CLINICAL BACTERIOLOGY

MT 3A (LEC) LECTURE BY: Maria Theda Ann B. Mondejar,RMT

Jamelarin A.C BMLS 2C
Laboratory should be informed as to which antimicrobial
agent(s) the patient is receiving.

● Collect specimens at the right stage of the disease.

SPECIMEN COLLECTION AND HANDLING ○ Acute phase (when the patient is
experiencing the symptoms of the disease),
is the most appropriate time to collect most
Chapter 11 DIAGNOSTIC MICROBIOLOGY 6 specimens
•Goal of specimen collector must be: ● Package the specimen in a container or transport
medium designed to maintain viability of the
TO MAINTAIN THE VIABILITY OF THESE ORGANISMS WITH MINIMAL
organisms and avoid hazards that results from
CONTAMINATION
leakage
•Three Components of Specimen Quality
○ Aseptic technique is required to prevent
1. Proper selection of the specimen contamination
2. Proper collection of the specimen ● The specimen container should be labeled with the
3. Proper transport of the specimen to the laboratory patient’s identification, with the date and time of
collection, and source of specimen with specific
anatomic site.
GENERAL CONSIDERATION IN COLLECTION OF ● The specimen must be promptly taken to the
CLINICAL SPECIMEN laboratory and immediately examined.
○ Specimens should be protected from heat
The universal precautions are followed throughout the and cold and promptly delivered to the
collection and handling process. laboratory so that the results of the analysis
✔Persons collecting and handling the specimens should wear will validly represent the number and types
gloves and a laboratory gown/coat. of organisms present at the time of
collection.
✔Eye protection should also be worn if splashing is a potential ● Notify the laboratory in advance if unusual
hazard pathogens or agents of bioterrorism are suspected
✔Accident and injuries should be reported immediately In case there is a delay:
✔Refrigeration at 4°C to 6°C offers a safe and dependable
HELPFUL HINTS REGARDING HANDWASHING method of storing many clinical specimens.

● Make sure that you have washed your hands ✔The length of time of refrigeration varies with the type of
sufficiently by: rubbing your soapy hands and specimen:
interlaced fingers together for as long as it takes you
● Swabs from wounds (except for anaerobic cultures),
to sing:
the urogenital tract, throat, rectum and samples of
○ Birthday Song twice feces or sputum can be refrigerated for 2- 3 hours.
● Urine specimens can be refrigerated for at least 24
○ all verses of “Twinkle, Twinkle Little Star” once
hours.
● Alternatively, you can use quick drying alcohol foam,
gel, or lotion. ✔ The length of time of refrigeration varies with the type of
○ quick dry in 15 seconds specimen:

● CSF from patients with suspected meningitis should

be examined at once.
BASIC PRINCIPLES OF SPECIMEN COLLECTION
● Specimens for viral isolation should be refrigerated
● Specimen must be properly selected immediately but never frozen.

○ Specimens should be collected at the proper CRITERIA FOR REJECTION OF SPECIMENS

site of infection, with as little external
CRITERIA FOR REJECTION OF SPECIMENS
contamination as possible by practicing
aseptic techniques. 1. The information on the requisition does not match the
● Select the correct anatomic site for collection of the information on the specimen label.
specimen using aseptic technique 2. There is no patient identification on specimen
● Collect the appropriate quantity of specimen container
● Specimen should be collected before the 3. Grossly contaminated specimen.
administration of any antimicrobial agent. 4. Specimen submitted in improper transport container
○ But if the culture has been taken after or the container is leaking
initiation of antibacterial therapy, 5. Excessive delay between collection and arrival of
counteractive measures such as adding specimen in the laboratory and specimen has not
penicillinase or diluting the specimen should been preserved.
be done. a. ideally within 30 minutes of collection
 b. preferably within 2 hours Wooden applicator stick
6. Inadequate amount of specimen to perform all test
● commonly used applicator
requested, like one swab was submitted with multiple
requests for various organisms. Nichrome or thin aluminum wire
7. 24-hour specimen for culture
● recommended applicator for collecting specimens
8. Specimen is dried up like dry swabs for culture. from the nasopharynx and urethra
9. Anaerobic culture is requested on specimen on which
anaerobes are indigenous
10. Some factors rendering a specimen inadequate. ➔ Swab collection systems are available that provide
a. sputum collected on a swab or in a tissue transport media and protect the specimen from
paper drying
b. stool with gross barium ➔ Lesions, wounds, and abscesses present many
problems to the laboratory
c. specimen placed in formalin ➔ The term wound is not an appropriate specimen label
d. broken or leaking container – exact anatomic site must be provided
11. Specimen submitted is not the proper specimen for ◆ Specimen is collected from the advancing
the test. margin of the lesion and should be collected
12. Clinically evident results cannot be obtained from a by needle aspiration rather than by swab
Foley catheter tip rather request a urine sample ➔ Before collection, area should be cleansed to
culture. eliminate as much of the commensal flora as
13. If a duplicate specimen is received, only one must be possible
processed unless the laboratory is notified and given ➔ Aspirated material should be placed into a sterile
reasons for processing the duplicate. Blood cultures tube or transport vial and not “squirted” onto a swab
are an exception

