CHAPTER 1: BACTERIALCELL STRUCTURE, PHYSIOLOGY,

METABOLISM, AND GENETICS Ribosomes; site Present in all Present in all

of protein
SIGNIFICANCE synthesis (non-
 Evolution membrane)
 Variation Size 70 S in size, consisting of 80 S in size,
 Role of clinical microbiologist 50 S and 30 S subunits consisting of 60S and
40S subunits
ROLE OF CLINICAL MICROBIOLOGIST Electron transport In the cell membrane if In the inner
 Culture organisms from specimens for energy present; no mitochondria membrane of
 Snapshot of specimen present mitochondria and
 Classification and identification of organism chloroplasts
 Possible cause of disease Sterols in Absent except in Present
 Prediction and interpretation of susceptibility cytoplasmic Mycoplasma spp.
 Improve treatment membrane
Plasma Lacks carbohydrates Also contains
BACTERIAL RELATIONSHIPS TO ISOLATION membrane glycolipids and
 Growth requirements of bacteria glycoproteins
 Allows microbiologist to select the correct medium for primary Cell wall, if Peptidoglycan in most Cellulose, phenolic
culture present bacteria polymers, lignin
 Increases likelihood of pathogen isolation (plants), chitin (fungi),
 Steps in bacterial classification other glycans (algae)
 Determine staining characteristics Glycocalyx Present in most as an Present; some animal
 Observe microscopic characterization of size and shape organized capsule or cells
 Determine metabolic biochemical reactions unorganized slime layer
Cilia Absent Present; see
ANTON VON LEEUWENHOEK description of flagella
 Dutch lens maker and biologist Flagella, if Simple flagella; Complex cilia or
 Discovered “Beasties” present composed of polymers of flagella; composed of
 “Father of Protozoology and Bacteriology” flagellin; movement by MTs, movement by
rotary action at the base; coordinated sliding
OVERVIEW OF THE MICROBIAL WORLD spirochetes have MTs microtubules
 Bacteria Pili and fimbriae Present Absent
 Parasites
 Fungi ILLUSTRATION OF PROKARYOTIC AND EUKARYOTIC CELLS
 Viruses

BACTERIA
 Prokaryotes
 No organelles
 Unicellular

COMPARISON OF PROKARYOTIC ANND EUKARYOTIC CELL

CHARACTERICTI PROKARYOTE EUKARYOTE
C
Typical size 0.4-2 um in diameter 10-100 um diameter
0.55-5 um in length >10 um in length
Nucleus No nuclear membrane; Classic membrane-
nucleoid region of the bound nucleus
cytosol
GEMONE
Location In the nucleiod, at the In the nucleus
mesosome
Chromosomal Circular; complexed with Linear; complexed
DNA RNA with basic histones
and other proteins
Genome: Plasmids, small circular In the mitochondria
extrachromosoma molecule of DNA and chloroplasts
l circular DNA containing accessory
information; most PARASITES
commonly found in  Eukaryotic
gram-negative bacteria;  Single or multicellular
each carries genes for  Motile or non-motile
its own replication; can  Categorized by flagella, pseudopodia, or cilia
confer resistance to
antibiotics FUNGI
Reproduction Asexual (binary fussion) Sexual and Asexual  Heterotrophic eukaryotes
Membrane-bound Absent All  Yeast
organelles  Unicellular, asexual reproduction
Golgi bodies Absent in all Present in some  Mold
Lysosomes Absent in all Present in some;  Most multicellular with sexual and asexual reproduction
contains hydrolytic  Filamentous
enzymes  Dimorphic
Endoplasmic Absent in all Present in all; lipid  Filamentous at room temperature
reticulum synthesis, transport  Yeast at human body temperature
Mitochondria Absent in all Present in most
Nucleus Absent in all Present in all
Chloroplasts for Absent in all Present in algae and
photosynthesis plants
 o Extremophiles
VIRUSES o Not encountered in clinical microbiology
 Smallest infectious particle  Eukarya
 Smaller than what is seen by light microscopes o More complex than prokaryotes
 DNA or RNA o Contain organelles
 May be single or double stranded
 Acellular COMPARISON of CELL STRUCTURE
 Obligate intracellular parasites  Eukaryotic cell structure cytoplasmic structure
 Require host cells for replication and are usually host and/or  Cell envelope structures
host cell specific o Plasma membrane
 Cell wall
VIRAL EMERGENCE  80S ribosomes (60S and 40S subunits)
 Becoming better known by  Prokaryotic cell structure
 DNA or RNA makeup  Cell envelope structures
 Host disease signs and symptoms o Plasma membrane (cell membrane)
 Chemical makeup  Prokaryotic cell structure
 Geographic distribution  Cell wall
 Resistance to lipid solvents and detergents o Gram-positive cell wall
 Resistance to pH and temperature changes o Acid-fast cell wall
 Antigenicity (serologic methods)
o Gram-negative cell wall
VIRAL CLASSIFICATION/TAXONOMY o Absence of cell wall
 Based on  Surface polymers
 Genome  70S ribosomes (50S and 30S subunits)
 Replication o Svedberg (S) units are sedimentation rates during high
 Virion structure speed centrifugation.
o Values are NOT additive, due to binding together resulting
 Taxonomy is based on in surface area loss.
 Genotype
o Base sequencing of DNA or RNA GRANULES AND ENDOSPORES
o Comparison of the base composition ratio to determine  Prokaryotes
degree of relatedness  Granules in the cytoplasm
 Phenotype o Glycogen
o Macroscopic and microscopic morphology o Poly-β-hydroxybutyrate
o Staining characteristics o Polyphosphates
o Nutritional requirements  Endospores
o Physiologic and biochemical characteristics o Bacillus and Clostridium produce endospores.
o Susceptibility or resistance to antibiotics or chemicals o Highly resistant to chemical agents, temperature change,
starvation, dehydration, ultraviolet (UV) light, gamma
GENERAL TAXONOMY radiation, and desiccation
o Not involved in reproduction
 Domain
 Kingdom GRAM NEGATIVE OUTER MEMBRANE
 Division (Phylum)  Lipopolysaccharide components
 Class  Antigenic O–specific polysaccharide
 Order  Core polysaccharide
 Family (-aceae)  Lipid A (also called endotoxin)
 Tribe  Outer membrane functions
 Genus (capitalized)  Acts as a barrier to hydrophobic compounds and harmful
 Species (lowercase) sp. singular or spp. plural substances
 Acts as a sieve, allows water-soluble molecules to enter
NOMENCLATURE through porins
 Genus species or Genus species or G. species  Enhances attachment to host cells
 Some genera have the same first letter so the first syllable is used
 Staph. for staphylococcus ACID-FAST CELLS WALLS
 Strept. for streptococcus  Waxy layer of glycolipids and fatty acids
 Esch. coli (bacteria)  Major component is mycolic acid.
 Ent. coli (parasite) o Strongly hydrophobic
 Difficult to Gram stain: lightly gram-positive
CLASSIFICATION BY PHENOTYPIC AND GENOTYPIC  Acid-fast stain
CHARACTERISTICS  Carbolfuchsin
 Species o Mycobacterium spp. and Nocardia spp. stain
 Subspecies (subsp.): phenotypic differences o Others decolorize with acid-alcohol
o Serovarieties (serovar)
o Biovarieties (biovar) BACTERIA LACKING CELL WALLS
o Phage typing: susceptibility to specific viruses  Mycoplasma
 Strain  Ureaplasma
 Species with different susceptibility patterns  Membrane contains sterols
o Example: susceptibility or resistance to penicillin  L-forms
 Genetic relatedness  Bacteria that have lost cell walls
 rRNA
 Led to some reclassification SURFACE POLYMERS
 Capsule
CLASSIFICATION BY CELLULAR TYPE: PROKARYOTES,  Organized polysaccharide or polypeptide structure
EUKARYOTES, AND ARCHAEOBACTERIA o Adherence to surface
 Three domains o Prevents phagocytosis
 Bacteria  India ink
o Classic prokaryotic cell encountered in clinical  Presents as clear halo-like structure
microbiology  Serologic typing
 Archaea  Sometimes remove capsule to detect somatic antigens,
usually by boiling
CELL APPENDAGES
 Flagella  Acridine orange
 External rotating filaments
 Common
o Lophotrichous
o Peritrichous
o Polar
 Pili
 Nonmotile long filamentous tubes
 Fimbriae
 Hairlike proteins used for adhesion
 Methylene blue

