Introduction to Microbiology

What is Microbiology? Study of microscopic organisms

– 5 Groups of microbes:• bacteria, fungi (yeasts & molds), viruses,

protozoa, algae

Clinical microbiology– study of microscopic organisms in

pathogenic (disease causing) processes in humans

Purpose of Clinical Microbiology To work with health care professionals to

provide accurate and rapid diagnostic aid and to manage information about infectious diseases– prompt diagnosis leads to early treatment– reduces morbidity (course of illness) and mortality

(death) To prevent or control the spread of pathogens

– In the community and/or hospital-acquired (nosocomial)

To track organisms’ resistance – to antimicrobial/antifungal agents

Basic Bacteriology includes:

Inoculation Incubation Isolation Inspection Interpretation Identification

Microbial life characteristics

All living things have similar “life characteristics” Respiration Reproduction Growth Motion Nutrition Excretion

Respiration Process organisms use to convert energy in

chemical bonds of O2 or an organic molecule to make ATP energy

Aerobic organisms use O2 during respiration organisms are incubated in ambient air

(room air) Anaerobic organisms

use organic molecules (N2) during respiration

intolerant of oxygen

RespirationOrganism can be:

Obligate aerobe Facultative anaerobe Microaerophilic Capnophilic Aerotolerant anaerobe Obligate anaerobe

Facultative anaerobic organisms capable of adaptive behavior can grow with or without oxygen

Microaerophilic organisms grow best at O2 tension and CO2

example: Campylobacter species

Capnophilic organisms require CO2 (5-10%) for growth

example: Neisseria /Haemophilus species

Respiration

Respiration

Aerotolerant anaerobescant growth in room air or 5-10%

CO2example: Clostridium carnis

Obligate (strict) anaerobecannot tolerate oxygenexample: Clostridium

hemolyticum

Generating Anaerobic Conditions Anaerobic chamber or glove box

Attached to N2 and Mixed gas tanks Gas-Pak anaerobic jar

Na borohydride + Na bicarbonate + citric acid +(add) H2O in the presence of a palladium catalyst forms

H2 + CO2 + N2

H2 + O2 (from the environment) H2O palladium catalyst becomes inactivated by water

reactivate by heating to 160-1700C for 2-4 hours indicator system: methylene blue/resazurin 90-100 minutes to achieve anaerobic conditions

Generating Anaerobic ConditionsAnaerobic chamber or glove box

Generating Anaerobic ConditionsA-Jar ANO2 Gas-Pouch

Generating Anaerobic ConditionsThe Gas-Pak

Generating Anaerobic ConditionsThe A-jar

Anoxomat

Anoxomat

Uses 3 cycles to flush jar Achieves anaerobic conditions in 1-3 minutes,

microaerophilic in 20 seconds Uses a rechargeable catalyst Uses gas mixture of: 10% CO2, 5% H2, 85% N2, Uses less gas than an anaerobic chamber

Generating Capnophilic Conditions CO2 incubator (5-10%)

tank is attached to an incubator, a regulator adjusts gas flow

CO2 packs (5%) similar principle as Gas-Pak

Candle jar (3%) place lit candle in jar with media; as flame

burns, it consumes O2 and produces CO2

Generating Capnophilic Conditions

Candle Jars CO2 Gas-pouch

Reproduction During bacterial reproduction, DNA is

replicated, the cell grows and then divides into 2 identical daughter cells changes in nucleotide sequences of the DNA

molecule may occur changes may be spontaneous or result of the

influence of external agents(i.e antibiotics) bacteria can also exchange genetic material

between other organisms

Reproduction Bacteria reproduce by binary fission

cell doubles in size, then divides into two identical daughter cells

doubling or generation time = time required for population of bacteria to double in number• varies from organism to organism• E. coli: 20 minutes• M. tuberculosis: 16-20 hours

Phases of Bacterial Growth Lag phase

growth does not begin immediately after inoculation to media

organisms are acclimating to their new environment

They are enlarging and producing energy Log (exponential) phase

each cell in population divides in two growth occurs exponentially organisms are most susceptible to damage

at this point, especially from antibiotics

Phases of Bacterial Growth Stationary phase

cells stop metabolizing as a result of food and nutrients depletion

growth stabilizes: rate of doubling equals rate of death

Death phase food is depleted and toxic waste product

build up more orgs are dying than doubling actual numbers of viable organisms decline

Phases of Bacterial Growth

Genetic Recombination Transfer of DNA from one organism to another

Include:

• Transformation

• Conjugation • Transduction

Transformation:

Free DNA fragments from dead Bacterium enter a related species across the cell wall

DNA fragments are exchanged for a piece of the recipient’s DNA

Ex: Strep pneumoniae, non encapsulated strains acquire a capsule

Transformation

Conjugation Transfer of DNA from one donor bacterium

to a recipient via a sex pilus Plasmids coding for antibiotic resistance

may also be transferred in this process.

