Microbiology of hematology

system
Milkias Abebe ( Assistant Professor)

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 Outlines
• Introduction
• Septicemia, Bacteremia, Toxemia
• Brucellosis
• Lyme Disease
• Relapsing Fever
• Rocky Mountain Spotted Fever
• Typhus

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 Objectives
At the end of this session, student will be able to:
 Describe the different diseases of hematology system

 Identify their causative agent

 Identify the pathogenesis of these microbial diseases

 Explore the diagnosis, treatment and prevention of these

diseases

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 Introduction
• Although the circulatory system is a closed system, bacteria
can enter the bloodstream through several routes.

• Wounds, animal bites, or other breaks in the skin and mucous

membranes can result in the rapid dissemination of bacterial
pathogens throughout the body.

• Localized infections may also spread to the bloodstream,

causing serious and often fatal systemic infections.

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 Septicemia, Bacteremia and
Toxemia

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 Septicemia, Bacteremia and Toxemia
• Septicemia refers to the presence of microbial infection of the
blood that causes illness.

• Bacteremia refers specifically to bacterial septicemia,

however many physicians use the terms bacteremia and
septicemia interchangeably.

• When bacteria remain fixed at a site of infection but release

toxins into the blood, the condition is toxemia.

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 Signs and Symptoms

• Septicemia is characterized by fever (over 38°C), chills, nausea,

vomiting, diarrhea, shortness of breath, malaise and changes in
mental status such as confusion and an future feeling of trouble.

• These signs and symptoms can progress rapidly to septic shock, a

condition of extremely low blood pressure resulting from dilation of
blood vessels.

• Decrease in body temperature, decrease in or absence of urine

output, rapid breathing, unusual blood clotting, increased heart
rate, anxiety, and death characterize septic shock.

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 Signs and Symptoms

• Mortality from septic shock may exceed 50%, depending on

the bacterium and the overall health of the patient.

• Bacterial septicemia can trigger petechiae minute

hemorrhagic skin lesions—on the trunk and lower extremities

• Septic bacteria can also invade bones, causing osteomyelitis

which is inflammation of the bone and its internal bone
marrow.

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 Figure: Petechiae, a sign of bacteremia. These skin lesions can be small and relatively
diffuse or may coalesce to form large black sores containing dead cells.

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 Signs and Symptoms

• Toxemia manifests differently according to the toxins involved.

• Exotoxins, which are released from living microbes, include

cell-killing cytotoxins and neurotoxins, such as botulism toxin
that prevents muscular contraction and tetanus toxin that
prevents muscular relaxation.

• Dying Gram-negative bacteria disintegrate, releasing

endotoxin, which is the lipid A portion of lipopolysaccharide
(LPS) from the outer layer of a Gram-negative cell’s outer
membrane, into the blood.

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 Signs and Symptoms

• A severe form of toxemia with septic shock is streptococcal

toxic-shock-like syndrome (TSLS), in which a patient’s blood
pressure drops quickly and the patient may suffer from
dizziness, confusion, difficulty in breathing, and a weak and
rapid pulse.

• The liver and kidneys may fail.

• Staphylococcal toxic shock syndrome is a similar condition

often associated with reproductive tract infections

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 Pathogens and Virulence Factors

• Depend on bacterial species.

• Bacteria that form capsules may resist phagocytosis or intracellular
digestion, allowing them to reproduce and metabolize in the blood.

• Other bacteria have the ability to capture iron, often by the use of
siderophores, which is necessary for bacterial growth, from carrier
proteins in the plasma or to destroy erythrocytes, releasing iron
from hemoglobin into the blood and making it available for
bacterial growth.

• By endotoxin: cause serious signs and symptoms because they

stimulate the body to release chemicals that trigger fever,
inflammation, diarrhea, hemorrhaging, blood coagulation, or shock.
How?

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 Pathogens and Virulence Factors

• Bacteria causing septicemia are opportunistic—normal members of

the microbiota that become pathogenic nosocomial; that is, they
are acquired in a health care setting.

• Gram-negative nosocomial bacteria include Pseudomonas

aeruginosa , Neisseria meningitidis, Escherichia coli, species of
Salmonella, Bacteroides.

• Gram-negative bacteria are more commonly associated with

septicemia

• Gram-positive bacteria such as Staphylococcus aureus and

Streptococcus pneumoniae can also be opportunistic causes of
septicemia.