 Lactophenol cotton blue

BACTERIA MORPHOLOGY
 Bacteria size
 0.4 μm to 2 μm
 Microscopic shapes
 Cocci
 Bacilli
 Spiral
 Calcofluor white

 India ink

COMMON STAINS  Endospore stain

 Gram stain

 Acid-fast stains GRAM STAIN

 Heat fix (methanol can be used)
 Crystal violet (1 min)
  Primary stain  Capnophiles
 Iodine (1 min)  Microaerophiles
 Fixes iodine
 Alcohol-acetone (quick on and rinse)
 Decolorizer
 Safranin (30 sec)
 Counterstain BACTERIAL GROWTH
Note: Rinse with water between steps  Generation time
 Time required for one cell to become two
ACID-FAST STAIN  Growth curve
 Ziehl-Neelsen method (heat)  Lag
 Kinyoun (detergent)  Log
 Carbolfuchsin  Stationary
o Primary stain (red)  Death
 Acidified-alcohol  Determination of cell numbers
o Decolorizer  Direct counts
 Methylene blue  Plate counts
o Counterstain  Density
 Auramine-rhodamine
o Fluorochrome stain BACTERIAL BIOCHEMISTRY AND METABOLISM
Appears yellow or orange under fluorescent microscope  Metabolism
 Fermentation and respiration
ACRIDINE ORANGE  Biochemical pathways from glucose to pyruvic acid
 Stains nucleic acid orange under UV light  Anaerobic utilization of pyruvic acid (fermentation)
 Bacteria  Aerobic utilization of pyruvate (oxidation)
 Living or dead  Carbohydrate utilization and lactose fermentation
 Useful in samples with low bacterial numbers
FERMENTATION AND RESPIRATION
CALCOFLUOR WHITE  Fermentation
 Compound binds to chitin in fungi  Anaerobic process carried out by both obligate and
 Bright apple-green and blue-white fluorescence facultative anaerobes
 Requires UV light  Electron acceptor is an organic compound.
 Less efficient in energy generation than respiration
OTHER STAINS  Respiration
 Methylene blue  Efficient energy-generating process in which molecular
 Metachromatic granules in Corynebacterium diphtheriae oxygen is the final electron acceptor
 Lactophenol cotton blue  Certain anaerobes can carry out anaerobic respiration.
 Stains fungal cell walls blue o Inorganic forms of oxygen act as the final electron
 India ink acceptors.
 Negative stain to visualize capsules
THREE MAJOR PATHWAYS
 Endospore stain
 Malachite green stains endospores
1. EMP GLYCOLYTIC PATHWAY
MICROBIAL GROWTH AND NUTRITION  Major pathway in conversion of glucose to pyruvate
 Major nutritional needs  Generates reducing power in the form of NADH2
 Carbon source for cellular constituents  Generates energy in the form of ATP
 Nitrogen source for proteins  Anaerobic; does not require oxygen
 Adenosine triphosphate (ATP) energy source for cell  Used by many bacteria, including all members of
functions Enterobacteriaceae
 Trace elements
 Phosphorus (P), sulfur (S), sodium (Na), potassium (K), 2. PENTOSE PHOSPHATE (PHOSPHOGLUCONATE)
chlorine (Cl), calcium (Ca) PATHWAYS
 Microbial growth and nutrition  Alternative to EMP pathway for carbohydrate metabolism
 Nutritional requirements for growth  Conversion of glucose to ribulose-5-phosphate, which is
o Autotroph rearranged into other 3-,4-,5-,6-, and 7- carbon sugars
o Heterotroph  Provides pentose for nucleotide synthesis
o Human pathogens  Produces glyceraldehydes-3-phosphate, which can be
converted to pyruvate
TYPES OF GROWTH MEDIA  Generates NADPH, which provides reducing power for
 Minimal medium biosynthetic reactions
 Nutrient medium  May be used to generate ATP (yield is less than with EMP
 Enriched medium pathway)
 Selective medium  Used by heterolactic fermenting bacteria, such as
 Differential medium lactobacilli, and by Brucella abortus, which lacks some of
the enzymes required in the EMP pathway.
 Transport medium
3. ENTNER-DOUDOROFF PATHWAY
ENVIRONMENTAL FACTORS INFLUENCING GROWTH
 Converts glucose-6-phosphate (rather than glucose) to
 pH
 Human pathogens generally grow at neutral pH. pyruvate and glyceraldehyde phosphate, which can be
 Temperature funneled into other pathways
 Psychophiles  Generates one NADPH per molecule of glucose but uses
 Mesophiles one ATP
 Thermophiles  Aerobic process used by Pseudomonas, Alcaligenes,
 Gaseous composition of the atmosphere Enterococcus faecalis, and other bacteria lacking certain
 Aerobes glycolytic enzymes.
o Obligate
o Facultative ANAEROBIC UTILIZATION OF PYRUVIC ACID (FERMENTATION)
 Anaerobes  Alcoholic fermentation
o Obligate  Homolactic fermentation
o Aerotolerant  Heterolactic fermentation
  Propionic fermentation There are many organisms that inhabit our environment. Most of these
 Mixed acid fermentation microorganisms are nonpathogenic
 Butanediol fermentation
 Butyric acid fermentation  Prokaryotes, including bacteria, do not have membrane-
enclosed nuclei and organelles
 Eukaryotes differ from prokaryotes in that they have
CARBOHYDRATE UTILIZATION AND LACTOSE FERMENTATION membrane-enclosed nuclei and organelles
 Enterobacteriaceae family  Viruses cannot be seen under an ordinary light microscope,
 Lactose differentiates organisms although their cytopathic effects on cells lines are visible.
o β-galactoside permease They are obligate parasites, and antibiotics are ineffective for
o β-galactosidase treatment of viral infections. Viruses have DNA or RNA, but
 Breaks bonds of glucose to galactose never both, in contrast to prokaryotes and eukaryotes.
– Releases glucose  Bacteria utilize two biochemical pathways, fermentation and
 Other organisms respiration (oxidation), to catabolize carbohydrates to
produce energy
BACTERIAL GENETICS  The major way bacteria are classified in the diagnostic
 Anatomy of a DNA and RNA molecule microbiology laboratory is the Gram stain reaction. Whether
 Double helix an organism is gram-positive (blue or purple) or gram-
 Phosphate-pentose sugar-nitrogen containing base negative (pink or red) is an important first step in identifying
o Deoxyribose or ribose bacteria and in determining appropriate antimicrobial therapy
o Purine (A or G) or pyrimidine (T or U and C)  Bacterial spores are formed as a result of harsh
 A=T C=G in DNA environments. They are a means of survival, not
 A=U C=G in RNA reproduction.

TERMINOLOGY CHAPTER 2: HOST-PARASITE INTERACTION

 DNA
A. ROLE OF THE USUAL MICROBIAL FLORA
 Storage of genetic information (genetic potential)
Origins of Microbial Flora
 Replication produces DNA copies.
 Fetus
 RNA
o Sterile until birth
 Produced by transcription of DNA
 mRNA  Exposure to environment leads to colonization
 Translation  Microorganism relationships
 mRNA is read by ribosome. o Symbiosis: two organisms living together
o Codon: a group of three nucleotides  Commensalism
o tRNA matches codon with anticodon o Microorganism benefits while host is not harmed
 Mutualism
 Protein
o Microorganism and host benefit
 Functional unit: expression of genetic potential
 Parasitism
BACTERIAL GENETICS o Microorganism benefits while the host is harmed
 Genetic elements and alterations
 The bacterial genome CHARACTERISTICS OF INDIGENOUS MICROBIAL FLORA
 Extrachromosomal elements  Indigenous flora
o Plasmids  Microorganisms commonly found on or in healthy persons
 Mobile genetic elements  Resident flora
o Mutations o Microorganisms that colonize an area for months or
o Genetic recombination years
 Transient flora
 Mechanisms of gene transfer
o Microorganisms temporarily colonizing a host
 Transformation
 Carrier state
o Uptake and incorporation of naked DNA
o Acute: short term
 Transduction
o Chronic: long term
o Transfer of genes by a bacteriophage
 Conjugation
FACTORS THAT DETERMINE THE COMPOSITION OF THE USUAL
o Transfer of genetic material from a donor to a recipient
MICROBIAL FLORA
strain of bacteria
 Restriction enzymes
 Specific nutritional factors
o Enzymes that cut DNA at specific sequences
 Antibacterial substances
o Bile, lysozyme, fatty acids
 Environmental factors
o Moist or dry
 Most microorganisms live in moist areas.
 Skin folds
 Low pH
 Female genital tract, gastrointestinal (GI) tract of
breast-fed infants
 Gaseous atmosphere
 Low oxidation/reduction potential

