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Lecture-1
Introduction
What is the microbiology?
Microbiology is the study of microorganisms - little, really little, critters (except for
The Bis One). And, please take a look at some relative sizes of different living cells at
Jim Sullivan's Page: Cells Alive! These "bugs" include: bacteria (that's the Latin
plural for bacterium); viruses (that's the non-Latin plural for virus - virii sounds
weird, so I don't say it); and, fungi (that's the Latin plural for fungus - which by now
you have guessed, or already knew, and may not be all that interested to know,
anyway). Microbiology is actually made up of several sub-disciplines which
individually may stand alone, because there is so much to learn in each. These
disciplines include: Immunology (the study of the immune system and how it works to
protect us from harmful organisms and harmful substances produced by them - is what
I, Marci, and Larry work on; Virology, the study of viruses, and how they function
inside cells - Marci does some of this, too; Pathogenic Microbiology, the study of
disease-causing critters and the disease process - is what Eric does; Microbial
Genetics, the study of gene function, expression, and regulation - is what Susan and
Del do - although Del mostly examines mutations in genes and substances which appear
to prevent mutations; Physiology, the study of biochemical mechanisms - is what Jim
and Clarence do. I'll focus on bacteria right now, not because this group of critters is
necessarily more interesting, but because I know very little about fungi, and because I
don't want to talk about viruses just now.
Bacteria are necessary for all life on this planet - for every known ecosystem including the human ecosystem! Without bacteria, there would be no life, as we call
life, on the earth. However, it is a good thing that most bacteria die-out. Here is why:
bacteria are single-cell organisms, which produce more of their kind by cell division,
alone. Therefore, if one begins with a single bacterial cell like E. coli for example, in 20
minutes there will be two, and 20 minutes later, four, etc., E. coli cells. At this rate,
even though most bacteria are several hundred-times smaller than we can see with our
naked eye (never seen a clothed eye), in only 43 hours, from that one cell at the
beginning, there would be enough E. coli to occupy the entire volume of the
earth (1,090,000,000,000,000,000,000 cubic meters)! In only about two
additional hours, these bacteria would weigh as much as the earth 6,600,000,000,000,000,000,000 tons! Bummer! Luckily for us, most bacterial cells
die because of the enormous competition for food, and because of other tiny
organisms which produce substances (antibiotics) that kill them - you know, like
penicillin, which is made by a particular fungus, the mold - Penicilliuni). Thank
goodness for that one, huh? Actually, many antibiotics are made by certain bacteria
too, and, we get many of our necessary vitamins and nutrients from bacteria by
allowing the bacteria to multiply in number, and isolating the things that they make,
that we cannot make. For example, amino acid supplements are available ("enriched"
bread simply means that the amino acid, lysine, which we absolutely need, but cannot
make ourselves, is added to the flour used to make the bread), to provide one
additional source which most people will eat. This amino acid is produced by certain
bacteria grown in huge vats (can be 20,000 liters at one time - that is about 1,500
gallons!), and purified for our use. Antibiotic production is similarly done.
With the advent of molecular genetics and recombinant DNA technology, bacteria
now play a very important role as producers of human substances. Since we have
learned how genes function, we are able to introduce a human gene into a bacterium
and have the product of the human gene expressed. Consequently, a hormone called
erythropoietin, which is necessary for the proper development of red blood cells
(erythrocytes), but very, very, difficult to isolate, is now available in high quantity.
People who do not have kidneys cannot make this hormone; however, because the
hormone has been cloned into bacteria, plenty of these hormones can be made,
purified, and given to these people. Human insulin can be similarly made. These are
only two examples of the many substances now available to treat human disorders
because of our understanding of bacteria
Microbiology of foods
The important concept here is that microbes that come with food products are adapted to
live on that food item or are transients. Thus, the microbes that come on the food from
farm are the most likely to be equipped with the enzyme systems capable of
degrading and thus spoiling the food.
What is spoilage? We discussed the general sociological-psychological aspects earlier in
lecture. In general microbiology terms, it generally relates to the total microbial load
along with the presence and activity of specific spoilage organisms. Fig 5.1 shows
that it usually takes growth of microbes present to > 107 to produce detectable (by
humans) odors, flavors, colors.
