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Title: Chair Microbiology, Virology, and Immunology


1
Chair Microbiology, Virology, and Immunology
CLINICAL AND
HOSPITAL MICROBIOLOGY
Lecturer Prof. S.I. Klymnyuk
2
METHODS OF EXAMINATION IN MICROBIOLOGY
BACTERIOSCOPIC BACTERIOLOGICAL SEROLOGICAL BIOLOGI
CAL ALLERGIC EXPRESS-DIAGNOSIS
3
BACTERISCOPIC METHOD
Neisseria meningitidis
4
BACTERISCOPIC METHOD
Neisseria meningitidis
5
BACTERISCOPIC METHOD
Neisseria gonorrhoeae
6
BACTERISCOPIC METHOD
7
BACTERISCOPIC METHOD
Yersinia pestis
8
BACTERISCOPIC METHOD
Bacillus anthracis
9
BACTERISCOPIC METHOD
Mycobacterium tuberculosis
10
BACTERISCOPIC METHOD
Streptococcus pneumoniae
11
BACTERISCOPIC METHOD
Clostridium pneumoniae
12
Bacteriological method
Isolation of pure culture
13
Bacteriological method
Neisseria meningitidis
14
Liquor
15
Serologic method
Agglutination test
16
Serologic method
Agglutination test
17
Serologic method
Agglutination test
18
Serologic method
IHAT
19
Serologic method
Ring precipitation test
20
Serologic method
Immunodiffusion (Ouchterlony) test
21
Serologic method
Mancinis test
22
Serologic method
CFT
23
Serologic method
ELISA
24
Serologic method
ELISA
25
Biological method
TBS in rabbit
TBC in guinea pig
26
Allergic method
Mantouxs test
27
Express-diagnosis
IFT
28
Clinical microbiology is the field of medical
microbiology, which study microbial diseases in
somatic departments of all specialties. There
are general tasks, which provide the help to
doctors in diagnosis, treatment and prophylaxis
of purulent complications, before clinical
microbiology
29
  • Features of purulent processes in non-infectious
    clinics
  • - polietiology
  • - non-specific clinical signs.
  • There are more then 2000 causative agents of
    purulent diseases.
  • The more frequent of them there are
  • - genera Staphylococcus, Streptococcus,
    Bacteroides, Prevotella
  • families Enterobacteriaceae (Proteus,
    Klebsiella, Escherichia, Serratia, Citrobacter,
    Hafnia etc.), Pseudomonadaceae, Neisseriaceae
    (Acinetobacter, Moraxella, Branchamella).
  • In urological and gynecological clinics -
    Mycoplasmas, Chlamydia.

30
Most of conditionally pathogenic bacteria belong
to normal human flora so its hard to determine
their etiologic role (etiological
significance). So, they can present normal
microflora of tested fluids and tissues and
contaminate them from environment. Thats why
for correct interpretation of examination results
its necessary to know composition of normal
microflora of tested samples. In that cases if
tested samples are sterile (blood, synovial and
pleural fluids, liquor, exudates) all microbes
which are present in them may be causative agents
of diseases.
31
Bacteria Frequently Present as Normal Flora
Occasionally Causing Overt Disease
Organisms Usual Locale Infectious Disease Process
Staphylococcus aureus Nose, skin In all areas of the body, nosocomial diseases, food poisoning
Staphylococcus epidermidis Skin, nose, vagina Endocarditis, nosocomial phlebitis, acne
Enterococci Feces Blood, wounds, urinary tract, endocarditis
Viridans streptococci Saliva Endocarditis
Peptostreptococ-cus sp Mouth, feces, vagina Abscess formation, gangrene
Neisseria sp Throat, mouth, nose Meningitis
Veillonella sp. Mouth, vagina Bacterial endocarditis, abscesses
32
Lactobacillus sp. Mouth, feces, vagina Bacterial endocarditis (rare), lung abscess (1 report)
Corynebacterium sp. Nasopharynx, skin, vagina Bacterial endocarditis
Mycobacterium (not Mycobacterium tuberculosis) Prepuce, clitoris, lung, feces, tonsils, Food, skin Suspected in some infectious disease processes
Clostridium sp. Feces, skin, environment, including food, Vagina Clostridia myositis, cellulitis, food poisoning
Enterobacteriaceae Feces, vagina, mouth, urethra Urinary tract, wounds, pneumonia, nosocomial scesses, meningitis, blood, peritonitis, enteritis, abscesses, etc .
