LABORATORY SAFETY

Sterilization, Disinfection, and Decontamination

 Sterilization: kills all forms of microbial life
 Disinfection: destroys pathogenic organisms
 Decontamination: removal of pathogenic microorganisms
 Affected by:
o Organic load o Temperature
o Type of organism present o pH
o Concentration and exposure o Humidity
time o Presence of biofilm
o Physical and chemical nature
of the surface

Methods of Sterilization
 Incineration
o 870O-980OC
o Safest method
o Prions, infective proteins
 Moist heat
o Steam under pressure (121OC and 132OC)
o Sterilize biohazardous trash and heat-stable objects
o Fastest and simplest
o Autoclave
 Dry heat
o Requires longer exposure times: 1.5-3 hours
o Higher temperature than moist heat: 160-180OC
o Glassware, oil, petrolatum, or powders
 Filtration
o Antibiotic solutions, toxic chemicals, radio isotopes, vaccines, and carbohydrates
o Liquids: cellulose acetate or cellulose nitrate membrane with vacuum
o Air: high efficiency particulate air (HEPA) filter (0.3 µm)
 Ionizing (gamma) radiation
o Disposables
 Chemicals
o Most common: ethylene oxide (used in gaseous form)
o Vapor-phase hydrogen peroxide: HEPA filters, metals, nonmetal devices (medical
instruments)
o Hydrogen peroxide gas plasma

Methods of Disinfection
 Physical methods
o Boiling: 100OC for 15 minutes (vegetative bacteria)
o Pasteurizing: 70OC for 30 minutes (food pathogens)
o Nonionizing radiation: UV light
 Chemical methods
o Alcohols: 60-90% ethyl or isopropyl alcohol solution
o Aldehydes: Formaldehyde and Glutaraldehyde (sporicidal in 3-10 hours)
o Halogens
o Peracetic acid: surface sterilization of surgical instruments
o Hydrogen peroxide: 3% hydrogen peroxide (inanimate objects)
o Quarternary ammonium compounds: bench tops or other surfaces
o Phenolics: derivatives of carbolic acid (phenol): Amphyl
 Antiseptics
o Iodine: widely used antiseptic
o 70% alcohol
o 1% Silver nitrate
Chemical Safety
 All hazardous chemicals in the workplace
be identified and clearly marked with a
National Fire Protection Association
(NFPA) label stating the health risk.
 Chemical hygiene plan: guidelines on
proper labeling of chemical containers
 Material Safety Data Sheets (MSDSs)
 Written chemical safety training and
retraining programs
 Fume hoods: protect against chemical odor,
but they are not HEPA-filtered to trap
pathogenic microorganisms

Fire Safety
 Types of fire extinguishers:
o Type A: trash, wood, and paper
o Type B: chemical fires
o Type C: electrical fires

Standard Precaution
 CDC 1987: Universal Precautions: to
reduce the risk of HBV transmission in
clinical laboratories and blood banks
 1996: Standard Precaution: blood and
body fluids from every patient be treated as
potentially infectious. Applies to blood and
all body fluids except sweat

Engineering Controls
 Laboratory environment
o Biohazard symbol should be prominently displayed on the laboratory doors and
equipment that contains infectious material
 Biologic Safety Cabinet
o Protect workers from aerosol exposure to infectious disease agents
o Air is sterilized either by heat UV light, or most commonly, by passage through
HEPA filter
o Class I-III according to the effective levels of biologic containment

o Class I
Allow room (unsterilized) air to pass into the cabinet and around the area
and material within, sterilizing only the air to be exhausted
Negative pressure
May be ventilated to the outside or exhausted to the work area
Causally operated with an open front
o Class II
Sterilize air that flows over the infectious material as well as the air to be
exhausted
Vertical laminar flow BSCs
Class IIA: self-contained, 70% of the air is recirculated into the work area
Class IIB: exhaust air is discharged outside the building, radioisotopes,
toxic chemicals, or carcinogens
o Class III
Affords the most protection to the worker
Completely enclosed and have negative pressure, most protection to the
worker
Infectious material is handled with rubber gloves that are attached and
sealed to the cabinet
  Personal Protective Equipment

