Bacterial Cell Structure,

Physiology, Metabolism
and Genetics

Presented by: Tom Anthony A. Tonguia, RMT, CSSO

JMCFI – MLS Department
MLS 3A Bacteriology
Objectives
1. Classify bacteria based their anatomy and physiology, mode
of replication, genetics, microscopic characteristics, and
classification.
2. Determine the nutritional and physiology requirements for
bacterial growth
3. Elucidate the bacterial growth curve and identity the factors
that influence each of the phases.
Table of contents

01 02 03
Overview of the Classification and Bacterial
microbial world taxonomy morphology

04 05 06
Microbial Growth Bacterial
Biochemistry and Bacterial Genetics
and Nutrition metabolism
01
Overview of the
microbial world
Overview
● The study of microorganisms by the
Dutch biologist and lens maker Anton
van Leeuwenhoek has evolved
immensely from its early historical
beginnings.
● Today we know that there are
enormous numbers of microbes in,
on, and around us in our
environment.
● Many of these microbes do not cause
disease.
Overview

Bacteria Parasites
• Protozoa are unicellular organisms
• Bacteria are unicellular organisms
within the kingdom Protista that
that lack a nuclear membrane and
obtain their nutrition through
true nucleus.
ingestion.
• They are classified as prokaryotes
• Some are capable of locomotion
(Greek: before kernel [nucleus]),
(motile), whereas others are
having no mitochondria,
nonmotile.
endoplasmic reticulum (ER), or
• They are categorized by their
Golgi bodies.
locomotive structures: flagella
• The absence of the preceding
(Latin: whiplike), pseudopodia
bacterial cell structures
(Greek: false feet), or cilia (Latin:
differentiates them from
eyelash)
eukaryotes
Overview
Fungi
• Fungi are heterotrophic eukaryotes
that obtain nutrients through
absorption.
• Yeasts are a group of unicellular Viruses
fungi that reproduce asexually.
• Most fungi are multicellular, and • Viruses are the smallest infectious
many can reproduce sexually and particles (virions); they cannot be
asexually. seen under an ordinary light
• Molds are filamentous forms that
can reproduce asexually and microscope.
sexually. • They are neither prokaryotic nor
• Certain fungi can assume both eukaryotic.
morphologies (yeast and • Many times we can see their effects
hyphae/mycelial forms), growing as on cell lines, such as inclusions,
yeast at incubator or human rounding up of cells, and syncytium
temperature and as the filamentous where these characteristics
form at room temperature become diagnostic for many viral
(Dimorphic fungi). diseases
02
Classification
and Taxonomy
Classification/Taxonomy

● Taxonomy (Greek taxes: arrangement; Greek nomos: law)

is the orderly classification and grouping of organisms
into taxa (categories).
● Taxonomy involves three structured, interrelated
categories:
○ Classification/Taxonomy, Nomenclature, and
Identification.
● It is based on similarities and differences in genotype and
phenotype.
Classification and Taxonomy
• Diagnostic microbiologists traditionally emphasize placement
and naming of bacterial species into three (occasionally four
or five) categories:

1. Family
2. Genus
3. Species
4. Strains
Classification and Taxonomy
• The following standard rules for denoting bacterial names are used.
• The family name is capitalized and has an “-aceae” ending (e.g.,
Micrococcaceae).
• The genus name is capitalized and followed by the species epithet,
which begins with a lowercase letter; both the genus and the species
should be italicized in print but underlined when written in script
(e.g., Staphylococcus aureus or Staphylococcus aureus).
• Often the genus name is abbreviated by using the first letter
(capitalized) of the genus followed by a period and the species
epithet (e.g., S. aureus).
Classification and Taxonomy
• To eliminate confusion, the first two letters or the first syllable are
used when two or more genera names begin with the same first
letter (e.g., Staph. and Strept. for when Staphylococcus and
Streptococcus are discussed).
• The reason for combining the two allows the species epithet to be
used for a different species in another genus.
03
Bacterial
Morphology
Bacterial Morphology
● Bacteria vary in size from 0.4 to 2 µm. They occur in three
basic shapes (Figure 1-5):
○ Cocci (spherical)
○ Bacilli (rod-shaped)
○ Spirochetes (spiral)
● Cocci may occur singly, in pairs (diplococci), in chains
(streptococci), or in clusters (staphylococci).
● Bacilli (plural of bacillus) may vary greatly in size and
length from very short coccobacilli to long filamentous
rods and the ends may be square or rounded.
● Bacilli with tapered, pointed ends are termed fusiform.
● Some bacilli are curved.
● When a species varies in size and shape within a pure
culture, the bacterium is pleomorphic.
● Bacilli may occur as single rods or in chains or may align
themselves side by side (palisading).
● Spirochetes vary in length and in the number of helical
turns (not all helical bacteria are called spirochetes)
Common Stains used for
Microscopic Visualization
● Gram Stain
○ Most commonly used stain in the clinical microbiology laboratory.
○ It places bacteria into one of two main groups: gram-positive (blue to purple) or
gram-negative (pink).
● Acid Fast Stain
○ Acid-fast stains are used to stain bacteria that have a high lipid and wax content in
their cell walls and do not stain well with traditional bacterial stains.
● Acridine Orange
○ Acridine orange is a fluorochrome dye that stains both gram positive and gram-
negative bacteria, living or dead.
○ It binds to the nucleic acid of the cell and fluoresces as a bright orange when a
fluorescent microscope is used.
● Endospore stain
○ The endospores appear green within pink-appearing or red-appearing bacterial cells
Common Stains used for
Microscopic Visualization
● Calcofluor White
○ Calcofluor white is a fluorochrome that binds to chitin in fungal cell walls.
○ It fluoresces as a bright apple-green or blue-white, allowing visualization of fungal
structures with a fluorescent microscope.
○ Calcofluor white was the original “blueing” used in high-volume laundries to whiten yellow-
appearing white cotton and other fabrics.
● Methylene Blue
○ Methylene blue traditionally has been used to stain C. diphtheriae for observation of
metachromatic granules.
○ It is also used as a counterstain in acid-fast staining procedures.
● Lactophenol Cotton Blue
○ Lactophenol cotton blue is used to stain the cell walls of medically important fungi grown in
slide culture.
● India Ink
○ India ink is a negative stain used to visualize capsules surrounding certain yeasts, such as
Cryptococcus spp.
RECAP
04
Microbial Growth
and Nutrition
Microbial Growth and Nutrition

● All bacteria have three major nutritional needs for growth:

○ A source of carbon (for making cellular constituents).
■ Carbon represents 50% of the dry weight of a bacterium.
○ A source of nitrogen (for making proteins).
■ Nitrogen makes up 14% of the dry weight.
○ A source of energy (adenosine triphosphate [ATP], for performing cellular functions).
● Smaller amounts of molecules, such as phosphate for nucleic acids and phospholipids of
cell membranes and sulfur for protein synthesis, make up an additional 4% of the weight.
Environmental Factors Influencing Growth

● Bacteria that grow on humans vary in their atmospheric requirements for growth.
○ Obligate aerobes require oxygen for growth.
○ Aerotolerant anaerobes, previously referred to as facultative aerobes, can survive in
the presence of oxygen but do not use oxygen in metabolism (e.g., certain
Clostridium spp.).
○ Facultative anaerobes can grow either with or without oxygen.
○ Obligate anaerobes cannot grow in the presence of oxygen.
○ Capnophilic organisms grow best when the atmosphere is enriched with extra
carbon dioxide (5% to 10%).
○ Microaerophilic bacteria require a reduced level of oxygen to grow.
Environmental Factors Influencing Growth

● Temperature influences the rate of growth of a bacterial culture.