Moraxella sp. Nose, genito-urinary tract Conjunctivitis, etc
33
Achromobacter sp. Nose, genitourinary tract, skin Meningitis, blood, urethritis, burns
Pseudomonas sp. Feces, skin Blood, burns, wounds, urinary tract, respiratory tract, meningitis
Alcaligenes faecalis Feces Blood, urinary tract, conjunctiva, respiratory tract, meningitis
Haemophilus sp. Nasopharynx, conjunctiva, vagina Laryngotracheobronchitis, meningitis, pyarthrosis, conjuncti vitis, genitourinary tract
Fusobacterium sp. Mouth, saliva, feces Infected human bites, gangrene
Bacteroides sp. Feces, mouth, throat Bactenal endocarditis, abscesses, mixed infections
34
Pure culture of conditionally-pathogenic microbes
may be causative agents of disease according to
the such signs -  microbes are present in tested
material from pathologic focus in the amount of
104-105 colony-forming units (CFU) in 1 ml or 1
g -  repeated isolation from the same material
the same culture -  increasing in the patients
serum antibodies to the autostrains or microbial
culture, which can be causative agent.
35
There are necessary roles before collection of
tested material -  to take material before
antibacterial therapy beginning or after some
time after antibiotic inoculation which is
necessary for its excretion from the organism (as
a rule 8-10 hours) - to take material from
infectious focus or examine proper
discharges - hold on to the strict aseptic for
the purpose to prevent contamination of the
specimen by microflora of environment
36
-  material is taken into the sterile boxes
clinical specimen with anaerobic bacteria must be
protected from atmosphere oxygen action - the
collection of an adequate specimen is useless if
the time between collection and culturing allows
the disease-producing organism to die (in another
cases its necessary to use the refrigerator or
special transport media) - isolation of viruses,
Rickettsia, Chlamidia is made in specialize
laboratories -  to clinical specimen a proper
document is added, which has data , which has
data for correct microbiological examination.
37
Examination of the blood
Bacteriemia, septicemia, septicopyemia
38

Bacteremia bacteria in the blood frequently
is accompanied by the onset of chills and fever,
an increase in pulse rate, and a drop in blood
pressure. Even in infections in which bacteremia
is a major aspect of the disease, the organisms
in the bloodstream are not always constantly
present in sufficient numbers to be grown from a
single blood specimen. Patients with such
infections may have to provide several blood
specimens before the causative agent can be
isolated. When an intermittent bacteremia is
suspected, it is routine to obtain three 10 to
20-ml blood samples over a 24 hour period to
maximize chances for isolation of the organism
39
Collection of a Blood Specimen In taking a blood
specimen for culture, one should be aware that
although blood is normally sterile, the skin that
must be penetrated is not sterile. Routinely, the
skin should be cleansed first with 70 to 95
alcohol to remove dirt, lipids, and fatty acids.
The site then should be scrubbed with a circular,
concentric motion (working out from the starting
point) using a sterile gauze pad soaked in an
iodophor. The iodine should be allowed to remain
on the skin for at least 1 minute before it is
removed by wiping with a sterile gauze pad soaked
with 70 to 95 alcohol. It must be emphasized,
however, that all this will be useless if the
person drawing the blood palpates the vein after
the cleaning process, thereby contaminating the
very site that had been cleaned. After cleansing
the penetration site, the blood can be withdrawn
using either a sterile needle and syringe or a
commercially available, evacuated blood
collection tube.
40
Media Inoculated With Blood Specimens Blood
always should be inoculated into the appropriate
medium at the bedside partially evacuated,
commercially available, blood culture bottles,
which contain 30 to 100 ml of a rich, liquid
medium such as brain-heart infusion or trypticase
soy broth is routinely used. If possible, 10 to
20 ml of blood should be taken from the patient
and inoculated into an approximately10 fold
excess of the blood culture medium when possible,
two such bottles should be inoculated. One is
vented to permit the growth of aerobic bacteria
(by inserting a sterile, cotton plugged needle
through the rubber stopper until the bottle has
filled with air), and the other is not vented to
allow the growth of anaerobic organisms. Special
media for aerobic and anaerobic culture are
available. Some commercially available bottles
are provided with a venipuncture set, which
allows the blood to be injected into the medium.
