CLINICAL BACTERIOLOGY

Important Events in the Development of

Microbiology
1590-1595 Hans Jansen developed the first compound microscope.
1676 Antonie van Leeuwenhoek discovered animalcules.
1786 Otto Friedrich Muller produced the first classification of
bacteria.
1798 Edward Jenner introduced the smallpox immunization through
cowpox inoculation.
 Important Events in the Development of
Microbiology
1838-1839 Theodor Schwann and Matthias Jakob Schleiden
introduced the cell theory.
1858 Rudolf Virchow proposed the theory of biogenesis.
1861 Louis Pasteur used the aerobic and anaerobic
terminologies in describing the metabolism of yeast.
1880 -described the weak strain of organisms as attenuated in
developing vaccines
1881- developed the anthrax vaccine.
1885 -produced the rabies vaccine.
1867 Joseph Lister published his work on antiseptic surgery.
1875 Ferdinand Colm used the genus name Bacillus in
classifying bacteria.

1881 Robert Koch cultured bacteria on gelatin.

1882 Robert Koch discovered the tubercle bacilli and proposed
Germ theory
Koch's postulates were first published.
1884 Elie Metchnikoff described phagocytosis.
Autoclave was developed.
Hans Christian Gram introduced Gram stain.
• 1887 Julius Richard Petri introduced the use of the Petri dish.
• 1890 Emil von Behring prepared antitoxins for diphtheria and
tetanus.
• 1895 Jules Bordet discovered the complement.
• 1896 Ronald Ross showed that malarial parasites are carried
by mosquitoes
• 1900 Walter Reed proved that yellow fever is transmitted by
mosquitoes.
• 1901 Karl Landsteiner discovered the blood
group system.
• 1906 Fritz Schaudinn and Erich Hoffman
showed that the Treponema pallidum
causes syphilis.
• 1907 Recognition of the role of protozoa in
the disease process
• 1909 Sigurd Orla-Jensen established the
importance of including the physiologic
characteristics of bacteria in their
classification
• 1910 Paul Ehrlich developed a
chemotherapeutic agent for syphilis.
• 1937 Edouard Chatton introduced living organisms as either
prokaryotes or eukaryotes.
• 1944 Selman Waksman discovered streptomycin.
• 1945 Alexander Fleming discovered penicillin.
• 1951 Max Theiler developed the yellow fever vaccine.
• 1953 Francis Crick and James Watson discovered the structure of
DNA.
• 1954 Rosalyn Yalow and Solomon Berson developed the
radioimmunoassay.
• 1962 Cornelius van Niel and Roger Stanier described prokaryotes.
• 1976 Recognition of archaeobacteria as a distinct microbial group
• 1980 Robert Gallo and Luc Montagnier identified and isolated HIV.

• 1983-1984 Kary Mullis developed polymerase chain reaction (PCR) -

• 1986 The first hepatitis B vaccine created through genetic engineering

was approveuse.
• 1995 Chickenpox vaccine was approved.
• Craig Venter, Hamilton Smith, and Claire Fraser released the first
complete g
• sequence of a microorganism utilizing Haemophilus influenzae .
BACTERIAL TAXONOMY
-formal system of organizing,
classifying, and naming living
things.
Carl von Linne, a Swedish
botanist, laid down the basic
rules for taxonomic categories
(binomial system).
• CLASSIFICATIONS
BACTERIAL TAXONOMY
NOMENCLATURE
• It is the naming of microorganisms according to established guidelines
provided by the
International Code of Nomenclature of Bacteria or the Bacteriological Code.
• In writing the genus name,
The first letter capitalized
species epithet (specific name),
• which begins with a lower-case letter.
• italized in print but underlined when written in script
• (e.g.. Staphylococcus aureus or Staphylococcus aureus).
• 3. When bacteria are referred to as a group, their names are neither
capitalized nor underlined
• (e.g., staphylococci,streptococci, etc).
BACTERIAL CELL
STRUCTURE,PHYSIOLOGY,
METABOLISM & GENETICS
BY: CATH JOY B. RONQUILLO, RMT
MICROBIOLOGY
subdivided into 4 groups:
•Viruses = are the smallest known organisms (gen. < 1um)
= consist of either DNA or RNA ( but never both)

