Eggs and egg products 219
Eggs and egg products
8.1 Shell eggs
8.2 Bulk liquid egg
Shell eggs
The following methods are recommended for the examination of shell eggs for
salmonellae on the basis of their successful use. Whenever possible an attempt
should be made to quantify the numbers of organisms present.
8.1
8
Disposable gloves should be worn during the examination of shell eggs.
Documentation
Record the following:
• The source of the eggs (i.e. shop, supermarket, farm gate, etc.).
• The type and size of the eggs (i.e. battery or free-range, small, medium, large, etc.).
• The name of the packer or producer.
• The packing and sell-by dates.
• The presence of visible cracks and/or faecal material adhering to the shell.
Equipment
Incubator set at 37 ±1°C
Incubator set at 41.5 ±1°C.
Media
Buffered peptone water (BPW)
Rappaport Vassiliadis soya peptone broth (RVS)
Selective agars: xylose lysine desoxycholate agar (XLD) and a second medium of
choice, e.g. modified brilliant green agar (BGA), manitol lysine crystal violet bile agar
(MLCB), a chromogenic agar, etc.
220 Section eight
Method 1 Individual eggs: examination without shell
disinfection
(Fig. 8.1)
Procedure
(a) Crack the egg against the top of a sterile screw-capped jar or disposable plastic
250 mL container holding 180 mL BPW and drop the contents into it.
(b) Homogenize the mixture by shaking the container.
(c) Drop the shell into a further 180 mL BPW contained in a separate screw-capped
jar.
(d) Incubate the BPW cultures at 37°C for 18 ± 2h.
(e) Subculture the BPW containing egg contents to XLD and a second medium of
choice and proceed with isolation of salmonellae as described in steps (c)–(f) of
Section 6.12, method 1.
(f) Subculture 0.1 mL of BPW containing the shells to 10 mL of RVS broth. Incubate
at 41.5°C for 20–24 h and proceed with the isolation of salmonellae as described
in steps (c)–(f) of Section 6.12, method 1.
A modified procedure to separate yolk and albumen is as follows:
1 Proceed as described in step (a) but with an empty jar or container.
2 Aspirate the albumen with a sterile 10 mL pipette and transfer to a tared sterile con-
tainer. Weigh the transferred albumen and add nine times the weight of BPW, then
mix. This forms a 1/10 homogenate. Continue as described above. Report the final
result in relation to the weight examined.
3 Repeat step 2 above with the yolk using a fresh sterile pipette.
4 Proceed with the isolation of salmonellae as described in steps (d) and (e).
The use of BPW is optional as homogenized egg is a good culture medium. BPW
prevents coagulation of the egg during incubation.
Method 2 Individual eggs: examination with shell
disinfection
(Fig. 8.2)
Procedure
(a) Wipe the shell with a large sterile cotton wool swab moistened with BPW and
then drop the swab into 180 mL of BPW in a sterile screw-capped jar or disposable
250 mL container.
(b) Wipe the shell with a large isopropyl alcohol impregnated wipe or cotton wool
ball soaked in 70% industrial methylated spirit (IMS), or immerse the egg in IMS.
Remove and allow to dry completely.
(c) Crack the egg against the top of a sterile jar or disposable plastic 250 mL container
holding 180 mL of BPW and drop the contents of the egg into it. Discard the shell.
(d) Incubate the BPW cultures at 37°C for 18 ± 2h.
continued
Eggs and egg products 221
(e) Subculture the BPW containing egg contents to XLD and a second medium of
choice and follow the procedure for isolation of salmonellae described in steps
(c)–(f) of Section 6.12, method 1.
(f) Subculture 0.1 mL BPW containing the swab from the shell to 10 mL RVS broth.
Incubate at 41.5°C for 20–24 h and proceed with the isolation of salmonellae
described in steps (c)–(f) of Section 6.12, method 1.
A modified procedure to separate yolk and albumen is as follows:
1 Proceed as described in steps (a)–(c) but drop the yolk and albumen into separate
sterile 60 mL or 250 mL containers.
2 Aspirate the albumen with a sterile 10 mL pipette and transfer to a tared sterile
container. Weigh the quantity and add nine times the weight of BPW, then mix.
