WEEK 1 – INTRODUCTION TO BACTERIOLOGY CORNELL METHOD

Lecture

History

Anton van Leeuwenhoek - first observations of bacteria at the microscopic level (30-266x magnification); father
of bacteriology and protozoology; drawings of cells in 1684

Huygens – free living protozoa fungi forms

• yeast
Robert Hooke (1667) - filamentous microscopic fungi; (end of 16th
century)
• mold (filamentous
used microscope with 3-500x magnification which helped him recognized cell
structure)
structure

Pietro Antonio Micheli - described 900 species of molds

Cicero and Fracastorius - fevers might be caused by minute

animals (contagium vivum); closest theory to modern
workflow:
pathogenesis
1. specimen collection (from site of
Jan Baptist van Helmont – spontaneous generation theory infection)
proponent; believed rat and mice could arise from container, rags, 2. direct microscopic examination
and wheat kernel after three weeks 3. culture (inoculate and incubate in
culture media) – propagation of
Louis Pasteur - when air is filtered through cotton wool, large bacteria no. help easily ID it (37ºC in
numbers of microorganisms are held back; disproved 18-24 hours)
spontaneous generation through his swan-neck flask experiment; 4. bacterial identification –
(1860) semisynthetic medium made up of ammonium salts, yeast, macroscopic and microscopic
ash, candy sugar; (1865) proposed that disease must airborne examination
5. biochemical test
John Tyndall - existence of heat-stable forms of certain bacteria;
removal of which involved the process of repeated heating and rest
classification of culture media
Ferdinand Cohn (1849) - staining of histological specimens; carmine 1. general purpose (basal media)
and hematoxylin (veggie dyes); (1872) basal medium (various – for growth of non-fastidious
additions could be made) bacteria (basic nutrients)
➢ gelatine and agar – solidifying agents (1882) 2. selective (inhibitors)
➢ silica gel media - chemolithotrophic bacteria 3. differential
4. enrichment
Robert Koch (1877) – methylene blue to stain bacteria (specific to a. broth/liquid medium - nutrients
bacterial cell wall); (1882) employing heat for the stain to penetrate the dissolved in water (turbid (+)
waxy envelope vs. clear (-))
➢ tubercle bacillus (Mycobacterium tuberculosis) – impenetrable
to stain due to its cell wall made up of fatty acid, mycolic acid; must apply heat first, waiting for steam
to come out, before staining, a process called acid-fast
staining fastidious bacteria – requires an
additional/special nutrient ex.
Hans Christian Gram (1884) – differential dye/stain Neisseria spp.: iron, Haemophilus
➢ primary stain – crystal violet: purple → Gram-positive spp.: hemin
➢ safranin – red → Gram-negative

Frederick Loeffler (1890) - demonstrate the presence of bacterial flagella (motility vs. non-motility)

1919 – ultraviolet microscope

Marton – Belgian physicist who invented electron microscope in 1934 (2-300,000x magnification)

1965 – scanning electron microscope

Julius Richard Petri (1887) - petri dish

Beijerinck (1898) - enrichment culture media

McIntosh and Fildes - anaerobic jar (for culture: aerotolerance)

Chamberland (1884) – autoclave (sterilization equipment)

Edward Jenner (1796) - Cowpox virus used to immunize a boy against smallpox

Ignaz Semmelweiss (1844) – childbed fever

Joseph Lister – covering wounds: wound dressing with chemicals

Metchnikoff (1882) - cellular immunity, phagocytes

Ehrlich (1891) - active and passive immunity

Charles Calmette – Bacille Calmette Guerin vaccine (against Mycobacterium tuberculosis)

Taxonomy

Classification - organization of microorganisms that share similar morphology, physiology and genetic
components into specific group/ “taxa”
➢ specie - collection of bacterial strains that share common physiologic & genetic features ex. Neisseria
meningitidis
➢ genus - comprises of different species with common and sufficiently different features ex. Neisseria
spp.
➢ family – “ceae”

Nomenclature - naming of microorganisms according to established rules & guidelines (Genus specie)