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 Pathogens and Virulence Factors

• Streptococcus pyogenes causes streptococcal toxic-shock-like

syndrome (TSLS).

• Streptococcal infections of the skin or wounds rather than of

“strep throat” are associated with TSLS.

• Streptococcal toxic-shock-like syndrome VS toxic shock

syndrome (TSS), which is associated with tampon use and
caused by Staphylococcus.

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 Pathogenesis

• Septicemia begins with direct inoculation of bacteria into the blood

such as can occur during medical procedures, via nonsterile needle
use by drug users, from an infection elsewhere in the body, or
through small abrasions in the respiratory or digestive tracts.

• Septicemia is commonly associated with prolonged venous needle

placement, the use of inadequately sterilized kidney dialysis
machines, surgical wounds, infected teeth, and urinary tract
infections.

• Nosocomial infections account for about half of all cases of

staphylococcal septicemia

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 Pathogenesis

• People with suppressed immune systems due to alcoholism, other

drug abuse, malnutrition, stress, or HIV infection cannot effectively
fight bacterial infections.

• Endotoxin activates nearly all nonspecific defensive responses of

the body:
• it activates complement via the alternative pathway, causes
coagulation (blood clotting), elicits inflammation, and initiates the
release of potent cytokines from macrophages, monocytes, B cells,
and other defensive cells when it binds to the cytoplasmic
membranes of these cells.

• Coagulation can be widespread and severe, a condition called

disseminated intravascular coagulation (DIC), which can be fatal.

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 Pathogenesis

• Released cytokines, including tumor necrosis factor (TNF),

interleukins (ILs), and platelet activating factor (PAF), normally
elicit defensive reactions at localized infections, but they
become life threatening when carried to an extreme
throughout the body in septicemia.

• TNF causes tissue damage and is pyrogenic (fever producing).

• IL-1 also triggers fever and causes the bone marrow to

release a large number of immature neutrophils, which attach
to and damage blood vessel walls.

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 Pathogenesis

• IL-6 and IL-8 damage circulating neutrophils and further injure

cells lining blood vessels, allowing plasma to escape the
vascular system, which quickly reduces blood pressure.

• The drop in blood pressure reduces blood flow and oxygen

delivery to vital organs, which can give way, resulting in death

• platelet activating factor is another potent trigger for

coagulation

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 Figure: Potential effects of endotoxin.
4/5/2023 What type of bacteria produces
Milkias A endotoxin? 22
 Diagnosis, Treatment, and Prevention

• Physicians diagnose septicemia based upon signs and

symptoms.

• Clinicians are able to culture bacteria from the blood

• Prevention of septicemia depends upon quick treatment of

infections, particularly in patients whose immune system is
impaired

• Newer treatments utilizing monoclonal antibodies against LPS

and TNF show some small success in mitigating the effects of
endotoxin

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 Brucellosis

• Bang’s disease, after microbiologist Bernhard Bang (1848–

1932), who investigated the disease.

• Malta fever, rock fever of Gibraltar, and fever of Crete, after

localized epidemics in those locales.

• Brucellosis is characterized by a fluctuating fever that spikes at

about 40°C every afternoon, giving the disease another of its
common names—undulant fever.

• The patient also has chills, sweating, headache, myalgia

(muscle pain), and weight loss.

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 Pathogen and Virulence Factors

• Brucella is a genus of nonmotile, aerobic, Gramnegative coccobacilli

that lack capsules.

• B. melitensis, which infects goats and sheep, B. abortus in cattle, B.

suis in swine, and B. canis in dogs, foxes

• In animal hosts, the Brucella lives as an intracellular parasite in

organs such as the uterus and placenta, but these organs are not
infected in humans. Why?

• Infections in animals are either asymptomatic or cause a mild

disease, though they sometimes cause sterility or spontaneous
abortion.

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 Pathogen and Virulence Factors

• As a Gram-negative bacterium, Brucella has endotoxin that

accounts for some of the signs and symptoms of brucellosis.

• The bacterium also has the ability to grow and multiply inside
phagocytic cells, evading antibodies and some antibacterial
drugs.

• Humans become infected either by consuming unpasteurized

contaminated dairy products or through contact with animal
blood, urine, or placentas in workplaces such as
slaughterhouses, veterinary clinics…

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 Pathogen and Virulence Factors

• The bacterium enters the body through breaks in mucous

membranes of the digestive and respiratory tracts.