Proceed as described in steps (d) and (e) of method 1. Report the final result in
relation to the weight examined.
3 Repeat step 2 above with the yolk using a fresh sterile pipette.
4 Proceed with the isolation of salmonellae as described in steps (d) and (e) of
method 1.
Record data
Separate shell from contents
Shell
Contents
180mL BPW
Incubate 16–20h at 37°C
180mL BPW
Homogenize
Incubate 16–20h at 37°C
Subculture 0.1mL
to 10mL RVS broth
Incubate 20–24h at 41.5°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Salmonella to Reference Laboratory
Report results
Read culture plates
Subculture colonies
to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Day 1
Day 2
Day 3
Day 4
Fig. 8.1 Examination of individual shell eggs without shell disinfection.
222 Section eight
Method 3 Batched eggs: examination without shell
disinfection
(Fig. 8.3)
Procedure
(a) Break the contents of six eggs into a tared stomacher bag. Weigh the contents and
then homogenize. Add an equal weight of BPW. Alternatively mix the egg con-
tents vigorously with an equal weight of BPW in a sterile wide-necked container.
(b) Decant, if necessary, into a sterile flask of 1L capacity or a large wide-necked
screw-capped container and incubate at 37°C for 18 ±2h.
(c) Put the shells into a sterile screw-capped jar containing 180 mL BPW and incubate
at 37°C for 18 ±2h.
(d) Subculture 0.1 mL of BPW shell culture to 10 mL RVS broth and incubate at 41.5°C
for 20–24 h.
(e) Proceed from steps (b) and (d) above with the isolation of salmonellae as
described in steps (c)–(f) of Section 6.12, method 1.
Disposable gloves should be changed after each batch of six eggs.
Method 4 Batched eggs: examination with shell
disinfection
(Fig. 8.4)
Procedure
(a) Wipe the shells of six eggs with a large sterile cotton wool swab moistened
with BPW and then drop the swab into 180 mL BPW contained in a sterile
screw-capped jar or disposable container of 250 mL capacity. Incubate at 37°C for
18 ±2h.
(b) Wipe the shells with a large wipe impregnated with isopropyl alcohol or a
cotton wool ball soaked in 70% IMS, or immerse the eggs in IMS. Allow to dry
completely.
(c) Break the six eggs into a tared stomacher bag and weigh. Homogenize the
contents then add an equal weight of BPW. Alternatively mix the egg contents
vigorously with an equal weight of BPW in a sterile wide-necked container.
Discard the shells.
(d) Decant the egg mixture, if necessary, into a sterile flask of 1 L capacity or a large
wide-necked screw-capped container and incubate at 37°C for 18 ±2h.
(e) Subculture 0.1 mL of the shell swab culture to 10mL of RVS broth and incubate at
41.5°C for 20–24 h.
(f) Proceed from steps (a) and (d) above with the isolation of salmonellae as described
in steps (c)–(f) of Section 6.12, method 1.
Disposable gloves should be changed before proceeding further.
Eggs and egg products 223
Separate shell from contents
Discard shell
Record data
Swab shell with BPW-soaked swab
Disinfect shell with IMS or isopropyl alcohol wipe
Shell swab
Contents
180mL BPW
Incubate 16–20h at 37°C
180mL BPW
Homogenize
Incubate 16–20h at 37°C
Subculture 0.1mL to
10mL RVS broth
Incubate 20–24h at 41.5°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Salmonella to Reference Laboratory
Report results
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Day 1
Day 2
Day 3
Day 4
Fig. 8.2 Examination of individual shell eggs with shell disinfection.
Bulk liquid egg
Raw (unpasteurized) and pasteurized liquid egg should be transported to the
laboratory and examined separately to ensure no cross-contamination occurs.
Raw bulk liquid egg
Sampling
Take samples from the raw egg balance tank immediately before pasteurization.
This will enable the most representative results on levels of contamination to be
obtained. If the balance tank has a sample tap, allow some of the egg to run to
waste to minimize contamination from the tap before taking the sample into a
sterile disposable container. Use sterile disposable dippers to take samples from
8.2
balance tanks without sample taps. Most processing plants will not allow glass
jars to be brought into the plant.