● Microorganisms have been categorized according to their optimal temperature for growth.
○ Bacteria that grow best at cold temperatures are called psychrophiles (optimal
growth at 10° to 20°C).
○ Bacteria that grow optimally at moderate temperatures are called mesophiles
(optimal growth at 20° to 40°C).
○ Bacteria that grow best at high temperatures are called thermophiles (optimal
growth at 50° to 60°C).
● Psychrophiles and thermophiles are found environmentally in places such as the Arctic
seas and hot springs, respectively
Environmental Factors Influencing Growth

● Temperature influences the rate of growth of a bacterial culture.

● Microorganisms have been categorized according to their optimal temperature for growth.
○ Bacteria that grow best at cold temperatures are called psychrophiles (optimal
growth at 10° to 20°C).
○ Bacteria that grow optimally at moderate temperatures are called mesophiles
(optimal growth at 20° to 40°C).
○ Bacteria that grow best at high temperatures are called thermophiles (optimal
growth at 50° to 60°C).
● Psychrophiles and thermophiles are found environmentally in places such as the Arctic
seas and hot springs, respectively
Growth Curve

● If bacteria are in a balanced growth state, with enough nutrients and no toxic products
present, the increase in bacterial numbers is proportional to the increase in other
bacterial properties, such as mass, protein content, and nucleic acid content.
● Measurement of any of these properties can be used as an indication of bacterial growth.
● Four phases of growth:
○ Lag phase
■ bacteria are preparing to divide
○ Log phase
■ bacteria numbers increase logarithmically
○ Stationary phase, in which
■ nutrients are becoming limited and the numbers of bacteria remain constant
○ Death phase
■ the number of nonviable bacterial cells exceeds the number of viable cells.
Determination of Cell Numbers

● In the diagnostic laboratory, the number of bacterial cells present is determined in one of
three ways:
○ Direct counting under the microscope
○ Direct plate counter
○ Density measurement
04
Bacterial Biochemistry
and metabolism
Metabolism

● Microbial metabolism consists of the biochemical reactions bacteria use to break down
organic compounds and the reactions they use to synthesize new bacterial parts from the
resulting carbon skeletons.
● Bacteria vary widely in their ability to use various compounds as substrates and in the end
products generated.
● Various biochemical pathways exist for substrate breakdown in the microbial world, and
the particular pathway used determines the end product and final pH of the medium.
● Diagnostic schemes analyze each unknown microorganism for:
○ utilization of various substrates as a carbon source
○ production of specific end products from various substrates
○ production of an acid or alkaline pH in the test medium
Fermentation and Respiration

● Bacteria use biochemical pathways to catabolize (break down)

carbohydrates and produce energy by two mechanisms:
○ Fermentation is an anaerobic process carried out by both obligate

and facultative anaerobes.

○ Respiration (not an act of breathing) is an efficient energy

generating process in which molecular oxygen is the final

electron acceptor.
Biochemical Pathways from Glucose to Pyruvic Acid

● The starting carbohydrate for bacterial fermentations or oxidations is

glucose.
● When bacteria use other sugars as a carbon source, they first convert
the sugar to glucose, which is processed by one of three pathways.
● These pathways are designed to generate pyruvic acid, a key three-
carbon intermediate.
○ Embden-Meyerhof-Parnas Glycolytic Pathway

○ Pentose Phosphate Pathway

○ Entner-Doudoroff pathway
Aerobic Utilization of Pyruvate (Oxidation)

● The most important pathway for the complete oxidation of a

substrate under aerobic conditions is the Krebs or tricarboxylic acid
(TCA) cycle.
● In this cycle, pyruvate is oxidized, carbon skeletons for biosynthetic
reactions are created, and the electrons donated by pyruvate are
passed through an electron transport chain and used to generate
energy in the form of ATP.
Carbohydrate Utilization and Lactose Fermentation

● The ability of microorganisms to use various sugars (carbohydrates)

for growth is an integral part of most diagnostic identification
schemes.
● The fermentation of the sugar is usually detected by acid production
and a concomitant change of color resulting from a pH indicator
present in the culture medium.
Anaerobic Utilization of Pyruvic Acid (Fermentation)

● Pyruvic acid is a key metabolic intermediate.

