CLINICAL BACTERIOLOGY – RMT 2024

INTRODUCTION TO BACTERIOLOGY (MOD 1)

Ms. Arbee Mae Castro, RMT, MLS (ASCPi )
02/07/22

BACTERIOLOGY
KOCH’S POSTULATES
The study of single-celled microorganisms that lack a true nucleus. → The agent must be present in every case of the disease.
Note: The key word here is “lack a true nucleus.” → The agent must be isolated and cultured in vitro.
→ The disease must be reproduced when a pure culture of the
HISTORY agent is inoculated into a susceptible host.
→ The agent must be recoverable from the experimentally-
Hippocrates (460-370 BC) infected host.
→ “Father of Medicine”
→ Founder of Medical Ethics Von Behring and Kitasato (1890)
→ Discovered that injection of animals with bacterial toxin
Aristotle (384-322) would result in the production of a substance in the
→ Proposed the “Spontaneous Generation Theory”. blood capable of preventing a disease which led to the
→ Spontaneous Generation Theory- states that living development of vaccines.
organisms could develop from non-living materials.
Edward Jenner
Hans and Zacharias Janssen (1950) → Father of Immunology.
→ “Dutch lens grinders”
→ Invented the first compound microscope. Joseph Lister
→ Founded Aseptic Surgery. *very important in
1660: Robert Hooke (1635-1703) Bacteriology*
→ Published "Micrographia”
Hans Christian Gram
1676: Anton Van Leeuwenhoek (1632-1723) → Employed Gram’s staining for microscopic bacterial cell
→ “Father of Microbiology” differentiation.
→ Invented the first microscope
Jules Bordet
1688: Francesco Redi (1626-1678) → Discovered Bordetella pertussis as the causative agent
→ Was an Italian physician who refuted the idea of for whooping cough.
spontaneous generation by showing that rotting meat
carefully kept from flies will not spontaneously produce Friedrich Loeffler
maggots. → Cultivated Kleb’s Loeffler’s bacilli.

1835: Agostino Bassi de Lodi Alexander Flemming

→ Showed that a disease affecting silkworms was caused → Discovered Penicillin from mold “Penicillum nolatum”.
by a fungus.
→ “Fungus” – the first microorganism to be recognized as
a contagious agent of animal disease. MICROBIAL TAXONOMY

1836: Theodor Schwann → Orderly classification and grouping of organisms into

→ Helped develop the “Cell Theory” “taxa” or categories.
→ Taxon means “arrangement”
1861: Louis Pasteur's (1822-1895) → Nomos means “law”
→ In addition to Francesco Redi, he also disproved the Note: Taxon + Nomos = Law of arrangement of microorganisms.
Spontaneous Generation Theory 3 CATEGORIES OF TAXONOMY
→ Proposed the “Germ Theory of Disease” – in which the → Classification
germs would be the one who would cause the disease. → Nomenclature
→ Identification
1876: Robert Koch (1843-1910)
→ “Father of Bacteriologic Technique” Proposed in Koch’s
Postulates in 1884 LINAEAN TAXONOMY
FORMAL RANK EXAMPLE
Domain Bacteria
Kingdom Prokaryotae
Division or Phylum Gracilicutes
Class Scotobacteria
Order Eubacteriales