COLLECTION PROCEDURES
● Specimens for microbiology cultures should be
collected in sterile containers except stool
● Stool specimens can be collected in clean, leak-proof
containers
● Swabs
Generally are not recommended for collection:
✔Do not provide sufficient quantity
✔Easily contaminated
✔Can become dried out leading to a loss of organisms
● Appropriate for specimens from the upper respiratory
tract, external ear, eye, and genital tract

Kinds of Swabs According to Tip Used:

Cotton – tipped swab
● most commonly used kind of swab
● some cotton-tipped swabs may contain fatty acids
that may be detrimental to microbial growth

Polyester-tipped swabs (Dacron or Rayon)

● With the incorporation of a small amount of silica gel
in a glass tube containing the polyester swab the
viability of group A streptococci is further maintained
for 3 days

Calcium alginate – tipped swab

● an excellent substitute for cotton-tipped swab,
since it is dissolves in certain solutions that are
compatible with the bacterial preservation
● should never be used where herpes virus is
anticipated, as it may inhibit replication of this virus
● not suitable for Chlamydia spp. as it may interfere
with extracting reagents used in antigen-antibody
detection

Kinds of Swab According to Applicator Used:

 ● most advantageous time to draw blood cultures will
be just before the anticipated rise of temperature
prior to fever spike* (some recommend drawing of
blood at time of febrile episode) and before antibiotic
administration
● collection of a single set of blood cultures is
unacceptable
● minimum collection of 2 (not more than 3) sets taken
in 24 – 48 hours period is required
● Adult = 10 – 30 ml of blood per culture (CFU/ml of
blood is frequently low)
● In infants and children = 1 – 5 ml of blood per culture
(concentration of microbes in blood is higher)
● generally accepted method of blood collection is the
30 ml one time collection
● 10ml from each of separate venipuncture sites, using
3 separate needles and syringes
● In cases of fever of unknown origin, 4 separate blood
cultures

✔ 2 drawn on each day of 2 days, will detect most causative

agents

● drawing of blood should be from different sites to rule

out contamination

Blood should be prevented from clotting with an

anticoagulant

● SPS, Liquoid (sodium polyanethol sulfonate)

Holding or Transport Medium ○ best anticoagulant – 0.025% to 0.05%
● SAS – sodium amino sulfate
● designed to prolong the survival of microorganisms
when a significant delay between collection and Other properties of SPS :
culturing cannot be avoided a.Anti-complementary
● Contains substances that do not promote
multiplication of organisms b.Anti-phagocytic
● Examples: c.inactivates aminoglycoside antibiotics
○ Stuart’s medium
d.neutralizes bactericidal effect of human serum
○ Cary and Blair medium
○ Amies medium
SPS may inhibit the growth of the following:
○ Transgrow medium (for Neisseria spp.)
● Neisseria spp.
Patient – collected specimen ● Gardnerella vaginalis
● Medical personnel should provide patients with ● Streptobacillus moniliformis
thorough instructions on how to collect the sample ● Peptostreptococcus anaerobius
✔inhibitory action of SPS can be counteracted with the
addition of 1.2% gelatin
BLOOD Blood Culture
Bacteremia ● Penicillinase or other ARD (antibiotic removing device
may be added to blood culture media to inactivate
✔ the presence of viable organisms in the blood
beta-lactam antibiotics (e.g. penicillin) and other
which may indicate the presence of a focus of
antibiotics present in patient’s blood
disease (pneumonia or liver abscess)
● Ideal dilution – 1:10 (1 part blood and 10 parts
✔ a transient release of bacteria into the bloodstream medium)
(after vigorous tooth brushing) ○ Ratio of blood to culture medium in adults is
Septicemia or sepsis 1:10
○ Ratio of blood to culture medium in
✔ indicates a situation in which bacteria or their neonates is 5:10
products (toxins) are causing harm to the host ● Use of 2 different bottles of blood culture media is
Collection recommended for optimal recovery of pathogens.
○ one vented (aerobic-TSB)
● venipuncture site should be cleansed with alcohol ○ unvented (anaerobic-thioglycollate broth)
then with 2% iodine or an iodophor and allowed to ● Blood cultures should be incubated at 35ºC
dry for at least 1 minute
 ● examined visually daily (with subcultures done at Sediments
intervals) for 7 days before reporting as negative or
✔smears (Gram stain, India ink)
no growth
○ But in patients suspected of having ✔ culture for culture of:
endocarditis, brucellosis and fungemia,
● Haemophilus influenzae
incubation should be prolonged for 2 – 4
● Streptpcoccus pneumoniae
weeks
● Neisseria meningitides
Growth is usually indicated by the following: ● Cryptococcus neoformans

