Assignment 2

Student number: 50719017

Question 1
1.1) Pure culture
A pure culture is a population of cells arising from a single cell. Pure cultures (PC) are
used to study a single type of cell species as it is quite difficult to study an individual cell
in a mixed culture. There are numerous techniques used to isolate pure cultures, a few
of them are mentioned below:
Spread Plate- when a mixture of cells is spread out on an agar so that each cell
grows into a single colony. A small volume of a diluted microbial solution
containing around 30-300 cells is placed in the center of an agar plate and
spread evenly over the surface using a sterile bent-glass rod. The dispersed cells
develop into individual colonies.
Streak Plate- The cell mixture is placed at the edge of the agar with an
inoculating loop or swab and then streaked out over the surface using one of
many patterns. Eventually, single cells drop from the loop as it is rubbed along
the agar surface and develop into individual colonies
Pour Plate- samples are diluted several times to reduce microbial population
sufficiently to obtain separate colonies when plating. When sample is of a known
volume, it is mixed with agar and poured into a sterile petri-dish. When agar
hardens, it fixes the cells in place and forms individual colonies which are
counted. Plates containing 30-300 colonies are counted


1.2) Photoautotrophs
These are microorganisms that use light energy and have CO
2
as their primary source of
Carbon. Because CO
2
has no hydrogen ions, autotrophs need alternative source of
energy. In this case, through the process called photosynthesis, photoautotrophs get
most of their energy from light.

1.3) Light Microscopy
This is probably the most used microscope. It is composed of a lens that magnifies the
sample and a light source that illuminates the sample. Modern light microscopes are all
compound microscopes; this means that the microscope has more than one lens and
the image magnified image will be further enlarged the other lenses. Resolution of
microscope is inversely proportional to wavelength of radiation used to illuminate
specimen. Light micro scopes are composed of an ocular, body, nosepiece, objective
lenses, stage, Adjustment knobs (coarse and fine), sub-stage condense, light source
and a light intensity controller. During preparation of samples, cells undergo fixation
(preservation and fixing specimen in position) and staining of the cell and cell structure
(this allows the observer to see the cell since most samples are colorless).

1.4) Bacterial Cell Wall
Bacterial cell wall is a rigid structure that lies outside the cell membrane. It is mostly
composed of peptidoglycan. The cell wall may shape the cell to into three distinct
shapes: Rod-like often called Bacillus, Spherical shaped called Coccus and rod shaped
bacteria which are curved called Vibrios. However, some bacteria may lack a distinctive
shape and are usually referred to as Pleomorphic. The composition of the cell wall helps
microbiologist determine whether a bacteria is gram positive or gram negative. The
difference between gram-positive cell envelope and gram-negative cell envelope is listed
below

1.5) Eukaryotic Cell Organelles
Eukaryotic Cell Organelles are:
a. Nucleus- contains genetic information. Controls cellular functions
b. Nucleolus-
c. Chloroplast- converts solar energy to chemical energy. Found mostly on
Phototrophs
d. Vacuole-saclike structure that stores food and provides water balance
e. Lysosomes- contains digestive enzymes that digests food or certain material
within the cell
f. Golgi apparatus- modifies, sorts and packages proteins from endoplasmic
reticulum
g. Cytoplasmic matrix- jelly like structure that contains cell organelles
h. Cilia and flagella- cell locomotion
i. Cell wall and pellicle- supports cell and provides protection for the cell
j. Plasma membrane- cells boundary from external environment. Regulates
material that enters and leaves the cell
k. Mitochondria- Produces energy of cell by converting food to ATP. Site of cellular
respiration.
l. Ribosomes- site of protein synthesis. Proteins are assembled from amino acids
m. Endoplasmic reticulum- serves as a transport way for materials to other parts of
the cell
n. Microfilaments-

