LECTURE 2: Ardhea Mustika Sari, S.T.P., M.

Sc
Department of Food Science and Technology

THE METHOD OF MICROBIOLOGY Universitas Sebelas Maret

MICROBIAL CULTURE
• due to microorganism’s size are very small, observation of single
microbe is difficult  obtaining information can be done by
examination population of microorganism
• Such populations are obtained by growing microorganisms, under
more or less well-defined conditions, as CULTURES
• Pure Culture  culture that contains only one kind of microorganism
• Mixed Culture  contains more than one kind of microorganism

• how to obtain microbial cultures?

 PROCEDURE TO OBTAIN MICROBIAL CULTURE
Isolation
• the separation of a particular microorganism
from the mixed populations that exist in nature
Cultivation
• the growth of microbial populations in
artificial environments (culture media) under
laboratory conditions.
CULTURE MEDIA
 A culture medium is a solid or liquid preparation used to grow, transport, and
store microorganisms
 Suitable culture medium  need for microbial growth and maintenance in
laboratory
 Isolation and identification need special media  identification microorganism in
water and food, industrial microbiology, testing for antibiotic sensitivity
 Although all microorganisms need sources of energy, carbon, nitrogen,
phosphorus, sulfur, and various minerals, the precise composition of a satisfactory
medium will depend on the species  because nutritional requirements vary so
greatly.
 Knowledge of a microorganism’s normal habitat often is useful in selecting an
appropriate culture medium  nutrient requirements reflect it’s natural
surrounding
DEFINED MEDIUM
 Medium that all components are
known
 Many chemoorganotrophic
heterotrophs also can be grown in
defined media  glucose ac carbon
source and ammonium salt as nitrogen
source
 Widely use in research
COMPLEX MEDIUM
Media that contain some ingredients
of unknown chemical composition
This media is useful as a single
complex medium  sufficiently rich
and complete to meet nutritional
requirements of many different
microorganisms
complex media often are needed
because the nutritional requirements
of a particular microorganism are
unknown, and thus a defined medium
cannot be constructed
TYPE OF MEDIA
enriched
• General purpose fortified by
special nutrient to encourage
the fastidious heterotroph
• Ex: Blood agar
selective differential
• favor the growth of • Media that use for distinguish
particular microorganisms between different group of
• For isolation specific bacteria
mikroorganism • permit tentative identification
• Ex: Endo agar, eosin of microorganisms based on
methylene blue agar, and their biological
MacConkey agar characteristics
• MacConkey agar is both
• differential and selective. 
contains lactose and neutral
red dye, lactose-fermenting
colonies appear pink to red
in color and are easily
distinguished from colonies of
non fermenters.
CULTURING MICROBES
The Five “I’s
Inoculation: Producing a pure culture
Isolation: Colony on media, one kind of microbe, pure culture
Incubation: growing microbes under proper conditions
Inspection: Observation of characteristics (data)
Identification: use of data, correaltion, to ID organism to exact
species
CULTURING MICROBES
Innoculation: Producing a pure culture
Introduce bacteria into a growth medium using “aseptic technique” to prevent
contamination. Tools: Bunsen burner, loop. Needle, etc.
CULTURING MICROBES

Innoculation: Producing a
pure culture
Introduce bacteria into a
growth medium using
“aseptic technique” to
prevent contamination.
Tools: Bunsen burner, loop.
Needle, etc.
CULTURING MICROBES
Isolation: Colony on media, one kind of microbe, pure culture
CULTURING MICROBES
CULTURING MICROBES-ISOLATION

Isolation: Colony on media,

one kind of microbe, pure
culture. Many colonies?
Use a needle, pick one, and
redo streak plate
CULTURING MICROBES-IDENTIFICATION

Blood agar : rich with nutrients, can see a difference, thus differential; much more
later
INCUBATION
Incubation: Allow organisms to grow
under the optimal conditions
Temperature, with or without oxygen
etc
INCUBATION

Incubation: Allow organisms

to grow under the optimal
conditions
Temperature, with or
without oxygen etc
Candle jar reduces oxygen
CULTURING MICROBES
Inspection: Observation, description
Colony Morphology, Microscopic examination (grams stain)
GRAM STAINING
MICROSCOPIC STUDY
Microscopic study: Gram + bacilli, Gram - bacilli
MICROBIOLOGY – CHAPTER 3
Microscopic study: Acid fast, and capsule
MICROBIAL GROWTH IN BROTH MEDIUM
ANY QUESTIONS???