USUAL MICROBIAL FLORA: SKIN

 Generally superficial organisms
 Skin surface and hair follicles
 Apocrine sweat glands
 Secrete substances metabolized by bacteria
o Release of odorous amines
 Normal flora
 Colonize skin surface
 Prevent pathogens from colonizing

POINTS TO REMEMBER
USUAL MICROBIAL FLORA: MOUTH
 Low oxidation reduction potential
 Anaerobes grow
 Buccal mucosa and tooth surface USUAL MICROBIAL FLORA: GENITOURINARY TRACT
 Production of acids by microorganism  Sterile sites
o Tooth decay  Kidneys
 Bladder
 Fallopian tubes
 Nonsterile sites
 Distal centimeter of urethra
 Vagina

USUAL MICROBIAL FLORA: RESPIRATORY TRACT

 Upper respiratory tract
 Mouth, nasopharynx, oropharynx, larynx
 Lower respiratory tract MICROBIAL FLORA AND DISEASE
 Trachea, bronchi, pulmonary parenchyma  Opportunistic infections
o Protected by ciliary epithelial cells and mucus  Cause disease when habitat is changed
 Normally considered sterile  May occur due to weakened immune system
 Trauma
 Introduce flora to sterile site
 Immunosuppression
 Immunosuppressive drugs
 Chemotherapy
 Radiation
 Immune defects

MICROBIAL FLORA AND PROTECTION FROM DISEASE

 Normal microbial flora
 Prime the immune system
• Anexic animals: germ free
 Sterile environments impair immune development.
 Microenvironment
 Microbial flora block colonization of pathogens.
• Antibiotics can reduce protection.

C. PATHOGENESIS OF INFECTION

MICROBIAL PATHOGENESIS
 Pathogenicity
 Ability of an organism to produce disease
 Opportunistic pathogens
 Usually do not cause infection
 Special circumstances
 True pathogens
 Organisms that cause disease in healthy
USUAL MICROBIAL FLORA: GI TRACT immunocompetent hosts
 Comprises esophagus, stomach, small intestine, and colon • Examples: Y. pestis and B. anthracis
 Stomach normally sterile  Iatrogenic infections
 Acidic pH  Occur from medical treatment or procedures
o Some exceptions
 Endospores, parasitic cysts, H. pylori
 Other pathogens enter in food particles
 Escape stomach and enter the intestine
o Colonize the small and large intestines
 Antibiotics
 Can significantly alter the usual flora

VIRULENCE  Secretory antibody
 Relative ability of a microorganism to cause disease  IgA proteases
 Degree of pathogenicity • Antigenic variation
 Numbers of organisms required to cause disease  Lactoferrin: binds free iron
 Virulence factors • Meningococci can use lactoferrin for iron.
 Traits that determine pathogenicity and virulence  Lysosomes
o Capsules • Prevent fusion
o Toxins • Escape phagosome
o Adhesive fimbriae
INVASION
RESISTING PHAGOCYTOSIS  Ability to penetrate and grow in tissues
 Phagocytes  Localized
 Major role in clearing bacterial infection • Few layers or in one body area
 Capsule  Disseminated
 Inhibit engulfment • Spread to distant areas and organs
 Prevent phagosome-lysosome fusion
EXOTOXINS
 Escape to cytoplasm
 Toxins
 Leukocidins
 Poisonous substances secreted by organisms
 Damage or kill leukocytes
 Exotoxins
 Inhibit chemotaxis
 Binding subunit
• Allows toxin to enter cell
 Toxic subunit
• Disrupts or destroys cellular function

ABILITY TO RESIST PHAGOCYTOSIS

 Prevention of phagocytosis
 Capsule
o Masks cell surface structures, inhibits complement
 Protein A
o Impairs opsonization of host antibodies
o Binds Fc portion of immunoglobulin (IgG), preventing
opsonization and phagocytosis
ENDOTOXINS
 Killing of phagocytes
 Lipopolysaccharide (LPS)
 Panton-Valentine leukocidin
 Cell wall component in gram-negative bacteria
o Causes discharge into cytoplasm, killing cell
 O-specific polysaccharide-core-lipid A
 Toxin activity
BACTERIAL STRUCTURES THAT PROMOTE ADHESION
 Lipid A
 Adhesive structures
 Effects
 Fimbriae (pili)
 Hypotension
 Surface polysaccharides
 Fever
 Enable attachment to host surface structures
 Initiates coagulation
 Increase ability to colonize

INTRACELLUALR SURVIVAL
 Circumvent host’s protective mechanisms
 PHAGOCYTIC CELLS
 Engulfing cells
 Neutrophils (PMNs)
 Macrophages
 Chemotaxis
 Chemically caused movement to a location
 Necessary to mobilize phagocytes to infection
 Diapedesis
HOST RESISTANCE FACTORS  Movement from blood vessels to tissues
 Physical barriers STEPS OF PHAGOCYTOSIS
 Mechanical barrier  Attachment
• Intact skin is effective against most pathogens.  Attachment of organism to phagocyte
 Cleansing mechanisms • Facilitated by opsonins
 Desquamation of skin  Ingestion
 Movement of liquids  Invaginates and engulfs particle
• Examples: Tears, urine, mucus secretion  Enclosed in phagosome
 Cilia • Fuses to lysosome
• Clearing of debris by locomotion  Killing
 Low pH  Increase in metabolic activity
 Stomach, vagina  Causes production of acids and hydrogen peroxide
 Release of enzymes
• Bacteriocidal
 Intracellular pathogens
 Circumvent this process

INFLAMMATION
 Chemical mediators increase blood flow causing
 Erythema
 Redness
 Edema
• Swelling
 Heat
 Pain
• Due to swelling
 Increases number of white blood cells (WBCs) in tissue

COMPONENTS OF INFLAMMATION

HOST RESISTANCE FACTORS

 Antimicrobial substances
 Fatty acids on skin
 Hydrochloric acid (HCl) in the stomach
 Lysozymes
 Immune proteins
• IgA
• Low-molecular-weight cationic proteins
 b-lysins
• Complement
 These synergize to
increase effectiveness of killing.
• Interferon
 Indigenous microbial flora
 Prevent pathogen colonization
• Bacteriocidins
 Inhibit closely related
bacteria
IMMUNE RESPONSES
 Innate immunity
 Natural or nonspecific immunity
o Physical barriers
o Chemical barriers
o Phagocytosis
 Adaptive or specific immunity
 Antibodies
 Lymphocytes
o B cells
o T cells
 T helper
 Cytotoxic
 PRIMARY AND SECONDARY ANTIBODY RESPONSES
 Primary
 Rapid appearance of IgM
 Peak in 2 to 3 weeks followed by decline
 Gradual change over to IgG or IgA antibodies
 Secondary (anamnestic immune response)
 Rapid increase in IgG antibodies
• Higher levels of IgG with prolonged elevation
• Higher specificity
 Somatic hypermutation

CELL-MEDIATED IMMUNITY (CMI)

 Protection from intracellular pathogens
 T helper cells
 Lymphokines (cytokines)
 Signal activation of macrophages and other
phagocytes
 Cytotoxic T cells
 Kill infected cells

MECHANISMS BY WHICH MICROBES MAY OVERCOME THE HOST

DEFENSES
 Induce immune tolerance
 Not recognized as foreign
 Immune suppression
 Actively destroy, inactivate, or limit the effect of the
immune response
 Antigenic variation
 Intracellular “hiding”

ROUTES OF TRANSMISSION AND EXIT

 Airborne
 Transmission by food and water
 Close contact
INNATE IMMUNE DEFENSES OF BODY  Direct contact
 Cuts and bites (nonarthropod)
 Wounds
 Arthropods
 Bites of insects
 Zoonoses
 Contact with animals

HUMORAL IMMUNE RESPONSE

 B cells
 Aided by helper T cells
 Immunoglobulins (antibodies)
 IgG: monomer
• 70%-75% of serum immunoglobulin
• Opsonizing antibody, crosses
placenta
 Immunoglobulin M (IgM): pentamer
• 10% to 15% of serum immunoglobulin
• Complement fixation
• First antibody produced
 Immunoglobulins (antibodies)
 Immunoglobulin A (IgA): dimer
• 15% to 20% of serum immunoglobulin
• Secreted at mucous membranes
 Immunoglobulin E (IgE): receptor bound
• Very low serum concentration
• Role in clearance of parasites and
allergies
 Immunoglobulin D (IgD): surface bound
• Very low serum concentration
• Role in signaling of B-cell receptors ROUTES OF ENTRY AND EXIT
  Make it very resistance to treatments
 Mycobacterial cell walls
o Lipid rich
 Enveloped viruses
o Lipid envelope can make them more susceptible
 Biofilms
o Protection by microbes living in communities
 Prions
o Most resistant infectious agent
o Naked pieces of protein without nucleic acids
o Transmitted through contaminated products
 Medical products
 Therapeutic devices
 Body fluids
 Food products
o Special methods for sterilization
 Routine methods are not sufficient

