#3 The goal for each method is to dilute out the colonies to a point where you can isolate individual

colonies. I would
imagine that using a streak-method for the spread plate method, you will be able to have decreasing densities of
colonies where as the pour-plate method will be more homogeneous. The pour-plate method also grows differently
since some colonies will be stuck in the middle of the agar.
*Using the spread method a small volume of a bacterial suspension is distributed evenly over the surface of an agar
plate using a smooth sterilized spreader (2). In the case of track plates, gravity is used to spread the inoculum down
the agar in a column forming a track (1).
For spread plates you make a series of dilutions of your bugs in a buffer, put a certain volume on an agar surface
and spread them using a spreader or glass beads. The bacteria then form colonies on the agar surface. The number
of colonies in 1 ml of initial medium equals the number on the plate times volume and dilution.
For the pour plates you dilute bacteria the same way, but add them to, and mix with, the melted medium and then
pour the mixture into your plates. The medium solidifies; the colonies grow throughout the volume of the plate.
The spread plate is generally used when calculating CFUs/ml, but the pour plate can be used as an alternative.
There is potential for contamination in whichever method you use. For a pour plate, many people contaminate the
media when adding the dilution of bacteria to it. For a spread plate, you can get contamination when spreading the
bacteria and the plate is open to the air.
One potential problem when doing a pour plate is that you can add your bacteria to the media before it has cooled
sufficiently, therefore killing some of the bacteria and affecting your CFUs/ml. You don't have this problem when doing
a spread plate. Another pain with the pour plate method is that you need a flask of media for each dilution you want
to plate, whereas with spread plates, you can pour your media in advance and then test several dilutions. This is
especially helpful when you have many plates to do. A third issue with pour plates is that if you don't swirl the media
enough after you add the dilution of bacteria, you won't get even distribution of colonies on a plate, making it pretty
hard to count accurate CFUs. (This is also a potential problem with spread plates)
I'm a fan of the spread plate over the pour plate....not because the pour plate is overly inaccurate, but because I've
seen more consistent results with spread plates. Plus, they can save you time.

Viable cell counting - viable cell count allows one to identify the number of actively growing/dividing cells in a
sample.

The spread plate relies on bacteria growing a colony on a nutrient medium so that the colony becomes

visible to the naked eye and the number of colonies on a plate can be counted.
Selective media can be used to restrict the growth of non-target bacteria.
The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air, for
example water samples.

Discussion
Normally it is very difficult to determine the actual number of microorganisms in a population. Thus, we calculate the
number of microorganisms by enumeration from the sample of population. Viable plate count is the method being
used most frequently in enumeration of bacterial population. We can either use pour plate method or spread plate
method for viable plate count. Colony forming unit (CFU)is introduced in viable plate count for the enumeration.
Viable plate count has it's limitations such as CFU only accounts the visible colony in enumeration, take some time
for the visible colonies to grow and too many colonies could cause error in the count. To overcome the overcrowded
problem, dilution is required. There are multiple choices for the dilution. To have a dilution of 1/100, we could either
adding 1mL of sample to a 99mL dilution blank or adding 1mL of sample from the 1/10 dilution to a 9mL dilution
blank, depending on the volume needed in enumeration.
#4 Standard Plate Count (SPC) is useful to monitor process control and determine sources of contamination, but is
not a true measure of specific risk pathogens in feed or of overall feed quality.
What are the limitations?

SPC measures most microbiological growth, but does not differentiate between the naturally occurring

bacteria, yeast, molds, etc. and the pathogenic or spoilage organisms.

While a high SPC may be used as an indicator of poor sanitation, inappropriate storage, or problems with

process control, it does not determine the presence of pathogens (to humans or animals).
A low SPC, likewise, does not guarantee samples are pathogen free.
SPC does not measure the entire bacterial population, but rather the number of microbes that grow on the

specific medium under particular growing conditions.

The type of bacteria that is present is not known - it might be good, it might be bad.
The medium/agar may not support growth of certain pathogenic bacteria.
It is difficult to distinguish between feed particles and bacteria.
It cannot be used on fermented ingredients like cheese.
Bacteria colonies may be too small to be seen. Conversely, the colonies can be overcrowded or clumped

together, increasing error in reporting.

Careful consideration must be given to the agar or medium being used, temperature and time of incubation,
length of time and storage conditions of samples, potential contamination of samples, proper dilution of the
sample to avoid overcrowding of colonies on plates, etc.

#2
A. Alternate streak patterns and different culture media
A variety of alternate streak patterns exist. Some are used for specific inocula, such as a urine specimen. The
patterns also differ in the number of sectors as well as in the number of times the loop is sterilized.
The four quadrant streak pattern would be recommended for use when large amounts of bacteria are expected in the
inoculum. The extra sector will provide additional dilution and increase the probability of isolated colonies on the
plate. The four quadrant streak plate is described in a variety of references, e.g., in Cappuccino and Sherman's

