Lecture 4 notes

PHYSICAL FACTORS FOR BACTERIAL GROWTH

They include Oxygen, carbon dioxide, temperature, moisture, pH, pressure and light.

Oxygen: In terms of oxygen utilization, bacteria are classified as follows;

 Strict (Obligate) aerobes: Depend solely on oxygen supply.
 Strict (Obligate) anaerobes: Do not need oxygen at all.
 Microaerophilic or aerotolerant: Require very low oxygen levels for growth but cannot
tolerate the levels of O2 present in an air atmosphere.
 Facultative anaerobes: Grow better in presence of oxygen but can still grow even in
absence of oxygen. They do not require O2 for growth although they may use it for
energy production if it is available.
Some anaerobes can tolerate low levels of O 2 (non-stringent or tolerant anaerobes), but others
(stringent or strict anaerobes) cannot tolerate even low levels and may die upon brief exposure
to air.

Temperature: The temperature that allows for most rapid growth during a short period of time
(12-24 hours) is known as optimum growth temperature.
In reference to temperature, the following classifications apply;
 Psychrophiles: Low temperature required 4-20°C
 Mesophiles: 20 – 40oC required 15-48°C
 Thermophiles: > 40oC required 42-68°C
 Extreme thermophiles: >68°C

Carbon dioxide: CO2 is required by all bacteria and usually available as a product of
metabolism. Slow growing or fastidious organisms may not generate enough CO2 so that this
must be supplied exogenously. The requirement may be increased by environmental change e.g.
caused by the transfer of bacteria from growth in vivo to culture in vitro.

Many pathogenic bacteria therefore require addition of 5 - 10% CO2 to the incubator for primary
isolation in vitro from clinical material.
 Carbon Sources
• Simple carbohydrates, sugars, proteins.
• Some organisms can fix atmospheric CO2
Some bacteria use CO2 as their major, or even sole, source of carbon (autotrophs). Other bacteria
require organic compounds as their carbon source (heterotrophs).

PH (acidity or alkalinity) - hydrogen ion concentration: For most bacteria, the optimum pH for
growth lies between 6.5 and 7.5 and the limits generally lie somewhere between 5 and 9.

Based on pH values requirements, bacteria are grouped into three broad categories.
• Acidophiles: Require pH values of 6 or less for maximum growth, and usually have pH
optimum of 2 - 3.5, and can grow in a range between pH 0.5 and 6.0 e.g. Thiobacillus
thioxidans
• Neutrophiles: Neutrophiles prefer pH values around neutrality (pH 7.0), i.e. pH range
between 6.5 - 7.5.
• Alkalophiles: Alkalophiles grow at pH values 7 - 12 with the optimum around 9.5

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Illumination: Photosynthetic bacteria (phototrophs) must be exposed to the source of

illumination since light is the source of energy.

Hydrostatic pressure: Bacterial growth may also be influenced by hydrostatic pressure.

Bacteria have been isolated from the deepest ocean trenches where the pressure is measured in
tons per square inch, and many of these organisms will not grow in the laboratory unless the
medium is subjected to a similar pressure

Salinity: Halophiles and osmophiles from the sea and other natural water bodies of high salinity
can grow only when the medium contains an unusually high salt concentration.

Water: Moisture is an absolute requirement for growth of all bacteria; at least 80% of the
bacterial cell mass is water. Bacteria require soluble nutrients for diffusion into the cell.
• Growth is inhibited in strong solutions.
• Bacteria with defective cell walls burst in very weak solutions.
Bacteria require water, and all nutrients must be in aqueous solution before they enter the cells.

Other requirements for Bacterial Growth

• Nitrogen source
• Essential nutrients
• Inorganic ions, iron

BACTERIAL REPRODUCTION AND GROWTH

Reproduction in bacteria is usually asexual. The modes of cell division in bacteria include binary
fission, budding, fragmentation and formation of conidiospores or sporangiospores.

Binary fusion: A single cell divides to form two new cells of equal size after developing a
transverse septum (cross-wall). Transverse binary fission is the most common and important
mode of cell division in the usual growth cycle of bacterial populations.

Budding: Some bacteria such as Rhodopseudomonas acidophila reproduce by budding, a

process in which a small protuberance (bud) develops at one end of the cell. The bud enlarges
and eventually develops into a new cell which separates from the parent.

Fragmentation: Bacteria that produce extensive filamentous growth, such as Nocardia species
reproduce by fragmentation of the filaments into small bacillary or coccoid cells, each of which
gives rise to new growth.