Clinical Bacteriology, CDU – BSMT2A

 Family Enterobacteriaceae DNA commonly
Genus Escherichia found in Gram
Negative Bacteria
Species or Epithet coli
Asexual REPRODUCTION Sexual and Asexual
Subtype Escherichia coli O157:H7 Absent MEMBRANE-BOUND All
ORGANELLES
Absent in all LYSOSOMES Present in all;
SPECIES contain hydrolytic
enzymes
Absent in all ENDOPLASMIC Present in all; lipid
→ Most basic taxonomic group.
RETICULUM synthesis, transport
→ Further divided into: MITOCHONDRIA
Absent in all Present in most
• Subspecies (subsp) Present in all RIBOSOMES (SITES OF Present in all
• Serovarieties (serovar) PROTEIN SYNTHESIS)
• Biovarieties (biovar) 70S = 50S and 30S RIBOSOME SIZE 80S = 60S and 40S
In the cell ELECTRON In the mitochondria
membrane; no TRANSPORT FOR and chloroplasts
mitochondria ENERGY
NOMENCLATURE Absent except in the STEROLS (IN THE Present
mycoplasma CYTOPLASMIC
→ Binomial System MEMBRANE)
Lacks Carbohydrates PLASMA MEMBRANE Contains
glycolipids and
Remember:
glycoproteins
→ The 1st letter of family name is capitalized and has an –
Peptidoglycan CELL WALL Chitin (fungi),
aceae ending. glycans (algae),
e.g., Enterobacteriaceae lignin (plants)
→ The 1st letter of genus name is capitalized. Present as capsule or GLYCOCALYX Present
→ The Species name begins with lower case letter. slime layer
→ Both the genus and the species should be italicized in Absent CILIA Present
print or underlined when written in script. Simple Flagella FLAGELLA Complex flagella;
(composed of movement by
→ Abbreviation- The 1st letter of genus capitalized followed
flagellin); movement sliding filaments
by a period and the species name follows. e.g., E.coli by rotary action
→ The first syllable of first 2 letter are used when 2 or more Present PILI AND FIMBRIAE Absent
genera names begin with the same first letter. e.g., Ent.coli
or Esch.coli
PROKARYOTIC/BACTERIAL CELL STRUCTURE
CLASSIFICATION OF BACTERIA
Cytoplasmic Structures
Phenotypic → Readily observable.
Characteristics 1. NUCLEUS – no membrane bound nucleus
→ Genetic make-up of an 2. MESOSOME – point of attachment of chromosomes
organism based on their DNA
Genotypic and RNA structure and
3. RIBOSOMES – consists of RNA and protein; site of
Characteristics homology.
protein synthesis
4. CYTOPLASMIC GRANULES/ INCLUSION BODIES –
→ Prokaryotes
storage deposits of polysaccharides
Cellular type → Eukaryotes
→ Archaeobacteria Examples:

 Babes-Ernst Bodies/ Volutin Granules/

PROKARYOTE CHARACTERISTIC EUKARYOTE Metachromatic Granules
0.4 – 2.0 µm in TYPICAL SIZE 10-100 µm in
 Corynebacterium diptheriae
diameter diameter
 Endospores
0.5 – 5 µm in length >10 µm in length o Found in Bacillus and Clostridium
No nuclear NUCLEUS Classic membrane  Clostridium has Calcium
membrane; found in bound-nucleus dipicolinate or dipicolinic
the nucleoid region
acid in it (composition of
of the cytosol
Located in the GENOME In the nucleus
inclusion bodies)
nucleoid at the
mesosome
Circular complexed CHROMOSOMAL DNA Linear complexed
with RNA with histones and
other proteins
Plasmids, small EXTRACHROMOSOMAL In mitochondria
circular molecule of DNA and chloroplasts

Clinical Bacteriology, CDU – BSMT2A

Cell Envelope Structures 2. Pili (singular pilus) –are non-motile, long, hollow protein
tubes made up of pilin that connects two bacterial cells
1. PLASMA MEMBRANE (CELL MEMBRANE)
- phospholipid bilayer 2 types of Pili:

a. Sex/fertility pili – for sexual conjugation by

2. CELL WALL (MUREIN LAYER)
transferring DNA from 1 cell to another
- maintains shape of cell and has high affinity to dyes.
Composed of N-acetylglucosamine and N-
b. Somatic/common pili – for adhesion of bacteria to
acetylmuramic acid host cell

3. GRAM – POSITIVE
• thicker cell wall. Contains teichoic acid 3. Fimbriae (singular fimbria) – are non-flagellar, sticky,
Characteristics:
proteinaceous, hair-like appendages that aids in adhesion to
→ Permeability – osmotic pressure is maintained
tissues and to surfaces. It contributes to the virulence of the
→ Plasmolysis – a cell in a saline solution shrinks
because water passes out bacteria.
→ Plasmoptysis – a cell in distilled water bursts

NOTE: Gram-negative organisms have an outer membrane. Its purpose is to

serve as primary permeability barriers to hydrophilic and hydrophobic
compounds. It is a bilayer glycopolysaccharide

4. CAPSULE
- made up of polysaccharides. Act as virulence factors
(the bacteria cannot easily be easily destroyed); Appear
as clear halo

Organism

EUKARYOTIC CELL STRUCTURE

1. Nucleus – has chromosomes which contains DNA. They are

covered with basic proteins called histones. It is bounded by a
Capsule bilayered lipoprotein membrane known as the nuclear membrane.