41
Identification of Blood Isolates Blood cultures
are incubated at 36C and observed daily for at
least 1 week for evidence of turgidity or
hemolysis. Gram's stains, streak plates, and
antibiotic susceptibility tests should be carried
out as quickly as possible after the observation
of visible growth in the original broth culture.
In the absence of obvious growth in 1 or 2 days,
blind subcultures on chocolate blood agar plates
may speed the appearance of obligately aerobic
organisms. Commercially available penicillinase
can be added to blood cultures from patients who
have received penicillin therapy. Penicillinase
preparations should be checked for sterility to
eliminate them as a potential source of
contamination. Resins incorporated in special
blood culture media can neutralize a broad
spectrum of antibiotics.
42
Once a Gram's stain has rendered some information
concerning the type of organism involved, special
supplementary or differential media should be
inoculated. MacConkey or eosin-methylene blue
plates should be streaked if gram negative rods
are present, and prereduced media should be
inoculated if obligate anaerobes such as
Bacteroides or Fusobacterium are suspected. The
finding of organisms that constitute the normal
flora or are frequent inhabitants of the skin
(eg, diphtheroids, Staphylococcus epidermidis.
Bacillus sp.) usually is viewed with suspicion,
unless the frequency of isolation or the clinical
setting indicates they did not arrive as
contamination during the collection of the blood
43
Pathogenesis of toxic shok
44
If microbes are absent after 10 days of
incubation it means, that inoculation results are
negative and blood is sterile.
45
URINE Most urinary tract infections are initiated
by organisms that gain entrance to the bladder by
ascending through the urethra, and they are more
common in women than in men In men, however, a
chronic infection of the prostate gland also can
be the source of a bladder infection (cystitis)
or a kidney infection (pyelonephritis). In both
sexes, most urinary tract infections are caused
by normal flora enteric organisms, among which
can be species of Escherichia, Klebsiella,
Fnterobacter, Proteus, Pseudomonas, and
Enterococcus.
46
Examination of urina
Etiology of urethritis
47
Major causes of urinary tract infections
48
Pathogenesis of genital tract infections
49
Major causes of vaginitis
Major causes of cervicites
50
Collection of Urine Specimens Urethral
catheterization can yield samples with minimal
contamination, but the danger of introducing
organisms from the urethra into the bladder
provides some risk to this procedure. Moreover,
microbial flora in the urethra, particularly in
men, can contaminate the specimen, leading the
microbiologist to an erroneous conclusion.
Therefore, catheterization is not performed
routinely for the collection of urine samples.
Instead, voided samples are obtained after
careful cleansing of the external genitalia.
However, the following considerations must be
adhered strictly if bacteriologic reports on
voided urine samples are to be meaningful.
51
First, all voided urine samples will contain some
bacteria, therefore, a quantitative assay for the
number of bacteria present must be carried out.
Second, this number will be grossly misleading
unless the exterior genitalia are carefully
cleaned to remove contaminating bacteria from the
female vulva and perinea area and from the male
urethral meats. The patient must be carefully
instructed in how to wash these areas with soap
and water, then rinse them thoroughly to remove
any residual soap. Women must be instructed to
keep the labia continuously apart during the
washing, rinsing, and voiding of urine. Urine
from either sex should be collected in a sterile
cup only after the first 20 to 25 ml has been
voided, because the flushing action of the
initial flow will remove many of the organisms
present in the urethra.
52
Media Inoculated With Urine Specimens All voided
urine samples, as well as most samples collected
by catheterization, contain some bacteria, so the
clinical diagnosis of an infection is based on
the numbers of bacteria in the urine.
Considerable experimental data have resulted in
the formulation of the following rules (1)101
bacteria or more per milliliter from a clean,
voided specimen indicates a urinary tract
infection (2) a value of 101 to 104 bacteria per
milliliter in a symptomatic patient requires a
second culture, and (3) 103 or fewer bacteria per
milliliter usually is not considered significant
in a voided sample. Single samples cannot be
considered to be 100 accurate, and it
frequently is advisable that duplicate samples
collected at different times be sent to the
diagnostic laboratory. Because of the need to
measure the bacteria present in a sample of
urine, several different techniques have been
devised to accomplish a rapid enumeration.
