Initial of colour, making it hard to see the

          Initial observations of
microorganisms are made with stained preparations to provide contrast as the
cytoplasm usually lacks of colour, making it hard to see the colourless cells
on the bright microscope background, called field. Stains are salt composed of
a negative and a positive ion. The colour of basic dyes is present in positive
ion, whereas acidic dyes are present in negative ion. Bacteria are slightly
charged at pH 7. Thus, coloured positive ion in a basic dye is attracted to the
negatively charged bacterial cell. Basic dyes, such as crystal violet,
malachite green and safranin are more commonly used than acidic dyes. Acidic
dyes are not attracted to most types of bacteria in which negative ion are
repelled by the negatively charged bacterial surface, resulting in stained
background instead.

          Differential stains enable the
observer to differentiate bacterial cells based on staining differences. The
Gram stain, uses air-dried and heat fixed smears, named for Hans Christian
Gram, is one of the most useful staining technique as it classifies bacteria
into two large groups which are gram-positive and gram-negative. Crystal
violet, the primary stain, stains both gram-positive and gram-negative cells
purple as the dye enters the cytoplasm of both cells. When the mordant, iodine
is applied, it forms large crystals with the dye that are too large to escape
the cell wall. The application of alcohol, decolourizing agent dehydrates the
peptidoglycan of gram-positive cells to make it more impermeable to crystal
violet-iodine. However, the effect on gram-negative cells is quite different.
Alcohol dissolves the outer membrane of gram-negative cells and leaves small
holes in the thin peptidoglycan layer through which crystal violet-iodine
diffuse. Since gram-negative cells are colourless after the alcohol wash, the
addition of safranin, the counterstain turns the cell pink. It provides a
contrasting colour between primary stain, crystal stain. Although a
gram-positive and gram-negative cell both absorbs safranin, the pink dye of
safranin is masked by the purple dye previously absorbed by gram-positive

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          Gram-positive cell wall consists of a
very thick, rigid peptidoglycan wall and a network of disaccharide chains,
glycan strands cross-linked to one another, both in the same layer as well as
between layers forming a three-dimensional mesh by short, elastic peptides. The
abundance and thickness may be the reason why crystal violet dye retains in the
gram stain technique. Cell wall of gram-positive cells also contains a
sugar-alcohol and phosphate polymer called teichoic acid. Teichoic acids which
are bound to the glycan chains or cell membrane are vital for cell viability.
Cell death may occurs if the genes for teichoic acid synthesis are deleted.
Despite that, the function of teichoic acids still remains unclear.

          Gram-negative cell wall is
differently structured when compared to gram-positive cell wall. The
peptidoglycan strands consists just a single layer or two making the cell more
susceptible to lysis. This is among the reason why it loses crystal violet dye
during the gram stain technique. Teichoic acid is also absent in gram-negative

          When essential nutrients are depleted
certain gram-positive bacteria form specialized “resting” cells called
endospores. Unique to bacteria, endospores are highly durable dehydrated cells
with thick walls and additional layers. Endospores are formed inside the
bacterial cell membrane. The process of endospore formation within a vegetative
cell takes several hours in a process called sporogenesis; when key nutrients
such as carbon or nitrogen source become scarce. Depending on the species, the
endospore might be located terminally, subterminally or centrally inside the
vegetative cell. When the endospore matures, the vegetative cell wall lyses;
killing the cell and the endospore is freed.

          Endospore cannot be stained by
ordinary methods, such as Gram staining, as the dye does not penetrate through
the wall of endospore. A commonly used endospore stain is Schaeffer-Fulton
endospore stain. Malachite green, the primary stain is applied to a heat-fixed
smear and is heated. The heat assists the stain to penetrate the endospore
wall. The smear is washed with decolourizing agent, water to remove the
malachite green from the cell, except the endospores. Safranin, the
counterstain is applied to stained portions of the cell other than the
endospore. In a well-prepared smear, endospores will appear in dark green
within pink cells. Since endospores are highly refractive, they can be detected
under the light microscope when unstained. However, they cannot be
differentiated when stored materials are present, without the aid of a special

          Endospores can remain dormant for thousands
of years. An endospore returns to its vegetative state by a process known as
germination. Germination can be triggered by physical or chemical damage of the
endospore coat. Enzymes present in endospore will break down the extra layers
surrounding the endospore, where water enters and metabolism resumes. Since one
vegetative cell forms a single endospore, in which after germination, remains
as one cell. Sporogenesis in bacteria is not a means of reproduction.

          Micrococcus luteus cells died relatively
rapidly when they were added to natural soil. Microscopic observation showed
that the cells were being physically destroyed by bacterial predators in the
soil. Two of these predators were responsible for the initial, main attack, and
they were isolated. The isolates on laboratory media lysed M. luteus cells in a
manner like the attacks that occurred in soil. Neither predator was obligate,
however, nor were they nutritionally fastidious. One of these bacteria produced
mycelium and conidia. Under nutritionally poor conditions it used slender
filaments of mycelium to seek out host cells. Micrococcus luteus has one of the
smallest genomes of free-living actinobacteria sequenced to date, comprising a
single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to
encode 2,403 proteins. The genome shows extensive synteny with that of the
closely related organism, Kocuria rhizophila, from which it was taxonomically
separated relatively recently. Despite its small size, the genome harbours 73
insertion sequence (IS) elements, almost all of which are closely related to
elements found in other actinobacteria. An IS element is inserted into the rrs
gene of one of only two rrn operons found in M. luteus. The genome encodes only
four sigma factors and 14 response regulators, a finding indicative of
adaptation to a rather strict ecological niche (mammalian skin). The high
sensitivity of M. luteus to ?-lactam antibiotics may result from the presence
of a reduced set of penicillin-binding proteins and the absence of a wblC gene,
which plays an important role in the antibiotic resistance in other
actinobacteria. Consistent with the restricted range of compounds it can use as
a sole source of carbon for energy and growth, M. luteus has a minimal
complement of genes concerned with carbohydrate transport and metabolism and
its inability to utilize glucose as a sole carbon source may be due to the
apparent absence of a gene encoding glucokinase. Uniquely among characterized
bacteria, M. luteus appears to be able to metabolize glycogen only via
trehalose and to make trehalose only via glycogen. It has very few genes
associated with secondary metabolism. In contrast to most other actinobacteria,
M. luteus encodes only one resuscitation-promoting factor (Rpf) required for
emergence from dormancy, and its complement of other dormancy-related proteins
is also much reduced. M. luteus is capable of long-chain alkene biosynthesis,
which is of interest for advanced biofuel production; a three-gene cluster
essential for this metabolism has been identified in the genome.