• Brucella travels inside phagocytic cells to organs of the body,

including lymph nodes, spleen, bone marrow, liver, and heart.

• Arthritis, splenomegaly (enlarged spleen), enlarged testes,

endocarditis, meningitis, and encephalitis may result.

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 Diagnosis, Treatment, and Prevention

• Daily undulating fever suggests a diagnosis of brucellosis.

• Diagnosis is complicated because Brucella is difficult to
culture in the laboratory.

• Serological tests are conclusive—a rising level of antibodies

against Brucella in the blood is diagnostic.

• Physicians treat brucellosis with combinations of antibacterial

drugs, including doxycycline and rifampin or streptomycin for
several weeks.

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 Diagnosis, Treatment, and Prevention

• An attenuated vaccine for animals exists, but physicians do

not administer it to humans because the vaccine sometimes
causes brucellosis.

• With pasteurization of dairy products, immunization of

uninfected domesticated animals, and the slaughter of
infected ones, the threat of brucellosis residents has been
reduced.

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 Lyme Disease

• A large (0.5 μm * 3-20 μm) spirochete—Borrelia burgdorferi

causes Lyme disease.

• vector-borne Lyme disease mimics many other diseases, and

its range of signs and symptoms is vast.

• Disease typically has three phases in untreated patients:

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 Lyme Disease

Phase 1
• An expanding red rash, which often resembles a bull’s-eye, occurs at the
site of infection within 3–30 days .

• About 80% of patients have such a rash, which lasts for several weeks.

• Other early signs and symptoms include malaise, headaches, dizziness,

stiff neck, severe fatigue, fever, chills, muscle and joint pain, and
lymphadenopathy (infected lymph nodes).

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 Lyme Disease

Phase 2
• Neurological symptoms (for example, meningitis,
encephalitis, and peripheral nerve neuropathy) and cardiac
dysfunction typify the second phase, which is seen in only
10% of patients.

• The final phase, seen in almost 80% of patients, is

characterized by severe arthritis that can last for years.

• Lyme disease is rarely, if ever, fatal.

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 Distinctive “bull’s-eye” rash of Lyme disease.
Such a rash is often seen in the initial phase of the disease
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(a) A characteristic bull’s eye rash of Lyme disease forms at the site of a tick bite.
(b) A darkfield micrograph shows Borrelia burgdorferi, the causative agent of Lyme
disease.

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 Pathogen and Virulence Factors

• B. burgdorferi has an unusual metabolism in that it does not use

iron in its enzymes or its electron transport chains.

• It uses manganese in place of iron, thereby by-passing one of the

body’s natural defense mechanisms—the lack of free iron in human
tissues and fluids.

• The bacterium can change its outer membrane proteins via genetic
rearrangement to emerge as antigenically different variants, making
it more difficult for the immune system to clear the organism from
the blood.

• When it dies, Borrelia releases an active endotoxin (lipid A) from its

outer membrane.

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 Pathogenesis

• Hard ticks of the deer tick genus Ixodes (ik-so¯de¯z) are the
vectors of Lyme disease.

• A deer tick lives for two years, during which it passes through
three stages of development: a six-legged larva, an eight-
legged nymph, and an eight-legged adult.

• Once during each stage it attaches to a vertebrate host for a

single blood meal.

• After each of its three feedings, the tick drops off its host and
lives in leaf litter or on brush.

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 Pathogenesis

• Transmission of Borrelia from a female tick to her offspring is

rare, so larvae that hatch in the spring are usually uninfected,
becoming infected during their first blood meal.

1. Over the winter, larvae digest their blood meals while Borrelia
replicates in the ticks’ guts.

• In the spring of their second year, the ticks molt into nymphs
and feed a second time, infecting new hosts with Borrelia via
saliva.

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 Pathogenesis

2.(Uninfected nymphs can become infected if they feed on an

infected host.)
• Laboratory studies have shown that infected ticks must
remain on a host for 36–48 hours in order to transmit enough
spirochetes to establish a Borrelia infection in that host.

• Nymphs drop off their hosts and undergo further

development into adults.

3. In the fall, adult ticks feed a final time

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 Pathogenesis

4. mate, lay eggs, and die. Adults infected with Borrelia infect
their hosts as they feed.
• Adult ticks are much larger than nymphs are, so humans
usually see and remove adults before they can transmit
Borrelia; thus, nymphs most often infect humans.

