MT 111 LECTURE 1: INTRODUCTION TO CLINICAL BACTERIOLOGY

Lectured by: Arbee Mae Castro, RMT, MLS (ASCPi)CM

Bacteriology
 Study of single-celled microorganisms that lack a true nucleus.

History
 Hippocrates (460-370 BC)- “Father of Medicine” and founder of medical ethics
 Aristotle (384-322)- Spontaneous Generation Theory (living organism could develop from non-living materials)
 1590: Hans and Zacharias Janssen (Dutch lens grinders) invented the first compound microscope.
 1660: Robert Hooke (1635-1703) published “Micrographia” (contains drawings and detailed observations of
biological materials that were observed under the microscope)
 1676: Anton Van Leeuwenhoek (1632-1723) “Father of Microbiology” (he was the first person to observe
microorganisms under the microscope)
 1688: Francesco Redi (1626-1678) was an Italian physician who refuted the idea of spontaneous generation by
showing that rotting meat carefully kept from flies will not spontaneously produce maggots
 1835: Agostino Bassi de Lodi showed that a disease affecting silkworms was caused by a fungus- the first
microorganism to be recognized as a contagious agent of animal disease
 1836: Theodor Schwann helped develop the “Cell Theory” (cell is the basic functional unit of all living organisms)
 1861: Louis Pasteur's (1822-1895)- Germ Theory of Disease (a germ causes infectious diseases)
o Just like Francesco Redi, he disproved the Spontaneous Generation Theory
 1876: Robert Koch (1843-1910)- “Father of Bacteriologic Technique”
o He also developed the “Koch’s postulates” (1884) which states that:
1. The agent must be present in every case of the disease.
2. The agent must be isolated and cultured in vitro.
3. The disease must be reproduced when a pure culture of the agent is inoculated into a susceptible host.
4. The agent must be recoverable from the experimentally-infected host.
o Koch’s postulates was the basis of the culture methods done in the laboratory
 1890: Von Behring and Kitasato discovered that injection of animals with bacterial toxin would result in the
production of a substance in the blood capable of preventing a disease which led to the development of vaccines.
 Edward Jenner- Father of Immunology because he was the first to institute vaccination against infectious diseases
 Joseph Lister- founded aseptic surgery
 Hans Christian Gram- employed Gram’s staining for microscopic bacterial cell differentiation
 Jules Bordet- discovered Bordetella pertussis as the causative agent for whooping cough
 Friedrich Loeffler- cultivated Kleb’s Loeffler’s bacilli
 Alexander Flemming- discovered penicillin from mold Penicillum nolatum

Microbial Taxonomy and Nomenclature

 Taxonomy- the orderly classification and grouping of organisms into taxa or categories. It comes from the Greek word
“taxon” meaning arrangement and “nomos” means law.
 Three categories of Taxonomy
o Classification- the categorization of organisms into taxonomic groups
o Nomenclature- naming of an organism by international rules according to its characteristics
o Identification- refers to the practical use of a clarification scheme
 Linnaean Taxonomy
Formal Rank Example
Domain Bacteria
Kingdom Prokaryote
Division or Phylum Gracilicutes
Class Scotobacteria
Order Eubacteriales
Family Enterobacteriaceae
Genus Escherichia
Species or Epithet coli
Subtype Escherichia coli O157:H7
 Species- most basic taxonomic group
Further divisions of species
o
 Subspecies (subsp)- based on phenotypic differences, these differences are readily observable
 Serovarieties (serovar)- based on serologic differences, involves immunology
 Biovarieties (biovar)- based on biochemical test result differences
 Nomenclature- binomial system, it would contain the genus and the species
o 1st letter of family name is capitalized and has an –aceae ending. Ex. Enterobacteriaceae
o 1st letter of genus name is capitalized. Species name begins with lower case letter. Ex. Escherichia coli
o Both the genus and the species should be italicized in print or underlined when written in script.
o Abbreviation- 1st letter of genus capitalized followed by a period and the species name follows. Ex. E.coli
o First syllable of first 2 letter are used when 2 or more genera names begin with the same first letter. Ex. Ent.coli or
Esch.coli