53
A standard platinum dilution loop (commercially
available) holds about 0.001 ml of liquid. Such a
loop can be used to streak a urine specimen
directly onto a nutrient-agar plate. If the loop
is calibrated monthly and compared with counts
obtained by the pour plate method, the overall
accuracy of the calibrated loop technique is
equivalent to that of a pour plate. A variety of
screening kits are commercially available for
suspected urinary tract infections. One, called a
paddle or dip slide type, has a selective agar
medium coated on one side and a nonselective agar
medium on the other side. The paddle is dipped
into the urine specimen, reinserted into its
sterile container, and incubated at 35C for 18
to 24 hours become counting the colonies (Fig.).
The amount of urine adhering to a paddle has-been
determined experimentally by the manufacturer,
and this kit is reported to be about 95 as
accurate as the pour plate procedure. Other kits
also are available, and it is likely that each
laboratory has its preferred method for
enumerating the microorganisms present in a urine
specimen.
54
Golds streak
55
Evaluation creteria 1. Bacteriuria degree not
more then 103 CFU/ml of urine testifies the
absence of inflammatory process. It is
contamination of urine. Bacteria in the same
amount which are found in the urine testify about
chronic persistent infection. 2. Bacteriuria
degree 104 CFU/?l testifies doubtful result.
Examination must be repeated. 3. Bacteriuria
degree 105 CFU/ml testifies the availability of
inflammatory process. 4. Changes of bacteriuria
degree during disease may be used for control of
the disease and therapy effectiveness.
56
Identification of Urine Isolates If a speedy
result is required, a Gram's stain of the
uncentrifuged urine specimen can be examined,
primarily because if one drop is allowed to dry
on a slide without spreading, the appearance of
one or more bacteria per oil-immersion field
(sometimes with leukocytes present) is indicative
of a total bacterial count greater than 10
microorganisms per milliliter of
urine. Blood-agar plates, as well as MacConkey
and eosin-methylene blue plates, should be
streaked with the urine specimen. Because members
of the Enterobacteriaceae are, by far, the most
frequent causes of urinary tract infections.
57
CEREBROSPINAL FLUID
Etiology of meningitis
58
The usual clinical signs of meningitis are
headache, fever, vomiting, and a stiff neck
however, many of these signs can be absent, or
not evident, in infants. There are several
specific organisms that are frequent causes of
meningitis, namely N. meningitidis, H.
influenzae, and S.
pneumoniae. In addition, other organisms, such as
M. tuberculosis and Cryptococcus
neoformans, less frequently cause meningitis.
Essentially any organism that gains entrance to
the fluid surrounding the brain and spinal cord
can grow and causes inflammation of the menings.
Such infections frequently are severe and, unless
promptly and adequately treated, can result in
the death of the patient in a matter of hours.
59
Specimen Collection of Cerebrospinal
Fluid Cerebrospinal fluid (CSF) is obtained by a
puncture into the lumbar region of the spine. It
is of utmost importance that the puncture site be
decontaminated in the manner described previously
for venipunctures to ensure that no contaminating
organisms are mechanically injected into the CSF.
The collected specimen should be placed into a
sterile screw-cap tube and delivered immediately
to the diagnostic laboratory.
60
Media Inoculated With Cerebrospinal Fluid A
diagnosis of meningitis usually is based on the
microbiologic findings in the CSF, chemical
determination of the total protein and glucose
present in the fluid, and its cellular content.
Because the total specimen frequently is only 1
to 2 ml., the sample must suffice for the
haematology, chemistry, and microbiologic
findings. Therefore, after the cell count, the
CSF is routinely centrifuged for 10 minutes at
1200 times gravity part of the supernatant is
used for the chemical assays, and the sediment is
the source for the bacteriologic evaluation. The
sediment from the centrifuged sample is
inoculated onto one blood and one chocolate
blood-agar plate. Both plates are incubated
aerobically under 5 to 10 CO2 at 35C, and
disks of hematin and NAD are added to allow the
growth of H. influenzae. Another method of
providing these required factors is to make a
single streak of S. aureus across the plate. The
staphylococci release these factors by lysis of
the red blood cells in the agar, and H.
influenzae will be found growing only as
satellite colonies adjacent to the growth of the
staphylococci. The chocolate-agar plate is
incubated under an atmosphere of 10 CO2. Both
nutrient broth and a special broth for the growth
of anaerobes should be inoculated with the CSF
sediment. All cultures should be inspected daily
and, in the event of growth broth media should
be subcultured onto an appropriate agar medium.