Identification - describe key features of microorganisms

➢ genotypic characterization – molecular method (PCR, genetic sequencing)
o genetic makeup; genes and nucleic acids
o DNA base composition ratio - G, C, A, T (nitrogenous bases); used as indicator of relatedness
o nucleic acid base sequence analysis - order of bases along RNA or DNA; strands; measure
of degree of relatedness
➢ phenotypic characterization
o readily observable characteristics (appendages, stain, temperature)
o macroscopic morphology - microbial growth pattern in culture media; colonial size, texture,
and pigmentation
o microscopic morphology - size, shape, intracellular inclusions or appendages, arrangement of
bacterial cells
o staining characteristics - ability of the organism to be stained with a particular stain
o environmental requirements - temperatures, presence or absence of oxygen, pH levels,
presence of ions and salts (halophilic organisms)

Host-Microorganism Interaction

1. Physical encounter between host and microorganism non-sterile specimens –

(exposure) – transmission occurs: reservoir → mode of with presence of normal
transmission → host; dependent on human activities flora from skin, GIT, RT
• intervening agents: vector (arthropods, insects), vehicle sterile specimens –
(fomites, inanimate objects) without normal flora
• reservoir: place of origin of infecting agent
• host: microbial colonization - persistent survival of microorganisms in surface of human body;
dependent in human defenses that protects internal tissues and organs
o skin – physical and chemical barrier
▪ dermal layer - physical barrier; capability to slough off; provide dry, acidic, and cool
conditions
▪ hair follicles and glands - produce acids, alcohols, and toxic lipids
▪ conjunctival epithelium (eyes) - tears (flushing action)
▪ skin associated lymphoid tissue (SALT) - specific and nonspecific immunity
o mucous membrane
▪ mucosal cell - capability to slough off; tight intercellular junction
▪ goblet cell – mucus production: protective lubrication; bacterial trapping; contains Ab;
provides antibacterial substances: lysozyme, lactoferrin, lactoperoxidase
▪ mucous associated lymphoid tissue - specific and nonspecific immunity

2. Microorganism colonization of host surfaces (incubation period) – propagation, multiply/colonization,

pathologic changes
• microorganism how normal flora
o colonizers/normal flora - microorganisms that inhabits turns into
human body pathogens: through
▪ resident - survive, thrive and multiply; presence site transfer, is
more permanent increase in
▪ transient - survive but not multiply on the surface population
o microbial colonization - last step in establishment of long-
lasting, commensal relationship between colonizer and human host; first step in developing
infection and disease
➢ factors contributing to successful colonization
o survival against environmental conditions - localization in moist area; protection within
ingested debris; expression of specific metabolic characteristics
o attachment and adherence to host cell surface – pili, adherence proteins
 o motility, production of subs. that may compete with host for acquisition of nutrients, ability to
coexist w/ other microorganism

3. Microorganism entry, invasion, and dissemination (clinical manifestation) – signs and symptoms
development, acute phase (early phase)
o Host
▪ factors contributing to disruption of physical barrier
1. trauma – wounds, abrasions, burns
2. inhalation – smoking, toxic gases
3. implantation of medical devices
4. other diseases
5. childbirth and overuse of antibiotics
▪ responses to microbial invasion
1. nonspecific response
a. phagocytes - cells that ingest and destroy foreign particle
polymorphonuclear (ex. macrophage (‘backups’, cleaners)
Neutrophils)
first cell on the scene
bone marrow- circulation bone marrow- circulation- tissues
days or less in survival several days to weeks
mediates immune system defenses
bacterium → receptors → phagocytosis → phagosome + lysosome → phagolysosomes → exocytosis
(soluble debris)
b. inflammation – swelling, redness, heat, pain
i. coagulation system
ii. complement system
iii. cytokines
2. specific response
a. antibody-mediated immunity
b. cell-mediated immunity
Cellular B cells T cells NK cells
Residence lymphoid tissues circulation &
lymphoid tissues
Functions produce Ab
Subtypes B cells helper T cells similar to
plasma cells cytotoxic T cells cytotoxic but do
B-memory cells suppressor T cells not require
presence of Ag
to function

o Microorganism
▪ infection - growth and multiplication of microorganisms that result in damage to host
▪ disease - infection produce notable changes in human physiology (signs and
symptoms begin)
▪ pathogens - microorganisms causing infections/diseases
▪ virulence factors - characteristics that enable them to cause disease
❖ attachment- microbial attachment to surface through different mot; pathogens vs
colonizers
❖ invasion - traumatic factors; direct actions of virulence factors
❖ surviving inflammation
• phagocytes
o production of capsule and toxins
o inhibit fusion of phagosome-lysosome
o resistance to lysosome
o active and rapid replication
• complement system
o capsule to hide surface molecules
o produce substances that inhibit complement activation or
destroy specific complement proteins
❖ microbial toxin - biochemically active substances released by microorganisms
that have a particular effect on host cells; can cause disease in the absence of
pathogens
• intoxication- ingestion of preformed bacterial toxin
• endotoxin - Gram (-) bacteria; produce toxins inside
• exotoxin - Gram (+) bacteria; specific and more limited effects than
endotoxins
 ▪ pathogenesis - first step in infection and disease development