ZOONESES OTHER FACTORS IN DISINFECTION

 Concentration of disinfecting agents
 Too concentrated or too dilute is not effective
o Following manufacturer’s directions is important
 Presence of organic material
 Inactive chemical agents
 Prevents interaction between chemicals and
microorganims
 Nature of surface being disinfected
 Will the chemical agent damage surface?
 Does time of exposure need to be altered?
 Contact time
 Length of exposure of agent to object
 Nature of microorganism can affect time required
 Temperature
 Can slow chemical reactions, increasing killing time
 Generally the higher the temperature, the better the killing
 pH
 high or low pH can inactivate agent
 Biofilms
 Communities of bacteria can have resistant protective
layers
 Compatibility of disinfectants
 Synergy
o Two work better together
 Antagony
o Two inactive each other
CHAPTER 4: CONTROL OF MICROORGANIMS o Bleach and quatemary ammonium compounds
A. DISINFECTIION AND STERILIZATION E.H. SPAULDING CATEGORIES OF MEDICAL MATERIALS
 Sterilization versus disinfection
 Sterilization is the destruction of all forms of life  Critical materials
o All or nothing process  Those that enter sterile or the vascular system
 Disinfection is elimination of a defined scope of o Must be sterile
microorganism o No spores
o Defined by Joseph Lister
 Semicritical materials
 Disinfectant  Contact with mucous membranes
 Chemical agents applied to inanimate objects o High level disinfection
 Antiseptic o tuberculocidal
 A substance applied to the skin to eliminate or reduce the
 Noncritical materials
number of bacteria present
o Contact with intact skin
FACTORS THAT INFLUENCE THE DEGREE OF KILLING o Intermediate to low level disinfection
 Type of organisms
 Number of organism METHODS OF DISINFECTION AND STERILIZATION
 Concentration of disinfecting agent
 Presence of organic material  Physical methods
 Nature (composition) of surface to be disinfected  Heat
 Contact time o Moist heat
 Temperature  1 atmosphere (atm) at 121 deg. C for 15 min
 pH o Dry heat
 Biofilms o Boiling
 Compatibility of disinfections and sterilants o Pasteurization
 Filtration
o Filters with various pore sizes
o Bacteria, mold, yeast generally larger than 0.45
TYPES OF ORGANISMS um
 Organisms vary in their ability to withstand chemical and
physical treatment  Radiation
 Endospores o Ionizing
o Coats rich in proteins, lipids, and carbohydrates with cores  Short wavelength: high energy (high
containing dipicolinic acid and calcium penetrance)
 o Gamma rays, electron beams: good for  Hypochlorite
disposables  Sodium hypochlorite (bleach)
o Nonionizing o Pros
 Long wavelength: low energy (low  Inexpensive and broad-spectrum killing power
penetrance) o Cons
o Ultraviolet (UV): good for surfaces  Requires long exposure time for sterility
 Corrosive and pH sensitive
CHEMICAL METHODS  Inactivation by organic matter
Chemosterilizers  Rapidly degrades (30 days max)
 Killing effect by:  Generally used for surface decontamination
o Reaction with components of the cytoplasmic membrane o 0.5-1% solution for surfaces with greater than 3 min
 Leakage and death exposure
o Denaturation of cellular proteins  Longer if organic material present
 Disrupt metabolism o 1.10 solution of 5.25% sodium hypochlorite for blood
o Reaction with thiol groups of enzymes spills
 inactivation
o Damage RNA and DNA DETERGENTS: QUATERNARY AMMONIUM COMPOUNDS (QUACS)
 Inhibit replication
 Cationic, surface-active agents
DISINFECTANTS AND ANTISEPTICS: A HISTORICAL  Surfactant: reduce surface tension
BACKGROUND o Disrupt cell membranes, causing leakage
 Germ Theory  Reduced effectiveness in hard water and soap
 Idea that microorganisms (germs) cause disease o Inactivated by excess organic material
o Not “evil spirits”  Some gram-negative organisms resistant
 Need to practice asepsis to prevent contamination  Pseudomonas spp.
 Semmelweis (1816-1865)  Generally used on noncritical surfaces
 Hand washing can prevent disease  Bench tops and floors
 Lister (1827-1912)
 Using chemicals to sterilize the air and in wound PHENOLICS
dressings
 Phenol (carbolic acid)
ALCOHOLS  Substituted with halogens, alkyl, alkyl, phenyl, or benzyl
groups
 Ethyl alcohol  Broad-spectrum activity but not sporocidal
 Isopropyl alcohol o Additive to detergents to disinfect
 Broad spectrum but not sporicidal  Stable and biodegradable
o Bacteriocidal, pseudomonacidal, tuberculocidal,  Active in presence of organic matter
virucidal  Disrupt cell walls and precipitate proteins
o To remove spores filter through.22 um filters  Used in disinfection of hospital, institutional, and
 Inactivated by organic material household environments
 Work by denaturing proteins  Chlorhexidine gluconate (0.5-4%)
o Must be used in 60% - 90% concentration  Disrupts cell membrane
o Must be allowed to evaporate from the surface  Precipitates cell contents
 Broad spectrum and effects can last for 6 hours
ALDEHYDES  Not generally effective against endospores and
nonenveloped viruses
 Formaldehyde  Can cause severe skin reactions in infants under 2 months
o 37% aqueous or as a gas of age
 Sensitive to pH
 Carcinogen and irritant
o Optimal range is pH 5.5-7.0
 Nontuberculocidal
 Not recommended on a routine basis  Hexachlorophene (3%)
 Glutaraldehyde  Primary effective against gram-positive bacteria
o Alkylation of RNA and DNA via alkylation of sulfhydryl  Interrupts bacterial electron transport
 Quick effectiveness (15-30 sec)
groups
o Longer for gram-negative organisms when effective
o Effective against bacteria, fungi, tuberculins, and
 Prescription only due to toxic effects
viruses
 Chloroxylenol (0.5-4%)
o Does not penetrate organic material well
 Used primarily against gram-positive bacteria
o noncorrosive
 Primarily in skin applications
 Unaffected by organics
IODOPHORS  Neutralized by nonionic surfactants and polyethylene
glycol (PEG)
 Tinctures
 Alcohol and iodine solution used as antiseptic TRICLOSAN
 Iodophor
 Iodine and neutral polymer carrier that increases slow  Disrupts cell wall
release of iodine  Primarily used as a hand wash and surgical scrub
o Require free iodine, therefore proper dilution is vital
 Not affected by organics
 Povidone-iodine (5%-10%)
 Affected by surfactants, emollients, and pH
o Exposure time greater than 30 sec
 Intermediate reaction time with excellent persistence
o Disinfectant only, not sporicidal
o Skin irritant, therefore, must be removed from skin HEAVY METALS
after use
 Rarely used due to toxicity and population
 Bacteriostatic
 Prevent the growth of bacteria
 Silver nitrate
 Used prophylactically for gonococcal conjunctivitis in
newborns
CHLORINE AND CHLORINE COMPOUNDS
GASES