•Bacteria = are larger, (0.1 – 5 um or more in length)

= contains both DNA & RNA but genetic information is not organized w/in
the nucleus
= most can freely divide except for some (strict intracellular parasites-
means limited genetic info & metabolic capabilities)

•Fungi = are larger & more complex than bacteria

= genetic material is organized w/in a nucleus
•Parasite = complexity is much varied (ranging from simple single-cell organisms
such as amoeba to highly complex multicellular organisms such as worms
& insects)
3 TYPES OF CELLS (3-domain system)
• Eukaryotic
• Prokaryotic
• Archeobacteria
Difference between Prokaryotic and Eukaryotic cell
 EUKARYOTIC CELLS STRUCTURE
A. Cytoplasmic Structures
1. Nucleus
2. Nucleolus
3. ER( endoplasmic reticulum)
-Rough and Smooth
4. Ribosomes
-attached to ER; 80’S in size; 2 subunits (60’s and 40s
5. Mitochondria
6. Lysosomes
7. Chloroplast
 EUKARYOTIC CELLS STRUCTURE
B. Cell envelope Structures
1. Plasma membrane
2. Cell wall
3. Motility organelles
a. cilia- shorter projections
b. Flagella- longer
 PROKARYOTIC CELLS STRUCTURE
A. Cytoplasmic Structures
1. Nucleoid/chromatin body
-DNA appears as a diffuse nucleoid w/o a nuclear membrane
-DNA consists of a single circular chromosome
2.Ribosomes
- Free in the cytoplasm
- 70’S in size; 2 subunits (50s and 30s)
3.Cytoplasmic granules/metachromatic granules
-storage deposits
4. Endospores/spores
-resistant to harsh environmental conditions
 PROKARYOTIC CELLS STRUCTURE
B. Cell envelope Structures
1. Plasma membrane
-bilayered lipoprotein; without sterols
-site of electron transport chain for energy production
2. Cell wall
-rigid structure; different types (g+,g-,acid fast)
3. Capsule
-virulence factor;helps in evading phagocytosis
4. Cell appendages
a. pili/fimbriae-hairlike structures; for attachment (w/ adhesins)
b. Flagella-organ of locomotion
Bacterial Flagellar Arrangements
1. Monotrichous
2. Amphitrichous
3. Lophotrchous
4. Peritrichous
5. Atrichous
 CELL WALL
A. Gram-positive cell wall
1. peptidoglycan/murein
-principal component of g(+)cell wall
2. Teichoic acid
3.Lipoteichoic
 CELL WALL
A. Gram-negative cell wall
1. Inner peptidoglycan layer
2. Outer membrane
-unique to g- cell wall
-contains LPS(Lipopolysaccharides)
-contains 3 regions:
a. core polysacharride
b. antigenic O-specific polyssacharide
c. lipid A/endotoxin
-responsible for fever and shock
3 MAJOR NUTRITIONAL NEEDS
1. Source of carbon
2. Source of nitrogen
3. Source of energy (ATP)
AUTOTROPHS
-can grow simply using CO2 as sole source of carbon

HETEROTROPHS
-require complex organic sources such as glucose

FASTIDOUS
-require additional nutrients such as vitamins, hemoglobin etc.
ENVIRONMENTAL FACTORS INFLUENCING GROWTH
A. PH
-pathogenic bacteria grow best at neutral pH
-culture media area adjusted to pH 7.0-7.5

B. TEMPERATURE
1. Psychophiles-(10-20 degrees celcius)
2. Mesophiles-(20-40 degrees celcius)
3. Thermophiles -(50-60 degrees celcius)

 Humans pathogens grow at 37

 Cultures are routinely incubated at 35-37
ENVIRONMENTAL FACTORS INFLUENCING GROWTH
C. GASEOUS COMPOSITION

1. Obligate aerobes
2. Microaerophilic
3. Facultative anaerobes
4. Obligate anaerobes
5. Capnophilic- requires increased CO2
6. Microphilic- requires decreased CO2
BACTERIAL GROWTH
• Binary fission:one cell divides into 2 cells
• Generation time/ doubling time- time required for once cell to divide
into two cells
BACTERIAL GROWTH (PHASES)

