A Presentation made as a part of my course on "Food Microbiology" (Bachelor's study)

1. FACTORS AFFECTING GROWTH OF
MICRO-ORGANISMS
By
Anas Ejaz Shaikh
13FET1006

2. Growth
 Growth is an orderly increase in the quantity of
cellular constituents.
 The ability of the cell to form new protoplasm from
nutrients available in the environment.
 In most bacteria, growth involves
(i) increase in cell mass and number of ribosomes,
(ii) duplication of the bacterial chromosome,
(iii) synthesis of new cell wall and plasma membrane,
(iv)partitioning of the two chromosomes,
(v) septum formation, and cell division.
 This asexual process of reproduction is called binary
fission.

4. The Bacterial Growth Cycle has Four Phases
time
logcfu/ml
4
8
12
lag
exponential
stationary
death

5. Budding in Yeast

6. Microbial Growth
Requirements

7. The Requirements for Growth
PHYSICAL REQUIREMENTS
– Temperature
– pH
– Oxygen
– Hydrostatic
Pressure
– Osmotic pressure
CHEMICAL REQUIREMENTS
(NUTRITIONAL FACTORS)
– Carbon
– Nitrogen, sulfur,
and phosphorous
– Trace elements
– Oxygen
– Organic growth
factor

8. Physical Factors Required for
Growth of Micro-organisms

9. 1) pH
 The pH, or negative logarithm of
hydrogen ion concentration, [H+],
of natural environments varies from
about 0.5 in the most acidic soils to
about 10.5 in the most alkaline
lakes.
 Optimum pH: the pH at which the
microorganism grows best.
 Most bacteria grow between pH 6.5
and 7.5
 Molds and yeasts grow between pH
5 and 6

10. According to their tolerance for acidity/alkalinity,
bacteria are classified as:
Acidophiles (acid-loving): grow best at pH 0.1-5.4
Neutrophiles: grow best at pH 5.4 to 8.0
Alkaliphiles (base-loving): grow best at pH 7.0-11.5

11.  Most acidophile organisms have evolved extremely efficient mechanisms to pump
protons out of the intracellular space in order to keep the cytoplasm at or near neutral
pH. Therefore, intracellular proteins do not need to develop acid stability through
evolution.
 However, other acidophiles, such as Acetobacter aceti, have an acidified cytoplasm
which forces nearly all proteins in the genome to evolve acid stability.
 Studies of proteins adapted to low pH have revealed a few general mechanisms by
which proteins can achieve acid stability.
 In most acid stable proteins (such as pepsin protein from Sulfolobus acidocaldarius),
there is an overabundance of acidic residues which minimizes low pH destabilization
induced by a buildup of positive charge.
 Other mechanisms include minimization of solvent accessibility of acidic residues or
binding of metal cofactors.
Acidophiles
Mechanisms of adaptation to acidic environments

12. Alkaliphiles:
 Microbial growth in alkaline conditions presents several complications to normal
biochemical activity and reproduction, as high pH is detrimental to normal cellular
processes.
 For example, alkalinity can lead to
denaturation of DNA,
instability of the plasma membrane,
inactivation of cytosolic enzymes as well as other unfavorable physiological changes.
 Thus, to adequately circumvent these obstacles, alkaliphiles must either possess
specific cellular machinery that works best in the alkaline range, or they must have
methods of acidifying the cytosol in relation to the extracellular environment.
 To determine which of the above possibilities an alkaliphile uses, experimentation has
demonstrated that alkaliphilic enzymes possess relatively normal pH optimums. The
determination that these enzymes function most efficiently near physiologically neutral
pH ranges (about 7.5-8.5) was one of the primary steps in elucidating how alkaliphiles
survive intensely basic environments.
 Because the cytosolic pH must remain nearly neutral, alkaliphiles must have one or
more mechanisms of acidifying the cytosol when in the presence of a highly alkaline
environment.

