CLINICAL BACTERIOLOGY

BIOSAFETY AND QUALITY CONTROL | LABORATORY

BY: EVONY VILORIA

BIOSAFETY AND QUALITY CONTROL IN that flows over the

MICROBIOLOGY LABORATORY infectious material
- Clinical laboratory specimens are and the air
potential hazards since they contain exhausted
infectious agents such as bacteria, viruses
and fungi. CDC recommended a 2 TYPES OF CLASS II CABINET
UNIVERSAL PRECAUTION which states that CLASS II A - it has fixed
“ALL CLINICAL SPECIMENS LIKE BLOOD opening. 70% of the
TISSUE. SEMEN. VAGINAL SECRETIONS, air is recirculated.
BODY FLUIDS WITH VISIBLE BLOOD, CSF, CLASS II B - it has a variable
AS WELL AS BIOFLUIDS SUCH AS sash opening: used
SYNOVIAL. PERICARDIAL. PLEURAL AND for chemicals, radio
AMNIOTIC FLUID MUST BE CONSIDERED isotopes and
INFECTIOUS". CDC also recommends the carcinogens.
use of PPE in handling these clinical CLASS III CABINET - it provides the
specimens. highest level of
safety to the worker.
CABINET CLASSES
CLASS I CABINET - It is an - the air coming into
open-fronted type of and going out of the
cabinet with cabinet is sterilized
negative pressure using HEPA filter
(ventilated cabinets) and the infectious
material within is
- It allows handled with rubber
unsterilized air to gloves that are
enter the cabinet attached and sealed
and circulate to the cabinet.
around the area, and
expose the material - it is used for BSL 4
within Only the air agents.
to exhausted is
sterilized using BIOLOGICAL SAFETY CABINET
HEPA filter. - it Is a device that encloses a working area
in such a way as to protect workers from
- It is used for BSL 2 aerosol exposure to infectious disease
and 3 agents. agents.
CLASS II CABINET - it is known as the - the air that contains the infectious
laminar flow BSC. material is sterilized, either by heat, UV
light or by passage through a HEPA filter.
- most commonly
used BSC in Clinical
Laboratory (class II
A)

- it sterilizes the air

using HEPA filter
1
 CLINICAL BACTERIOLOGY
BIOSAFETY AND QUALITY CONTROL | LABORATORY
BY: EVONY VILORIA

EXAMPLES: HIV, Bacillus anthracis,

Yersinia pestis, Toxoplasma, Salmonella,
Shigella

3. BIOSAFETY LEVEL 3 AGENTS

- These are the agents for aerosol
transmission

-The lethal pathogens, the air movement in

the laboratory must be controlled to
contain the infectious materials.
EXAMPLE: Mycobacterium tuberculosis,
Francisella tularensis, Coxiella burnetti,
systemic fungi, St. Louis encephalitis virus

4. BIOSAFETY LEVEL 4 AGENTS

- These agents that cause life threatening
infections.

- Maximum containment and

decontamination of all personnel and
materials before leaving the area is
observed.
EXAMPLE: Arbovirus, arena virus, filo
CLASSIFICATION OF BIOLOGIC AGENTS
virus, corona virus, small pox virus.
BASED ON HAZARDS
1. BIOSAFETY LEVEL 1 AGENTS
PERSONAL PROTECTIVE EQUIPMENT (PPE)
- it include those that have
no-known.potential for Infecting healthy DONNING
people. it is used in laboratory teaching 1. GOWN
exercises of students. 2. MASK OR RESPIRATOR
EXAMPLES: Bacillus subtills(biological 3. GOGGLES OR FACE SHIELD
indicator of oven), Mycobacterium 4. GLOVES
gordonae, Naegleria gruberi
DOFFING
2. BIOSAFETY LEVEL 2 AGENTS 1. GLOVES
- these are the agents acquired by ingestion; 2. GOGGLES OR FACESHIELD
percutaneous or mucous membrane 3. GOWN
exposure. 4. MASK OR RESPIRATOR
5. WASH HAND OR USE AN
- it include all the common agents of ALCOHOL-BASED HAND SANITIZZER
infectious diseases. IMMEDIATELY AFTER REMOVING ALL PPE

- personnel are required to change their INFECTION CONTROL

clothes with the recommended lab clothing - all laboratory related accidents MUST BE
before going to their specific stations. reported immediately to the head of the
laboratory and safety officer.

