Pengenalan asas mengenai Bacteria - FULL INFO-

1. Classification, Nomenclature, Laboratory Identification
The Gram-Positive Cell
As previously mentioned, Gram-positive bacteria are characterized by their
blue-violet color reaction in the Gram-staining procedure . The blue-violet
color reaction is caused by crystal-violet, the primary Gram-stain dye,
complexing with the iodine mordant. When the decolorizer is applied, a slow
dehydration of the crystal-violet/iodine complex is observed due to the closing
of pores running through the cell wall. Because the crystal-violet is still present
in the cell, the counter stain is not incorporated, thus maintaining the cell's
blue-violet color. If you recall, most cell walls contain peptidoglycan, a
molecule made of amino acids and sugar. A distinguishing factor among Gram-
positive bacteria is that roughly 90% of their cell wall is comprised of
peptidoglycan and a Gram-positve bacteria can have more than 20 layers of
peptidoglycan stacked together to form the cell wall. That's pretty thick!
Examples of common Gram-positive cells are Staphylococcus aureus and
Streptococcus cremoris, a bacterium used in dairy production.

2. GRAM-POSITIVE COCCI
The Gram-positive cocci are grouped together based on their Gram-stain
reaction, thick cell wall composition, and spherical shape. Most of the
organisms in these groups are members of the Micrococcaceae family All of
the organisms in these groups are non-endospore forming chemosynthetic
heterotrophs. We will discuss only the clinically relevent bacteria from
Micrococcus and Staphylococcus of the Micrococcaceae family.
Streptococcus and Enterococcus(formerly a species of Streptococcus) are
discussed as well because of the many diseases they inflict on humans. The
chart below shows the paths to identification of the genera discussed.
GRAM-POSITIVE RODS
The Gram-positive rods in this section will be divided into three distinct
varieties based upon their ability to produce endospores and their
morphological appearance:
• ENDOSPORE-FORMING
o BACILLUS
• REGULAR, NON-ENDOSPORE-FORMING

3. o LACTOBACILLUS
o LISTERIA
o ERYSIPELOTHRIX
• IRREGULAR, NON-ENDOSPORE-FORMING
o CORYNEBACTERIUM
These bacteria are ubiquitous in nature and most are aerobic or facultatively
anaerobic.
The Gram-Negative Cell
Unlike Gram-positive bacteria, which asuume a violet color in Gram staining,
Gram negative bacteria incorporate the counterstain rather than the primary
stain. Because the cell wall of Gram(-) bacteria is high in lipid content and low
in peptidoglycan content, the primary crystal-violet escapes from the cell when
the decolorizer is added. This is because primary stains like to bind with
peptidoglycan- something the G(-) cell has very little of. Gram(-) bacteria cause
a lot of problems because many species are pathogenic. This pathogenic
capability is usually associated with certain components of their cell walls,
particularly the lipopolysaccharide (endotoxin) layer. Remember the Black
Plague which wiped out a a third of the population of Europe? It was caused by
the tiny G(-) rod, Yersinia pestis. Most enteric (bowel related) illnesses can
also be attributed to this group of bacteria. Some of my favorites are
Salmonella food poisoning. If you choose, you can directly access the diseases
section of this tutorial to learn more.
MISCELLANEOUS GRAM-NEGATIVE RODS
VIBRIO
CAMPYLOBACTER
HELICOBACTER

4. NEISSERIA
The Neisseria genus consists of aerobic, non-spore-forming Gram-negative
coccobacilli which inhabit the mucous membranes of many animals (and
humans). These non-motile microbes require a moist environment and warm
temperatures (human body temperature range) to achieve optimum growth. An
important means of identification of Neisseria species is the oxidase test, for
which all members test positive. Additionally, Neisseria grow well on
chocolate agar containing antibiotics that inhibit growth of Gram-negative
bacteria, Gram-positive bacteria, and molds. The two most clinically significant
members of the genus Neisseria are N. gonorrhoeae and N. meningitidis.
N. gonorrhoeae
Infection by the diplococcoid bacterium N. gonorrhoeae is referred to as a
gonococcal infection. Gonorrhea is transmitted between humans through
intimate contact of the mucous membrane. This sexually transmitted organism
can be carried by men and women for many years without any sign or
symptoms. In infected males, the disease is characterized by a urethral
discharge of pus and can eventually result in other complications such as
prostatitis and periurethral abscess. The incubation period of the bacterium can
last from a day to a week. Females with gonorrhea exhibit vaginal discharge,
abdominal pain, and abnormal non-menstrual bleeding. Ironically, the
widespread use of birth control devices such as the pill has actually increased
the number of gonococcal infections in the United States. Use of the pill can
lower the glycogen concentration of the vaginal membrane. This environmental
change inhibits the growth of acid-producing bacteria, such as Lactobacillus,
which are the natural flora of the vagina. The vaginal pH soon becomes less
acidic and a variety of organisms are able to grow there. As with most other
sexually transmitted diseases, gonorrhea is prevalent in young adult and
homosexual populations. This disease may sound really bad, but it is treatable.
N. gonorrheae is sensitive to ultraviolet radiation, drying, and antibiotics.