61
Identification of Isolates from Cerebrospinal
Fluid Because meningitis frequently presents an
emergency situation, it is imperative that a
tentative diagnosis is made as soon as possible.
It is mandatory that the sediment from the
centrifuged CSF be subjected to Gram's stain and
examined microscopically. Because the number of
organisms often is small, it is recommended that
at least 30 minutes be spent for such an
examination. If organisms are seen, additional
procedures sometimes can be used to substantiate
immediately a tentative identification. The most
common of these are to carry out a
coagglutination reaction using latex beads with
known specific antiserum or to stain with
specific, fluorescence-labelled antiserum.
Capsular antigens of certain streptococci, N
meningitidis, and H influenzae can be present
even in the absence of bacteria on the Gram's
smear, and using latex bead agglutination
procedures may speed up the diagnosis of
meningitis.
62
Spinal fluid from a possible case of tuberculosis
meningitis should be stained for acid fast
organisms, and a possible infection by C.
neoformans can be diagnosed tentatively using wet
mounts of spinal fluid sediment mixed with India
ink or nigrosin to demonstrate the large capsules
surrounding the yeast cells. A latex bead test
for cryptococci also is available. An evaluation
of a patient's inflammatory response also aids in
the diagnosis of a meningeal infection. In
general, polymorphonuclear leukocytes predominate
in the CSF in acute bacterial infections, whereas
meningitis resulting from fungi, Leptospira, or
M. tuberculosis is characterized by the presence
of lymphocytes.
63
RESPIRATORY TRACT AND MOUTH
64
(No Transcript)
65
Because of the myriad normal resident flora in
the upper respiratory tract, the isolation of
lower respiratory tract infectious agents can be
difficult and contusing. This is complicated
further by the occasional presence of small
numbers of potential pathogens such as
pneumococci, meningococci, streptococci,
Staphylococcus aureus, Haemophilus influenzae,
or enteric organisms that are indigenous to the
upper respiratory tract.
66
Specimen Collection From the Respiratory
Tract The microbiologist must be certain that
lower respiratory tract specimens represent
sputum that has been brought up by a deep cough.
However, it may not be possible to obtain a good
sputum sample from a young child, a debilitated
older person, or someone who is comatose. In such
situations, other procedures must be carried out
to obtain a specimen from the lower respiratory
tract. One technique is transtracheal aspiration,
which, as shown diagrammatically in Figure 1,
uses a needle and tube inserted into the trachea.
This technique also overcomes the problem of
contamination from the oropharynx. On some
occasions, sterile saline solution is injected
through the tube before aspiration.
67
Transtracheal aspiration A pillow should be
placed beneath the neck to permit maximum
extension or the neck. After cleansing the skin,
a 14 gauge needle is inserted into the trachea,
and a polyethylene tube is passed through the
needle into the lung 1 he needle is withdrawn,
and the tube is connected to a syringe containing
3 ml to 4 ml of physiologic saline. The saline is
injected into the lung and immediately with drawn
for culture.
68
Media Inoculated With Respiratory Tract
Specimens All throat swabs should be kept moist
until delivered to the laboratory. Special media
are used for the isolation of specific pathogens,
and the laboratory should be informed by the
clinician what range of pathogens is possible.
For instance, sheep blood-agar plates are
sufficient for the isolation of ?-hemolytic
streptococci, but S. aureus, Streptococcus
pneumoniae and Neisseria meningitidis grow better
on chocolate blood agar in the presence of excess
CO2 A suspected C diphtheriae would be
inoculated additionally on a Loeffler's
coagulated-serum slant and a potassium
tellurite-agar plate. To isolate and identify
Bordetella pertussis from a suspected case of
whooping cough, special medium would be
inoculated from a swab.
69
A swab containing a possible H. influenzae would
be streaked on a chocolate-blood agar plate.