2
 CLINICAL BACTERIOLOGY
BIOSAFETY AND QUALITY CONTROL | LABORATORY
BY: EVONY VILORIA

- for hiv testing, serum samples SHOULD Institute of

BE examined at an interval of 6 weeks, 3 Standards and
months, and six months. Technology (NIST)
- Calibrate
HAZARDOUS WASTES periodically
- Are substances which singly, or in 2. CARBON DIOXIDE - the percentage of
combination, pose a significant present or INCUBATOR CO₂ must be
potential threat or hazard to human health checked daily
or to the environment and require special 3. CENTRIFUGE - the speed or
handling, processing, or disposal because revolution per
they are flammable, oxidizers, explosive, minute(rpm) must
reactive, corrosive, toxic, infectious, be checked twice a
carcinogenic, bioconcentrative persistent in year using a
nature, potentially lethal, an irritant or a TACHOMETER.
strong sensitizer.
4. CULTURE MEDIA - all media should
be checked based
DISPOSAL OF HAZARDOUS WASTE
on their
- All materials contaminated with
performance and
potentially infectious agents must be
sterility - records
decontaminated before disposal using
shoul be
an autoclave, incinerator, or alternative
maintained for 2
waste treatment methods.
years.
- Pipettes, swabs, and other glass objects 5. REAGENTS - reagents should
should be placed inside a firm cardboard be tested daily
contains before disposal. with both positive
and negative
- Sharp objects (needles, scalpels, slides) controls.
are paced in containers that are autoclaved 6. ANTIMMICROBIAL - susceptibility
or incinerated. SUSCEPTIBILITY testing of control
organisms is
QUALITY CONTROL IN A MICROBIOLOGY usually done daily
LABORATORY for 20-30 days
7. PERSONNEL - all tests
QUALITY CONTROL COMPETENCE performed on
-as the measures designed to ensure the patuents must be
medical reliability of the laboratory data. subjected to
proficiency teting
twice a year.
INSTRUMENTS THAT REQUIRES QUALITY
CONTROL MEASUREMENTS 8. STOCK CULTURES - an organism
should be
1. THERMOMETER - each subcultured twice
CALIBRATION thermometer must after thawing to
be checked before return it to a
use, against a healthy state
reference **organisms tored
thermometer form in freezer should
the National
3
 CLINICAL BACTERIOLOGY
BIOSAFETY AND QUALITY CONTROL | LABORATORY
BY: EVONY VILORIA
be kept at -70℃
SPECIMEN COLLECTION, TRANSPORT
AND PROCESSING
- Specimen collection, handling, and
transport are the most critical step in
diagnostic medicine because the results
that the laboratory provides to the
clinicians greatly depend on the quality of
the specimen.
- Proper skin cleansing prior to blood
cultures and spinal taps decreases the
presence of contaminants in the specimen.
The specimen must be collected from the
actual site of infection whenever possible.
- The laboratory can make accurate and
useful determinations only if a specimen
has collected and transported
GENERAL GUIDELINES FOR SPECIMEN
COLLECTION
Specimens should be collected during the
acute or early phases of an illness.
The correct anatomic site must be
identified/selected for collection of the
specimen.
The site of infection should be cleansed
properly before collection.
The quantity of the collected specimen
should be sufficient for diagnostic testing
If possible, specimen should be collected
before antibiotics are administered.
Specimens should be submitted on two
swabs, one id for direct smear, second is for
culture. Swabs never recommended for
anaerobic culture.
For lesions, wounds, and abscess, the
specimen is collected from the advancing
margin of the lesion preferably by needle
aspiration rather than by swabs.
Aspirated specimens must be placed into a
sterile tube or transport vial and not
squirted onto a swab.
You must instruct well the patient in
specimen collection for example in urine
and feces.
The specimen must be labelled accurately.
SPECIMEN CONTAINERS AND OTHE
MATERIALS FOR COLLECTION
Specimens for microbiology cultures
should be collected in sterile container.
Swabs are used for specimens for the upper
respiratory tract, external ear, eye, and
genital tract.
Swabs are not recommended for routine
collection because they are easily dried out.
If swabs are used, Dacron or calcium
alginate swabs are preferred rather than
cotton-tipped ones
Cotton-tipped swabs may have excessive
fatty acids which are inhibitory and toxic
to some bacteria.
SPECIMEN LABELING:
- write the patient information including:
Patient's complete name and identifying
number must be put in the label.
- the age and birthdate should be included
if they are part of the protocol
- the label should include the date and time
of collection.
- identification of mislabeled specimen or
requisition should not be done over the
telephone.
SPECIMEN TRANSPORT:
1. Specimen should be transported
immediately to the laboratory. Preferably
within 30 minutes- 2 hrs.
2. Specimens should be maintained as
much as possible to its original state when
it was collected when transporting it.
3. For anaerobes, transport should not take
more than 10 minutes.
4. For CSF transport should be done within
15 minutes.
5. All specimens should be leak proof.
6. All specimen containers should be
placed in sealable, leak-proof plastic bags.
4
 CLINICAL BACTERIOLOGY
BIOSAFETY AND QUALITY CONTROL | LABORATORY
BY: EVONY VILORIA

