MICROBIOLOGY

CLINICAL BACTERIOLOGY LECTURE

○ Apply to human blood and other body fluids w visible

OUTLINE blood but does not apply to feces, nasal secretions,
I. Laboratory Safety B. Sterilization saliva (unless they contain visible blood)
A. Possible Routes for C. Disinfection ● Standard precautions
Infection D. Antiseptics ○ Set of preventive measures applied to all patients
II. Exposure Control Plan E. Biocides that are designed to reduce the risk of infection in
III. Universal and Standard F. Liquid Decontaminants the health care setting
Precautions VIII. Fire Safety ● Potentially infectious
A. Safety Guidelines for A. Type A ○ Blood, tissue, body fluids, secretions, excretions
Laboratory B. Type B (except sweat)
IV. Personal Protective C. Type C
Equipment D. Type D A. POSSIBLE ROUTES FOR INFECTION
V. Engineering Controls E. Class K 1. No food or drink
A. Biosafety Levels IX. Chemical Safety 2. No smoking
B. Biosafety Cabinets A. Material Safety Data 3. No cosmetics
VI. Post Exposure Plan Sheet 4. Protective eyewear must be worn
VII. Disposal of Hazardous B. Hazardous Chemicals 5. Wear face shields
Waste 6. Clothing: gowns, aprons lab coats, closed shoes
A. Autoclave 7. Hair should be tied up
8. Wash hands frequently and thoroughly
9. Eyewash stations: 100 feet or 10 secs away from any area in
the lab
I. LABORATORY SAFETY
10. Emergency stations: shower
11. Respirators: available where there is air contamination
A. POSSIBLE ROUTES FOR INFECTION 12. Mouth pipetting: ingestion of microorganisms; use pipette
● Airborne bulbs/automated devices
○ Aerosols from centrifugation of unstopped tubes or 13. Glassware and sharps: in puncture-resistant containers
from heating cultures or specimens too rapidly, 14. Centrifuges: minimize the production of aerosols
removing stoppers from tubes, leakage from a
container that holds contaminated specimens IV. UNIVERSAL AND STANDARD PRECAUTIONS
● Ingestion
● Protective laboratory clothing disposable gloves, eye
○ Infection as a result of failure to wash hands or
protection, face masks
eating, drinking, smoking, applying cosmetics, or
● Barrier protection: to prevent skin and mucous membrane
pipetting w the mouth
contamination
● Direct inoculation
● Gloves: worn when there is potential for skin contact w
○ Infection from needlesticks, broken glass, animal
potentially infectious materials; worn in phlebotomy and
bites, small scratches on the dingers
capillary puncture
● Mucous membrane contact
● Facial protection and body covering: worn when splashes
○ Infection if the organism can directly enter through
w blood and other body fluids
the mucous membranes (conjunctiva of the eye)
● High-efficiency particulate air respirators: should be fit
● Arthropod vectors
tested for each person; those who encounter mycobacteria
○ Infectious sources include ticks, fleas, and
through contact w either patient or specimen
mosquitoes
● Lab employees who come in contact w body fluids should be
offered hepa B vaccinations for free
II. EXPOSURE CONTROL PLAN
Exposure control plan V. ENGINEERING CONTROLS
● The risk of exposure to infectious agents for all job
● To protect employees from the hazards that may occur during
classifications and explains exposure-reduction methods must
the performance of lab procedures
include:
● All labs must adhere to a minimum of Biosafety Level 2
○ Safety education
guidelines
○ Universal precautions and safety standard
precautions
○ Engineering controls A. POSSIBLE ROUTES FOR INFECTION
○ PPE Table 1. Biosafety levels.
○ Disposal of hazardous waste
○ Post Exposure procedures BSL Function
1 No known pathogenic potential for immunocompetent
III. UNIVERSAL AND STANDARD PRECAUTIONS individuals
● Centers for Disease Control (CDC) Bacillus subtilis
○ Universal precautions as recommendations that Most undergraduate lab courses operate at this level
describe handling of clinical specimens by health Adherence to std lab techniques
care personnel; 1987 2 Level 1 + lab coats, protective gloves, limited access,
● Clinical Laboratory and Standards Institute (CLSI) decontamination of all infectious waste, biohazard signs
○ Universal precautions as set of preventive measures Apparatus includes partial containment equipment
designed to reduce the risk of transferring HIV, hepa Lab acquired infections: HBV, HIV, Staphylococcus, enteric
B virus, and other bloodborne pathogens in the pathogens such as Salmonella and Shigella
health care setting 3 Level 2 + special lab clothing, controlled access
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 Mycobacterium tuberculosis, Brucella, Coccidiodes
immitis, Rickettsia, arbovirus
Air movement must be carefully contained
4 Level 3 + entrance through a separate room, street clothing à
lab clothing
Maximum containment
Filovirus, arenavirus