Classification of Bacteria
1. Phenotypic characteristics- readily observable characteristics of a bacteria/organism
2. Genotypic characteristics- genetic make-up of an organism based on their DNA and RNA structure and homology
3. Cellular type
o Prokaryotes- cells that lack a “true” nucleus and no membrane-encased organelles. Ex. Bacteria
o Eukaryotes- cells with a “true” nucleus and membrane-encased organelles. Ex. Fungi, algae, protozoans
o Archaeobacteria- grows under extreme environmental conditions, not encountered in clinical microbiology
CHARACTERISTIC PROKARYOTE EUKARYOTE
Typical Size 0.4-2.0 µm in diameter 10-100 µm in diameter
0.5-5 µm in length >10 µm in length
Nucleus No nuclear membrane; found in the Classic membrane bound nucleus
nucleoid region of the cytosol
Genome Located in the nucleoid at the mesosome In the nucleus
Chromosomal DNA Circular complexed with RNA Linear complexed with histones and other
proteins
Extra chromosomal DNA Plasmids, small, circular molecule of the In mitochondria and chloroplasts
DNA commonly found in the Gram
Negative bacteria.
Reproduction Asexual Sexual and Asexual
Membrane bound organelles Absent All
Lysosomes Absent in all Present in some; contain hydrolytic enzymes
Endoplasmic Reticulum Absent in all Present in all; lipid synthesis, transport
Mitochondria Absent in all Present in most
Ribosomes (sites of protein Present in all Present in all
synthesis)
Ribosome site 70S= 50S=30S 80S=60S=40S
Electron Transport for energy In the cell membrane; no mitochondria In the mitochondria and chloroplasts
Sterols (in the cytoplasmic Absent except in Mycoplasma Present
membrane)
Plasma Membrane Lacks Carbohydrates Contains glycolipids and glycoproteins
Cell wall peptidoglycan Chitin (fungi), glycans (algae), lingnin
(plants)
Glycocalyx Present as capsule or slime layer Present
Cilia Absent Present
Flagella Simple flagella (composed of flagellin) Complex flagella; movement by sliding
movement by rotary action filaments
Pili and Fimbriae Present Absent

Prokaryotic/Bacterial Cell Structure

 Cytoplasmic structures
1. Nucleus- no membrane bound organelles
2. Mesosome- point of attachment of chromosomes
3. Ribosomes- consists of RNA and protein, site for protein synthesis.
 4. Cytoplasmic Granules/inclusion bodies- serves as storage deposits of polysaccharides
o Babes Ernst bodies/volutin granules/metachromatic granules- found in Corynebacterium diphtheriae
o Endospores- found in genus bacillus and clostridium
 Cell Envelope structures
1. Plasma Membrane (Cell Membrane)- composed of phospholipid bilayer
2. Cell Wall (Murein layer)- maintains shape of cell and has high affinity to dyes.
o Composed of N-acetylglucosamine and N-acetylmuramic acid
o Gram positive- thicker cell wall, contains teichoic acid
o Gram negative- has an outer membrane which serves as primary permeability barriers to hydrophilic and
hydrophobic compounds and is a bilayer lipopolysaccharide
o Characteristics:
 Permeability- osmotic pressure is maintained
 Plasmolysis- a cell in a saline solution shrinks because water passes out
 Plasmoptysis- a cell in distilled water would burst
3. Capsule- made up of polysaccharides, acts as virulence factors and appear as clear halo under microscope
o The organism cannot be killed directly by the WBCs or any cells that fight the bacteria that invades the body
4. Slime layer- also made of polysaccharides (similar to capsules) but are more diffuse layers surrounding the cell.
 Cell Appendages
1. Flagella- are exterior protein filaments that rotate and cause bacteria to be motile. This causes the bacteria to have a
“true” motility. It is made up of a protein called flagellin.
o Classification according to number and arrangement of the flagella on
bacterial cell:
a. Atrichous- no flagellum
b. Monotrichous- single flagellum at one pole
c. Amphitrichous- flagellum on both poles of the cell
d. Lophotrichous- bundle of flagella in one or both poles of the cell
e. Peritrichous- flagella surrounds the entire organism
2. Pili (singular pilus)- are non-motile, long, hollow protein tubes made up of pilin that connects two bacterial cells
o Two types of Pili:
a. Sex/fertility pili- for sexual conjugation by transferring DNA from 1 cell to another
b. Somatic/common pili- for adhesion of bacteria to host cell
3. Fimbriae (singular fimbria)- are non-flagellar, sticky, proteinaceous, hair-like appendages that aids in adhesion to
tissues and to surfaces. It contributes to the virulence of the bacteria.