• Introduced by a tick’s bite, Lyme disease spirochetes move

from the site of infection through the blood and lymph.

• They can accumulate in joints and, along with antibodies

directed against them, trigger arthritis

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 Diagnosis, Treatment, and Prevention

• Diagnosis of Lyme disease, typically based on observations of

its usual signs and symptoms

• diagnosis is confirmed with ELISA and western blot tests to

demonstrate the presence of antibodies against Borrelia.

• People for recreation: hiking, picnicking, and working

outdoors in areas where Lyme disease is prevalent should
take precautions to reduce the chances of infection,
particularly during May to July(9-11), when nymphs are
feeding.

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 Diagnosis, Treatment, and Prevention

• People who must be in the woods should wear long-sleeved

shirts and long, tight-fitting pants, and should tuck the cuffs of
their pants into their socks to deny ticks access to skin.

• Repellents containing DEET (Diethyl-meta-toluamide), which

is toxic to ticks, should be used.

• As soon as possible after leaving a tick-infested area, people

should thoroughly examine their bodies for ticks or their bites.

• Attached ticks should be removed using forceps/tick extractor

.

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When removing a tick that has already attached itself

• Use blunt tweezers to gently pull near the site of attachment until
the tick releases its hold on the skin.

• Avoid crushing the tick's body and do not handle the tick with bare
fingers. This could release bacterial pathogens and actually increase
your exposure. The tick can be killed by drowning in water or
alcohol, or frozen if it may be needed later for identification and
analysis.

• Disinfect the area thoroughly by swabbing with an antiseptic such

as isopropanol.

• Monitor the site of the bite for rashes or other signs of infection.

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(a) This black-legged tick, also known as the deer tick, has not yet attached to the
skin. (b) A notched tick extractor can be used for removal. (c) To remove an
attached tick with fine-tipped tweezers, pull gently on the mouth parts
until the tick releases its hold on the skin. Avoid squeezing the tick’s body, because
this could release pathogens and thus increase the risk of contracting Lyme disease

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 Diagnosis, Treatment, and Prevention

• Health departments recommend that outdoor

enthusiasts wear light-colored pants while
hiking in areas where Ixodes is endemic. Why?

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 Relapsing Fever

• Borrelia spp. also can cause relapsing fever.

• Two of the most common species are B. recurrentis, which

causes epidemics of louse borne relapsing fever, and

• B. hermsii, which causes tick borne relapsing fevers.

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 Relapsing Fever

• These Borrelia species are transmitted by the body louse

Pediculus humanus and the soft-bodied tick Ornithodoros
hermsi, respectively.

• Lice acquire the spirochetes from human reservoirs, whereas

ticks acquire them from rodent reservoirs.

• Spirochetes infect humans when Borrelia in the vector’s saliva

or excreta enter the skin rapidly as the vector bites.

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 Relapsing Fever

• In both louse- and tick borne relapsing fevers, bacteremia

usually occurs after the initial exposure, leading to a sudden
high fever (39–43 °C ) typically accompanied by headache and
muscle aches.

• After about 3 days, these symptoms typically subside, only to

return again after about a week.

• After another 3 days, the symptoms subside again but return

a week later, and this cycle may repeat several times unless it
is disrupted by antibiotic treatment

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 Relapsing Fever

• The diagnosis of relapsing fever can be made by microscopic

observation of spirochetes in blood, using darkfield
microscopy .

• For louse borne relapsing fever, doxycycline or erythromycin

are the first-line antibiotics.

• For tickborne relapsing fever, tetracycline or erythromycin are

the first-line antibiotics.

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A peripheral blood smear from a patient with tick borne relapsing fever. Borrelia appears
as thin spirochetes among the larger red blood cells. (credit: modification of work by
Centers
4/5/2023 for Disease Control and Prevention)
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 Rickettsias

• Gram-negative, highly pleomorphic bacteria (may be cocci, rods, or

threads)
• Chlamydias and rickettsias, that are obligate intracellular
pathogens, meaning that part of their life cycle must occur inside
other cells called host cells.

• When not growing inside a host cell, Chlamydia and Rickettsia are
metabolically inactive outside of the host cell.

• They cannot synthesize their own adenosine triphosphate (ATP),

and, therefore, rely on cells for their energy needs.

