SAN PEDRO COLLEGE MICROBIOLOGY

Respiratory Therapy Department

Charisa Antonette S. Huelva – 2B

CULTURING PROCEDURES same type of bacterial cells because they all are
descendants of the first cell
Inoculation  the end result of inoculation and incubation is isolation of
 a process of cultivating microorganisms by introducing a the microbe in macroscopic form
tiny sample (the inoculum) into a container of nutrient  isolated microbes may take the form of separate colonies
medium, which provides an environment in which they (discrete mounds of cells) on solid media, turbidity in
multiply broths
 any instrument used for sampling and inoculation must be  also known as subculturing, involves taking a tiny bit of
sterile growth from an isolated colony and inoculating a separate
 an observable growth that appears in or on the medium medium
after incubation is known as culture
 the type of media depends on what you are culturing
 samples can be taken everywhere
 the sample is placed into a container of sterile medium that
provides microbes with the appropriate nutrients to sustain
growth
 involves using a sterile tool to spread the sample on the
surface of a solid medium or to introduce the sample into a
flask or tube
 selection of media with specialized functions can improve
later steps of isolation and identification
 some microbes may require a live organism as the
inoculation medium

Technique: inoculate a large area with few bacteria. Spread

out the cells so they are far apart

Inspection
 the cultures are observed macroscopically for obvious
growth characteristics (color, texture, size) that could be
useful in analyzing the specimen contents
 slides are made to assess microscopic details such as cell
shape, size, and motility
 staining techniques may be used to gather specific
Incubation information on microscopy morphology
 the next step after inoculation  examine growth
 the inoculated medium placed in a temperature-controlled Colony Morphology – appearance of a colony
chamber (incubator) for multiplication or growth of - size, shape, texture & color are characteristics of the
microorganisms bacteria
 signs of microbial growth: cloudiness, sediment, or color Microscopic Morphology – take a sample of colony & look
 “appearance of colonies” is the most common at it under the microscope
manifestation of growth on solid media especially with - with staining techniques, you can make out the shape, size
bacteria and fungi and motility of the bacterial cell
 an incubator can be used to adjust the proper growth
conditions of a sample
 setting the optimum temperature and gas content promotes
multiplication of the microbes over a period of hours, days
and even weeks
 produces a culture – the visible growth of the microbe in
the medium

Identification
 a major purpose of the 5 I’s is to determine the type of
microbe, usually to the level of species
 summaries of characteristics are used to develop profiles of
the microbes isolated from the sample
 information can include relevant data already taken during
Isolation
inspection and additional tests that further describe and
 In order to separate one type of bacteria from another, you differentiate the microbes
must isolate each cell from one another
 specialized tests include biochemical tests to determine
 one isolated bacterial cell will multiply and grow into a metabolic activities specific to the microbe, immunologic
mound of cells called a colony. A colony consists of all the tests, and genetic analysis
 How can you tell what kind of microorganism you have? –
test their biochemistry or cell metabolism:
- kind of sugars they digest
- products given off during growth
- enzymes they have
- mechanisms for deriving energy
- use or no use of oxygen
These are biochemical tests – used to figure out exactly what
species of bacteria you have.
1. Pure Culture/Axenic Serial Dilution
 i  a series of sequential dilutions used to reduce dense culture
s of cells to a more usable concentration
a  each dilution will reduce me the concentration of bacteria
by a specific amount

TYPES OF CULTURES
container of medium that grows only a single known
species or type of microorganism
 frequently used for laboratory study
 allows precise examination
 control of one microorganism MEDIA PREPARATIONS
 can have second-level culture called subculture, which is
Media – “providing nutrients in the laboratory”
from a well-isolated colony transferred into a separate
- extremely varied in nutrient content and consistency
container of media and incubated
- specially formulated for a particular purpose
2. Mixed Culture
- mostly designed for bacteria and fungi
 is a container that holds two or more differentiated species Culture Media
of microorganisms - a solid or liquid preparation used to grow, transport, and
store microorganisms
- much of microbiology depends on the ability to grow and
maintain microorganisms in the laboratory
- microorganism’s normal habitat reflects its nutritional
requirements
Uses:
- it is important to grow microorganisms outside the body
for the following purposes:
 to identify the cause of infection from the clinical
sample so that proper treatment can be given
 to study the characteristics or properties of
microorganisms
 to prepare biological products like vaccines, toxoids,
antigens, etc.
METHOD OF ISOLATING PURE CULTURES Nutrients in Media
 Water – to dissolve materials to be transported across
Streak Plate Method
cytoplasmic membrane
 Small droplet of culture or sample spread over surface of
 Source of C – required for the construction of all organic
the medium with an inoculating loop
molecules
 Uses a pattern that thins out the sample and separates the - Usually glucose
cells
 Source of N
 Used to isolate a single type of bacteria - Proteins: broken down into amino acids
 Spreads out original “parent bacteria” in a sparse pattern - Beef extract: broken down into nucleic acids
that, after growth, results in individual colonies  Buffer System – most bacteria grow at pH 7.0 which is
 After incubation, the 4th quadrant of your plate should have achieved by using a buffer system
dots - Carbonate buffer
 These small “dots” are individual colonies, and represent - Phosphate buffer
millions of bacteria of the same type  Source of minerals – required in small amounts
- Iron
- Sulfur
- Phosphorous