5. Because chlamydia infection is often associated with a gonococcal infection, a
regimen of ceftriaxone and doxycycline is used to kill both organisms.
LABORATORY INDICATIONS:
• Oxidase +
• Glucose fermentative
N. meningitidis
It doesn't take a genius to figure out that N. meningitidis causes meningitis,
inflammation of the membranes covering the central nervous system. The
different strains of N. meningitidis are classified by their capsular
polysaccharides. This bacterium is the second leading cause of meningitis in
the United States. Early symptoms may include headache, fever, and vomiting.
Death can quickly follow due to endotoxin shock or focal cerebral involvement.
Infection doesn't always lead to death, however. The organism can often
assume a carrier status with very few carriers actually developing the disease.
Infected patients can be treated with penicillin, while rifampin may be used
prophylactically as a means of preventing the disease state in carriers.
ENTEROBACTERIACEAE
Members of genera belonging to the Enterobacteriaceae family have earned a
reputation placing them among the most pathogenic and most often
encountered organisms in clinical microbiology. These large Gram- negative
rods are usually associated with intestinal infections, but can be found in almost
all natural habitats. They are the causative agents of such diseases as
meningitis, bacillary dysentery, typhoid, and food poisoning. As well as being
oxidase negative, all members of this family are glucose fermenters and nitrate
reducers. In most cases, the pathogenicity of a particular enteric bacterium can
be determined by its ability to metabolize lactose. Non-utilizers are usually
pathogenic while the lactose utilizers are not. Because many different species in
this family can cause similar symptoms, biochemical tests are crucial to the
identification, diagnosis, and treatment of infection. We will discuss the twelve
genera of the Enterobacteriaceae family which are most commonly encountered
in the clinical laboratory:
• ESCHERICHIA COLI

6. • SHIGELLA
• EDWARDSIELLA
• SALMONELLA
• CITROBACTER
• KLEBSIELLA
• ENTEROBACTER
• SERRATIA
• PROTEUS
• MORGANELLA
• PROVIDENCIA
• YERSINIA

7. Gram-staining Procedure
Gram-staining is a four part procedure which uses certain dyes to make a
bacterial cell stand out against against its background. The specimen should be
mounted and heat fixed on a slide before you procede to stain it. The reagents
you will need to successfully perform this operation are:
• Crystal Violet (the Primary Stain)
• Iodine Solution (the Mordant)
• Decolorizer (ethanol is a good choice)
• Safranin (the Counterstain)
• Water (preferably in a squirt bottle)
Before starting, make sure that all reagents, as well as the squirt-bottle of water,
are easily accessible because you won't have time to go get them during the
staining procedure. Also, make sure you are doing this near a sink because it
can get really messy. Wear the appropriate lab attire.
STEP 1: Place your slide on a slide holder or a rack. Flood (cover completely)
the entire slide with crystal violet. Let the crystal violet stand for about 60
seconds. When the time has elapsed, wash your slide for 5 seconds with the
water bottle. The specimen should appear blue-violet when observed with the
naked eye.
STEP 2: Now, flood your slide with the iodine solution. Let it stand about a
minute as well. When time has expired, rinse the slide with water for 5 seconds
and immediately procede to step three. At this point, the specimen should still
be blue-violet.

