After such a process has evolved, monitoring of processing instruments
to insure that proper time and temperature levels
have been attained is of much greater value than the continued checking for bacterial populations.
Yet, the packer must always remember that his process
is equipped to handle a specified type and level of contamination
and that it will be inadequate if raw materials having unexpectedly large numbers of organisms
having increased resistance are encountered.
Procedures for the determination of total mesophilic aerobes are described as :
-
Weight 25 grams of sample utilizing aseptic technique,
( This means sterilizing everything used to handle the sample to prevent contamination)
-
Place the 25 grams into a sterile blender with 250 ml sterile peptone water blank
and blend well (about 2 minutes). This is the first dilution is 1:10 for the homogenate.
-
Pipette 1.0 ml of this solution to another sterile 9 ml water blank.
Cap the bottle and shake well. This is the second dilution and is 1:100 for the homogenate.
-
Using a clean pipette, transfer 1 ml of the second dilution to another sterile 9 ml
peptone water blank. Cap and shake.
This is the third dilution and is 1:1000 for the homogenate.
-
Set up five sterile petri plates and from the three dilutions,
pipette the following quantities to a petri-plate : (each to a different plate)
From the first dilution - 0.1 ml.
From the second dilution - 0.1 ml and 1.0 ml.
From the third dilution - 0.1 ml and 1.0 ml.
Label the plates with a wax pencil.
-
Add Plate Count Agar to the plates.
Be sure the agar is cool enough to hold in your hand before you do this !
Swirl the plates gently to mix the sample with the agar.
Allow to harden and store inverted.
Incubation may be at 35℃ for two days (as total mesohpilic count)
or 7℃ for 10 days (as total psychrotrophic count).
-
Count the plate(s) that has between 30 and 300 colonies.
- Calculate the dilution factor and report the results as number per gram of sample.
Coliforms and Escherichia coli
Tests for coliform organisms are of value
in assessing the sanitary condition of the raw matreials and of the final product.
For this test, suitable dilutions are inoculated into fermentation tubes of LST
（Lauryl Sulfate Tryptose）broth and after these have incubated at 35℃ for 1-2 days,
inoculating loopful of solution into fermentation tubes of BGLB
（Brilliant Green Lactose Bile） broth from all LST cultures showing gas production.
After incubating at 35℃ for one to two days,
gas producing tubes are indicative that coliform bacteria are present,
that the meat product may have been exposed to fecal contamination,
that unfit raw materials may have been used, or that the product has received unsanitary handling.
While several additional test, such as fermentation tubes of EC broth, 45.5℃ incubating; streaking loopfuls of material onto EMB (Eosin Methylene Blue) agar plates
from all EC cultrues showing gas production and the IMViC reactions,
are needed to conclusively identify the fecal organism,
Escherichia coli, positive results show that a sanitation problem exists.
Procedures for the rapid detection of coliforms and E. coli
using pour plate method are described as following:
-
Use the same dilution and pipetting procedure as for total plate counts.
-
Use Violet Red Bile agar instead of Total Plate count agar.
-
After the agar hardens in the petri plates, pour a layer of agar on top of the hardened agar.
This creates a more anaerobic environment for coliforms and E.coli.
Incubating the plates at 35℃ for 1 - 2 days.
-
Growth of purplish-red subsurface colonies
indicates the presumptive presence of those organisms.
-
For confirm test that inoculating into fermentation tubes of LSTB （incubating at 35℃） and EC (incubating at 45.5℃) for 1-2 days.
Gas production from LSTB tubes and EC tubes i
ndicates the presence of coliform and E. coli, respectively.
A Coagulase-Positive Staphylococci─Staphylococcus aureus
Staphylococci are widely distributed in man's environment
and occur in varying numbers in air, dust, water, milk, feces, staphylococcal infections.
When animals are beaten, mistreated, or injured,
such mishandling often results in the production of deep-seated abscesses
containing viable staphylococci as well as other organisms.
The single most important source of S. aureus in foods is man.
Man is an major host for this species, most often carrying the organisms on his hands,
arms and other skin areas, nasal passages, throat and hair.
S. aureus, in addition to producing enterotoxin when it grows in food,
is associated with a variety of pyemic infections in man
ranging from localized pus-containing lesions such as infected cuts, abrasions, pimples
and carbuncles to those that are more generalized and systemic.