Biohazard label
● Used to identify those areas of the lab where infectious
specimens or cultures are stored/present
Figure 1. Biohazard label.
Sterilization
● Done by heat, UV light, passage of air through HEPA filter
BIOSAFETY LEVEL 2 CRITERIA FOR MICROBIOLOGY
PRACTICES
● Limit access to lab when working w infectious agents
● Decontaminate work surfaces at least once a day and after
any spill
● Use mechanical pipetting devices
● Do not eat, drink smoke, or use cosmetics. Do not store food
in biological refrigerator.
● Wash hands thoroughly
● Minimize the creation of aerosols
● Wear lab coats, gowns, smocks and remove before leaving
the lab
● Avoid skin contamination by covering cuts w occlusive
bandages and using gloves
● Do not use needles that are bent, cut, or recapped
● Handle M. tuberculosis at BSL 3 in Class II BSC while
wearing a solid-front gown and personal respirator (or
face-folding mask); use sealed safety cups for centrifugation
● Handle mild-lime fungi in BSC; plated media for myco should
be sealed w a cellulose band or cellophane or labelling tape
(prevent dispersion of spores)
B. BIOSAFETY CABINETS
CLASS I BSC
● Provide partial containment for procedures involving
moderate- and high-risk microorganisms (BSL 2 and 3)
● Open-fronted, negative-pressure, ventilated cabinets
● Unsterilized air enters and circulates w/in the cabinet
● Exhaust air from cabinet is filter by HEPA filter
CLASS II BSC
● Provide partial containment for procedures involving
moderate- and high-risk microorganisms (BSL 2 and 3)
● Sterilize bot air entering and circulating w/in the cabinet and
the exhaust air
● Vertical laminar-flow biological cabinets
● Open-fronted; have HEPA filter, recirculated airflow w/in the
workspace
● Exhaust air is filtered, too, by the HEPA filter
● Type II-A
○ Self-contained w 70% of the air recirculated
○ Not required to be vented; acceptable for low- to
moderate-risk agents
○ Most hospital microbio labs use this type
● Type II-B
○ Must be vented w 30% of the air exhausted from the
cabinet and 70% recirculated back into the room
CLASS III BSC
● Provide the highest level of safety
● All air entering and leaving the cabinet is sterilized w a HEPA
filter
● Supply air: drawn through the HEPA filter
● Exhaust air: filtered through 2 HEPA filters
● System is entirely enclosed
● Infectious materials: handled w rubber gloves
VI. POST EXPOSURE PLAN
● All lab accidents or safety incidents: reported to the lab safety
officer or supervisor
● Immediate and appropriate medical treatment
● Administration of immunizations, hepa B immune
immunoglobulin, hepa B vaccinations
● Collection of serum samples at 6 weeks, 3 mos, and 6 mos
for antibodies to HIV or abraded skin exposure to HBV or HIV
● Documentation of the accident w a report
● Corrective actions
VII. DISPOSAL OF HAZARDOUS WASTE
● Microbiological waste must be decontaminated before
disposal
● Needles and sharps should be placed in puncture-resistant
containers
● Contaminated materials should be placed into two leak-proof
plastic bags
● Contaminated pipettes, swabs, and glass should be placed
into puncture-resistant burn boxes
A. AUTOCLAVE
● Decontaminates that uses saturated steam at 121oC and 15
psi of pressure for 1 hr to sterilize
● Infectious medical waste: 132oC for 1/2 -1 hr
● Monitored periodically
● Bacillus stearothermophilus: spore indicator; weekly
monitoring
B. STERILIZATION
● Physical or chemical process that kills all microorganisms,
including spores
● Moist heat or steam under pressure: autoclave
● Dry heat: 171oC for 1 hr, 160oC for 2 hrs, 121oC for 6 hrs
● Liquid: glutaraldehyde, 6-30% hydrogen peroxide, 6-8%
formaldehyde, chlorine dioxide
C. DISINFECTION
● Destroys most microbes but does not kill spores
● Moist heat: 75-100oC
● Liquid: glutaraldehyde, 3-6% hydrogen peroxide, 1-8%
formaldehyde, chlorine compounds, 70% isopropyl alcohol
● Liquid household bleach
● Quaternary ammonium compounds: benzalkonium chloride
D. ANTISEPTICS
● For skin or tissue; not to be substituted for a disinfectant
● Alcohols: 70% ethyl or isopropyl alcohol; not against spores
● Iodophors: iodine combined w an organic carrier molecule;
povidone-iodine and poloxamer-iodine
● Hexachlorophene: bactericidal and broad spectrum but may
be toxic
E. BIOCIDES
● Chemical agents that inactivate microorganisms; either -static
and inhibit growth of the microbe or -cidal and kill the target
organism
F. LIQUID DECONTAMINANTS
● 70% EtOH or 10% bleach (NaClO)
● For 10 mins to increase ability to decontaminate
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 ● Question 2
VIII. FIRE SAFETY ● Question 3
Answer 1
● Ignition sources: open flames; heating elements; spark
Answer 2
gaps, from light switches or static electricity; electrical
Answer 3
instrumentation