a. hemolysis Supernatant

b. gas bubbles in the medium ✔test for the presence of antigens

c. turbidity ✔for chemistry examinations

d. colony formation on top of blood layer

e. clot or pellicle formation THROAT SWAB
● most abundant normal flora of the throat
● Newer (faster) methods of blood culture ○ alpha hemolytic Streptococci
a. Isolator – lysis centrifugation blood culture ● most common pathogen of the throat
system (for intracellular organisms) ○ Streptococcus pyogenes or group A beta –
b. Self – contained subculture hemolytic streptococcus which should be
diagnosed with the following precautions:
○ Septi – Chek a modification of the
biphasic a.)patient’s throat should be swabbed from the posterior
pharynx, uvula to the tonsil on both sides of the throat
○ Vacutainer agar slant blood culture
medium ● any areas of purulence
● Bactometer - Detection of the presence of ● inflammation
microorganism through changes in electrical ● ulceration
impedence
a. Bactec System – Detection of CO2 emission
which is the end product of metabolism

CEREBROSPINAL FLUID

•Streptococcus pyogenes or group A beta – hemolytic

Collection and Handling streptococcus which should be diagnosed with the following
● Collection of CSF is by aseptically inserting a needle into precautions:
the subarachnoid space between L3 and L4. b.)swab is inoculated to a soybean – casein digest agar
Three to four tubes are usually filled: containing 5% sheep blood