13. Mechanisms of cytosolic acidification in alkaliphiles
 Alkaliphiles maintain cytosolic acidification through both passive and active
means.
 In passive acidification, it has been proposed that cell walls contain
acidic polymers composed of residues such as galacturonic acid, gluconic
acid, glutamic acid, aspartic acid, and phosphoric acid. Together, these
residues form an acidic matrix that helps protect the plasma membrane from
alkaline conditions by preventing the entry of hydroxide ions, and allowing for
the uptake of sodium and hydronium ions.
 The most characterized method of active acidification is in the form of
Na+/H+ antiporters. In this model, H+ ions are first extruded through the
electron transport chain in respiring cells and to some extent through
an ATPase in fermentative cells. This proton extrusion establishes a proton
gradient that drives electrogenic antiporters—which drive intracellular Na+
out of the cell in exchange for a greater number of H+ ions, leading to the net
accumulation of internal protons. This proton accumulation leads to a
lowering of cytosolic pH.

14. Organism Minimum pH Optimum pH Maximum pH
Thiobacillus thiooxidans 0.5 2.0-2.8 4.0-6.0
Sulfolobus acidocaldarius 1.0 2.0-3.0 5.0
Bacillus acidocaldarius 2.0 4.0 6.0
Zymomonas lindneri 3.5 5.5-6.0 7.5
Lactobacillus acidophilus 4.0-4.6 5.8-6.6 6.8
Staphylococcus aureus 4.2 7.0-7.5 9.3
Escherichia coli 4.4 6.0-7.0 9.0
Clostridium sporogenes 5.0-5.8 6.0-7.6 8.5-9.0
Erwinia caratovora 5.6 7.1 9.3
Pseudomonas aeruginosa 5.6 6.6-7.0 8.0
Thiobacillus novellus 5.7 7.0 9.0
Streptococcus pneumoniae 6.5 7.8 8.3
Nitrobacter sp 6.6 7.6-8.6 10.0

15. 2) Temperature
 According to their growth temperature range, bacteria
can be classified as:
Psychrophiles : grow best at 15-20oC
Psychrotrophs : grow between 0°C and 20–30°C
Mesophiles : grow best at 25-40oC
Thermophiles : grow best at 50-60oC
Hyperthermophiles: grow best at & above 80oC
 Typical Growth Rates and Temperature
– Minimum growth temperature: lowest temp which
species can grow
– Optimum growth temperature: temp at which the
species grow best
– Maximum growth temperature: highest temp at which
grow is possible

17. Psychrophiles:
Arthrobacter
Psychrobacter
Halomonas
Pseudomonas
Hyphomonas
Sphingomonas
Mesophiles
:
Listeria monocytogenes
Pseudomonas maltophilia
Thiobacillus novellus
Staphylococcus aureus
Streptococcus pyrogenes
Streptococcus pneumoniae
Escherichia coli
Clostridium kluyveri
Thermophiles
:
Bacillus stearothermophilus
Rhizomucor pusillus
Chaetomium thermophile
Humicola insolens
Thermus Aquaticus
A novel application of
Thermus Aquaticus is in PCR
(Polymerase Chain Reaction)

18. The Temperature Scale of Microbial Growth

19. 3) Oxygen
 Aerobes: require oxygen to grow
 Obligate aerobes: must have free oxygen for aerobic
respiration (e.g. Pseudomonas)
 Anaerobes: do not require oxygen to grow
 Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)
 Microaerophiles: grow best in presence of small amount of
free oxygen
 Capnophiles: carbon-dioxide loving organisms that thrive
under conditions of low oxygen
 Facultative anaerobes: carry on aerobic metabolism when
oxygen is present, but shift to anaerobic metabolism when
oxygen is absent
 Aerotolerant anaerobes: can survive in the presence of oxygen
but do not use it in their metabolism
 Obligate: organism must have specified environmental
condition
 Facultative: organism is able to adjust to and tolerate
environmental condition, but can also live in other conditions