          Escherichia coli (E. coli) bacteria
normally live in the intestines of people and animals. Most of the E. coli are
harmless and actually are an important part of a healthy human intestinal
tract. However, some E. coli are pathogenic, meaning they can cause illness,
either diarrhea or illness outside of the intestinal tract. The types of E.
coli that can cause diarrhea can be transmitted through contaminated water or
food, or through contact with animals or persons. E. coli consists of a diverse
group of bacteria. Pathogenic E. coli strains are categorized into pathotypes.
Six pathotypes are associated with diarrhea and collectively are referred to as
diarrheagenic E. coli. The worst type of E. coli, known as
E. coli O157:H7, causes bloody diarrhea and can sometimes cause kidney failure
and even death. E. coli O157:H7 makes a toxin called Shiga toxin and is known
as a Shiga toxin-producing E. coli (STEC). 
There are many other types of STEC, and some can make you just as sick
as E. coli O157:H7. One severe complication associated with E. coli infection
is haemolytic uremic syndrome (HUS). The infection produces toxic substances
that destroy red blood cells, causing kidney injury. HUS can require intensive care,
kidney dialysis, and transfusions.

          Bacillus cereus is a facultatively anaerobic spore forming bacteria (Albrecht, 2017). This
bacterium is harmful to humans as it can cause foodborne illness while some of
the strains can be beneficial as probiotics for animals and it
is being reported to be a cause of serious and potentially fatal
non-gastrointestinal-tract infections (,
2018). The B.cereus pathogenicity is
intimately associated with the production of tissue-destructive exoenzymes. The
major hurdle in evaluating B. cereus when isolated from a clinical specimen is
overcoming its stigma as an insignificant contaminant. Besides of food
poisoning and severe eye infections, this bacterium has been incriminated in a
multitude of other clinical conditions such as anthrax-like progressive
pneumonia, fulminant sepsis, and devastating central nervous system infections,
particularly in immunosuppressed individuals and intravenous drug abusers. Its
role in nosocomial acquired bacteraemia and wound infections in postsurgical
patients has also been well defined, especially when intravascular devices such
as catheters are inserted. Primary cutaneous infections mimicking clostridia
gas gangrene induced after trauma have also been well documented. B. cereus
produces a potent ?-lactamase conferring marked resistance to ?-lactam
antibiotics. Antimicrobials noted to be effective in the empirical management
of a B. cereus infection while awaiting antimicrobial susceptibility results
for the isolate include ciprofloxacin and vancomycin.

         There are many outbreaks of food
poisoning attributed to Bacillus cereus have been reported recently and all
have been associated with cooked rice. Tests were made to assess the heat
resistance of B. cereus spores in aqueous suspension, the growth of the
organism in boiled rice stored at temperatures in the range 4-55° C., and the
effect of cooking and storage on the growth of the organism in boiled and fried
rice. The spores of B. cereus survived cooking and were capable of germination
and outgrowth. The optimum temperature for growth in boiled rice was between
30° and 37° C. and growth also occurred during storage at 15° and 43° C. During
the incubation period, the diarrhea lasts for 6-15 hours while emetic lasts for
30 minutes to 6 hours. The symptoms are watery diarrhea, abdominal cramps,
nausea and vomiting. The duration of this illness is usually lasts 24 hours.

          Even more, when vegetative cells of
certain bacteria such as Bacillus cereus is subjected to environmental stresses
such as nutrient deprivation, they produce metabolically inactive or dormant
form-endospore. Formation of endospore circumvent the problems associated with
environmental stress and helps them to survive. During unfavourable conditions especially
when carbon and nitrogen become unavailable endospores can form within
different areas of the vegetative cell.

Based on the
results, Micrococcus luteus is a
gram- positive bacterium which in purple colour rod shaped. The shape of Escherichia coli can be observed as a pink
colour rod shaped and give a result of gram-negative. Bacillus cereus shows results of gram- positive bacterium which is
purple colour and rod shaped. The improvement way in this experiment is wipe
gently after each staining to prevent from wiping off the bacterium. Besides,
the bacterium should be away from fire when heating to prevent the bacterium
from dead.

          Several hints and precaution steps
need to be taken into consideration in this experiment. Avoid making thick
smears as more time is required to decolourize thick smears. Do not blot the
slide vigorously with paper towel or you may rub off the Gram stain. If the
Gram’s iodine is yellow or pale mean it has lost its potency and should be
discarded. Cultures used to prepare the smear should be less than 24 hours old.
If too much decolourizing agent is applied, a lot of crystal violet-iodine
complex will be removed from the gram-positive cell walls.


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