 Postexposure Control
o All laboratory accidents and potential exposures must be reported to the
supervisor and safety officer
o Prophylaxis: Hepatitis B virus immunoglobulin (HBIG) or HBV booster
immunization in the event of exposure to Hepatitis B
o Postexposure prophylaxis: Neiserria meningitidis (possible disease transmission)
o HIV testing

Classification of Biologic Agents Based on Hazard

 Biosafety Level 1 (BSL-1)
o no known potential for infecting healthy people and are well define and
characterized
o Bacillus subtilis and Naegleria gruberi
 Biosafety Level 2 (BSL-2)
o Most commonly being sought in clinical specimens
o Agents of infectious disease, HIV, HBV, Salmonella organisms, and several more
 Biosafety Level 3 (BSL-3)
o Handling of material suspected of organisms unlikely to be encountered in a
routine clinical laboratory
o Mycobacteium tuberculosis, Coxiella burnetii, and mold stages of fungi
o Primarily transmitted by infectious aerosol
 Biosafety Level 4 (BSL-4)
o Exotic agents that are considered high risk and cause life-threatening disease
o Marburg virus or Congo-Crimean hemorrhagic fever
o All procedures are performed under maximum containment (Class III BSC)
o Usually handled by health or research laboratories

SPECIMEN MANAGEMENT

Appropriate Collection Techniques

 Acute (early) phase of an illness
 Within 2-3 days for viral infections
 Before antimicrobials, antifungals, or antiviral medications are started

Specimen Transport
 Within 2 hours of collection
 Specimen containers: leak-proof; transported within sealable, leak-proof, plastic bags
with a separate section for paperwork
 Bags should be marked with a biohazard label
  Special preservatives or temperature-controlled, or holding media

Specimen Preservation
 Boric acid: urine
 Polyvinyl alcohol (PVA) and buffered formalin: stool for ova and parasite examination
 Transport or holding medium
o Stuart’s medium and Amie’s medium: common
o Charcoal: added to absorb fatty acids
 Anticoagulants
o 0.025% Sodium polyanethol sulfonate (SPS): usually used
o Heparin: viral cultures; may inhibit the growth of gram-positive bacteria and yeast
 Should not be used:
o Citrate
o Ethylenediaminetetraacetic acid (EDTA)
o Others

Specimen Storage
 Refrigerator temperature: 4OC
 Ambient (room) temperature: 22OC
 Body temperature: 37OC
 Freezer temperature: -20OC or -70OC
 Urine, stool, viral specimens, sputa, swabs, and foreign devices (catheters): 4OC
 Serum for serologic studies: 1 week at -20OC
 Tissues or specimen for long-term storage: -70OC

Specimen Labeling
 Patient’s name  Date and time of collection
 Identifying number (hospital or  Source
sample number)  Initials of the individual who
 Birth date collected the sample

Specimen Requisition
 Patient’s name  Ordering physician
 Hospital identification number  Exact nature and source of the
 Age and DOB specimen
 Sex  Diagnosis
 Collection date and time  Current antimicrobial therapy

Rejection of Unacceptable Specimens

 Information on the label does not match the request or the specimen is not labeled at all
 Specimen has been transported at the improper temperature
 Specimen has not been transported in the proper medium
 Insufficient quantity of specimens
 Specimen is leaking
 Transport time is more than 2 hours post collection or specimen was not preserved
 Specimen was received in a fixative (formalin), which kills any microorganisms present
 The specimen has been received for anaerobic culture from a site known to have
anaerobes as part of the microbiota (vagina, mouth)
 The specimen is dried
 Processing of specimen would produce information of questionable medical value (Foley
catheter tip)

Specimen Processing
 When multiple specimens arrive at the same time, priority should be given to those that
are most critical: CSF, tissue, blood, and sterile body fluids
 On arrival, time and date should be recorded
 When a specimen is received with multiple requests, but the amount of specimen is
insufficient to do all of them, the microbiologist should call the clinician to prioritize the
testing
Gross Examination of Specimens
 All processing should begin with a gross examination of the specimen
 Areas with blood or mucus should be located and sampled for culture and direct
examination
 Stool: barium (chalky white color)
 Note the status of the specimen: bloody, clouded, clotted