PHYSICAL STATES OF MEDIA

1. Liquid Media
- water-based solutions that do not solidify at temperatures
above freezing and that tend to flow freely when the
container is tilted
- termed also as “broths, milks, or infusions” – are made by
Pour Plate/Loop Dilution dissolving solutes in distilled water
 in this technique, some of the colonies will develop deep in - growth appearance: throughout the container, dispersed,
the medium itself and not just on the surface cloudy, or flaky
- examples:
 nutrient broth (beef-extract + peptone)
  Methylene blue milk and Litmus milk (whole milk and - contains one or agents that inhibit the growth
dyes) of a certain microbe or microbes
 Fluid thioglycollate – slightly viscous broth used to - very important in primary isolation of a
determine patterns of growth in oxygen specific type of microorganism
2. Semisolid Media - hasten isolation by suppressing the unwanted
- exhibits a clot-like consistency under ordinary room background organisms and allowing growth
temperature due to presence of solidifying agent of the desired ones
- used to determine the motility of bacteria and to localize a - examples:
reaction at a specific site  Mannitol salt agar (MSA) – contains
- examples: high concentration of NaCl (7.5%)
 Motility test medium – with a small amount of agar as inhibitory
 Sulfur indole motility (SIM) medium – with small  MacConkey Agar & Hektoen enteric
amount of agar (0.3-0.5%) agar (HE) – contain bile salts as a
- test for physiological characteristics used in selective agent and inhibitory factor
identification Differential Media
3. Solid Media - grow several types of microorganisms but are
- provide a firm surface on which cells can form discrete designed to bring out visible differences
colonies among those microorganisms
- good for isolating and culturing bacteria and fungi - differences: types of chemicals contained in
- Two forms: the media and the ways that microbes react to
 Liquefiable solid media – reversible solid media them
- contains solidifying agent that changes its physical - dyes are considered to be effective for
properties in response to temperature differential agents because their pH indicators
 Non-liquefiable solid media – do not melt that color in response to the production of an
- materials included are rice grains, cooked meat media, acid or a base
egg or serum media 4. Specimen Transport Media
- used to maintain and preserve specimens that
CHEMICAL CONTENT OF MEDIA have to be held for a period of time before
clinical analysis or to sustain delicate species
1. Synthetic that die rapidly if not held under stable
- chemically defined composition condition
- contain pure chemical nutrients and 5. Assay Media
molecular content - Used by technologists to test the effectiveness
2. Non-synthetic or complex of antimicrobial drugs and by drug
- its composition is not definable by an exact manufacturers to asses the effect of
chemical formula disinfectants, antiseptics, cosmetics, and
- from extracts from animal or plant tissues preservatives on the growth of
including such materials as ground-up cells microorganisms
and secretions 6. Enumeration Media
- other examples: blood, serum, meat extract, - used by industrial and environmental
infusions, milk, soybean digests, and peptone microbiologists to count the numbers of
- nutrient both, blood agar, and MacConkey organisms in milk, water, food, soil, and
Agar presents rich mixture of nutrients for other samples
microbes with complex nutritional needs
1. General-purpose media

FUNCTIONAL TYPE
- designed to grow a broad spectrum of
microbes that do have special growth
requirements
- nonsynthetic (complex) and contain a mixture
of nutrients that could support the growth of
variety of bacteria and fungi
- examples: nutrient agar and broth, brain-heart
inclusion, and trypticase soy agar (TSA)
TSA – a complex medium that contains
partially digested milk protein (casein),
soybean digest, NaCl and agar
2. Enriched medium
- contains complex organic substances such as
blood, serum, hemoglobin, or special growth
factors
Growth factors
- provided in species in order to grow
- organic compounds such as vitamins and
amino acids that the microbes cannot
synthesized themselves
- “fastidious” for bacteria requires growth
factors and complex nutrients
Blood Agar – made by adding sterile animal
blood (sheep) to a sterile agar base
3. Selective and Differential Media
- cleverest and most inventive media recipes
- designed for special microbial groups
- have extensive applications in isolation and
identification
- permits preliminary identification of a genus
or even a species
Selective Media