 Ethylene oxide
 Best for plastic and heat sensitive materials
o Explosive hazard
o 450-700 mg liter at 55-60 deg C for 2 hours
o Humidity best at 30%
 Kills through alkylation of nucleic acids
 Vaporized hydrogen peroxide (H2O2)
 Periacetic acid
 Both are bactericidal, fungicidal, tuberculocidal, virucidal
and sporicidal
 When used together contact time required is shortened
ENVIRONMENTAL PROTECTION AGENCY (EPA) REGULATIONS ON
CHEMICAL SURFACE DISINFECTANTS
 Regulate use, sale, and distribution of antimicrobial pesticide
products
FRONT PANEL
 Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA)
Require appropriate labels based on lab test data
o
INFORMATION ON DISINFECTANT LABELS
HYGIENIC HANDWASHING/WATERLESS HAND RUBS
BACK PANEL
 Goal is to eliminate transient flora
 Also to protect the skin with resident flora
 Hand washing
 Remove physical dirt
 Before and after patient contact or objects
 No visible soiling
 Use waterless liquid or gel
o Fast-acting antiseptic
 Small volume, quick acting
SURGICAL HAND SCRUB/WATERLESS SURGICAL HAND RUBS
 Goal is to eliminate transient flora and most resident flora
 Prevent surgical infections
 Broad spectrum, fast acting, persistent
 FDA guidelines
 Reduce bacteria by 1 log10 on each hand after 1 minute
on the first day and blow baseline after 6 hours
 Reduce bacteria by 2 log10 on each hand after 1 minute
on the end of the second day
 Reduce bacteria by 3 log10 on each hand after 1 minute
on the end of the third day
B. MICROBIOLOGY SAFETY
SAFETY PROGRAM FOR THE CLINICAL LABORATORY
 Address biologic hazards
 Describe safe handling, storage, and disposal of chemicals
and radioactive substances
 Outline laboratory or hospital policies in the event of
emergencies
 Perform initial safety training for all employees and update
annually
 Teach correct techniques for lifting and moving objects
SAFETY FROM INFECTIOUS AGENTS
 Occupational Safety and Health Administration (OSHA)
 Safety training for potentially exposed employees
 Goal is to protect workers
o 1991 Bloodborne Pathogen Final Standard
 Revised in 2001 in conformance with the
Needlestick Safety and Prevention Act
 Exposure control plan
WORK PRACTICE CONTROLS
 Required by OSHA
o Determine tasks that may result in occupational
hazards
o A plan to investigate exposure and prevent
reoccurrences
o Methods of compliance with universal precautions
o Engineering and work practice controls
o Personal Protective Equipment (PPE)
o Guidelines for workplace cleanliness PERSONAL PROTECTIVE EQUIPMENT
o Guidelines for handling and disposal or regulated
waste
o A training program for all employees
 Universal/standard precautions
 Developed in 1985, redone in 1996
 All blood and body fluids are treated as infectious
o Includes nonintact skin and mucous membranes
o The lone exception is sweat
 Precautions address
o Handwashing
o Gloves, mask, eye protection, face shield
o Lab coats
o Appropriate sharps disposal
o Environmental controls
 Provide procedures for care, cleaning, and
disinfection of surfaces
 Product name, brand, or trademark
 Ingredient statement (concentration or strength)
 “keep Out of Reach of Children”
 EPA registration number and establishment number
 Precautionary statements
 Hazards to humans and domestic animals
 First aid
 Environmental hazard
 Physical or chemical hazard
 Directions for use
 How to use the product
 Application sites and rates
 Worker protection issues
 Aftercare
 Equipment
 Treated surfaces
 Cleaning supplies
 Storage and disposal
 Transmission-based precautions
 Added precautions that are used when the patient is
known to be or suspected of being infected or colonized
with an infectious agent that requires extra measures to
prevent spread or transmission of the agent
 Categories
o Contact precautions
 Examples: MRSA, Clostridium difficile
o Droplet precautions
 Examples: Neisseria meningitides,
Bordetella pertussis
o Airborne precautions
 Examples: Mycobacterium tuberculosis
 Engineering controls
 Controls designed to isolate or remove hazards from
the workplace
o Some examples are eye wash stations, safety
showers, eye shields
 Laboratories
o Negative air pressure
o Limited access
o Insect prevention
 No recapping or breaking of contaminated needles
 Disposal of needles in puncture-resistant containers
 Procedures minimize splashing and the generation of air
droplets
 Specimens transported in containers with secure lids
 Prevent leakage of infectious materials
  Gloves, lab coats, masks, respirators, face shields, safety
glasses
 Must be accessible and worn when potential for
exposure exists
 Must be removed before leaving the work area
 PPE and BSC class I
 BSL-3
 Potential aerosol transmission
 Agents may have serious lethal consequences
 PPE, BSC Class II or III, negative-pressure rooms
 BSL-4
 Dangerous and exotic pathogens
 PPE, BSC Class III, negative-pressure rooms
 Decontamination of room and personnel after use

BIOLOGIC RISK ASSESSMENT

HAZARDOUS WASTE
 Hazards  Clinical laboratory is responsible for the proper handling and
 Two major sources disposal of all of the waste it generates
o Processing of patient specimens  The Clinical Laboratory Standards Institute (CLSI), Clinical
o Handling active cultures Laboratory Waste Management Approved Guideline, 2 nd
 Modes of infection edition, addresses
 Direct contact  Chemical, infectious, radioactive, sharps,
 Inhalation multihazardous, and nonhazardous waste
 Ingestion  The reduction of waste generation, volume, and toxicity
 Needle stick of unavoidable wastes
DISPOSAL OF INFECETIOUS WASTE
RECOMMENDATIONS FOR LABORATORY SAFETY  Must follow local, state, and national regulations on the
IMPROVEMENTS disposal of infectious waste
 Usually autoclaving or incineration
 Know that the bacteria must be handled in the laboratory, do  Place in appropriate containers labeled with biohazard
not remove items from the lab symbol
 Use dedicated writing utensils and supplies that stay at work
stations HAZARDOUS WASTE REDUCTION
 Be aware of what organism they are working with and what  Substitute less hazardous chemicals when possible
the signs and symptoms are if they get infected with one of  Develop procedures that use less of a hazardous chemical
these organisms  Recycle chemicals when possible
 All should be trained and proficient in biosafety practices and  Segregates infectious wastes from uncontaminated trash
techniques  Substitute micromethodology to reduce volume of chemicals
 Always wear lab coats over personal clothing reagents as well as infectious waste

CENTERS FOR DISEASE CONTROL AND PREVENTION’S (CDC) EMPLOYEE RIGHT TO KNOW
Guidelines for Safe Work Practices  Provides for a chemical hygiene plan
 Employees should have a through working knowledge of
 Identify the hazards associated with an infectious agent or chemicals used
material  All hazardous chemicals must be labeled with National Fire
 Identify the activities that might cause exposures to the agent Protection Association (NFPA) hazard rating diamond
or material
 Consider the competencies and experience of laboratory HAZARDOUS CHEMICALS COMMONLY USED IN THE
personnel LABORATORY
 Evaluate and prioritize risks (evaluate the likelihood that an Flammables
exposure would cause a laboratory-acquired infection (LAI)
 Methanol
and the severity of consequences if such an infection occurs)  Acetone
 Develop, implement, and evaluate controls to minimize the  Ethanol
risk for exposure Potential or Proven Carcinogens
 Formaldehyde
SAFETY FROM INFECTIOUS AGENTS  Aniline (crystal violet) stain
 Auramine-rhodamine (Truant) stain
 Processing of patient specimens Irritants and Corrosives
 Labeling samples with known infectious agents  Hydrogen peroxide
 Window period  Acids: HCl, H2SO4, Acetic Acid
o Patients who have not tested positive or have yet  NaOH
to be tested are hazardous
o Universal precautions are vital MATERIAL SAFETY DATA SHEETS (MSDS)
 Generally, specimens are processed in a biosafety  Sheets provided by manufacturer
cabinet  Name, address, telephone of manufacturer
 Working with actively growing cultures  Nature of chemical, name and hazardous ingredients
 Frequently wash hands to avoid exposure  General characteristics of chemical, signs and symptoms
 Wear appropriate PPE of exposure, primary route of entry
 Bandage wounds  Precautions to take in using chemical and control
 Prevent exposure when determining microbial odor measures
 Appropriate engineering controls  Emergency and first aid procedures
 Spill cleanup procedures
BIOLOGICAL SAFETY CABINET (BSC)  Disposal recommendations
 Protects from aerosol transmission of organism
 Three types HAZARDOUS CHEMICALS INVENTORY
o Class I  Current inventory of hazardous chemicals
o Class II  Must be updated annually
o Class III  Corresponding MSDS all present and updated
BSLs  Sources
 BSL-1  29 CFR Part 1910, Subpart Z, Toxic and Hazardous
 Well classified and not know to cause disease substances, OSHA
 Standard PPE  National Toxicology Program Annual Report on
 BSL-2 Carcinogens
 Moderate potential hazard  International Agency for Cancer Research Monographs
  Manufacturers’ safety data sheets (SDSs)
LAB SAFETY FOR HAZARDOUS CHEMICALS
 Fume Hoods
 Prevent inhalation of fumes
 Evaluate annually for face velocity and operation
 Acid/base spill kits
 Flammable spill kits
 PPE stored in a designated area for spills
 Fire extinguishers with appropriate labels
 Employees trained in symbol recognition and use
 Aspirated material should be placed into a sterile tube
or transport vial
PATIENT COLLECTED SPECIMENS
 Educate patients with through instructions
 Should be instructed by appropriate medical personnel
in verbal and written forms
 Attach printed instructions in multiple languages with
pictures
 It should not be assumed a patient knows how to collect a
specimen

