منتنمت

1. Microbial Growth requirements: Nutritional
requirements, Physical requirements and
types of culture media

2. Microbial Growth and Nutrition
• All bacteria have the following major nutritional needs for growth:
oA source of carbon (for making cellular constituents).
oA source of nitrogen (for making proteins).
oSmaller amounts of molecules, such as phosphate for nucleic acids
and phospholipids of cell membranes synthesis.
oVarious metals and ions for enzymatic activity.

3. Nutritional Requirements for Growth:
• Bacteria are classified into two basic groups according to how they meet
their nutritional needs.
1. Autotrophs (lithotrophs): Environmental bacteria
2. Heterotrophs (Chemoorgantrophs): Pathogenic bacteria
Autotrophs:
• They are able to grow simply, using carbon dioxide as the sole source of
carbon, with only water and inorganic salts required in addition.
• They obtain energy either photosynthetically (phototrophs) or by
oxidation of inorganic compounds (chemolithotrophs).
• They contain environmental bacteria.

4. Heterotrophs:
• They require more complex substances for growth.
• They require an organic source of carbon, such as glucose.
• They obtain their energy by oxidizing or fermenting organic substances.
• They include all bacteria that inhabit human body.
• They are divided into two groups based on nutritional needs:
1. Fastidious bacteria: requiring additional metabolites such as vitamins,
purines, pyrimidines, and hemoglobin supplied in the growth medium (e.g,
Haemophilus spp, Streptococcus spp).*
2. Non-fastidious bacteria: requiring simple substances such as source of
carbon (glucose) and source of nitrogen (peptone) (e.g Pseudomonas
aeruginosa, Enterobacteriaceae).

5. Environmental Factors Influencing Growth:
There are three environmental factors influence the growth rate of
bacteria, include:-
 pH
 Temperature
 Gaseous composition of the atmosphere
pH:
• Most pathogenic bacteria grow best at a neutral pH.
• Only exception, V. cholerae requires alkaline pH.

6. Temperature: *
1. Psychrophiles: Bacteria that grow best at cold temperatures (<0 ° C to 20° C).
They contains Polyunsaturated fatty acids in their cell membrane which gives fluid
nature to the cell membrane even at lower temperature.
Examples: Vibrio marinus, Psychroflexus
2. Psychrotrops (facultative
psychrophiles): Can grow at 0 ° C
with an optimum of 20-30° C.

7. 3. Mesophiles: Bacteria that grow best near human body temperature (25-40° C).
Most human pathogens are mesophilic bacteria.
Examples: E. coli, Salmonella, Klebsiella, Staphylococci, Streptococci.
4. Thermophiles: Bacteria that grow best at high temperatures (45° to 85° C).
They contains saturated fatty acids in their cell membrane so their cell membrane
does not become too fluid even at higher temperature.
Examples: Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus
5. Hyperthermophiles: Bacteria that grow best at high temperatures (80° to >
100° C).
Examples: Thermodesulfobacterium, Pyrolobus fumari, Thermotoga.

8. Gaseous composition of the atmosphere:*
Bacteria that grow on humans vary in their atmospheric requirements for
growth.
 Obligate aerobes: Grow only in the presence of oxygen (e.g., Ps. aeruginosa).
 Aerotolerant anaerobes: can survive in the presence of oxygen but do not use
oxygen in metabolism (e.g., Clostridium perfringens).
 Obligate anaerobes: They cannot grow in the presence of oxygen (e.g.,
Bacteroides spp, Clostridium spp).
 Facultative anaerobes: They can grow either with or without oxygen (e.g.,
most pathogenic bacteria).

9. Other factor for growth:
Growth in Salt concentration: Most of pathogenic bacteria grow in isotonic
solution (so-called non-halophilic), but some grow in hypertonic solution
(called-halophilic).
Halophilic: organisms that grow in high salt (NaCl) concentration (e.g., Vibrio
parahaemolyticus, Staphylococcus spp).
Capnophiles: Organisms grow best when the atmosphere is enriched with
extra carbon dioxide (5% - 10%) (e.g., Haemophilus spp, Neisseriae spp and
Streptococcus pneumoniae).
Microaerophiles: Bacteria that require a reduced level of oxygen to grow
(5%), e.g., Campylobacter spp, Helicobacter pylori.

10. Types of culture media:
• Culture medium used for growing the pathogen in the laboratory, which
contain all essential nutrients, ions, and maintain the correct pH and osmotic
pressure.
• Culture media are classified based on consistency into three types
(solid, semisolid and liquid media):
• Solid media:
• These are solidified by incorporating a gelling agent such as agar (1.5-2% w/v).
• They are used mainly in petri dishes (plate) and also in tubes or bottle.
• solid medium is used to isolate and identify discrete colonies of each
organism present in the specimen.
Agar is polysaccharide extract obtained from seaweed and is commonly used to solidify
culture media. Its setting temperature of 32–39 ° C and melting temperature of 90–95 ° C

11. Semi-solid media:
• This type of medium containing a small amount of agar (0.4–0.5% w/v).
• Semi-solid media used as followed
• Transport media
• Motility
• Biochemical tests.
Broth (liquid) media:
• These type of culture media do not contain agar.
• Fluid media are most commonly used as enrichment where organisms are likely to
be few e.g. blood culture.
• They are also used for biochemical testing e.g. peptone water sugars for MR-VP
test.