A. TYPE A XI. REFERENCES

● Water Fire Extinguisher ● APA format
● Used on combustible materials for fires in paper, wood,
rubber, sloth, plastics
● Extinguish fire w cooling effect

B. TYPE B
● Carbon dioxide fire extinguisher
● Used on extremely flammable liquids or electrical fires
including fires of oil, kerosene, gasoline, paints, fats, grease,
solvents
● Extinguish fire by eliminating oxygen

C. TYPE C
● Dry chemical extinguishers
● Used in electrical fires in wiring and other electrical sources or
equipment
● Extinguish fire bc chemicals do not conduct electricity

D. TYPE D
● Combustible metals
● Used for fires involving combustible metals: Na, K, Mg, Na-K
alloys
● NaCl salt wc forms a metal layer over the fire wc occludes the
oxygen
● Graphite, sodium carbonate based, copper based

E. CLASS K
● Dry and wet chemical extinguishers
● Used for kitchen fires involving combustible materials such as
oil or fat
● Extinguish fire by using various wet or dry chemical agents

IX. CHEMICAL SAFETY

● Every compound or reagent must have precautionary labels

A. MATERIAL SAFETY DATA SHEET

● Characteristics of hazardous compound chemicals

Table 2. Sections of MSDS

Section Information
I Manufacturer’s name and address, emergency tel. no., tel
no. for info about preparation
II Hazardous ingredient’s identity info, components, specific
chemical identity common name, PEL
III Physical/chemical characteristics
IV Fire and explosion hazard data
V Reactivity data
VI Health hazard data: routes of entry
VII Precautions of safe handling and use
VIII Control measures

B. HAZARDOUS CHEMICALS
● Corrosive: causes visible destruction or irreversible damage
to human skin on contact
● Toxic: serious biological effects after inhalation, ingestion, or
skin contact w relatively small amts
● Carcinogenic: ability of chemical to induce a malignant tumor
● Ignitable: any chemical that can burn and includes both
combustible and flammable liquids
● Explosive: reactive and unstable substances that readily
undergo violent chemical change

X. REVIEW QUESTIONS
● Question 1
3