FIRE SAFETY  Urine

 R- Rescue: remove anyone from danger  Clean-catch midstream urine specimen
 A- Alarm: pull fire alarm, call to report fire o First morning urine preferred
 C- Contain: close doors to contain fire o Cleanse external genitalia
 E- Extinguish: use proper fire extinguisher to extinguish small o Reduce indigenous flora
fires o Void first portion and collect middle portion
 Fire evacuation plan must be posted o Rinses urethra
 Appropriate drills should be conducted  Similar procedure for catheters
 Sputum
THERMAL INJURIES  First morning specimen is preferred
 Thermal gloves up to shoulder for autoclaves  Difficult to collect
 Warning signs for hot liquids/instruments o Contamination from oropharyngeal flora
 Freezer burns o Sputum versus saliva and nasal secretions
 Liquid nitrogen or ultra-low-temperature freezers  Expectorated sputum
o Wear thermal gloves for handling materials o Rinse mouth with water
o Expectorate with aid of a deep cough into a sterile
STORAGE OF COMPRESSED GASES container
 Flammable and nonflammable gases  Induces sputum
 Secured and stored in vented areas o Aerosol of solution that stimulates coughing
 Locate away from open flames and heat sources  Stool
 Metal cal to prevent breakage of pressure valve when  Specimen of choice for gastrointestinal pathogens
not in use  Bacteria: three specimens
 Proper transportation equipment used when moves o One a day for 3 days
between locations  Parasites: three specimens
o Over 10 days
MISCELLANEOUS SAFETY CONSIDERATIONS  Antigen screens usually only require one specimen
 Back Safety  Never taken from toilet or contaminated with urine
 Use legs to lift, not back  Commercial collection
 Ask for help or use a cart when load is too heavy o 1:3 ratio of stool to preservative
 Use good posture  Barium contamination invalidates sample
 Stay physically fit o White chalky substance
 First aid training
 All personnel should be trained in cardiopulmonary SPECIMEN LABELING
resuscitation (CPR) and other life saving first aid  Proper identification attached to container
 Immunizations  Name
 Hepatitis B vaccination  ID number
 Room number
CHAPTER 6: SPECIMEN COLLECTION AND PROCESSING  Physician
 Culture site
BASIC PRINCIPLES OF SPECIMEN COLLECTION  Date of collection
 Time of collection
 Fundamentals
 Collect specimen in acute phase of infection TEST REQUISITIONS
o Before antibiotics are administered
 Information
 Select correct anatomic site
 Patient’s name
 Use proper technique
 Patient’s date of birth and gender
o Minimal contamination
 Patient’s room number or location
 Collect appropriate quantity
 Physician’s name and address
 Pack to maintain viability and prevent leakage
 Specific anatomic site
 Label specimen accurately
 Date and hour of specimen collection
 Transport or store specimen promptly
 Diagnosis or relevant history
 Notify lab in advance if unusual pathogens or
 Antimicrobial agents
bioterrorism agents are suspected
 Transcriptionist of orders
 Collect procedures
 Thorough requisitions can assist microbiologist to suspect
 Sterile containers
pathogens that may alter media used for maximum recovery
 Swabs are not recommended (quantity nonsufficient
 Electronic process should be designed with input from the
(QNS) and drying)
microbiologist
o Cotton (can be toxic to some bacteria), Dacron, or
 Laboratory should communicate to the individual ordering the
calcium alginate
test any requested test that is not recommended
o Can be used for
o Upper respiratory tract (URT), external ear,
SAFETY
eye, and genital tract  Transport specimens in leak-proof secondary containers
 Transport media can be used to prevent drying
 Specimen and papers kept separate
 Lesions, wounds, abscesses
 Specimens should contain needles or sharps
 Need exact anatomic sites
 Must handle specimens with proper personal protective
 Collect from needle aspiration from advancing line of
infection equipment (PPE) and engineering controls
o Clean area to remove contaminants
TRANSPORT OF SPECIMENS  QNS
 Transport within 30 minutes of collection  Transport time longer than 2 hours with no
 Preferably within 2 hours preservatives
 Specimens  Specimen received in fixative/wrong preservatives
 Use immediately if possible  Anaerobic culture for specimens in which anaerobes
 Some pathogens can be stored at room temperature if are indigenous
temperature sensitive  Specimen is dried up
 Refrigerate  More than one specimen from the same source on
 Freeze same day
o Exception: blood culture
 One swab submitted with multiple requests for various
organisms
 Expectorated sputum
o Less than 25 white blood cells (WBCs), greater
SPECIMEN STORAGE than 10 epithelial cells per low power field, and
 Specimens not transported or processed immediately mixed bacterial flora
 Call for recollection
Specimen Storage Guidelines  Never discard specimen until contact has been made
Refrigerate Room Temperature  Must document the reason for rejection
Catheter tips (IV) Abscess, lesion, wound  If physician requires culture
CSF for viruses Body fluids  Must document possibility of compromised results
Ear: outer CSF for bacteria  May occur in situations where recollection is not
Feces (unpreserved) Ear: inner possible or very invasive
Feces for Clostridium difficile toxin Feces (preserved)
(up to 3 d; >3 d storage at -70 deg. MACROSCOPIC OBSERVATION
C)  Swab or aspirate
Sputum Genital  Stool consistency (formed or liquid)
Urine (unpreserved) Nasal, N/P, throat  Blood or mucus present
Tissue  Volume of specimen
Urine (preserved)  Fluid: clear or cloudy
 Help determine adequacy and any special tests needed
PRESERVATIVES
 Specimen types using preservatives MICROSCOPIC OBSERVATION
 Urine  Determines quality of specimen
o Boric acid  Can give microbiologists and physician an indication of the
o Maintain colony counts infectious process involved
 Stool  May provide presumptive identification
o Refrigerate for up to 2 hours  Rapid information
o Longer than 2 hours use Cary-Blair transport  Can be useful in initial treatment
media  Helpful for media selection
o Clostridium difficile toxin assays
o Refrigerated for 48 hours or freeze at -70 deg. PRIMARY INOCULATION
C if longer  Nonselective media
o Ova and parasite exams  Support growth of most nonfastidious organisms
o Special fixatives used for preserving specimen  Sheep blood agar
 Selective media
ANTICOAGULANTS  Support growth of one type of organism but not another
 Sodium polyanethol sulfonate (SPS) o Columbia nalidixic acid (CAN) agar
 Concentration must not exceed 0.025% (wt/vol)  Differential media
o Most bacteria  Allows grouping of microbes based on demonstrated
 Heparin characteristics of the media
 Viral cultures and mycobacterium from blood  Sheep blood agar, MacConkey agar
 Coagulants not used  Enriched media
 Citrate  Contain growth factors added to nonselective media to
 Ethylenediaminetetraacetic acid (EDTA) allow fastidious organisms to grow
o Chocolate agar
TRANSPORT MEDIA  Enrichment broth
 A liquid medium designed to encourage small numbers
 Holding or transport media of organisms to grow
 Contain substances that do not promote growth of the  Suppress other flora present
microorganisms but ensure preservation o Lim broth (enhances group B strep)
o Stuart’s transport medium
 Broth media
o Amie’s transport medium
 Supplement to agar to detect small numbers of
o Some transport media contain charcoal to absorb aerobes, anaerobes, and microaerophiles
the fatty acids given off by cotton swabs o Thioglycolate broth
 Direct inoculation
 Blood to blood culture bottles CULTURE MEDIA SELECTION (picture)
 Neisseria gonorrhoeae specimens to JEMBEC Sytem
o Additional specimen should also be provided for SPECIMEN PREPARATION
direct examination and Gram stain  Direct inoculation
 Pus, urine, sputum, sterile body fluids
PACKAGING INFECTIOUS SUBSTANCES (picture)  Concentration to improve yield
 Large volumes
LEVELS OF SPECIMEN PRIORITY (picture) o Peritoneal, pleural, continuous ambulatory
peritoneal dialysis (CAPD)
UNACCEPTABLE SPECIMENS/SPECIMEN REJECTION  Fluid greater than 1ml
o Centrifuge 20 min at 3000 x g
 Suboptimal specimens for rejection
o Can filter if fluid is thin enough
 Requisition information does not match specimen label
 Inappropriate transport container or leakages  Swabs
 Two swabs
 o One for direct smear, one for culture
 Tissue homogenization
 Grind up tissue for culture
ISOLATION TECHNIQUES
 Isolation streak
 Four quadrants
o Allows grading of relative concentration of
organisms
 Quantitative isolation
 Loops with specific volumes are struck down the center
o Center spread over the area of the plate
STREAK TECHNIQUES (picture)
INCUBATION CONDITION
 Most cultures grow between 35 deg. C and 37 deg C
 Oxygen conditions depend on organisms
 Aerobic
 Anaerobic
 Capnophilic
 Microaerophilic
 Time required
 Most held for 48 to 72 hours
 Some held 5 to 7 days
Picture of tubes
ISOLATION OF UNUSUAL OR FASTIDIOUS BACTERIA (picture)
CULTURE WORKUP
 What is the specimen source?
 Does this source have normal biodate?
 If normal biodata, what do they look like?
 What are the most likely pathogens?
 What is the colony morphology for these pathogens?
 Which media is demonstrating growth, and what is the
purpose of the media?
 Does it require a full workup to genus and species?
NONROUTINE SPECIMENS
 Is the specimen likely to contain low or high numbers of
microorganisms?
 If low, concentration of specimen is advantageous from
a large amount of specimen
 If extremely low, how important is it to enhance them,
such as the presence of one organism in a specimen
that should be sterile?
o Use broth
 Are organisms likely to be fastidious or nonfastidious?
 Important factors
o Temperature, atmosphere, and length of
incubation
 Is any normal biota associated with the specimen?
 Does the specimen contain any preservatives or growth
inhibitors that must be counteracted?
 What is a reasonable amount to culture
 Are all areas of the specimen homogeneous, or will the
portion chosen for culture affect the results?
 Is the objective to select a single agent from a mixed culture?
 Is there a need to culture both external and internal surfaces?
COMMUNICATION OF LABORATORY FINDINGS
 Accurate and timely information to health care professionals
 Preliminary results as they are available
o Depends on the situation
 Clearly interpret results to avoid confusion
 Avoid technical jargon and abbreviations
 Critical values
 Must be reported immediately
o May indicate a life-threatening situation
EXAMPLE OF CRITICAL VALUES IN MICROBIOLOGY (picture)
CHAPTER 7: MICROSCOPIC EXAMINATION OF MATERIALS FROM
INFECTED SITES
 Christian Gram
 Scottish surgeon
 1880: cluster-forming cocci in purulence
 1884: developed Gram stain
 Rapid response on direct examination
 Confirm submitted material is representative
 Identify cellular components and debris of inflammation
to estimate the probability of infection
 Identify specific infectious agents
 Guide physicians to early treatment with antibiotics
 Develop epidemiologic data
 88% of the time physician has correct presumptive
identification (ID)
 Other instances
 Lab provides additional assistance on treatment
 Change treatment if presumptive ID incorrect
 Support or refute initial ID
PREPARATION OF SAMPLES
 Smears from swabs
 Do not use swabs used to inoculate media
o Must do smear from separate swab
 Always collect two swabs
 Prepare by rolling swab back and forth over slide
o Do NOT rub over surface