12. Culture media are also classified basis on purpose or application
into five-types, including:-
• Basic (simple)
• Enriched
• Selective and Enrichment
• Differential (indicator)
• Transport

13. Basic (simple) media:
• These are simple media such as nutrient agar and nutrient broth that will support
the growth of bacteria that do not have special nutritional requirements.
• They are often uses in:
 Preparation of enriched media
 To maintain stock cultures of control strains of bacteria,
 Sub-culturing pathogens from differential or selective media before to performing
biochemical and serological identification tests.

14. Enriched media:
• A growth medium that contains added growth factors, such as blood, lyzed
blood, serum, vitamins, yeast extract, and other special growth factors.
• They are use for isolation of fastidious organisms.
• Examples enriched media and fastidious bacteria:
• Blood agar (Streptococcus spp)
• Chocolate agar (Haemophilus spp)
• Buffer charcoal yeast extract agar (Legionella pneumophila)

15. Selective media:
• These are solid media which contain substances (e.g. bile salts or other
chemicals, dyes, antibiotics) which inhibit the growth of some bacteria
but allow others to grow. Examples:
• MacConkey agar (Enterobacteriaceae)
• Thiosulfate bile salt sucrose agar (V. cholerae)
• Crystal violet blood agar (Streptococcus. pyogenes)
• Kanamycin or neomycin blood agar (Streptococcus. agalactiae)

16. Enrichment medium:
• This term is usually described fluid selective media which contain
substances that inhibit the growth of unwanted organisms. Examples:
• Alkaline peptone water (V. cholerae).
 Selenite F broth or Tetrathionate broth (Salmonella spp).
Differential (Indicator) media:
• These are media to which dyes (indicator) or other substances are added
to differentiate microorganisms.
• Indicator (dye) changes the medium color when acid is produced
following fermentation of a specific carbohydrate.
• Note: Many media used to isolate pathogens are both selective and
differential or both enriched and differential.

17. e.g, MacConkey agar, Thiosulfate bile salt sucrose agar, Xylose lysine
deoxycholate agar
Note: Blood agar have no chemical indicator but it is also differential media that is used to
differential the bacteria according to their hemolysis reactions:
A: Beta-hemolysis: there is clear zone of complete hemolysis around colonies.
B: Alpha-hemolysis: Partial hemolysis, there is green color around colonies
C: Gamma-hemolysis: there is no hemolysis and changing of color around colonies.
CLED
XLD
MacConkey agar Blood agar

18. Reaction
Sugar
Indicator
Media
Neutral or alkaline
Acid
Pale yellow (non-lactose
fermenter)
Pink (lactose fermenter)
Lactose
Neutral red
MacConkey agar
Pale yellow (non-lactose
fermenter)
Pink (lactose fermenter)
Lactose
Neutral red
Deoxycholate citrate
agar (DCA)
Pink (non-fermenter)
Yellow (CHO fermenter)
Xylose
Sucrose
Phenol red
Xylose lysine
deoxycholate agar
(XLD)
Pale color (non-lactose
fermenter)
Yellow (lactose
fermenter)
Dark Pink (lactose
fermenter)
Lactose
Bromothymol
blue (BB)
Andarade +
BB
Cysteine lactose
electrolytes deficient
(CLED)
Pale green (non-lactose
fermenter)
Yellow (sucrose
fermenter)
sucrose
Bromothymol
blue
Thiosulfate citrate
bile salt sucrose agar
(TCBA)
Examples for some differential media

19. Transport media:
• These are mostly semisolid media that contain substances to prevent the
overgrowth of commensals and preserve the viability pathogenic organisms.
• They are used when specimens cannot be cultured immediately after collection.
• e.g.,
• Cary-Blair medium (use for enteric pathogens)
• Amies medium (use for Neisseria gonorrhoeae)
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20. Basic Concept of Bacterial
Metabolism

21. Metabolism
.A
Basic Concepts of Metabolism
.B
Glycolytic Pathways
.C
Fermentation
.D
Respiration

22. A. Basic concept
1. Definition:-
•
Metabolism refers to all the biochemical reactions that occur in a cell or
organism.
•
There are consist of two processes:
.1
Catabolism: The processes by which a living organism
obtains its energy and raw materials from nutrients.
.2
Anabolism: The processes by which energy and raw
materials are used to build macromolecules and cellular
structures (biosynthesis).

