Whilst these  last few suggestions are fine for the surface, the numbers of bacteria inside eggs are low. You need to not think of this as a food but as what it was really designed for-a carrier of the developing embryo. It is vital the chicken stops infection so eggs have

1. Enzymes such as lysozyme that stop growth

2. Antimicrobial peptides-notably a specific Beta defensin called gallin

3. High IgY antibody levels.

Salmonella Enteritidis and Pullorum/Gallinarum are adapted for transovarian transmission and survival in eggs.

Neither I or other researchers on vertical transmission of Salmonella use enumeration for eggs as it is not a reliable measure. If bacteria enter via a crack etc. and grow to a high level your nose will tell you before culture.

when ever you are using a selective medium for isolation of bacteria from specimen like egg  enrichment with selenite F broth is better (18 hours incubation).if you are plating the sample directly on selective medium , enriched medium like blood agar also should be used simultaneously because inhibition in selective medium may not allow the growth of pathogen.

Egg contents are  (and generally remain) sterile due to the physiology of the laying hen. If they were not sterile no egg would ever hatch (and produce young chicks) as they are incubated for 21 days at around 37 C! Cracked and soiled eggs are the main risk factors. See also attached article.

contents of egg will be sterile. but egg shell will be cntaminated with faecal matter while laying.. That is why boiled egg is safe to consume than raw egg.

Eggs soiled with faecal matter are (in the EU at least) not allowed to be sold for human consumption. Consumers should not accept them.

Thanks for you answers, egg content can be infected in two ways vertically when the egg is formulated in the hen's oviduct, and horizontally when eggs being laid since the egg pores are wider than size of bacteria. However, many research proved the transmission of Salmonella from eggshell to content. My question actually is can I conclude that eggshell and content free from Salmonella in which the result I have obtained after isolating them on a brilliant green agar negative 0 growth, but I didn't enrich the Salmonella  before streaking them on a plate. Also, would that bias  my result if Salmonella needs to be enriched before culturing 

Enrichment eliminates any possibility of enumeration since the population levels are now different than they were (much higher) from the indigenous counts in the original food item. In other words, a plate count after enrichment is useless data unless you are simply evaluating the ability of enrichment media to increase targeted microbes to higher levels (but then you should be starting with a standardized low level).

Salmonella Enteritidis is known to infect the ovaries of laying hens and lead to internalized contamination from transovarian infection (ova/yolk is contaminated as it is released from the ovary and is contaminated before being enclosed in the shell with egg white.  However, good luck trying to isolate these as estimates are that this occurs perhaps in 1-of-20,000 retail (store bought) eggs. This information has also helped push the regulations that intact shell eggs in stores must be refrigerated (to reduce the possibility of Salmonella in internally-contaminated eggs to grow to even higher levels).  A large outbreak of salmonellosis in the USA a few years ago was attributed to the use of contaminated feed in young poults that were then distributed to hen laying facilities resulting in a greater proportion of internally contaminated eggs than normal and higher levels of consumer illness. One event that has helped push the FDA into tighter regulations on the safety of animal feeds (since they ultimately affect humans). External contamination is another issue and can be abated by washing of eggs....although conditions may allow surface bacteria to gain entrance into the porous egg shell (i.e., warm egg placed in cold environment causes contraction of contents and negative pressure at the pores to draw microbes into the shell).

Peter is right. Salmonella Enteritidis contamination of eggs is actually quite rare-although possibly more common than 1 in 20000 where vaccination of laying hens is not practised. If you are doing this is the UK then you are unlikely to find Salmonella in commercially produced eggs (Lion Mark) as they are from vaccinated hens.  The pronounced decline in human salmonellosis in UK is a consequence of this. The infection rate for Salmonella Pullorum is higher as vertical transmission is important in tis serotype. It is however not relevant as a zoonotic infection and is virtually eradicated in the US/UK. Other seotypes vary rarely infect chicken eggs-though some phage types of Typhimurium can in occasion and some (DT8) can be associated with commercial duck egg production of which there has been a UK & Ireland outbreak in recent years.

If you are going to try to look at Salmonella in eggs content the best way is to either break contents into selenite enrichment overnight then plate on your selective media of choice-XLD or BG would be my choice. If you are interested in total contamination then place the egg in a sterile jar with selenite and shake the whole thing up to break the shell.

I am not aware of E. coli entering an intact egg in the hen or after laying. Anything will be surface faecal contamination or in damaged eggs. Although some avian pathogenic E. coli (APEC) can infect the ovaries and oviduct, there is no indication of vertical transmission. In this case surcease swabbing an whatever method you use for coli will be fine.

Finally I would forget enumeration-numbers are always low. The egg is a strongly anti-microbial environment containing both enzymes and antimicrobial peptides. Numbers are always low. 

If you look at my profile there are a number of papers in there that have the methods for Salmonella in eggs.

I should add even faecal contamination is very unlikely to give any Salmonella-there is so little now in UK layers unless you are sampling 1000s of eggs you are unlikely to find much. The importance of faecal contamination in commercial eggs production is actually low as eggs are routinely cleaned in many countries. I think looking at coli in eggs is  a pretty spurious source of foodborne infection.

