Clinical microbiology

Dr. Jayesh Patidar
www.drjayeshpatidar.blogspot.com
CLINICAL
MICROBIOLOGY

BACTERIOSCOPIC
BACTERIOLOGICAL
SEROLOGICAL
BIOLOGICAL
ALLERGIC
EXPRESS-DIAGNOSIS
METHODS OF EXAMINATION IN MICROBIOLOGY

Neisseria
meningitidis
BACTERISCOPIC
METHOD

Neisseria
gonorrhoeae
BACTERISCOPIC
METHOD

BACTERISCOPIC METHOD
Yersinia pestis

Bacillus anthracis
BACTERISCOPIC
METHOD

Mycobacterium tuberculosis

Streptococcus pneumoniae

Clostridium pneumoniae

Isolation of pure culture
Bacteriological method

Neisseria meningitidis
Bacteriological
method

Agglutination test
Serologic method

Ring precipitation test
Serologic method

Immunodiffusion (Ouchterlony) test
Serologic method

Mancini’s test
Serologic method

TBC in guinea pig
TBS in rabbit
Biological method

Mantoux’s test
Allergic method

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

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.

Most of conditionally pathogenic bacteria belong to
normal human flora so it’s hard to determine their
etiologic role (etiological significance).
So, they can present normal microflora of tested fluids
and tissues and contaminate them from environment.
That’s why for correct interpretation of examination
results it’s 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.

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

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

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

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 patient’s serum antibodies to the
autostrains or microbial culture, which can be causative
agent.

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;

- 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 it’s 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.

Examination of the blood
Bacteriemia, septicemia, septicopyemia

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

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.

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.

Identification of Blood Isolates
Blood cultures are incubated at 36°C 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.

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

If microbes are absent after 10 days of incubation it
means, that inoculation results are negative and blood is
sterile.

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.

Examination of urina
Etiology of urethritis

Major causes of urinary tract infections

Pathogenesis of genital tract infections

Major causes of vaginitis Major causes of cervicites

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.

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.

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.

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 35°C 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.

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/мl 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.

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.

CEREBROSPINAL FLUID
Etiology of meningitis

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.

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.

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 35°C, 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.

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.

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.

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.

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.

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.

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.

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
37°C 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.

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

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.

Identification of Respiratory Tract Isolates
The appearance of the colony on sheep blood agar and the
use of the Gram’s 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.

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.

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

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.

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 36°C.

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‖.

Criteria of microbial growth evaluation
(streak with tampon onto plate agar):
І – very poor growth – growth only in liquid media, on plate agar
growth is absent;
ІІ – small growth - about 10 colonies;
ІІІ – moderate growth – 11 - 100 colonies;
IV – great growth – more then 100 colonies.
І-ІІ degree - contamination,
ІІІ-IV - etiological significance of real microbe.
Contamination level 105 CFU/g is critical, it testifies the role of
bacteria in development of infectious process.

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.

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.

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.

Concentration of microbes in human digestive tract

Main group of bacteria in the gut
Anaerobic bacteria
Aerobic and facultativly
anaerobic bacteria
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

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.

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.

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.

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 35°C to 37°C, it
should be streaked on standard, differential media such as
MacConkey or eosin-methylene blue and deoxycholate agar
plates. Hektoen enteric or xylose-lysine-deoxycholate 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

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.

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.

Clinical microbiology

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