23. 2. Oxidation-reduction:
•
In metabolic pathways, we are often concerned with the
oxidation or reduction of carbon.
•
Reduction and oxidation always occur together.
•
In a reduction-oxidation reaction (redox reaction), one
substance gets reduced, and another substance gets oxidized.
•
The molecule that gets oxidized is called the electron donor,
whereas, the molecule that gets reduced is called the electron
acceptor.

24. .3
EnzymaticPathways for Metabolism
•
Metabolic reactions take place in a step-wise fashion until
the formation of the final product takes place.
•
Each step requires its own enzyme.
•
The sequence of enzymatically-catalyzed steps from a
starting raw material to final end products is called an
enzymatic pathway (or metabolic pathway).

25. .4
Co-factors for Redox Reactions
•
Enzymes that catalyze redox reactions typically require a cofactor to “shuttle”
electrons from one part of the metabolic pathway to another part.
•
There are two main redox cofactors:

NAD (Nicotinamide adenine dinucleotide)

FAD (Flavin adenine dinucleotide)
NAD(oxidized) + H+  NADH(reduced)
FAD(oxidized) + H+  FADH(reduced

26. .5
ATP: A “currency of energy” for many cellular reactions
•
ATP (Adenosine Triphosphate). It is a nucleotide with three
phosphate groups linked in a small chain.
•
The last phosphate in the chain can be removed by phosphokinase
(the ATP becomes ADP)
•
During catabolism, energy released from the oxidation of carbon is
captured and used to synthesize ATP from ADP and phosphate.
.1
C6H12O6 + 6 O2 = 6 CO2 + 6 H2O+ Energy
.2
ADP + Phosphate + Energy = ATP + H2O`

27. B. Glycolytic pathways:
•
The three major biochemical pathways bacteria used by
break down glucose to pyruvic acid are:
.1
The Embden-Meyerhof-Parnas (EMP) glycolytic
pathway
.2
The pentose phosphate pathway
.3
The Entner-Doudoroff pathway

28. .1
The Embden-Meyerhof-Parnas (EMP) glycolyticpathway:
•
Major pathway in conversion of glucose to pyruvate
•
Generates reducing power in the form of NADH+H (2
molecules).
•
Generates energy in the form of ATP (2 molecules).
•
Anaerobic (does not require oxygen)
•
Used by many bacteria, including all members of
Enterobacteriaceae

29. 2. The pentose phosphate pathway:
•
Alternative to EMP pathway for sugars metabolism
•
Conversion of glucose to ribulose-5-phosphate
•
Provides pentoses for nucleotide synthesis
•
Generates NADPH
•
May be generate ATP , which is less than with EMP
pathway.
•
Used by bacteria which lacks some of the enzymes
required in the EMP pathway.

30. 3. Entner-Doudoroff Pathway
•
Generates one NADPH molecule
•
Generate one ATP molecule per glucose
•
Aerobic process
•
used by bacteria which lacking certain glycolytic enzymes.

31. C. Sugars fermentation:
•
The term "fermentation" refers to the breakdown of a sugar (such
as glucose or maltose) to pyruvic acid and then, usually, to lactic
acid.
•
Fermentation is also called the glycolytic cycle (glyco = sugar, lytic =
breakdown).
•
Glycolytic cycle is the process by which facultative bacteria generate ATP
in the absence of oxygen.
•
If oxygen is present, the pyruvate produced by fermentation enters the
Krebs cycle (tricarboxylic acid cycle).
•
Glucose pyruvate ATP+CO2 + H2O
Glycolytic Krebs
cycle

32. Note:
•
Facultative and anaerobic bacteria ferment but aerobes, which can grow only
in the presence of oxygen, do not.
Fermentationpathways
.a
HomolacticacidFermentation:
Pyruvic Acid -----> Lactic Acid, eg. Streptococci, Lactobacilli
b. Heterolactic acidfermentation:
Pyruvic Acid------ Lactic acid & Ethanol, e.g., some Lactobacilli
c. Mixed acidfermentation:
Pyruvic Acid ---> acetic acid, succinic acid, formic acid, ethanol, e.g.
Escherichia coli, Salmonella.
(Methyl red test)
d. Butanediol fermentation:
Pyruvic acid---- acetylmethylcarbinol “acetoin”, e.g., Klebsiella. (VP test)

33. D. Respiration:
•
For aerobic respiration, the oxygen acts as the hydrogen acceptor in the
final steps of energy production.
•
For anaerobic respiration, NO3, SO4, CO2 can serve as terminal
electron acceptors.

34. Ironmetabolism:
•
Iron, in the form of ferric ion, is required for the growth of bacteria
because it is an essential component of cytochromes and other enzymes.
•
To obtain iron for their growth, bacteria produce iron-binding
compounds called siderophores, which have very high binding affinity.
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