8. STEP 3: This step involves addition of the decolorizer, ethanol. Step 3 is
somewhat subjective because using too much decolorizer could result in a false
Gram (-) result. Likewise, not using enough decolorizer may yield a false Gram
(+) results. To be safe, add the ethanol dropwise until the blue-violet color is no
longer emitted from your specimen. As in the previous steps, rinse with the
water for 5 seconds.
STEP 4: The final step involves applying the counterstain, saffranin. Flood the
slide with the dye as you did in steps 1 and 2. Let this stand for about a minute
to allow the bacteria to incorporate the saffranin. Gram positive cells will
incorporate little or no counterstain and will remain blue-violet in appearance.
Gram negative bacteria, however, take on a pink color and are easily
distinguishable from the Gram positives. Again, rinse with water for 5 seconds
to remove any excess of dye.
After you have completed steps 1 through 4, you should dry the slide with
bibulous paper or allow it to air dry before viewing it under the microscope.

9. STAPHYLOCOCCUS
Clinically, the most important genus of the Micrococcaceae family is
Staphylococcus. The Staphylococcus genus is classified into two major groups:
aureus and non-aureus. S. aureus is a leading cause of soft tissue infections, as
well as toxic shock syndrome (TSS) and scalded skin syndrome. It can be
distinguished from other species of Staph by a positive result in a coagulase
test(all other species are negative).
The pathogenic effects of Staph are mainly asssociated with the toxins it
produces. Most of these toxins are produced in the stationary phase of the
bacterial growth curve. In fact, it is not uncommon for an infected site to
contain no viable Staph cells. The S. aureus enterotoxin causes quick onset
food poisoning which can lead to cramps and severe vomiting. Infection can be
traced to contaminated meats which have not been fully cooked. These
microbes also secrete leukocidin, a toxin which destroys white blood cells and
leads to the formation of pus and acne. Particularly, S. aureus has been found to
be the causative agent in such ailments as pneumonia, meningitis, boils,
arthritis, and osteomyelitis (chronic bone infection). Most S. aureus are
penicillin resistant, but vancomycin and nafcillin are known to be effective
against most strains.
Of the non-aureus species, S. epidermis is the most clinically significant. This
bacterium is an opportunistic pathogen which is a normal resident of human
skin. Those susceptible to infection by the bacterium are IV drug users,
newborns, elderly, and those using catheters or other artificial appliances.
Infection is easily treatable with vancomycin or rifampin.
LABORATORY INDICATIONS:
• Anaerobic glucose fermentation with acid production
• Catalase +
• Nitrate +
• Coagulase +

10. STREPTOCOCCUS
The Streptococcus genus consists of Gram-positive, aerobic bacteria which
appear as chains under microscopic observation. The organisms in this genus
are characterized by a coccus appearance, a thick cell wall, and aerobic action
on glucose. Four different classification systems exist for this important
microorganism:
CLINICAL
• Pyogenic Streptococci
• Oral Streptococci
• Enteric Streptococci,
HEMOLYSYS
• alpha-hemolysis
• beta-hemolysis
• gamma-hemolysis
SEROLOGICAL-Lancefield (A-H), (K-U)
BIOCHEMICAL(physiological)

11. GROUP A
The first group in the Lancefield classification system includes only one
species of Streptococcus, S. pyogenes. This particular opportunistic pathogen is
responsable for about 90% of all cases of pharyngitis. A common form of
pharyngitis is "Strep throat" which is characterized by inflamation and swelling
of the throat, as well as development of pus-filled regions on the tonsils.
Penicillin is usually administered to patients as soon as possible to quell the
possibility of the infection spreading from the upper respiratory system into the
lungs. Once in the lungs, the infection could give rise to pneumonia. Some
cases also develop into rheumatic fever if left untreated. Other diseases linked
to S. pyogenes are skin infections such as impetigo, cellulitis, and erysipelas.
LABORATORY INDICATIONS:
• Catalase -
• Beta-hemolysis
• Bacitracin sensitive
GROUP B
The B classification of Lansefield also includes only one bacterium, S.
agalactiae. For years this bacterium has been the causative agent in mastitis in
cows. Currently, it has been found to be a cause of sexually transmitted
urogenital infections in females. Although infection is easily treated with
penicillin, proper diagnosis is necessary for women nearing labor because the
infection can easily spread to the child via the birth canal.
LABORATORY INDICATIONS:
• CAMP +
• Beta-hemolysis
GROUP D
Type D Streptococcus is the next clinically important bacterium because of the
multitude of diseases it is known to cause. Although many are harmless, the
pathogenic strains cause complications of the human digestive tract. This group
has recently been reclassified into two divisions: Enterococcus and non-
Enterococcus. The Enterococci include E. faecalis, a cause of urinary tract
infections, and E. faecium, a bacterium resistant to many common antibiotics.
Diseases such as septicemia, endocarrditis, and appendicitis have also been
attributed to group D Strep. Fecal matter from infected individuals is a source

