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General Concepts in

Microbiology 2
Anchela Y. Uy-Biag, MD, MBA, DPASMAP
Content Outline
• Microbial Growth
• Cultivation Methods
• Media selection and isolation
• Metabolism
• Methods of detection and identification
– Microscopy and Staining
– Antigen detection
– Antibiotic susceptibility
Microbial Growth
Growth of Microbes
• Increase in number of cells, not cell size
• One cell becomes colony of millions of cells
• Control of growth is important for
– infection control
– growth of industrial and
biotech organisms
Bacterial Division
• Bacteria divide by binary fission usually
perpendicular to the length axis and thereby
two new cells are produced
• Alternative means
– Budding
– Conidiospores (filamentous bacteria)
– Fragmentation
Fig. 7.13
Generation Time
• Time required for cell to divide/for population
to double
• Average for bacteria is 1-3 hours
• E. coli generation time = 20 min
– 20 generations (7 hours), 1 cell becomes 1 million
cells!
For a unicellular bacterium, the cell number increase
exponentially with base 2 as seen in the table below:
Plotting growth on graphs

Linear growth

Exponential growth
Standard Growth Curve
Phases of Growth
• Lag phase - making new enzymes in response to new
medium
- the cell devision is delayed due to how the
inoculum has been treated

• Log phase - exponential growth


- as fast as the soluble nutrients permit
- the doubling time can be determined here
- most sensitive to drugs and radiation during this
period
Phases of Growth
• Stationary phase –
– nutrients becoming limiting or waste products
becoming toxic
– an essential nutrient has ceased
– pH-changes due to end products
– death rate = division rate
• Death phase – death exceeds division
– due to some toxic substance excreted from the
bacteria
Growth in Colonies

• A pure culture contains only one species or strain.


• A colony is a population of cells arising from a single
cell or spore or from a group of attached cells.
• A colony is often called a colony-forming unit (CFU).
Streak Plate
Measuring Bacterial Growth
Serial Dilutions
• Direct Measurements of Microbial Growth
• Plate counts: Perform serial dilutions of a sample
Standard Plate Count

Inoculate
Petri plates
from serial
dilutions
2 methods:
Pour Plate
Spread Plate
Plate Count
After incubation, count
colonies on plates that have
25-250 colonies (CFUs)
Direct Measurements of Microbial Growth

Filtration
Direct Measurements of Microbial Growth

Direct microscopic count


Direct Measurements of Microbial Growth
Measuring Microbial Growth

Direct methods
• Plate counts
• Direct microscopic count
Indirect methods
• Turbidity
• Metabolic activity
• Dry weight
Estimating Bacterial Numbers
by Indirect Methods

Turbidity
Metabolic Activity
Dry Weight
Factors Affecting Bacterial Growth
The Requirements for Growth:
Physical Requirements
• Temperature
– Minimum growth temperature
– Optimum growth temperature
– Maximum growth temperature
Temperature

Figure 6.1
Temperature Optima
• Optimum growth temperature is usually
near the top of the growth range
• Death above the maximum temp. comes
from enzyme inactivation
• Mesophiles most common group of
organisms
• 40ºF (5°C) slows or stops growth of most
microbes
Psychrotrophs/Psychrophiles

Figure 6.2
Oxygen Requirements

• Obligate aerobes – require O2


• Facultative anaerobes – can use O2 but also
grow without it
• Obligate anaerobes – die in the presence of
O2
The Requirements for Growth:
Physical Factors - Chemical Requirements

Oxygen (O2)
Toxic Forms of Oxygen
• Singlet oxygen: O2 boosted to a higher-energy
state
• Superoxide free radicals: O2–
• Peroxide anion: O22–
• Hydroxyl radical (OH)
pH
• Most bacteria grow between pH 6.5 and
7.5
• Acid (below pH 4) good preservative for
pickles, sauerkraut, cheeses
• Acidophiles can live at low pH
The Requirements for Growth:
Physical Factors / Requirements
• Osmotic pressure
– Hypertonic environments, increase salt or sugar,
cause plasmolysis
– Extreme or obligate halophiles require high osmotic
pressure
– Facultative halophiles tolerate high osmotic pressure
The Requirements for Growth:
Physical Requirements