It is for this reason that employees must wear gloveswhen handling processed meats
and that individuals with infected scratches or wounds, boils, or respiratory infections
should not be allowed to have direct contact with meat products.
The cause of the food poisoning by S. aureus is due to a specific protein
formed during the growth of the organism in certain kinds of foods
such as barbequed, cooked or roast beef, pork, chicken and turkey, hams meat mixtures,
milk products, egg products, salads, and cream-filled products.
Staphylococci are generally associated with foods that have been heated and recontaminated.
The organisms are 〝opportunists〞
whose growth is dependent upon finding a food high in protein and salt
that is held at a desired incubation temperature -- at least 10℃ or higher,
and a substrate that is relatively free of competing bacteria.
Even then not all strains of S. aureus produce enterotoxin;
those most likely to produce the poisonous protein are pigmented and coagulate plasma.
These pathogenic staphylococci are readily destroyed by heat
but their enterotoxins are much more heat resistant.
The most common type of enterotoxin, Type A, is the most frequently involved in outbreaks
but is the most sensitive to heat -- showing a 5% decrease in reaction in one minute at 60℃.
By contrast, Type B retains activity even greater heating at 60℃ for as long as 16 hours
or 100℃ for 30 minutes.
The presence of coagulase positive staphylococci in processed meats is very undesirable.
Thus, every effort should be made to identify and eliminate their source.
To examine for coagulase positive staphylococci, testing is much more involved.
Suitable amounts of material should be placed in tubes of TSB （trypticase soy broth）
containing 10％ NaCl and incubated for 2 days at 35℃.
Then loopfuls of this material should be streaked onto Staphylococcus 110 medium plates.
After 2 days at 35℃, typical colonies should be picked
and after these have given visible growth on agar slants,
inoculated into brain heart infusion (BHI) broth and incubated for 18-24 hours at 35℃.
Then, after adding 1/2 ml of reconstituted coagulated plasma with EDTA,
the BHI broth tubes should be examined for clot formation within a 4 hour period.
Microbial Standards for Meat and Meat Products
What are the purposes of microbiological standards for meats:
I think most microbiologists would agree that they can serve a useful prupose
in three main respects:
- Control risk from pathogenic organisms.
- Ensure that the food has not been grossly contaminated.
- To give the expectation of reasonable storage life.
For protection against pathogenic hazard,
the choice is receptively simple and frequently the statement will be that the food;
i.e., zero tolerance.
Detection of pathogens and toxins can pose some very difficult problems
for the microbiologist and to categorically state that no pathogen is present is impossible.
An alternative is to chose some more readily detectable indicator organism
from the same source as the pathogen and limit the numbers of the indicator.
The best known example is the use of the coliform count
or sometimes the Escherichia coli count as an insurance against enteric pathogens.
The assumption is that if the E.coli count is low,
it is likely that enteric pathogens are absent or at least present in numbers so low
that they do not pose a hazard.
It is difficult, to say the least,to get agreement as to the exact level for a standard.
The microbiological standards and guidelines for various meat products in different places
are shown as in Table 1.
The presence of coliforms or E.coli is no easier to evaluate.
These organisms may multiply in meat and excessively high numbers
may be encountered after a time
but there numbers do not necessarily reflect the sanitary conditions
under which the product was originally produced.
I do not want to leave the impression that these determination are of no use
but they do have to be applied with thoughtfulness and common sense.
Where a microbial index is essential an objective foundation for the standard
should be established by demonstrating a relation between the standard
and the hazard to which it relates.
Spoilage and Shelf-life of Various Meat Products
Microorganisms require nutrients, water and sometimes special environment of: (1) pH,
(2) salt concentration, (3) presence or absence of air, and (4) temperature.
Microorganisms have these requirements so that they can grow and reproduce,
as do all living things. When proper conditions exist, and they do with virtually all meats,
then microorganisms grow, reproduce, and cause spoilage.
The microbes associated with spoilage of various meat products are shown as in Table 2.
In attempting to maximize shelf-life, the uncontrollable elements
are the level of contamination the product receives
and the storage temperature at which it is held. Remember two important rules:
(1) the fewer the contaminates, the longer the shelf-life,
and (2) the colder the storage temperature, the longer the shelf-life.
The number of microorganisms can be controlled through adequate sanitation.
Refrigeration, to the extent that it is mechanically feasible and cost-wise possible,
can be controlled. But packaging techniques also affect shelf-life
and the way spoilage manifests itself.