● tube 1 – chemistry and serology c.)a bacitracin disk test is placed directly onto a blood agar
● tube 2 – for microbiology plate with a purified subculture
○ The specimen for microbiological d.)plates are incubated at 35ºC for 18 – 24 hours with re-
examination should be placed in sterile, incubation of 1 day if no growth, before being discarded as
screw – capped and airtight tubes. negative
● tube 3 – for hematology (differential count)
● tube 4 – if available, for microbiology
NASOPHARYNGEAL SWAB
➔ Collection and Handling should be transported ● recommended for the isolation and detection of
immediately to the laboratory without refrigeration carriers
◆ If a delay is unavoidable the specimens ● nichrome or alluminum wire – recommended
should be incubated no longer than one applicator for collecting specimens from the
hour or left at room temperature nasopharynx and the urethra
◆ specimens for viral studies should be:
✔refrigerated up to 24 hours
✔or frozen at -70ºC if a longer delay is anticipated
SPUTUM
Processing of CSF
●
Specimen are centrifuged and decanted Sputum specimens are often collected for the diagnosis of
bacterial pneumonia
● Lower respiratory tract specimens are among the most ● A single specimen that has yielded a negative result
difficult specimens to collect adequately because they is not usually sufficient to exclude bacteria or
are contaminated with oropharyngeal flora parasites
● For this reason, they are one of the least clinically ● if bacterial infection is suspected – 3 specimens
relevant specimens received for culture should be collected (once a day for three days)
● Other specimens, such as blood or a bronchoalveolar ● if parasites are suspected – 3 specimens collected
lavage (BAL), may be more accurate in detecting the within 10 days – microscopic detection of ova and
etiologic agent parasites
● When sputum specimens are submitted to ● Stools for viral culture must be refrigerated if not
microbiology, the laboratory should be informed of inoculated to media within 2 hours
whether the specimen is expectorated or induced ● Stools for detection of Clostridium difficile cytotoxin
● Expectorated sputum should be refrigerated for maximum of 48 hours until
○ rinse mouth with water and expectorate with tested
the use of deep cough ● 1 – 2 grams or walnut – sized specimen in a clean
○ single specimen for detection if bacterial waxed cardboard or plastic container is sufficient for
lower respiratory tract infection most procedures
○ for fungal or mycobacterial infections, 3 ● Specimens should be delivered within 1 hour.
separate early morning specimens are ○ If there is a delay of > 2 hours, the specimen
appropriate should be placed in a transport medium
● Induced sputum using aerosol induction (Cary – Blair)
○ patients breathe aerosolized droplets of a ● If culturing for Shigella spp.:
solution that stimulates cough reflex ○ Rectal swab may be recommended by
○ Saliva is most of the time cultured inoculating at bedside
mistakenly. ● laboratories often use two – vial system
Collection ✔one vial contains formalin for
concentration
Other means of obtaining sputum:
✔one vial contains a fixative for preparing
✔ gastric aspirate – for acid fast bacilli
stained slides such as Polyvinyl alcohol with
✔ transtracheal aspiration zinc
✔ transthoracic needle biopsy ● A rectal swab may be substituted as a specimen for
bacterial or viral culture as long as fecal material is
✔ broncoscopy with aspiration or biopsy
visible on the swab.
✔ thoracentesis
✔The swab should be placed in Cary – Blair
✔ open lung biopsy or Stuart’s transport medium
✔ bronchoalveolar lavage (BAL) Staining of Smears
✔ protected catheter bronchial brushing and etc. ● Gram staining is not routinely done since gram (-)
bacilli are usually seen in stool. However, it may help
● Sputum should be collected from a deep cough
in the detection of certain etiologic agents:
○ saliva is most of the time cultured
mistakenly a. presence of gram (+) cocci in chains and
clusters
● First morning specimen is preferred
● The patient needs to understand the difference ● Suggestive of enterococcal and
between sputum, saliva, and nasal secretions staphylococcal infections
● Sputum for mycobacterial culture should be added b. thin, comma – shaped gram (-) bacilli
with: ● Campylobacter and Vibrio
○ N-acetyl-L-cysteine – for digestion of ● Smear for Gram staining is not routinely done since
mucus gram (-) bacilli are usually seen in stool. However, it
may help in the detection of certain etiologic agents:
○ 2% NaOH – for decontamination
c. Budding with or without hyphal elements
● Yeast infection
● Anaerobic cultures are not done on expectorated d. large numbers of large and thin gram (+)
sputum specimens except by percutaneous bacilli
aspiration or by protected bronchial brush ● Clostridium difficile
● According to Bartlett’s Classification suitable Acid fast staining should also be used to detect
sputum specimens should have: Cryptosporidium species and Mycobacteria.
✔ less than 10 epithelial cells/lpf more than
25 PMNs/lpf
URINE
FECES
Collection and handling Collection

● Specimen of choice for the detection of ● Midstream clean-catch urine – routinely used
gastrointestinal pathogens
 ○ First morning is preferred since it provides a f. Presence of an organism in any quantity
more concentrated sample obtained by suprapubic aspirate is
○ Patients need to cleanse the external significant
genitalia to reduce indigenous flora ● Neisseria spp. inhibited by fatty acids and salts,
○ Patients are asked to void without collecting therefore, calcium alginate and cotton swabs should
the first portion of the urine flow and instead NOT be used for specimen collection
to collect the middle portion ● When genital cultures are subjected to Gram staining,
○ First portion of urine flow washes acid alcohol is recommended as decolorizer
contaminants from the urethra, and the especially if Neisseria spp. are suspected
midstream portion is more representative of ● Culture in BAP, MSA, EMB, and CHOC – Gas Pak if
the urine in the bladder suspecting for Neisseria spp. and Haemophilus spp.
● Suprapubic aspiration – routinely used ● MTM or TM with VCN – medium that supports growth
● Catheterization of Neisseria spp.

Handling Men

1. Urine should be cultured within 2 hours after ● Exudates maybe expressed from the urethral orifice
collection. or a small diameter swab may be inserted 3 to 4 cm
2. Urine may be stored as long as 24 hours under into the urethra.
refrigeration(4°C). ● Specimen should be plated immediately on the
3. Preservative – boric acid appropriate media and not allowed to dry or be
● Gram stain of uncentrifuged specimen to exposed to cold temperature.
determine significant bacteriuria ● A direct gram stained smear should be prepared.