On arrival at the laboratory, defrost samples of frozen egg in a refrigerator
at 0–4°C or at room temperature for 2–3 h. Examine raw egg in 25mL samples.
Many samples of raw egg are likely to contain at least one Salmonella spp.
Where a sample is positive for salmonellae, a most probable number (MPN)
estimation may be performed as described in Section 6.12, method 6.
224 Section eight
Record data
Separate shells from contents
Shell (×6)
Contents (×6)
180mL BPW
Incubate 16–20h at 37°C
Equal volume BPW
Homogenize
Incubate 16–20h at 37°C
Subculture 0.1mL
to 10mL RVS broth
Incubate 20–24h at 41.5°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Salmonella to Reference Laboratory
Report results
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Day 1
Day 2
Day 3
Day 4
Fig. 8.3 Examination of batched shell eggs without shell disinfection.
Method 1 Enrichment culture for Salmonella spp.
Procedure
(a) Add 25 mL of the raw liquid egg to a jar containing 225 mL of BPW plus 5 mg of
novobiocin/L and 10 mg of cefsulodin/L.
(b) Incubate at 37°C for 18 ±2h, then subculture 0.1mL to 10 mL of RVS broth.
(c) Incubate RVS broth for 20–24 h at 41.5°C and subculture on XLD and a second
medium of choice. Proceed with isolation of salmonellae as described in steps
(c)–(f) of Section 6.12, method 1.
Eggs and egg products 225
Record data
Swab six shells with BPW/cotton wool swab
Disinfect six shells with IMS or isopropyl alcohol wipe
Separate shell from contents
Discard shells
Shell swab (¥6)
Contents (¥6)
Equal weight of BPW
180mL BPW
Incubate 16–20h at 37°C
Equal weight of BPW
Homogenize
Incubate 16–20h at 37°C
Subculture 0.1mL
to 10mL RVS broth
Incubate 20–24h at 41.5°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Plate on XLD and another
medium of choice
Incubate 20–24h at 37°C
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Salmonella to Reference Laboratory
Report results
Read culture plates
Subculture suspect
colonies to slope
Incubate 5h at 37°C
Slide agglutinate
Identify serotype
Day 1
Day 2
Day 3
Day 4
Fig. 8.4 Examination of batched shell eggs with shell disinfection.
Pasteurized bulk liquid egg
Council Directive 89/437/EEC [1], on hygiene and health problems affecting
the production and the placing on the market of egg products, specifies the tests
to be performed on heat treated liquid egg and egg products when sampled at
the production premises. The microbiological tests are a mesophilic aerobic
colony count (30°C), Enterobacteriaceae count and absence of Staphylococcus
aureus in 1g and Salmonella in 25 g or mL. For statutory purposes internationally
recognized methods of examination should be used. The provisions of this
directive have been implemented in the UK in the Egg Products Regulations
1993 [2]. In addition to the microbiological criteria, these regulations specify an
alpha-amylase test to ensure that the product is adequately pasteurized (see
Method 7).
Sampling
Take the sample from the pasteurized egg holding tank as close to the
pasteurizer as possible by the procedure agreed on site. Samples of frozen egg
should be defrosted in a refrigerator at 0–4°C or room temperature for 2–3h on
arrival at the laboratory.
226 Section eight
Method 2 Examination for Salmonella spp.
Examine a 25 g or 25 mL portion of sample by Section 6.12, methods 1, 2 or 3.
Method 3 Enumeration of Salmonella spp.
Procedure
(a) Take a sufficient quantity of sample to test 6¥18mL by the multiple tube method
(Section 5.7, method 1).
(b) Add 18 mL to each of six jars containing 180mL of BPW.
(c) Incubate for 18 ±2 h at 37°C.
(d) Subculture 0.1 mL from each jar to separate 10 mL volumes of RVS broth.
(e) Incubate for 20–24 h at 41.5°C and subculture on XLD and a second medium of
choice. Proceed with the isolation of salmonellae as described in steps (c)–(f) of
Section 6.12, method 1.
