVB-N), are useful for assessing fish
freshness.
Regulation 2019/627 sets the limits for TVB-N and includes the microdiffusion method
described by Conway y Byrne (1933) among the approved methods of analysis. This is a
quantitative and simple method, which is based on the volatilization of the basic nitrogen
compounds in a basic solution. The volatile compounds diffuse into the boric acid solution
to form boric acid salts that are reduced to HCl-salts during titration.

(TVB-N) limit values for certain categories of fishery products (Regulation 2019/627)
Unprocessed fishery products shall be regarded as unfit for human consumption
where organoleptic assessment has raised doubts as to their freshness and chemical
checks reveal that the following TVB-N limits are exceeded:

TVB-N limit values

Fish species (mg/100 g of flesh)
Sebastes spp, Helicolenus dactylopterus, Sebastichthys capensis 25
Pleuronectidae family (except halibut: Hippoglossus spp) 30
Salmo salar, Merlucciidae family, Gadidae family 35
Whole fishery products used for fish oil for human consumption 60

Procedure
The Conway method, involving microdiffusion of an extract deproteinized by
perchloric acid, is used.
Preparation of the sample
- Weigh 10 g  0.1 g (from 100 g samples from at least three different points) and mix
them together by grinding.
- Mix with 90 mL perchloric acid 0.6 N and homogenize for 2 min in a blender.
- Filter or centrifuge at 3,500 rpm for 10 min and collect the supernatant.
The extract obtained can be kept for at least seven days between 2 ºC and 6 ºC.
Quantification of TVB-N: Method described by Pearson (1968).
- Add 2 mL of a 3 g/100 mL boric acid solution containing the pH indicator (1 g/100 mL
phenolphthalein solution) into the inner chamber of the Conway unit.
- Add 1.5 mL of the previously obtained extract into the outer chamber, followed by the
addition of 1.5 mL of saturated potassium carbonate, and immediately close the
Conway unit with a silicone-sealed lid.
- Gently rotate clockwise and anticlockwise and incubate at room temperature for 3 h at
37 ºC or overnight at room temperature.
- Titrate the TVB-N in the inner chamber with HCl 0.01 N or 0.05 N until the green-
colored solution turns pink.
- Calculate the concentration of TVB-N in the tested sample normalized against a blank
control. The concentration shall be expressed in mg/100 g.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-19

(VTS-VT0) x NHCl x 14 x [(VHClO4 + Ws) /VE]

TVB-N (in mg/100 g) = x 100
Ws
Where VTS: Volume of HCl for sample titration (in ml)
VT0: Volume of HCl for blank titration (in ml)
NHCl: HCl Normality
14: Molecular weight of N
VHClO4: Volume of perchloric acid added to sample
VE: Volume of sample extract added to the outer chamber of Conway plate (in ml)
Ws: Weight of sample (in g)

Results

Sample: FISH 2

Data obtained:
Titrate the TVB-N in the inner chamber with HCl 0.001 N:
- sample: 0,5 ml HCl
- blank: 0 ml HCl

TVB-N = [ (0,5-0)*0,001*14*[(45+10)/1,5]*100 ] /10 = 5,775 mg/100g

Interpretation and decision about the product:

The TVB-N limit value for hake is 35mg/100g; we have detected 5,775mg/100g in sample
''fish 2'', so it fits the microbiological criteria.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-20

9. DETERMINATION OF HYDROXYMETHYLFURFURAL (HMF) IN HONEY

Hydroxymethylfurfural (HMF) is not an intrinsic component of honey, but is easily
formed from sugars, in the presence of acids and / or by heating.
Small quantities of HMF can be found in honey due to the acid catalysed dehydration of
hexoses. The final content will depend on the initial content, storage time, temperature,
and honey composition, mainly acidity and humidity.
Long-term storage can lead to accumulation of HMF, which occurs in parallel with the
loss of diastase and invertase activity. The HMF content of honey is used as an index of
the degree of freshness and can also be used to detect the fraudulent addition of
inverted sugar, since this process involves heating the product, or inadequate heating of
honey.
Aldehydes derived from furan, the main one being hydroxymethylfurfural, react with
barbituric acid and para-toluidine to give a red compound determined at 550 nm.
The Spanish legislation authorizes a maximum of 40 mg of HMF/kg of honey (RD 1049/2003).
Reagents
a) Barbituric acid solution. Dissolve 500 mg of barbituric acid in distilled water by
heating slightly over a water bath at 100°C. Make up to 100 mL with distilled water.
b) Para-toluidine solution. Place 10 g of para-toluidine in a 100 mL volumetric flask;
add 50 mL of isopropanol, and 10 mL of glacial acetic acid. Make up to 100 mL with iso-
propanol. This solution should be kept in a dark place and renewed daily.
c) Oxygen-free distilled water. Heat to boiling and bubble with nitrogen the distilled
water. Then, allow to cool.
Procedure
1. Sample preparation
Honey sample (S)
- Weigh about 2 grams (± 0.01 g) of honey sample and place it into a beaker.
- Dissolve in 10 mL of oxygen-free distilled water by gently manually stirring the solution
with a spatula. Do not heat.
Heated honey sample (HS)
- Weigh about 2 g (± 0.01 g) of honey sample and place it into a beaker.
- Dissolve in 10 mL of oxygen-free distilled water by heating in a magnetic stirrer for 2 min.
Floral variety honey
- Weigh about 2 g (± 0.01 g) of one floral variety of honey into a beaker.
- Proceed as in “honey sample”.
Academic year 2021/2022 Laboratory work. Food Hygiene and Safety II-21

2. Photometric determinations
Honey sample (M)
- Pipette 2.0 mL of the sample solution (M) to each of two tubes and add 5.0 mL p-
toluidine solution to both.
- Add 1.0 mL of distilled water to one tube (blank value) and 1.0 mL of barbituric
acid solution to the other with gentle shaking.
Heated honey sample (MC)
- Pipette 2.0 mL of the heated honey sample solution (MC) to a tube and add 5.0
mL p-toluidine solution.
- Add 1.0 mL of barbituric acid solution to the tube with gentle shaking.
Floral variety (O)
- Pipette 2.0 mL of the floral variety honey solution. Proceed as in “honey sample”.
Carry out immediately and complete in 1-2 min. Measure the absorbance of the sample
against the blank as soon as the colour intensity has reached a maximum (3-4 min after
adding the barbituric acid solution), using 1 cm cuvettes at 550nm.
Calculation and expression of results
The content of HMF is calculated as follows:
Absorbance x 19.2
mg of HMF/100 g =
Path length of the cuvette (1 cm)

Express the results in mg of HMF per 100 g of honey.

Results
Sample No. (M): 2 Floral variety (O): ROMERO
Data obtained:
A1: 0,51 ; A2: 0,36 ; A3 (heated): 0,399

*1: mg of HMF/100 g = (0,51*19,2)/1 = 9,79 mg HMF/100g = 97,9 mg HMF/kg

*2: mg of HMF/100 g = (0,36*19,2)/1 = 6,91 mg HMF/100g = 69,1 mg HMF/kg
*1: mg of HMF/100 g = (0,399*19,2)/1 = 7,66 mg HMF/100g = 76,6 mg HMF/kg

Interpretation and decision about the product:

The Spanish legislation authorizes a maximum of 40 mg of HMF/kg of honey, so the
results are unsatisfactory. Honey number 2 could not be commercialized, we have to
check to see what the problem is and correct it.

*Some floral varieties are heather (brezo), holm oak (encina), rosemary (romero),
eucalyptus (eucalipto), and orange blossom (azahar).