7. Culture isolates must be triple-packecl and can be refrigerated; if more than 48

before being shipped. hours, it should be at -70°C.
8. Specimen bags should be marked with
biohazard label. PRESERVATIVES - BORIC ACID
maintains the
TRANSPORT REQUIREMENTS OF appropriate colony
SPECIMENS FOR MICROBIOLOGICAL counts for urine
TESTS specimen at room
TRANSPORT SPECIMEN temperature for
CONDITION 24hrs
Immediately at room Body fluids, bone, - preservatives
temperature inner ear, corneal should not be added
scraping, foreign on fecal specimens
bodies, gastric TRANSPORT OR - these media
aspirate, suprapubic HOLDING MEDIA maintains the
aspirateof urine, viability of
prostatic samples microorganisms
Within 15 mins. at CSF present in a
room temperature specimen but do not
allow their
Within 2 hrs at Blood or bone
multiplication.
room temperature marrow specimens
- Charcoal
Within 24 hrs at Abscess, outer ear, sometimes added to
room temperature conjuctiva absorbed fatty acids
specimen, genital present in the
tract specimen, specimen that could
hhair nail skin kill fastidious
scrapings, organism like
respiratory tract Neisseria
specimens, tissue gonorrhoeae and
specimens Bordtella pertussis.
Immediately at 4℃ Gastric biopsy EXAMPLE:
Within 2 hrs at 4℃ Urine sample from Stuart’s medium,
catheter Amies meium, Cary
Within 24 hrs at Rectal swab, stool Blair Transport
4℃ culture, clean Medium,
voided mistream Transgrow, JEMBEC
urine. (John E. Martin
Biological Chamber)
SPECIMEN PRESERVATION ANTICOAGULANTS - 0.025% SPS is used
- If transport of the specimen to the for blood culture
laboratory is delayed, or if it will not be media
processed immediately, the specimen can - Reasons of using
be maintained with the use of preservatives, 0.025% SPS
holding media and even culture media. 1. To inhibit
Stool specimens for C. difficile toxin assay phagocytosis and
should be collected without a preservative complement
5
 CLINICAL BACTERIOLOGY
BIOSAFETY AND QUALITY CONTROL | LABORATORY
BY: EVONY VILORIA