Figure 6.4
The Physical Requirements for Growth

• Moisture-
• Hydrostatic Pressure-
• Radiation-
Nutritional Factors - Chemical
Requirements
• Carbon
– Structural organic molecules, energy source
– Chemoheterotrophs use organic carbon sources
– Autotrophs use CO2
• Nitrogen
– In amino acids and proteins
– Most bacteria decompose proteins
– Some bacteria use NH4+ or NO3–
– A few bacteria use N2 in nitrogen fixation
Nutritional Factors - Chemical
Requirements
• Sulfur
– In amino acids, thiamine and biotin
– Most bacteria decompose proteins
– Some bacteria use SO42– or H2S
• Phosphorus
– In DNA, RNA, ATP, and membranes
– PO43– is a source of phosphorus
Nutritional Factors - Chemical Requirements
• Trace elements
– Inorganic elements required in small amounts
– Usually as enzyme cofactors
– Vitamins- organic substances and growth factors
– Organic compounds obtained from the environment
– Vitamins, amino acids, purines, and pyrimidines
– Nutritional Complexity
– Locations of Enzymes
– Adaptations to Limited Nutrients
Sporulation / Endospores
• Formation of endospores in Bacillus, Clostridium
• and G+ genera
• Can Survive long periods of drought
• Axial nucleoid
• Endospore septum grows
• Spore coat and Exosporium
• Germination- 3 stages:
• 1) Activation, 2) germination, 3) outgrowth
Culture Media
• Culture medium: Nutrients prepared for
microbial growth
• Sterile: No living microbes
• Inoculum: Introduction of microbes into medium
• Culture: Microbes growing in/on culture medium
• Synthetic media
• Defined synthetic media
• Complex media
Culture Media
• Chemically defined media: Exact chemical
composition is known
• Complex media: Extracts and digests of yeasts,
meat, or plants
– Nutrient broth
– Nutrient agar
Agar

• Complex polysaccharide
• Used as solidifying agent for culture media in
Petri plates, slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100°C
• Solidifies ~40°C
Anaerobic Culture Methods
• Reducing media
– Contain chemicals (thioglycollate or oxyrase) that
combine O2
– Heated to drive off O2
Anaerobic Culture Methods

• Anaerobic
jar

Figure 6.5
Anaerobic Culture Methods
• Anaerobic
chamber

Figure 6.6
Capnophiles Require High CO2

• Candle jar

• CO2-packet

Figure 6.7
Selective Media
• Suppress unwanted
microbes and
encourage desired
microbes.

Figure 6.9b–c
Differential Media

• Make it easy to distinguish colonies of


different microbes.

Figure 6.9a
Enrichment Media
• Encourages growth of desired microbe
• Assume a soil sample contains a few phenol-
degrading bacteria and thousands of other bacteria
– Inoculate phenol-containing culture medium with the soil
and incubate
– Transfer 1 ml to another flask of the phenol medium and
incubate
– Transfer 1 ml to another flask of the phenol medium and
incubate
– Only phenol-metabolizing bacteria will be growing
METABOLISM
Focal Metabolites
• Precursors where biosynthetic origins of
building blocks and coenzymes can be
traced
• These are:
o Glucose-6-phosphate
o Phosphoenolpyruvate
o Oxaloacetate
o α-Ketoglutarate
Metabolic Pathways
• Growth by Carbon
– Calvin cycle
– Used by autotrophs (carbon dioxide as sole
source of carbon)
– CO2, ATP and NADPH are used to
reductively convert pentose-5-phosphate to
2 molecules of triose phosphate
Metabolic Pathways
• Growth by Nitrogen
– Nitrogen fixation: required for continuation
of life
– Bacteria and cyanobacteria with
nitrogenase enzyme complex
– N2 is reduced to 2 molecules of NH3 (widely
used nitrogen source)
Peptidoglycan
• Polymer consisting of sugars and amino
acids that forms a mesh-like layer forming
the cell wall
• Sugar component consists of alternating
residues of β-(1,4) linked N-
acetylglucosamine and N-acetylmuramic acid
• Thicker in Gram-positives than Gram-
negatives
• Penicillins act on transpeptidases which form
the crosslinks
Peptidoglycan
Synthesis