• R. rickettsii causes Rocky Mountain spotted fever, a life-threatening

form of meningoencephalitis

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 Rickettsias

• R. rickettsii infects ticks and can be transmitted to humans via a bite

from an infected tick

• Another species of Rickettsia, R. prowazekii, is spread by lice. It

causes epidemic typhus, a severe infectious disease common during
warfare and mass migrations of people.

• R. prowazekii infects human endothelium cells, causing

inflammation of the inner lining of blood vessels, high fever,
abdominal pain, and sometimes delirium.

• A relative, R. typhi, causes a less severe disease known as murine or

endemic typhus,

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 Rocky Mountain Spotted Fever

• A number of arthropod-borne bacteria cause rashes in

humans.

• One of these is Rocky Mountain spotted fever (RMSF), caused

by a rickettsia rickettsii .

• In most cases (90%), RMSF manifests with a non-itchy, spotted

rash on the trunk and appendages, including the palms and
soles.

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 Rocky Mountain Spotted Fever

• In about 50% of patients, the rash develops into subcutaneous

hemorrhages called petechiae (pe-te¯ke¯ -e¯).

• Patients with RMSF also have fever, headache, chills, muscle

pain, nausea, and vomiting

• In severe cases, the respiratory, central nervous,

gastrointestinal, and renal systems fail.

• Infections of the brain may also occur, producing language

disorders, confusion, spasms, coma, and death.

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 The rash in a case of Rocky Mountain spotted fever.
Petechial rash

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 Pathogen and Virulence Factors

• intracellular parasite that possesses a cell wall of

peptidoglycan and an outer membrane of lipopolysaccharide,
which has little endotoxin activity.

• Outside of a host cell, R. rickettsii is unstable and dies quickly;

therefore, it requires a vector for transmission from host to
host.

• This vector is a hard tick of the genus Dermacentor

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 Pathogen and Virulence Factors

• Once it is in a host, Rickettsia enters the host’s cells by

stimulating endocytosis.

• Inside a phagosome, it secretes an enzyme that lyses the

phagosome membrane, releasing the bacterium into the
host’s cytosol before a lysosome fuses with the phagosome.

• As a result, Rickettsia avoids being digested

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 Pathogenesis

• R. rickettsii is typically dormant in the salivary glands of its tick

vectors; only when the arachnids feed for several hours is the
bacterium infective.

• The active bacterium is released from a tick’s salivary glands

into the mammalian host’s circulatory system, where it infects
a cell lining a small blood vessel.

• In rare cases, humans become infected following exposure to

tick feces or to tissues and fluids from crushed ticks

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 Pathogenesis

• R. rickettsii secretes damage to blood vessels, which allows

blood to escape, resulting in low blood pressure and
insufficient nutrient and oxygen delivery to the body’s organs.

• Almost 5% of patients die, even with treatment.

• Patients recovering from life-threatening acute Rocky

Mountain spotted fever may experience paralysis of the legs,
hearing loss, and gangrenous secondary infections with
Clostridium,that necessitate the amputation of fingers, toes,
arms, or legs.

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 Pathogenesis

• Dermacentor ticks transmit R. rickettsii among humans and

rodents, which act as reservoirs.

• Male ticks infect female ticks during mating.

• Female ticks transmit rickettsia to eggs forming in their

ovaries in a process called transovarian transmission.

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 Diagnosis, Treatment, and Prevention

• Serological tests such as latex agglutination and fluorescent

antibody stains have been used to confirm an initial diagnosis
based on a rash on the soles or palms, sudden fever, and
headache following exposure to hard ticks.

• Nucleic acid probes of specimens from rash lesions provide

specific and accurate diagnosis,

• Physicians treat Rocky Mountain spotted fever with

doxycycline, tetracycline, or chloramphenicol.

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 Diagnosis, Treatment, and Prevention

• Prevention of infection involves wearing tight-fitting clothing,

using tick repellants, promptly removing attached ticks, and
avoiding tick infested areas, especially in spring and summer,
when ticks are most hungry.

• It is impossible to eliminate the ticks in the wild, in part

because they can survive without feeding for more than four
years.

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 Typhus
• Typhus a group of diseases caused by rickettsias transmitted by
arthropod vectors

• There are several forms of typhus, namely, louse borne epidemic

typhus caused by R. prowazekii, flea borne endemic typhus caused
by R. typhi, chigger (chi·guh) borne scrub typhus caused by Orientia
tsutsugamushi, and several other quite rare forms.