Thick sputum that is to be cultured for
Mycobacterium tuberculosis usually is thinned by
digestion in 4 NaOH and a mucolytic agent at
37C for 1 hour, followed by high-speed
centrifugation (2000 times gravity for 30
minutes). Other digestion procedures also have
been reported. These procedures result in a
concentration of the tubercle bacilli and the
destruction of most contaminating organisms. It
should be recognized that these digestion
procedures also destroy the tubercle bacilli, and
the time and temperature should not be extended
beyond the recommended limits. After
centrifugation, sediment material can be used to
inoculate media, such as Lowenstein-Jensen medium.
70
ORGANISMS COMMONLY ISOLATED FROM RESPIRATORY
TRACT SAMPLES AND SPECIALISED PROCEDURES USED FOR
THEIR IDENTIFICATION
Organism Special Procedures
Streptococcus group A hemolytic Sensitive to commercially available bacitracin disks, catalase negative, fluorescently-labelled antibody or conglutination
Streptococcus pneumoniae Sensitive to optochin disks lethal for mouse in 18 hours
Staphylococcus aureus Vogel Johnson medium ferments mannitol (coagulase positive)
Haemophilus influenzae Streak blood plate and check for hematin and NAD requirement
Neisseria meningitides Grow in Thayer Martin medium
Bordetella pertussis Bordet-Gengou agar plates
Corynebacterium diphtheriae Loeffler's coagulated serum and potassium tellurite plates
71
Organisms causing upper respiratory tract
infections that may cause lesions in the throat
should be obtained with a cotton swab and
streaked on a suitable medium as soon as possible
or used for direct antigen detection.
Nasopharyngeal cultures usually are obtained with
a cotton swab on a bent wire, which can be passed
through either the nose or the mouth, carefully
bypassing the tongue and oropharynx.
Nasopharyngeal cultures are especially important
for detecting carrier states for meningococci,
Corynebacterium diphtheriae, group A -
?-hemolytic streptococci, and H influenzae. The
last organism also can cause an acute
epiglottitis, but initial treatment for that
infection is based on clinical evaluation and
must be initiated before laboratory isolation
would be possible.
72
Identification of Respiratory Tract Isolates The
appearance of the colony on sheep blood agar and
the use of the Grams stain are the most powerful
tools available for a presumptive identification
of a potential pathogen. If tuberculosis is
suspected, acid-fast stains should be made on the
centrifuged sediment obtained from the sputum
digestion procedure. Many other specialized
procedures are available, and the choice depends
on information received from the clinician and on
the appearance of the initial isolates.
Fluorescein-labelled antibody or latex particles
with attached antibody directed against group A
streptococcus provides rapid identification of
these organisms.
73
WOUNDS AND ABSCESSES Pus and exudates from an
infected wound or open abscess would be expected
to contain the etiologic agent of the infection.
However, in open wounds, skin and soil
contaminants almost invariably are found that,
under appropriate growth conditions could outgrow
the true infectious organism, resulting in an
erroneous laboratory report.
74
Skin infection
75
Infections in which anaerobes are the predominant
pathogens or are commonly present
Region Type of Infection
Head and neck Brain abscess, Otogenic meningitis extradural or subdural, Empyema,Chronic otitis media, Dental infection
Pleuropulmonary Pneumonia secondary to obstructive process,Aspiration pneumonia, Lung ahscess, Bronchiectasis, Thoracic empyema
Intraabdominal Liver abscess, Pylephlebitis , Peritonitis, Appendicitis, Subphrenic abscess, Wound infection after bowel surgery or trauma, Liver abscess
Female genital Puerperal sepsis, Postabortal sepsis, Endometritis, Tuboovarian abscess
Other Perirectal abscess, Gas forming cellulitis, Gas gangrene, Breast abscess
76
Collection of Specimens from Wounds and
Abscesses Whenever possible, a sterile
syringe and needle should be used to collect
specimens from wounds and abscesses. The use of a
swab is routinely unsatisfactory because of the
limited amount of material collected by this
method, making it difficult or impossible to
isolate the etiologic agent or agents. It also is
important to remember that wounds and abscesses
arc commonly infected with obligately anaerobic
bacteria, which quickly die on a swab that are
exposed to the atmosphere. Therefore, all
aspirates should be transported to the laboratory
in special tubes containing oxygen-free gas. Such
containers, which can be obtained commercially,
usually contain a few drops of 0.0003 resazurin,
an oxidation-reduction indicator that turns pink
if air contaminates the bottle. Burns often are
infected with opportunists such as Pseudomonas
aeruginosa, enteric organisms, and staphylococci,
and yeast, make isolation of the definitive
infectious agent extremely difficult.
