Microorganisms are studied through a process involving 5 basic techniques: inoculation, incubation, isolation, inspection, and identification. Specimens are collected and inoculated into nutrient media to promote growth. After incubation, isolated colonies are examined microscopically and through biochemical tests to identify the microbe. A variety of media types are used for different functions like selecting for certain microbes or enabling differentiation between types.

1. THE METHODS FOR
STUDYING
MICROORGANISMS

2. Specimen collection: Nearly any object, common
ones are body fluids and tissues, foods, water
or soil. Specimens are removed by some form
of sampling device. This may be a swab,
syringe, or a inoculation needle and
inoculation loop that holds, maintains and
preserves the microbes in the sample.

3. The Five basic techniques to grow,
examine and characterize
microorganisms
 INOCULATION
 INCUBATION
 ISOLATION
 INSPECTION
 IDENTIFICATION

4. Inoculation(producing a culture)
 To cultivate or culture, one introduces a tiny
sample(the inoculum)into a container of a
nutrient medium which provides an
environment in which they multiply
 Selection of media with specialized functions
can improve later steps of isolation and
identification.

5. Incubation
 To adjust the proper growth conditions of a
sample
 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

6. Isolation
 The end result of inoculation and incubation in
macroscopic form
 The isolated microbes may take the form of
separate colonies (discrete mounds of cells)
on solid media, or turbidity in broths

7. Methods for isolating bacteria
 Streak method
-a small droplet of culture or sample is spread
over the surface of the medium according to a
pattern that gradually thins out the sample and
separates the cells spatially over several
sections of the plate

8.  Loop Dilution/ pour plate
-the sample is inoculated serially into a series of
cooled but still liquid agar tubes so as to dilute
the number of cells in each successive tube in
the series. Inoculated tubes are then plated
out into sterile plates and are allowed to
solidify. The end result is that the number of
cells per volume is so decreased that cells
have ample space to grow into separate
colonies

9.  Spread plate technique
-A small volume of liquid diluted sample is
pipetted onto the surface of the medium and
spread around evenly by a sterile tool (like a
hockey stick).Like the streak method, cells are
pushed into separate areas on the surface so
that they can form individual colonies

10. Media: Providing Nutrients in the
lab
3 Primary levels of media classification:
1. Physical form
2. Chemical composition
3. Functional type

11. Physical state
(medium’s normal
consistency
Chemical composition
( type of chemicals a
medium contains)
Functional type
(purpose of medium)
1. Liquid
2. Semisolid
3. Solid(can be
converted into liquid
4. Solid (cannot be
liquefied)
1. Synthetic (chemically
defined)
2. Nonsynthetic (not
chemically defined)
1. General purpose
2. Enriched type
3. Selective
4. Differential
5. Anaerobic growth
6. Specimen transport
7. Assay
8. enumeration

12. Physical states of media
 Liquid media- are water based solutions that
do not solidify at temperatures above freezing
and that tend to flow freely when the container
is tilted. These media, termed broths, milks or
infusions, are made by dissolving various
solutes in distilled water. Growth occurs
throughout the container and can then present
a dispersed, cloudy or particulate appearance.

13.  Semisolid media- exhibit a clotlike consistency
because they contain an amount of solidifying
agent(agar or gelatin) that thickens them but
does not produce a firm substrate. Semisolid
media are used to to determine the motility of
bacteria and to localize a reaction at a specific
site.(0.3-0.5% agar) ex. SIM(hydrogen sulfide
production and indole reaction)

14.  Solid media- provide a firm surface on which cells can form
discrete colonies and are advantageous in isolating and
culturing bacteria and fungi.
2 forms
1. Liquefiable- contain a solidifying agent that changes its
physical properties in response to temperature. Agar a
complex polysaccharide isolated from the red alga
Gelidium. It is solid at room temperature, and it melts at the
boiling temperature of water (100). Ex. Nutient agar(beef
extract and peptone, as well as 1.5% agar by weight)
2. Nonliquefiable –they do not melt. They include materials
such as rice grains (to grow fungi), cooked meat
media(anaerobes), and potato slices

15. Chemical content of Media
 Synthetic- contain pure organic and inorganic
compounds
 Nonsynthetic/compex media-contain at least
one ingredients that are not chemically
definable- extracts of animals, plants or
yeasts, ground up cells, tissues and
secretions, milk and peptone . Blood agar,
nutrient broth and MacConkey agar.