1.6) Nutrient uptake by bacteria
a) Passive diffusion
This is a process where molecules move from a region of high concentration to
region of lower concentration due to thermal agitation. It is dependent on size of
concentration gradient between the interior and exterior of the cell. A large
concentration gradient is needed for efficient nutrient uptake and only small
molecules may move across membranes through passive diffusion
b) Facilitated diffusion
A process that uses a permease to increase rate of diffusion. It carries molecules
from high concentration to low concentration. It requires no metabolic energy and a
smaller concentration gradient than passive diffusion.
c) Active transport
Active transport runs against concentration gradient and the use of metabolism is
required. ABC transporters are used to transport molecules to from low
concentrations to higher. ATP is the energy source
d) Group translocation-
A process whereby organic molecules are modified while being transported.
1.7) Bacterial growth curve
This is graph that shows the rate of growth of micro-organisms in a closed system. It
consists of four phases:
a) Lag phase- period of inactivity. No growth is observed
b) Exponential phase- period where microbes are growing at a logarithmic rate.
c) Stationery phase- period where death rate = growth rate.
d) Death phase- period where death rate is higher than growth rate

1.8) Pasteurization
Method of physical control of microbes involving heating solutions to temperatures below
their boiling point. It does not kill microbes but it lowers microbial concentration. Consists
of three types;
a) Low temperature long time (LTLT)- heating solutions for 30 minutes at 62
b) High temperature short time (HTST)- heating solutions for 15 seconds at 72
c) Ultrahigh-temperature (UHT)- heating solution for 2-3 seconds at 150-160


QUESTION 2
2.1) a) 1867- Pasteur proves that micro-organisms dont arise from spontaneous generation
Although Francesco Redi disproved that spontaneous generation occurred in large
organisms in 1668, people still believed that spontaneous generation existed.
Spontaneous generation was a belief that micro-organisms could suddenly be created
from inanimate objects. This was a popular belief especially under the Christian society
since this belief supported Creation by God. However, Louis Pasteur disproved this
theory through a simple experiment. He used a swan neck flask filled with medium and
boiled the solution to kill all microbes present within the flask. He melted the top of the
flask and shaped it into an s-shape to allow air through yet preventing microbe carrying
dust particles from contaminating the solution. After incubation of media, no microbes
were present within the sample. This proved that spontaneous generation existed for
most scientists. Of course Christians than were still skeptical about the disproval of
spontaneous generation. I still question though whether Pasteur would be credited for
disproving spontaneous generation if there were spore forming microbes within the
sample

b) 1867- Lister publishes his work on antiseptic surgery
After Ignaz Semmelweis proved that child-bed fever was spread by doctors and could be
prevented by proper hand sanitation, Joseph Pasteur realized that he could use
Semmelweis concepts in preventing patient from dying from infections during surgery.
His work on antiseptic surgery was vital as it was highly likely that a lot of patients would
have avoided going to the doctor because they might have developed a mentality that
no one lives after surgery. By using phenic acid, Lister prevented wound infections
which in turn reduced patient death from these infections. His work on antiseptic surgery
ha saved a lot of lives, and work may still be used on this very day.


c) 1929- Fleming discovers penicillin
After Ehrlich proposed that chemotherapeutic agents, which are substances that could
inhibit or kill microbes in a host without seriously harming the host are existent, a lot of
research began on finding such substances. Alexandra Fleming mistakenly made one of
the most famous microbiological discoveries in this science. He discovered Penicillin, a
substance produced by the fungus Penicillium notatum. This fungus accidentally landed
on an exposed petri-dish before it was inoculated with bacteria. Penicillin has a selective
toxicity towards the cell wall of micro-organisms. This is due to a presence of
peptidoglycan within the cell wall which is inhibited from being synthesized by penicillin.
This may weaken the cell wall which may result in osmotic lysis. Alexander Fleming,
together with Howard Florey and Ernst Chain, received the Nobel Prize in 1945 for the
discovery and production of penicillin

d) 1981- HIV is recognized
Possibly the most known microbe in the modern society, HIV is an immune weakening
virus which targets t-helper cells. These cells express the cd4+ receptor which is the
preferred receptor for HIV. It was initially thought that this virus lead to GRID (Gay-
related immune deficiency), but this was later rectified to AIDS (Acquired Immune-
deficiency syndrome), after the realization that the virus also affected heterosexual
partners. Although it is believed that the virus us a descendent of SIV (virus similar to
HIV that affects other primates), there is no clear knowledge on how the virus entered
and mutated into infecting humans. Currently, there is no cure or vaccine for HIV;
however treatment to avoid HIV from escalating to Aids is available.

e) 2003- the SARS Epidemic
Possibly the most epic epidemic in the last 20 years, the SARS outbreak reminded us
the importance of Microbiology. Severe Acute Respiratory Syndrome was a viral
epidemic caused by the Coronavirus. These are large, spherical viruses with a
membranous envelope and a characteristic halo of peplomers. SARS is probably a true
Zoonotic virus that had somehow mutated and infected humans. It is unknown how this
virus came into contact with humans however it is proposed that it may have been
transmitted through the virus original host; the civet cat (which is a delicacy in Southern
China). SARS infected 8500 people and killed 800 patients in 27 countries. The index
case of SARS was from a single health care worker from Guangdong, China.