22. 4) Hydrostatic Pressure
 Water in oceans and lakes exerts pressure
exerted by standing water, in proportion to its
depth
 Pressure doubles with every 10 meter increase
in depth
 Barophiles: bacteria that live at high pressures,
but die if left in laboratory at standard
atmospheric pressure
 E.g.:Photobacterium, Shewanella, and Colwellia

23. 5) Osmotic Pressure
 Environments that contain dissolved
substances exert osmotic pressure, and
pressure can exceed that exerted by
dissolved substances in cells
 Hyperosmotic environments: cells lose water
and undergo plasmolysis (shrinking of cell)
 Hypoosmotic environment: cells gain water
and swell and burst

24. Plasmolysis

25. Halophiles
 Salt-loving organisms which require moderate to large
quantities of salt (sodium chloride)
 Membrane transport systems actively transport sodium
ions out of cells and concentrate potassium ions inside
 Why do halophiles require sodium?
1) Cells need sodium to maintain a high intracellular
potassium concentration for enzymatic function
2) Cells need sodium to maintain the integrity of their
cell walls

26. Responses to Salt

27. Chemical Factors
Required for Growth of
Micro-organisms

28. Eleme-
-nt
%
dry
wt
Source Function
C 50
organic
compounds or
CO2
Main constituent of cellular material
O 20
H2O, organic
compounds, CO2,
and O2
Constituent of cell material and cell
water; O2 is electron acceptor in aerobic
respiration
N 14
NH3, NO3,
organic
compounds, N2
Constituent of amino acids, nucleic
acids nucleotides, and coenzymes
H 8
H2O, organic
compounds, H2
Main constituent of organic compounds
and cell water
P 3
inorganic
phosphates
(PO4)
Constituent of nucleic acids,
nucleotides, phospholipids, LPS,
teichoic acids
S 1
SO4, H2S, So,
organic sulfur
compounds
Constituent of cysteine, methionine,
glutathione, several coenzymes

29. K 1 Potassium salts
Main cellular inorganic cation and
cofactor for certain enzymes
Mg 0.5
Magnesium
salts
Inorganic cellular cation, cofactor for
certain enzymatic reactions
Ca 0.5 Calcium salts
Inorganic cellular cation, cofactor for
certain enzymes and a component of
endospores
Fe 0.2 Iron salts
Component of cytochromes and
certain non-heme iron-proteins and a
cofactor for some enzymatic
reactions
Ele-
ment
%
dry
wt
Source Function
Trace elements - Mn, Co, Zn, Cu, and Mo.

32. CULTURE MEDIA FOR GROWTH OF
MICRO-ORGANISMS

33. Introduction
 Culture medium provides similar environmental and nutritional conditions that
exist in the natural habitat of the microbe.
 Most culture medium contains water, a source of carbon & energy, source of
nitrogen, trace elements and some growth factors.
 Besides these, optimum pH, oxygen tension and osmolarity too have to be taken
into consideration.
 Some of the ingredients of culture media include:
1. Distilled or demineralised water should be used.
2. Peptone is a byproduct of protein digestion. Proteins are often obtained from
heart muscle, casein, fibrin or soya flour and is digested using proteolytic
enzymes such as pepsin, trypsin or papain.
3. Casein hydrolysate is obtained from hydrolysis of milk protein casein using HCl
or trypsin.
4. Meat extract is obtained by hot water extraction of lean beef and then
concentrated by evaporation.
5. Yeast extract is prepared from washed cells of bakers’ yeast and contains wide
range of amino acids, growth factors and inorganic salts.

