Unit 1 Chapter 1

Bacterial Cell Structure

CLS 3303

Clinical Microbiology

Taxonomy Defined as the orderly classification & grouping of organisms into

categories Kingdom, Division, Class, Order, Family, Tribe, Genus and

Species ( these are the formal levels of classification) Family = “Clan”; has “–aceae” ending Genus = “Human last name” Species = “Human first name”

When in print, genus and species are italicized. (Staphylococcus aureus)

, When written genus and species are underlined. (Staphylococcus aureus)

To abbreviate organism names: use first letter capitalized of the genus followed by a period and the species epithet. ( i.e S. aureus)

Nomenclature

Staphylococcus sp. is used when referring to the genus as a whole when the species is not identified.

“sp.” – singular (Staphylococcus sp.)

“spp.” – plural (Staphylococcus spp.)

Classification by Cellular Type

Bacteria Identification – test each bacterial culture for a variety of metabolic characteristics and compare the results with known results.

All organisms are either “prokaryotes”, “eukaryotes”, or “archaeobacteria”

Classification by Cellular Type: Prokaryotes

PROKARYOTES - bacteria

Do not have a membrane-bound nucleus

DNA is a single circular chromosome and RNA are free in the cytoplasm

Have both cell (plasma) membrane AND cell wall.

Have no mitochondria, endoplasmic reticulum (ER) or Golgi bodies

Classification by Cellular Type: Eukaryotes

EUKARYOTES - fungi, algae, protozoa, animal cells, and plant cells

Cells have nuclei that contains DNA and are complex

Most cells do NOT have a cell wall (Fungi have cell walls made chitin)

Classification by Cellular Type: Archaeobacteria

Resembles eukaryotes

Found in microorganisms that grow under extreme environmental conditions

Cell wall lacks peptidoglycan

See chart on page 5 for comparisons of Prokaryotes and Eurkaryotes

Prokaryotic & Eukaryotic Cell Comparison

Bacterial Cell Wall

Gram Positive (G+) Cell Wall

Very thick protective peptidoglycan layer

Many G+ antibiotics act by preventing synthesis of peptidoglycan

Consists of cross-linked chains of glycan

Also contain teichoic acid and lipoteichoic acid

Unique structure makes these bacteria G+ by protecting against the decolorizing step in Gram staining

Gram Negative (G-) Cell Wall

Two layers; outer is much thinner than G+ cell walls

Outer wall contains several molecules, including Lipid A which is responsible for producing fever and shock in infections with G- bacteria

The thin walls allow the decolorizer to enter the cell and take out the crystal violet stain.

(G+) and (G-) Microorganisms

G+ cocci in clusters→

G- bacilli (rods)→

When identifying bacteria, remember that rods can sometimes be short and look like cocci, but cocci do not look like rods

Acid Fast Cell Wall

Mainly Mycobacteria and Nocardia Have a G+ cell wall structure but also a waxy layer of

glycolipids and fatty acids (mycolic acid). It is hydrophobic and affects permeability

Waxy layer makes them difficult to gram stain (More than 60% of the cell wall is lipid)

Cannot be decolorized by acid-alcohol, hence the name “acid fast”

Bacteria is pink

Background is green or blue

Absence of Cell Wall

Mainly Mycoplasma and Ureaplasma

Lack of cell wall results in a variety of shapes microscopically

Contain sterols in cell membrane

Surface Polymers: Slime Layers

Some bacteria produce slime layers

Made of polysaccharides

Inhibit phagocytosis and also help to attach to the host.

Surface Polymers: Capsule

Some bacteria produce a capsule

Protect the bacteria from phagocytosis

Capsule usually does not stain, but can appear as a clear area (halo-like)

Cell Appendages

Flagella – exterior protein filaments that rotate and cause bacteria to be motile

Polar

– Extend from one end

– Can occur singly or in multiple tufts

Peritrichous

– Flagella found on all sides of bacteria

Pili (fimbriae) – hairlike projections that aid in attachment to surfaces

Examples of Flagella

Bacterial Morphology

Microscopic Shapes Cocci (spherical)

Bacilli (rod-shaped)

Spirochetes (helical)

Groupings Singly

Pairs

Clusters

Chains

Palisading

Bacterial Morphology (cont’d)

Size and length

Short

Long

Filamentous:

Fusiform: bacilli with tapered, pointed ends

Curved

Pleomorphic: variance in size & shape within a pure culture

Other Common Bacterial Stains: Acridine Orange (fluorochrome dye)

Stains nucleic acid of both G+ and G- bacteria, either living or dead; used to locate bacteria in blood cultures and other specimens where background material obscures gram stains

Other Common Bacterial Stains: Methylene Blue

Stain for Corynebacterium diphtheriae to show metachromatic granules and as counter-stain in acid-fast stain procedures