1. Lag phase
2. Log phase
3. Stationary phase
4. Death or decline phase
 Bacterial Genetics:
1. Genotype
2. Phenotype
3. Bacterial genome/bacterial
chromosome
4. Plasmid
TRANSPOSONS
Transposons- usually located in plasmids; mobile elements that carry
antibiotic-resistance genes
• BRING UNEXPECTED TWISTS TO DNA
 Bacterial Genetics:
6. Insertion sequences-small mobile nucleic acid strands capable
of activating or inactivation genes
7. mutations-changes that occur in the DNA code
8. Point mutation- change in one nucleotide base; leads to a
change in a single amino acid within a protein; single base is
replaced with different one
9. Frameshift mutation- one or few nucleotide pairs are deleted
or inserted in the DNA
1. Base substitution
one base is inserted in place of another
takes place at the time of DNA replication

Frame shift mutation

insertion or deletion of one or two base pairs, disrupting the phase of the triplet-
encoded DNA message.

1. Missense mutation- when the base substitution results in a codon that simply
causes a different amino acid to be inserted

2. Nonsense mutation- when the base sustitution generates a termination codon

that stops protein sysnthesis prematurely
 GENETIC RECOMBINATION
GENE TRANSFER OR GENE EXHANGE BETWEEN INDIVIDUAL BACTERIAL
CELLS (HORIZONTAL TRANSFER)
• MECHANISMS:
1. Transformation- uptake and incorporated of naked DNA into
bacterial cell
2. Transduction- transfer of bacterial genes by bacteriophage
3. Conjugation- transfer of genetic material from a donor to
recipients strain which requires close contact between 2 cells
-mediated by the formation of a sex plus
-plasmids and chromosomal genes can be replicated and transferred
by this method.
Bacteriology:

CONTROL OF MICROORGANISMS
& SPECIMEN COLLECTION AND
HANDLING
STERILIZATION
-removal/killing of all forms of life

DISINFECTION
-removal/killing of pathogenic organism but not
necessarily bacterial or other spores
*disinfectants:applied to inanimate objects
*antiseptics: applied to living tissues
 FACTORS THAT INFLUENCE THE DEGREE OF
KILLING
1. Types of organisms
2. Number of organisms
3. Concentration of disinfecting agent
4. Amount of organic soil present
5. Nature of the surface to be disinfected
6. Temperature of pH of process
7. Length of contact time
8. Type of water available (hard vs soft)
9. Biofilms(community of bacteria)
10. Compatibility of disinfectants
Types of organisms according to resistance to
killing
1. Bacterial spores most resistant
2. Mycobacteria
3. Non-lipid viruses
4. Fungi
5. Bacteria (vegetative cells)
6. Lipid viruses (enveloped) least resistant
Device Classification
1. Critical materials- invade sterile tissues; require
sterilization
2. Semicritical materials-come in contact with
mucuos membranes; require high level
3. Noncritical materials- come in contact with
intact skin; require intermediate-level to low-
level disinfection.
 Device Classification

High Level Disinfection with sporicidal activity

Intermediate Level Disinfection Tuberculocidal but not sporicidal
Low-level Disinfection Non-sporicidal & non-tuberculocidal
METHODS OF DISINFECTION AND
STERILIZATION

I. Physical Methods
A. Heat

1. Incineration (870-980 °C)

omaterials are burned to ashes

2. Moist heat/ heat under pressure (autoclave)

osteam is put to 15psi and achieves 121 °C
o15mins exposure
oAll microorganisms are destroyed including spores
o 132°C for 30-60mins
A. Heat
3. Dry heat (oven)
oRequires longer time and higher temperature
o160-180 °C for 1.5-3hrs
oUsually employed for glasswares

4. Boiling
o100 °C in 10mins
oOnly disinfection is achieved (tuberculocidal)

5. Pasteurization
o63 °C for 30mins or 72°C for 15 secs (tuberculocidal)
oUsually employed in the food industry
II. Filtration

 Used for liquids and air via thin

membrane filters & applied to
heat sensitive solutions like
vaccines and antibiotics
III. Chemical Methods
*Use mainly as disinfectant and some are chemosterilizer