2. Nucleolus – round, refractile body which is the site of ribosomal

5. SLIME LAYER RNA synthesis. It is located within the nucleus
- similar to capsules but are more diffuse layers
surrounding the cell. Also made up of polysaccharides 3. Endoplasmic Reticulum – is a system of membranes that
occur throughout the cytoplasm

Cell Appendages Divided into two:

 Rough Endoplasmic Reticulum

1. Flagella – are exterior protein filaments that rotate and
covered with ribosomes which gives it a “rough”
cause bacteria to be motile. This is referred to as “true”
appearance. It is the site of protein synthesis
motility. It is made up of a protein called flagellin
 Smooth Endoplasmic Reticulum
Classification According to number and arrangement of the flagella on no ribosomes. It doesn’t synthesize proteins but it
bacterial cell: synthesizes phospholipids

a. Atrichous – no flagellum 4. Golgi Apparatus – modify and package proteins sent by the
b. Monotrichous – single flagellum at one pole rough endoplasmic reticulum
c. Amphitrichous – flagellum on both poles of the
cell
5. Ribosomes – where protein synthesis occurs.
d. Lophotrichous – bundle of flagella in one or both
6. Mitochondria – main site of energy production
poles of the cell
e. Peritrichous – flagella surround the entire 7. Lysosomes – contains hydrolytic enzymes for degradation of
organism macromolecules and microorganisms within the cells

Clinical Bacteriology, CDU – BSMT2A

7. Peroxisomes –break down hydrogen peroxide

8. 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

9. Cell Wall – provides rigidity and strength to the exterior of the

cell. Most eukaryotic cells don’t have cell walls except for fungi. Sarcina
Eight cocci arranged in cuboidal pattern
Rep. organism
10. Cilia – short projections that extend from the cell surface and Sarcina lutea
used for locomotion

2. BACILLI (sing. Bacillus)- rod shaped

BACTERIAL MORPHOLOGY

1. COCCI (sing. Coccus) spherical in shape Diplobacilli Streptobacilli Coccobacilli

Bacilli in pairs Bacilli in chain Bacilli in short rods
or ovals
a. Diplococci- Neisseria gonorrheae Rep. Organism: Rep. Organism: Rep. Organism:
b. Streptococci- Streptococcus pyogenes Mycobacterium Bacillus subtilis Escherichia coli
c. Staphylococci- Staphylococcus aureus tuberculosis
d. Tetrads- Gaffkya tetragena Can either be in
e. Sarcina- Sarcina lutea snapping or
slipping
diplobacilli

Fusiform bacilli Palisading bacilli

Having one blunt and one Bacilli usually divide in the
pointed end and typically same plane and are solitary,
associated in pairs (fusiform but can combine to form
figure) diplobacilli, streptobacilli, and
palisades
Rep. Organism: Rep. Organism:
Diplococci Streptococci Fusobacterium fusiforme Corynebacterium diptheria
Cocci in pairs; coffee-bean Cocci strings
shaped
Rep. Organism: Rep. Organism:
Neisseria gonorrheae Streptococcus pyogenes
Can Either be extracellular or
intracellular diplococci

Staphylococci Tetrads
Colonies of cocci that are Groups of four organized in
irregular (grape-like) the same plane
Rep. Organism: Rep. Organism:
Staphylococci aureus Gaffyka Tetragena

Clinical Bacteriology, CDU – BSMT2A

3. SPIROCHETES - Spiral/helical. Jump long axis that bends PHASES OF BACTERIAL GROWTH
when in motion
1. Lag Phase/Physiologic Youth Phase – little or no growth
Ex. Treponema pallidum 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

4. SPIRILLUM - Long axis remains rigid when in motion

5. VIBRIO - Curve rod-shaped, comma shaped

Ex. Vibrio cholera

NUTRITIONAL REQUIREMENTS
1. Carbon – for synthesis of cellular component
a) Lithotroph/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’s why the media for culturing
mycobacteria is egg based

8. Environmental Requirement – oxygen, temperature, pH

GENERAL RULES I. OXYGEN

1. All cocci are gram positive except Neisseria, Veillonella, a) Obligate aerobe – only survive in the presence of 15-21
and Moraxella group. % O2 and 0.03%CO2; mostly it’s oxygen
2. All bacilli are gram negative except Bacillus, b) Obligate anaerobe – lives only in the absence of O2;
Clostridium, Mycobacterium, Corynebacterium, Listeria, environmental requirement:
 5-10% hydrogen
Nocardia, Erysipelothrix, Lactobacillus, Kurthia, Rothia.
 5-10% CO2
3. All cocci are non-motile and non-spore-formers.
 80-90% Nitrogen
4. All encapsulated organisms are non-motile.  0% oxygen
5. Bacillus and Clostridium are spore-forming bacteria. C. Facultative Anaerobe – Generally AEROBIC but can
6. Spiral organisms are very hard to stain but they are gram survive in the absence of O2
negative. D. Aerotolerant Anaerobe – An ANAEROBE that can
survive in the presence if O2
E. Microaerophile – requires only 5% of O2
F. Capnophile – Requires 5-10% CO2