o Preserves morphology and relationship of
organisms
o Gets organisms off both sides of the swabs
SMEAR PREPARATION FROM SWABS (picture)
PREPARING INFECTED MATERIALS FOR VISUAL EXAMINATION
(picture)
SMEARS FROM THICK LIQUIDS OR SEMISOLIDS
 Immerse swab in specimen for several seconds
 Prepare a thin spread on glass slide
 Too thick is bad for staining
 Smears from thick, granular, or mucoid materials
 Get thick and thin areas, crush granules
 Use two-slide technique
SMEARS FROM THICK LIQUIDS OR SEMISOLIDS (picture)
SMEARS FROM THICK, GRANULAR, OR MUCOID MATERIALS
 Two –slide technique
 Place portion of sample on labeled slide
 Press second slide label side down on top
o Flattens or crushes components
 Rotate glass surfaces against each other
 Pull slides smoothly away from each other
TWO-SLIDE TECHNIQUE (picture)
SMEARS FROM THIN FLUIDS
 Urine, cerebrospinal fluid (CSF), other fluids
 Drops on slide and mark reverse of slide
 Do not spread unless too turbid
 Marking helps find sample
 Stain slide
 Cytocentrifuge preparations are preferred method
SMEAR FROM THIN FLUIDS (picture)
CYTOCENTRIFUGE PREPARATIONS
 Deposits cellular elements and microorganisms as a
monolayer
 Clears protein using a filter pad to clear background
 Enhances morphology and concentrates sample, making
viewing faster
CYTOCENTRIFUGE TECHNIQUE
 Bowl is clipped onto slide; bowl contains a lid to “close” the
sample (preferred)
 Place a small aliquot of fluid 0.1 to 0.2 ml in the
cytocentrifuge holder
 Spin for 10 minutes
 Remove slide and smear if too thick
 Fix sediment in 70% alcohol for 5 minutes
CENTRIFUGE TECHNIQUE (picture)
  Computer systems
STAINS  Microorganism description
 Simple stains  Can describe microorganisms in a way that morphology
 Color forms and shapes can be used with specimen type and prevalence to
 Wright-Giemsa imply the likelihood of a specific agent
 Differential stains
 Coloring specific components TERMINOLOGIES FOR DIRECT EXAMINATION (picture)
 Gram; acid fast, calcofluor white
 Probe mediated stains
 Directed at specific organism identification EXAMINATION OF PREPARED MATERIAL
 Antibody or DNA probe stain  In clinical infections, 105 colony-forming units (CFUs) are
common
STAIN FOR INFECTED MATERIALS (picture)  Two types of infections
 Monomicrobial
GRAM STAIN PRINCIPLES  Polymicrobial
 Gram positive o Require more investigation
 Retain crystal violet (CV) because it binds teichoic o Anatomic location along with clinical symptoms
acid o More common in surgical wounds, aspiration
 Iodine replaces chlorine in stain molecule with iodine to pneumonias, perirectal abscesses, turbo-ovarian
link it to cell wall abscesses
 Gram negative
 Thin walls with lipopolysaccharide (LPS) CHARACTERIZATION OF BACKGROUND MATERIAL
 Alcohol-acetone decolorizer  Examine material
o Damages the thin lipid walls and washes out CV  Note characteristics (thick, blood, etc.)
 All non-stained elements get safranin dye (pink)  Scan under low power (x2.5 to x10) objective magnification
obj)
GRAM STAIN PRECAUTIONS  Homogenous or heterogeneous
 Gram stain may vary due to  Are pathogens evenly distributed or in only one field
 CV being rinsed too vigorously
 Failure to use iodine CHECKLIST OF MATERIAL EXAMINATION
 Decolorized too vigorous or prolonged  Is there evidence of contamination by normal (resident)
 Insufficient decolorizing microbial flora?
 Safranin applied too long  Epithelial cells, bacteria without inflammatory cells,
 Morphology may be altered other debris
 Antibiotic use  Is necrotic (amorphous) debris in the background?
 Evidence of destruction and remains of tissue
ACID-FAST STAINING  Are unexpected structures present?
 Carbofuchsin binds to mycolic acid
 Kinyoun stain SEARCH FOR MICROORGANISMS
 Cover with carbolfuchsin fro 5minutes  Scan for more detail at x40 or x60 obj
 Wash slides with running tap water  Use x100 obj for final evaluation
 Decolorize with acid alcohol until color removed  Examine more than one area of the smear
(fast)  Should find more than one organism
 Wash in running tap water  What kind of site? Sterile?
 Flood slides with methylene blue counterstain for  Do not over-interpret the findings
1 minute  Strict criteria for microbial morphotypes should be
 Wash with tap water and allow to air dry applied
 Wait for additional test or staining if necessary
CALCOFLUOR WHITE STAIN
 Calcofluor white is a colorless dye that binds to cellulose and EVALUATION OF ANTIBIOTICS
chitin  Is there evidence of purulence?
 Fluoresces when exposed to long-wavelength ultraviolet (UV  Red blood cells (RBCs), neutrophils, necrosis
light)  Is there a single most probable etiologic microorganism?
 Add 1 to 2 drops of calcofluor white to smear  Presumptive ID based on morphology
 Is there infection polymicrobial or monomicrobial?
MODIFIED WRIGHT-GIEMSA STAIN  What is the mixture of organisms?
 Fix smear with alcohol (methanol)  Will suspected pathogens be susceptible?
 Dip in fixative solution five times, 1s each  S. aureus, Haemophilus sp., β-lactamase
 Dip slide in solution I same as above  Enterococci-resistant to single antibiotics
 Grain excess  Bacteroides and pseudomonas: resistant to
 Dip slide in solution II same as above aminoglycosides
 Drain excess  Fungus: unresponsive to antibacterial antibiotics
 Rinse with tap water
SPECIAL HANDLING
 Modify tests when special consideration
MICROSCOPES
 Haemophilus sp. Switching to chocolate agar
 Brightfield compound light microscope
 Increase incubation time for slow growers
 Primary scope used
 Add special media
 Darkfield microscope o Legionella, mycobacterium
 Primary for spirochetes
 Parasites seen
 Dissecting microscope
 Order an ova and parasite exam
 Large parasites
o Strongyloides stercoralis
 Fluorescent microscope
 Special applications and fluorescent stains GRADING AND CLASSIFYING MATERIALS
 Electron microscope  Prevent culturing of normal flora-contaminated specimens
 Very specialized, usually for nonculturable viruses  Prevent use of antibiotics that may not be necessary
 Most important in sputum specimens
OBSERVING MICROBIAL PATHOGENS (picture)
 Criteria for separating samples before culture or
evaluation
TERMINOLOGY FOR DIRECT EXAMINATION
 Murray-washington method of assessment
 Important for communication
 Across countries and within staff
  Greater than 10 staphylococcal exterotoxin C (SECs)
(unacceptable); greater than 25 polymorphonuclear
meutrophils (PMNs) per x10 field (significant)
 Heineman’s method
 Emphasizes ratio of SECs and PMNs
CONTAMINATING MATERIALS
 Criteria for rejection
 Less than 25 PMNs/low power field (LPF)
 Greater than 10 SECs or mixed bacteria/LPF
 Gram smear report
 Quantitate contaminating materials
o 1+ = light; 2+ = moderate; 3+ = moderately heavy;
4+ = heavy
 Request new culture
 Brief evaluation, identification on know pathogens
observed
o Example: Neisseria spp.
 No antibiotic susceptibility unless known primary pathogen
LOCAL MATERIALS
 Do the materials fit with what should have been collected?
 Less than 25 PMN/LPF
 Less than 10 contaminating SEC/LPF
 Cellular or fluid elements local to sample
 Respiratory secretions: mucus, macrophages, goblet
cells, ciliated columnar cells (picture)
 CSF: cellular element
 Cavity fluid: macrophages, mixed white blood cells (WBCs),
mesothelial cells (picture)
 Wounds: blood and proteinaceous fluid
 Cervix: mucus, columnar epithelial cells, goblet cells,
leukocytes
 Prostate secretions or semen: spermatozoa and mucus
PURULENCE
 Criteria
 Greater than 25 PMSs/LPF
 Mucus and/or heavy proteinaceous material
 Gram stain smear
 Only organisms associated with WBC, mucus, or
exudates
 1+ (≤1 organism/oil-immersion field (OIF)), 2+ (few
organisms/OIF), 3+ (moderate number/OIF), 4+
(many/OIF)
 Contaminating materials should be ≤1+ none or few
 Culture ID guidelines
 Note findings and correlate with smears
o Do they match?
o Identify specific bacteria if possible by
morphology
o S. pneumonia from other streptococci
 Antibiotics susceptibility testing
 S. aureus, gram-negative bacilli, or others as
appropriate or requested
MIXED MATERIALS
 Criteria
 Greater than 25 PMN/LPF
 Less than 10 epithelial cells or contaminating
bacteria/LPF
 Local secretions
 Gram smear report
 Quantitate only those organisms intimately associated
with purulent exudates
 Must request a new specimen if presence of purulence and
uninterpretable culture results
SAMPLE REPORT OF DIRECT EXAMINATION (picture)
QUALITY CONTROL (QC) IN DIRECT MICROSCOPIC
INTERPRETATIONS
 Monitor smear and culture interpretation
 Ongoing; tests correlation
 Explain any discrepant results
 Provides continuing education and improvement
 Improve specimen collection
 Track to technologist or clinic to remedy problems
CHAPTER 8: USE OF COLOBY MORPHOLOGY FOR THE
PRESUMPTIVE IDENTIFICATION OF MICROORGANISMS
INTRODUCTION
 Colony morphology
 Characteristics and form of bacterial colonies
 Compare with direct examination
 Distinctive patterns can distinguish some pathogens and
facilitate presumptive identifications
IMPORTANCE OF COLONIAL MORPHOLOGY AS A DIAGNOSTIC
TOOL
 Importance of colonial morphology
 Provide a presumptive diagnosis in times of critical
need
o Best guess giving type of specimen and what is
seen before confirmatory tests
 Enhance quality of patient care through rapid results
and cost-effectiveness
o Prevents time-consuming testing that yields few
results
o Help jump-start therapy
 Play a significant role in quality control
o Helps maintain accuracy of commercial or
automated systems
o Troubleshoot mixed cultures or errors in
commercial systems
INTIAL OBSERVATION OF CULTURES
 Observe colony morphology
 18 to 24 hours postculture
o Age of culture may affect size and characteristics
of colonies
INTERPRETATIONS OF CULTURES
 Initial distinctions between gram-positive and gram-negative
isolates (selective)
 Blood agar: morphology, grows most microorganisms
 Chocolate agar: most organisms and fastidious
organisms
 MacConkey’s agar: gram-negative organisms,
particularly enteric
 Differentiation using plated media (differential)
 Lactose fermenters (pink)/non-lactose fermenters
(colorless)
o Escherichia coli / Citrobacter: dry pink colonies
o Klebsiella / Enterobacter-like organisms: large
mucoid pink colonies
 Comparative analysis of the culture media is very important in
initial interpretation
HEMOLYSIS
 Hemolysis of blood agar
 Observation in the media immediately surrounding or
underneath the colony
 Most important is presumptive identification of strep
 Use transillumination
o Light source behind the plate helps visualize
hemolysis
TRANSILLUMINATION
 The use of transillumination to determine whether colonies
are hemolytic. The technique can be used for MacConkey
also to see slight color differences in non-lactose fermenters
HEMOLYSIS
 Two major types
 α hemolysis: partial clearing of blood that results in a
green discoloration of the medium
o Examples: Streptococcus pneumonia, certain
viridians strep
 β hemolysis: complete clearing of blood cells around
the colonies
o Examples: S. pyogenes, S. agalactiae, Listeria
monocytogenes
 Non-hemolytic colonies are sometimes referred to as y
hemolytic
o Examples: Enterococcus faecalis (formerly called
“Group D Strep”), Staphylococcus saprophyticus,
and Sthaphylococcus epidermidis
FORM OR MARGIN
  Edge of colonies
 Smooth, filamentous, rough or rhizoid
o Examples: Basillus anthracis is filamentous;
diphtheroid colonies have rough edges
 Swarming: hazy blanket of growth on surface
o Proteus spp.