34. Types of culture media
I. Based on their consistency
a) Solid medium
b) Liquid medium
c) Semi solid medium
II. Based on the constituents/
ingredients
a) Simple medium
b) Complex medium
c) Synthetic or defined medium
d) Special media

35. Special media
– Enriched media
– Enrichment media
– Selective media
– Indicator media
– Differential media
– Transport media
– Media for biochemical reactions
III.Based on Oxygen requirement
- Aerobic media
- Anaerobic media

36. Solid media – contains 2% agar
 Colony morphology, pigmentation, hemolysis
can be appreciated.
 Eg: Nutrient agar, Blood agar
Liquid media – no agar.
 For inoculum preparation, Blood culture,
continuous culture.
 Eg: Nutrient broth
Semi solid medium – 0.5% agar.
 Eg: Motility medium

37. Agar
Universally used for preparing solid medium
Obtained from seaweed: Gelidium
No nutritive value
Not affected by the growth of the bacteria.
Melts at 98°C & sets at 42°C
2% agar is employed in solid medium
Other solidifying agents
Egg yolk and Serum
Serum containing medium such as Loeffler’s
serum slope
Egg containing media such as Lowenstein
Jensen (LJ) medium and Dorset egg medium are
solidified as well as disinfected by a process of
inspissation.

38. Solid medium
Semi-solid
medium
Liquid
medium

39. Simple media / basal media:
Those bacteria that are able to grow with minimal requirements
are said to nonfastidious and those that require extra nutrients
are said to be fastidious.
Simple media can support most non-fastidious bacteria
Eg: Nutrient Broth, Nutrient Agar
 NB consists of peptone, meat extract, NaCl, water
 NB + 0.5% Glucose = Glucose Broth
 NB + 2% agar = Nutrient agar
 Agar conc. Reduced (0.2 - 0.5%) = Semi-solid medium
Nutrient Agar Nutrient Broth

40. Complex media
 Media other than basal media.
 They have added complex ingredients such as yeast extract or
casein hydrolysate, which consist of a mixture of many chemical
species in unknown proportions
 Provide special nutrients
Eg: blood agar, peptone-beef extract agar

41. Synthetic or
Defined media
Media prepared
from pure chemical
substances.
exact
composition is
known
Used for special
studies, eg.
metabolic
requirements
Eg: peptone water-
(1% peptone +
0.5% NaCl in
water)

42. Special Media

43. Enriched media
 Enriched media are used to grow nutritionally exacting
(fastidious) bacteria
 Addition of extra nutrients in the form of blood, serum,
egg yolk etc, to basal medium makes them enriched
media.
 Blood agar, chocolate agar, Loeffler’s serum slope etc
are few of the enriched media.

44.  Blood agar is prepared by adding 5-
10% (by volume) to a basal medium
such as nutrient agar or other blood
agar bases.
 Since blood cannot be sterilized, it has
to be collected aseptically from the
animal.
 Human blood must be avoided since it
may contain inhibitory substances
including antibiotics.
 After the blood agar base is
autoclaved, blood is added to the
medium at temperature just above the
solidifying point of agar.
 The mixture is then poured on to the
plates and allowed to solidify.
 Blood agar is useful in demonstrating
hemolytic properties of certain bacteria.

45. Chocolate agar is also known as heated
blood agar or lysed blood agar.
The procedure is similar to that of blood
agar preparation except that the blood is
added while the molten blood agar base is
still hot.
 This lyses the blood cells and releases
their contents into the medium.
This process turns the medium brown,
hence the name.
 This medium is especially useful in
growing Hemophilus sp and Neisseria sp.
Serum for medium can be obtained from
animal blood but must be filtered through
membrane or seitz filter before use.