Further, remember Louis Pasteur who considered the spoilage of beer and wine to be
"diseases" and tried to invent methods to inhibit or kill off the specific spoilage
organisms or "disease" causing organisms. Remember this from Intro to General
Micro as well as what we covered previously in Pasteurization. For the microbiologist,
it is important to detect the SSO's or indicator organisms.
MILK
Market milk excludes colostrums, containing mainly proteins (antibodies of the
mother), and is rich in fat, protein, lactose and water Fat is mainly triacylglycerols
(95%) with contributions from phospholipids, free fatty acids and diacylglycerol.
Protein is mostly caseins (phosphoproteins) along with whey proteins (albumins and
globulins). While lactose is the major sugar, citrate is also present, and the non-
protein nitrogen is mostly urea with contributions from amino acids and vitamins.
Caseins can be separated from milk by making the milk (pH 6.5) acid to pH about 4.6
and getting the proteins to their bioelectric point. At this pH they precipitate out and
this is one of the important principles in cheese making we will visit later in the
course. Further milk has lactoferrin and lactoperoxidase, which provide minimal
protection from spoilage. Adding H2O2 in non-refrigerated milk (see paragraph on
Africa) can increase the inhibitory effect of lactoperoxidase, which would increase
shelf life in non-refrigerated conditions.
Milk microbes come from: udder interior, teat exterior, and the milking and milk
handling-transport equipment. All of which get exposed to milk and can develop a
milk selected micro flora. Milk taken from health cows should have zero to 102 to
103 bacteria/ml. These are mainly micrococcus, streptococci and Corynebacterium
bovis. Cows suffering mastitis should be excluded from milking because these cows
produce milk with 108 bacteria/ml along with PMN's.
But an non-symptomatic cow could produce 105 bacteria/ml...and account for a
higher microbial count in a milking.
Mastitis causing bacteria include Staphylococcus aureus, Esherichia coli,
Streptococcus agalactiae, Strep, uberis, Pseudomonas aeruginosa and
Corynebacterium pyogenes. The first three are human pathogens, and in addition
other human pathogens can cause mastitis:
Mycobacterium bovis and M. tuberculosis. Infected cows should be removed from
the herd, treated with antibiotics (injection directly into the udder) and monitored
until they are cured. The udder exterior is less problematic in dry months. Heavily
contaminated teats can contribute 105 bacteria/ml. Thus, dairy farmers use procedures
to reduce this contribution to the microbial load of milk: clean facilities and bedding
for the cows, washing the teats with disinfectant treated water, keeping the milking
room and equipment very clean. Since 1982 in the UK the Milk Marketing Board
pays farmers for the milk based on it's bacterial count. This is not done in the USA.
Milk in USA is paid based on milk fat content and tested for antibiotics as well as
microbes.
Milk is heat treated (after homogenization): batch pasteurization = 62oC, 30 min;
High Temperature Short Time (HTST, heat exchanger) = 72oC for 15 sec; Ultra High
Temperature (UHT, heat exchanger) = 133oC for 1 sec. The temperatures/times prior to
the 1950 were less and based on M. bovis, but in the 1950's it was discovered that
Coxiella burnetii (causes Q fever) was more heat resistant, so these
temperatures/times are based on that standard. UHT is commercially sterile and
results in a milk product not requiring refrigeration although it is not a "botulism
cook", but the redox potential of milk is too high for Clostridium botulinum spores to
germinate. Alkaline phosphatase tests pasteurization. This enzyme is easy to assay,
present in all milk and is inactivated by pasteurization. The test takes only a few
minutes.
Thermodurics surviving pasteurization are Gram positives spore formers and nonspore formers such as Micrococcus, Microbacterium, Enterococcus and
Lactobacillus as well as a few strains of the Gram negative Alcaligenes tolerans.
But, the main spoilage is due to psychrotrophic bacteria: Gram negative rods
introduced post-pasteurization: Pseudomonas, Alcaliegens, Acinetobacter and
Psychrobacter. Milk should have a shelf life of 10+ days, many dairies keep milk for 2
weeks to test for shelf-life. Spoiled milk is coagulated, smelly, acidic. Milk products:
there are many! Figure 5.3 shows the groups. Remember that there are about 1,500 types
of cheeses alone! Letters on the arrows indicate the type of post milking food
preservation processing.