Direct Microscopic Examination

 Assess the quality of the specimen
 Can give an early indication of what may be wrong with the patient
 Workup on the specimen can be guided by comparing what grows in culture to what was
seen on the original smear
 Usually not performed on throat, nasopharyngeal, or stools specimens
 Most common stain in bacteriology: Gram staining
 Most common direct fungal stains: KOH, PAS, GMS (Grocott’s methenamine silver
stain), calcofluor white
 Most common direct acid-fast stains: auramine rhodamine, Ziehl-Neelsen, Kinyoun

Phases of Growth Media

 Broth
o Liquid medium
o Nutrients are dissolved in water and bacterial growth is indicated by a change in
the broth’s appearance from clear to turbid
o May contain pH indicators (phenol red): change in color in the presence of
metabolites
o Thioglycollate broth: semisolid medium: provides an indication of the type of
organism present based on oxygen requirements
 Agar
o Solid medium: combination of a solidifying agent added to nutrients and water
o Agarose: most common solidifying agent (melts at high temperatures: > 95OC but
resolidifies after temperature falls below 50OC)
 Biphasic medium
o both liquid and solid phase

Selection of Culture Media

 Nutritive Media
o Contains nutrients that support the growth of most nonfastidious organisms
o Support growth of a wide range of microorganisms
o Considered nonselective
o Differential
o Tryptic soy agar, and nutrient agar plates (bacteria), or Sabouraud’s dextrose agar
(fungi, blood or chocolate agar)
 Supplemental or Enrichment Medium
o Contains specific nutrients required for the growth of bacterial pathogens
o Buffered charcoal-yeast extract (BCYE): L-cysteine and other nutrients for the
growth of Legionella pneumophilia
 Enrichment Broth
o Back up broth
o Specialized broths used to enhance the growth or organisms present in low numbers
o Detection of anaerobes in aerobic culture or organisms that may be damaged by
previous or concurrent antimicrobial therapy
o Thioglycollate broth (anaerobes), brain-heart infusion broth (BHIB), tryptic soy
broth (TSB), LIM (Todd Hewitt broth: colistin and nalidixic acid), gram-negative
broth (enteric gram-negative organisms)

 Selective Media
o Support the growth of one group organisms but not another by adding
antimicrobials, dyes, or alcohol
o MacConkey agar: crystal violet: inhibits gram-positive organisms
 o Columbia agar: colistin and nalidixic acid: inhibits gram-negative organisms
o Can also be differential media if, in addition to their inhibitory activity, they
differentiate between groups of organisms
 Differential Media
o Employ some factors that allow colonies of one bacterial species or type to
exhibit certain metabolic or culture characteristics
o MacConkey agar: differentiates between gram-negative bacteria that cannot
ferment lactose
 Brain-heart Infusion
o Broth or agar
o With or without blood
o Carbohydrate: dextrose: readily accessible source of energy
o BHI broth: often used as a major component of the media developed for culturing
patient’s blood for bacteria
o Bacterial susceptibility
 Chocolate Agar
o Hemoglobin, hemin (X factor), nicotinamide adenine dinucleotide (NAD, V
factor): released during lysis
o Neisseria gonorrheae, Hemophilus spp. (neither grows on sheep blood agar)
 Columbia CNA with Blood
o Columbia agar base: 3 peptone sources and 5% defibrinated sheep blood
o Differentiates bacterial colonies based on hemolytic reactions
o CNA: colistin and nalidixic acid
 Gram-Negative Broth
o Selective broth
o Cultivation of gastrointestinal pathogens from stool specimens and rectal swabs
o Sodium citrate and sodium deoxycholate (bile salt)
o Mannitol: primary carbon source
 Hektoen Enteric Agar
o Bile salts
o Dye: bromthymol blue (pH indicator) and acid fuchsin
o Carbohydrates: lactose, sucrose, and salicin
o Ferric ammonium citrate: detection of H2S
o Differential medium
 MacConkey Agar
o Dye: crystal violet
o pH indicator: neutral red
o Carbohydrate: lactose
 Phenylethyl Alcohol Agar
o Sheep blood agar with phenylethyl alcohol (inhibits gram-negative bacteria)
o Provides nutrients for common gram-positive cocci (enterococci, streptococci,
and staphylococci)
o Should not be used for the interpretation of hemolysis
 Sheep Blood Agar
o Supports growth for all but the most fastidious clinically significant bacteria
o Protein source (tryptones), soybean protein digest (contains natural carbohydrate),
sodium chloride, agar, and 5% sheep blood
o Interpret hemolysis
 Modified Thayer-Martin Agar
o Enrichment (chocolatized blood) and selective medium (antibiotics)
o Colistin (gram-negative), vancomycin (gram-positive), nystatin (yeast), and
trimethoprim (Proteus spp)
o Martin-Lewis agar: asamycin (nystatin) and higher concentration of vancomycin
 Thioglycollate broth
o Enrichment broth or semisolid media
o Casein, yeast and beef extracts, and vitamins: enhances the growth of most
organism
o Resazurin: oxidation-reactin indicator
o Dextrose, vitamin K, hemin: used to modify the basic formula
o 0.075% agar: prevents convection currents from carrying atmospheric oxygen
o Thioglycolic acid: reducing agent
  Xylose-Lysine Deoxycholate Agar
o Selective and differential for Shigella and Salmonella
o Salt and sodium deoxycholate
o Phenol red: indicator
o Carbohydrates: lactose, xylose, and sucrose
 Eosin Methylene Blue (EMB) Agar
o Carbohydrate: lactose
o Indicators: Eosin Y and methylene blue
o Differentiation of lactose and non-lactose fermenting enteric bacilli
 Salmonella-Shigella Agar
o Inhibitor: brilliant green
o Carbohydrate: lactose
o Indicator: neutral red
o Ferric citrate and sodium citrate
o Selective for Salmonella and some Shigella spp.
 Thiosulfate Citrate-Bile Salts (TCBS) Agar
o Indicator: bromthymol blue
o Yeast extracts, bile salts, citrate, sucrose, ferric citrate, and sodium thiosulfate
o Selective and differential for Vibrio spp.
 Trypticase Soy Broth
o All-purpose enrichment broth
o Can support the growth of many fastidious and nonfastidious bacteria
o Used for subculturing various bacteria form primary agar plates