4. Outcome – recovery, chronic/disability, death

o Encounter & entry – incubation stage (no s/s)
o Colonization & entry – prodromal stage (s/s, highly communicable)
o Invasion & dissemination – clinical stage (peak of s/s), then stage of decline (s/s subsides)
o Outcome – convalescent stage (full recovery, chronic infection, death)

Definition of Terms

 acute phase - early stage of a disease preceding the adaptive phase of the immune response
 anaerobe - organism that does not require oxygen for life and reproduction
 antibody - protein or immunoglobulin molecule characterized by specific amino acid sequence produced by
the host as a result of a specific antigenic stimulation
 antigen - substance that produces sensitivity and initiates an immune response
 antisepsis - destruction of microorganisms to prevent infection
 bacteremia - presence of viable bacteria in the blood, as evidenced by their recovery in blood cultures
 bactericidal - antimicrobial that kills a microorganism
 bacteriocin - proteins produced by some bacteria that inhibit the growth of other strains of the same organism
or related specie
 capnophile - microorganism that grows best in the presence of carbon dioxide
 disinfection - removal of microbes that may cause disease from an environment
 disinfectant - substance designed to be used on inanimate objects to kill or destroy disease-producing
microorganisms
 etiologic agent - microorganism causing a disease.
 fastidious - hard to grow; requires additional growth factors.
 genotype - genetic makeup of an organism
 Gram-positive bacteria - bacteria that retain the crystal violet–iodine complex and appear blue-black on
Gram-stained smears
 Gram-negative bacteria - bacteria that do not retain the crystal violet–iodine complex, stained red by the
safranin counterstain
 halophilic - “salt-loving”; an organism that grows best in media with an increased concentration of NaCl
 immunocompetent - ability of an immune system to mobilize and deploy its antibodies and other responses
to stimulation by an antigen
 immunocompromised - describe an individual with deficient function of the immune system
 immunosuppression - describe the state of an immune system that is suppressed
 latent phase - permits the infection to evolve without any obvious external symptoms
 mesophile - organism that grows best in moderate temperature, neither hot nor cold
 microaerophile - microorganism that grows in conditions of reduced oxygen and increased carbon dioxide.
 microaerophilic - microorganisms that require environments containing concentrations of oxygen lower than
that present in the atmosphere
 microbial load - total number of organisms present
 obligate aerobe - microorganism that requires oxygen for growth
 obligate anaerobe - microorganism that can live and reproduce only in a strict anaerobic environment (0%
oxygen)
 nosocomial infection - infection acquired within 72 hours of a stay in a health care facility
 opportunistic infection - disease caused by a microorganism with low virulence that becomes pathogenic in
a host with low immunologic resistance
 pathogenicity - ability of a microorganism to cause disease
 phenotype - observable or measurable characteristics of an organism
 sepsis - systemic response to bacterial infection
 resistant strain - not inhibited by the usual systemic concentrations of the antimicrobial agent with normal
dosage schedules
 susceptible - implies that an infection caused by the bacterial strain tested may be appropriately treated with
the dosage of antimicrobial agent recommended for that type of infection and infecting species
 intermediate - implying that the agent might be effective for infections located at body sites where the drugs
are physiologically concentrated, or when a high dosage of drug can be used
 zoonosis - disease that humans acquire from exposure to infected animals or products made from infected
animals
 zoonotic - pertains to diseases that can be transmitted from animals to humans
 Laboratory

A laboratory personnel must learn to know:

• what hazards exist
• the basic safety precautions
• how to apply the basic rules of common sense