MEAT
This section does not cover fish and seafood but most muscle based animal products
(cattle, poultry, sheep, pig and goats) which are produced from our surplus of plant
proteins: it takes 2 kilos of grain to produce 1 kilo of chicken, 4 for pork, 8 for beef.
Meat has a high nutrient content the animal is sacrificed, muscles undergo rigor
mortis during which the muscles oxygen supply is cut off and they use stored
glycogen as a source of energy producing lactic acid. The redox falls and pH lowers to
5.5 with the production of about 1% lactic acid. If rigor mortis does not go to
completion two meat defects can occur: DFD (dark, firm, dry) meat with high pH
(due to stress or exercise just before slaughter) and PSE (pale soft oxidative) of
mainly pig meat wherein glycogen use rapidly to decrease pH while the muscle is
still warm.
Meat in a healthy animal contains no microbes. All come from contamination after
slaughter. Fresh, cleanly slaughtered meat should be < 10 bacteria/kg. Most microbes
come from skin and hair (dust raised during skinning) and the gut (huge load of
microbes). How the animals were raised determines the degree to which the skin/hair are
contaminated and whether there are fecal microbes on the hide. During dressing out the
animal, contamination can come from the knives and processing equipment. After
skinning, the carcasses are washed with treated hot water (chlorinated or acidified
with lactic acid). This decreases bacterial numbers 1 to 3.5 logs. The carcass is then
chilled. Surface numbers are then 102 to 104 bacteria/cm2. Carcasses are held at <
lOoC from primary processing and transport. Only psychotropic microbes can have a
spoilage effect.
Poultry primary processing processes large numbers of birds/hr, which leaves little
time for sterilization, disinfection of equipment. From the farm to plant,
contamination from feces to feathers is common. In the plant the birds are hung by
their feet on lines, stunned and killed by carotid artery cut. Close proximity allows
flapping wings to contribute to the spread of microbes from bird to bird. The birds are
then scalded by a 50oC water dip (to loosen feathers), the scalding water has a high
microbial count.
After scalding, the birds are mechanically de-feathered (by rubber fingers), then
eviscerated and chilled in water. Use of counter current flow and chlorination in
scalding and chilling waters lowers contamination rates. (Note the methods used in the
video in class).
Spoilage of Meat is done mainly by Gram-negative bacteria and yeasts all of which
must be psychrophilic/psychrotrophic. The first sign of spoilage is an off odor
(human nose is indeed sensitive) at 107 bacteria/cm2 due to the production of acids,
esters, sulfur containing molecules and amines (usually produced later). Vacuumpacked meats tend to have no oxygen and the spoilers are Gram positives that can
ferment Lactobadllus, Carnobacterium and Leuconostoc this comes with sour
odors characteristic of acids.
FISH
Fish is extremely perishable and therefore requires freezing and
maintenance of subzero temperatures until product is sold. Thus, almost
all of the fish in supermarkets are processed (dressed) as soon as they are
caught (factory ships) or shortly thereafter on shore and frozen.
Fish come with a high microbial load in the slime layer (skin), gills and gut.
These are almost always Gram negatives: Pseudomonas, Shewanella,
Psychrobacter, Vibrio, Flavobacterium and Cytophaga. Sometimes Gram
positives are present but are restricted to coryneforms and micrococcus. All
these microbes are either psychrophilic or psychrotrophic and therefore
adept at growing at refrigerated temperatures. Several foodborne diseases are
associated with fish. Crustaceans and Mollusks share the same problem.
Further since mollusks are filter feeders, they can concentrate toxins and
pathogens. They generally can only be taken from waters that are shown to
have low incidence of fecal contamination. We will discuss some of these as
carriers of certain diseases later.
Spoilage of fish is rapid, the pH of fish flesh remains high, pH > 6. The
bad-fishy spoilage comes from the reduction of trim ethylamine oxide
(TMAO) to trim ethylamine (TMA) which stinks like other amines! TMAO
is an osmotic regulatory molecule in most seafood and fish and is therefore
present in quantities: Fish and seafood also have good quantities of amino
acids which can be fermented or converted to other amines and sulfur
compounds (mercaptans). Microbes responsible for most spoilage are
Pseudomonas, Shewanella, Photobacterium and Vibrio.