Environmental Requirements
 Oxygen and Carbon Dioxide Availability
o Aerobic: uses oxygen as the terminal electron acceptor and grow well in room air
o Anaerobic: oxygen is inhibitory or lethal
o Facultative anaerobic: grow in the presence or absence of oxygen
o Strictly aerobic: cannot grow in the absence of oxygen
o Microaerophilic or microaerobic: grow only in low levels of oxygen
(approximately 20% or less)
o Aerotolerant: grow slowly and poorly in the presence of oxygen
o Capnophilic: grows best with higher oxygen concentrations (5-10%)
 Temperature
o Similar to those of internal human host tissues and organs
o 35-37OC
o Cold enrichment: Listeria monocytogenes and Yersinia enterocolitica (4-43OC)
 pH
o Near neutral pH: 6.5-7.5
 Moisture
o Increased atmospheric humidity enhances the growth of certain bacterial species

Incubation Conditions
 Fungi: 28-30OC
 Bacteria, viruses, and acid-fast bacillus: 35-37OC
 Aerobes: grow in ambient air: 21% oxygen and a small amount (0.03%) of carbon dioxide
 Anaerobe: cannot grow in the presence of oxygen
o Anaerobes jar, bags, or chambers: 5%-10% hydrogen, 5-10% carbon dioxide, 80-
90%nitrogen, and 0% oxygen
 Capnophiles (H. influenzae and N. gonorrheae): require increased concentrations of
carbon dioxide (5-10%) and approximately 15% oxygen
o achieved by candle jars (3% carbon dioxide), carbon dioxide incubator, chamber
jar, or bag
 Microaerophiles (C. jejuni, H. pylori): grow under reduced oxygen (5-10%) and
increased carbon dioxide(8-10%)
BACTERIAL GENETICS, METABOLISM, AND STRUCTURE

Bacterial Genetics
 Nuclei Acid and Organization
o Nucleic acids: hereditary information resides or is encoded
o Two major classes of nucleic acids
o Prokaryotes: no membrane bound organelles: genetic material is not enclosed in a
nucleus
o Eukaryotes: genetic material enclosed in a nuclear envelope

CHARACTERISTICS PROKARYOTES EUKARYOTES

DNA within a nuclear membrane No Yes

Mitotic Division No Yes
DNA associated with histones No Yes
Chromosome number One More than one
Membrane-bound organelles No Yes
Size of ribosome 70S 80S
Cell wall containing peptidoglycan Yes No