● Bacteria process pyruvic acid further using various fermentation
pathways.
● Each pathway yields different end products, which can be analyzed and
used as phenotypic markers.
○ Alcoholic fermentation
■ This is the pathway used by yeasts when they ferment glucose to
produce ethanol.
○ Homolactic fermentation
■ All members of the Streptococcus genus and many members of
the Lactobacillus genus ferment pyruvate using this pathway
Anaerobic Utilization of Pyruvic Acid (Fermentation)

○ Heterolactic fermentation
■ Some lactobacilli use this mixed fermentation pathway, of which, in addition to
lactic acid, the end products include carbon dioxide, alcohols, formic acid, and
acetic acid.
○ Propionic acid fermentation
■ Propionic acid is the major end product of fermentations carried out by
Propionibacterium acnes and some anaerobic non–spore-forming, gram-
positive bacilli
○ Mixed acid fermentation
■ Members of the genera Escherichia, Salmonella, and Shigella within the
Enterobacteriaceae use this pathway for sugar fermentation and produce a
number of acids as end products—lactic, acetic, succinic, and formic acids
○ Butanediol fermentation
■ Members of the genera Klebsiella, Enterobacter, and Serratia within the
Enterobacteriaceae use this pathway for sugar fermentation
Anaerobic Utilization of Pyruvic Acid (Fermentation)

○ Heterolactic fermentation
■ Some lactobacilli use this mixed fermentation pathway, of which, in addition to
lactic acid, the end products include carbon dioxide, alcohols, formic acid, and
acetic acid.
○ Propionic acid fermentation
■ Propionic acid is the major end product of fermentations carried out by
Propionibacterium acnes and some anaerobic non–spore-forming, gram-
positive bacilli
○ Mixed acid fermentation
■ Members of the genera Escherichia, Salmonella, and Shigella within the
Enterobacteriaceae use this pathway for sugar fermentation and produce a
number of acids as end products—lactic, acetic, succinic, and formic acids
○ Butanediol fermentation
■ Members of the genera Klebsiella, Enterobacter, and Serratia within the
Enterobacteriaceae use this pathway for sugar fermentation
05
Bacterial Genetics
Genetic Elements and Alterations

● The bacterial chromosome (also called the genome) consists of a

single, closed, circular piece of dsDNA that is supercoiled to fit inside
the cell.
● It contains all the information needed for cell growth and replication.
● In addition to the genetic information encoded in the bacterial
chromosome, many bacteria contain extra information on small
circular pieces of extrachromosomal, dsDNA called plasmids.
● They are not essential for bacterial growth, so they can be gained or
lost.
Mutations

● Certain pieces of DNA are mobile and may jump from one place in the
chromosome to another place.
● These are sometimes referred to as jumping genes.
● The simplest mobile piece of DNA is an insertion sequence (IS)
element.
● It is approximately 1000 base pairs long with inverted repeats on each
end.
● The main effect of IS elements in bacteria is that when an IS element
inserts itself into the middle of a gene, it disrupts and inactivates the
gene
Mechanisms of Gene Transfer
● Transformation
○ the uptake and incorporation of naked DNA into a bacterial cell
○ Once the DNA has been taken up, it can be incorporated into the bacterial genome by recombination
● Transduction
○ The transfer of bacterial genes by a bacteriophage (virus-infected bacterium) from one cell to another
○ A bacteriophage consists of a chromosome (DNA or RNA) surrounded by a protein coat. When a phage
infects a bacterial cell, it injects its genome into the bacterial cell, leaving the protein coat outside.
● Conjugation
○ Conjugation is the transfer of genetic material from a donor bacterial strain to a recipient strain.
○ Close contact is required between the two cells. In the E. coli system, the donor strain (F+) possesses a
fertility factor (F factor) on a plasmid that carries the genes for conjugative transfer.
● Restriction Enzyme
○ Bacteria have evolved a system to restrict the incorporation of foreign DNA into their genomes.
○ Specific restriction enzymes are produced that cut incoming, foreign DNA at specific DNA sequences.
 Specimen
Collection
and Processing
Presented by: Tom Anthony A. Tonguia
Table of contents