activation. -70℃
2. To - fecal specimen for
neutralized Clostridium difficile
aminoglycosie may be kept at -70 ℃
antibiotic. if the storage will be
3. To longer than 3 days
neutralized the
bactericidal
effect of plasma. STORAGE REQUIREMENTS PRIOR TO
SPECIMEN PROCESSING
SPECIMEN STORAGE STORAGE SPECIMENS
- if specimen cannot be processed CONDITIONS
immediately after transporting them to the Plate as soon as * Body fluids
laboratory, they must be stored under possible * Bone specimens
temperature conditions. * Gastric biopsy
*Suprapubic aspirate
1. REFRIGERATOR - for stool, urine, of urine
TEMPERTURE sputum, swabs and * Prostatic cancer
viral specimens. 24 hrs at room * Abscess
(4℃) temperature * Outer ear
- if the delay of
transport is longer specimen
than 2 hrs, the * For inner ear
specimen can be specimen - 6 hours
placed in Cary Blair at room temperature
transport medium. * Eye and conjuctiva
- specimen suspected specimens
for anaerobic bacteria * Genital tract
should never be stored specimen (for
in a refrigerator. urethral specimens,
- incubator put them in JEMBEC
temperature (35℃) at 35 ℃
- CSF SHOULD BE immediately upon
KEPT AT THIS receipt in the
TEMPERATURE laboratory)
2. AMBIENT - it is for anaerobic * Upper respiratory
(ROOM) culture, sterile body tract specimens
TEMPERATURE fluids, genital * Tissue specimens
specimens, ear & eye Indefinitely at room * Hair, nail or skin
(22-25℃) temperature scrapings
swabs
3. FREEZER - serum for serological Immediately at 35℃ * Blood
TEMPERATURE studies are frozen up * Bone marrow
specimens
(-20℃ OR -70℃) to 1 week (-20℃) * Corneal scrapings
- tissues or specimens CSF (although,
that are to be stored Six hours at 35℃
immediate
for a long time period processing is
should be kept at recommended)
6
 CLINICAL BACTERIOLOGY
BIOSAFETY AND QUALITY CONTROL | LABORATORY
BY: EVONY VILORIA

24 hours at 4℃ * Lower respiratory questionable medical value.

tract specimens 11. More than one specimen from the same
* Clean-voided source (except for blood) and from the
midstream urine same patient is submitted on the same day.
* Straight catheter 12. A single swab is submitted with
urine multiple requests for various organisms.
* Indwelling 13. Expectorated sputum in which the
catheter urine Gram stain reveals <25 WBCs and > 10
* Foreign devices epithelial cells.
such as catheters
72 hours at 4℃ * Rectal swab SPECIMEN PRIORITIZATION
* Stool culture - when multiple specimens arrive at the
same time, priority should be given to those
who have critical conditions such as CSF,
SPECIMEN REJECTION tissue, bloo, and sterile fluid.
- all rejected specimens require the - Urine, throat, sputum, stool or wound
acknowledgement of the person incharge drainage are specimens that can be stored
of collecting the specimen. and processed later.
- specimens that are impossible to recollect - Acid fast bacilli (AFB), viral and fungal
or would require the patient to go invasive can be processed by batch.
procedure may need to be processed
regardless of the conditions of the LEVELS OF SPECIMEN PRIORITIZATION
specimen. LEVEL DESCRIPTION SPECIMENS
1 Critical/Invasive Amniotic
REASONS FOR SPECIMEN REJECTION fluids,
1. The information on the label does not blood,
match the information on the requisition brain, CSF,
slip. heart valves,
2. The specimen is transported at an pericardial
improper temperature. fluids.
3. The transported specimen has not been 2 Unpreserved Bone, feces,
placed in the proper medium. sputum.
4. The quantity of specimen is not sufficient 3 Quantitation Catheter tip,
(QNS) for testing. required urine, tissue
5. The transport of specimen exceeds the 4 Preserved Urine, feces,
two hours' post-collection period. and swabs
6. The specimen is preserved in a fixative in holding
(formalin). media
7. The specimen has been reserved for
anaerobic culture from a site known to SPECIMEN PROCESSING
have anaerobes as part of the indigenous > GROSS EXAMINATION
microbiota (vagina and mouth). - stool specimens should be examined for
8. The specimen has already dried up upon evidence of barium, cloudiness, or clotting.
transporting to the laboratory.
9. The specimen is leaking. > PREPARATION SMEARN
10. The specimen being processed has a > DIRECT MICROSCOPIC EXAMNIATION
7