NAM: N-acetylmuramic acid NAG: N-acetlyglucosamine


METHODS OF DETECTION
AND IDENTIFICATION
Labs Used in Diagnosis of
Infectious Diseases
1. Morphologic identification of the agent in
stains of specimens or sections of tissues

2. Culture isolation and identification of the


agent

3. Detection of antigen from the agent by


immunoassay
Labs Used in Diagnosis of
Infectious Diseases
4. DNA-DNA or DNA-RNA hybridization

5. Detection and amplification of organism


nucleic acid in patient’s specimens

6. Demonstration of meaningful antibody or


cell-mediated immune responses to an
infectious agent
A properly collected
specimen is the single most
important step in the
diagnosis of an infection
Rules about Specimen Collection
1. Quantity of material must be adequate
2. Sample should be representative of the
infectious process
3. Contamination of the specimen must be
avoided by using only sterile equipment and
aseptic precautions
4. The specimen must be taken to the lab and
examined promptly
5. Meaningful specimens should be secured
prior to antimicrobial treatment
Methods of Identification
• Microscopy and Staining
• Culture systems
• Antigen detection
• Molecular diagnostics
Microscopy and Staining
STAIN REMARKS
Gram stain Most widely used
Acid-fast stains Identify mycobacteria
(Ziehl-Neelsen,
Kinyoun stain)
Immunofluorescent Fluorescein-labeled
antibody (IF) staining antibodies
Used for Bordetella or
Legionella
Gram and Acid-Fast Staining
Microscopic Appearance/Phenotypic appearance of some organisms

Genus Shape

Bacillus Rods

Sporolactobacillus Rods

Clostridium Rods or filaments


Desulfotomaculum Rods or filaments

Sporosarcina Cocci in tetrads or


packets
Thermoactinomycetes Branched filaments
Culture Systems
MEDIA INDICATION
Blood agar (sheep blood) Standard
Chocolate agar Neisseria and
Haemophilus
MacConkey or EMB Gram-negative rods
Brucella agar Obligate anaerobes
Bordet-Gengou or Bordetella spp
charcoal-containing
Brain-heart infusion agar, Fungi
inhibitory mold agar,
Saboraud’s dextrose agar
Antigen/Antibody Detection
• Enzyme-linked immunosorbent assays
(EIA)
• Agglutination tests
• Western blot immunoassay
ENZYME-LINKED IMMUNOSORBENT ASSAY

medical-dictionary.thefreedictionary.com
Molecular Diagnostics
• Nucleic acid probe
– hybridization of a nucleic acid sequence matched
with complementary RNA or DNA

• Identification using bacterial 16S RNA


– Labeled probes specific for 16S RNA are mixed

• Polymerase chain reaction (PCR)


– Amplify small amounts of DNA using DNA
polymerase
– Identified by labeled probes
Disk Diffusion Susceptibility Test

Identifies to
which
antibiotic the
organism is
susceptible or
resistant to
Minimum Inhibitory
Concentration
- Measures the exact concentration of antibiotic
necessary to inhibit bacterial growth

eglobalmed.com, rapidmicrobiology.com
Importance of Normal Flora
• M. tuberculosis, Salmonella typhi and Brucella
species are always pathogenic

• Many bacteria and fungi reside normally in


the body as part of natural flora

• Knowledge of normal flora helps you to decide


if you’ll believe the culture results
Thanks for Listening!

Any questions?