01 02 03
Basic Principles of Preservation, Transport, Specimen
Specimen Collection Storage of Speciments
Processing

04
Culture Workup
Objectives
1. Point out the specific considerations and requirements for
specimen collection, transport, and processing
2. Enumerate the biosafety requirements to ensure personnel
and environmental safety in specimen collection, transport,
and processing.
3. Create a process flow for specimen collection, transport, and
processing.
Introduction
● The laboratory can make accurate and useful determinations only if a specimen
has been collected properly.
● The specimens to be analyzed are likely to contain living organisms; the goal of
the specimen collector must be to maintain the viability of these organisms
with minimal contamination.
Collection Procedures
● Specimens for microbiology cultures should be collected in sterile containers
except for stool specimens, which can be collected in clean, leakproof
containers.
● Generally, swabs are not recommended for collection because they do not
provide sufficient quantity, are easily contaminated, and can become dried out
leading to a loss of organisms.
● Swabs are appropriate for specimens from the upper respiratory tract,
external ear, eye, and genital tract.
● The tips of swabs may contain cotton, Dacron, or calcium alginate.
● Swab collection systems are available that provide transport media and
protect the specimen from drying.
Collection Procedures
● Lesions, wounds, and abscesses present many problems to the microbiology
laboratory.
● The term wound is not an appropriate specimen label, and the exact anatomic
site must be provided.
● The specimen is collected from the advancing margin of the lesion and should
be collected by needle aspiration rather than by swab.
● Before the specimen is collected, the area should be cleansed to eliminate as
much of the commensal flora as possible.
● Aspirated material should be placed into a sterile tube or transport vial and not
“squirted” onto a swab.
Collection Procedures
● Urine
○ Clean-catch midstream urine specimen
● Sputum
○ Sputum specimens are often collected for the diagnosis of bacterial
pneumonia.
○ Lower respiratory tract specimens are among the most difficult
specimens to collect adequately because they are contaminated with
oropharyngeal flora.
○ First early morning specimen is preferred
● Stool
○ A rectal swab can be submitted for bacterial culture as long as fecal
material is visible on the swab.
Safety
● It is imperative that specimens collected for microbiology not pose a safety
hazard to the individuals who handle them.
● Leaking containers and specimens with needles attached present the greatest
hazards.
● All specimens must be transported in leakproof secondary containers.
● Transporting personnel should refuse to transport specimens without the
protection of a secondary container.
Labeling and Requisitions
Specimen Storage
● Some specimens that will not be transported or processed immediately can be
maintained by being stored under certain conditions.
● The individual responsible for storing the specimen needs to be informed as to
the best storage environment for each specimen type.
● Some specimens, such as urine, stool, sputum, swabs (not for anaerobes),
foreign devices such as catheters, and viral specimens can be maintained at
refrigerator temperature (4°C) for 24 hours.
Preservatives
● Two specimen types in which preservatives can be used are urine and stool.
● Boric acid is used in commercial products to maintain accurate urine colony
counts.
● The systems are designed to maintain the bacterial population in the urine at
room temperature for 24 hours and are useful for collection of urine
specimens at distant locations.
● Stool specimens for bacterial culture that are not transported immediately to
the laboratory can be refrigerated; if the delay is longer than 2 hours, the
specimen can be added to Cary-Blair transport media.
Anticoagulants
● Anticoagulants are used to prevent clotting of specimens, including blood, bone
marrow, and synovial fluid.
● Organisms bound up in clotted material are difficult to isolate.
● The type of anticoagulant used and the concentration are important because
some anticoagulants have antimicrobial properties.
● Sodium polyanethol sulfonate (SPS) is the most common anticoagulant used
for microbiology specimens.
Holding or Transport Media
● Another way to maintain the integrity of the specimen from the time of specimen
collection until laboratory processing of the sample is with the use of holding or