Quantitative cultures may assist in the
interpretation of laboratory findings. Specimens
of burned tissue and any drainage material should
be sent to the laboratory for culture and
evaluation.
77
Media Inoculated With Wound and Abscess
Specimens In general, obligate anaerobes, such as
those in the genera Clostridium, Bacteroides,
Eubacterium, Fusobacterium, and Actinomyces, must
be considered. Numerous specialised media can be
used successfully for the growth of the obligate
anaerobes. Most contain whole or lysed blood from
sheep, complex infusions such as brain-heart
infusion or chopped meat, vitamin supplements
such as yeast extract and additional vitamin K,
and, in broths, a reducing agent such as
thioglycolate or cysteine with 0.1 agar added to
reduce convection cur-rents. Because most wound
infections or abscesses contain multiple
organisms, the use of liquid media alone is not
satisfactory. In fact, if isolated colonies are
obtained on agar plates, little is gained by the
examination of broth cultures. However, agar
plates must be incubated in an anaerobic jar (a
jar from which all oxygen has been removed) or a
similar device. Because most infections are
caused by mixtures of aerobic and anaerobic
bacteria, blood-agar plates as well as selective
and differential media (eg, eosin-methylene blue
or Mac Conkey) also must be inoculated and then
incubated aerobically at 36C.
78
Identification of Wound Isolates The multiplicity
of genera that can be found in a wound makes it
difficult to list firm rules for their
identification. A Gram's stain of all specimens
should be observed first. The results of
microscopic examination may provide in-formation
that will aid in a decision regarding which media
should be inoculated and under what conditions
the culture should be incubated. It is no simple
affair to differentiate between the true
etiologic agents of wounds and abscess infection
and the contaminants that go along for the ride.
79
Criteria of microbial growth evaluation
(streak with
tampon onto plate agar) ? very poor growth
growth only in liquid media, on plate agar growth
is absent ?? small growth - about 10
colonies ??? moderate growth 11 - 100
colonies IV great growth more then 100
colonies. ?-?? degree - contamination, ???-IV
- etiological significance of real microbe.
Contamination level 105 CFU/g is critical, it
testifies the role of bacteria in development of
infectious process.
80
FECES Gastrointestinal illnesses usually are
characterized by diarrhea or the presence of
blood, mucus, and, in certain cases, white blood
cells in voided stools. Many such disturbances
are cases of food poisoning resulting from the
ingestion of a preformed toxin. Symptoms of such
intoxication rarely last beyond 24 hours, and
treatment usually is confined to the intravenous
replacement of lost fluids and electrolytes.
81
A bacteriologic examination of food suspected of
causing an illness would be more likely to yield
informative data concerning the etiology of
intoxication than would an examination of a
faecal specimen. For example, a Gram's stain
revealing large numbers of staphylococci,
together with a history and the clinical symptoms
of staphylococcal food poisoning, would provide
strong circumstantial evidence that the
gastroenteritis was due to the ingestion of food
contaminated with staphylococcal enterotoxin.
82
A similar situation would be seen in food
poisoning due to Clostridium perfringens. In both
cases, the organisms would be present in large
numbers in the contaminated food however,
because the staphylococcal enterotoxin is more
stable to heat inactivation than are the
staphylococci themselves, it would not be unusual
to see large numbers of staphylococci in a Gram's
stain (of a heated cream soup) and yet not be
able to culture significant numbers of organisms
from the suspected food. On the other hand,
because the C. perfringens enterotoxin is
produced only during sporulation, large numbers
of viable organisms might be found in a similar
situation. It is likely that many cases of
gastro-enteritis are of viral origin.