Nonchromosomal Elements
 Plasmids
o exist as double stranded, closed,
circular, autonomously replicating
extrachromosomal genetic elements
o Number per bacterial cell varies
o Composed of several genes
 Transposable elements
o “Jumping genes”
o Pieces of DNA that move from one
genetic element to another (plasmid
to chromosome or vice versa)
o Unable to replicate independently
o Do not exist as separate entities in
the bacterial cell
o Two types: simple transposon or
insertion transposon (IS) and
composite transposon
 Episomes
o capable of replication independently of the host chromosome
o Considered part of the bacterial genome
o Not as stable as the chromosome and may be lost during replication

Replication and Expression of Genetic Information

 Replication
o Binary fission: production of 2 daughter cells from one parent cell
o Genome must be replicated so that each daughter cell receives an identical copy
of functional DNA
o Must occur quickly and accurately

Genetic Exchange
 Transformation
o Recipient cell uptakes of naked (free) DNA released into the environment when
another bacterial cell (donor) dies and undergoes lysis
o Haemophilus, Streptococcus, and Neisseria
 o Antibiotic resistance, ability to cause disease
o Not limited to organisms of the same species

 Transduction
o DNA form two bacteria may come together in one cell allowing recombination
o Mediated by viruses capable of infecting bacteria (bacteriophage)
o Generalized transduction or specialized transduction

 Conjugation
o Occurs between 2 living cell,
involves cell-too-cell contact,
requires mobilization of the
donor bacterium’s chromosome
o Intercellular contact is not well
characterized in bacterial species
o E.coli: mediated by sex pilus

o Plasmids: must be replicated

o Transposons: transposition: process by which genetic elements excise from one
genomic location to another
Bacterial Metabolism
 Fueling
o Utilization of metabolic pathways involved in the acquisition of nutrients from the
environment
o Acquisition of nutrients
Simple diffusion: water, oxygen, and carbon dioxide
Active transport: most common methods for uptake of nutrients: sugars,
amino acids, organic acids, inorganic ions
Group translocation: requires energy but chemicals transported undergoes
chemical modification (sugars, purines, pyramidines, and fatty acids)
o Production of metabolites
Embden-Meyerhof-Parnas (EMP) Pathway (glycolysis)
Tricarboxylic acid (TCA) cycle
Alternate pathways: Entner-Doudeoroff (degrades gluconate and glucose)
and Pentose Phosphate Pathway
o Energy Production
Produces the energy required for nearly all cellular processes (nutrient
uptake and precursor production)
Accomplished by the breakdown of chemical substrates (catabolism
coupled with oxidation-reduction reactions)
Generation of ATP: substrate-level phosphorylation (oxidative
phosphorylation)
 Generates ATP from ADP
 Aerobic respiration: uses oxygen as the terminal electron acceptor
 Anaerobic respiration: final electron transport acceptors other than
oxygen
 Biosynthesis
o Uses the precursor products in dozens of pathways to produce a variety of
building blocks (amino acids, fatty acids, sugars, and nucleotides)
o Depends on anabolic metabolism
 Polymerization and Assembly
o Assembling of the building blocks into macromolecules (lipids,
lipopolysaccharides, polysaccharides, proteins, and nucleic acids)
o Synthesis of macromolecules is driven by energy and enzymatic activity in the cell

Structure and Function of the Bacterial Cell

 Bacteria: single-cell prokaryotic microorganisms
 Fungi and parasites: single-cell or multicellular eukaryotic organisms
 Viruses: dependent on host cells, infectious organisms, not considered cellular organisms
 Prions: abnormal infectious proteins, not considered living cells

CHARACTERISTICS PROKARYOTES EUKARYOTES

DNA within a nuclear membrane No Yes

Mitotic Division No Yes
DNA associated with histones No Yes
Chromosome number One More than one
Membrane-bound organelles No Yes
Size of ribosome 70S 80S
Cell wall containing peptidoglycan Yes No
Bacterial Morphology
 Cocci: circular
 Coccobacilli: ovoid
 Bacilli: rod shaped
 Fusiform: pointed end
 Curved
 Spiral
 Gram stain
o Gram positive
bacteria: stain
deep blue or
purple
o Gram negative
bacteria: stain
pink to red