46. Enrichment media
 Liquid media used to isolate pathogens from a mixed culture.
 Stimulate growth of desired bacterium
Inhibit growth of unwanted bacterium
 Media is incorporated with inhibitory substances to suppress the unwanted
organism → increase in numbers of desired bacteria
 Eg:
Selenite F Broth – for the isolation of Salmonella, Shigella
Tetrathionate Broth – inhibit coliforms
Alkaline Peptone Water – for Vibrio cholerae
Selenite F Broth Tetrathionate Broth Alkaline Peptone water

47. Selective media
 The inhibitory substance is added to a solid
media
 Increase in number of colonies of desired
bacterium
Eg:
 Deoxycholate citrate medium for dysentery
bacilli
 Mac Conkey’s medium for gram negative
bacteria
 TCBS – for V. cholerae
 LJ medium – M. tuberculosis

48. Thiosulfate citrate
bile salts sucrose (TCBS)
agar
Mac Conkey’s medium

49. LJ mediaDeoxycholate citrate
agar

50. Indicator media
 contain an indicator which changes its colour when a
bacterium grows in them
 Eg:
Wilson-Blair medium – S. typhi forms black colonies
McLeod’s medium (Potassium tellurite)– Diphtheria bacilli
Wilson-Blair medium McLeod’s medium

51. Urease medium
Urease producing bacteria
Urease
Urea → CO2 + NH3
NH3 → Medium turns pink

52. Blood agar:
shows three types of Hemolysis
α Hemolysis
β Hemolysis
γ Hemolysis

53. Blood Agar and Hemolysis
Certain bacterial species produce extracellular enzymes that lyse red blood
cells in the Blood agar (hemolysis).
These hemolysin (exotoxin) radially diffuses outwards from the colony (or
colonies) causing complete or partial destruction of the red cells (RBC) in the
medium and complete denaturation of hemoglobin within the cells to colorless
products.
Alpha hemolysis: Partial lysis of the RBC to produce a greenish-gray or
brownish discoloration around the colony. α hemolysis is due to the reduction
of RBC hemoglobin to methemoglobin in the medium surrounding the colony.
Many of the alpha hemolytic streptococci are part of the normal body flora.
But Streptococcus pneumoniae which is also alpha hemolytic causes serious
pneumonia and other deadly infectious disease.
Beta Hemolysis: Complete lysis of Red Blood Cells, causing a clearing of
blood from the medium under and surrounding the colonies
Gamma or non hemolysis: No hemolysis of RBC. No change of the
medium under and surrounding the colonies.

54. Differential media
 Substances incorporated in it enabling it to
distinguish between bacteria.
 Eg: Mac Conkey’s medium
– Peptone
– Lactose
– Agar
– Neutral red
– Taurocholate
 Distinguish between lactose fermenters & non
lactose fermenters.

55. MacConkey agar:
 Lactose fermenters – Pink colonies
 Non lactose fermenters – colourless colonies

56. Transport media
 Media used for transporting the samples.
 Delicate organisms may not survive the time taken for
transporting the specimen without a transport media.
 Eg:
– Stuart’s medium – non nutrient soft agar gel containing a
reducing agent & charcoal (to neutralise inhibitory
substances)
used for Gonnococci
– Sach’s buffered glycerol saline is used to transport feces
from patients suspected to be suffering from bacillary
dysentery.
– Cary Blair medium and Venkatraman Ramakrishnan
medium are used to transport feces from suspected
cholera patients.
– Pike’s medium is used to transport streptococci from throat
specimens.

57. Stuart’s Medium
Buffered Glycerol Saline

58. Anaerobic media
 Anaerobic bacteria need reduced oxidation –
reduction potential and extra nutrients. Such media
may be reduced by physical or chemical means.
Boiling the medium serves to expel any dissolved
oxygen.
Addition of 1% glucose, 0.1% thioglycollate, 0.1%
ascorbic acid, 0.05% cysteine or red hot iron filings
can render a medium reduced.
 Robertson cooked meat that is commonly used to
grow Clostridium spps medium .
Thioglycollate broth contains sodium thioglycollate,
glucose, cystine, yeast extract and casein
hydrolysate.
Methylene blue or resazurin is an oxidation-
reduction potential indicator that is incorporated in
the medium. Under reduced condition, methylene
blue is colourless.

60. Study of Micro-organisms
using PDA Medium

77. Study of Oligotrophic
Bacteria on Nutrient-deficient
medium