Type Source Possible Injury

Biological Infectious agents Bacterial, fungal, viral, or
parasitic infections
Sharp Needles, lancets, and broken Cuts, punctures, or exposure to
glass bloodborne pathogens
Chemical Preservatives and reagents Exposure to agents that are
poisonous, caustic, or
carcinogenic
Radioactive Equipment and radioisotopes Damage to a fetus or
generalized overexposure to
radiation
Electrical Ungrounded or wet equipment Burns or shock
and frayed cords
Fire/explosive Open flames and organic Burns or dismemberment
chemicals
Physical Wet floors, heavy boxes, and Falls, sprains, or strains
patients

Hazard Communication Standard – ensure that all

laboratory personnel have a thorough working knowledge
of the hazards of the chemicals with which they work;
states that all clinical laboratory personnel have a need
and a right to know the hazards and identities of the
chemicals to which they are exposed when working
➢ employee right-to-know: OSHA laboratory chemical
hygiene plan and hazard communication standard
• NFPA 704 – a symbol system used to inform
firefighters of the hazards they may encounter
when fighting a fire in a particular area

Chemical Hygiene Plan – includes guidelines on:

• proper labeling of chemical containers
• manufacturers’ material safety data sheets
(MSDSs) – information provided by the
manufacturer or distributor for hazardous
chemicals.
• written chemical safety training and retraining programs
➢ fume hood - prevent inhalation of toxic fumes; protect against chemical odor by exhausting air to the
outside, but are not HEPA-filtered to trap pathogenic microorganisms

Fire Safety. Post fire evacuation plans – essentially blueprints for finding the nearest exits (+alarms and
extinguishers) in case of fire; fire drills conducted quarterly or annually

Fire Type Composition of Fire Type of Fire Extinguishing

Extinguisher Material
Class A Wood, paper, or Class A Water
clothing
Class B Flammable organic Class B Dry chemicals, carbon
chemicals dioxide,
Class C Electrical Class C foam, or halon
Class D Combustible metals None Dry chemicals, carbon
dioxide,
Class K Grease, oils, fats Class ABC or halon
❖ Rescue — rescue anyone in immediate danger
❖ Alarm — activate the institutional fire alarm system
❖ Contain — close all doors to potentially affected areas
❖ Extinguish/Evacuate — attempt to extinguish the fire, if possible; or evacuate, closing the door

Operating fire extinguisher. Pull pin; Aim at base of fire; Squeeze handles; Sweep nozzle, side to side
Electrical Safety. Electrical cords should be checked regularly for fraying. All plugs should be the three-prong,
grounded type. All sockets should be checked for electrical grounding and leakage at least annually. No
extension cords should be used in the laboratory.

Biosafety – also known as infection control, understanding chain of infection is necessary

• infectious agents – consist of bacteria, fungi, parasites, and viruses
o early detection and treatment of infectious agents can stunt their opportunity for growth
• reservoir – a place where an infectious agent can live and possibly multiply (humans, animals, insects,
fomites, blood/body, fluids)
o disinfecting the work area kills the infectious agent and eliminates the reservoir
➢ fomites – equipment and objects that act as reservoir
• portal of exit - a way to exit the reservoir to continue the chain of infection (nose, mouth, mucous
membranes, specimen collection)
o sealed biohazardous, waste containers, sealed specimen, containers, hand hygiene, standard
precautions\
• means/mode of transmission – a way to reach a susceptible host
o hand hygiene, standard precautions, PPE, patient isolation, (transmission-based precaution)
➢ direct contact: an unprotected host touches or is touched by the reservoir
➢ droplet: the host inhales material from the reservoir, such as aerosol droplets from an infected person.
➢ airborne transmission: dried aerosol nuclei circulating on air currents or attached to dust particles are
inhaled.
➢ vehicle: contaminated food or water is ingested.
➢ vector: parasites such as malaria are transmitted by a mosquito bite.
• portal of entry – a means to enter the reservoir; can be the same as the portal of exit (nose, mouth,
mucous membranes, skin, unsterile equipment)
o hand hygiene, standard precautions, PPE, sterile equipment
• susceptible host - can be another patient during invasive procedures, visitors, and health care
personnel when exposed to infectious specimens or needlestick injuries
o immunizations, patient isolation, nursery precautions, healthy lifestyle

Handwashing
1. (Remove jewelry). Stand in front of the sink. Do not lean on the sink with clothes.
2. Use paper towel to cover the water control and turn on the water.
3. Wet hands thoroughly. Allow the water to flow from arms to fingertips.
4. Apply soap to hands (preferably antimicrobial).
5. Wash the palm, back, and wrist of each hand using strong, frictional, circular movements.
6. Interlace fingers and thumbs and move hands back and forth for ten seconds.
7. Rub nails against the palm.
8. Rinse hands thoroughly.
9. Dry hands well.
10. Use paper towel to turn the water off.