Transduction DNA is transferred from one cell to another

by a bacteriophage (bacterial virus)

Genetic Recombination

Conjugation

Transduction

Motion

Movement of microorganisms is accomplished by either flagella, pseudopodia or cilia

Bacteria move by means of flagella– classification can be based on location of

the flagella

Flagella Location Atrichous

no flagella

Monotrichous single flagellum at either end

Amphitrichous flagella at both ends; singly, in pairs or clumps

Peritrichous flagella surrounding the entire organism

Flagella Location

Nutrition Bacteria obtain nutrients by taking up small

molecules across the cell wall Nutrition + Respiration ATP (energy) Nutrition is obtained from the culture media

organic and inorganic materials adequate moisture to maintain osmotic pressure

• Most bacteria requires an isotonic/hypotonic environment

pH must be properly adjusted bacteria: 6.5-7.5 fungi: 5-6

Hypotonic movement

http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/hypotonic_flash.html

Metabolic Pathways

Bacteria can use 3 pathways for glucose degradation

Metabolic pathways

The result of any metabolic process is: Hydrogen ions are transferred to

compounds of higher redox potential The result is release of ATP energy.

Embder-Meyerhoff Pathway

Glycolytic or anaerobic pathway (fermentation)

Glucose is degraded without the presence of oxygen

End products are acids which are detectable by pH changes:

A. acetoin (VP pos bacteria) (mildly acidic) B. Mixed acids (MR pos bacteria) (pH<4.4) Examples: Enterics, Anaerobes

Entner-Douderoff Pathway

Aerobic pathway Oxygen is required for glycolysis End products are weak acids Examples: non-fermenting GNR

Warburg-Dickens (Hexose Monophosphate shunt)

Aerotolerant Nonoxidative bacteria that are capable

of growing in the presence of oxygen but grow better anaerobically

Examples: Aerotolerant anaerobes

Excretion

Removal of waste products For bacteria, this is usually

accomplished by either active or passive transport

Cellular Organization

Cell is the basic unit of life All living cells are divided into 2 groups:

Eukaryotesanimal, plants, fungi, protozoans, and algae

Prokaryotesbacteria

Cellular OrganizationProkaryotes vs. Eukaryotes Prokaryotes (bacteria)

genetic material is not enclosed within membrane DNA is not associated with histone proteins cell walls contain peptidoglycan no membrane-bound organelles

Eukaryotes (fungi, parasites, plants, animals) genetic material is within a membrane and

organized into chromosomes DNA is associated with histone proteins Cell walls(plant,fungi, algae)/No cell walls

(animal,protozoans) Cell walls have no peptidoglycan contain nucleus and membrane-bound organelles

Bacterial cell wall Rigid structure that gives the organism its

shape Contains peptidoglycan (a.k.a murein)

mucopolysaccharide Prevents the cell from bursting in hypotonic

conditions Maintains osmotic pressure within the cell Types of cell walls

– based on their staining ability Gram positive Gram negative Acid fast

Gram-Positive Cell Wall Composed of dense layer of peptidoglycan

and teichoic acids Gram stain: resist decolorization and retain

crystal violet dye (purple) Thick peptidoglycan (murein) layer (60-90%

of G+ cell wall) forms rigid structure Teichoic acids

link the cell wall peptidoglycan to cell membrane regulate the movement of ions into and out of the

cell Triggers immune response (i.e initiate

inflammation)

Gram-Positive Cell Wall

Gram-Negative Cell Wall Gram stain: easily decolorized and absorbs

counterstain, safranin (pink) Cell wall consists of two layers

thin inner layer of peptidoglycan (10-20% of G- cell wall)

outer membrane of proteins, phospholipids and lipopolysachharides (LPS)

Outer membrane is a barrier to toxic substances(I.e penicillin G) is a sieve, allowing water soluble molecules and

proteins into the cell provides attachment sites to allow adhesion to

host cells and triggers immune response (i.e fever, inflammation)

Gram-Negative Cell Wall

Cell Walls

Lipopolysaccharide (LPS)

Lipopolysaccharide (LPS) contains 3 regions antigenic O-specific polysaccharide

• specific to each species core polysaccharide

• common to all Gram negatives inner lipid A (endotoxin)

• causes fever and shock associated with Gram negative sepsis

Acid-Fast Cell Wall Contains small amounts of peptidoglycan and

large amounts of glycolipids (mycolic acid) Mycolic acid waxy lipid (60% of cell wall)

Make cell wall impermeable Difficult to stain, appear as “ghost cells” on Gram

stain Block entry of chemicals and cause organism to

become more resistant to phagocytosis

Acid fast stain: resist decolorization by acid-alcohol mixture, retain carbol fuchsin (red) Ex: Mycobacteria, Nocardia species

Bacterial Taxonomy Comprise of classification, nomenclature, and

identification Classification is the arrangement of organisms

into specific groups based on their common morphologic, physiologic, and genetic traits classically based on phenotypic behavior (i.e.,

biochemical reactions, etc.) newer techniques for classification are based on

genotypic characteristics (i.e. how closely the organisms are genetically related)

NomenclatureBinomial system

Binomial System Two-name systemGenus and species

Genus: name given to related organisms- ALWAYS capitalized first letter- Can be abbreviated

Species: name given to each type of organism within a genus

- NEVER capitalized Either italicized or underlined

- Ex. Proteus (genus) mirabilis (species) or P. mirabilis System is constantly changing

- Neisseria catarrhalis Moraxella catarrhalis Branhamella catarrhalis Moraxella catarrhalis

Classification: Kingdom Division Class Order Family Tribe Genus Species