ELEVATION
 Raised, convex, flat, umbilicate or umbonate
 Raised: raised flat top
 Convex: dome shaped
 Flat: not raised
 Umbilicate: convex with depressed center (pitting); S.
pneumoniae (if no capsule)
 Umbonate: convex with protruding nipple; dipththeroids

DENSITY
 Transparent, translucent, opaque
 β-hemolytic strep translucent (e.g., Group B)
 Group B strep: semiopaque (bull’s-eye colony)
o Also most staph and gram-negative rods
COLOR
 White, gray, yellow buff
 Coagulase negative staph are white
 Enterococcus and most gram-negative rods are gray
 Micrococcus and Neisseria are yellow or off-white
 Diphtheroids are buff

CONSISTENCY
 Determine by touching a colony with a loop
 Brittle (splinters), creamy, dry, waxy, or sticky
 Sticky: entire colony comes off the plate
 S. aureus: creamy
 Neisseria: sticky
 Nocardia: brittle
 Streptococci: dry
 Diphtheroids: dry and waxy

PIGMENT
 Inherent characteristics of special organisms
 Pseudomonas aeruginosa: green or green metallic
sheen
 Serratia marcescens: brick red
 Kluyvera: blue
 Chromobacterium violaceum: purple
 Prevotella melaninogenica: brown-black anaerobe

ODOR
 Distinctive odors that help identify organisms
 S. aureus: old sock
 P. aeruginosa: fruity or grape-like
 Proteus mirabilis: putrid
 Haemophilus spp.: musty basement
 Nocardia spp.: freshly plowed field

ORGANISMS IN LIQUID MEDIA

 Streamers
 Vine-like growth in media, puff-ball-like
o streptococci
 Scum-like growth
o yeast
 Turbidity
 Overall cloudiness of liquid
 Gas bubbles present
o Enterics