Microbiology, A Laboratory Manual, 8th ed. (3). Sometimes, cultures will be streaked on enrichment media or various
selective and differential media. For instance, a culture which is expected to have a gram-negative pathogen will be
streaked on a MacConkey agar plate, which inhibits the growth of gram-positive organisms.
B. Incinerating material on transfer loopsflaming
Reusable microbiological loops and needles are sterilized by flaming. A Bunsen burner is traditionally used for this
process. Most microbiology manuals show the microbiologist positioned with his/her hand above the burner, with the
loop placed into the flame. To avoid possible contact with the flame, the microbiologist might consider placing his/her
hand below the flame with the loop/needle above the hand in the flame. The flame of the Bunsen burner should be
adjusted to blue, with the darker blue cone of cooler air visible in the center of the flame. The loop or needle should
be placed into the hotter part of the flame and kept there until it glows red. There is a possible aerosolization hazard
if the loop or needle contains liquid or a bacterial clump. These loops and needles should be placed into the heat
slowly so that the moisture evaporates rather than sputters.
If an incinerator such as a Bacti-Cinerator is used to sterilize the loop, the loop is to remain inside the incinerator for 5
to 7 seconds. When warmed up (which will take 5 minutes), the temperature inside the incinerator is 815C. The
incinerator will take 5 to 10 minutes to warm up to working temperature.
C. Several techniques decontaminate transfer loops between sectors of a streak plate: flame, dig into agar, flame
once and rotate loop
A variety of methods exist for removing organisms from the loop between sectors. Beginning students are generally
taught to sterilize the loop between each sector by incinerating and then cooling the loop. Clinical microbiologists
practice a variety of methods. Some flame once after the initial sector and then rotate the loop so that the next
sectors can be streaked with an unused side of the loop. Other laboratorians (as clinical microbiologists name
themselves) stab the loop several times into the agar to clear the loop between sectors.
#8 Koch reported the use of a medium containing 7.5% sodium chloride as a selective agent for the isolation of
staphylococci in 1942. (5) The results were confirmed and improved by Chapman in 1945 by the addition of this salt
concentration to Phenol Red Mannitol Agar, as Staphylococcus aureus usually ferments mannitol. (3) Non-pathogenic
staphylococci usually show less luxuriant growth on this medium after the incubation period.
A sodium chloride, concentration of 7.5%, is nearly ten times the usual concentration seen in most media. It serves
to inhibit most organisms except staphylococci in mixed flora specimens. The beef extract and peptones supply the
essential elements carbon, nitrogen, and sulfur. Mannitol is added to show the fermentation capabilities of the
organisms. Acid production as the result of fermentation of this sugar results in the formation of colonies with a yellow
zone. Those staphylococci that do not ferment mannitol show a purple or red zone around the colonies.
A "selective" medium is one which encourages the growth of desired organisms, usually by discouraging the growth
of undesired organisms. A "differential" medium is one that offers visible indication of a physiological property, such
as the fermentation of a carbohydrate. Mannitol salt agar(MSA) was developed by microbiologists seeking a method
for isolating Staphylococcus aureus, a pathogen frequently transmitted by contaminated food. This medium contains
7.5% salt, which is inhibitory to most bacteria other than staphylococci. While most species of Staphylococcus are
capable of growing in this high salt concentration, S. aureus is also capable of fermenting the carbohydrate mannitol.
MSA contains the pH indicator phenol red, which has an orange color near neutrality but turns bright yellow at acidic
pHs. On MSA, colonies of S. aureus are yellow and may even turn the medium around the colony yellow due to the
drop in pH around the colony of a mannitol fermenter.
#9

*Repeated freezing and thawing may cause cell damage to the meat, releasing nutrients, making them more
available to bacteria. Repeated thawing increases the time that the meat is at a temperature that bacteria can grow.
It's the same as leaving the meat out for some time, except it is done in small stages. The bacteria are not destroyed
by freezing, nor is any toxin they may produce. Many food poisonings also occur because meat is stored incorrectly
in the freezer. If you are putting unfrozen meat into a freezer, make sure it is not touching any frozen meat, as it will
cause it to thaw out and refreeze. If this is done a few times, what you could have is spoiled meat.
The colder you keep the food, the more likely you are to prevent spoilage from molds and bacteria, and the slower
the enzymes within the food work to chemically change it. But.. The problem is that the water in the food ruptures
the cell membranes as it expands into ice crystals. This effect is more pronounced in meat than in vegetables and
fruits according to Harold McGee:
The intercellular spaces [in meat], which are often large and which contain some water but very little else, freeze first,
and then draw water out of the cells by osmosis. This in turn increases the concentration of dissolved substances
within the cell, which further lowers the freezing point. Parts of the cell interior do not freeze at all.
Refreezing is bad for three reasons. First, by refreezing food you multiply the damage to it--any cells that escaped
rupture the first time the food was frozen are at risk of being ruptured the second time. Second, when food has been
frozen and thawed out, it has larger pockets of liquid within it than the first time due to the ruptured cells. When the
food is refrozen, the larger pockets of liquid can freeze into much larger ice crystals, which can tear through many
more cell membranes and lead to more damage to the food. (The best way to avoid cell damage, incidentally, is
flash-freezing, which produces smaller ice crystals, minimal cell damage, and maximum freshness.)
The third and most important reason not to refreeze is increased risk of spoilage due to microorganisms. Many
people thaw food by letting it sit at room temperature for several hours, giving the microorganisms in it time to get
busy and partially spoil the food before it's refrozen. The problem is particularly pronounced in large pieces of meat
such as a turkey, some parts of which may be at or near room temperature for hours during thawing. That's why
turkeys should be thawed in a sink filled with water--the water equalizes the temperature and makes for faster
thawing. Alternatively, you can thaw in the refrigerator, which is slower but retards spoilage by keeping the meat cool.
Even so you're likely to have some multiplication of microorganisms. If you refreeze and rethaw, you've subjected the
food to double the microorganism growth and double the fun.
#10 Also, because any bacteria that is on the grinding equipment can be mixed into the meat as it is ground, which is
why it is so imporant that this equipment be properly cleaned and sanitized.
because a steak or roast only has it's surface area available for bacteria to grow on. Because ground beef has been
ground up, any bacteria on the outside of the meat get mixed up with the inside & spread to any available surface of
the ground beef meaning they can grow & multiply faster (Check this)
If you think about it it has alot more surface area, each little chunk of Beef has alot of surface as compared to a
steak. So therefore there is alot more space for bacteria to grow.