2.2) Microbiological stains are used to identify cells in a light microscope. Micro-organisms
are mostly colorless so the use of stains is necessary for enabling microbiologists to see
these microbes in a microscope. There are two types of dyes normally used for staining
namely; Basic dyes (positively charged and binds to negative ions) and Acid dyes
(negatively charged and binds to positive ions). There are various types of stains used to
stain microbes. Here are a few examples:

Simple staining- a single stain is used here. It is easy to use, hence the name given to
this type of staining. The sample is covered with a stain and left for a certain period of
time. The excess stain is than washed off with water and the sample is dried. Basic dyes
are normally used in this procedure

Differential staining- Developed by Christian Gram, this type of staining groups bacteria
into two groups- gram positive and gram negative. Bacteria are stained with a basic
dye(crystal violet). This is than followed by exposing the sample to an iodine solution
that acts as a mordant. The sample is than decolorized by washing it with ethanol or
acetone. Gram-positive bacteria will remain crystal violet after being washed with
ethanol or acetone whereas gram negative bacteria will become colorless. Safranin is
then added to the sample, this leaves gram-positive bacteria stained dark purple and the
gram-negative bacteria will assume a pink-red color.

Acid-fast staining- used for certain bacterial species that do not bind readily to simple
stains. The bacterium is heated with a mixture of basic fuschin and phenol. After the
penetrations of fuschin, the cell will retain the color even after it is washed with ethanol
and acetone. Non-acid fast bacteria will be decolorized and will assume a blue color
after being counterstained by methylene blue.

Negative staining- used for staining specific structure, it reveals the presence of a
capsule that surrounds many bacteria. The bacteria are mixed with India ink or nigrosin
dye. The bacteria are than air-dried after being smeared on a thin film on a slide. Since
the ink and dye particles cannot penetrate the bacteria or it capsule, a blue-black
background will be observed. This background will enhance the appearance of the
capsule, which will be expressed through a light region. However, the image will show a
distortion in the shape of the bacteria

Spore staining- endospores are not easily stained by most dyes but will remain colored if
they are successfully stained. The Schaeffer-Fulton method is sometimes used to stain
spores. In this method, the bacteria are stained by heating it with malachite green.
Malachite green is a strong stain than can penetrate the endospore. After staining, the
bacterium is than washed with water and then counterstained with safranin. The
endospore will appear resting in a pink-red cell.

Flagella staining- staining of flagella by using mordants like tannic acid and potassium
alum and finally staining by use of pararosaniline or basic fushin. This is used to
increase visibility of flagella.


Question 3
Control of microbes is a very important part of microbiology. In this section we address concepts
such as sterilization, inhibition of microbial growth and prevention of microbial contamination.
There are three general categories of microbial control, namely- Physical, chemical and
antibiotic control.

Physical
Physically controlling micro-organisms is achieved through the use of temperature, filtration and
radiation. Unlike chemical and antibiotic control of microbes, physical control of microbes is not
dependent on the type of microbe present within a solution or item. Physical control has no level
of selective toxicity so it will have a relatively same mode of action regardless of whether a cell
has a type of cell wall, Dna structure etc. Therefore, such control is only used on inanimate
material and would be extremely hazardous to the human body. Examples of physical control
are mentioned below. These examples will be briefly explained