• Cases of fleaborne endemic typhus, also called murine typhus,

occur in small numbers in the southern regions of California and
Texas.

• epidemic typhus, the most important of the typhus group of

diseases.

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 Epidemic Typhus

• The disease epidemic typhus is caused by Rickettsia

prowazekii and is transmitted by body lice, Pediculus
humanus

• Epidemic typhus is characterized by a high fever and body

aches that last for about 2 weeks.

• A rash develops on the abdomen and chest and radiates to

the extremities.
• Severe cases can result in death from shock or damage to
heart and brain tissues.

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 Epidemic Typhus

• When a bacteremic patient is bitten, the organism is ingested

by the louse and multiplies in the gut epithelium.

• It is excreted in the feces of the louse during the act of biting

the next person and auto-inoculated by the person while
scratching the bite.

• The infected louse dies after a few weeks, and there is no

louse to louse transmission; therefore, human infection is an
obligatory stage in the cycle.

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 Epidemic Typhus

• Epidemic typhus has played an important role in human

history, causing large outbreaks with high mortality rates
during times of war or adversity.

• During World War I, epidemic typhus killed more than 3

million people on the Eastern front.

• In recent years, most outbreaks have taken place in Burundi,

Ethiopia, and Rwanda

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 Epidemic Typhus

• Laboratory diagnosis of rickettsial diseases is based on

serologic analysis rather than isolation of the organism.

• Although rickettsiae can be grown in cell culture or

embryonated eggs, this is a hazardous procedure that is not
available in the standard clinical laboratory.

• A PCR based assay to detect R. rickettsii DNA

• Of the serologic tests, the indirect immunofluorescence and

enzymelinked immunosorbent assay (ELISA) tests are most
often used.

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 Epidemic Typhus

• The WeilFelix test is not good because its specificity and

sensitivity are too low.

• The WeilFelix test is based on the cross reaction of an antigen

present in many rickettsiae with the O antigen polysaccharide
found in P. vulgaris OX2, OX19, and OXK.

• The test measures the presence of antirickettsial antibodies in

the patient’s serum by their ability to agglutinate Proteus
bacteria.

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 Epidemic Typhus

• The specific rickettsial organism can be identified by the

agglutination observed with one or another of these three
different strains of P. vulgaris

• Prevention of typhus is based on personal hygiene and

“delousing” with DDT.

• A typhus vaccine containing formalin killed R. prowazekii

organisms is effective and useful in the military during
wartime

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 Murine (Endemic) Typhus

• is caused by Rickettsia typhi and is transmitted by the bite of the rat

flea, Xenopsylla cheopis, with infected rats as the main reservoir.

• Clinical signs and symptoms of murine typhus include a rash and

chills accompanied by headache and fever that last about 12 days.

• Some patients also exhibit a cough and pneumonia-like symptoms.

• Severe illness can develop in immunocompromised patients, with

seizures, coma, and renal and respiratory failure.

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 Murine (Endemic) Typhus

• Clinical diagnosis of murine typhus can be confirmed from a

biopsy specimen from the rash.

• Diagnostic tests include indirect immunofluorescent antibody

(IFA) staining, PCR for R. typhi, and acute and convalescent
serologic testing.

• Primary treatment is doxycycline, with chloramphenicol as the

second choice.

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 Culturing blood
• Blood culture is required when bacteraemia (septicaemia) is
suspected.

• Mycobacterium tuberculosis (HIV-associated tuberculosis),

leptospira species, Borrelia species, rickettsiae, and Bartonella
bacilliformis

FUNGI
• Candida albicans and other yeasts, e.g. Cryptococcus
neoformans, and occasionally Histoplasma capsulatum and
other fungi that cause systemic mycoses.

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Collect blood and inoculate culture media

• Whenever possible blood should be collected before

antimicrobial treatment has started.

• When the patient has recurring fever, collect the blood as the
temperature begins to rise

• Choice of culture media: Columbia agar and Columbia broth

diphasic medium with added SPS (sodium polyanethol
sulphonate), also known as Liquoid.

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 Right: Diphasic blood culture medium. Left:
Inoculated diphasic medium
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• Thioglycollate broth medium is recommended to isolate strict
anaerobes should an anaerobic infection be suspected.