Other Common Bacterial Stains: Lactophenol Cotton Blue – fungal stain

Other Common Bacterial Stains: Calcuflour White – fungal stain

A fluorochrome that binds to chitin in fungal cell walls

Apple-green or blue-white with a fluorescent microscope

Other Common Bacterial Stains: India Ink

Negative stain for capsules, surrounds certain yeasts

Other Common Bacterial Stains: Endospore stain

Heat is used to help the primary stain (Malachite green) into the spore. The spore stains green

The counter stain, (safranin) stains the rest of the organism

Microbial Growth and Nutrition Needs

Source of carbon for making cellular constituents

Source of nitrogen for making proteins

Source of energy (ATP) for cellular functions

Smaller amounts of other molecules

Nutritional Requirements for Growth

Autotrophs (lithotrophs)

Able to grow simply, using only CO2, water and inorganic salts

Obtain energy via photosynthesis or oxidation of inorganic compounds

Occur in nature and do not normally cause disease

Nutritional Requirements for Growth

Heterotrophic

Require more complex substances for growth

Require an organic source of carbon and obtain energy by oxidizing or fermenting organic substances

All human bacteria fall in this category

Within this group, nutritional needs vary greatly

Types of Growth Media

Minimal medium – simple; not usually used in diagnostic clinical microbiology

Nutrient medium – made of extracts of meat or soy beans

Enriched medium – nutrient medium with extra growth factors, such as blood

Selective medium – contains additives that inhibit the growth of some bacteria while allowing others to grow

Differential medium – contains additives that allow visualization of metabolic differences in bacteria

Transport medium – holding medium to preserve those bacteria present but does not allow multiplication

Environmental Factors Influencing Growth

pH – most media is between 7.0 and 7.5

Temperature – most pathogens grow at body temperature; grown at 35° C in the lab

Environmental Factors Influencing Growth

Gaseous composition Obligate aerobes – require oxygen

Obligate anaerobes – cannot grow in the presence of oxygen

Facultative anaerobes – can grow with or without oxygen

Capnophilic – grow better with extra CO2 (5 -10%)

Microaerophilic- grow better in low oxygen environments ( about 20%)

Campylobacter spp. require 5 – 6% oxygen

Bacterial Growth

Reproduce by binary fission

Can be fast (as little as 20 minutes for E. coli) or slow (as long as 24 hours for M. tuberculosis)

Determination of the Number of bacterial cells

Direct counting under microscope: estimate the number of bacteria in a specimen. Does not distinguish live or dead cells

Direct plate count: grown from dilutions of broth cultures. Counts viable cells only. Colony Forming Units (CFU/mL)

Density measurement: (turbidity) bacterial broth culture in log phase

Bacterial Biochemistry and Metabolism

Metabolic reactions cause production of energy in form of ATP

Identification systems analyze unknown specimens for:

Utilization of variety of substances as a source of carbon

Production of specific end products from various substrates

Production of acid or alkaline pH in the test medium

Fermentation

Anaerobic process in obligate and facultative anaerobes

The electron acceptor is an organic compound

Does NOT require oxygen

Oxidation (Respiration)

More efficient energy-generating process

Molecular oxygen is the final electron acceptor

Aerobic process in obligate aerobes and facultative anaerobes

Metabolic Pathways

Main one is Embden-Meyerhoff

Convert glucose to pyruvic acid, a key intermediate

Generates energy in the form of ATP

Metabolic Pathways

From pyruvic acid: Alcoholic fermentation (ethanol) (ex: yeast)

Homolactic acid fermentation (lactic acid) )ex: strep)

Heterolactic acid fermentation (lactic acid, CO2, alcohols, formic and acetic acids

Propionic acid

Mixed acid fermentation (lactic, acetic, succinic, and formic) (ex: e-coli and salmonells)

Butanediol fermentation: (ex: Kleb, enterobacter & serratia)

Butyric acid fermentation: (ex: obligate anaerobe)

Metabolic Pathways

Main oxidative pathway is the Krebs Cycle, resulting in acid and CO2

Carbohydrate Utilization & Lactose Fermentation

“Sugars” = carbohydrates

Lactose fermentation – key component in identification schemes

Lactose is converted to glucose, so ALL lactose fermenters also ferment glucose

Genetic Elements and Alterations

Plasmid

Extra piece of DNA

Code for antibiotic resistance and other virulence factors are often found on plasmids

Sometimes passed from one bacterial species to another. This is how resistance is acquired.

Plasmid Replication

Genetic Elements and Alterations

Mutations

“They don’t always read the book”

Changes that occur in the DNA code

Results in changes in the coded protein or in the prevention of its synthesis

References

http://media.photobucket.com/image/micro/lovitex2000/Micro%20biology%20lab/b1cf.jpg?o=81

http://nhscience.lonestar.edu/biol/wellmeyer/bacteria/capsules3.jpg

http://www.iccb.state.il.us/pt3/images/sci/mod11/bacillus_subtilis.jpg