Clinical Bacteriology, CDU – BSMT2A

 II. TEMPERATURE MICROBIOLOGICAL CULTURE
- method of multiplying microbial organisms by letting
A. Psychrophilic/Cryophilic – cold temp 15-20ᵒC them reproduce in a culture medium under controlled
B. Mesophilic – 30-37ᵒC; most pathogens belong here laboratory conditions.
C. Thermophilic – warm temperature about 50-60ᵒC - one of the primary diagnostic methods in the
D. Hyperthermophilic/Extreme Thermophilic – 80-100ᵒC microbiology lab

Biochemical Identification of Bacteria:

III. pH
1. Utilization of variety of substances as a carbon source.
most bacteria require neutral or slightly alkaline pH 7.0-7.5 2. Production of specific end products from various
substrates.
A. Acid-loving – Lactobacillus acidophilus requires pH 3.0; 3. Production of an acid or alkaline pH in the test medium.
tomato juice agar is used
B. Alkali-loving – Vibrio requires pH 8-10; use Alkaline
Peptone Water as a culture medium CULTURE TYPES

1. Pure Culture – made up of organisms from one specie

2. Mixed Culture – made up of organisms from different
BACTERIAL REPLICATION species
3. Stock culture – pure culture used as a supply for
4 Stages in Bacterial Replication: research
1. Unwinding of supercoiled DNA
2. Unzipping of DNA I. Types of Culture Medium According to CONSISTENCY OR
3. Synthesis of new DNA strands PHYSICAL STATE:
4. Termination of replication
1. Liquid culture medium – no agar or any solidifying
*Bacterial multiply via Binary Fission* (2 identical daughter cells) agent
2. Semi-solid culture medium – contains 0.5-1% agar
GENE TRANSFER 3. Solid culture medium – has 2-3% agar

3 Mechanisms of Genetic Transfer: II. Types of Culture Medium According to Manner of

1. Bacterial transformation DISPENSING:
2. Bacterial transduction
3. Bacterial conjugation 1. Plated Culture Media– in petri dishes
2. Tube Culture Media – in test tube

III. Types of Culture Medium According to COMPOSITION:

BACTERIAL BIOCHEMISTRY AND METABOLISM
1. Synthetic/chemically - defined culture medium – the
Bacteria use biochemical pathways to catabolize carbohydrates exact amount of components are known.
(CHO) and produce energy by: 2. Complex Culture Medium – composition varies from
batch to batch
1. FERMENTATION- anaerobic utilization of pyruvic acid 3. Tissue Culture Medium – for organism that can’t grow
on artificial culture media; use cancer tissues
2. OXIDATION – aerobic utilization of pyruvate

IV. Types of Culture Medium According to FUNCTION OR

USE:

BACTERIAL METABOLIC PATHWAYS 1. SIMPLE OR BASAL CULTURE MEDIA/GENERAL ISOLATION

The starting carbohydrate for bacterial fermentation or oxidation is CULTURE MEDIA
glucose. Bacteria break down glucose to pyruvic acid by 3 2. ENRICHMENT CULTURE MEDIUM
metabolic pathways: 3. ENRICHED CULTURE MEDIUM
a. (Used for growing fastidious organisms)
1. Embden-Meyerhof-Parnas (EMP) Pathway - anaerobic b. Organisms that require additional nutrients for
growth
2. Pentose Phosphate Pathway - alternative to EMP
4. SELECTIVE CULTURE MEDIA
3. Entner-Duodoroff Pathway - aerobic process a. Used to select growth of desired organisms and
inhibits the use of others

Clinical Bacteriology, CDU – BSMT2A

5. TRANSPORT MEDIUM
a. Transporting samples to and from the lab
i. STUART’S MEDIUM
ii. CARY-BLAIR MEDIUM
6. DIFFERENTIAL CULTURE MEDIUM
a. Used for glucose fermenters for Enterobacteriaceae
family

Clinical Bacteriology, CDU – BSMT2A