12. for isolation and identification techniques. Once identified, Group D Strep can
be treated with ampicillin alone or in combination with gentamicin.
LABORATORY INDICATIONS:
• Hydrolysis of bile esculin (dark brown medium)
-this indicates the ability of the bacteria to tolerate bile from the liver
• Growth in high salt conc.
OTHER IMPORTANT STREP
Streptococcus pneumoniae
Because its surface carbohydrate antigens do not correspond to a specific
Lancefield group, S. pneumonia is discussed separately. Although not given a
letter designation, S. pneumoniae can be considered a Pyogenic (pus-
producing) strain of Strep. It can be distinguished from other Pyogenic bacteria
by its high sensitivity to Optochin (no growth zone of inhibition). This
bacterium causes pneumonia (obviously!), meningitis, and otitis media. It also
demonstrates alpha-hemolytic growth on blood agar.
Viridans Group
The Viridans Streptococci, consisting of S. mutans and S. mitis, are alpha-
hemolytic bacteria. These bacteria inhabit the mouth. In fact, a large percentage
of tooth decay can be attributed to S. mutans.
BACILLUS
Bacillus represents a genus of Gram-positive bacteria which are ubiquitous in
nature (soil, water, and airborne dust). Some species are natural flora in the
human intestines. When grown on blood agar, Bacillus produces large,
spreading, gray-white colonies with irregular margins. A unique characteristic
of this bacterium is its ability to produce endospores when environmental
conditions are stressful. The only other known spore-producing bacterium is
Clostridium. Although most species of Bacillus are harmless saprophytes, two
species are considered medically significant: B.anthracis and B. cereus.

13. B. anthracis
B. anthracis is the bacterium which causes anthrax in cows, sheep, and
sometimes humans. Anthrax is transmitted to humans via direct contact with
animal products or inhalation of endospores. Under the microscope, B.
anthracis cells appear to have square ends and seem to be attached by a joint to
other cells. The spores are best observed when the bacterium is cultered on
artificial media. Sources of infection are usually industrial or agricultural and
the infection is classified as one of three types:
• CUTANEOUS INFECTION (95% of human cases)
• INHALATION ANTHRAX (rare)
• GASTROINTESTINAL ANTHRAX (very rare!)
LABORATORY INDICATIONS:
• Nonhemolytic (sheep blood agar)
• Non-motile
• Gel hydrolysis -
• Catalase +
B. cereus
Unlike B. anthracis, B.cereus is a motile bacterium which can cause toxin-
mediated food poisoning. It is known to inhabit many kinds of food including
stew, cereal, and milk. Most recently, however, it has been found in fried rice.
The two toxins released by the bacterium lead to vomiting and diarrhea,
symptoms similar to those of Staphylococcus food poisoning. Because toxin
production usually takes place after the infected foods are cooked, proper cold
storage of food is recommended immediately after preparation.
LABORATORY INDICATIONS:
• Hemolytic (sheep blood agar)
• Motile
• Gel hydrolysis +
• Glucose, maltose, & salicin fermentative
• Catalase +

14. Biochemical Tests, Media, Techniques
CATALASE TEST
Some bacteria and macrophages can reduce diatomic oxygen to hydrogen
peroxide or superoxide. Both of these molecules are toxic to bacteria. Some
bacteria, however, possess a defense mechanism which can minimize the harm
done by the two compounds. These resistant bacteria use two enzymes to
catalyze the conversion of hydrogen peroxide and superoxide back into
diatomic oxygen and water. One of these enzymes is catalase and its presence
can be detected by a simple test. The catalase test involves adding hydrogen
peroxide to a culture sample or agar slant. If the bacteria in question produce
catalase, they will convert the hydrogen peroxide and oxygen gas will be
evolved. The evolution of gas causes bubbles to form and is indicative of a
positive test.