Mechanism of action:
1. Reaction with components of the cytoplasmic membrane
2. Denaturation of cellular proteins
3. Reaction with thiol(-SH) groups of enzymes
4. Damage to RNA and DNA
III. Chemical Methods
A. Alcohols
oMost common: Isopropyl and ethyl (70%)
oNon-sporicidal, non-tuberculocidal
oDenatures proteins

B. Aldehyde
1.Formaldehyde
2.Glutaraldehyde
ogermicidal and sporicidal
oKills through inactivation of DNA and RNA
oSterilizer for medical equipment
o“cold sterilization”
III. Chemical Methods
C. Halogens
1.Iodine(mainly used as antiseptics)
oNon-sporicidal,non-tuberculocidal

2. Chlorine and Chlorine compounds (tuberculocidal)

oHypochlorous acid oxidizes bacterial enzymes
oExamples: household bleach, water treatment
III. Chemical Methods
D. Heavy Metals
*precipitates proteins, lyses cell membranes
*action is primarily bacteriostatic/slowly bactericidal

1.Mercuric chloride- merthiolate

2.Silver nitrate – gonococcal conjunctivitis
III. Chemical Methods
E. Detergents
oQuaternary ammonium compounds
oDisrupts cell membrane
oNon-sporicidal, non-tuberculocidal

F. Phenolics
oDisrupts cell wall
oPrecipitate proteins
oSome may have tuberculocidal acitivity
oEx.lysol
III. Chemical Methods
G. Gases

A. Ethylene oxide- alkalytes

nucleic acids in the spore and
vegetative cell
B. H202
C. Periacetic acid (PAA)
SPECIMEN COLLECTION AND
HANDLING
Basic Principles of Specimen Collection
If possible, specimens should be collected during the acute phase of
infection and before the administration of antibiotics.
It must be initiated by written order followed by selection of a site of
a culture
Culture the infecting agents and avoid the usual flora
Results must be compared with suspected diagnosis carefully
 Appropriate Collection Techniques
PATIENT COLLECTED SAMPLES:
 Must be adequately instructed
 If possible a written instruction must
be given while the procedure is being
verbally instructed
 I. ASPIRATES AND TISSUES
 Must be collected using sterile technique
 best specimen
 Skin must be disinfected before collection of
blood or other spxn.
 Tissue sample must be protected from drying
 Aspirate from unopen wounds is superior from
an open draining wound
 II. SWABS
 Used for URTI spxn
 Used as last resort for wound spns
1. Clean the wound
2. Explore the wound
3. Obtain a fresh material
4. Obtain adequate quantity
III. URINE
 Perianal region must be washed
 Mid-tream catch
 IV. SPUTUM
 Specimen for LRT(lower respiratory tract)
 Remove denatures by rinsing mouth; gargle
with water
 Expectorate deep cough
 First morning specimen
 Aspiration may be necessary if
expectorating is not possible
 V. STOOL
 Kits/ containers should be provided
 Patients should be instructed to avoid
contamination
 Use of sterile containers may not be necessary*
PRESERVATION, STORAGE & TRANSPORT
MAJOR CONCERNS:
1. Overgrowth
2. Death of organisms due to temperature and pH change
3. Inaccurate quantitation of organisms
4. Loss of organisms due to drying
5. Protection from oxygen for anaerobes
6. Safety for transporter
Use of Preservatives:
Boric Acid: urine
Phosphate buffered saline: stool

Use of Anticoagulants:
Sodium polyanetholsulfonate (SPS);most common
Sodium amylsulfate (SAS)
Heparin : viral culture
Citrate and EDTA should not be used
 Use of Holding and Transport Media
• *keeps the organism viable while inhibiting
replication

Examples:
Carey-Blair
Modified-Stuart transport media
Amies
Storage of Specimens:
Refrigeration (4 °C): urine, viral
blood spn, catheters, swabs
Incubator (37 °C) : CSF, bacterial
blood spn, cultures on agar
plates
Room temperature (25°C): spns
for fungus isolation
Freezing (-70 °C) : if processing
will be delayed more than 4 days
 Labelling and Rejection of Specimen
I. Requisitions
II. Unacceptable/Sub-optimal spns
1.non-matching requisition and specimen
2. Leaking specimen
3. Syringes with needles attached
4. Stools contaminated with urine or barium
5. Anaerobes from inappropriate sources/containers
6.Unpreserve specimen older than 2 hours
7. Refrigerated blood cultures for bacteriology
8. dried-up specimen
9. Specimen in formalin
10. Specimen in non-sterile container
11. QNS Specimen
12.Inadequate specimen
Bacteriology:

DETECTION METHODS
Universal Precautions
1. Barrier protection-gloves, masks, lab gown, puncture resistant
biohazard container(color)
2. Engineering controls – protects while working(BSC) from aerosols
a. Heat
b. UV
c. HEPA(High efficiency particulate air) – removes 0.3um particle
BSC classes
CLASS DESCRIPTION

Class 1 Open(unsterilized air circulates), Negative

pressure
Exhaust air sterilized by HEPA filters

Class II Both air entering, circulating and exhaust is

fertilized(routine)

Class III Entirely enclosed, infectious materials are

handled with rubber gloves
(highest level of safety)
One HEPA- sterilizes supply air
Second – for exhaust air
 Biosafety levels(BSL) for Infectious
Agents
Type Usual agent Precaution

BSL 1 Not pathogenic in adhere to lab standard

immunocompetent(undergr
aduate)
B. subtilis
BSL 2 Lab acquired/pathogenic Level 1/Class 1 and 2 BSC
HBV, HIV,staph, enterics and protective barriers

BSL 3 Air movement must be Level II/BSC II(controlled

controlled(MTB, access)
Coccioides,Rickettsiae) Can be treated

BSL 4 Research lab Level III – no Tx available

Exotic virus-
filovirus,arenavirus
DIRECT EXAMINATION TECHNIQUES
 Smear Preparation

I. Tissues- performed using

touch preparations
 Smear Preparation

II. Swabs-must be rolled

back and forth across a
dry, clean slide
 Smear Preparation
III. Aspirate and body fluid
Single Drop Smear- If high numbers of organisms
are expected; thick specimen

Centrifuged smear- If small number of organisms is

expected
Layered smear- used for low-volume specimens
Cytocentrifuged Smear-an aliquot of the specimen
is spun directly into a slide
 STAINING
I. Gram Stain- g(+) or g(-)
oCell wall structure determines the G/S characteristics
REAGENT PURPOSE G(+) CELL WALL G(-) CELL WALL
Crystal violet Primary stain Purple Purple

Grams Iodine Mordant Purple Purple

Acetone-Alcohol Decolorizer Purple Colorless

Safranin Red Counterstain Purple Pink

 STAINING
II. Acid Fast
oUsed primarily to detect Mycobacterium species
oFuchsin Stains (2 methods):
o Kinyoun method- also known as the cold method;uses a higher conc. of
detergent to facilitate statining
o Ziehl-Neelsen method-hot method; uses heat to facilitate staining
REAGENT PURPOSE ACID FAST CELL WALL NON-ACID FAST CELL
Carbolfuchsin Primary stain Red Red

Acid-Alcohol Decolorizer Red Colorless

Methylene blue Counterstain Red Blue

oFluorochrome Stains
oAuramine-rhodamine
Acid-fast organism
fluoresce yellow or orange
under a fluorescent
microscope
 STAINING
III. Other Staining Methods
a. Methylene blue- use to observe
metachromatic granules in Corynebacterium
diptheriae

b. Acridine Orange- fluorochrome dye that stains

both g+ and g- organism both living and
dead.(highly sensitive)
 Microscopic Examination of Unstained
Specimens
1. Saline Mount
-to determine the biologic activity of
microorganism including motility or reactions to
certain chemicals or serologic reactivity in specific
antisera.
 Microscopic Examination of Unstained
Specimens
2. Hanging-drop procedure
-uses a hanging-drop slide ( a thick glass slide with a central concave well)
-same purpose with the saline mount but with a deeper field of focus and
without the distorting effects from the weight of the coverslip.
-generally used to observe motility
 Microscopic Examination of Unstained
Specimens
3. Darkfield examination
-used to visualize organisms that are invisible by
brightfield microscopy and stain only with great
difficulty
-particularly useful in demonstrating spirochetes
The large number of epithelial cells in groups
1 through 4 indicates contamination.