transport media
○ Stuart’s or Amie’s transport medium is commonly used.
● Some transport systems contain charcoal to absorb fatty acids given off by the swab
that can be detrimental to the survival of Neisseria gonorrhoeae and Bordetella
pertussis
● In certain situations, direct inoculation to culture media at the time of specimen
collection (bedside inoculation) is optimal for isolation of the pathogen.
● Blood is usually placed into a broth culture medium immediately after collection.
● Synovial and peritoneal fluids also can be inoculated into blood culture broth bottles
at the bedside.
● Besides the blood culture bottles, additional specimen should be sent to the
laboratory in a container for Gram stain preparation.
● Specimens for N. gonorrhoeae can be placed directly onto a commercial transport
system such as the JEMBEC system
Holding or Transport Media
● Another way to maintain the integrity of the specimen from the time of specimen
collection until laboratory processing of the sample is with the use of holding or
transport media
○ Stuart’s or Amie’s transport medium is commonly used.
● Some transport systems contain charcoal to absorb fatty acids given off by the swab
that can be detrimental to the survival of Neisseria gonorrhoeae and Bordetella
pertussis
● In certain situations, direct inoculation to culture media at the time of specimen
collection (bedside inoculation) is optimal for isolation of the pathogen.
● Blood is usually placed into a broth culture medium immediately after collection.
● Synovial and peritoneal fluids also can be inoculated into blood culture broth bottles
at the bedside.
● Besides the blood culture bottles, additional specimen should be sent to the
laboratory in a container for Gram stain preparation.
● Specimens for N. gonorrhoeae can be placed directly onto a commercial transport
system such as the JEMBEC system
Unacceptable Specimens and Specimen Rejection
● The analytical phase of the laboratory testing process begins as the specimen
is received in the laboratory.
● On receipt in the laboratory, the specimen needs to be examined to ensure that
it has been properly selected, collected, and transported.
● Performing tests on specimens that are of poor quality would yield misleading
information that might result in misdiagnosis and inappropriate therapy.
● The microbiology laboratory must establish and publish the criteria for
specimen rejection.
Macroscopic and Microscopic Observation
● Notations from the macroscopic observation should include the following:
○ Swab or aspirate
○ Stool consistency (formed or liquid)
○ Blood or mucus present
○ Volume of specimen
○ Fluid—clear or cloudy
● A direct microscopic examination is a useful tool that provides rapid
information.
● In critical situations, such as meningitis, the direct microscopic examination
can be used to guide therapy choices when therapy must be initiated before
culture results are available.
Types of Culture Media
● Nonselective media support
○ the growth of most nonfastidious microbes.
● Selective media support the growth of one type or group of microbes but not another.
○ contain inhibitory substances such as antimicrobials, dyes, or alcohol. for gram-
positive organisms.
● Differential media
○ allow grouping of microbes based on different characteristics demonstrated on the
medium.
○ Media may be differential and nonselective (e.g., sheep blood agar is nonselective but
differentiates organisms on the basis of hemolysis)
● Enriched media
○ contain growth enhancers that are added to nonselective agar to allow fastidious
organisms to flourish.
● Broth media
○ can be used as a supplement to agar plates to detect small numbers of most
aerobes, anaerobes, and microaerophiles
Types of Culture Media
● Nonselective media support
○ the growth of most nonfastidious microbes.
● Selective media support the growth of one type or group of microbes but not another.
○ contain inhibitory substances such as antimicrobials, dyes, or alcohol. for gram-
positive organisms.
● Differential media
○ allow grouping of microbes based on different characteristics demonstrated on the
medium.
○ Media may be differential and nonselective (e.g., sheep blood agar is nonselective but
differentiates organisms on the basis of hemolysis)
● Enriched media
○ contain growth enhancers that are added to nonselective agar to allow fastidious
organisms to flourish.
● Broth media
○ can be used as a supplement to agar plates to detect small numbers of most
aerobes, anaerobes, and microaerophiles