Eukaryotic Cell Structure

1. Nucleus- has chromosomes which contains DNA. They are covered with basic proteins
called histones. It is bounded by a bilayered lipoprotein membrane known as the
nuclear membrane.
2. Nucleolus- round, refractile body which is the site of ribosomal RNA synthesis. It is
located within the nucleus.
3. Endoplasmic Reticulum- is a system of membranes that occur throughout the
cytoplasm.
o Rough Endoplasmic Reticulum- covered with ribosomes which gives it a “rough” appearance; site of protein
synthesis
o Smooth Endoplasmic Reticulum- no ribosomes. It doesn’t synthesize proteins but it synthesizes phospholipids
4. Golgi Apparatus- modify and package proteins sent by the rough endoplasmic reticulum
5. Ribosomes- found in the RER, where protein synthesis occurs.
6. Mitochondria- main site of energy production.
7. Lysosomes- contains hydrolytic enzymes for degradation of macromolecules and microorganisms within the cells.
8. Peroxisomes- break down hydrogen peroxide which is toxic to the cell.
9. Plasma Membrane- regulates transport of macromolecules into and out of the cell. Also contains cholesterol which
keeps the membrane fluid. The polar heads of phospholipids are hydrophilic and the nonpolar tails are hydrophobic
which lie in the center of the plasma membrane.
10. Cell Wall- provides rigidity and strength to the exterior of the cell. Most eukaryotic cells don’t have cell walls except
for fungi.
11. Cilia- short projections that extend from the cell surface and used for locomotion of eukaryotic cells.
Bacterial Morphology (classified according to shape)
1. Cocci (sing. Coccus) spherical/circle
o Diplococci- cocci in pairs, kidney bean/coffee bean-shaped; Ex: Neisseria gonorrheae
 Intracellular diplococci- the bacteria is found within the WBC also known as neutrophil/PMN; when observed
under the microscope, it would indicate that the patient has an active infection.
 Extracellular diplococci- the bacteria is found outside the WBC; since they are cocci in pairs, they would look like
kidney bean-shaped or coffee bean-shaped; it would indicate that the patient is undergoing therapy or treatment.
o Streptococci- cocci in long chains; Ex: Streptococcus pyogenes, causative agent of strep throat
o Staphylococci- cocci in grape-like clusters; Ex: Staphylococcus aureus, causative agent of food poisoning
o Tetrads- cocci that divides in 2 planes to form 4 groups; Ex: Gaffkya tetragena
o Sarcina- cocci that divide in 4 planes to form 8 groups; Ex: Sarcina lutea
2. Bacilli (sing. Bacillus)- rod shaped
o Diplobacilli- bacilli in pairs; Ex: Mycobacterium tuberculosis
 Non-sporing snapping diplobacilli- if the organism appears in V-shaped
 Non-sporing slipping diplobacilli- if the organism appears parallel to each other
o Streptobacilli (sporing streptobacilli)- bacilli in long chains; Ex: Bacillus subtilis
o Coccobacilli (non-sporing coccobacilli)- short rod shaped bacilli; Ex: Escherichia coli
o Fusiform bacilli- bacilli with tapered/point ends; Ex: Fusobacterium fusiforme
o Palisading bacilli- aligned side by side
3. Spirochetes- spiral/helical; long axis bends when in motion; Ex. Treponema pallidum
4. Spirillum- long axis remain rigid when in motion
5. Vibrio- curve rod-shaped, comma shaped; Ex. Vibrio cholerae
Diplococci Streptococci Staphylococci Tetrads Sarcina