16. Media to suit Every Function
1. General purpose media- are designed to grow as
broad spectrum of microbes as possible. As a
rule, they are nonsynthetic and contain a mixture
of nutrients that could support pathogens and
nonpathogens alike. Ex. BHI, TSA
2. Enriched medium- conains complex organic
substances such as blood serum, hemoglobin or
special growth factors that certain species must
have inorder to grow. Bacteria that require
growth factors and complex nutrients are
fastidius. Ex. Blood agar to grow fastidius
streptococci and other pathogens. Thayer Martin
medium- for neisseria

17.  3. Selective medium- contains one or mre
agents that inhibit one or more agents that
inhibit the growth of a certain microbe or
microbes but not others and thereby selects or
encourages microbe nd allow it grow.MSA
contains a concentration of NaCl that is quite
inhibitory to most human pathogens except
staphyloccoccus.

18. Selective media, agents and
functions
Medium Selective agent functions
Mueller tellurite
Enterococcus faecalis
broth
Phenylethanol agar
Tomato juice agar
MacConkey agar
Salmonella/shigella agar
Lowenstein-Jensen
Sabourauds agar
Potassium tellurite
Sodium azide
tetrazolium
Phenylethanol chloride
Tomato juice, acid
Bile, crystal violet
Bile, citrate, brilliant
green
Malachite green dye
Ph 5.6(acid)
C. diptheriae
Fecal enterococci
Staph and Strep
Lactobacilli from saliva
Gram negative enterics
Salmonella and shigella
Mycobacteria
Fungi, inhibits bacteria

19. Differential media
 Grow several types of microorganisms and
are designed to display visible differences
among those microorganisms. Differentiation
shows up as variations in colony size or color,
in media color changes or in the formation of
gas bubbles and precipitates

20. medium Substances that
facilitate
differentiation
Differentiates between
Blood agar Intact red blood cells
Manitol,phenol and 7.5%
Types of hemolysis
Species of stap. NaCl
Hektoen enteric agar
Spirit blue agar
Urea broth
Sulfur indole motility
Triple sugar iron gar
Nacl
Bromthymol blue, acid
fushin,sucrose, salicin,
thiosulfate, ferric
ammonium citrate, and
bile
Spirit blue dye and oil
Urea, phenol red,
thiosulfate, iron
Triple sugars, iron and
phenol red dye
Also inhibits the salt-
sensitive species
Salmonell, shigella and
othe lactose fermenters
and nonfermenters
Dyes and bile also inhibit
gram positive bacteria
Bacteria that use fats
from those that do not
Bacteria that hydrolyze
urea to ammonia
H2S gas producers from
nonproducers
Fermentation of sugars,
H2S production

21. medium Substances that
facilitate differentiation
Differentiates between
XLD agar
Birdseed agar
Lysine, xylose, iron,
thiosulfate,phenol red
Seeds from thistle plant
Enterobacter,
escherichia, proteus,
providencia, salmonella
and shigella
Cryptococcus
neoformans and other
fungi

22. Miscellaneous media
 A reducing medium contains
substance(cystine or thioglycollic acid) that
absorbs oxygen or slows the penetration of
oxygen in a medium, thus reducing its
availability. Reducing media are important for
growing anaerobic bacteria or determining
oxygen requirements.

23. Transport media
 Are used to maintain and preserve specimens
that have to be held for a period of time for
clinical analysis or to sustain delicate species
that die rpidly if not held under stable
conditions.

24. Assay media
 To test effectiveness of antimicrobial drugs and
by drug manufacturers to assess the effect of
disinfectants, antiseptics, cosmetics and
preservatives on the growth of microorganisms

25. Enumeration media
 Used by industrial and environmental
microbiologists to count the numbers of
organisms in milk, water, food, soil and other
samples

26.  A culture may exist in one of the following
forms:
1. Pure
2. Mixed
3. contaminated

27. Inspection
 Cultures are observed macroscopically for
obvious growth characteristics(color,
texture,size) that coild be useful in analyzing
specimen contents. Slides are made to assess
microscopic details such as shapes, size, and
motility. Staining techniques may be used to
gather specific information on microscopic
morphology

28. Identification
 A major purpose of the 5 I’s is to determine the
type of microbe, usually to the level of species.
Specialized tests include biochemical tests to
determine metabolic activities specific to the
microbe.