Gram Stain of Uncentrifuged Specimen

1. simplest and most rapid method for detecting Women
significant bacteruria
● Cervical specimen are obtained by the physician with
● ✔ Presence of 1 organism/oil immersion field the aid of a speculum.
(examining 20 fields) correlates with
● Lubricants should not be used as this are lethal to
significant bacteriuria (more than 105 Neisseria gonorrhoeae.
CFU/ml)
● Specimen should be plated immediately on the
2. Colony count can be performed by pour plate appropriate media and not allowed to dry or be
method or calibrated loop method and dip slide exposed to cold temperature.
method
● A direct gram stained smear should be prepared.
-colony count is done on BAP
Calibrated loop dilution factor
-0.001ml or 1µl 1:1000 or 1/1000 Collection and Culture of Anaerobic Specimens
-0.01ml or 10µl 1: 100 or 1/100 ● Specimen for anaerobic culture are aspirated with a
Formula of colony count sterile needle and syringe to avoid contact with
normal flora.
CFU/ml = NC X RDF ● Specimens are swabbed and immediately put into an
= # of colonies X reciprocal of the dilution factor evacuated tube filled with oxygen – free CO2 or
nitrogen (“gassed – out tube”) containing an agar or
Interpretation of culture results: broth indicator system or other anaerobic transport
a. Colony counts of > 105 CFU/ml (100,000 device like:
CFU/ml) for 1 or 2 bacterial species are
✔Anaerobic culturette
clinically significant of infection
✔Bio – Bag
b. Colony counts of > 105 CFU/ml for 3 or more
bacterial species most likely represent ✔Anaerobic Pouch
contamination
✔GasPak Pouch
c. A colony count of > 103 /ml of a bacterial ● Never use dry swab or a regular aerobic transport
specie from a midstream urine of a male is media.
also considered clinically significant.
Requirements
d. A colony count of 102 – 103/ml of 1or 2
bacterial species from females with lower 1. exclusion of oxygen
urinary tract symptoms may also be 2. low redox potential
clinically significant. 3. prevention of oxidation

e. Colony counts of < 50,000 CFU/ml are seen

in contaminated specimens.

Methods of Promoting Anaerobiosis

Anaerobic jars
 ● Brewer Jar ✔ A pre-reduced anaerobically sterilized
○ Oxygen is removed by means of electrically (PRAS) agar is distributed under anaerobic
heated platinized catalyst with the electrical conditions as a thin layer around the inner
connection outside the jar. walls of test tubes
● Torbal Jar
✔ Tubes are rolled and cooled until the
○ Similar to the brewer jar but uses a rubber O
melted agar forms a thin layer
ring rather than Plasticine and a catalyst
active at room temperature thus requiring
no electrical heating. Culture Media
○ Thioglycollate Broth – the only medium
● Gas Pak Jar capable of supporting the growth of three
○ This is the most convenient and widely used groups of organisms:
anaerobic jar.
✔Aerobes – surface medium
Components:
✔Microaerophiles – sub-surface
I. hydrogen and C02 generator
✔Anaerobes – bottom
envelope - activated with water
II. palladium – coated aluminum ○ Sodium thioglycollate –substance which
pellets – catalyst provides low redox potential
III. methylene blue or resazurin
indicator strip –upon exposure to
atmospheric O2 turns blue or pink

Principle and Results:

I. With water added to the CO2 and
H2 generator envelope and pellets
catalizing the formation of water
from H2 and O2, anaerobiosis is
achieved
II. iIndication of anaerobiosis :
● production of heat
● moisture inside the jar
● decolorization of indicator
strip (white or colorless)
● Jars utilizing the “evacuation – replacement”
system

✔air is removed by drawing a vacuum of 25

inches of mercury

✔the jar is filled with oxygen – free gas such

as nitrogen between evacuation of air

✔anaerobiosis is achieved more quickly with

this method but less convenient for routing
use

Methods of Promoting Anaerobiosis

● Glove Box or Anaerobic Chamber
✔an enclosed system which consist of a
large clear plastic, airtight bag or chamber
filled with oxygen – free gas mixture of
nitrogen, hydrogen and CO2

✔chambers allows materials to enter

through an air lock

✔Anaerobiosis is maintained by palladium

catalyst and hydrogen gas in the chamber.
✔The operator uses gloves or sleeves that
form airtight seals around his or her arms to
manipulate items inside the chamber.

● Roll Tube Technique