Class no 1.

RECOURCES:
Microorganisms can carry out different biochemical reactions to get nutrients. Diversity in nutritional
requirements, ability to release multiple enzymes is often used in microbiological diagnostics.
BACTERIAL METABOLISM
NUTRITIONAL REQUIREMENTS
Microorganisms require a lot of compounds in their metabolism;
1. Carbon - required by all bacteria for growth: autotrophs and heterotrophs.
a) Autotrophs use carbon dioxide (CO2). Autotrophy (chemotrophy, chemoautotrophy or chemolithotrophy)
is a unique form of metabolism found only in bacteria. It’s a form of metabolism, in which inorganic compounds
are oxidized directly (without using sunlight) to yield energy. This metabolic mode requires energy
for CO2 reduction. Bacterial photosynthesis (CO2) - a light-dependent, anaerobic mode of metabolism. Carbon
dioxide is reduced to glucose, which is used for both biosynthesis and energy production. Depending
on the hydrogen source used to reduce CO2, both photolithotrophic and photoorganotrophic reactions exist
in bacteria.
b) Heterotrophs use complex organic compounds (carbohydrates, amino acids). It is biologic oxidation of organic
compounds, such as glucose and other sugars, to yield adenosine triphosphate (ATP) and simpler organic
(or inorganic) compounds, which are needed by bacterial cells for biosynthetic or assimilatory reactions. Two
main groups of heterotrophic microorganisms exist: prototrophs - require only one rather simple compound
for growth; auxotrophs - require more than one and more complex compound for growth.
2. Inorganic ions (phosphate, potassium, magnesium, nitrogen, sulfur, sodium).
3. Various organic nutrients (various carbohydrates, amino acids, short-chain fatty polypeptides, vitamins,
purines, pyrimidines): auxotrophs and prototrophs.
4. Electron donors (ammonia, nitrites, hydrogen sulfide) – as a source of energy.
5. Electron acceptors (oxygen, pyruvate, lactate).
OXYGEN REQUIREMENTS
1. Obligate aerobes The tolerance to oxygen is related to the ability
2. Obligate anaerobes of the bacterium to detoxify superoxide and
3. Facultative aerobes/anaerobes hydrogen peroxide, produced as by-products
4. Microaerophilic bacteria of aerobic respiration (special enzymes: superoxide
5. Aerotolerants dismutase, peroxidase, catalase).
ENERGY METABOLISM
A. Aerobic respiration (respiratory metabolism) – type of heterotrophic metabolism that uses oxygen (O2 serves
as the final electron acceptor) and in which 38 moles of ATP (adenosine triphosphate) are derived from
the oxidation of 1 mole of glucose, yielding 380,000 cal (aerobes).
B. Anaerobic respiration – another heterotrophic mode of metabolism in which a specific inorganic compound
other than O2 serves as a terminal electron acceptor, e.g. nitrogen, sulfur; it produces fewer ATP molecules
(34 moles of ATP)(facultative aerobes/anaerobes, anaerobes).
C. Fermentation – another type of heterotrophic metabolism in which an organic compound is the terminal
electron (or hydrogen) acceptor; less energy is generated from this incomplete form of glucose oxidation (only
2 moles of ATP), but the process supports anaerobic growth (anaerobes).
Breakdown of glucose in fermentation:
glucose - pyruvic acid - lactic acid
glucose - acetaldehyde - ethanol
BACTERIAL DIAGNOSTICS
THE CRUCIAL STEPS IN MICROBIOLOGICAL DIAGNOSTICS Pure culture is always necessary!
1. Proper collection of specimens.
2. Appropriate microbiologic media (simple, enriched, selective etc.).
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3. Growth conditions (O2 and CO2 concentrations, temperature, humidity).
DIAGNOSTIC MEANING OF BACTERIAL METABOLISM
Most bacteria are identified and classified on the basis of their reactions in a series of biochemical tests based on
bacterial metabolism and its products. Some tests are used routinely for many groups of bacteria
(e.g. oxidase test, nitrate reduction, amino acid and carbohydrate breaking down enzymes). Others are restricted
to a single family, genus, or species (e.g. coagulase test for staphylococci).
Most often used biochemical features: saccharolytic activity (carbohydrate line, methyl-red reaction,
Voges-Proskauer reaction), proteolitic activity (liquefaction of gelatin, clotted serum test), decarboxylation,
deamination, ammonia or hydrogen sulfide or indole production, breaking down of urea. Bacteria can
be characterized by the use of single-enzyme tests, such as catalase test, or coagulase test; or by the use of tests
based on the presence of metabolic pathways (API=Analytical Profile Index, ATB, Phoenix and Vitec automated
systems).
METHODS OF IMMUNOLOGICAL DETECTION OF MICROORGANISMS
1. Detection of microbial antigen with known antiserum:
a) Capsular swelling reaction.
b) Slide agglutination tests.
2. Identification of serum antibodies:
a) Direct agglutination/hemagglutination.
b) Complement fixation test.
3. Other tests used to identify serum Ag (antigens) or Ab (antibodies):
a) Enzyme-linked immunosorbent assay (ELISA).
b) Enzyme immunoassay (EIA).
c) Direct and indirect fluorescent-antibody tests.
d) Latex agglutination tests.
ENVIRONMENTAL FACTORS INFLUENCING GROWTH
Factors that should be controlled during cultivation of bacteria:
1. Nutrients
• Autotrophs – able to grow simply, using carbon dioxide as the sole source of carbon, with only water and
inorganic salts. Autotrophs obtain energy either photosynthetically (phototrophs) or by oxidation
of inorganic compounds (chemotrophs). Autotrophs occur in environment.
• Heterotrophs – require an organic source of carbon, such as glucose, and obtain energy by oxidizing
or fermenting organic substances. All bacteria that inhabit the human body are heterotrophs.
• Simple or complex requirements for organic molecules:
• Fastidious bacterium (auxotroph) – has lost the ability to synthesize certain substances required
for its growth and metabolism; e.g. Haemophilus influenzae; Fastidious bacterium require growth factor –
an organic compound necessary for the growth of a given pathogen unable to synthesize it (e.g. heme, NAD,
vitamin K).
• Non-fastidious bacterium (prototroph) – able to synthesize its required growth factors requiring only
inorganic substances for growth; e.g. Escherichia coli;
2. pH
• Acidophiles – multiply under highly acidic growing optimally around a pH of 10; e.g. Vibrio
conditions optimum pH about 3.0; e.g. cholerae.
Helicobacter pylori.
• Neutrophiles – optimum pH 6.0-8.0; e.g.
Staphylococcus aureus.
• Alkaliphiles – microbes capable of survival in
alkaline (pH roughly 8.5-11) environments,
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3. Temperature
• Psychrophiles – extremophilic bacteria
or archaea which are cold-loving, having an
optimal temp. for growth at about 15°C or lower,
a maximal temp. for growth at about 20°C and a
minimal temp. for growth at 0°C or lower;
Arthrobacter sp., Psychrobacter sp.
• Psychrotrophs are cold-tolerant bacteria
or archaea that have the ability to grow at low
temperatures, but have optimal and maximal
growth temperatures above 15 - 20°C; Bacillus
sp.
• Mesophiles – best growth at 30-37°C; e.g. Staphylococcus aureus.
• Thermophiles – best growth at 50-60°C; e.g. Alicyclobacillus.
• Hyperthermophiles – thrive in extremely hot environments - from 60 °C upwards, e.g. Sulfolobus solfataricus.