Boiling at 100 Celsius for 30 minutes kills most everything except endospores. Boling is
normally used to disinfect water and objects that tolerate boiling water.
Autoclaving is the most effective and, most efficient method of sterilization. Steam is used to
sterilize items and therefore autoclaving is useful if temperatures are above 100. Normally,
121/ 15 psi is the standard temperature employed. Autoclaving is used to sterilize glassware,
bio-hazardous wastes, surgical dressing.
Pasteurization, developed by Louis Pasteur, is the subjections of substances to temperatures
lower than its boiling point. This reduces the number of micro-organisms present within the
sample and may drastically increase the shelf life of these substances. Low temperature long
term (LTLT) is pasteurization done at 63 for 30 minutes. High temperature short term (HTST) is
pasteurization done at 72 for 15 seconds. Ultrahigh-temperature sterilization is pasteurization
done at 140-150 for 1-3 seconds. The larger the sample, the high the temperature needed for
sterilization. Duration decreases as temperature is increased.
Dry heat is used on material that cannot withstand wet conditions. Duration heat application
and time are basic principles for sterilization. Items are placed in an oven at 160/ 2 hours and
170/1 hour. This is not as effective as the use of moist heat.
Low temperatures are used to either slow microbial growth or prevent microbial growth. Hence
this may not necessarily kill micro-organisms. Substances are either refrigerated or frozen and
these actions will slow the rate of growth or prevent growth respectively. Subjecting items to low
temperatures may increase the shelf life of these items but some may be contaminate with
psychrophiles when refrigerated, therefore, refrigeration is useful for short term storage.

Filtration generally removes micro-organisms from solution. This is useful for solutions that are
sensitive to heat. There are two types of filters. Depth filters consist of fibrous or granular
materials bonded together into a thick layer filled with twisting channels with small diameters.
The microbial solution is vacuumed through the filters where cells are removed via physical
screening, entrapment or also by absorption. Membranous filters are 0.1 mm thick and
consists of pores 2 m in diameter. They are made of cellulose acetate, cellulose nitrate,
polycarbonate, polyvinylidene fluoride and other synthetic material. they remove most
vegetative cells but not virus as virus are smaller than 2 m. I imagine that filtration is only
useful for liquids that may lose certain properties if they are exposed to other methods of
physical microbial control. Air can also be sterilized through filtration through the use of Laminar
flow biological safety cabinets.
Radiation is the use of radiation to kill micro-organisms. It destroys or kills nucleic acids. Either
Ultra-violet or ionization radiation can be used for sterilization. Ultraviolet radiation can only
1
sterilize surface as it cannot pass through these surfaces.260 nm is lethal enough to kill
microbes. Ionization radiation




Chemical control
Chemical methods of controlling microbes the most used methods by the public. These are
chemical substances that act as disinfectants or antiseptics because they cannot readily kill
bacterial endospores. Unlike physical agents, chemical agents have to cause less harm to
animals as possible, they have to have a pleasant odor or have rather be odorless. The
chemical agent has to remain stable during storage as it would be pointless and expensive to
use a full dose chemical agent at once due to its instability. It should be noted though that some
chemical agents are used regardless of whether they meet the above standards. Unlike physical
agents again, there is potential for microbes to develop resistance towards these agents. An
advantage of these chemical agents though is their quick ability to disinfect and prevent
microbial growth on surfaces. Some are animal friendly, so minimal or no harm to tissue cells
will be observed when they are used.
Examples of chemical agents include: Phenolic, Halogens, Alcohols, Heavy metals, Quaternary
Ammonium compounds, Aldehydes and Sterilizing gases.


Antibiotic control
Antibiotics are used as chemotherapeutic agents. These are microbial products that inhibit or kill
micro-organisms. They have to exert as minimal damage as possible towards host cells yet
have high toxicity towards pathogens and therefore have to have a high level of selective
toxicity. Side effects may occur if the drug doesnt have a high level of toxicity.
They may inhibit microbial growth or even destroy microbes. The minimum concentration
needed to inhibit growth is the Minimal inhibition Concentration (MIC) and the minimum
concentration needed to kill microbes is the Minimal Lethal Concentration (MLC). Antibiotics can
occur naturally, they can be synthetic or semisynthetic (naturally occurring but are modified to
increase their effect). Mechanisms of action of antibiotics are:
a) Inhibition of cell wall synthesis
b) Inhibition of nucleic acid synthesis
c) Inhibition of protein synthesis
d) Disruption of cell membrane
e) Inhibition of metabolic activities
A problem about antibiotics is that resistance towards these drugs are ever increasing, which
means that certain micro-organisms are becoming resistant to pharmacological methods used
by these drugs.


REFERENCES
Prescott L.M; Harley J.P; Klein D.A. 2005. Microbiology 6
th
edition.
www.textbookofbacteriology.net
http://highered.mcgraw-hill.com
RESULTS
40 = 40
20 = 60
------------------------------
Total = 100 / 100 (100%)
COMMENTS
Excellent
1