Formation of conidiospores/sporangiospores: Species of the genus Streptomyces and related

bacteria produce many spores per organism by developing cross-walls (septation) at the hyphal
tips. Each spore gives rise to new organism.

GROWTH OF BACTERIA
The term growth as commonly applied to bacteria refers to the changes (increase) in the total
population rather than an increase in the size or mass of an individual organism.

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The most common means of bacterial reproduction is binary fission. Thus, if we start with a
single bacterium, the increase in population is by geometric progression:
 1....2.....4.....8......16......32.....................
 1....21......22.......23.......24.........25...........2n
The rate of growth is limited by:
 the availability of nutrients
 temperature
 ability to remove toxic products
The time required to divide is called the generation time.
 for most organisms, it is measured in minutes
 Fast - as little as 10 min. generation time (Vibrio vulnificus) as long as 24 hr.
(Mycobacterium tuberculosis)

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Bacterial Growth Curve

Lag Phase
• No growth
• No cell division has started
• The cells are adapting to environment
• Active synthesis of enzymes and other constituents and metabolic processes are now
taking place.
Log (i.e. logarithmic) /Exponential Phase
• Maximal growth rate
• Increased metabolic rate
• Rapid reproduction/ Increased cell number and increased total cell mass
• Antibiotics most active
Stationary Phase
• Slow growth then stops due to nutrient depletion and waste accumulation
• Equilibrium of dying cells and new cells i.e Rate of reproduction equals rate of cell death
• Cell variation is taking place as spores form and gram negative bacteria test
negative. Decline/Death Phase
• Caused by decreased viability leading to cell death due to toxic waste accumulation
• There is decrease in selective permeability leading to lysis
• Ability to reproduce decreases
• Death rate exceeds reproduction

ISOLATION OF MICROORGANISMS IN PURE CULTURE

To study the properties of a given organism, it is necessary to handle it in pure culture free of all
other types of organisms.
To do this, a single cell must be isolated from all other cells and cultivated in such a manner that
its collective progeny also remain isolated.

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Plating
Unlike cells in a liquid medium, cells in or on a gelled medium are immobilized. Therefore, if
few enough cells are placed in or on a gelled medium, each cell will grow into an isolated
colony. The ideal gelling agent for most microbiologic media is agar, an acidic polysaccharide
extracted from certain red algae. A 1.5–2% suspension in water dissolves at 100°C, forming a
clear solution that gels at 45°C. Thus, a sterile agar solution can be cooled to 50°C, bacteria or
other microbial cells added, and then the solution quickly cooled below 45°C to form a gel.

Once gelled, agar will not again liquefy until it is heated above 80°C, so that any temperature
suitable for the incubation of a microbial culture can subsequently be used.

Three techniques
1. Pour-plate method, a suspension of cells is mixed with melted agar at 50°C and poured
into a Petri dish. When the agar solidifies, the cells are immobilized in the agar and grow
into colonies. If the cell suspension is sufficiently diluted, the colonies will be well
separated, so that each has a high probability of being derived from a single cell. To make
certain of this, however, it is necessary to pick a colony of the desired type, suspend it in
water, and re-plate. Repeating this procedure several times ensures that a pure culture
will be obtained.

1 ml 1ml 1ml 1ml

Original 9 ml H2O (10–1 dil) 9 ml H2O (10–2 dil) 9 ml H2O (10–3 dil) 9 ml H2O (10–4
dil) Sample

1.0 ml Mix with warm 1.0ml

Agar and pour

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2. Streak-plate technique - the original suspension can be streaked on an agar plate with a
wire loop. As the streaking continues, fewer and fewer cells are left on the loop, and
finally the loop may deposit single cells on the agar. The plate is incubated, and any well-
isolated colony is then removed, re-suspended in water, and again streaked on agar. If a
suspension (and not just a bit of growth from a colony or slant) is streaked, this method is
just as reliable as and much faster than the pour-plate method.

Steps in a Streak Plate

An example of a streak plate

3. Spread plate technique, a small volume of dilute microbial suspension containing a 30–
300 cells is transferred to the center of an agar plate and spread evenly over the surface
with a sterile bent-glass rod. The dispersed cells develop into isolated colonies. Because
the number of colonies should equal the number of viable organisms in a sample, spread
plates can be used to count the microbial population.