Kapnophiles –need CO2.

4. Gaseous composition of the atmosphere

• Obligate aerobes – require oxygen as an electron acceptor; growth restricted by limited availability
of oxygen; e.g. Bacillus sp.
• Microaerophiles – require a reduced level of oxygen to grow; e.g. Campylobacter sp., Helicobacter sp.
• Obligate anaerobes – require a substance other than oxygen as an electron acceptor; growth is inhibited
by oxygen; e.g. Actinomyces sp., Bacteroides sp.
• Aerotolerant organisms – survive (no growth and multiplication) in the presence of atmospheric oxygen;
e.g. Lactobacillus sp.
• Facultative anaerobes – can grow either with or without oxygen; e.g. Escherichia coli.
• Capnophiles – grow best when the atmosphere is enriched with extra carbon dioxide (5% to 10%);
e.g. Campylobacter sp., Helicobacter sp.
5. Salt concentration and ionic strength of medium
Most bacteria tolerate wide range of external pressure. However, bacteria in:
• Hypertonic solutions – there is high osmotic pressure which removes water from cell, causing shrinkage
of cell membrane (plasmolysis).
• Hypotonic solutions – there is low osmotic pressure which causes water to enter the cell. Microbe may lyse
or burst if cell wall is weak.
Because of specific requirements there is division into:
• Osmophilic organisms – require higher osmotic pressure; e.g. Aspergillus, Saccharomyces, Micrococcus.
• Halophilic organisms – require higher salt concentration; Archea.