Staining Techniques
 Gram stain
o Hans Christian Gram
o Rapid presumptive identification of microorganisms
o Gives important clues related to the quality of specimen
o Gram-positive: basic dye (crystal violet)
o Gram-negative: allows the crystal violet to wash out easily with the decolorizer
alcohol (acetone)
o Considered a differential stain

GRAM POSITIVE GRAM NEGATIVE

Thick peptidoglycan layer Thinner peptidoglycan layer

Numerous techoic acid cross-linkages Outer lipid bilayer (dehydrated during decolorization)

 Acid-Fast stains
o Specifically designed for a subset of
bacteria whose cell walls contain long-
chain fatty acids (mycolic acid): render
the cells resistant to decolorization even
with acid alcohol decolorizers
o Differential stain
o Mycobacteria: most encountered acid-fast
bacteria

Bacterial Cell Components

 Divided into:
o Outer cell structure and its appendages:
cell envelope
o Cell’s interior
 Cell envelope
o Outer membrane (gram-negative)
o Cell wall: peptidoglycan macromolecules
(murein layer)
o Periplasm (gram-negative)
o Cytoplasmic or cell membrane: encloses the cytoplasm
o Outer membrane
Contain essential enzymes and other proteins located in the periplasmic
space
Bilayer structure: composed of lipopolysaccharides
Significant role in the ability to cause disease
 Porins: protein structures, water-filled structures that control the passage
of nutrients and other solutes (antibiotics) through the outer membrane
Murein lipoproteins: facilitate attachment of the outer membrane to the
next internal layer (cell wall), serve as adhesions for attachment to a host
cell or as transporters
o Cell Wall
Peptidoglycan or murein layer
Bacterial cell shape and strength
Protects against mechanical disruption
Offers some barrier to the passage of larger substances
Composed of disaccharide-pentapeptide units (N-acetylglucosamine and
N- acetylmuramic acid)
Thicker in gram-positive bacteria
o Techoic acids
glycerol or ribitol phosphate polymers combined with various sugars,
amino acids, and amino sugars
Present in gram-positive bacteria
Sometimes linked to N-acetylmuramic acid others (lipotechoic acid) are
linked to the next underlying layer: cellular or cytoplasmic membrane
o Mycolic acids
Mycobacteria
Waxy substances within the murein layer
Makes the cell more refractory to toxic substances (acids)
o Periplasmic space
Typically found in gram-negative bacteria
Bounded by the internal surface of the outer membrane and the external
surface of the cellular membrane
Contains the murein layer
Consists of gel-like substances that assist in the capture of nutrients form
the environment
Contains several enzymes involved in the degradation of macromolecules
and detoxification of environmental solutes (antibiotics)
o Cytoplasmic (inner) membrane
Present in both gram-negative and gram-positive bacteria
Deepest layer of the cell envelope
Heavily laced with various proteins and enzymes vital to cellular
metabolism
Additional osmotic barrier
o Cellular appendages
Capsule
 exterior to the murein layer of gram-positive bacteria, outer
membrane of the gram-negative bacteria
 High molecular weight polysaccharides
 Protects the bacteria from attack by components of the human
immune system
 Facilitates and maintains bacterial colonization on biologic and
inanimate surfaces
Fimbriae or pili
 Hairlike, proteinaceous structures
 Extend from the cell membrane into the external environment
 Serve as adhesins to help bacteria attach to animal host cell surfaces
 Sex pilus: E.coli; serves as conduit for the passage of DNA during
conjugation
Flagella
 Composed of flagellin
 Bacterial motility
 Monotrichous: one end
 Lophotrichous: present on both ends
 Amphitrichous: single flagellum present on both ends of the cell
 Peritrichous: entire cell surface is covered by flagella
 Cell interior
o Cytosol
Nearly all other functions not
conducted by the cell membrane
occur Contains enzymes
Site of protein synthesis
Granular appearance:
polysomes and inclusions
(storage or reserve granules)
 Glycogen: storage form of glucose
 Polyphosphate granules: storage form of
inorganic phosphates
o Nucleoid
Highly coiled DNA intermixed
with RNA, polyamines, and
various proteins
o Plasmids
Other genetic element that exist independently in
the cytosol
o Endospores