If this study is based in a developing industry I'll give the same advice as I have in Africa- work on pooled samples of 50 eggs or more from each source-farm. market, supermarket etc. to work out if there is a problem.

Thanks Peter, and Paul for your answers, and helpful comments. My other concern also is different selective medium for each type of enterobacteriaceae bacteria need to be used, or one medium such violet Red Bile Glucose Agar is enough to assess the microbial quality of table eggs sold in the market. 

Regards 

Mohammed 

Sorry- I could not answer that as it stands. It really depends on what the research question you are trying to address. 

The answers are quite complex. If you take an aliquot of each sample (0,1g, 1 g, 10 g or 25 g) followed by an enrichment procedure you may always obtain semi-quantitative result. If the sample is positive in 10 g but negative in 1g you may mathematically think that you have at least 0,1 Salmonella or E. coli / g. It is obvious that if you are doing an enrichment of a single sample you can not count the number of a specific bacterial cell in the sample and the result must be expressed in a two classes system: positive or negative in the amount of sample you have taken.

If you need Quantitative values, enrichment would not be the way to go. Also are you planning to look at the exterior of the egg or egg content?. If you are looking atthe exterior, use a known quantity of sterile PBS ad allow for the egg to be soaked and then collect the wash, make series of dilution and plate on MacConkeys, Hectone Enteric and Chromogenic agar plates. If have to use only one plate for Salmonella enumeration use Chromogenic agar [plates (expensive but will provide good data: search on Google you will find the details). Tentative confirmation using Polyvalent anti-O antisera that causes agglutination with 30 Sec at room temperature.

Mac will give you an idea as to E. coli numbers and can firm that it is E. coli by inoculating betaglcuronidase tubes and looking for color change from clear to yellow. You can use the same tube to run Indol test using Kovac's. If it is betaglucronidase positive and indol positive it is 99.(% E. coli.

If you are looking at the content, then homoginize the contents make dilutions and plate similarly. As this would be very viscous may be initial dilution could be 1 to 1000 and go from there.

All the best

Whilst these  last few suggestions are fine for the surface, the numbers of bacteria inside eggs are low. You need to not think of this as a food but as what it was really designed for-a carrier of the developing embryo. It is vital the chicken stops infection so eggs have

1. Enzymes such as lysozyme that stop growth

2. Antimicrobial peptides-notably a specific Beta defensin called gallin

3. High IgY antibody levels.

Salmonella Enteritidis and Pullorum/Gallinarum are adapted for transovarian transmission and survival in eggs.

Neither I or other researchers on vertical transmission of Salmonella use enumeration for eggs as it is not a reliable measure. If bacteria enter via a crack etc. and grow to a high level your nose will tell you before culture.

Thanks Nammalwar I did the same approach, in which the sample has been homogenised with PBS and serially diluted, however, I have plated them on a brilliant green agar for Salmonella, and eosin methylene blue to recover E.coli and other faecal organisms. 

First, I think it's important to point out that you don't usually try to 'enumerate' bacteria after enrichment.  The final number after enrichment does not reflect the number in the native/original food item, so it is countless to enumerate.  For pathogens, it is simply a matter of determining presence/absence after enrichment, which is the whole idea of enrichment, to allow growth of low numbers to higher numbers that are more readily capable of being detected.

Second, for something that is likely to be present at such low levels, you would likely have to 'pool samples' to be tested/enriched.  So you would probably need to pool the contents of many eggs, and even then you may not readily detect internalized Salmonella that might only be present in less than 1-in-10,000 (or more) eggs. 

Third, if one was trying to study this aspect of internally-infected (Salmonella) shell eggs, it might be better to isolate ovary-infected hens and test eggs collected from them instead of testing retail/store eggs where the infected eggs are 'diluted' when mixed with many more eggs from non-contaminated birds. 

Peter

I agree. At the height of the UK epidemic in the 1980s the levels of Enteritidis in eggs was between 1:500 to 1:5000. Experimentally it is difficult to infect an egg via normal (oral) infection routes with SE (it can be done with Pullorum). It is a rare event and salmonellosis from eggs often occurs as an outbreak (when an infected egg is used in a raw/lightly cooked dish where many eggs are used-so in catering) rather than sporadic cases (like Campylobacter from chicken meat).  Surveillance of Salmonella egg production here in the UK is very strict (via a National Control Plan) and detection of SE in a flock means any eggs cannot be sold as Grade A table eggs. They can either be pooled and pasteurised and sold to bakeries etc. (at a much lower rate) or it is more usual to depopulate the house and start again.

you can enrich you material in peptone water then streak on BPA agar media for salmonella and EMB and Mac Agar for E.Coli

As usually initial number of salmonella in eggs is very low, direct platting after serial dilution increases the possibility for salmonella to be lost . In addition salmonella cells may be injured and have no ability to grow in selective media directly so enrichment recovers injured bacteria and increases population levels to make them much higher  and easy to detect