Non-sporing Non-sporing
Sporing Non-sporing
snapping slipping Spirochetes Vibrio
Streptobacilli coccobacilli
diplobacilli diplobacilli

General Rules in Gram Staining

1. All cocci are gram positive except Neisseria, Veillonella, and Moraxella group.
2. All bacilli are gram negative except Bacillus, Clostridium, Mycobacterium, Corynebacterium, Listeria, Nocardia,
Erysipelothrix, Lactobacillus, Kurthia, Rothia.
3. All cocci are non-motile and non-spore-formers.
4. All encapsulated organisms are non-motile.
5. Bacillus and Clostridium are spore-forming bacteria.
6. Spiral organisms are very hard to stain but they are gram negative.

Phases of Bacterial Growth

1. Lag Phase/Physiologic Youth Phase- little or no growth, physiologic youth phase
2. Log/Exponential Phase- maximum rate of bacterial multiplication. During this phase, the bacteria is most susceptible
to antimicrobials due to high metabolic activity.
3. Plateau/Stationary Phase- number of bacteria alive is equal to the number of dead bacteria
4. Decline Phase/Degradation Phase/Death Phase- increase in number of dead bacteria

Nutritional Requirements
1. Carbon- for synthesis of cellular component
a. Litotroph/Autotroph- carbon source is from Carbon dioxide
b. Organotroph/Heterotroph- carbon source is from organic material i.e., glucose, lactose
2. Nitrogen- for protein synthesis
3. ATP- for energy
4. Water- bacteria is 70% water
5. Salt- halophilic organisms require salt for growth
6. Minerals- requires magnesium, sodium, potassium, iron
7. Additional or Other Requirements
a. X Factor/hemin- derived from hemoglobin degradation
b. V Factor/NAD/Coenzyme 1- produced by some bacteria (Staphylococcus aureus) and yeast
c. Protein- for Mycobacteria which requires increases amount of protein that is why the media is egg based
8. Environmental Requirements- oxygen, temperature, pH
I. Oxygen
a. Obligate aerobe- only survive in the presence of 15-21% O2 and 0.03% CO2.
b. Obligate anaerobe- lives only in the absence of O2. Their environmental requirement is 0% oxygen, 5-10%
hydrogen, 5-10% CO2, and 80-90% Nitrogen
c. Facultative anaerobe- generally aerobic but can also survive in the absence of O2
d. Aerotolerant anaerobe- an anaerobe that can survive even in the presence of O2
e. Microaerophile- requires only 5% of O2
f. Capnophile- requires 5-10% CO2
II. Temperature
o Psychrophilic/Cryophilic- cold temp. 15-20ᵒC
o Mesophilic- 30-37ᵒC; most pathogens belong here
o Thermophilic- warm temp. about 50-60ᵒC
o Hyperthermophilic/Extreme Thermophilic- 80-100ᵒC
III. pH- most bacteria require neutral or slightly alkaline pH 7.0-7.5
o Acid-loving- Lactobacillus acidophilus requires pH 3.0; tomato juice agar is used
o Alkali-loving- Vibrio requires pH 8-10; use Alkaline Peptone Water as a culture medium

Bacterial Replication
 4 Stages in bacterial replication and bacteria replicates/multiplies by binary fission (two identical daughter cells)
1. Unwinding of supercoiled DNA- this is required so that enzymes and cofactors involved in replication can access the
DNA molecule at the site where the replication process will originate.
2. Unzipping of DNA- Once the replication site is exposed, unzipping of the DNA begins.
3. Synthesis of new DNA strands- involves the enzyme DNA polymerase
4. Termination of replication- occurs when the two replication forks meet, resulting in 2 complete chromosomes.