Bacterial Cell Count and Bacterial Mass

The colony counting method: The number of living cells in a given culture or material can be
determined by means of the colony counting method. The samples are diluted logarithmically by
a dilution factor of 10. Using the pour plate technique, each dilution is mixed with 1 ml of liquid

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agar and poured out in a plate. In the surface inoculation method, 0.1 ml of each dilution is
plated out on a nutrient agar surface. The plates are incubated, resulting in colony growth. The
number of colonies counted, multiplied by the dilution factor, results in the original number of
viable bacterial cells (CFU = colony forming units).
Bacterial mass: The bacterial mass can be established by weighing (dry or wet weight). The
simplest way to determine the mass is by means of photometric adsorption measurement. The
increases in mass and cell count run parallel during phase C on the growth curve.

CULTURE MEDIA
For good identification and valid laboratory results, there is need of broad selection of media for
culture of micro-organisms.

Culture media: Mixtures of nutrients that the microbe need to live. Also it provides a surface
and the necessary moisture and pH to support microbial growth. A culture media neutralize any
toxic materials produced by the growing organism.
The grown organisms should be incubated in the correct atmosphere, at the optimum temperature
and for an adequate period of time.
Uses:
1. Isolation and identification of micro-organisms
2. Performing anti-microbial sensitivity tests
Common ingredients of culture media
 Peptone
 Meat extract
 Yeast extract
 Mineral salts
 Carbohydrates
 Agar
 Water
Peptone: Hydrolyzed product of animal and plant proteins: Free amino acids, peptides and
proteoses (large sized peptides). It provides nitrogen; as well as carbohydrates, nucleic acid
fractions, minerals and vitamins.
Meat extract: supply amino acids, vitamins and mineral salts.
Yeast extract: It is bacterial growth stimulants.
Mineral salts: these are:
 Sulfates as a source of sulfur.
 Phosphates as a source of phosphorus.
 Sodium chloride
 Other elements
Carbohydrates: Simple and complex sugars are a source of carbon and energy. Carbohydrates
assist in the differentiation of bacteria. Eg. Sucrose in TCBS agar differentiates vibrio species.
Lactose in MacConkey agar differentiates enterobacteria.
Agar: It is an inert polysaccharide of seaweed. It is not metabolized by micro-organism.
Property
 It has high gelling strength; high melting temperature (90-95 oC); low gelling temperature
 It forms firm gel at 1.5% W/V concentration.
 It forms semisolid gel at 0.4-0.5% W/V concentration.

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Uses:
 Solidify culture media
 May provide calcium and organic ions to inoculated bacteria.
Water
Deionized or distilled water must be used in the preparation of culture media.

TYPES OF CULTURE MEDIA

Media is classified into (5) five classes depending entirely on;
i. The ingredients the media contains
ii. What types of micro-organisms are capable of growing in it?
The five classes of media are;
1) Basic media
2) Enriched
3) Enrichment media
4) Selective media (inhibitory media)
5) Differential (indicator media)
Basic /Simple / All-purpose media
These are simple culture media, which can be in solid or liquid form. They contain the basic
nutritional requirements for the bacterial growth and do not require special nutrients.
Uses
 To maintain stock cultures of control bacterial strains
 To subcuture pathogenic bacteria from selective/differential medium prior to performing
biochemical or serological tests. Eg. Nutrient Broth; Nutrient Agar
 Used in setting of drug susceptibility testing
 Used for sub-culturing micro-organisms for selective and differential media
 Used in preparation of enriched media
Enriched media
Media that are enriched with whole blood, lysed blood, serum, peptones, special extracts or
vitamins to support the growth of pathogenic bacteria.
Uses
 It is used in growth of fastidious organisms that requires the extra nutritional growth
requirements. Such organisms are Haemophillus influenzae, Neisseria Spps,
Streptococcus Spps, TB, Corynebacterium Diphtheriae etc.
Example
 Blood agar medium (BA)
 Sheep blood agar medium (SBA)
 Chocolate blood agar media (CBA)
 Cysteine lactose electrolyte deficiency medium (CLED)
 Lowenstein Jensen medium (T.B)
 Loeffler’s media (C. diphtheriae)
 Robertson cooked meat medium (anaerobic organisms)
Enrichment media
Fluid media (broth or liquid form) that favors growth or suppresses growth of some
microorganism due to the production of toxins in the media.
Uses