(f) From the number of jars shown to contain salmonellae, calculate the MPN of the
organisms/g of sample from Table 5.5 (p. 119). The MPN of organisms present
may be estimated in the range from one to >10 per 100 mL.
Method 4 Aerobic colony count
UK legislation allows the use of either a pour plate technique or a surface spread tech-
nique. Methods 5.3, 5.4 and 5.6 are suitable. Incubate the plates at 30°C for 72 ±3h.
Method 5 Enterobacteriaceae
Examine the sample by the method described in Section 6.7, method 1.
Method 6 Staphylococcus aureus
The legislation requires absence of Staphylococcus aureus in 1 g; therefore an enrich-
ment method is required (although the UK legislation specifies a colony count
technique). Section 6.14, method 4 is suitable for detection by enrichment.
Eggs and egg products 227
Method 7Alpha-amylase test
This is a test for the efficiency of the pasteurization process. The time/temperature
combination used for the process should inactivate the enzyme alpha-amylase pres-
ent in the egg so the starch added during the test will not be broken down and will give
a blue coloration with iodine. The presence of large numbers of Bacillus spp. may
cause a false ‘fail’ result due to the presence of bacterial amylases. The test is only
applicable to whole liquid egg.
Reagents
Fresh starch solution: weigh out analytical quality soluble starch to the equivalent of
0.7 g dry starch. Mix with a small quantity of cold water to produce a thin cream.
Transfer this to about 50 mL of boiling water and boil for 1 min. Cool rapidly. Add
three drops of toluene and make the volume up to 100 mL with water. Store at 4°C.
Discard the solution 14 days after preparation.
0.001
M iodine solution: prepare a stock 0.1M solution by dissolving 3.6 g of potassium
iodide in 20 mL of water. Add 1.27g of iodine and make up to 100mL with water. This
solution is stable for a period of 6 months. Just before use, prepare a working solution
by diluting 1 mL of stock solution to 100 mL with water.
15% (w/v) trichloracetic acid solution.
Procedure
(a) Weigh 15g of liquid egg into a flask and add 2mL of fresh starch solution.
(b) Mix well and incubate in a water bath at 44 ±0.5°C for 30min
(c) Allow mixture to cool, then transfer 5 mL to a test tube containing 5 mL of 15%
trichloracetic acid solution. Mix well.
(d) Add 15 mL distilled water. Mix well.
(e) Either filter the mixture through Whatman
®
no. 12 fluted filter paper, discarding
the first few drops of filtrate, or centrifuge the mixture. Transfer 10mL of clear
filtrate or supernatant to a test tube containing 2 mL of 0.001 M iodine solution.
(f) Examine the colour of the solution. A blue-violet colour indicates the presence of
starch and thus the absence of alpha-amylase. To quantify the colour use (i) a
spectrophotometric standard with an optical density of 0.15 when compared
against water in 1 cm cells at a wavelength of 585 mm, or (ii) a disc 4/26 in a
Lovibond
®
colour comparator. If the test solution colour is greater than three
on the disc it indicates the presence of starch and thus the destruction of the
alpha-amylase by the heat treatment applied to the egg.
Controls
Include control samples with every batch of tests. Raw liquid egg serves as a positive
control and boiled liquid egg as a negative control.
Glassware (flasks, tubes, pipettes) for use in this test should not be used for any other
purpose and should be kept separate from other glassware. It should be carefully and
thoroughly cleaned to remove substances that may interfere with the test.
References
1 Commission of the European Communities. Directive 89/437/EEC. Hygiene and
health problems affecting the production and the placing on the market of egg
products. Off J Eur Communities 1989; L212, 22.7.89, 87–100.
2 Great Britain. Statutory Instrument 1993 No. 1520. The Egg Products Regulations.
London: HMSO, 1993.
8.3
228 Section eight
. a large wide-necked
screw-capped container and incubate at 37°C for 18 ±2h.
(c) Put the shells into a sterile screw-capped jar containing 180 mL BPW and. test 6¥18mL by the multiple tube method
(Section 5.7, method 1).
(b) Add 18 mL to each of six jars containing 180 mL of BPW.
(c) Incubate for 18 ±2 h at