Gene Transfer (3 Mechanisms Of Genetic Transfer)

 Bacterial transformation- “Free” or naked DNA found in the environment is taken up by a bacterial cell. The DNA
taken up by the bacteria will take 1 of 3 courses:
1. It is integrated into existing bacterial genetic material.
2. It is degraded.
3. It may replicate in the cytoplasm of the bacterial cell if it is a compatible plasmid.
 Bacterial transduction- this is where a virus injects DNA into the bacterial cell and once the virus injects DNA into
the bacterial cell, it may undergo:
o Lytic cycle- the replication of the bacterial chromosome is disrupted
o Lysogenic cycle- the bacteriophage DNA is incorporated into the bacterial genetic material
 Bacterial conjugation- DNA is transferred from one cell to another and this can be done by the sex pili

Bacterial Biochemistry and Metabolism

 Bacteria use biochemical pathways to catabolize carbohydrates (CHO) and produce energy by:
o Fermentation- anaerobic utilization of pyruvic acid. The end products of fermentation are lactate, butyrate,
ethanol, and acetoin which accumulates in the culture media.
o Oxidation- aerobic utilization of pyruvate. The most important pathway is TCA or Krebs Cycle and this cycle results
in the production of acid and evolution of CO2.

Bacterial Metabolic Pathways

 The starting carbohydrate for bacterial fermentation or oxidation is glucose. Bacteria break down glucose to pyruvic
acid by 3 metabolic pathways:
 1. Embden-Meyerhof-Parnas (EMP) Pathway- an anaerobic pathway; major pathway in conversion of glucose to
pyruvate and generates majority of the ATP needed by bacteria.
2. Pentose Phosphate Pathway- alternative to EMP pathway for carbohydrate metabolism. Glucose is converted to
ribulose-5-phosphate. ATP yield is lower as compared to EMP.
3. Entner-Duodoroff Pathway- an aerobic process which converts glucose-6-phosphate to pyruvate and
glyceraldehyde phosphate

Microbiological Culture
 Method of multiplying microbial organisms by letting them reproduce in a culture medium under controlled laboratory
conditions. It is the isolation of bacteria by the medical technologist, letting them grow in-vitro in the laboratory.
 One of the primary diagnostic methods in the microbiology lab

Biochemical Identification of Bacteria:

1. Utilization of variety of substances as a carbon source.
2. Production of specific end products from various substrates.
3. Production of an acid or alkaline pH in the test medium.

Culture Types
1. Pure Culture- made up of organisms from one specie
2. Mixed Culture- made up of organisms from different species
3. Stock culture- pure culture used as a supply for research

Culture Medium
 Nutrient medium used for growing microorganisms (isolating microorganisms)
 Types of Culture Medium According to Consistency or Physical State:
1. Liquid culture medium- no agar or any solidifying agent
2. Semi-solid culture medium- contains 0.5-1% agar
3. Solid culture medium- has 2-3% agar
 Types of Culture Medium According to Manner of Dispensing:
1. Plated Culture Media- dispensed into petri dishes
2. Tube Culture Media- dispensed into test tube
 Types of Culture Medium According to Composition:
1. Synthetic/chemically-defined Culture Medium- the exact amount of components are known
2. Complex Culture Medium- composition varies from batch to batch
3. Tissue Culture Medium- for organism that can’t grow on artificial culture media; uses cancer tissues like HeLa cells
 Types of Culture Medium According to Function or Use:
1. Simple or Basal Culture Media/General Isolation Culture Media- for non-fastidious microorganisms (organisms
that don’t require additional nutrients)
2. Enrichment Culture Medium- enhances growth of certain organisms
3. Enriched Culture Medium- for growing fastidious organisms (organisms that require additional nutrients for
growth)
4. Selective Culture Media- selects growth of desired organism and inhibits the growth of other
5. Transport Medium- for transporting samples to and from the lab; Ex: Stuart’s Medium, Cary-Blair Medium
6. Differential Culture Medium- differentiates glucose fermenters for Enterobacteriaceae family