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 Used for culturing micro-organisms that are to be sub-cultured to selective media e.g.
DCA (Deoxycholate Citrate Agar), XLD (Xylose lysine deoxycholate agar), SS, TCBS
(Thiosulphate citrate bile salts) etc.
Examples
 Selenite F broth for salmonella and Shigella Spps
 Alkaline peptones water broth for Vibrio. Cholera, Yersinia. Pestis
 Tetrathionate broth for Salmonella and Shigella spps.
Selective media (inhibitory media)
Media which contain inhibitory substances (e.g. Antibiotics) that prevent or slow down the
growth of bacteria other than pathogens for which the media are intended.
Uses
 Used to inhibit growth of organisms other than the microorganisms in which the media is
designed for.
 Used for culturing specimen from a site with normal floras to prevent growth of
unwanted micro –organisms.
Examples
 DCA (Deoxycholate Citrate Agar) for Salmonella. Shigella Spps
 SS (Salmonella / Shigella) media for Salmonella/Shigella Spps
 TCBS (Thiosulphate citrate bile salts) for Vibrio. cholerae
 Buztler media for Campylobacter Spps.
 Xylose lysine deoxycholate agar (XLD) for Salmonella/Shigella Spps.
Differential media (indicator media)
Media to which indicator substances are added to differentiate bacteria e.g. dyes or any other
changeable substances that can be visibly changed as a result of metabolic activities of a
particular micro-organism, which can be shown with an indicator (change of colour) or blood
cells reaction (haemolysis). Examples include
MacConkey media – Contain indicator (neutral red) which changes to pink after fermentation of
carbohydrates (LACTOSE), differentiating lactose fermenters e.g. (E. coli, Klebsiella organisms
from non-lactose fermenters e.g. pseudomonas spps, proteus spps.
SBA (Sheep blood agar) -Differentiate haemolytic organisms from non-haemolytic organisms e.g.
Streptococcus. pyogenes.
TCBS (Thiosulphate citrate bile salts) -Has an indicator Bromothymol blue which changes to
yellow after fermentation of sugar (sucrose), differentiating sucrose fermenters from non –
sucrose fermenters e.g. Vibrio. cholerae

Choice of culture media

The selection of culture media will depend on:
1. The major pathogens to be isolated, their growth requirements and the features by which
they are recognized.
2. Whether the specimens being cultured are from sterile sites or from sites having normal
microbial flora.
3. The cost, availability and stability of media.
4. The training and experience of laboratory staff in preparing, using and controlling culture
media.
Forms of culture media
 Solid culture media

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 Semisolid culture media

 Fluid culture media
Solid culture media
 Plate cultures in petri dishes
 Stab/slope cultures in tubes and bottles
Uses:
Description of bacterial colonies in terms of
 Size (diameter in mm)
 Out line (circular, entire, wavy, indented)
 Elevation (flat, raised, low convex and dome shaped).
 Transparency (transparent, opaque, and translucent).
 Surface (smooth (mucoid) and shiny, rough and dull).
 Colour (colourless, white, pink, and pigmented)
 Changes in medium E.g. Haemolysis in Blood Agar Blackening of medium due to
hydrogen sulphide production.
Semisolid culture media
Uses:
 As an enrichment media
 As motility media e.g mannitol motility test media
Fluid culture media
Bacterial growth in fluid media is shown by turbidity in the medium.
Uses
 As an enrichment media
 As biochemical testing media
 As blood culture media

Characteristics of representative media used to cultivate bacteria

Blood agar - Complex medium used routinely in clinical labs. Differential because colonies of
hemolytic organisms are surrounded by a zone of clearing of the red blood cells. Not selective.
Chocolate agar - Complex medium used to culture fastidious bacteria, particularly those found
in clinical specimens. Not selective or differential.
Glucose-salts - Chemically defined medium. Used in laboratory experiments to study nutritional
requirements of bacteria. Not selective or differential.
MacConkey agar - Complex medium used to isolate Gram-negative rods that typically reside in
the intestine. Selective because bile salts and dyes inhibit Gram-positive organisms and Gram-
negative cocci. Differential because the pH indicator turns pink-red when the sugar in the
medium, lactose, is fermented.
Nutrient agar - Complex medium used for routine laboratory work. Supports the growth of a
variety of non-fastidious bacteria. Not selective or differential.
Thayer-Martin - Complex medium used to isolate Neisseria species, which are fastidious.
Selective because it contains antibiotics that inhibit most organisms except Neisseria species.
Not differential.

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