Donning PPEs
1. First put on the gown, and tie it at your neck and waist.
2. Place face protection over your nose and mouth.
3. Fit it to your nose and mouth. Then hold the mask in place with one hand while you place the straps
over your head with your other hand. Make any final adjustments as needed.
4. When goggles and face shields are needed, put them on after the mask, and adjust them for proper fit
as needed.
5. Gloves are donned last; pull them securely over the cuffs of your gown.

Doffing PPEs
1. Gloves are the most contaminated PPE; remove them first.
2. Pull off the first glove using your other gloved hand so that the first glove ends up inside out in the still-
gloved hand.
3. Remove the second glove by sliding your ungloved finger inside the glove of the other hand and pulling
off the glove without touching the outside of the glove.
4. Dispose of the gloves in a biohazard container.
5. If you wear goggles or a face shield, remove them by touching only the band or ear pieces.
6. Untie your gown, and remove it by touching only the inside of the gown.
7. Dispose of the gown in a biohazard container.
8. Remove the mask by touching only the ties or bands.
9. Dispose of the mask in a biohazard container.

Disposal of Hazardous Waste

1. Biohazard containers
2. Sharps containers
3. Discard shelves, carts, bins, etc.—contaminated culture tubes and glassware used to store media and other
glassware should be placed in these areas for decontamination and washing.
4. Trash cans—any noncontaminated materials, paper, or trash should be discarded in these containers.
Under no circumstances should laboratory waste be disposed of in trash cans.

biologic safety cabinet - a device that encloses a workspace in such a way as to protect workers from aerosol
exposure to infectious disease agents.
• Class I cabinets allow room (unsterilized) air to pass into the cabinet and around the area and material
within, sterilizing only the air to be exhausted. They have negative pressure and may be ventilated to
the outside or exhausted to the work area and are usually operated with an open front.
• Class II cabinets (vertical laminar flow BSCs) sterilize air that flows over the infectious material, as well
as air to be exhausted. The air flows in “sheets,” which serve as barriers to particles from outside the
cabinet and direct the flow of contaminated air into the filters, most common type.
• Class III cabinets afford the most protection to the worker. Air coming into and going out of the cabinet
is filter sterilized, and the infectious material within is handled with rubber gloves that are attached and
sealed to the cabinet

classification of biologic agents based on hazard

• biosafety level 1 (BSL-1) agents include those that have no known potential for infecting healthy people
and are well defined and characterized
o basic teaching, research
o GMT
o none; open bench work
• BSL-2 poses a moderate risk to adults; unlikely to spread through community; effective treatment
available
o primary health services; diagnostic services, research
o GMT plus protective clothing, biohazard sign
o open bench plus BSC for potential aerosols
• BSL-3 can cause disease in healthy adults; may spread to community; effective treatment readily
available
o special diagnostic services, research
o as level 2 plus special clothing, controlled access, directional airflow
o BSC and/or other primary devices for all activities
• BSL-4 can cause disease in healthy adults; poses a lethal risk and does not respond to vaccines or
antimicrobial therapy
o dangerous pathogen units
o as level 3 plus airlock entry, shower exit, special waste disposal
o Class III BSC, or positive pressure suits in conjunction with Class II BSCs, double-ended
autoclave (through the wall) filtered air

classification of infective microorganisms by risk group

• risk group 1 (no or low individual and community risk) - a microorganism that is unlikely to cause human
or animal disease.
• risk group 2 (moderate individual risk, low community risk) - a pathogen that can cause human or animal
disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock, or the
environment. laboratory exposures may cause serious infection, but effective treatment and preventive
measures are available, and the risk of spread of infection is limited.
• risk group 3 (high individual risk, low community risk) - a pathogen that usually causes serious human
or animal disease but does not ordinarily spread from one infected individual to another. Effective
treatment and preventive measures are available.
• risk group 4 (high individual and community risk) - a pathogen that usually causes serious human or
animal disease and that can be readily transmitted from one individual to another, directly or indirectly.
Effective treatment and preventive measures are not usually available.