• If wishing to culture for S. Typhi only, the use of ox-gall

medium is recommended

• Blood for culture must be collected and dispensed with great

care to avoid contaminating the specimen and culture
medium.

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 2. Examine the specimen microscopically

• Gram smear: To detect Gram positive and Gram negative

bacteria, particularly when the patient is an infant or young
child.

• Ziehl-Neelsen smear: To detect AFB when the patient has

AIDS or suspected HIV disease

• Giemsa or rapid Field’s smear: To detect borreliae, or

parasites such as trypanosomes, malaria parasites, and
microfilariae.

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 3. Examine and report the cultures

Diphasic culture (Columbia agar and broth)

• Using a hand lens, examine twice daily (up to 7 days or 4 weeks
when brucellosis is suspected) for microbial growth, indicated by
colonies

• Subculture on blood agar, chocolate agar, and MacConkey agar.

• Incubate the blood agar and MacConkey agar plates aerobically and
the chocolate agar plate in a carbon dioxide atmosphere (candle
jar).

• Depending on the bacteria seen, further biochemical test (e.g. for

coagulase, catalase, oxidase, urease, and motility).

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Milkias A
Gram Stain Reagents

Crystal violet – initial stain

Iodine – mordant/binding agent
Alcohol – decolorizer
Safranin or diluted basic or carbol fuchsin –
counterstains

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Then….

Oil (100X): Neutrophils with Gram-positive

cocci in clusters
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Gram Stain: Urethral Discharge
Urethritis Neisseria gonorrhoaea

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Do All Bacteria Stain with the Gram Stain?

Organisms that do not stain

Chlamydia & others - too small & intracellular
Spirochetes - many are too thin

Mycobacteria - waxy cell wall

Poorly staining
Some anaerobes
Legionella

Brucella & others

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Gram Negative Rods on MacConkey Agar (2)

Lactose non-fermenter Lactose fermenter

Serratia Escherichia
marcescens coli

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93
Pseudomonas aeruginosa

Important properties
− Blue-green diffusible
pigment
− Fruity, grape-like odor
− Tends to be very antibiotic
resistant
− Primarily a water organism
but is found as normal GI
flora in about 20 - 25% of
people.

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94
Brucella

 Grows on Blood agar

and Chocolate agar;
discrete colonies are
seldom seen in one day.
 Once evident colonies
are small, translucent Initial Identification
soft & easily emulsified. Oxidase positive
 Non hemolytic on Blood Urease positive
(Rapid urease
agar positive)
Indole negative
Catalase positive
Cultures should be handled using
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Biosafety Level 3 (BSL-3) practices
Identification Tests for Enteric Gram-negative Rods

 Triple sugar iron (TSI)

agar or Kligler’s Iron
agar (KIA)
 Sugar fermentation TSI-
glucose 1X, lactose 10X,
sucrose 10X
 Sugar fermentation KIA -
glucose 1X, lactose 10X
 Iron salts in both detect H2S

 Phenol red indicator -

yellow in acid

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ANTIMICROBIAL SENSITIVITY (SUSCEPTIBILITY)
TESTING

 The test is used to measure the ability of the drug to

inhibit or kill pathogens in vitro. I.e. it is used to select
effective antimicrobial drugs.
 Sensitivity test is performed:
 For organisms with variable antibiotic sensitivity (un
predictable sensitivity) E.g. Shigella
 For non responding patients after taking adequate
therapy.
 For patients whose immune system is depressed
 For relapsing cases (reappearance of disease)

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Figure: Removing colonies from a primary culture Figure: Checking the turbidity of the test suspension
plate to make a suspension of the test organism. against the turbidity of a chemical standard.

Figure: Swabbing the surface of the susceptibility

Figure: Avoiding using too much inoculum by
testing agar. The plate is swabbed in three directions,
pressing and rotating the swab against the side
rotating the plate approximately 60° to ensure even
of the tube
distribution.
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Figure: Placing antimicrobial discs on the Figure: Measuring the zones of inhibition in mm. The
inoculated plate. end of inhibition is where growth starts.

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Reading Plates (2)

Zone measurements compared to CLSI

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Guidelines to interpret susceptibilities
101
Example of Interpretive Standards

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 Seminar Presentation Title
• Please refer your group assignment given to
class representative

• Based on your topic prepare ppt and share to

each other

• It will be presented In the class

103
 Thank You!

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