83
Concentration of microbes in human digestive tract
84
Main group of bacteria in the gut

Bacteroides (B. fragilis), Prevotella, Veilonella, Lactobacillus (L. acidophylus, L. brevis), Clostridia (C. perfringens, C. tetani, C. botulinum, C. sporogenes), Peptococcus, Peptostreptococcus, Actinomyces E. coli, Enterococcus, Citrobacter, Klebsiella, Proteus, Providencia, Pseudomonas, Alcaligenes, Bacillus (B. subtilis, B. Brevis), Lactobacillus, Enterococcus, Corynebacteria, Fungi, Candida
Aerobic and facultativly anaerobic bacteria
Anaerobic bacteria
85
Specimens from Intestinal Contents In culturing
intestinal contents, the choice of material to be
taken from the patient is obvious, although best
results are obtained when the faecal specimen is
collected during the acute stage of an episode of
diarrhea. If a specimen contains blood or mucus,
these should be included in material to be sent
to the laboratory. When a sterile swab is used
instead of a faecal specimen, the swab must be
inserted past the anal sphincter and rotated
several times before being withdrawn.
86
It is a common misconception that the
microorganisms found in faeces are rather hearty
and those special precautions to preserve the
viability of suspected pathogens are not
required. Nothing could be further from the
truth! Unless faecal specimens can be taken
directly to the laboratory for culturing, they
should be refrigerated or placed in a stool
preservative containing a buffer that will
maintain the pH near neutrality. One such
preservative uses about equal parts of sterile
glycerol (containing 0.033 M phosphate buffer, pH
7.4) and faeces. A pH indicator also can be
included to ensure that a drop in pH does not go
unnoticed. Failure to use a preservative will
result in the death of many of the enteric
pathogens, especially the Shigella and, to a
lesser extent, the salmonellae. Faecal specimens
of l to 2 g are adequate for bacteriologic
procedures.
87
Media Inoculated With Intestinal Specimens The
major intestinal flora consists of obligately
anaerobic gram negative rods, including organisms
in the genera Bactericides, Fusobacterium,
Eubacterium, and Clostridium. All these can cause
serious abscesses but, with the exception of the
enterotoxins from C perfringens and Clostridium
difficile, none of the obligate anaerobes has
been implicated in gastrointestinal disease
characterized by diarrhea. Therefore, unlike the
processing of blood or abscess specimens, it is
not usual to culture faecal specimens under
anaerobic conditions.
88
When species of either Salmonella, or Shigella
are the possible pathogens, it is advisable to
inoculate an enrichment medium that will
selectively permit the growth of these organisms
over that of the normal gram-negative flora. Many
such media are available, and it is probable that
some diagnostic laboratories use various
modifications of these media. Two of the more
common enrichment media are a tetrathionate and a
selenite F medium, both of which arc commercially
available. After incubation of the inoculated
enrichment medium for 12to 16 hours at 35C to
37C, it should be streaked on standard,
differential media such as MacConkey or
eosin-methylene blue and deoxycholate agar
plates. Hektoen enteric or xylose-lysine-deoxychol
ate plates also can be used. Many other
differential and selective media are available
that can be used for the isolation of the
pathogenic Enterobacteriaceae, and it is likely
that diagnostic laboratories vary somewhat in
their preference of one medium over another
89
Identification of Fecal Isolates Because of the
large numbers of facultative, gram negative rods
that make up the normal intestinal flora, the
isolation and identification of the
morphologically similar Shigella and salmonellae
requires the use of selective and differential
media as well as considerable experience in
working with these organisms. As outlined,
numerous kits are available for use in
identifying members of the Enterobacteriaceae.
One widely used kit, termed the API 20E, consists
of a plastic strip containing wells of dehydrated
media and appropriate indicators. The wells are
inoculated with a suspension of the unknown
organisms and, after 24 hours, a numerical value
is assigned to each positive reaction. Using the
sum of these values, a complete identification
can be made from tables that accompany the kit.
90
The identification of staphylococci from a faecal
specimen is i moderately easy task it the
laboratory has been instructed that the clinical
symptoms are compatible with those of a
staphylococcal enteritis Large colonies on
selective sheep blood agar plates showing Fs
hemolysis and grape like clusters of gram
positive cocci should be tested additionally for
coagulate production and, if specific antiserum
is available, for the production of enterotoxin.
Intestinal infections by yeast, such as species
of Candida, or any of the many parasitic protozoa
worms are diagnosed by the direct microscopic
examination of a faecal specimen.
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