15. CITRATE TEST
The citrate test is used to determine the ability of a bacterium to utilize citrate
as its only source of carbon. Bacteria can break the conjugate base salt of
citrate into organic acids and carbon dioxide. The carbon dioxide can combine
with the sodium from the conjugate base salt to form a basic compound,
sodium carbonate. A pH indicator in the medium detects the presence of this
compound by turning blue (a positive test).
COAGULASE TEST
Like the mannitol salts agar, the coagulase test is another
method for differienting between pathogenic and non-
pathogenic strains of Staphylococcus. Bacteria that produce coagulase use it as
a defense mechanism by clotting the areas of plasma around them, thereby
enabling themselves to resist phagocytosis by the host's immune system. The
sample in question is usually inoculated onto 0.5 ml of rabbit plasma and
incubated at 37 degrees celsius for one to four hours. A positive test is denoted
by a clot formation in the test tube after the allotted time.

16. OPTOCHIN TEST
The optochin test is a presumptive test that is used to identify strains of
Streptococcus pneumoniae. Optochin (ethyl hydrocupreine) disks are placed on
inoculated blood agar plates. Because S. pneumoniae is not optochin resistant, a
zone of inhibition will develop around the disk where the bacteria have been
lysed. This zone is typically 14mm from the disk or greater.
OXIDASE TEST
Cytochrome oxidase is an enzyme found in some bacteria that transfers
electrons to oxygen, the final electron acceptor in some electron transport
chains. Thus, the enzyme oxidizes reduced cytochrome c to make this transfer
of energy. Presence of cytochrome oxidase can be detected through the use of
an Oxidase Disk which acts as an electron donator to cytochrome oxidase. If
the bacteria oxidize the disk (remove electrons) the disk will turn purple,
indicating a positive test. No color change indicates a negative test.

17. UREASE TEST
Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form
carbon dioxide, ammonia, and water. Members of genus Proteus are known to
produce urease. Urease can be detected by plating bacteria onto an amide
containing medium, specifically urea. When urea is broken down, ammonia is
released and the pH of the medium increases (becomes more basic). This pH
change is detected by a pH indicator that turns pink in a basic environment. A
pink medium indicates a positive test for urease.
MEDIA
CHOCOLATE AGAR
Chocolate agar is a nutrient medium which is used in culturing fastidious
organisms such as Haemophilus species and Neisseria. It is comprised of sheep
blood that provides the X and V factors necessary for Haemophilus growth.
Chocolate agar, however, does not reveal hemolysis data, so species

18. differentiation among the members of Haemophilus must be performed in
another manner.
EOSIN METHYLENE BLUE (EMB) AGAR
EMB agar is a differential medium used in identification and isolation of Gram-
negative enteric rods. EMB agar also inhibits the growth of Gram-positive
organisms. The differential basis of this medium involves two indicator dyes,
eosin and methylene blue, that distinguish between lactose fermenting and non-
lactose fermenting organisms. Lactose fermenters form colonies with dark
centers and clear borders while the non-lactose fermenters form completely
coloroless colonies.
MACCONKEY AGAR
MacConkey agar is probably the most popular solid differential medium in the
world. It is mainly used in identification of lactose fermenting, Gram-negative
enteric pathogens and for inhibiting growth of Gram-positive organisms.
Bacterial colonies that can ferment lactose turn the medium red. This red color
is due to the pH indicators response to the acidic environment created by

19. fermenting lactose. Organisms that do not ferment lactose do not cause a color
change.
MANNITOL SALTS AGAR
A common medium used for the isolation of pathogenic staphylococci is the
Mannitol Salts Agar. The high salt concentration of this medium inhibits the
growth of most other organisms. Pathogenic staphylococci not only grow on
the medium, but they also produce acid from it. This acid production turns the
pH indicator from red to yellow. Non-pathogenic staphylococci can grow on
the medium but produce no acid from it.
SMEAR PREPARATION
The preparation of a smear is required for many laboratory procedures,
including the Gram-stain. The purpose of making a smear is to fix the bacteria
onto the slide and to prevent the sample from being lost during a staining
procedure. A smear can be prepared from a solid or broth medium. Below are
some guidelines for preparing a smear for a Gram-stain.

20. 1. Place one needle of solid bacterial growth or two loops
of liquid bacterial growth in the center of a clean slide.
2. If working from a solid medium, add one drop (and only one drop)
of water to your specimen with a water bottle. If using a broth
medium, do not add the water.
3. Now, with your inoculating loop, mix the specimen with the water
completely and spread the mixture out to cover about half of the
total slide area.
4. Place the slide on a slide warmer and wait for it to dry. The smear is now
ready for the staining procedure.