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6. Moisture
BACTERIOLOGICAL MEDIA
I. Classification based on consistency:
• Liquid: beef extract, peptone water, nutrient broth;
• Solid: agar-agar medium, MacConkey’s medium, Chapman’s medium;
II. Classification based on nutritional component:
• Synthetic: Chapman’s medium, MacConkey’s medium;
• Natural: beef extract, medium with potato or yolk;
III. Classification based on functional use or application:
• Basal: agar-agar medium, broth, peptone water;
• Enriched – enriched media are used to grow fastidious bacteria; e.g. blood agar, chocolate agar, Loeffler’s
serum slope;
• Selective – are designed to inhibit unwanted commensal or contaminating bacteria and help to recover
pathogen from a mixture of bacteria. Any agar media can be made selective by addition of certain inhibitory
agents that don’t affect the pathogen. Various approaches to make a medium selective include addition
of antibiotics, dyes, chemicals, alteration of pH or a combination of these e.g. Wilson-Blair medium, SF medium;
• Differential – media containing particular organic compound and indicator. If a given microorganism breaks
down the organic compound, indicator will change medium’s colour; e.g. Kligler’s Iron Agar, carbohydrate line;
• Selective-Differential – media that cause the colonies of particular type/species of microorganisms to have
distinctive appearance; e.g. Chapman’s medium (Mannitol Salt Agar), MacConkey’s medium;
• Transport – media used for specimen transport; prolong time of transport to 24 hrs; bacteria stay alive
(but not multiply); e.g. Portagerm, Haemomedium/Meningomedium;
• Diagnostic-Transport – media used for specimen transport that allow the preliminary differentiation
of microorganisms; e.g. Uricult;

PLANTING
• Clinical material may contain various microorganisms;
• Planting the sample under one set of conditions allows the selected group of microbes to produce
colonies, but many other may be overlooked;
• Each clinical sample should be planted on a set of various media and incubated in various conditions;
Types of growth of bacteria in liquid media:
• Sediment – e.g. Staphylococcus sp., Streptococcus sp.
• Turbidity – e.g. enteric rods
• Superficial growth – e.g. Bacillus sp.
Types of growth of microorganisms on solid media:
• „O” growth – restricted growth; colony has well defined edge (non-moving bacteria), e.g. E. coli.
• „H” growth – wide spread, spreading of bacteria because of the presence of high numbers of flagella (moving
bacteria), e.g. Proteus sp.
METHODS OF ISOLATION OF BACTERIA IN A PURE CULTURE
1. STREAK-PLATE METHOD
• Streaking on agar with a wire loop (three/four-way streak-plate inoculation), 1
• Last line of inoculum deposits single colonies on the agar surface,
• Pick such a single, well isolated colony and suspend it in water, 2
• Or replant/streak it on the solid medium to get the pure culture;
2. DILUTION METHOD (less reliable)
• Prepare serial dilutions of the bacterial suspension,
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• Plant sample of each dilution on the solid media to get the pure culture.

STANDARD GROWTH CURVE OF MICROORGANISMS IN A CULTURE

a - lag phase – during this stage, bacteria get d - death phase – Because there are so many
acclimated to their environment. They need time to bacteria, they can't move to find further resources.
get used to their nutrients, so growth is slow during Their food supply runs out, waste products build up,
this phase. and the bacteria die.
b - log phase – once the bacteria have adapted,
they begin to multiply exponentially. During this
stage, their numbers double even every 20-30
minutes.
c - stationary phase – Eventually, the number of
bacteria is so large that the food supply begins to
decrease. At this point, the rapid growth of the log
phase stops.